Growth Substance and Cell Elongation Citation Bonner, James Frederick (1934) Growth Substance and Cell Elongation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/86ND-SX71. https://resolver.caltech.edu/CaltechETD:etd-11252003-083628 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: Biology Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Unknown, Unknown Thesis Committee: Unknown, Unknown Defense Date: 1 January 1934 Record Number: CaltechETD:etd-11252003-083628 Persistent URL: https://resolver.caltech.edu/CaltechETD:etd-11252003-083628 DOI: 10.7907/86ND-SX71 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 4658 Collection: CaltechTHESIS Deposited By: Imported from ETD-db Deposited On: 26 Nov 2003 Last Modified: 03 Oct 2019 23:37 Thesis Files Preview PDF (Bonner_j_1934.pdf) - Final Version See Usage Policy. 13MB Repository Staff Only: item control page

GROWTH SUBSTANCE AND CELL ELONGATION Thesis by James Bonner. In Partial Fulfillment of the Requirements for the Degree of Doctor: of Philosophy OALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 1934 —Foreword,— It is with the deepest gratitude that this paper is dedicat~ sed to the memory of Dr.H.5,Dolk, under whose guidance the author be~ gan the study of growth substance.To Dr.Dolk is due in addition a great deal of the credit for the Laboratory of Plant Physiology which has made this type of work possible at the California Institute of Technology. | | The author expresses his appreciation to Dr.F.¥.Went both for his stimulating teaching,his many suggestions,end his indispen~ able advice. | To Dr.K.V¥.Thimann, with whom a portion of this work was carried out, the author gives the most sincere thanks,not only for actual assistance,but also for the unfailing supply of good ideas and useful information, The work upon respiration was done with the equipment of Dr.R. Emerson whom the authow desires to thank for his advice and suggestions as well as for the use of his apparatus, For the growing of a large number of Avena plants and for much other valuable assistance the author is greatly indebted to Mrs. F,Dolk. Finably,the author expresses to all of the members of the Staff of the Biology Department his appreciation for the criticism, suggestions, and assistance, which have been of continuous value during the prosecution of this work. PART I. PART II. PART III. PART IV. TABLE OF CONTENTS, The Study of Cell Elongation before the Discovery of Growth Substance a) Introduction | b) Osmotic Pressure of the Cell Sap e) Elasticity | 4) Plasticity @) Permeability £) Intussusception g) Imbibition Pressure The Study of Cell Elongation Since the Discovery of Growth Substance a) Discovery and Isolation of Growth Substance b) The Avena Seedling and Coleoptile c) The General Relation of Growth Substance to the Coleoptile a) Growth Substance and the Deformability of the Coleoptile @) Otjects of the Present Work A General Discussion of the Materials and Methods a) Plants b) Growth Substance c) Units The Hormonal Nature of Growth Substance a) Introduction b) Methods ¢) The Amount of Growth Substance Entering the Plant a) The Amount of Growth Corresponding to a Given Amount of Growth Substance at 27°. a On WU F F NY Fe 10 il 13 15 17 17 18 19 20 22 PART IV. PART VI. ii The Hormonal Nature of Growth Substance (cont.) e) f) 2) h) i) The a) ») ¢) a) The Amount of Growth Corresponding to a Given Amount of Growth Substance at 15°. The Composition of the Cell Wall. The Volume of the Cell Walls The Ratio of Cell Wall Constituents Produced to Growth Substance Used. Conclusions Relation of Wall Formation to Gell Elongation Introduction | Methods Experimental Results Conclusions Conditions Governing the Elongation of Excised Coleoptile Sections, a) >) e) a) @) f) 8) h) i) Introduction Methods Elongation in Water Alone Stirring Former Position of Section in the Coleoptile Growth Substance Concentration Fructose | Inhibition of the Aerobic Metabolism Conclusions 30 31 33 35 52 53 Bu 55 59 61 62 62 63 64 67 aii PART VI. The Relation of Hydrogen Ions to Elongation. a) Acid Growth and Acid Curvatures b) The Nature of the Phenomena c) Acidity of the Coleoptile during Growth a) Dependence of Acid Growth on Growth Substance @) Discussion and Conclusions PART VIII. Growth and Respiration. a) Introduction b) Methods c) The a) The Goleoptile as a Whole Elongation and Respiration during the ‘Grand Period Distribution of Growth and Respiration in the Coleoptile Rapidly Growing Zone Changes of Growth and Respiration with Time Effect of Cyanide upon Growth and Respiration Action of Growth Substance upon Growth and upon Respiration Effect of Temperature on the two processes Characteristics of Growth and Non-growth Respiration The Action of Growth Substance e) Discussion f) Summary CONCLUSIONS LITERATURE 74 76 85 87 93 104 111 113 117 120 121 122 Leu 128 132 136 138 152 154 PART 1. THE STUDY OF CELL ELONGATION BEFORE THE DISCGVERY OF GROWTH SUBSTANCE 2. Introduction The study of cell elongation is as old ag the study of plant growth for the reason that the. visible incrense in size of a plant organ is principally due to cell elongation. The first fruit- ful theory, however, concerning the nature of the processes and forces involved was enunciated by Sachs (1874). According to Sachs, the cell wall is elastioally stretched, by the pressure of the cell contents. ue to this stretching, the particles of which the wall is composed become further apart and new ones are then set down between them. This relieves the pressure of the wall and the cycle repeats itself ad infinitum or as long as the aell contents tan exert, prosaure on the wall. The theory contains the essential points, a) elastic stretching of the cell wall by internal pressure, b) intus— susceotion of new wall material. The first point was regarded as substantiated by the fact that as found by DeVries (1874), Laurent (1885) and others, the amount of elastic stretching of growling tissues is greatast at the time of fastest growth. The second point was based upon the work of Wigeli (1858) who had concluded that the growth of call walls ss well asretarch grains is by intussusception. It may be said at once, however, that the theory of Sachs has been fruitful principally because of the investigations which have been instigated for its attack or defence. Schwendener and Krabbe (1895) failed to find the proportionality between turgor stretching and growth rate —2e described by DeVries, and Klebs (1886) could not find the least elastic extension in growing filamenis of Zygnema, Wortmam (1587) and Noll (1887) found as did Sachs, that elastic stretching is essential to growth. (Goncerning the intussusception of nev material in the cell wall also, a divided opinion is to be found in the olcer literature. Wortmann found that slowly growing portions such as the upper side of a geotronically stimulated stem rapidly thicken their cell walls, and concluded that thickening and consequent decrease of elasticity is the cause of the eventual cessation of growth. Krabbe (1887) and Schwendener and Krabbe (1893) went so far as to claim that all growth is due to an active intussusception without the intervention of turgor atretching. Sufficient has been said to maxe it clear that from the peginning thera has been a consideraole variance between the views of the several investigators. A methodical examination of the general theories which have been put forth will be made and an attempt made to evaluate cach. b. Osmotic Pressure of the Gell Sap The primary cause of cell elongation is elastic stretching dae to the osmotic pressure. It is necessary to make it clear that altho cell elongation in the normally growing plant may be dus to the maintenance of a given property, for example osmotic pressure, at a sufficiently high value, nevertheless it is technically often more goavenieat to work with increases in growth rats and increases in the given property, for example, of osmotic pressure. Yor this reason the conves and concave sides of tropistically stimulated organs have been mich used. Altho there might once have been ob- jection to this procedure, there cannot be now, since as is clear ~3— from cur present knovledze of tropisms, there is no fundamental differance between tropistic growth response and norval cell elong~ ation. DeVries believad to have found that the osuntic value of the cell sap is higher in faster growing regions. Seley, 29) caer experiments came to the same conclusion. Krauss (1882), Mewehen (NBO Phillips (1920) failed to find this increase. Pfeiffer (1892) even found that hindering of elongation brought about an in-— crease of osmotic value and this was supported by Pringsheim (1906). More recently Warner (1928) working with geotropicaliy stimulated organs has shown quite definitely that there is an increase of sugar on tha coneave side, but that there is also an increase of acids on on the convex side. Borowikow (1913, 1914) found that variations of temperature or of acidity which decreased the growth rate of cells, correspondingly increased their osmotic value. The inverse relation between growth rate and ormmotic value has heen supported vy Reed (1925), and Fernald (1925, by c¥foscopic measurements. Ursorung and Blum (1924) investigated the matter somewhat further and obtained: finally a conclusive anewer. They found that altho tha two sides of a ceotrooically stimelated organ possess almost the same osmotic value, the convex side has a much higher suction force, which can then only se interpreted as due to lower wall resistance. It is clear from thelr work that increases of osmotic value cannot be the direct cause of increases of growth rate. yn &. Elasticity Zhe primary cause of growth 1s an increase in the elasticity of the cell wall. It has already been pointed out that Klebds (loc. cit.) failed to find any elastic extensibility in growing cells. Wortmann (loc. cit.), however, held that the gradual thickening of the cell wall reduced 1lts elasticity to the point where slongation by the pressures of the cell contents could no longer take place, i.e. that decreases in elasticity decrease growth. Noll (loc. cit.) supoosed that under the influence of the protoplasm the wall was kept con- tinually in an elastic condition to permit growth. Increases of erovta rate ound then be due to increases of elasticity. Overbeck (1920) studied geotropically stinulated roots whose curvatures were prevented. These roots when freed gave extraordinarily swift "schnell Kriimmungen", which, however, were almost completely removed by im mediate plasmolysis. By a later plasmolysis only a small portion of the curvature covld be removed. He concluded that during geo- trosic stimulation a one sided increase in elasticity had occurred which permitted the "schnell Kritwmng" and that the curvature was then gradually "fixed", DeHaes (1928) found that normally growing Avena coleoptiles possessed higher elasticity than coleoptiles whose growth had been slowed dow. by decapitation (see Part II). ‘These cases show that high elasticity often, Dut not always, accompanies fast growth. Heyn (1931) has finally settled the question. He has shown that in the Avena coleoptile and therefore probably in other cases increases in elasticity are results rather than primary causes of growth. This will be discussed in detail in Part II. lige a. Plasticity Tne primary S782 of growin is a plastic stretching of the feih wall. Laurent(loc.cit.) vostulated an enzyme achion upoa the cell wall which should result in a plastics condition.He vas,hovever,umable to fortify his position by experiment. Klebs(los.cit.deas willing to admit that the action of the mratoolasm mignt increase the wall plas ticity and showed that in algal filjaments the elongation was almost entirely irreversible. Ziegenspeck(1920)found great plasticity and little elasticity in root hairs,Pfeffer(1903) considered a "di a tion of the cohesion of the cell wali"to be neccassary for elongation, He showsd directly that this softening was a result of the action of the protoplasm.It is again, however,due to the work of Heyn(1931) and of Heyn snd Van Cverbeselr(1931) that a final solution of the quec— tion of plestic versus elastic stretching has been finally obteined. Thig work will be disgowesed in part II. 5a dg. Permeability Ths primary cause of growth ip an increase in the perme— ability of the cell membranes. Several investigators have expressed the opinion that increases in cell permeability should increase growth rate. TrB&adle (1910-18) thought that in tropistic reactions the primary effect of the stimulus was one upon permeabllity, and this view has been supported by Small (1919), by Braunér (1924) and by VanDillewijn (1927). It is well established that large changes of permeability result from the action of various stimuli. For axan ile light increases permeability according to the work of Blackman and Payne (1918), Hoagland and Davis (1923), and Brauner (1924), to men¢étion only a few works less open to objection than those of Lepeschkin (1909) and Tréndle (1910) which are usually quoted. Light prebably inercases the water permeability of the pulvinal cells of Fhaseolus, according to M. Brauner (1932). Movements of the leaves of sensitive plants and of sensitive stamens as those of Sparmannia are quite certainly accompanied by increase of the protoplasmic vermeability and the consequent expression of cell sap (Binning, 1953 a, b). It is therefore not at all unlikely that changes of permeability do occur both in normal growth and in tropisms. Such changes would be of value for cell elongation only if, for examole, water permeability oe the factor limiting slongation, and there is nO Treason to believe that this is,in general, the case. In Part Vi it will be show that it can hardly be the case in the Avena cole- optile. Changes in the direction of permitting an exosmosis as in the case of stamens would be actually detrimental to cell slon- gation. a. Intussusception Zhe primary cause of growth is an "active wall growth”. This vhrase has meant in general growth by forcing of new wall material in between the particles already deposited. As pointed aut by Pfeffer (loc. cit.) and by Strassburger (1898) not all growth could be of this kind since wall growth is in many cases by apposition, in which case increase in surface exient must be by some kind of stretching. Wigeli (loc. cit.) first put forward the theory that growth of the cali.wall is by Intussusception. This was in-part based upon his erroneous belief that the growth of starch grains is of the same kind. Pfeffer cites some cases which seem to be certainly the rasult of some Tactive growth", for sxeample the wrinkling of cell walls in cells no longer stretched. Krabbe (loc. cit.) supe. experimental ver! ported active grovth without giving it any strong eupaerwé. Ureprung f cation, and Blum (loc. cit.) have advanced active growth as important, prin- cipaliy because they failed to see how turgor, at its minimum in rapidly growing cells, could nave any bearing. upon elongation. Rentschler (1929)similarly adopted it as a last alternative. More recently S8ding (1934) and Strugger (1932, 1933, 1934) have supported active growth, but in both cases this is merely an opinion based on no convincing evidence. Against the general importance of intussus— ception may be cited the work of Pringshein (8886) and the work of Heyn which has shown that cell elongation is possible by an irrever-— sible increase of cell wall area in the absence of any possibility ot active growth. It is still necessary, however, to demonstrate in each case whether or not the formation of new cell wall is an integral part of the elongation process. -7- £, Imbibition Pressure The primary cause of growth is the imoibition pressure of the protoplasm. Loyd (1916), Calabeck (1927), Ulehla and Moravek (1922), McDougal (1925), Walter (1924), and most recently Struzger (1934) have supported various aspects of the general theory that dnereases of the iubivition pressure of the preteplasa or ather biocolloids are the primary causes of inereases in cell elongation. This theory has often taken the form of postulation of a machanism for the increase of the preasure of the cell. contents on the wall without an increase in the osmotic value of the cell. It is clear, however, and it has been emphasized by Walter (loc. eit.), that the vapor pressure of water in equilibrium with the cell sep will under all conditions be equal to tham in equilibrium with the proto- plasm. Otherwise water will pass from one to the other until such an equilibrium is estavliahed. Moreover the imolbition pressure which the protoplasm exerts on the cell wall must oe exactly equal to that exerted by the cell sap on the protoplasm and to the pressure with which the cell wall constrains the cell contents. Consider then “hat result an increase in imbibition pressure of the protoplasm might have. So far as increased uptake of water by the cel] is concerned, water will. be taken up until the protoplasm is again in equilivtrium with the ceil sap. In general for cells in the elongation stage and hence with very small amounts of protoplasm, this amount of water is negligible, and hence the elongation which it can bring about is negligible. There remains, however, the pos— sibility that increase in the hydration of the protoplasm may in- fluence, for exacple, the water permeability. This has been suggested Fn by Walter on the basis of the fact that Borowikow (loc. cit.) found the same effect of ionic lyotropic series upon growth of Helianthus hypocotyls as is commonly found for erfects upon vermeanility. DeHaan (1933), Dellingshausen (1933), and Forster (1933) have some evidence to show that increases in protoplasmic hydration increase permeability both to water and to salte.. It has been slraady pointad out, nowever, that there is as yet no good reason to believe that effects upon permeability have a direct relation to celi elongation. Van de Sande Bakhuysen (1930) has advanced the opinion that hydration of the cell wall as well as of the protoplasm is of importance in cell elongaticn. It is possible to visualize an eifect of hydration upon the conesive properties of the cell wall and hence upon its plastic stretching, but as yet such an effect and ite direct reletion te cell clongation has not been demonstrated. There can be no doubt but that some of the changes of growth rate which have been interpreted as due to changes in the hydration of the protoplasm have been actuaily due to changes in the actual water supply. This may be the case in the work of Walter (1924). One of the principal factore which should influence proto- plasmic imbibition is the pH of the protoplasmy it will be showm in Part VI that so far as the Avena coleoptile is concerned the effect of pH woon growth rate is not due to an effect upon orote— plasmic inbibition. Changes of imbibition pressure of the proto- plasm cannot therefore be of general significance in the control of growth. 3. PART II. THE STUDY OF CELL ELONGATION SINCE THE DISCOVERY OF GROWTH SUBSTANCE 2, Discovery and Isolation of Growth Substance The study of cell elongation has taken on a new aspect since the isolation by Went (1925) of a special substance whose presence in the plant cell is essential to elongation. The existence of special substances for the regulation of plant growth was first advanced by Duhemel (1758). Sache (1879) brought forth a long list of facts indicating the existence of a special flower forming, roct forming, leaf forming etc. substances, and proposed that correlation, i.e. the effect of one part on the growth of another, is in general mediated by diffusible substances. Sachs was, however, unable to supply any Livm experimental. evidence as tu theixy existence... Haber~ landt (191%~1919) went a step further. He demonstrated that a sub- stance is stored in the leptone of potato tubers which is able tas diffuse. into tuber sections without lepteme and coring about cell division there. Ricca (1916) showed that a substance which can pass through a tube filled with water controls the response of leaves in Mimosa, The idea of special substances was therefore not entirely new aven io 1910. then Boysen Jensen proposed the existence of such a substance for the control of cell elongation, especially in the Avena coleoptile. Paal (1914, 1919) soon established the fact of correlation, with respect to slongation, by a diffusible substance. F, Went (loc, cit.) worked out a method of quantitative determination and obtained the substance free of the plant, and Kégl, Haagen Smit, and Erxleben (1933) orepared the substance in the crystalline forn. —1d.. b. The Avena Seedling and Coleontile It has been shown by a variety of vorkera (Cholodny, 1927; Sdding, 193th ; Wielsen, 19305 Boysen—Jensen, 1931; iaibach, 1933; Van der Vey, 1933) that tail eubetance! or one of similar physiolo-— gical action is widely distributed through the plant kingdom, probably universally. However, the following discussion will limit itself to the Aveng coleoptile with which the greater part of the work has been dons, Before emoarking upon a detailed discussion of the growth of this orgen, it would to well, however, to give a brief survey of the morphology and anatomy of the Avena embryo, seedling, and especisily of the coleoptile. Reference may ve made to the work of Avery (1528, 1936) who has investigated the anatomy in some detail. The enbryc is at the base of the seed, rather at one side and opposite the groove. The embryonic axis consists at. the upoer end of short internodes bearing whorls of leaf primordia. Below this is the insertion of the scutellum or cotyledgon. The embryonic leaves and growing point ere surreunded by the conical coleoptile. At the scutellar node are the buds of two lateral roots. Below the scuteliar node is the primary roota and its coleorhiza. A large branched pro- canbial bundle proceeds from the scutellum to the scutellar node and nence inte the stele of the embryonic axis. The vascular system of the coleoptile consists of two laterel bundles arising from the stele. After germination (Fig. I) the root consists of the ugpal exarch vascular system with one large central metaxylem vessel. at the seuteliar node there is an abrupt transition to an endarch vas- cular system. The region between the coleoptile insertion and the scutellar insertion, commonly knomm as the mesocotyl is, then, of an Coleopti le et herd leat later leaves je Coleapts lar node i }e— mer istemal region at heed taternede with SeyleHar beadle and Stele , Y nary root Son Prt Y fissure 1.Radial Section through Col- eoptile and Seed. Figure 2.0ross Section throuzhn vucleoptile Showing Location of Vascular Sundles,. —-Lli-— essentially stem structure and is to be regarded as the first inter- node, A meristematic region at the top of this internode causes ites elongetion and at the same time elongates both the scutellar bundle and the stele, with the result thet the two run parallel in the mature internode. Only at the level of coleoptilar insertion does the scutellar bundle enter the stele in the seedling. The coleontilar oundes diverge from the stele at the point where the scutellar bundie turns down inte it. The vaswular bundles. of the coleoptile are small with few xylem groups and comparatively well developed snicem. A bud is in general oresent in the axil of the coleoptila, which is therefore to be considered as a leaf. The leaves within the coleoptile are supplied with bundles in part from the stele and in part from the scutellar insertion. The coleontile is elliptical in cross section (Fig. IJ) perpenduvlar with the long axis ‘Seamues& bo the plane of the seed. It is, of courga, hollow and ccuslets in general of about six cell layers. The ianer- most and outermost layers are of somewhat smaller cells than those of the inner parenchyma. The elongation of the coleontile, from a yom. or Lease, to WO or 50. mnm., is unaccommaniad by cell division (Tetley and Priestley, 1927). During this time also the diameter of the coleoptile changes but little. In the Avena colaoptile one has then a relatively pare example of growth by cell elongation. &, General Relation of Growth Substance to the Qoleuptiis In relation to growth and growth substance in the cole- optile the following facts have been established: 1) The growth substance is produced in the tip of the coleoptile and proceeds downwards toward the base. (Paal, 1913, 1919; Dolkx, 1930; lent, 1928) 2) 3) 4) 5) -l2- If the tip is remaved, the top of the renaining stump con— -mences to produce growth substance after the lapse of a definite tima (Dolx, 1930; Tsi Tong Li, 1930). This has been Called "ehysiological regeneration", If, after decapitation and reappearance of growth substance ia the upper zones, these zones are also removed the growth of the coleoptile falls practically to sero (Dolk, 1930). Application of growth substance to the lower zones of old coleoptiles which have stopped growing, causes under certain conditions, the recommencement of growth (Went, 1928). The response of the coleoptile to phototropic and geotrooia stimali is conditioned by the presence of growth substance (Dolx, 1926, -1930; Yent, 1928; DuBuy, 1933). After decani- tation the ability of the upper zones to raact to phototropic and gaotropic stimuli is last but returns at the same time as the new. growth substance. From 3, 4, and 5 it has been fairly concluded that the growth substance is not merely an accelerator of growth but that wrtkhet at no growth can oocur. 6) 7) 8) 9) Growth substance disappears continuously during growth. Only a small portion of the amount produced by the tip can be recovered from the lower zones. The movement of growth substance is a polar phenomenon (Yent, 1925; Van der Vey, 1932). In inverted coleoptile cylinders no downward transport occurs. The application of growth substance to decapitated aoleoptilas deformability. results in an inergase of their Manigeday (deyn, 1931). The one sided application of agar blocks containing growth substance to the stuups of decapitated coleontiles causes a growth curvature whose magnitude is over a considerable range proportional. to the amount of growth substance in the agar block (Went, 1928). Tuis is the oasis of the quantitative growth substance test (Van der Yey, 1932; Nielsen, 1930; Dolk and Thimann, 1932). an] Fae 10) The growth substance is not an enzyne ont a thermostable, lipoid soluole, unsaturated acid of molecular weight 323, and having the formula ©, gHa0%- Toasse are the essential points with respect to the relation of growth substance and grewth., Mere detailed discussions of various aspects will be given in the later sections. a. Growth Substance and the Deformability of the Coleoptile The problems .of.celi elongation may now be attacked through an examination of the properties of the coleoptile waich are altered under the influence of growth substance. The work of Heyn (1931), Heyn and Yan Overbeek (1931) and of S8ding (1929, 1931) has shown that some times after a coleoptile is given growth substasce. the elasticity and plasticity of the coleoptile are increased. ‘The fundamental experiment ia this: comperadle seta of coleoptiles ars more or lass freed af growth substance by sllowing them to atand for two hours after decapitation. One set is then supplied with growth substance and the other set not. The growth substance is alloved to ast for some time, say two hours; the coleoptiles are plasmolized to remove whatever resistance might be due to the proto- plasm, end the exteasibility under an applied force determined. Heyn found that both the reversible and irreversible extensibility ara increased after the action of growta substance sccompenying growth, He was able to goa further than this, however, by giving coleoptiles growth subatance out no water supply. In this case the elasticity recained relatively constant. This he considered to show that in- creases in elasticity are the result of elongation and that the pri- mary action of growth substance upon the coleoptile ia in increasing plactiat y. QToot vote, Later workee Kaql has Showy phyt oue of more Closely related Sebs lt auces way also be active, ~1y. This experiment is af importance because it shows that increases in elasticity are principally the result of growth, not the cause. Séding independently at about the same time and using some- what similar methods came also to the conclusion that more rapidly growing coleoptiles have more extensible cell walls than do more slowly growing ones. The inersase in derormability by growth substance is well illustrated by experiments in which weights are hung upon the ends of of horizontally placed turgid coleoptiles. Under the conditions of Heyn and Yan Gvernsek the reversible pending was slightly aifected, Neen tinagmactiaiaglN, the irreversible greatly affected, by the presence of growth supstance. Dn aioe caliente tin teal This experiment was also done independently by Séding. Note: The turgescent coleoptiles used in bending experiments nave the formal requirements of growth (according to Heyn) i.e. turgidity and increased wall plasticity. Nevertheless, unless they are supplied with water they do not grow. It seems probable that the effect of higher wall plasticity, decreasing the turgorand hence increasing the suction force is in per- mitting the uptake of water and accompanying increase in area of the cell wall. Increases in elongation would ba then due not directly to the increase in deformability of the cell wali but to the increase in suction force of the cell. As already mentioned, the work of Ursprung and Blun has shown that suction foree is at a maximum in the most rapidly growing regions of roots. It has been tacitly assumed that increases in irreversible deformability in reality are due to increases in plasticity. A supporter of “active growth" might object that these increases in deformability may be due to the rapid intussuscestion of wall particles during the action of growth substance Lb 1.@s Weidetendavdumap to 2 temporary excess of new wall over actual slon— gation and hence lowered turgor. It has not, in fact, been shown that growth substance does not eitect the deposition of new wall material. Heyn (1931) originally thought that the fact that growth subatance dees not affect the fixation of elastic stretohing indicated that it did not affect wall formation but hehas since (1934) vhowm that the process of fixation is in all Lil ihood only a relaxation of elastically stretched layers. &. Oojects of the Present York The information concerning cell elongation and ite con- nection with growth substance before tha undertaking of the prasent work have been outlined. With respect to cell elongation itself it. is clear that, between the two possible alternative mechanismg, plastic stretching or intussusception, a final decision has not as yet deen vossible. I+ is kmown, only, that growth substance brings guout an increase in colesptile deformability. Concerning the actual jearned: ateps of growth suostence action nothing has been Sowad, The first questions which 14 seems necessary to answer in the study of this problem are: 1) Does growth substance act as a true hormone, i.e. indirectly and in small amounts, or can it take part as a constituent, gor example, in wall formation? 2) Does growth substance influence formation of cell wall or does 1t influence proverties of the cell. wall? In this way ea final decision between intussusception of new material and plastic stretching may be obtainad. 3) Does growth substance influence the wall directly or does it influence functions of the protoplasm? 4 ~Lo~ 4) If growth substance acts through functions of the protoplasm, what is the nature of the action? These questions will be dealt with in the following sections. -1]- PART Til. A GENHRAL DISCUSSION OF THE MATERIALS AND METHODS a. Plents The Avene scedlings used in these experiments were of the pure line "Sieges Hafer" suoplied by Dr. Akermann ef Svaldv. They were grom in the dark rooms of the plant physiclogy laboratory, at the humidity (85 to 9G per cent) shown by Went to be suitable for the carrying out of growth substance activity teats. The temerature was held constant to 0.2° during the course of an experiment but varied occasionally between 25° and 27° during the course of three years. Only red light was used in the carrying out of all of the experluenuts in order to avoid phototropic curvatures of the plants. Illuminating gas, minute traces of which causes great irregularities in the grovth of the seedlings, t not present in the oulilding, The husted seeds were soaked in water for 4 or for 12 hours, germinated for 24 hours upon moist filter paper and then planted either in glass holders for tests (ent, 1925) or ina mixture of sand and leaf mold, Ait the total age of about 8 hours the coleogtiles were 2 to 3 om. tall and ready for use. b. Growth Substance It has been shown by Nielsen (1940) that considerable amounts of growth substance are produced in cultures of the mold, -higopus Sainus. The conditions for production of growth substance have been more thoroughly investigated by Bonner (1932) and by Thimann and Dolk (1933). The growth substance used in the present experiments -18~ was obtained from the large scale culture of this orgeniem, the preparation and purification being carried out ty Dr. K. ¥. Thimann. The mold wes grow upon a peptonas-glucase culture medium and care was taken to keep the conditions serobic, since this greatly increases the yield.. At. the end of threes days the mycelium was filtered off, the medium concentrated under diminished pressure to 1 per cent of its volume, filtered, acidified, and extracted six to seven times with peroxide free ether. The bulk of the ether was distilled off, the residue taken up in a smell volume of distilled water, boiled free of ether, and the solution chillea. Chilling causes the pre- cipitation of a rather lerge quantity of olly material which was filterad off. The filtrate is a growth substance preparation of rather high purity thich may be used directly for experiments. It has an activity of about 1078 mes. ver plant unit (see below) as compared with cyrstalline growth substance which has an activity of about 1. 5) es. per plant unit. later in the course of the ex- periments as will be indicated preparations of higher activity vere used. The author is greatly indebted to Dr. K, ¥Y. Thimann for these growth suostance preparations. c. Unita One growth substacsee unit, as used in the remainder of this paper, corresponds to that defined by Dolk and Thiaans (1932). It is that emount of growth suostance which when present in 1 cc. of solution will give after being wixed with lcc. of 3% agar end nade into agar blocks of 10.7 roa? , a curvature of 1° upo: the stancard decapitated Avena coleoptile under the standard conditions, of this laberatory. C PAK aed Thi mann, 1932) ~19-~ PART IV. THE HORMONAL NATCRE OF GROWTH SUBSTANCE a. Introduction The first. question to be answered concerns ths relation of the amount of growth substance in the plant ‘end amowsss of the various substances underzoing transformation during slongation. In this cart, it will be determined whether or not thera is any simple stoichiometric relation between the number of molecules in a given amount of cell wall and the number of molecules of growth sub- stance necessary to produce an. equivalent amount of elongation. This determination involves the reasonable assumption that the new cell wall formed during the grovth suostance has the same constitution as thet already deposited. This assumption is supported in Section 1°) of thie part. The experimental work is divided into five heads corresponding to the oleces of information necessary for the cal- culation of the desired relation. These heads are as follows: 1) The amount of growth substance entering the plant under the experimental conditions. 2) The amount of elongation brought about by a given smount of growth substance. 3) The actual increase in cel. wall during a given amount of slongetion. 4) The composition of the cell wall and hence the increase of each Constituent during a given amount of elongation. 5) The increase in volwne of cell wal: during a given anount of @longation. This is an independent method of arriving at the result given by 3. ~20— In addition the elongation produced by a given amount of growth substance under difrerent experimental conditions will be determined for comparison, i Method Growth measurements. The growth measurements were carried out with en automatically controlled motion picture camera. ‘The films, after development, were projected with a final enlargement of 100 times upon a screen, on which the images of the plants could be measured with considerable accuracy. The Leitz camera was coerated wth a "De Bouter intermittent Clinostat", which in turn was operated oy an electric clock. A general description of this type of clincatat is given by F.a.F.0. Yent (1929). The two plants which were photo~ erephed upon cach film were placed ebout one meter from the camera with their roots in water. Beside each plant was placed a millimeter scale which wae included in the photograph and from which the growth increnents were measured. Since the etiolated coleoptiles are very sensitive to light of wave length shorter than 5300 A°, the photozraphs were taken in orange light, using Yratten filier No. 24, which cuts off mast of the light shorter than 5450 A°, Eastman supersensitive panchromatic film wag used with an exposure of five seconds, The experiments were carried out in a constant temperature room at 27°, The relative humidity was kept at 90%, with a variation of not sore than 3-4%. Since the standard conditions for testing the activity tests of growth substance preparations by the curvature method aged in this laboratory include a temperature of 25°, a second room was kept at this temperature and the activity tests carried out in it. —Zi- g. The Amount of Growth Substance Bntering the Plant It has recently been found (Thimann and Bonner, 1932) that the rate at which growth substance passes from solution in agar into the decapitated coleaptile is directly provortional to its concentration in the agar, and may be expressed by the aquation ax, ~ at oes where x is the amount in a block of volume v. From experiments in which agar dDloe@ka containing growth substance after having yvreduced curvatures end placed upon fresh decapitated colecptiles, the rate constant of passage was determined. From the value of this constant the fraction of growth substance initially present which would paas out of blocks of different volumes was calculated, and shown to agree with the experimental results. For clocks oi ae standard size this fraction is 15%. I+ was also shown that for blocks of small volume a very large fraction of the growth sujstance initially pregont passes out, which accounts fer the belief, held by earlier investigators, that all of the growth substance passes from block to coleoptils. While the above work vas concerned with blocks placed uni- laterally wnon coleoptiles, the oresent experiments are concerned with vertical growth in which blocks are placed flat upon the cole-~ optile stump. Since the srea of contact is more than doubled, it might be expected that the proportion of growth substance passing inte the plant in this case would also be more than dowbled, Tor the evaluation of this oroportion, large numbers of plants were decapitated and supplied with agar blocks containing a imown amount of growth substance. precisely as waa later dona with the planta ~t2— whose growth rates were to be measured. At the expiration of 105 minutes, the Dlocks were removed, placed unilaterally upon decapi- tated test planta, and tha resulting curvatures meawared, <A. curva— ture 6 is, as pointed out by Thimann and Bonner proportional to the concentration of grawth substance in the agar block. Since blocks wera made from a growth substance solution of «now strength, they would, if placed won test plants, give an initial curvature 9. From the curvature 9 given by the second test plant, the amount of growth substance in the block at the beginning of the second test is also known. Hence, the fraction of the initial quantity of growth substance which passed out of the block while it ramained flat upon the coleoptile is given by 205 1, Table 1 gives a serias of determinations of @,, and of this fraction, for different values of ©). Tha mean value of the fraction passing out is 34d, (Insert Table 1) a. 8 unt of Growth Correspondi to a Given Amount of Growth Substanaa at 27° Although it has been shows that the response of caleoptiles to unilateral application of growth substance is linear over a certain renge,. the rasponse to symastrical application has not been quanti-— tatively followed. The response to different amounts of growth substance contained in agar blocks laid flat upon coleoptiles waa therefore measured in order to determine whether this was also linear, and if so, over what range. ~23- Preparation of plants. Ooleoptiles exhibit their maximo rate of growth when they are about 43 om. long (Konygeberger, 1922; see aleo below) and the rate of growth is not greatly different for plants a little shorter. Since for these experiments it is only necessary to Choose Dlants of similar growth rates, straight plants of lengths between the above limits were chosen. With the aid of a sharo blade the tip of the coleoptile was cut at a point 5-6 ma. from its anex, and the first leaf dram out until only 2-3 mo. remained and cut off flush with the stum of the coleoptile. This olece of leaf was allowed to remain, in order to prevent the gelatin from flowing into the cylinder. The plants were then removed to the dark room containing the apparatus, and placed in position beside the millimeter. scales. If the plants were to be swoplied with growth substance this was done by fastening, with 15% gelatin, an agar block containing growth substance in the desirad concentration upon the stump. The clinostat was next started and the first picture taken quickly as possible, which was in general 2-5 minutes after decapitation. Two pictures, separated oy a two minate interval, vere taken each 15 minutes. The values for the grovth increments in the subsequent tables are obtained from the means of these two picturas. Rate of grovth of decapitated soleoptiles. The rate of growth of decapitated coleoptiles, unsupplied with growth substance from an outside source, was first determined. Figure III gives grephnically the results obtained from the mean values of 6 plants, the rate of growth in millimeters per 15 minutes being plotted against time. The rate of growth may be seen to decrease steadily after 0:2 15 minutes 0.1 in’ mm. per Lo Grewth ca a a Tr Time after decapitation in minutes figure 3.Growth Rate of Secapltated Coleoptiles not Sup- plied with Growth Substance. ~2y. decapitation, for 105 minutes. After this time the rate increases rather sharsly, and reaches a constant level which is, however, not go high as the original, An inspection of the actual data (Table 2) (Insert Table 2) reveals that the mean values, plotted in Figure TII, do uot accurately represent the growth of the individual plants. In general, the plants tend to drop gradually to s minimum and mount quickly to the new constant level, out this is obscured by the mean, since the minima are somewhat spread out. This sudden vhysiological regeneration of a growth substance-producing region in the stump will be considered in the next section. It is also an indication that growth substance production by zones of the coleoptile celew the tip cannot take place until alow cancentration ls reached. The growth of Avena coleoptiles after decapitation has been previously studied by Dolk (1926) and by Heyn (1931). These investi- gators found exactly the same type of response as in the present case, put since they worked at lower temperatures (21° and 23° respectively) they found the minimum to occur somewhat later. As mentioned before, the appearance of new growth after the minimum is due to the or duction of growth substance oy tha uppermost region of the coleoptile stum, which previously did not produce growth substance. The growth of a decapitated coleoptile before the miniamin represents, then, the growth due to whaiever growth substance was left in the plant after removal of the tip. Hate of growth of plants supplied with low concentrations of growth substance, In the next series of experiments, plants were supolied wth agar blocks containing 9.9 and then 14.5 "vlant mits" -25- of growth substance, Now, the rate of passage of growth substance from block to plant depenis woon the concentration of growth substance da the block. Heuce, even by allowing the Dlock to remain for a vary long time upon a coleoptile, the complete transference of the growth substance to the plant would be impossible. It was found mach more convenient to remove the block after a definite time and to determina, as in the previous section, the amount of growth substance which had actually entered the plant. Yor comparison with plain decapitated coleoptiles, the cloaks were allowed to remain 105 minutes, that is, until physiological regeneration would have ooaurred if the plants had not ceen supplied with growth mibatanes. The mean growth rates of these plants are showin Figure IV, in which the curve for decasitated colsontiles without growth substance is inserted for comparison. The data are included in Table 3. The 9.9 (Insert Tabla 3) unit curve is somewhat irregular, Dut falls, in general, although mere slowly thas that of plain decapitated colooptiles, to a rather low value after 3 hours without exhibiting any minimun, Tse 14.5 unit curve, is, until removal of the block, nearly constant ata value slightly higher than that of the initial growth rate of plain de-~ caplitated colsoptiles. After removal of the block the rate falls through & minimum and Tises to 4 constant value. The minimum is ast sa low and is reached one half hour earlier than the minimum of plain de- capltated coleoptiles. he shape of the curve resembles creatly, however, that of Figure III. 15 minutes a bo I per in Growth 36 60 90 120 150 180 210 240 Time after decapitation. rn minutes Figure 4.Grvowth Fetes of Coleeptiles Supplied with Small mantities of Grama Suostance.Curve A, 9.9 units of Growth Substance; Curve B, 14.5 units of Growth Substance; Curve 6, no Growth Substance. ~26— The coleaptile recomrences the production of growth substance in this case, when it has reached the minimum growth rate, which is after 3 hours. The total growth between decapitation and thia minimum is due (a) to the added growth substance (i.e. given up by the block), (b) to the residual growth substanes left in the plant after decapi- tation. It is clear that the growth due only to the added growth substance is the tetal growth from the time of decapitation to the oiniman, less the amount of growth of vlain decapitated coleoptiles to thelr minimum. If 1t be assumed, as seems justified, that at the end of 3 hours the 9.9 unit curve also begins to mout toward a new constant level, the growth due to the 9.9 wnit block may be determined in the same way. Growth rates of plants sugplied with higher concentrations of growth substance. The rates of growth due to successively higher concentrations of growth substance was next studied, and Table 3 gives the means of the determinations at each concentration. The growth rates of four of the concentrations are plotted in Figure ¥. In each case, except one at very high concentration, the addition of more grov.n substance to the plant results in an increase of growth, In eléeral, also, the response to a large concentration of groath suvstance is immediate, that is, even in the first period the growth is larger than that of plain decapitated coleoptiles. After removal of the block the curves drop to a low constant value which is approximately the same in each case, The time between the Temoval of the block and the attainment of this Low value is, hovever, longer the higher the concentration. The fact that this final constant in mm per IS minutes Growth 30 60 90 120 150 180210 240°. 270 300] Time after decapitation.in minutes : Pigure Sadrowth Rates of Decapltated Coleuptiles Supplied with Large Quantities of Growth Suvstance.Curve A, 19.3 Units of Growth Substance;Curve 3, 29.0 Units of Growth Suvstance; Curve C, 39.5 Units of Growth Substence; Curve D, 115.9 Units of Growth Subsatence. -2]- level is lower for plants supplied with growth substance than for the plants of Figure III may be attriouted to the greater utilisation of other necessities of growth in the presence of more growth sub stance. None: of the curves of Figure ¥V exhibit a clear minisoun. Heyn has stated that the removal of the block ig exactly similar to removal of the original tip, and that the production of new growth substance comuences at a. definite time later. If this be true, no minima would oe expected with the higher concentrations of growth sudatance, since the growth substance left after the removal of the block would give rise to a rather large amowmnt of growth. -Thia growth would obscure the minimum. The quantitative relation between grovzth end growth substance which may be obtained from the present data with the aid of a differant assumption, however, lead to the belief that Heyn's interpretation is not the correct one, and that the true reasons for the absence of minima are tnose which will be Drought forward oelow. The linear relationship between gronth and growth substance. The zones which produce growth suosstance after physiological regener-— donot wh the presence ation af a tip, produce any appreciable amount. This suggests that the growth substance has, as a secondary function, the inhibition, py its presence in sufviciently iigh concentrations of the production of growth subetance by the lower zones. In a plain decapitated cole-~ optile or in one supplied with a small ancunt of growth substance, removal of the tip, or of the agar block, is followed by transport of all of the small amount of residual growth substance from the top ~e8- zones to the lower (Yen der Wey, 1932). The result is that when the concentration of growth substance in the too of the stump has become Leow anough to permit regeneration, the erorth rate of the culeoptile as a woole has become very small, and the growth eurve therefore yasses through a minim. I:, however, a decapitated coleoptile is supplied With a large amount of growth substance, a different result wouleé be expected. After removal of the block, the growth subatance which is left in the woper zones is. transported to the lover, but these zones are unable to utilise such a large amount as quickly as it is orought in. The result is that while the concentration in tha uppermost zone falls to a value sufficientiy low te permit re~ generation, the concentrstion in the lower zones remeins high, they grow continuously, and no minimum in the total growth rate ig ap- parent. The essential difference between the situation of plants supplied with lerge and “th small amounts of grovth substance is, then, one of distribution. In accordance with this view there is, for plants supplied with large concentrations of growth substence, no exact time at which growth due to the growth suostance from the block ceases, and that due to growth substance from the regenerated tip commences. As an approximation 1% can de assumed that the time at which tha growth reaches its final low constant level is the time at which the growth substance from the block is used up. The correction is in any event email. The total growth of these clants from time of decapitation to establishment of the final level, less that due to the residual growth substance, represents, then, in each case the grorth due to ~29- “the amount of growth substance which has entered the plant, and this amount is, as already pointed out, proportional to the concentration apolied. - Table 4 gives the growth thus obtained from each concentra— tion of growth substance. From the total growth, from the decapitation (Insert Table 4) to the final level, was subtracted the total growth, to their minimm, of the decapitated coleoptiles of Figure III. For comparison of the growth per unit Yo growth substance, this value is in each case also given. ‘For the 4 lowest concentrations this ratio, is within a few per cent, constant. Above 29 units the ratio decreases somewhat, that is, a further addition of growth substance produces proportionately less growth. Above 58 units the ratio decreases sharply; in fact 116 units gives but little more growth. Figure VI summarizes the amounts of growth from Table 4, plotted against growth substance entering ‘ the plant, and shows that the growth response of coleoptiles to growth substance is, up to 10 wits (i.e. 29 units applied) linear, while above 20 units (i.e. 58 units applied) more growth substance, on the average, produces little increase in growth. For comparison the curvatures pro- duced by unilateral growth are also plotted against amount of growth substance entering the plant (dotted line). The limiting amounts can be seen to be much higher for straight growth than for curved growth. This is probably due principally, as pointed out by Du Buy and Neurn- bergk (1932), to a relatively slow non polar transport of growth sub- stance, partwolar/y et ¢éhe cut surface, ww \ CURVATURE, 10° SS ® e e 6 * = 2 ° ee = / ve = e — = | f c — ° — Oo naa . 10 20 30 40 UNITS OF G.S. GONE INTC PLANT. Fisure GeAmount of Growth Réasulting from the Entrance of pifferent Amounts of Growth Substance into the Plant. Gurve A, Vertileal Growth at <7 C.Curve 3, Curved Growth at 25 CeCurve ¢, Vertical Growth at 15 ¢. Wen &. The Amount of Growth Cerresponding to a eiven Amount of owth stance at Lae G, Ee It is of interest to know whether under different conditions one molecule of growth substance brings about the same amount of growth. Agcordingly a complete set of measurements anslogous to the preceding wee carried ong 129 lower temperature, i.e. at 15°. For this purcoss a thermostat having a central chamber surrounded oy a water bath was constructed, The central chamber, made of copper for efficient heat transfer, was eqoipped with double glass doors through which the plants were vnotegraphed without difficulty. Humidity of the inner chamber was controlled by the presence of solid KCl, which kept the humidity et about 80%. The outer chamber, filled with water, was cooled by ice and regulated thermostatically to within 0.2° of the desired temperature, A thermometer in the inner chariber showed that within 2 to 3 minutes after closing of the double glass doors the chambter took on the temperature of the outer vath. Table 5 gives the results of measurements analogous to thase recorded in Table 3. At fifteen degress the minimun is reached by decagitated olants in two hours instead of in 105 minutes as at 27°, end in the sussequent three hours no regeneration is to be found. This is in agreement with the work of Tsi Tung Li (1930) who found that regeneration is delayed at lower temperatures. It also simplifics the calculation of elongation ner wait crowth substance since the only correction is that for the residual growth suostance. Tsble 6 (Insert Tables 5, 6, & 7) suncarizes the growth per unit of applied growth subatence as did eple 4, The amount of growth substance passing into the plant was -31- next determined in the seme way as was done for 27°, The teste for growth substance left in the blocke were, of course, carried out at 25°, Table 7 shows that the fraction passing out of the block at 15° in 2 hours is even slightly larger than that passing out at 27° in 105 minutes, i.e. the uptake of growth substance by the plant is practically independent of the temosrature. The significance of this fact is not clear since as found by Van der Wey (1932), the transport of growth substance has a relatively high temerature coefficient. It is vossivble that it is due to limitation of rate of entry of growth substance by the rate of nassage across the block- plant surface, a diffusion process of low temperature coefficient. In Figure V1.. is plotted the elongation ner unit of growth substance entering the plant at 15° as well as at 27°. It may be seen that the amount of elongation per molecule of growth substance is more than twice as great at 27° as at 15°. £. The Composition of the Cell Wall in order to convert growth, in terms of coleoptile length, into amounts of material actually laid down, the relation between coleoptile length and amount of cell wall material must be knom. The meferial used in these experiments consisted of cole— eptile cylinders, with tips removed, either of young plants of the size used in the previous work, or of old plants from which the Sioa isaves would soon break. It was found that altho the weight of "celi-walls" ver wn. was greater in the older plants, yet the composition of the cell walls was the seme. The old plants were more convenient to use, since they vrovided much more material with which to work. =74— Table 8 gives the dry weights per mo. of a number of samples of young coleoptiles. The weights depend cansiderably woon the con ditions of drying, since although samples dried at the same tine (100° for 24 hours) check fairly well, there is more variation bse- tween samples dried at different tines. Nevertheless, the dry weignt per mm. is. relatively constant. This constancy of dry waight per mn. might hewe been expected, since the coleoptile is, excent for the tip, ofjvery nearly uniform diameter, which varies but little from plant te plant. The dry weight oper mn. of coleoptiles grown at 15° was found to be practically the same as that of coleoptiles grown at 25°, The next step wae te ebtain gell walle free of eytoplasm and cell sap. In arder to accomplish this, samples were thoroughly ground, and extracted several times with cold water. This was folloved by two hours of extraction with repeated portions of Act water. The residue was retained upon a coarse filter, through which the colloidal solution obtained from the washings readily passed. Thig fraction which is not dissolved or suspended in water, can be considered as the cell wall. It was dried to constant veight at 95°. Tsvle 9 gives the amounts of this fraction obtained from severel samples of young coleoptiles. (Ineert Tables & & 9) Extraction of the cell wall fraction for several hours with ether gave no decrease in dry weight, and it was therefore concluded that no appreciable amount of fatty substances were present. The pectic substances were extracted by the method of Nanji and Norman (1928) in woich the cell wall samples are heated at 80° for 24 hours with 0.5% ammonium oxalate. Results for tyo such extractions are ~32- included in Table 10. (Insert Table 10) The amount of cellulose present in material of this kind may be determinad approximataly by the ‘ernde fiher!. The samples were heated successively with 2% 60,4 “th 2% KOH and with alcohol, and the residus dried and weighed. Fentosans may remain to a small extent as impurities, as has been shown by Lildtke (1931). For m- lignified tissues, however, the crude fiber is principally cellulose. The degradation was also carried out in steps woon young and o14 coleoptiles, in order to compare the composition of the two. Thesa results are included in Table 10. It may be seen that the amount of subatance removed at each step doea not depend unon the age of the coleoptils. . Since this is ths case, the ratio of the water- insoluble fraction to the cruise fiber was determined several times upon old coleoptiles (sce Fable 19). Determinations of the nitrogen in the samples were carried out Dy the micro-Kjeldahl method. An approximuation to the per cent of protein present in the wall may be obtained by multiplying the per cent of nitrogen by 6.2. The nitrogen found in two samples of eal] walls, together with the calenlatad protein, is ineluded in Table 10. A summary of the results is given in Table 11, in wich the different fractions of the cell wall are expressed as percentage {Insert Table 11) of the total average dry weight. It might seem at first sight rather surprising that only about 12% of the coleoptile may be considered as consisting of celluloses, but that such large amounts of hemicellulose -33- and proteins are present in the cell wall. Link (1929) has analysed Zea seedlings, of an age comparable to that of tha Avena plants used nere, and found that only 14-18% of their total dry weight is pure cellulose, and that 10-12% is pure xylan, It will be assumed that the increment. of length produced vy the action of growth substance is composed of normal tissue, that is, that the processes of cell wall formation keep up with the processes of cell~vall elongation, irrespective of whether thase two sats of processes are connected or not. That this is actualiy the case is indicated by the following experiment: coleoptiles were supplied every 2 hours for 14 hours with fresh agar blocks containing 100 units of growth substance, At the end of this time the coleoptiles were cut off, dried, and weighed, together wlth normal control cols optiles. Tha results were as follows: The plants supplied with growth substance increased in length an average of 1.9 em. (12 olants) while normal coleoptiles in the same time inercased by 1.0 om. (6 plants), The dry welght of the coleoptiles supplied by the growth substance WAS 5.28107° mg. per m., while that of normal coleoptiles carried out at the same time was, in two samples, 5.1 and 5.29107° mg per mmm. gz, The Volume of the Cel. Walls oe namanes A determination was made of the volume of new cell walls which sppear as the result of the action of a known amount of growth substance. Since the estimation of the area of the cell wall ia at best rather uncertain, the coleoptile will for the present purposes be considered as a hollow cylinder. Tsis simplifies the calculations without greatly affecting the result. In tha case of longitudinal 3h. growth, only the longitudinal walls need be considered. The inside and catside diameters, as well as the diameters of the individual celle of a large number of soleeptiles wore measured under a microscope with a calibrated eye-oiece micrometer, The nunioer of cell layers were counted and found to be fairly constant. Txble le (Insort Table 12) gives the maan values of these measurements. Figura VII shows a section, drawn to seale, througn a diagrammatic coleoptile of the dimensions given in Table 12, The scircunference of seach row of walls may be easily found. The mean circumference, when divided by the diameter of the ceils accupying this articular row, gives the nuwiber of cells of this kind present, and hence the number of radial cell- walls. The total length of cell walls found in this way, gives, when tiplied oy 1 mm., the area of cell wall in 1 ma of colsoptile. Thase calculations are givea im Table 13. The value obtained for (Insert Table 13) the total wall area is clearly a minimam figure, since it will be increased by (a) irvegularities in the shaves of the cells (hb) inter- cellular spaces, and (c) the vascular osundles, which are made up of smaller cells. Measurements of the cell wall thickness gave a mean value of o.4A, the outermost layer of cuticularizsd walls not deing included, From these values for aren and thickness, the volume of the cell wall will be calculated. Figure 7.fortion of a Cross Section through a Diagramatic Coleoptile. -35~ h, tio of Cel] Yall Constituents produced to Grovth Subatance used ¥rom Figare VI, it is clear that at 27° the response of the coleoptile is linear for quantities of growth substance smaller than about 35 units. Ye will therefore consider the response to 29.9 units, whieh is well within this range. fable 4 shows that the addition of 29 units produces a growth of 2.66 mm, From analysis of the coleoptile, (Table 9) it was found that the weight of cell walle in 1 mm. is 1. 64x107° mg. Consider for the moment that only the actual cellulose of the cell-~ wall hasbeen laid down by the action of growth substance, if such a reaction, in fact, takes place. This assumption is to some extent justified by the consideration that the principal skeletal material of the coleoptile is certainly the crystallised cellulose. It is clear that this procedure will lead to a minimum value for the carbo- hydrate deposited as a result of growth substance action. Table il has shown that the cellulose (crude fiber) constitutes 42% of the cell wall, and hence the amount of cellulose deposited as a result of 29 units is 2. 66x1. 64x10" x0. 42 = 1.83107" mz, Now cellulose is made up of glucose residues (C,H, 9.) of molecular weight 162, and 1 millimole contains 6.06x10°° such residues. Therefore, the number of such glucose residues laid down as cellulose under tha action of 29 units is 1.832107" x 1 x 6.06x10°° = 6.suai0t® residues. rl ~36- In order to arrive at a figure for the amount of growth substance involved, it is necessary to use the value given by Kael end Haegon-Smit (1931), since, while their subatance is not necessarily identical with that used here, it is closely similar in action, and was obtained by them in the crystalline state. According to then, one unit is equal to 1/30,000,0C0 mg. Thea wit which they use is defined as giving a 10° carvature, whereas ours is defined as giving a curvature of 1°. The blocks which they use have a volume of 2 van? and hence by the procedure of Table 4 of Thimann and Bonser (1932), 53% of the growth substance passes out of the block during their test. The amount passing out of the block in our test ia 15%, and hence we need to have 4 times their amount in our block to give the seme curvature. Qur unit is than Ux 3.3x107° x 0.1 = 1.3x107° mg. 8 3.82107! mg. were used. In this experiment 29 x 1.3x107 Table 1 shows that when blocks sre vlaced flat upon coleoptiles, as in thie experiment, 34% of the growth substance present in the block passes into the plant, that is, in this case, O.34 x 368x107! = 1.32207! sae. pass into the plant. The mean molecular weight of the growth sub- stance obtained by Kgl and Haagen-Smilt 1s 328, and this figure is checked by the value obtained in this laboratory. The number of molecules of growth substance entering the plant is then 6.06x10°° x 1.3x107! x shy = 2.9010" molecules. Ye have, then, from the above, 2.4ax10"4 molecules of growth sub- ra - 1 stance for 6.8x10°° molecules of CEH 95? that is, ona mwolecule of 9 J- erovth eubetance haa acted in the laying down of 3x10? 0,8, 0, rasidues. Since this ratio is greatly different from 1, it is at once clear that the growth substance is not involved directly in the deposition sither of cellulose, or of total cell—wall carbohydrate. We will now compare the numser of molecules of growth substence with the number of micellws of cellulose deposited. As Katz (1928) points out, the micelles of fibers, since they are not visible even with ultraviolet light, mst be shorter than 500 A’. Katz gives the most orobadle size of the micelles as 60 ~ 110 A®. The unit cell of cellulose is 8.7 x 7.9 x 10.3 A®, and contains four glucose residues (Meyer and Mark, 1928). If we tale the micelles as 87 x 4 x 103 A®, a figure which accords with that of Katz, they each contain 2000 glucose residues, Since 1 growth substance molecule aete in the deposition of 3210? glucose residues, it acts, therefore, in the deposition of 2 2000 3x10? x or 140 micelles. Since ‘here js no reason to believe that the micelles of cellulose in young tissues, such as coleoptiles, are any larger than those of the cotton and ramle fibers, with which the micelle size deter- minations wore made, it ie clear that there cannot be any one to one correspondence between growth substance molecules and micelles formed in growth, A second, lass reliable, method for determining the mamber of molecules of cellulose laid down by one molecule of growth sub- stance is as follows: It has been found that the total area of longitudinal cell walls per mum. of coleoptile length was 40.3 ome and the mean wall 3a thickness was 0.4 Hence the volume of the cell walle is -2 _ Wo.3 x Ueto! = 1.610107 om? As above, the unit of celiulose structure is the block containing four gluccses residues, and having dimensions as follows: =[c 8.7 X 7.9 x 10.3 AS, or a volume of 7.082107? ma’, The specific gravity of cellulose will not be greatly different from that of the other osll wall material, and we can assume the cell wall material to consist, therefore, of 42% cellulose by volume. Hence 1 ma. of coleoptile containg 1, 61x107° x 35g =x 1079 x 0.2 = 0. 96x10? © wit blacks. Since each block conteins four hexose residues, there are 3. ¢eni0'© hexose residues per mu. of coleoptile. An increase in length of 2.66 mm., the result of the ap- plication of 29 units of growth substance, therefore involves the 6 x 2.66 or 10.2x107° eroduction of 3. 82x10" ner hexoce residues, Since no allowance has teen made for the volume taken up by water in the wali, this figure may be somewhat too high. Considering the approximate nature of the determination of the cell wall volume, the agreement between this figure and that obtained above from the waigat of the wall must be regarded as satisfactory. Using the avove fieure, deposition we ind that one molecule of growth substance leads to the weebemavven of 46x10" hexose residues, instead of 3x109 as in the first cal- culation. Since from the sbove 1% is scarcely pessible to assign a stoichiometrical relationship between the growth substance and the materials of the cell wall, another possibility was investigated, -39- namely that the growth substance is distributed in a monomolecular layer over the surface of the cel., and is thus by some means able to promote the elongation of the cell wall area which it covers. According to the analyses of Kégl and Haagen-Smit, the growth substance has the approximate formula Cy Hae, Since it is also known to be an wBaturated acia (K8el, Erxleben, and Hanagen Smit, 1933) one may apply Langmir's (1913) measurements on surface films of the fatty acids, from which an unsaturated Cg acid will have a maximum area of 4. 6x1072? om”. As stated above, the cell wall has an area of 40.3 mm per mm. of coleoptile, and hence 2.66 mm, have an area of 107 tm”, The number of molecules of growth substance involved is 2.3x10'1, which from the above value, can only cover a maximum area of Q. Um, Therefore, to form a monomolecular layer only on tie surtace of the new cel. wall laid down, each molecule of growth sub— stance would have to cover approximately lLOOOtimes its own maxinum area, In order to cover the surface of all the growing cells it would have to cover even more, Now, for the growth substance to “act by producing any change in permeability, it must, presumably, produce a change in surface activity; to do this it must form at least a monomolecular layer upon the cell wall, which is clearly impossible. Furthermore the measurements of Rideal (1925) and of Langmuir (1927) show that the rate of pas.age of water from a liquid surface into the vapor is greatly decreased by the presence of suriace films of fatty acids. Thus it is likely that any effect of growth substance upon permeability to water would be in the direction of decreasing rather than increasing it. The theory that the action of growth lio. suostance is directly one of permeadility change is thus given the appearance of being extremely improbable. follows: 1) 2) 3) The results of the present section may be surmarized ac Ore growth substance molecule, at 27°, brings about elongation sccompanied by the following changes &) moleculgs of glucose to cellulose 3x10? ») micelles of cellulose formed 1.4xic® Gc) molecules of pectin formed 7x103 d) molecules of protein ( in the cell wall) formed, mol. wt. 35000 3.9n10° e) molecules of carbohydrate converted to hamicellulose 1.610" f) molecules of pentose converted into pentosans g.7x10", Since there is so little growth substance in comparison with the structural material it seems certain that growth substance ig a true hormone and not itself a structural material. There is. moreover, far toc little growth substance present 40 react estoichlometrically with any of the constitvents of the cell well and thus alter its properties. The theory of VanOveroeek (1933) that growth substance by adsorption on the micelles of cellulose alters the interfacial tension between them and the intermicellar fluid is out of the question. There is insufficient growth substance to form a monomolecular layer on the protoplasmic membranes. Moreover, growth sub-— stance should decrease rather than increase the water parme- ability of surfaces on which it is adsorbed. It is therefore not likely that growth suostance effects growth through an increase of water permeahility. Qne growth substance molecule causes less than one half as much elongation at 15° as at 27°, altho the amount of cole- optile formed per unit of elongation is the same. It is therefore impossible thet one growth substance molecule causes the intussusception of a given amount of wall material. ie TABLE 1. PERCENT O2 GROWTH SUBSTANCE FASSING Tol0 PLAN FROM BLOONS Fiat UPOoo THE COLEOPTIL: STUMP Units avolied Unite remaining ris passed out Nunher (Initiel vossible curvature) (Gurvature on of bilock of ne — Go 2” epplication) go - 3 Plants 8. 20 Fl s109 i By 14,u 9.0 3755 Vy 14,4 9.8 32.0 15 19.2 13.0 32,4 21 19.2 12.0 3745 16 Mean %b passed out of bloex au TABTE 2 4 3 ei TITHE GROWTE SUBSTAN LES UNSUPPLIED nal eS GROFWTE RATES OF DECAPITATED COLEOPT 1 inute mi 1 Time after decanitation in SO 104 126 15 60 x it ri ‘uya ST /um Plant No. ay uns 74 PL dog de a) Oo hi} fia 4 o im ~ et pik ne) 0 0.14 o.14 6 0.16 * 8 * oOoG a 3 ON ed Mo a my mid et rit © eo s . * aA * QO aa ooo © OVe eeu oN QA et Om a oe € + 7 e 8 * aoaog aoc So 3. S&S ay icy hae rtri O oaeg o > es ¢ * * 2 Oo co om 2 Oo fe 9) fa) re) re) re) O.08 Oy eS OY) St ee iy Gaoog OO © & a * * & Oo coc © Pee OL Pts SO PO rod is 4) a © CO rir co, * & a ° 2 ooo oe ae) o at ovat ont o eto m om OO rt m e . * : * 2 * Oao OOo mo O ar Lo TAL Ms me a . * ry e * + Qo @® MoO oO oi ONO rep PO oN Cd pei mi CU red ~ * a > a € . * oO a oae co) al mo on ai] [Soa BYUSLAAOUT qyaoayg rat a) Ti-1 TI~<c IV-1 a | Iv—2 VI-1 VITI~ loti’) - al. oz £t°o {to ole otra ist uy Gi Py pos . oe) On OL et ml et ooo tT" GT°O «za ALO OF-O 2t°O «ST"O TT°O «170 2t"O {TO 50°O ADO Cd a S a JQ SLeOAT ao. te, 4 o age TS°O Gk°O 6b °O TE"O 12°C BetQ Ge7O Bere f2°O O20 “kano ante 61°70 GeO Se"a HE7O GEC STO GzO azo Led 22°C :1°O ST°O Ceo Gere Gero st°o «eT°O «4T’aO 1T*O 6t"e 26°O 90°C 1O°O TO"O ETC L970 -86°O «60°C ~bO°C 9T°O WTO At’O 2L°O =IE"O Get ost Got OSE SkT Semutm ut uot eq tawoep Voit HONVISAOS ORO ah Gyre LAG 16° aro Te°O eo ae" fh °4 Ox°O Ge°O $2" GEto 6T°O Tee £t°G &T°O 10°O HOrO Nt SOT 18gye GUTy, HEMOED 0270 06 92 °O um ng 7) 32°0 4 OGL Pe OOD a od LN oes oot ed OU OOo aN OY GNAGEAATC 2O SONSOLINE SHG WRQNN STITRGORIO) 30 SHEVE DLAORD FOVARAY & FIG gapNupu GT dea Syuseuer0uT 4aosg w O rl oi ian Mw Or ar a mA ts eeoRTon ea me oN ity Ht pi o> “ADOTa at 9 ag yO yumeny Lb. TABLE 4, GTA GaOVTH AND ALOTSIONaL GROWTH RESULTING FROM APPLICATION OF GROWTH SUBST AUCE POR 105 MINUTES Units women Zotal ercwth Tire Additional growth avowtt epolied 1 oats (e-< tent) (see text) {see text) Unite asoliea . Gis Min. rm. 0 6 0.71 105 9) 9,9 3 1.54 180 0.83 6,084 14.5 10 2.0% 189 1.32 3,091 19.3 7 2.57 180 1.86 0,096 29.0 7 4437 195 2,66 0,092 35.5 6 3.91 217 3.20 0.081 43,5 7 4.05 217 3.34 jeOT8 56,0 G 4,98 225 L,27 0.074 79.0 4 4 a6 225 25 0.054 116.0 5.22 eho Wal 0.039 ot" 90°09 coro 200 20°0 Z0°0 ane elo 90°6 agro 2o°s Z0"O 20°0 G22 qT"A 50°70 20°7 Z0°0 20°0 c0"0 OT2 — €9°0 20°70 Z0’O Sor Lt 3T°O GSTTO 4GT*O LTO Gere TT°O 2£f°D “E°O atte Gtve love oT°O IT°9 E€°9 eT°O 20°9 £0°G 4O"G 90°C LEO 2070 2070 £9°O 40°O 907O 20"O £O"°O ¢0°2 2070 4£a°"0 G6t OST SOT O4L GET T2"0 St’ tr" so°° Lovo 7o"0 dal 2 og 30 al ec. £270 STO It’) Ob’) 30°O 10°O GOT seqnuym Zt woTys,taeoep Jeyse £0°H f£O'O Z20°r 20°9 20° 20°99 2o°0 ZoO°s — gore 20°e 20°09 oef STS o2°0 T2'O st°a LTO £T°O 4T"O et7G STG 60°70 OT°O 6O°C IT°O 06 GL outE HORVGSEOS HLAOd “To ee G PIS¥a Gora HOO Z0"O go"9 20°O 20°O Q oO m 6t°C ot"s oT"O ate ET°O 4L°O aq ies + Oh o00 a — eet eter et a * oan 6 P= 8G 10°09 90°O 20°O Zorn 20°0 Z20°o LT*a 61 ‘OG 1T°O T2°0 12°90 9T"O “uypU GT /am BgdeUIeZOUF YyMoID G BOT L He 6 S°02 g G*2T 6 ~"9 -6 avo a LF cas) Ch Gn RD. Omi Wy a DA Nv B8qteTa 4ooTa tt "3°23 jo gO °orr yuNoTy _L6_. TABLE o. POTAL GROWTE AND ADUIGIGUAL “ROUTH NUSULTING FacH APPLICATION Gr gROWTH SUBS ANGE FOS. 120 MINUTES. 15°C. nita 30, Of Total growth Tine Adcitional growth Growth _ Apolied plants Titi « win, ria. Applied g.s. ie) g G.9k 120 — _ 6.1 g 1.418 150 3.2a4 0.039 12.5 g 1. ho 180 0.46 0.037 20.9 9 1.75 225 9,81 9,039 5u 7 2.39 300 1.45 e087 10& 5 %eQ3 345 2.49 0.045 GasLe 7. SLOCKES Fuad UPON COLZGPIiL: STUUPS. 19° 6, Uaits 4, . Units pas.ed passed out 2... Cest : 5 Per ; eee Pla <8" snolied Units revaining out of blocix of block Plante 1 12.7 7.9 Lg 379 12 F-4 1le.7 §,3 hu 24.0 12 3 12.7 8.5 ue 33.0 le h 12.7 7.8 Log 38.7 12 5 12.7 8.0 L.7 47.0 12 Means Lee 3.8 ayy TABLE &. DRY NHIGHTS OF COLaGPDILE Aggregate length of Heignat in Weight ver 5 coleoptiles in mm. me, gramsxl0 7 392 10.9 5.09 % 18.0 5,17 18.0 5.46 349 19.2 3+ 91 Log 25.8 2.32 Log 26.0 0.35 207 12.3 5-95 132 &. 3. 6.30 173 1G, 6.08 179 hl.o 6.50 336 17.6 5,25 389 17.0 425 643 33-9 5.23 200 ic.7 5255 406 17.6 5,81 300 17-1 5.75 om yet Te ark 5. onl fi. WRIGHT G2 CLLL VaLLS PROM YOUNG COLSOPTILES Aggregate length velent of Jedent of cell of coleoptiles cel. wall wall ver ans in mw... in meg. in gramsx10™ Ligg 8.10 1.63 635 1.4 1.79 087 16.9 1.56 ae) 12.8 1.72 731 11.3 1.55 637 10.5 1.65 TABLE 10 sae , Neight after Veizcht after VYeleht after Veight of e zh ae extraction extraction extraction Total _ Gell walle ith mith with " Protein oxalate 2% H,S0), 2% Made éc.3 20.4 11.4 8.6 ——saw —mae: 47.6 Ya,g 25.0 17.4 — — 21.0 9.7 — _—— art 1i.l —_- ewe 22.8 10.3 ~~~ — 14.4 _- — 0.322 1.93 19.5 ~— ~ 0.592 2.08 —ly5-- RABLE 12 SUMMARY OF GOMPOSTSION OF GOLSOPSTEG TMLL “abt Fraation y OF Cell wala % total coleogtile Removed ny oxalate 8 (pectic suostances) 2.3 Removed by dilute acid 2g 2 less protein (hesiceliviosa) ° Renoved by dilute al-cali (further pentosans) 10 c.8 Froteina 12 3.1 Residue (cellulose) he 11.9 100 28.3 BL ne TABLE 12 MEAN VAGUES Gs DIALETSHS AND CELL S125 OF JOLAOPTIT LES Gutgide dianster Insiae diameter sunbar of cell rows Diameter oi epidermal cells Diameter of sud-coldermal cells Diameter of varenchyma cé.is 1,18 roa, Q.75 mun. Q 0.G21 mma. 0.9024 wa, 0.045 an, Diameter of inner aermal cells 0.924 mn, TABLE 13 GALCULALTON OF Das. Cia “ALL AREA Pix tl. Gr GOLEOPYILE Cell row Dianeter Gircunference No. of cells 20. of Geils . of wall. 720. in row x length of (cire fdian. ofcells) cell wall Eoidernis 1.18 3.71 cubside wall 173 3,63 Soideriad s i “ inside 1.14 3.58 peeoid 146 3.50 ; 13 3.28 Parenchyma 1 1.05 3,14 &7 4,02 a0 2.70 3 U. 82 2.58 103 2.47 Inner dernual Ge 76 20359 layer “ere en nome 21.68 1é.02 wt: . a yy ea a fy AA me a fotal area per ma, of coleaptile = 21.05 + 1lb.02 = 20.25 mm, PART V. THE RELATION OF WALL FORMATION 10 CELL ELONGATION a. introduction in. the general introduction it wae pointed out that active wall growth by intussusception has been advocated since the time of Krabbe and that fixation of elastic stretching by intussusception forms an intezral part of the theory of Sachse. Skding and Strugger among the present investigators of cell elongation advocate intuse susception, altia in neither case has any special evidence concerning this process been put forth. In Part IV it was shomm that one srowth subatance molecule does not coring about the formation of any given amount of cell wall, More comclicated actions of srowth svoctance apon intussusception might, however, be envisaged, and it is necessary $9 eetaclish whether or not intussusception takes place, by a direct measurement of the amount of cell wall formed under different conditions during the action of grovth substance, db. Methods Sections cut from colaoptiles and placed in growth sutstanse solutions of suitable concentration, elongate consideravly, as shown in Table 14, which also shows that similar sections placed in water (Insert Table 14) glone elongate mich less. ‘The several conditions zoverning this elongation will be congidered in detall in Part VI, but the fact thet 1t does occur will be made use of at this point. These sections, since they have no sxternal source of nutrient, must, if they form cell wall, accomplish it oy a transformation of materials already ~53~ within the cell. Except for the material lost by respiretion, their total dry weight must remain constant, and it is necessary to determine only that part of the total dry weight which may be cousidered as cell wall. For this purpose essentially the same method was used as in Part IV, i.e. the method develoved by Hansteen-Cranner (1914) sor the separation of cell contents from cell wall. The procedure was as follows; sections, in most cases 3.7 mn. long, were cut with & two bladed cutter irom coleoptiles acproximately 5 mm. below the apex. 10 such sections were placed in growth substance solution of the optimal concentration, i.e. 10 waits per oc., and 10 sections into water alone. After the dasired time of growth sucstance action the elongation of the sections was measured under a binocular micro scope with a eyeoiece nicrometer. They were then placed in previcusly dried and weighed micro-gooch crucibles. The shape and dimensions of these crucibles are shown in He in the bottom of each crucible had besn placed a small mat of acid washed asbestos, the mat washed firmly into place, and the whole dried to constant weight at 95°. The sections, once in the erucible, were thoroughly ground against the sides of the vessel. They were then washed successively with 40 to 50 portions of eold water and en equal amount of boiling. water, During the first few washings large amounts of colloidal material, ‘presumably cytoplasm and cell sag, were removed, The later washings were Clear, and the residue was then, according to Hansteen-Cranner and to tha resnites of Part IV to ba considered as cell wall. The cell walls were dried to constant weight at 95°. All weighings were performed uoon a Kuhlmann micro-talance cagable of estivating to 0.001 mg. The use of such a balance was necessary in view of the small amounts of material which it was desired to determine. bet ie a ca eee ee ee a pe 5 aa N N sw - 1CM. Figure 8.uwicro-Gooch Crucible. ~5u. Gg. Experimental Results A few series of duplicate datermlnations were first made in order to eatablish the accuracy of the method. In Table 15 it {Insert Tavle 15) may Oe sean that the agreement between duplicate determinations was in al] cases quite satisfactory. In sunsecuent tables, then, only the means of the duplicate or triplicate estimations mada in each case will ve given. If elongation is due to intussusception in the cell wall, one would expect the amount of cell wall per section to iucraase approximately in the game ratio as the length. Tavle 16 shovs that (Insert Table 16) at 25°, this la in fact the case, In the mean the ratio of the wall weight of the two sets of sections having different slongations is almost exactly equal to the ratio of their lengths. This is a sucvport of the assnwuption made in Fart IV that at ordinary tesmeratures elongation ia accompanied by wall formation. It will also oe show in Part VIII that at this temperature the total dry welgnt closely parallels coleoptile length over most af the coleoptile grand period. The fact that at 259 these processes fit well in with one another isa reflecte: in the fact that this temparcture is clase to the on- timum for the coleoptile. Slongation is not. of necessity, however, paralleled oy formation of cell wall. Coleoptile sections were allowed to extend at 2°C., It was found that, as shown in Taole 17, considerable elongation (Insert Table 17) 55 took placa at this temperature under the influence of growth subatance if a oreliminary hour at 25° was allowed, Under these conditione, altace 12 to 14 per cent of olongation took place, there waa little or no difrerence in the wall weight of clongated and non elongated seotions. If growta susstance solutions be made also 1 per cent in fructose, greater elongation takes place than in growth substance alone (see Part VI.). (“At the same time, as shown in Table 18 much (Insert Tsble 18) more cell wall is formed than corresponds to tha increase in length. At 2°, however, fructose has no affect uoon the formation of cell wall. The interest of this is two fold. In the first vlace it shows that the old supposition of Yortmann (see above) that decreases in elongation are due to formation of excessive cell wall cannot be in ali casas correct. In tne second place it shows again that cell wall formation bears no coustant relation to slongation. a. Gonciugions The results of the praesent section may be summarized as follows: 1) At 25° elongation may or may not te paralleled by cell wail formation, depending upon the matrititive condition of the acleoptile. 2) At 2° cell wall formation eaases, altho elongation continues. 3) Active growth by intussusception of new material as a possible mode of growth substance action is therefore excluded, 4 bla i, wwe a aE es ME Pare Hater. (Bach value is a mean of from 12 to 15 seations) Grovth of Qoleoptile Sections in Growth Substance Solution and in Solutlon etrawth % Growth %® Growin in @ hours in 4 heuwrs in 2 hours Water 3 4 9 Vater 3 5 7 Water 3 5 iz Geowth n Substance 1 ah 35 Growth Substance 13 26 45 Sab stance ~5T- fable 15, Buplicate Determinations of (ell Walls Grucible / Exot. 1. a3 3, 4, Weighs of Gsll Walls per 16 Ssestions.Mes. 1. 0.75 8.73 0.75 9.69 2, 0.77 0.75 0.72. 6.70 3, 0.73 0.78 0.74 4,69 +6 5. Or09 Table 16, Increase in Gell Wall Yeient Durine Elongation of Sections at 25°0. (Growth Substance Action for 6 Hours.) Foot. Im.in @.8. In.in Water tarerth in Growth in Wtoin as (Arbitrary units) {Achitrary units) G. Se Water (Mes. 1. 4,19 3,80 13.2 2.6 0,714 2, Yu 3.75 19.2 1.7 0.766 3. 4,50 4,o2 21.6 6.3 0,865 h, 4, 39 3.87 13,7 4.6 0.829 5, 4,56 4, 88 23,3 “ig 0, 847 Rxperinent i. 2. 3s 4, 5s Means Lengts in 6.8. 1.23 1.18 1.12 1.24 1.37 1.45 Length in Vater detent da 3S. 1.47 1.18 1.13 1.18 1.18 1.16 Weight in Faber Weoin Mater 8.009 0.650 0. 766 0,7 0. 758 so Jaqen UPSR Jagey UT UT ( en’ e°l «be JP BOLL ty UBER 66°O 36°T cog °O O61 °6 2 TT aT &e°t on970 Geb"o 2 a ay’t 6 GLG"O Gel"d Fa Ts aot ee o#1°0 GBT "T 3 on g2°Tt 22° GéL°G OZO°T g é. (Say )seqeg (S94) °S*S FeqeR UT °g°g UE ale 20°H oad *G al’¢ i rr | die 93°h = GE 66"¢ 32°§ G22 66°f #8" GE ST “SD OP 4g FE *D uP ay ‘ Steg UE WSteg Uaeoig Z qyMoig % Jeqge_ UE GIFUST “S°g UT fue] ‘dhe, yay ates bon eg A ng ts "ST STaRL tO°T ort Suee i OG" T OTT OLL°O a) 3°0 a? bb’ Oo TUT nO #.3°O 2 act go'T Itt 36s"0 Liga qe 9°cT £OrT 20°T TT6 He's a"t HST COT oT sn6"O ah6"G 6°¢ z°ct Joqem UF44 deqem Up wy (BR jaegey (Sa SO <egcg UT "gS °9 UF sma eet eeomeeepite ar pumanand g°y Ut SE "erg Uta UF quetey UE Ta ToR WLOTH % Ugg % 75 *einoH Og (897uQ Sreag Tony) Spt Se BaCTIR [OS Soueqsque Ujkoig Pie Gsojonag GE UST VeUCT” gutang tietay Tien Ties ut esterouy OL7s Oty *G 6L°€ 61" h ol’ 02°47 "€ care 9th "2 13° Gia *T (8eTUn Asea47q47) 7e4eg UT wqysueT "S°O UE uqsuey *4axg Astcs ne tie 3Gj WOlAoy SSweysqEs YywoIh God Je VioTises Jo Woryesuely, “Atmng FUstem (tea (180 UF sseesouy “LE STaBz -59- PART VI. CONDITIONS GOVERNING THE ELONGATION OF EXCISED COLECPTILE SECTIONS a, Introduction The preceding sections have showm clearly that the effect of grovth substance is not one upon intussusception of new material. Since, however, growth substance does influence the deformability of the coleoptile (Heyn, loc. cit.) the only alternative is that there is an increase in the plasticity of the cell wall. An effect upon plasticity might oceur either directly upon the wall, aa for example suggested by VanOverceek (193%) or indirectly as a result of the action of growth substance upon functions of the protoplasm. The theory of VanOveroeck has been shown above to be untenable. It seems a priori highly unlikely that so few molecules of growth sub- atance as are actualiy present in the plant should exert any direct eitect upon the oroperties of the cell wall. One experiment which was done to investigate this possibility might be mentioned. If growth sucstance acts upon the wall directly, the only function of the orotovlasm and cell contents would be to axert the necessary stretching force. Removal of turgor ami lbs replacement by a coi parable longitudinal tension should equally well bring about elan— gation under the influence of growth substance. The turgor of cole- optiles was removed by plasmolysis in KNO, and longitudinal tension b) replaced by 1G gran weights. According to Heyn (loc. cit.) such weights suffice to bring apout considerable elongation of plasmolized cole- cptiles. The coleoptiles carrying these weights were then suspended =60— 3%. Any KOO, elastic elongation permiited to cease and growth substence added to the solution, Not the least increase in elongation took piace in the plasmolised celsoptiles, altho similarly susyended but turgid coleoptiles responded immediately. The most likely, altho not ot necessity correct, oar only explanation of this result is that the action of growth suustance is upon the protoplasm and that the latter mist be in contact with the cell wall in order to bring about the plasticity increase. In the present part it will be, however, rigorously proven that the action of growth substance does involve the intervention of runctions of the yroteplasm, especially of res piration. Bafore going to this it will be necessary to describe in detail same of the techniques used in the succesding parts. be Methods For the study of proceeses of a metabolic nature it is convenient to have the experimental object suspended in solution, the composition of which may be readily altered as desired. For this reagon it was determined whether or not portions of colecptiles would elongate in growth svuostance solutions. Table 19 has show that they do and that the elongation in water slone is much less. The principal circumstances influencing thia slongation will next be described. the mageurements of elongation were made in two ways: a) sections 3.7 mu. long were cut with a special two bladed cutter, and placed in the solutions. These sactions could be measured con- yenieatly with a 10x binocular and eyepiece mioromotor to abaut 1.3 per cent. This is somewhat smaller than the variation between in dividual sactions of a supposedly comparable series, as is shown -61- in Table 19. The individual variation is particularly large if (Insert Table 19) the average elongation is also large. <A horizontal microscope equip- ped with a stand mounted uwoon a micrometer screw was used for a large portion of the measurements. Coleoptile sections were placed upon thin glass rods having a diameter just equal to the inside diameter of the coleoptile. These rods ware in turn imbedded in paraffin in- side of rectangular glass vessels through wiich the elongation of the sections, in this case originally 1 om. long could be readily followed. No essential difference between the measurements carried out in these two ways was discovered. In all. of. the experiments avoul to be described the plants were decapitated two hours before using in order to obtain them relatively free of growth suostance. ¢. Blongation in Yater Alone The growth of sections of coleoptiles, prepared in the manner already described, and imvmarsad in nure watar was first in- vestigated. Taole 20 gives the results of a typical series of measure (Insert Table 20) ments, and also a conparacsla series of measurements of sections with their sases in water, but their tips in air. It may be seen that the growth rate of the sections completely immersed in water fells steadily until sftar 6 hours it raaches a very low value. There is no sudden rise after two hours corresponding to a production of growth substance by the "physiological tip" as is the case with the sections whose tips were in air. The fact that any growth took place oo in the sections immersed in water is to be attributed orincipally to growth substance in them when they were removed from the plants. Table 14 has shown that the elongation of sections in growth sub-— stance may be marked, being as large ay 29% in four hours and 53% in 2h hours. ad. Stirring it was conceivable that with these seotiong immersed in solutions, the rate of diffusion of growth substance to the cut surface which it enters, or the rate of diffusion of oxygen to the tissus, might prevent a maximum crowth response. Therefore meagure— “ents of thea grewth rates of sections both with and without stirring of the solution by alr were mais, These results are given in Table 2] (Insert Table 21) and show that stirring of the solution is not necessary. 8, Former position of Section in the Coleontile The effect of the original position in the coleoptile of a given section uoon its response to grayth substance vas then investi- gated. That such an effect exists is showm by Taole 2c, which gives (Insert Table 22) the per cent growth ver hour of sections from the toys and bottans of a series of previously decapitate< coleoptiles. Similar measure- ments made upon colsoptiles divided into more sections showed that the two 3.7 si. sections nearest the apex of a 2c~3 om. decanitated plant have almost equal reactivity. The lower zones showed, as in Taple 22, a lower ability to grow in the presence of growth substanse, 63. Tt has been known from measurements upon the growth rates of entire coleoptiles (marked into zones with ink or paper marks) that the Lower zones: do grow mora slowly then those nearer the ton. This nas been attributed, however, to a lac« of growth substance in the lower zones woich must receive it through a long portion of coleopiile aatively using the srowth auostance. That this ia not the only factor.is shown here directly, since it is clear that the cells at the base of the coleaptils show a much smaller growth ressonse to growth. susatance than those nearer the top. In the present work, wnless otherwise stated, only the two sections from tha top wera used, £.. Growth Substance Ooncentration The effect of concentration of the growth substances solution upon growth of the sections was deteruined, and is shown in Table 23. (Insert Table 23) From Taosle 23 it is evident that there is an optimam growth suostancs concentration in the resion of 10 units per ac. Golesoptiles immersed in concentrations 49 great as.60 units per ¢c. show 8 sirinkags after 4 hours and at the end of 24 hours have frequently lost their tarcidity due, avparently, to a toxic erfect of the high concentration of growth oatance. A decrease in growth in very low growth substance concen trations waa also found. A simole consideration will show thas only in the case of the 0.01 unit selution oan this be due to an insurficient quantity of growth substance, and that even in the 0,1 unit solution the decrease mist be due directly to the low concentration. Prom the data of Part IV 6.00535 ec. of a 29 anit per ee. solution can give © his torty a key h concen trations iw wort duc To cas purities we the crvde growth substance preparatuns osed bere since try StaMerne groetl Substauce “B" alse exhib és Toxrety we he lo concentrations —bl under thees conditions e maximum of 7.85 mm, of goleaptile slongation. locc. of a O.1 unit var cc. solution could then give a maximum of 5,05 mn. total coleoptile elongstion. Since in general in the presant cage 12 sections were placed in 4 sc. of solution, thesa sections should be able to elongate a mariman total of 22 mm., or 459% ver section, woich is mush larger thau he 18% gbeerved, a portion of which is, moreover, dug to growth substance initially in the ssction. In the case of the 0.01 unit solution, however, each section should be able to elongate. med 5.9% more than controls in pure water, and Table 23 shows that the inerease is only 3% greater. g. Fructose The presence of a nutrient in the growth substance solution increases the elongation consideradly. For this purpose iructose was found to give more consistent results than glucose, Table 24 (Insert Table 24) gives an exauwule of the effect of 1 per cent fructose upon the salon gation. In general in the later experiments, however, fructose was not used since it proved quite favorable for the grovth of misro- organisms. The pH of the external solution exerts a marked effect uvon the growth rate of eoleeptile seetions. A datailed analysis of the effect of pH will be reserved for Part VII. h. Inhibition of the Aerobic Metabolism Upon the theory that the action of growth substance is a simmle physical change of cell wall, for exarple oy decreasing directly in some way the viscosity of the substance in which cellulose wicelles —-65— are imoedded (Heya, 1931), one would hardly expect the action to ve atooped by the presence of narcotics or evanide. If, however, growth substance denends for ita action upon orocesses of a matabolic nature, narcotics or cyanide snould inhibit this action. I+ was easily demon- streted that both KCN and phenyluzthnane stop growth. Teble 25 gives (Insert Table 25) aine@en 2 gumary of two experiments with different concentrations of KON and of Fhenylurethane and shows how marked is the stooping of growth. Further experiments will be given in Part VIII, It might be mentioned that potassiwua mono-lodsacetate as well as sodium fluoride also innioit the elongation af sections. Experiments were then carried out to determine if a cane asction between grovth and cell oxidation exists. The action of growth gacstance in solutions under an atmosphere of cure aitrosen was first investigated. Gomaercial Ny was passed over reduced copper in an electric furnace at 690°S. The gas was then cooled by passage through vwashoottles, and ourpoled through the solution containing the soleoptile sections, A prelininary experiment shoved that growth substance is not @fiscted in ts activity oy prolonged passage of Np through it. The sections were freed of On by treatment with Ne for two. hours pefore introduction of the growth substance solution. After this preliminary tvo hours, sufficient growth substance was introduced to mae the solution 10 wnits per cc., and the buobling was continued for four hours more. The sections were than measured, a oortion of them placed in growth substance solution in air, and tha ramainder in pure H,0, in air, Table 26 shows the results of 5 exoerinents. (Iasert Table 26) -66- The coleoptiles were not marmed by the prolonged lack of Onfis BOwn by the fact that they grav normally upon being supplied with both growth suostance and oxygen. However, a man growth of only 3% took place in N,, although in air from Table 23 20% growth would take glacs. That even this 3¢ grevth takes place ls to ve abtriduted prabably toa 9, remaining in the sections. Therefore, normal growth Yails to take olace in Np. Since the sections do not elongats, when placed in ture HA0, ta any extents than if immersed in H.0 without tha prelininary growth sabstance-N, treatment, sither the action of erouth substance does not take place or the growth substence is not taken up by the sections. In order to show unequivocally that imhbition of aerobie metavolisem inhibits actual elongation under the influenco of growth substance, the following experiment was undertaken; tvo lots of sections were placed vertically upon glass pins; wpon the apical ends of one set were placed ager clocks containing no growth sub- atance. Upon the apical ends of the other set vere placed blocks containing 1200 units per cc. ‘he wections were then left ab 25° in a saturated atmosphere for 2 hours. At the end of this time, 2 oortion of the sections which hai had no growth sucstance were piaced in water, aud the remainder placed in a 10 unit per cc. solutioa of growth substauce. The plants which had had 1200 unit blocks were distributed among the following solutions: a) KON 3x07 5 ») KON ix107) c) Phenylurethane 0.05% a) Vater =67- table 27 gives the results of one of five experiments which ylelded ciosely similar results. There is no question but that grovth substance passed into the plants which had 1200 unit blocks pinca these sections elongated more in water than did those which had plain agar, and were also placed in water. This elongation brought about cy zrowth substance already in the plant ia, hovevar, clearly inhibited by both KON amd phenylurethans. (Insert Table 27) Table 27 shows that sections containing growth substance elongate when placed in water. Since the sections whose elongation in growth substance solution was inhioited by Ny did not elongate to any great extent whan subsequently placed in water they had not taken up growth substance, The uptake of growth substance as well « : 5 as the actual action of growth subatance is, dependent woon aerobic metabolien. i. Gonclusions In thie section the technique of using excised sections of coleoptiles in the study of cell elongation has been described. The principal points are: 1) There is ao "physiological regeneration" in sections immersed in water. 2) The original nosition of the section in the coleoptile greatly influences its suJsequent response to growth substance, As would be expected, sections from the most rapidly growing zone of the coleoptile exhibit the greatest response. 3) There is an optimun growth substance concentration of 10 units/ec. \\) Fructose increases elongation in growtn wiostance solution, 5) Slongation under the influence of growth subatance is inhite ited by ZON,oy phenylurethane,or by an atrasphere of pure nitrogen. It is demonstrated that both the untake and the action of growbh wibatanse are connected with the aerobic metadoLian, fable 19 MEASUREMENTS OF SEOTICNS ITH BINOCULAR NIGROSGOPE Section Original Growth Substance Growth Substance Humber. Jength & hours $ hours wim. TA nua. 1 3.70 4,00 4,25 2 3.65 4.00 4. 35 3 3,70 4.10 4. 35 4 3.70 4,35 475 5 3.75 l20 4.50 6 3.70 425 4,5 7 3.70 4.210 435 5 3,65 4,00 425 g 3.15 4.15 4.50 10 3.70 4.40 4.35 1 3.70 4,00 425 12 3.70 4.00 L,10 Means 3.70 + .01 4,15 = .03 Wolo = 05 Table 20 GROWTH OF GOLEOPTILE SHOTIONS IN PURE WATER Plants % Growth per hr. ehr, 43 hr. hr, LB, hhr. 6 br, a . (flops in water) 1.8 1.6 2.2 0.7 0.6 6.4 2 6 2.5 2.5 4.0 44z WT 3.3 (Tops in air) =70~ Table 21 GROWTH RATES OF COLHOPTILE SEOTIONS IN GROWTH SUBSTANCE SOLUTION WITH AND WITHCUT STIRRING BY AIR % a sa 4 Tota Exot. Sections % grown Bae 2 nr, after € 2@hr. hr. 6hr. Shr. i0hr. 20 ur, 1 (no air) 7 & 6 3 1 9.2 16 2 (no air) 6 9g 4 3 2 9.3 19 3 {ao alr) 7 7 3 z — — 16 4 (air) 5 7 5 2 — — 22 5 (air) 7 6 4 1 i 4 14 6 (air) 7 & 5 3 —_ — 2) Mean of 1, ; __ _. a 2, and 3 &.0 4,3 2.7 ~ L7.0 Mean of hi, ; Growth substance = 10 ufee Taole 22 GROWIH RATES OF TOP AND BOTTOM SECTIONS OF COLZOPTILES IN GROWTH SUBSTANCE SOIUTION % Growth per hour Lhr. 2hr. 3ir. Uhr. Ahr. 6G br. Zhe. Top sections 4.5 42 4.3 7 2.7 2.4 2.4 Bottom ssctions 1.0 1.0 1.1 1.4 2.4 0.9 ued Growth suostance = 10 uj/ee TABLE 23 EFFLCT OF GROW?H SU:STANCZ GCONCENTRATICN UPON GROWSH OF COLEOPTILE SEOTICNS (Zach value mean of 50-150 sections). Growth Sudstance d + concentration omin 33 Standard Units. | 2 hours 4 hours 24 hours 8) 3.3 2.1 o.4 io 4,3 8.0 7-2 20 7.4 10.8 15.4 10 11.7 19.9 31.0 1 8.4 15.7 27.0 0.1 6.5 12.1 17.5 0.01 LA 7.0 1565 0.€O 3.3 5.6 11.9 TABIE 24 EFFECT OF FRUCTOSE ON GROWTH OF SECTIONS Exnt. Solution Growth in @ 4 ar. Sibr. 2h ar, 14 frus., 10 ufce —— 235 27 10 ufes 11 13 16 1 1% fruo, — 9 14 H,0 4 7 8) 1% frac., 10 ufec 12 23 33 10 ufec 10 14 15 2-14 true. 2 3 6 H,0 2 3 6 l@ fruc., 10 ufee 13 21 35 5 10 u/ee 1h 18 20 1# fruc. 5 7 10 H,9 6 7 & TABLE 25 INHIBITION OF GROWTH OF COLMOPTILE SECTIONS BY KON AND PHENYLURUTHANE Solution g Growth Solution % Growth &. S. alone \ 23 g. 8. alone 20) ga, + 2x10 N KCN 5 g.s. + 0.001% phenylurethane 23 g.s. + xlo7? Nu KON 2 z.9. + 0.01% " 14 ges. + 2x10? 3 KCW ~4 g.s. + 0.1% " £8. + 2x10°° N KON ~ 3 H,0. + 0.1% " H,0 + exi07~ HN KOM - TABLE 26 Hret. scctions ie Wt Pius INHIBITION OF GROWTH OF COLEOPTILE SECTIONS BY NITROGEN No. of % Grovth after 4 nr. in Na+ 2.8. 20 26 25 33 46 45 a reef Un Fate * % Gronth after 4 Growth after 20 hr. in airtg.» 20 nr. in airtho 17 — 21 — — 3.8 2s 8.0 13 Wo5 Sln this experiment the sections were not first freed of 0, -T3- TABLE 27 INHIBITIGN OF HIONGATION (AFTER GROWTH SUBSTANOE UPTAKE) By KON AND PHENYLURETHAWE Subssquent Agar Blog aolution (2 hr. water agar water water agar 1G ufes 3x10 7N KON #8. agar bea agar axl07 3 Kom ; . 6.05% Fhenyl- ef. agar urethane &.8, Agar water $ Slongation Sections during plesk action 15 } 3.0 15 15 15 15 15 5.0 ; Sucesquent 2.2 3.4 d.1 18.1 27.6 33.2 1.8 2.1 3.9 2.4 367 520 U1 49 5.5 11.2 12.8 25.5 -PE FART VII. THE RELATION OF HYDROGEN IONS TO ELONGATION &. Acid Grovth and Acid Surveturas The study of cell metabolism in reletion te growth substance was commenced with en investigation of the relation of hydrogen icns tu growth from the following considerations. It has long been knovm (for example Borowikow, 1913) that acids mney considerably inerease the growth rate of plants immersed in them. This fact has been el- sborated unon by Strugger (1932, 193%, 1934) and extended into a general theery of cell elongetion. According to Strugger, suitaole increases of acidity, either by cellular metabolism or by experimental interference, increase the inbibition pressure of the protoplasm and this in turn brings about an inerease in growth rete. Ags pointed cut in the general introduction there 1s no gaad reason ta believe that an increase in the imbibition pressure of the protoplasm would, as such, have any marked effect upon growth rate. It remains, however, possitle that chenges either jin cell wall hydration| ewe in some other or property of the protcplasm determined vy its puymay influence the growth rate. It seemed desireasle therefore to investigate the wicle probles of the relation of acidity to slongation with a view not only to the theory of Strugzer but also in order to discover any possible connections with growth suostance. Stmugger has described the effect of acids, in certain concentrations, im inereaning the growth rate of Hellenthus roots and hypocotyls, The growth rates of Helianthus roots ars, secording to Strugcer, minimal when they are slased in cuffers of pH about 5.1. =] Bae a Upon each side of this minimus the srovth rates rise te a maxima: and fall again to low values at pH 3 and pH 8. ‘The curve of growth rate against pH shows then two mamima, By renovel of strips of epidermis of ona side of hypocotyls, and by then placing these hypocotyls in burvers of suitable pH, Gtrugger was able to cause large curvatures avay frou the point of entry of the Duffer. Buffers which caused fast growth caused large curvatures, and this was interpreted as signifying that the parenchyma o: the side near the point of entry of the acic grows faster than the parenchyma of the coposite side. These carvatures will be called for convenience "acid curvatures" in the following discussion. Strugger. advanced the hypothesis that cell slongation 1s intimately connected with the pH of the cell protoplasm relative to its isoelectric point. According to this hypothesis, roots and hyoocotyls show a.minimwa growth rates at pH 5.5, oecause this is the isoelectric point of their protoplasn. Wita change of acidity on either side of this point an incrsase of growth rate results. Dolk andi Thimann (1932) found that if growth substance is supplisd to tha Avena coleootilée in a solution burtered at an alkaline oH ite effect in causing call elongation is abnormally wnall. Ji is & matter of general experience both in this laboratory and in others (Boysan-Jensen, 1932; Kagel and HaagenSait, 1931), that solutions of grovth subetaice muat be acid in order to give the maximun effect # vpern cell elongation. This fact has suggested that there might be soma relation between growth suvstance and the "acid growth" of Strugzer. Such a relation, if found to oxiat, could terhaps lead to some elu- sidetion of the mechanism of the action of the growth substance it- art self. The prasent @wek deals therefore vith the "acid grovth' and -76- Vaoid eurvatureal under the following heade: a) The neture of the ohenumenon. b) the acidity of the coleoptile during growth. > c} The dependence of acid growth on growth substance. ob, The Nature of the Phenomens If an entire section of a srowing root, hysocotyl, colecotile, or other organ is placed in a certain solution, the effect of this external solution woon the growth rate of the organ will depend greatly upon the speed with which the selution penstrates from the cut ends to the interior cells, The epidermis with its cutinized wall is relatively imperneable. Hence 2 small effect of a dissalved sudatance uoon growth rate dogs not demonstrate unabiguously that the substance has a small effect upon the growth rate of a single cell. It de neceasary to distinguish between transvort and actioa of the substance concerned. It is possible to simplify the problem of transport to a certain extent by the removal of the enidermis from one or both sides of the ergan. The dissolved suvstancs, acid, for example, may then penetrate directly the entire lensth of paren— chyma. If acid accelerates the growth rate of the parenchyma, and if the epidermis is removed from only one side, a curvature away froin the side without evidermis will result, because the cells nearest the woukd will increases their -rowth rates before those on the sida avay from the wound. Such a curvature will, nowever, tend to dis appear after a time when ali of the cells have been reached by the acid and slongate at tha same rete. If, hawavar, the parancnoyme inereasas its grovth rate without a corresponding increase in the growth rate of the eifidersis, a permanent curvature will result since epidermis hinders growth on one side and not on the other, This, as will be shown, is what occurs in the case of an Avena coleoptile which has had its epidermis removed from one side and which is vlaced in a sufficiently acia buffer solution. Liethods. For carrying out of acid curvatures, coleoptiles (depapitated 2 hr. before) were cut off from thelr mesocatyls and placed in rows upon short glass roils having diameters equal to the inside diameters of the hollow coleoptiles. Thess rods penetrated, of course, only a mm. or two into the lumen of the plants and in no way iaterfered with the curvatures which were ootained, The rods were in turn mounted upon glass strips (19 rods per strip) and the entire arrangement could then pe »olaced in a petri dish containing the solution under investigation. The removal of strips of epidermis was easily executed sy merely stripping the lattar off with a pair of small forceps. The strips which were removed wera avout one half ma. broad and were al<ays taken from one of the narrov sides of the slightly elliptical coleontile. At the expiration of the experimental period shadow photographs of the resultant curvatures were taken through the petri dish. 24 should be mentioned that altho the cole~ optiles were placed horizontally and altho they are normally very sensitive to geotropic stimuli, they di4 not under these conditions exhibit any eceotropic response whatsoever, and remained quite hari- zontal. The strips of epiderms which were reaoved were not of con- stant breadth and this caused a variation of the curvatures given by a series of othervise rather comparable plants. Table 26 confirms (Insert Table 28) Strugger's opservation of acid curvatures and shovs the extent of ~78- this variation in two series of plants, one with largs angles and one with small angles, Because of the rather large errors, it is of course necessary to base conclusions upon several experiments, In the following discussion curvatures away from the side without epi- dermis will be designated as 4 Melivaine's phosphate, citric acid make psssi ble buffers ware used since they the use of the same materials over a wide range of pH. The bufiere uere used, at different times, betweon the molalities of 0.0025 and 0.1 and were, hence, considerably below the osmotic value of the cells, which are in equilibrium with adout 0.25 molal E03. dell wall plasticity and elasticity were measured in the manner descrived by Heyn (1931). ‘The coleoptiles were fastened hori- zontally by their sases on saort glass rods, which were in turn mounted in vertical rows on supports. at uniform filstances fro... the end of the supports, weights of 250 mg, were placed and fastened with a small amount of lanolin. The ccoleoptiles were then placed in a sa- turated atmosxnhere under a bell jar to pravent drying. Prelininary experiments showed that if the riders were allowed to remain one half hour, large differences ia the curvatures of the plants infil- trated with different burviers develoved. Shadow photograghs were taken, the weights renoved, and the curvatares photographed again in two minutes. The curvature remaining after renovai of the weights, is the plastic portion, i.e. that due to irreversible stratching of tha cell walls. Tne dirference between the curvatures “hen the weights are still upon the plants and the plastic portion is the elastic or reversivle curvature. ~T9- Experimental. The acid curvatures appear in as short a time as 20 minutes ani after @ how's they changes relatively little (Table 25). For comparative purposes, then, all curvatures vere (Insert Table 29) measured aftar two hours. These curvatures were very large if the plants were placed in a buffer of pH 4.1 or less. External acidities of greater than pH 4.1 proved to oe toxic aftar more than tao hours. Plents vlaced in buffer solutions of pH 7 showed little if any cur- yature, Tne causa of these curvatures will now tbe considered. Ti has already been pointed out that in order for a permanent curvature to resilt it is necessary that there be a difverenca in elongation of epidermis and varenchyma, I+ was even conceivasle that the curvatures might be fue to an active contraction of tha epidermis. The behavior of isslated. strips of epidermis in buffers of different pH was therefore determined. The growth rate measurements were made with a horizontal microscope and rectangular glass vessel in the manner previously described. Epidermis was cotained by the removal of strips about one half mu. broad from ordinary plants. These etrips were fastened to emall hooks with aid of drops of warm wax, and were then suspended in tha observation vagseals. One-half gram weights were placed on the bottom ends of the strips to keep then under tension. Table 20 shows that by comparison with tne (Tnsert Tabla 30) growth of plants having much of the epidermis removed (see below) the growth of the epidernal strips is very slight; neither doses this growth denend narticulariy upon the pH of tha medium. However, the epidermis undergoes no shortening, and therefore this hypothesis FO concerning the acid curvatures is at once rulad ont. It was then necessary to loos for a difference of growth of the parenchyma in acid amd in neutral solutions. In order to have "parenchyma! as comparable as possible with the parenchyma of the plants of the curvature experiments the foi owing device wes used. ‘The epidarmis was renovad as equally as possible from tvo sides of the plants to be used. Since any email asynaetry in the remaining epidermis was found to cause curvatures which prevented accurate measurerent of the longitudinal growth, the plants were placed upon thin glass rods which traversed the entire Inner betes of the holiow goleoptile. This arrangement effeotually pre— vented cu.vatures without having any appreciable efiect updn the growth rate. Table 31 compares the growth rate at pH 4.1 and at pH (Insert Table 31) 7.2, and the results presented are characteristic of a large mambar of measurements. In buffer of pH 7.2 the growth is small and of the same order as that shown by Table 30 for the epidermis. Under these circumatances, then, no curvatures would be expected, In baffsr of pH 4.1, the growth during the first two hours is 14 times that in pH 7.2. It may also be conserved that the principal portion of the grovth takes place during the first 2 hours, just as was shown to be the case for the curvatures (Table 29). The acid curvatures are thus found to be due to difference in growth of epidermis and parenchyma. In solutions woich are neutral or nearly so curvatures do not occur, because the growth of the parenchyme is slowed domm to about thai of the epidermis, ~Z1- Biftegt of acid on the propertion of the coleoptila. There are various ways in whien acid might vring absut ite effect on the growth rate. Among the possibilities ara: &) ® swelling of thes walls of the parenchymatous calls: bo) an increase in the water perneability of the parenchymatous celle, by which an inereascd rate of water uptake would be made possible; c) an increase in the plasticity of the cell walls. It a "swelling" of the walls of the parenchyma is responsibvile for the acid growth reaction, curvatures should result as well in plesmolized as in unplasmolized coleoptiles. The epidermis of young coleoptiles was removed from one sids. These plants were placed in 10% XNO, solutions which at the same time were buffered at pH 4.] and 3 7.2. Table 32 shows that there is no large curvature in @ither set (insert Table 32) of plants. It appears therefore that the acid growth reaction depends upon the maintenance of turgor pressure, The rate of deplasmolysis in hypotonic solutions may be taken as some measure of water permesoility. (See the racent critical review by DeHaan, 1933). In the present case coleoptile sections were allowed to plasmolize in 10% KNO_, and then to depleemolize in 3 the ueual buffers of pH li.l aad 7.2. he doplasmolysis took place on a welled slide, and was followed under the microscopes. In both cases the rate of deplasuolysis was very rapid, most of the cells vpeing completely filled out in less than a minute. There was no observable difference between the behavior in oH 4.1 and that in GH 7.@. It is dlfficult to see how any small differesce which may om BE aw exiget between celis so extremely permeable to ater as these, can account for differences in srovwth rate, Phe effect of acid on cell-wall slastioity vas next investi- gated. In order to avoid amoiguous effects of injury as well as the longitudinal transport proolen, the coleoptiles were in this case infiltrated with the solutions by means of a vacuwn pump. Table 33 (Insert Table 33) soos that infiltration does affect the initial grewth rate in the cane menner as doos renoval of the enidermis. After a time of action of the buffer solutions of 1 to 4 hours, cell wall plesticity and elasticity were determined in the menner already described. Table 34 shows the results of five experiments waich demonstrate clearly (Insert Table 34) that under the present conditions, the difference in the action of pH 4.1 and oH 7.2 is sccompenied by a considerasie difference in the plastieity of the cell wall, i.e. they are more olastic in the acid solution. In every case the difference in the elastic portions of the curvature is small. The effect of acid uoon plasticity is to be stressed since it closely resembles the affect of growth substance upen plasticity which has veen described by Heyn. It has often been attempted to determing whether curvatures, either tropis{t® or as in Strugzer's case, due to acid solutions, are cue to Tactive growth" or to elastic bending, by veasuring the portion of the curvature whics is removed uvon plasmolyeis, Trindle (1917) and Overbeck (1926) found that the first stages of troepistic curvatures diseppear upen glasmolysie whereas the Later, larger -g3— curvatures are irreversible. Strugger concluded that sicee his acid curvatures wera only slightly reversitle by plasmolysis that they concerned "davei keinesfails aine blosse Uverdehnungserscheinung, sordern es idigt wirkliches Membranwachstum durch Intussusception. vor". Tne irreversioility of a curvature upon plasmolysis does net, however demonstrate that the curvature was dus to growth by intunsuseontican, In the first place, irreversible curvatures may also be chtained by plastic slongation with or without accompanying intussusception. In the second place, in the Avena coleoptile at least, the curvatures “which remain after plasmolysis are complicated by the tension which exists between evidermie and parenchyma cefore plasmolysis. Measure- ments showed that incolaoptiles of the standard length (2 to 3 em.) the eoldermis contracts 10 te 15 per cent if it is rexoved from the lant (see alsa Heyn, 193%). Normally tharefore it is undar con. pldera.le tension. If verfectly streieht colecotiles having the ecidermis removed from one side are plasmolizcd they sometimes snow negative curvatures of as much as 55 degrees due to the greater con- traction of the remeining epidermis than of the parenchyma. It ig nob sefs to conclude, without an examination of this factor of tissue tension, that a curvature is plastic or that it is due to intussus— ception by the fact that it is not removed upon plasmolysis. C. Acidity of the Golecstiie Guring Growth Mathod . iciorimatrice determinations of the cell sap showad normal . a 4 that ander wM conditions the pH is in the neighborhood of 6. However, of colorimetric determinations did not permit, surticiently accurate measurements of small. differences which might exist between portions = Fille of tha coleoptile having different growth rates, Therefore electro— metric methoce were resorted to. ‘The quinhydrone electrode. was used. The advantages of this electrode over the aydrogen electrade for the present purpose have veen pointed out by Gundel (1933). Briefly they are: a) since Hp ia strongly reducing in the groscnce of platinun clack, relatively large changes of the oxidation-reducticn potential of the sag may be caused, and this is partialiy avoidad by the use of the quinhydrone electrode; b) the passage of H, througn the sep drives out CO, and mey change the pH if the carvonate buffer system is of inmportenca. Gonsliderable difivlculty was experienced st first in oblaising reproducitle platinum electrodes. However, the following methed of cleaning the electrodes was found to be satisfactory. Toney vere firat dipped for a few minutes in hot aqua regia and then balled for several minutes in a selaticn of sodium biesolfite. “Vth this metood of cleaning, the taree electrodes which were used for seach determination agreed in general to within 1] milli-volt. A caLomel- saturated KGL reference Kalf-cell and. a saturated KCL bridge vere used. Fraruant checks af the notenti«l of the quinhydrone call ware made by mens of-standard HCL solutions and always. gave within 1 millivelt of the expected value. The potn@tiel of the cell sep was found to change with time. The sap appeared to become. more acid. Tails drift was reduced, altho not completely eliminated, by heating the colecotiles to 100° 0. for 2 minutes before crushing them. Jor comparative purposes, therefore, the readings after 10 minutes were aroitvrarily taken, For cowparison, a few measurecients were wade with the aicro glass electcode (McInnes, 1929). -85— The determinations were carried oat in the following monner. <5 to 30.3.1 ws. coleoctile sections were heated over a boiling wyater bath for tao minutes, They were then ground in a porcelain disa, diluted with an equal volume of water (which vas found ta cause ac sismifgicant change of the potential), a smell anount of Hastman cuine hydrome added, the vessel put in plece in a 25° 0, thermostat, connected with the K01 bridge, and the potential with each of 3 platinum electrodes determined with a Leeds and ilorthrup type K notentiometer. Experimental. Fer a comparison of ragidly growing ana slowly growing zgon<s of the plent, the colecntiles ware decapitated sad the Toilesing gortions removed: a) the 4.1 wn. gone beginning about 5 ma, from the tin (zone of maximum erowth rate; Went, 1928); bo) the 3.1 mu. zone next to the mesocotyl (zone of minixnum erouth rate, except, of course, for the extreme apex). Table 35 zives the results of a muriber of measurements. In each (Inser} Table 35) experiment a considerasle amount. oY variation is found suong secples while would be expected to be the same. This is prosably due ta reaction of the quinhydrone with the metabolites of the sap, since the determinations made with the glass electrode (where reactions of the metabolites with the measuring system are excluded) vere rather sore constant. Table 35 shows that the “tops” are slightly aitha not significently more basic then the "bottome"., ais result ia in accord with general experience, altho the difference is smaller than has been sometimes coserved. Sachs, far examole, found in 1862 that the «ost acical portion of a growing root is basic, the zone of maximum ~86- growth rate ‘alrost newtrel" and the more basal portions "decidedly acid", Gundel more recently found that in the roots of Vicia faba, hypocotyls of delianthus, and epicotyls of Fhaseolus, there is an inereace of acidity in the oldar portions. it is not safe to conclude, from the avove only, that there is no change of pH during the action of growth substance. As shown by Thimann (1934) the upoer sone containgy more growth substance then does the lower and this is as would be expected, since 1t is nearer the source, i.e. it is nearer to the tin. However, as has already been polnted out even with the same growth suosiance supply the lower gone grows more slowly. A comparison of the same zone with different growth substance supplies was therefore made. Solutions a) of 1% fructose and lo anits growth substance per oc. and 0) water elone were used. Table 36 shows that again the pH of the rapidly groving (Insert Table 36) sections is slightly, altna hardly slgnizicantly more basic than that of the slowly growing sections. This conclusion vas again ecorraoorated with measurements made with the glass electrode. It is impossible to correlate an increased rats of cell elongation due to growth substance acticn with an increase of cell acidity, at least so far as measurements made upon crushed cells go. The oH which has been determined is mainly dependent upon that of the cell sap, out this sap is mixed with the protoplasm as well as with the esll walls. How far the pH af the orotopliasm and cell walls afvects that of the sap cannot ve said, but it seems likely that ereat differences batween the protoplasmic acidity of fast growing and slow growling sections vould ve reflected by at least a small -87- chanze in that of the poorly ouffered sap, Whatever difierence is found, however, lizs in the opposite direetion from thes which would be expected if pH chongoe play any part in the control of aural growth. Increase of cell acidity is not, then, a prerequisite to or an accompanyuent of normal growth. The significance of the acid growin reaction ils therefore to be sought elsewhere. This will be considersd in the following section. ad. The Deveudence of Acid Growth on Growth Substance I: one considers the relation of acid growth to the growth normally brought about in the Avena coleoptile by growth substance, the various possibilities may oe included under the following heads: a) acid action is entirely unrelated to that of growth substance; t) acid acts by cringing about the same cellular changes as Circuits oo does growth substance, but short wabs the steps for which growin substance is necessary; c) acid acts through an influence upoa growth substance which then causes growth in the normal manner, whatever this may be. The first possibility will be neglected for the time being. Sirugser seems to consider the second possibility as likely. He found that uncer all conditions the objects with which he worxed showed a much increased protoplasmic viscosity with acidity, and considered that the inereass in viscosity and increase in imbibitioa pressure leads to an inerease in growth rate. dna the course oz the present study the protoplasmic viscosity of Avena coleoptiles was studied py Hever's plusmolysis form and time nethod (the method uged py Strusger). The viscosity was found to remain within tne ~fi— rather consideraole uncertainty of the method, unchanged duriag cell @longaticn, ana to be indevendent ox the action of growth substance. In the case o: Avena, then, large changes of orotoulasmic viscosity are not of necessity associated with growtn (althe they sonstimes occur; seo Langs, 1953) end the general significance of such changas may consequently be questioned. It was shown in a previous section that at least one of the ultimate effaets of acid upen the coleoptils is identieal with that of erowth suostance, namely an increase of cell wall plasticity, It has also been ssown above, however, that the action of growth substance probably does not involve a production of acid in the cell. Therefore the supclying of acid to the cell does not result errewtin in a simple shortSusvame or the processes for wnich growth suostance is necessary. More complicated actions of acid in short eabtins these vrocesses might be conceived. It will now ba showm, nowever, that in the case of the Aveng coleoptile at laast, placing the plant in an acid medium results in the conversion of growth substance already in the cell from a relatively inactive fonized form to an unlonized form which is active in causing cell elongation, and that “acid growth!’ is completely desenient woon growth substance. (a) If agar blocks are placed upon one side of decapitated coleostiles, curvatures result, which are within certain limits pro- portional to the amount of growth sunstance in the olocks CHent, 1923). However, as orey.ously pointed out, it was found oy DJolk and Thimasan (1932), that if the agar block containing zrowth substance is vuflered at a neutral or alkaline pH the curvatures are mich smaller than would ~89— be expected, Hor exawole, trom thelr unouolished data, a growth sub- stance solution which when acidified and made into agar blocks gave l2.0 degrees of curvature; when made into agar vlocks with puffer eof pH 7.0 gave only 3.3 degrees. It follows that srowth substance solutions must oe made acid in order to cbtain large effects upon growth, dhe reason for thign as already mentioned, that growth sup— atances is a yweak acid and tne undissociated. fora has apparentiy a greater growtn potency than does the salt. Table 47 compares the growth rates of plants (with their epidermises removed) in several solutions. It may bea seen that (Insert Table 37) a) growth substance. in acidified but unbuffered solution greatly increases the growth rate over tnat in distilled water; b) growth substance in acid burfer (pH 4.1) causes an even further ineresse, c) in ouffer of pH 7.2 the growth rate is very small and is not ereatly increased by the addition of growth substance. In brief, growth substance is of considerable use to the plant only when it is given in the undissociated form. This provides a basis for the <ffect of acid woon growth if it can be shown that there is dissociated zrowth substance in the plant which can be converted into tne active form. (b) The garlier attesots to extract zrovth substance from the lower portions of colecptiles have always given negative resulis. Snis, as is now apparent, is ocecause the amount present is rather qmall, and the usual wechnique, that of placing the plants upon agar ~90- vlocks end aliowing the grovth substance to diffuse out (Vent, 15928) does not permit or the acewnulation of enough plants on one block to give a reauily detectuvle amount. In onder to obtain anounts which may be determined oy the curvature assay in the usual manner, it is aecessary to 2rind up a larze number of plants, extract the pulp, and concentrate the solution. The work of Thimaan (1934) hae show that if chlorcform is used as the solvent, and the solution is acidified during the extraction, zromth substance may be readily obtained from coLleoptiles. Table 3&8 shows that about 5 plant units Wehiuudausea Watton GAh per plant are obiained in this way, and this egrees with (Insert Taole 38) the amounts found oy Thimann. The point of interest in the present connection is that there is extractasle growth susstance present in the plant. Jt has already becn shown that noraally about 94 per cent of tis srowth sutstance will be in the inactive or slightly active salt form (because of the pH of the plant). 1+ will later be shom that woen a coleootile is placed in an acid solution its internal pH deoreeses, although not to that of the external solution. Therefore more O1 its own erovin suustance is availaple to the plant when it is placed in such an acid butler. (c) The minimum of crowtn rave reached by plants after decapi- tation has seen interoreted as due to the using wp of the residual srowta substance after reacval of the source (the tip) anid a subse quent “physiological regeneration" of the tiv. According to this view, acid curvatures made with freshly decapitated plants should oe _91- larger than those mada with plants decacitated 2 hours previously. Tasle 39 shovs that this is actually tha case. (Ihsert Table 39) (a) The per cent inhibition of ‘acid growin" and of "zrowth suvstance growth" oy HON is of the same order. Table 4O gives the (Insert Zavle 4) mean results from several expsriments in which the erosth rates of plents (with the epidermis removed from the two sides) in various solutions is compared. It wiil se secn that the change of "acid growth" with time resexbles that of "sravth substance growth". Grovth suostance gives in the first hour, however, considerably more zrowth, than doas acid. In cempering the growth rates in HON of various cancentrations, omiy the first hour may be safely used, since damage and loss of turger may result after this time. From fable 41 it may (Insert Table 41) ~@ seen that the two kinds of growth are stopoed uy HON concentrations srovth is sonewhat less sensitive Other experiments have Vive cndiated ON Te D/A" RVD of the sane order altnoe the acia than do the crowth suustanee growth. that the uptase oo growth suistance is more sensitive to HG! than is the action, and the acid growth, interpreted as due to growth suostance is conegimed only with the action of erowth substance already present in the individual cells. Henee we find a somewhat smaller sensitivity to HON in the case of the acid growth, The acid curvatures are also partially altho not comoletely inhioited by HON. whe change of acid curvatures witha pH may next be considered. favle L2 gives the results of 5 typical experiments over the range (Insert Table 2) from oH 2.5 to pH &. The trend of the curvatures is the same in all of the experiments sut the abolute values of the angl.s vary cona- siderably. This variation, the cause of which is wnieterm:ned, occurred in all of the large nurber of experiments wiich were carried out. ihe mean values from ail of the experiments do uot differ significantly from those of the five experiments of Tasie 42, and for convenience only the latter will ce considered here. The change of curvatures with DH is illustrated graphically by Fie. TX, curve B. Tn contrast with the above mentioned douple maxinnan curve obiained by Strugger for delianthus roots, this curve is of an § typs, and resemoles rather strikingly the titration curve of an acid. Fig. 1K {carve A) gives also the titration curve of an acid of pK 4.8) that of growth substancez Dolk end Thimaun, 1932). Jao order to compare the tre curves it is convenient to reduce them to the same ordinate at some carticular pH, and to observe how they change on eithsr side of this point. Ourve C is constructed so that at pH 6.1 (that of the cell sap) the percentaze curvature is L.,2 or equal to the per cent or wakéa sociated acid of pK 4.8 remaining in a solution of pk 6.1. Suppose that as alraady indicated the acid curvatures are due to transformation, at sone goint inside of the plant, of growth sun- stances Grom an inactive salt form to active acid forma, and that thea active growth substance thus produced causes the growth which results in curvature. ‘le would expect that the ph in the plant woula not be % Nondiszociation Degrees Curvature Figure 9.Relation of Acid Curvetures to pH. Titration Curve of Acid saving pK 4.8 (Curve &) Gurve OaGhange of Actual Curvature with gle - "-" Gurve C,% Change of Curvature (See Text. ) —93~ exactly that of the outsice buffer solution, since the buffer has first to venetrate the plant, and thea to overcome whatever buffering Gupacity the vlant may possess. Tharefore, the per cent curvature snoula not change as rapidly with pH of the external oufier as dogs the oer cent non-dissociation of an acid dissolved in the buffer itself. This is just what is found in FigurelX, at leas., so far as tne acid side of pH 6.1 is concerned. Upon the basic side the curvatures are so smali that it is impossivle to draw any conclusion. It was possiole to show directiy that the acidity insias of the plant ¢oes uot change exactly with that of the buffer. This was done by measuring colorimetrically the pH of the crushed coleoptiles (previously washed tree of ouffer left uson the surface) after the execatioa of acid cur- vatures. favle 43 zives a characteristic set of these values together (Insert Table 43) with those expected from Figure IX. If these pointys are plotted on ¥Yigore IX, they are seen to lie reasonacly close to the titration curve. If the actual acidity of the plant is considered, then the per cent curvature increases as it should if it is the formation of usionized growth susstance which causes the curvature, a. Discussion and Conclusions It is difficecvlt to reconcile the present findings “th those of Strigezer for Helianthus. In the case of Avena the change of acid growth rate with pH ise determined oy the change ai the disso- Glation of the week acid, growth substance. In Helianthus according to Strugser the change of acid growth with pH sives a douple maximcm oh curve between pH 3 and pH 3. BZorowikow (1913) who alse worked with Helianthus obtained resiite woich may be more eagily unueratcad in tne light of the present work.. He found larce stimulations of srowth in acid solutions. These stivulations were decreased by the adsition of neutral salite to the solution. The stimulation given by bases apd on the other hand was either small or absent; in general, gg rather sporadic in its appearance. He expleincd this, since he felt that it would te suitable to find two maxima, as dus to the fect that a vasic solution with a growing plant. in it is always a mixture of salt and base because of the CG, evolved. oy the plant. His rescits might equally well be explained as due to the absence of any par- ticular stimulation by bases. In the course. of the present work a few -ensurementse of the acid curvatures of botn Helianthus hypocotyls and Pisum epicotyls have been made. Tavle 4Y shows that under the (Insert Table 4+) present conditions the same genera. change of curvature wita pH tekes placo ac was vound for Avena, and the different recults of Strugser are probably due to a fundamental difierence 91 technique. The behevior of Helianthus is according to Strugger different in otner respecte from that of Avena. He oslieves to have found that hypo- cotyls grow faster under anserobic conditions than in the presence of air, atic has interpreted this as due to the production ot acids during anserosic metaboliem. ‘Thig is, as already siavn, the reverse of the situation in Avena. It has also been impossible to find the chanzes in protoplasmic viscosity which Strugger hac associated with the pH and isoelectric coint or the protoplasm, ~K. The work of the present section was undertaken with the hone that it might lead to an elvcidstion of the manner in which srovth gsauostance acts in oringing about growth. This ebucidation would have been possisle if the effect of acid had ceen, as mentioned above, to bring about the same cellular changes as does growth subrtance, but with the shortcutting of the ctepe for which growth suostancs is neeied. The facts presented show, however, thet the effect of acid on zrowth is, in the case of Avena at any rate. only by way of the eifect of acid on growth aostence already present in the plant. It is also shown that in the normal plant, as well as in the action of growth suostance usen sections cut from the plant, there is no increase of acidity as the result of or during the action of groanth substance. It would appear then that there are at least two ways connected wi ti. growth substence in which the growth rate of an Avena colecptile cen ce increased, Ths firet is the way in wich 1t is done in the normal plant, by an increase of the total amount of growth suvsstance present, Unly a small portion of this will be in the active non-dissociated term at the pH of the plant, but oy increasing the totel amount present the amount present as nonedissociated acid will alse be increased, The second vay in woiecs the growth rete may be increased is oy coaversion of some of the inactive zrowth substence salt already present in the olant inte the acid. This is what occura in the “scid erowth" reacti.n, when the cells of the plant are actually made more acid, ana the icnization of grovth substance reduced. It revains possiule that there are some other effects of acid wcoon the colacctile out it is clear that se far as the growth rate is concerned these other effects, of which a small change in protoplasmic viscosity may be one ~Sh— are of minor significance under the present ccuditions. The cifferent results perhaps of Struvger, due puaededy to didierences in material or othor experivental conditions, are orobakly due to the elevation of other Yactors follows; 1) 2) 4) 5) 6) 7) to importance, Tae results o1 the present section may be suusarized as The growtn of the Avena colecptile in bur. er solutions of different pH hes bean investigated. The formation of cur— vatures in acid solutions by plants naving the eoldermis removed from-one side has been confirmed. Acid solutions increase the grovth rete by increasing the cell wall plasticity. Increass ef cell wall plasticity is knows to result frou the action of growth substance. The increase of growth rate in acid ouffers is inhibited by HGH concentrations of tae same order as those which ine hioit the increase of crawth rate due to growth substances, There is na increase in ceil acidity ander the influence of grovtn suustance. Tneare is growth suostance present in the vlant even after the hormone producing tle is removed. The increase of growth rate with decrexse of internal pH Tollews closely, the inerease of non-dissociated acid with decraass of pH in a solution of the weak acid, growth sub-— stance, From the foregoing it is concluded that the increase of growth rate in acid buffers is due to the conversion of gravth sucetence already in the clant from an inactive salt fom: to an active nondisscelated Yorn. TABLE 29 SAMPLE SER1G5 Or CURVATURES PRO! IPDIVIDUAL PLANTS (Time of issersion; 2 hours) olution Curvature of plant in desress pH 4.3 gy 71 29 23 50 73 66 73 #80 76 pH 5.2 l@ 11 19 9 18 13 1G 24 16 le TABLE 29 CHANGE OF CURVATURES WITH TIME pH 4.1 (Curvatures in degrees) iiccpt. 2 hr. 4 hr, o hr. & hr. I 51 54 Sy 5h TI Ly 4 Uy i III ky. Uy yu he TABLE 30 sean 63 27 14.3 $1.5 SoFECT Of AVID AND NEUTRAL SUYSURS ON GROWTH OF HFIDER AL STRIPS Expt. pi let br. @nd us. 0. striog I bed uel 0.2 4 11 4 0-5 0.0 4 III 7.2 0. 0.2 4 IV 7.2 0.1 0.2 4 ee 7.0 3.1 6 Plants wlth "“eyldermis removed", -~93- TABLE 3). EEFOOT OF ACID AND NEUTRAL DUPFERS Of GROTH RATE OF PLATS SAVING ERIDEHIS RPIOVED Fach BOT SIDES “i Biongation ner hour Suiter Lhr. 2hr. Ghr. Ghr. Shr. hr, 48+Bhants pH 7.2 0.3 o.4 0.3 O.4 0.3 0.2 16 TABLE 32 ACID CURVATURES OF PLASMOLIZED CULEOFTILES® Guryatures ia pH 4.1 Gurvetures in pH 7.2 a Flas:nolized Jot plassiolized risemolized Hot plasmolized I +4, 20 —7ye +0.2° —4.0° Ii 44,09 —68° +3, 3° +2. 0° Svhece slants were young (coleokpiles 1 to 2 om. tell) ani therefore did sot exhiscit the plasmuclysis cur~ vyatures discussed in this part. TABLE 33 EFYECGT OF INYILATRATION UFON THE GROWTH Rate OF CULEOP iiLs SECTIONS IN BUzkER SOLUTIONS Growth in % per ar. during ; Hu lar. 2@hr. 3Zhr, 4Ubr, hr. 6 hr. Plants Infiltrated 4,1 5.8 3.2 1.5 ied 0.8 0.3 le. Not =" 41 3.2 3.4 2.6 2,0 1.9 1.3 le Infiltrated 7.2 0.6 0.2 G.1 0.2 0.3 u.2 12 Rot 4 7.2 Q.9 0.5 0.6 0.7 0.5 0.2 12 ~99- TABLE 34 EYSEC? OF ACTU UPON THE PLASTICITY OF OOLECPOILES fice jn Elastic curvature BElestic Survature buffer in degrees in degrees hr. pH OY.T DH 7.2 ob LL oH 7.2 growth is Expt. Plants T ll, 12 «4,2 O.1 4 ~—— — 24.5 4.3 II Ll, 11 6.9 90.1 2 6.3 5.0 25.7 4.3 III 10, 8 2.5 0.0 1.5 6.3 4.8 13.2 3.9 Iv 12, 10 1.5 0.0 1 4d 45 13.5 2.1 V 10, 9 1.6 4.0 1 5.5 4,7 16.6 2.7 TEBLE 35 CIDITY OF PAST GRIVING AND SLOW GROWING SEOTIONS OF COLEOPTILES guinnydrone electrode Glass alestrode Pot, with celomel half cell, volts Pot. with 1.07 HCL, volts Exot. ifogg! IZottoms!t Expt. Mops! "Bottoms! I 0.109 0.116 I U.eb7 0.279 o.10h O.1¢8 — 0.281 II O.1u0 0.101 II 0.287 u,esu G.099 0,097 0.287 0.279 6.094 0.097 ; 0.094 pu 6.17 6.04 Til 0.055 0.087 9.085 0.091 Iv 0.102 Q.112 0.097 G.100 v 0.102 G.107 9.091 ©.096 0.096 0.096 VI 0.093 0,094 6.099 0.100 Mean 0.096 2 9.002 0.100 7 0.002 pi = aE Ey AO 6.067,03 5.994,03 pH OF SHGTIONS OF AVUNA COLEORTILES IN 1% FAUCTOSE PLUS GROWIH SUBSTANGE AND IN DISTILLED H,0.° 2 Exot. Potential in volta No in growth In £0 cmeaeel substance I 0.090 609k 0.988 0.089 II 0.084 0.988 IIz 0.092 0.094 9.9090 0.094 IV 0.090 u.092 0.089 0.091 Mean 0. 089%,001 9.0914, 001 pH 6,184.02 6.14+.02 “Time in Selvtigne = 4 hours TABLE 37 EFFECT OF GROWDH SUBSTANCE UPON GROWLH RATES IN ACID AND NEUTRAL SOLUTIONS Growth in % per hr. during Solation Plants ihr. 2 hr, Sars g.8. in distilled H,0 18 10.9 3.4 1.2 oH 4.1 buffer 18 6.9 3.1 1.6 s,8. in pH 4.1 buffer 18 12.0 4.2 0.6 pH 7.2 bafier 18 0.3 0.4 0.3 gs. in pH 7.2 burver 18 G18 1.0 o.3 Szrowth suostance concentration = 10 units/ee -101— TABLE 38 EXTRACTION OF GRO TH SUBSTANCE FROM LOVES PORLIONS OF COLEQPOILSS WITH HOOL 3" No, of Vol. of Vol. of final . Vol. of a planta. Bool, solution Angle blogiss_ é:3./olant Rr . o rans - Oh 4s Ree 0.2500 34 agar 42 Lounen 4.7 waits TABLE 39 EFFECT OF PREVIOUS DECAPITATION UPON ACID CURVATURE oH 1 a Planve decap. Piants decan, uxol. 2 bus, vefore immediately I 59° 160° Ii 2° 110° TABLE Lo GROWTH RATE OF PLANTS WITHOUT EPIDERMIS . owth per hr, during i 4 eee Solution Elents unr, Zhe, ghr, Uhr. four é hr. g.8, 10 uf/ee 18 10.9 3.4 1.2 0.6 0.3 0.2 pH 4a 18 6.9 4.1 1.6 1.9 0.6 0.5 "ee 1g 2.3 9.9 uu o.4 0.2 0.2 +5 Gil L.,| id 0.8 ~i.3 oH ie 6x10" 4 le 0.7 -—.1 S. +80 } 12 3.2 .e oH Le Cax107 WN 12 3.9 -L.0 Bob, ao) 12 6.5 2,9 pH 4.1 asia 4} ie 5.4 0.5 ~1h2- TABLE 42 PER GEN? TMHISITION OF GROWIH SUBSTANCE AND ACGIO GROWTH BY HON % inhibition in first hour Solution HON strength _% Solution HCN strength 4% oH LL ea1o74 90 BB smio7t 93 pH 4.1 leo 43 g.8, leelo74 71 pH 4,1 exio7t 22 g.8. exio7 4 Yi TABLE he VARTATION OF AGID QURVATURE WITH pH Exot. pH 2.5 3.4 4, 4.8 5.2 5.7 G4 7.2 7.9 1 70 56 37 #15 — 9 6 1 2 2 & 83 We 28 19 10 7 3 2 3 101 80 76 21 15 16 7 \ 2 4 7774 O45 5 2 4 5 107 999 7H aD 7 1 2 Means 89 768 58 27 #19 (Ile 6.4 2.2 1.6 me wo37 87 130 9 eT 3 ge ~193— TABLE 43 INTERVAL pH OF COLECPTILES APTER ACID CURVATURUS pH of buffer 2.45 3.4 4.1 4.7 5.7 @4 7.2 7.9 oH of plant 4.7 4.8 5.2 5.7 5.9 6.1 6.3 6.9 pH expected =p 3g) 519 5.57 5.9 6.2 6.6 7.0 from curva TABLE tH CHANGE OF ACID CURVATURES 41TH pH Ii! HiLIANTAUS HYPOCOTYLS AND FISUM EPICOTYLS Plants pH Wal 4s 5.2 2.8 6.4 (Garvature in dex.) Pisum 6 77 Yl 22 17 7 Helianthus 6 111 50 27 22 g Le ~19h_ PART VITIL GROWTH AD RESPIRATION A. Introduction In Part VI it was shown that aerobic metabolism is istimately associated with clongation wider the influence ef growth substance. Tne present section is therefore concerned with the respiration of the coleoptiie, particularly in its relation to elongation. This work was unuaertaksn wlth the hove that slongation woulda exhivit a close correlation with some relatively accessiole property of the resgiration, for example with its magnitude or its respiratory qio- tient. It may be said at once, however, that this was not the case and that the work of the present section, while revealing sevaral points of interest and defining more clearly the devendense of slon— gation woon respiration, has not resulted in any solution of the ul~ timate fashion in which respiration is essential to growth. Since the subject of respiration is rather far removed from the suojecits of the prededdag parts, it will ce necessary to make a snort review of the literatare. It has long been known that the growth of an aerobic plant depends upon the existence of its aerovic respiration. Sefore the end of the aineteenth century, Wieler, Aubttitm, ond others had dem nstrated that grovth of the higher plants in generel ceases or is greatly dimiaished in air of less (1903) thea 0.1 to 3 per cent oxygen. Pfefferpexpressed thase viadings simply in the statement that respiration is a "formal necessity" (1370) of growth. Sachs held essentially tne same view. Sachs) as well as others also resglizea that there 1s a certain amount or correlation = a— between the intensity of the respiration of a given olant and the intensity of ita growth processes, Thus the respiration of a dormant seca is iow wat thet ai the developing segedling is nigh. ‘This con sideration lead several investigators to detersine the course of respiration aver the "grand period” of growth. De Saussure (1833) measured the respiration ar gerninating saads, but more extensive studies vere maie by Mayer (1878) and tiachawi(1576). They round that the rate of respiration vasses through a grand period sinilar tO that of growth rate. ‘This observation has ceen abundantly confirmed in ore recent years, for exauple by Stahlfelt (1926), Kidd, ‘Jest, aad Srlegs (1921) and oy Hatenrichter (1926) for the higher vlants and oy a large number of worsers {see De Bosr, 1928) for fungi. ‘ It has been shown by many, bezinaing with Malpighi and Gedlews ki Seatautsuwe thst very low oxygen pressures decrease net only growth out also respiration, ana that pressures of oxygen somewhat higher than that prevailing in the atmosphere often sowaass the rates of : (seuntys, ise), eoth sete of processeas Changes of temperature have been also. found to oring goout qualitatively similar changes in growth and in rea piration. Pfeffer pointed out, however, that the cardinal points for growth are Trequentl» diferent from those of the respiration of: the game or aniga, or example, the rate of respiration eften continues to inoreuse after growth has passed through its option termerature. Thig is well shown in the recent and thorough work of Mack (1930) upon the growth aad respiration of wheat. There cannot be then any Girect cosresvondence oetween wacnitude of respiration and magnitude of growth rate, -106~ snere nave been few attempts to analyse further whatever correlation may exist vatween growth and resoiration, Pfecfer has contented s.mself with designating respiration as a formal necessity of srowth, Sachs took the view generally accepted that raspiration is ¢ssentio. to growth in that 1t furnishes the necessary energy and goiuted out that respiration would be ale to produce as a by z , - (1934) product the organic acids shown by De Vries to be @asential for the maintenance of the turgor of the cell. Jit has even been suggested thes the principal function ef respiration is. the production of osmotically active substances. It has long been assumed, rather gratuitously, that the bissyatheses and the mechanical work of srowth utilize an anoreciable if sot a cunsiderasle portion of che energy liberated by the res- plration of the growine plant. This assumption 1s refiecte: in the statement comsonly found in textbooks (erg. Kostychev, "Plant Res- piration") that respiration is essential for zrovth pecauss of the onergy required in the Lotter preccss, In recont yoars 1% has boon desongirated that the major portion if not ali of the energy given off oy resplration ultixately apoears as heat. Since this is. the case, it is clear that resoiratory cnergy has not entered into syn thetic processes except in so far as needed to furnish energy of activation (hence for the overcoming of "potential hunoe"). This latter energy will of course appear ultimately as heat. Un the other hand, enerey which agsears as heat may, it is true, previously have done useful mechanical work. Jonsider for example the worse of plastic stretching of an Avena coleobtile. Such work goes into what may 09 considered as the overcoming of friction of the particles = LO Pa of cell wali. However, work used wp in overcoming friction appears as heat. “se are hence unacle to decide fron caloriuetry whether or aot respiratory energy nas dons mechanical work of this nature. The question of the enersy of growth can ve asked in at least imo ways: a) in what form does the energy lost from the organism and ite watrients agpear; and b) in what form docs the energy of respiration appear? These two quastions cannot ve anewvered with complete satisfaction at present, but the nearest aporoach to the answers may be found in the recent work of Algera (1932). This investigacor has consiructe: a compensation calorimeter of con- siderable precision and has measured the neat evolved at constant venperature oy a culture of Aspersillus niger growing on glucose and armonie, At the sawe time tha v0, producticn of the culture was meanured, and the initial and fimal heate of oonmuction of tho modiun and myceliu: estimated. Only this one work will te considered here since technicslly it is superior :o those of Mollisra (1922), Terroine and “harmser (1922) and Doyer (1615) to mention only a few of the investigsiions on this suoject. question a) will be comsidere? fivet, uring six days wie medium lost 8510 calories, end the myceliua increased by 5606 eglories. Therefore 4204 calories remain to be accounted for. 3299 calerios oppeared as heat, an excess of 95 calories or 0.5 ger cent of the total heat transfer, and within the experimental. error, It is clear that no considersble portion of the energy lost oy the medium nas gone into any sort of physical energy of ‘cell organization", which is not recoverable oy combustion. ihe answer to the other cuestion demands a knowledge of the respiratory quotient auring tne time of G0, evolution, but unfortunately this quantity was not determined by Molliard, Terroine and YVurmeer, or by Algera, If the quotient is taken as that of complete glucose combustion, tha usual procedure, one .inds thet 3518 calories sere evolved by the respiration. This is impassible since only 3204 caloriés at siost were lost from the cutrient and syceliwa. cenes itis clear that the respiratory quotient umst have a value above one and that. seach mol. of GO, does not come from a complete coxvbustion of one sizth mol of glucose tut that a portion of the GO, comes from fermentation of some xind.. Tamiya (1932) has shown that the respiratory cuotient of Agpergilius orgWee on glucose is ove: 1 and Puriewitsoh found that the R.q. of A, niger on glucose is in the asighucrhood of 1,1. If the d&... were in this case about 1.1 the energy oF res— piration would almost exactly balance the heat evolved and hence the logs of energy of the nutrient and selution. It should be remarxed that this fuilure to establish the H.q. before calculation of the energy of respiration from evolved GO, may lead to serious error. For examole Doyer calculated that the heat released by of wheat $s eed hs { s respiration during six days of germination,was avout two tines the energy lost from the ceedling as determinea vy heat ov concustion, Stiles ans Leach (1933) neve found large varlaticns ia the R.a. of all types of seeds during the course of devel pment and it is certainly aujugtifisble to assame a quotient of unity as "as done by Dover, Sime the energy of vasepliration cames rather close to balencing the heat evoived, it is clear that no major portion of it is stored as chemical energy of the plant material formed. In fact, LCQ— Algera even calculatcs that the formation of protein from glucane and “Hy yields energy. The ensrey involved in the synthesin of protein from esaino acide (as in a seedling) mey likewice ve precumed to be smali. The formation of cellulose, which composes ov par cent of the mycelium of Aspergillus accorcing to the analysis of Terroine, (a) froutmann, Bonnst and Jacquot, is slightiy endothermic, but the free energy oc cellulose formation cannot oe calculated without a better gnovled se of the concentrations in the cell. Since havever,. tae cellulose is continually removed fram solution, conditions should favor synthesis and the energy required oe small or none. When permissible methods are uped, dt ia found, then, that within experimental ervor all of the restiratory energy is liscerzted as heat. However, as already vointed out, 1t cannot be ssid what portion of this energy may oot have verfarmed. some useful function in growth ocfore its final agpearance as heat. “where is, however, no question of an appreciaole portion of the energy bDeinz peraanently S$. faras mechanical world is concerned deflected ta tne synthesis of new cell material. {a calculation af deting elugdtion oF the colevptile the nav: work, ancunts to at most 0.4 per cent of the respiratory snerey®, Tt sees clear therelure that some relation other than Considerations oue@ based gricarily upon energy wanddkhans cust connect respiration with ceil elongation. * Footnote: Consider a section 4.5 am. long. It will have a cross- sectional area of aporoximately O.16 wean (Teble 12). In two hours it may eloneate roughly 0.45 ms. (Paole 53) in buffer of pH 4.1. The callsof such a section nave an gemotic value of approximately iC atmospheres, The pressure exerted upon the cell wall may be men lees (Ursprimg and Blum, 1924). Taking 10 atmospheres as a maxiouid value and. assuming constant internal pressure, the pdv work ~L1O-- done during elongation 1s therefore 10 x 1.01x10° x 3.1 x G45 x 6.16 x ixld™? = 2.3x109 eres = 0.5us107* calories. Sed at Doring the sane tre nours 5.0 wn” of oxygen (a@ measured sstandard conditions of temperature and pressure) will have deacon coneumed (facole 54), Therefore the mols of oxygen used have been a } _ =f 3.9 XS sa = 1-3e1G © mole O,. 2.2 x10 4 (Table 2) During the same two hours the respiratory quotient was 1.0% The saecoption will be made that. the extra GG, was from a feruentation yielding energy negligivle in comparison with that yielded by the eerobiec respiration, Therefore 1.3x107! x¢ = U.22x107! mols hexose have been burned. faking the heat of combustion of glucose as a rough measure of the energy liberated in: the cell, mt bee . tee . 680,000 x O.,22x10 te O.15"10 ~ calories have been licverated. The ratio of vdv mre done to energy liverated lg therefore malt 2 AuxlO 1.5x10-2 ~ U.36x107°, or 0.4 per cent. In practically all ar the works which have been mentioned thus far, "growth" has consisted of a complex of cel. division, cel.. elongation, and cell differentiation. Hither the growth of the entire plant, or in a few cases of the isolated root or stem apex has been considered. Another common fsiling is showm in the experimente of Mack (1930) in which the growth of the wheat "shoot" (presumably given by the Length of the coleoptlle) was compared with the respiration of the entire seedling. It is clear that a closer investigation of the relation of growth to respiration must, if possible a) differ— entiate vetween the different stages of grovth, b) consider the to -111- processes in the same organ, or if toesisle in the same tissue, It ia with these considerations in mind that the oresent study of the growin and rergiration of the Avena coleoptile has been undertaken, The oceservacle srovth of this coleoptile,as alread: pointed out, pro- ceeds almost entirely ty cell elongation, The present work is divided inte two parts, 1) the distri- bution of growth and respiration ooth over the grand veriod of the coleoptile and in the coleoutile itself at the stage at which it is in general ured for experiuental studies on growth and tropisns; 2} & more varticular study of the relation of the two vrecesses in the most raocidly growling region of the coleoptile. b, Methods. The measurements of respiration were carried cut according to the manometric methods of Varburg (1925). ‘whe vessels which were used consisted of two sete of six cach. One. seat, having volwies in the neighborhood of lo cc., possessed centre] siksli vells and. side receptacles for reagents to be added to the rerpiring tissues an during the course orf experinenté. the other set of wessels coasessed Ba8 volumes of about 5 co. and could hence be used for the measurements of smaller gas -xchanges than was cos iole with the first set. Uniess otherwise steted, all experiments upon respiration were carricd out in a thermostat at 25° 0. The usual shaking device (“arbdurg, 1926) was aped Tor azitation of the vessel contents during the experiments. ALL of the manipulations were carried out under red light in order to avoid possivule ohovotroplc stinulation, altho it is true that ao sigoifi.ent effects of smali amounts of ordinary light woon the rate of respiration could os found. ~Lie- Ii is necessary to have the gleces of tissue small enough to insure that the rate of diffusion of oxygen into then does not dimit the rate of oxygen uptake. Genevois (1927) has calculated. that plant tissues under conditions very sivilar to those used here may be O.4 mu. thick before the rate of oxygen diffusion becomes liniting. fae tissue of the coleopiile is not over U.4 mn. thick since this organ is holiow. It was found, however, that the respiration of ths Leaf, normally inside the colsoptile, de mich aucelerated ig this dee? be removed ana given free access to oxygen. The srowing and respiring tissues which were used vere of courss suomit.ed to seme injury as they vere cut from the coleoptila. inat & moderate smount of cutting has little influence upon the rese piration of plant tissues has been shown by Stahlfelt (1926b) ana by Genevois (1927). Table 44 shows that the respiration of these (Insert Table 45) ¢ sections im ouly decreased §% ner cent oy a four fold increase in the nu ver of cut eurfaces and that a doutling of the nuniber of cut surfaces nas litile effect. Itis safe to conclude that the measured rate Of oxygen wolake is e fair eutimate of the oxysen wotake of the same seatiin in the intaet plant. As with other proverties of the coleoptile, the resplration rate varies from day to day. ‘able 54 gives a good idea of the extent of this variation. One reason for this undovbtedly lies in sligh difierences in tne age of the sections used. Other governing factors may lie in the oretreatment of the ceedlinsse or in the unknown factors causing variation of the growth suostence activity frou day to day (Xégl1, 1933b). The duplicate sameles fron plants of the same day are w=] A moh more comparable as shown in Taole Lg, (Insert Taole 46) Seo grovtn gubetance oreparations were used. The "crade growth suvstance" had an activity of avout exio7® mgs per plant unit, while the "purified growth substance" had an activity of about sx107! mgs, oer plant unit. Unless otherwise stated the first three 4.5 mm. sections below the 6 mm, tiv of coleoptiles 2.5 to 3 cm. tall were used. These will oe snc hereatter as standard sactions. ° ; of C. “The Cc lee tile ssa Whe le j Period. During the Gran > ~ahoneatluu gnd Reselratloo Sos oar nas m4 {yin It is in the first vlace desirable to know if the changes in respiration durlag the erand period of colsoptile growth give any supoort toa bas view thal there ls an intimate connection between cell slongation and ouyesa consumption. The grand curve of growth of the Avega coleogtile has been determined many times for exanple by Hothert, (1896), Siero (191%), and ay XondBeberzer (192°). The ehanzas of colsootile oxyeen consumption duriag this cericd have not ceen dater- mined, but such deterninationsa have oaea made woon other plants, Mever (1675) aad Rischwri (1876) found thai the grand curve of res- nivration of germinating seeds was "similar throughout" to the grand curve of growth, Mors recently Kidd, Yest, and Brises (1921) have shown that in Helianthus the respiration cer unit dry weight follows closely the increase in dry weizht ver unit dry weight. Hafenrichter (1923) on the other hena found that in thopbean, the maximun rate of G05 evolution per plant ocours eariier than the maximun rate of sagot elongation. Tis author novever cosparad the respiration of the entire plant with the growth of the suoot alone, which is, as already ocinted -ll- out, not nerniasszole. axperimental. {00 seeds were hulled and soaxed for 12 hours. AL cone end of this time a rigid selection fer tices nearly in the same stage was made and tne selected seeds claced upon moist paner to serminate. «Av the exgiration of 24 hours, another selection vas made and the selected sesis planted. Saumles of plants were taken samples eacn le hours, and their lengths seasured. Each of tasse (yas then suspended in 3 cc. of pufrer, DH 4.1, placed in a manoveter vessel, and its resgiration rate measured over half hour intervals for 2 hours. «At the end of this time the dry weights of two of the samples were detersine: after drving at 90° 0. for four hours. The altroven contents of the other twa samoles were estimated oy the micro-kjeldahl method. The weight of nitrogen determined in this way, gives, when miltislied by 6.1, sn ascroximate value for the weight of orotein oresent in the olant. it is impracticable to separate tne coleoptile rrom tne very young emoryo. Therefore at the sees of 12, 2h, 36, and 4S nours the entire embryo witn the exeection of the scutellum was dissected out and used. At 48 sours the entire evoryo was compared with the soot alone. The entire shoot was then used et 4S, 69, and 72 hows. At fe hours tie entire shoact was commared with the coleoptile alone, ani the coleactile alone uscd thereafter. Ta all cases the duplicata deterninations of respiration, dry weight, and altrogen agreed quite satisfactorily. Tavle 47 gives a summary of the data obtained. {Insert Table 47). Between the ages of 39 and lg hours tha root oreaks from the coleorniza and elongates raovidly. This accounts for the large imerease ia length, and apparently, for the large inersace im res— -115- piration of the entire emoryo at 43 hours. Betwsea Jo and 106 hours the taird leaf (Avery, 1930) vreake through the colesutile. At this tine the rave of slonzatioa of the colaoptile fallea awiftly te G. from the data of Taole 47 it is possible to calculate the aoproximats values of the respiration rate, dry weight, and oroteln content of the coleoptile alone over the entire grand period. The values oi each quantity cefore 72 nours give ooviously maximua figures, above waien tacese of the coleostile cannot be. At 72 hours the coleootile possesees 52,5 per ceat of the entire shoot respiration, 85.5 ner cent of the entire shoot length, 50.5 per cent or the dry weight, and 4o,5 per cent of the total protein. One can assuma that as a tirst approximation these values hold over the course of development of the shoot before 72 hours. At 4. hours the relation betyveen phoot anid entire emoryo is complicated sy the oresence ot roots. Setysen 3) and 48 hours the respiration per unit dry welgat and per unib protein increases markedly, indicating that the resciration of young roots is intenss, However, it will oe notised that the respiration per unit dry weight of the 48 hour shoot is aluost the sane as that of the 36 hour embryo. Sinee the ratio of shoot dey weight to emoryo dry weight in the 360 hoar embryo is know, the apvroxinate values of the snoot respiration may be calculated for 3u, 2, and le hoars. Itshould o¢ enghasized that the calculation of these ap- proximate values is permissibie only since in any event the quantities concerned are rather small ia co-narison wth their mazsitudes later in the grand curve, Table 4S gives the sucrarized data for the (Insert Table 4g) coleontile alona, The rate of oxygen consumption goes through a PER 12 HOURS. RESPIRATION OF COLECPTILE. ARBITRARY UNITS ~, GROWTH AVE GM owe ee Figure 10.Grevth and Respiration During the Grand reriod of the Goleoptile.e Curve A,Rate of Respiration at end of each le sour Period. Curve 3,Totel Growth During Hach le Hour rerlods ~116— yery clear grand period, inereasing etesdily until crowth of the cole@cotile ceases, and then falling off. Rate of oxygen consumption is olotved together with total elongation nex noriod, asuinnt time in Sigare X. The parallelis:. between the two ourves is very marked. avery change of slope of one curve is accompaniad oy 2 change in slova of the same sign in the other. At the time at which the Leaf venetrates the tip or tne colgoptile and growth of the latter falls to zero, the resolration faile, altho only 6 per cant. At sost then 6 par cent of the resolration might oe directly involved in growth. Tne comarieon of total coleoptile rospiration with total solecptile slongation is only one of the ways in which the data may o@ considered, If one considers, however, the respiration per walt doy waisot, it may be seer thet with the exception of the to earliest periods, there is a-steady falling aff with increase in the age of tne slant, i.e. the maximan of the grand perio. oceurs very early, is agreecent whth the findines of Kidd, “est, and Brisgs (1921) ani Stalfelt (1926). One might compare the respirations rer wilt dry welght with the totel elongation or with the growth rate. In either case the maxioum rate of respiration ia earlier than the me«imum growth and the parallelism of figwe X is not angarent. Table 47 shows that wie Pespiration per wilt protein is reagvkably constant, and falls ort anly sligntly with age, whereas the respiretion ser cnit dry weight salis of. much more swiftly. This points to the fact that in general, comparisons of respiratory rate should :¢ made unen the basis of the living matter nresent and not uson the dry weight. This was pointed ae gat long oy Falladin£ z0, Betvees 48 hours and 96 hours the coleoptile increases its length oy 26 times, Despite this fact the dry weight ver unit length ~117- remains almost constant and the percentage of protein falls only to — one half of its original value. There has taken place then not only synthesis of cell wall but also of protoplasm. The normal growth of the coleoptile consists not only of vacuolation and water uptake by protoplasm already present, but is accompanied under these conditions by protoplasm formation. In fact, inepection of Table 47 reveals that under the conditions elongation, respiration, dry weight, and protein parallel one another rather closely. This is, as already pointed out, probably #a related to the fact that the temperature used is near the optimum for the growth of Avena, since at this temperature one would expect the various processes to fit rather accurately to~ gether. Another point to which attention should oe directed is the continued increase of dry weight after cessation of coleoptile growth. This is in accord with the fact that coleoptiles become much stiffer with time after cessation of growth. Under the present conditions, then, the rate of total ree— _ piration of the coleoptile parallels qualfitatively the amount of total elongation. This may indicate that the two processes are either directly ennnected or that they are effected in the same way by a common factor. It may, however, only indicate that protoplasm is formed during elongation and that respiration per unit protovlasm is rather constant. - G. Distribution of Growth and Respiration in.the Coleoptile The preceding section has shown that during the grand period the . of coleoptile, growth is paralleled qualitatively by the grand period of coleoptile respiration, altho there is no quantitative relation. ~11g- | This section will deal with the distribution of growth and respiration over the length of the coleoptile. The comparison in this case is between sections of physiologically different age instead of between coleoptiles of different age as in the previous section. The growth distrioution in the Avena coleoptile was direst: determined by Rothert (1893) but it has been more extensively studied by Went (1928). In plants of the ordinary experimental age (about 3 em. tall) the srowth rate rises from 0 at the extreme apex to a maximum about 10 mm. lower. From this point to the. node of the coleoptile insertion the growth rate falls steadily. Fig. XI gives an example of this distribution taken from Went. Exverimental. Plants of as nearly the same height as possible were selected, tips exactly 5 mm. long removed, and the leaves ex— tracted in the usual manner. The balance of the coleoptilse was then divided into two six mm. zones and the base or remainder. The respiration rate of each set of sections was determined separately. The dry weights and microkjeldehl nitrogens of similar lots of sections were also determined. Results. Table 49 gives the distribution of respiration in three typical experiments. Altho there are minor variations in each experiment, in the mean the respiration is fairly constant over (Insert Table 9) the entire coleoptile length. The reason for this constancy of res— piration through the coleoptile could ve explained as due to the synthesis of vrotoplasm during elongation as discussed in the pre— ceding section. Table 50 shows that in fact there is more protein (Insert Table 50) -119- per mm. in the base than in the top, altho due to the increase of dry weight toward the bottom, the per cent of protein is somewhat higher in the tip. The respiration per unit protein falls offcontinuously from the tip to the base, the values being 24.6, 22.3, 19.1, and 17.7 respectively. This is just as was found in the previous section for the grand period of respiration, where the respiration per unit protein decreased continuously with the age of the coleoptile during the later stages, and ig an examole of the fact that the cells of the tip are "physiologically younger" than those of the base. This decrease of respiration per unit protein might in fact be added to the manifestations of “aging” summarized by DuBuy. and Nuernbergk (1932). The distribution of growth rate in the coleoptile was interpreted by Went as due to the interaction of two factors, a) growth substance, present in excess in the tip and limiting in the pase, b) "Zell sbreckunguaberial" (including all factors operating from the base) present in excess in the base and limiting in the tip. It has been shown above that neither in the grand period nor in the distribution over the coleoptile is there a quantitative correlation between respiration per mn. and growth per mm, This makes it in possible that respiration or respirable material is the second factor ‘of Went, For example, if there is sufficient respiration to allow of the growth rate of the fastest growing zone, then since the rate of respiration is the same in the tip or slower growing zone, res—. piration cannot there be limiting. Table 49 shows that the increase of respiration by carbohydrate (fructose) is small in every section and hence itis probable that ordinary nutrients are not the second ee e A 2 mS, ta oe oe 22 = / \ == 3 \, ° , 8 \ ° Sf \, , mt) 20 30 DISTANCE FROM APEX, M.M, Figure 11.Distribution of Growth and of Respiration in the Coleoptile. Curve A,Rate of Respiration per mem. Curve B, Rate of Respiration per mg.Protein. Curve C,Growth Rate, (After Went.1928). —120- factor. It is true however, that of the small increases in respiration upon the addition of fructose, that of the tip was greatest and that of the base least. In conclusion it should be pointed out that the two factor interpretation of growth distribution is in reality somewhat oversimolified, since it has been demonstrated in Part VI that the lower zones give a lower response to a given concentration of growth substance than do those higher | up. a. The Kap: dl Y Growing Z ore Changes of growth and respiration with time. The changes of growth rate and respiration rate of coleoptile sections with time have been determined many times, always with aporoximately the same result. Figure XII shows the course in a typical experiment in which coleoptile sections were suspended in puffer of pH 4.1 made up to 10 units of growth substance per cc. (Here as later unless otherwise stated the purified growth substance of activity 3x107! mgs. per plant unit was used). The growth rate falls steadily until in the eighth hour it has fallen to 2 per cent of its original value. The rate of respiration, on the other hand, after an initial period of sinking remains rather constant at about 82 to sl per cent of its initial value. It is, however, during the most rapid fall of growth rate that the fell of respiration takes place. From this it is clear that if there is any quantitative relation between growth and res- piretion, then at most about 17 per cent of the latter is essential to the former, since 62-83 per cent of the respiration can go on with very little accompanying growth. | The fact that the respiration is well maintained for several hours shows that there is no question here of deficit of nutrients. 100 50 OF FIRST HOUR GROWTH & RESPIRATION 4% % — 1 2 3 4 5 6 7 8 HOURS AFTER CUTTING Figure 12,Changes in the Growth Rate and the Respiration Rate of Coleoptile Sections with Time.Curve A,Respiration Rate. Curve B,Growth Rate. -121- This is in accord with the fact shown in Teble M9 that the addition of fructose to the medium has but little effect upon the respiration. Effect of cyanide upon growth and respiration. It hasbeen previously shown that growth and respiration are inhibited by cyanide to about the same extent. A quantitative examination of the inhibition has now been made to determine more orecisely the relation. Sections were suspended in buffer of pH 4.1 which had been made up to a concentration of 10 units per cc. in growth substance, The desired quantity of freshly prepared 0.1 molal HCN wae then added, The growth rates of a vortion of the sections were measured with the horizonval microscope, and the respiration rates of the remainder determined in the usual fashion. The inhibition of growth is expressed in per cent of the growth of controls not receiving HON. This is also true of all of the respiration inhibitions with the exception of those using 5x107> molal HCN. Here uoon account of the small inhibition it was found more satisfactory to measure the same somple before and after the addition of HON. It should be noticed that in these experiments HON solutions are used in vessels containing KOH in the alkali wells. This causes a falling off of the inhibiton with time as the HON goes over into the KOH, The inhibition was found to be rather constant over the first hour, and in these experiments only the first hour is considered, since even during the second hour the higher concentrations proved toxic. (Concerning the use of HCN with vessels containing alkali see also Emerson, 1928). Tables 51 and 52 gives the results of several experiments. In general the inhibiton of growth is rather reproducible. The (Insert Tables 51 & 52) 100 \ INHIBITION , 2 2 4 6, 8 HON CONCENTRATION. MOLAL x 10 Figure 13.Inhibition of Growth and of Respiration by HON. Curve A,Inhibition of Respiration Rate. Curve B,Inhibition of Growth Rate. | | -l22~- inhibition of respiration is much more variable especially in the lower concentrations as is shown by the large errors. Figure XIII presents the data of Tables 51 and 52 graphically. ‘The two inhibitione parallel one another rather closely altho there is a suggestion of smaller HCN sensitivity of respiration at 5x0? molal and of smaller HCN sensi- tivity of growth at usi974 molal. It seems fair to conclude that a given reduction of respiration by HCN is accompanied by a comparable reduction in the rate of elongation. Action of growth substance uvon growth and upon respiration. The activity of growth substance in promoting cell elongation depends principally upon the undigsociated acid present (Part VII). However, the pH of the cell is such (6.1) that only 4 to 5 per cent of the growth substance which it contains is present in the active form. If the cell acidity is increased by placing the section in an acid buffer solution, more of the cell's own growth substance becomes active and its rate of elongation is increased even without the addition of more growth substance. If the sections be placed in a neutral or basic solution, on the other hand, the cell acidity becomes less than normal and more of its growth substance is changed to the inactive form with the result that the rate of elongation of the section is decreased. Table 53 gives a summary of the growth rates (Insert Table 53) under different conditions of acidity and of growth substance. (The sections of Table 53 were not infiltrated but merely placed in the different. solutions). The purpose of the present section is to com— pare the rates of respiration under tie se different conditions. -123- Table 54 presents the results of 11 experiments which demonstrate that there is no appreciable difference in the rate of oxygen uptake at vH 4.1 and 7.2 despite the different growth (Insert Table 544) rates in the two cases. This is in agreement with Gustavson (1920) who found no effect of pH upon the rate of mould respiration. In tinge, section the maximum portion of the respiration which might be directly due to elongation was shown to be less than 6 per cent. In another section this portion was found to be less than 16~17 per cent. The present experiment shows, however, that if such a respiration exists, its oxygen consumption is negligible in com parison with the total. ah earhier Paper (Beaner, 1933) Inpwewmerbeus pare it was shown that certain growth substance preparations cause an increase in the rate of respiration. It was suggested that altho this might prove to be a property of the hormone itself, it might on the other hand be due to associated impurities. It can be shown by the use of a number of different growth substance preparations that the latter supposition is the correct one. For example, consider the two preparations, "bulk CCl, extract! activity ox107° mgs, per plant unit, and tyrucine filtrate", activity 3x107! mgs. per plant unit. Table 55 shows that while the former gives a (Insert Table 55) considerable increase in oxygen uptake, the latter has no effect. The stimulating effect of the crude growth substance preparation to is probably due to a specific stimulating substance and not) the -124~ nutritive action of associated organic material for the following reasons: a) it is shown that fructose which should also exhibit a nutritive action has but little effect on respiration; ‘b) in the above mentioned paper (Bonner, 1933) it was shen that the stimulating action of the crude extract is des- broyea by oxidation with H505- It has been found by Schwabe (1932) and by Burge, Wickshire, and Estes (1928), that amino acids in small amounts cause considerable increases in the respiration of various plants, as Elodea, Fontinalis, Vallisneria, and Spirogyra. Amino acids cannot be present in the growth substance preparations which have been extracted with ether. However, the effects of two amino acids in concentrations near the optimum given by Schwabe were investigated and the results are given in Table 56, together with the effect of fructose for comparison. Under the present conditions alanine has no effect and histidine is slightly toxic. (Insert Table 56) Effect of temperature on the two processes. Another ex- temnal factor whose effects upon the two processes might be compared is temperature. The literature concerning the effect of temperature upon growth as well as that concerning respiration is tremendous and only a few of the references will be made here. Rateroye/QNEUpmdnDe Candolle (1835) first determined the cardinal temperatures for thegrowth of a variety of seedlings. This type of work was extended by Sachs (1879), Képpen (1870) and others. More recently interest has centered upon the eifect of temperature upon the actual growth rate. Leitch (1916) found that -125- the rate of root growth in Pisum possesses a Q,, of 1.9 to 2.0 be— tween 10° GC. and 25° C. Somewhat higher values were found by Lehen- bauer (1914) for Zea maigw. Talospobseined results similar to those of Leitch for roots of Leptdim. Balls (1908) found that between 15° and 30° ¢. the rate of growth of fungal hyphae has a Q,, of 2 to 3. On the other hand there are examples of lower tamperature coefficients for elongation. Hamada (1930) gives data from which it may be cal- culated that the Qo of elongation of the normal Avena coleoptile between 15° and 25° ¢. is 1.7. The Qo is still lower if Longer periods are considered since coleoptiles grow actually longer at 15° than at 25°, “Mack (1930) has shown that the ho (at the oxygen pressure of orbsdary air) of Triticum shoots between 15° and 25° C. is about 1.5 and that the G5 detween 10° and 25° is 2.4. It is also of interest that the Qo of the reaction time of phototropic response in the Avena coleoptile has been found by M. DeVries (1914) to be about 1.5. The earlier literature an the effect of temperature upon respiration has bean summarized by prefcerfin the statement "respiration exhibits no distinct optimum lying within Limits of temperature which can be endured for prolonged periods", and contrasts this with growth which in general shows a distinct and relatively low optimum. Kuyper (1910) investigated the matter in some detail, and found that "Van't Hoffts rule" held over the range of 0° to 20° ©. for the respiration of Pisum and Triticum, Measured over short periods, (one hour) res— piration increased steadily up to 45° C., or far over the optimum ‘growth temperature. Measured over longer periods the respiration showed an optimum at about 35° C., due to progressively greater -126- injury of the plant at higher temperatures. A careful study of the effect of temperature upon the respiration of Vicia faba has been made by Navez (1929), who found Q,9 values of 2.3 and 2.7 for the intervals 15° to 25° C., and 10° to 20° C. respectively and these values for the Qo are those usually found for respiration. Goncerning the relative effects of temperature changes on ‘the two processes of growth and respiration, it has already been mentioned that Pfeffer pointed out the general lack of correspondence between the cardinal points of the two processes, and suggested that at the higher temperatures the two might compete for nutritive substance. The data of Mack (1930) show an increase in temperature is more effective in increasing the respiration than in increasing the growth rate of the shoots of wheat. Kidd, West, and Briggs (1921) believe that their data point to a lower temperature coefficient of growth than of respiration. However, their measure was that of relative increase in dry weight and is hence not comparable with the other studies mentioned above. The work of Hamada upon Avena seedlings and that of Mack on Triticum has shown that the Qo of elongation of the coleoptile between 10° and 25° C. is distinctly below 2, while the Qo of res piration is markedly higher. It will now be shown that this is also the case for coleoptile sections growing in growth substance solutions. The growth and respiration of comparable lots of sections at two temperatures Maa compared, using in every case the plants of the same day. Since all that was desired was a comparison of the effect of lowered temperature upon the two processes, only two temperatures were used, namely 25° and 10° C, Two thermostats were -127- used at the. same time, the one being kept at 10° 0. by the cooling unit of an electric refrigerator; aed the other being maintained at 25° C. Comparable sections were suspended in buffer of pH 4.1 made up to 10 units of growth substance per cc. One half of the sections were then placed in vessels in the 10° ©. thermostat, and the other half in vessels at 25° C. Measurements of the pressure decrease were made each half hour, and at the end of the desired period, the sections were removed and measured under the binocular microscope with a calibrated sye piece micrometer. It is to be noted that in these exoeriments the sections were 3.5 mo. long whereas those of the previous experiments were 4.5 mm. long. Table 57 presents a few typical results. As longer periods are considered, the ratios of the amounts of elongation and of the total amounts of respiration at the two temperatures, both decrease, altho the former decreases much more. In the case of respiration it may be seen that this is due to the fact that the respiration at 25° sinks with time whereas that at 10° actually increases a small | amount. In the case of growth the ratio decreases because elongation continues for a longer time at 10°, i.e. elongation continues at 10° after it has almost ceased at 25°. Measurements of the hourly growth rate showed that in fact after the fourth hour the growth at 10° is actually faster than that at 25°. The Qs of the first hour was also found to be somewhat higher than that of the first two hours, but in no case was it over 2.6. (Insert Table 57) ~lhfa- Table 58 gives the summary of a number of 2 hour determin- ations of respiration rate and elongation rate at the two temp- eratures , together with the qo's calculated with the aid of the assumption that the change of rate with temperature is linear over the range considered.The work of Navez(loc.cit.) and of Kuyper (loe.cit) shows that this assumption 1s justified in the case of respiration at least, articularly since an aproximate value is all that iff desired for comparison with the work of other in- vestigators.The & , of respiration over this range 1a 2.6 and hence well within the range found by Navez and by Kuyper.The &o Of elongation is however hide and thus similar to the values founda by Mack and by Hamada. It is of interest that Heyn and Van0verbeek obtained a Qo of about 1.6 for the rate of plastic bending of Avena colsoptiles after gpowth substance entry from agar blocks. —128- Characteristics of Growth and Non-Growth Respiration. (a) The HON sensitivity. I+ was next desired to find out if the respiration of a coleoptile section is qualitatively changed in any respect by the intensity of its growth. The sensitivity to HON was the first characteristic of the respiration which was examined. It has been found by Genevois (1927) that the sensitivity of the respiration of the stems of Lathyrus to KON decreases as the stems become older. At the same time the respiration per unit dry weight decreases in a manner similarto that described earlier for coleoptiles. The res- piration of a section from a 108 hour o1d coleoptile will, of course, be less than that of an 84 hour section since as shown in Table 48 the respiration per unit length is less. Table 59 shows that the _ (Insert Table 59) sensitivity to cyanide is also hess. ‘Sections which have ceased growth due to age have then a smaller proportion of their respiration in- hibited by a given concentration of HCN than do younger, actively growing sections. Sections respire at the same rate in solutions of pH 4 as in solutions of pH 7, altho the growth rates are different. This offers therefore an opportunity to compare the sensitivity of sections to HON, the sections having the same rate of respiration but different growth rates. Cyanide inhibition is proportional to the amount of free HCW present in a solution, probably because only HCN enters thea cell. Therefore in comparing the inhibition at two different vE's it is important to consider only the fraction of the total cyanide present as free HCN in the two cases. In all of the present experi- ments cyanide was added as fresnly made up HCN. The pK of HCN is, at -129- 25° 6., 9.14. Therefore in a solution buffered at pH 7, less than one per cent of the cyanide is present as CN ion. At both pH 4 and pH 7, then, the inhibition will depend upon practically all of the added cyanide. Table 60 presents the results of 5 experiments. (Insert Table 60) At the lower HCN concentration, the inhibition exhibits the same variability previously noted. At the higher concentration the in- hibition is remarkably constant from one experiment to the next. At both of the HON concentrations there is a quite significant difference between the sensitivities in the two buffer solutions. Moreover it is interesting to note that the per cent inhibition at pH 7 is of the same order as that shown in Table 59 for sections at pH 4 whose growth has ceased due to age. | The cyanide sensitivity of elongation is practically the same at pH 7 as at oH 4 (Table 61). This is an added confirmation (Insert Table 61) of the supposition that the elongation process is essentially the same under the two conditions. The cyanide sensitivity of elongation lies between that of respiration at pH 4 and that of respiration at pH 7, altho somewhat closer to the former. | From this experiment st “ad be said that respiration at pH 4 does differ somewhat from that at pH 7 and that the former kind of respiration is accompanied by the faster elongation. Respiration at pH 4 also resembles elongation somewhat more closely in this one characteristic than does respiration at pH 7. -130- (b) The Respiratory Quotient. The preceding section has shown that the respirations of fast growing and slow growing sections differ in at least one respect, namely in HON sensitivity. Another characteristic property of a particular type of metabolism is its respiratory quotient. The value of this quantity in fast and slow growing sections was therefore studied. Most of the determinations of the respiratory quotient of plants since the days of Bonuier and Mangin (1884) have concerned themselves with germinating seeds and changes in the respiratory quotient due to changes in the food reserve. In relatively few cases have the respiratory quotient of the embryo alone been examined. That there may be large differences between the quotient of the entire seedling and that of the young plant alone is well shown by the work of Murlin (1933), who found that while the respiratory quotient of the entire plant and seed (Bicinus) was 0.3 to 0.4, thatof the young plant alone was 0.8 to 1.1. The quotients were determined according to the method described by Warburg (1928). The method consists essentially in measurements of a) the gas which may be driven out of the tieaue and solution by acid at the beginning of the experiment ("preformed 60") ; b) the net gas exchange and gas drivmoff by acid at the end of the experiment; c) the oxygen consumption during the experiment. Coleoptile sections were suspended in the solution to be used and placed in the vessels having side arms. In vessels for the determination of a) and b) no alkali was placed in the well, but 0.4 cc of 4 molal H,S0, was placed in the side arm. At thedesired moment this acid could be tipped into the main body of the vessel and the ‘change in pressure noted after the reestablishment of temperature -131- equilibrium. There is a small volume change of the solution upon addition of the acid, but this was corrected for. For the deter- mination of ¢ vessel containing KOH in the alkali well and acid in the side arm was usec. This acid was tipped in at the same time as that for the determination of b, in order to measure the oxygen uo- take during death of the sections as well as during the main body of the experiment. | The determination of the respiratory quotients revealed a peculiar fact which has however, so far as can be seen at present, no relation to the action of growth substance and whose significance has not yet been studied further. The respiratory quotient is markedly higher for sections in acid buffer than for sections in neutral buffer. Table 62 shows the results of a series of experiments, each lasting two hours. In buiter of pH 4 the quotients are very constantly more than 1. In buffer of pH 7 they are more irregular but are markedly pelow 1. It uust be emphasized that the fact that during the main body of the experiment most of the 0, is driven off by the buffer of pH 4 and a portion of it is retained by the buffer of pH 7 does not affect directly the determination of the quotient since in both cases all of the co, is ultimately driven off with excess acid. The buffer was only 0.025 molal whereas the final acidity of o¢ the solution was 0.85 molal. In order to make clear the method of calculation as well as to illustrate the lack of effect of growth substance upon the respiratory quotient, Table 63 gives in full the data of three typical six hour experiments. The total elongation of comparable sections for the same period is included for comparison. In the nearly unbutfered fructose solution the quotient is pemmayn unity as would be expected. Growth substance has no effect. In buffer of oH 4 ~132- the quotient is very high. Growth substance, altho increasing the growth has no effect upon the quotient. _ *Strugger (1932, 1933) has ubggested that fermentation favors growth by cell elongation. The high quotient in buffer of pH 4 might indeed be construed as due to a sort of a fermentation. However, such a high quotient is not essential to growth as is shown in Table 63. (Insert Tables 62 & 63) Moreover, if fermentation were favorable to growth one might expect that anaerobic conditions ciould stimulate growth or that HON in suitable amounts should, by inhibition of the "Pasteur reaction" increase the growth rate. An increase of growth rate by either anaerobic conditions or HCN has never been observed in the couree of these investigations. The action of growth gub stanc . Altho respiration is necessary for elongation, the decrease of elongation, for example, by decrease of temperature or increase of pH, is not proportionally the same as the decrease of respiration. This could be due to a consecviive fusion of the characteristics of several, component processes, only one of which is a respiratory process, into the characteristics of the elongation process as a whole. An attemcot was therefore made to separate these component processes from one another. The steps involved in the bringing about of elongation by the application of growth substance may be divided into those of uptake, subsequent action, amd actual elongation. It has deen shown in Part Vi that respiration is necessary for the passage of growth substance into a coleoptile section. Nevertheless the Qo of thie process was shown in a previous section to be approximately 1. I+ -133- is Likely that the rate of passage of growth substance into the cole— optile is limited not by the rate of transport within the section, which has the high Q,) of 2.6 (Van der Vey, 1932) but by diffusion across the cut surface, which should have a low 20° If the tem perature coefficient of entry be measured by subsequent elongation in water at 25° (Table 64) or by plasticity at 25° (Table ol) it is (Insert Table 64) alse found to ve one. However, the elongation of sections in solution is not limited by the rate of entry of growth substance as was shown in Part VI where it was demonstrated that the growth rate of sections in growth substance solution is greater than that of sections already full of growth substance from previous treatment with growth substance containing agar blocks. It has been assumed by Heyn (loc. cit.) that if growth sub— stance agar blocks are placed upon plants without water supply, an Saction" of growth substance takes place AD results in increased plasticity and, if the plant is then placed in water, an increased growth rate. veeubte. ‘That some small amount of such "action" does take place is possible. A large part of this "action" must however, ‘take place later for the following reasons; a) Table 65 shows that if sections without water are allowed to take up growth substance, the subsequent elongation is still greatly (Insert Table 65) influenced by pH of the solution. These sections are already full of growth substance, which if action takes place apart from elongation, should have acted. Nevertheless elongation depends upou the presence -154- -of undissociated growth substance, which has veen shown (Part VII) to ve essential for elongation as a whole. b) Growth substance action has been shown (Part IV) to be of necessity indirect, i.e. chemical and would then ve likely to have a ho greater than 1. The Qo of growth substance effect prior ta actual bending or elongation is, however, 1, i.@. that of growth substance entry ¢) The Qo and cyanide sensitivity of elongation after the entry of growth substance from agar blocks are’ similar to the Qo and cyanide sensitivity of elongation as a whole (Tables 66 and 58). (Insert Table 66) Therefore the limiting process of elongation takes place after block action, i.e. must take place during the actual deformation of the wall. d) That the actual increase of wall plasticity takes place largely during wall deformation is not in conflict with the experi- ments of Heyn. He found that coleoptiles without water and with or without growth substance showed the same extension onder tension if they were immediately plasmolized. increases in wall plasticity depended upon the turgidity of the plants. One is forced then to the conclusion that a continuous action of growth substance is necessary for increases in wall plasticity and hence for elongation. This increase in plasticity is not se- parzble from the actual extension process which it makes possible, at least so far as the present methods are concerned. Since this is the case, it is impossible to determine, for example, the Qo of -135- snerease in wall plasticity, as this Qo may be "blended" with that of wall stretching which is presumably close to one. It is therefore quite possivle that the Qo of actual action is as high as that of respiration, although a direct measurement is impossible. This indication that growth substance action is a metabolic process continuous during wall extension makes more understandable the result of Part IV., i.e. that the "efficiency" of growth substance possesses a S10 of 2.2, That this temperature coefficient is almost identical with that of respiration further suggests that the wall extension dus to one growth substance molecule depends directly uson the magnitude of a respiratory process. Concerning the process of action a few in-vitro experiments have been made. ‘The effects of growth substance upon the dehydro- genase system of coleoptiles was extensively investigated using the usual Thunberg technique. A slight increase in the reduction time of methylene blue in the presence of growth substance was found, but °Footnote: It may readily be seen how two coupled processes of different temperature coefficients may yield an intermediate one. For example consider the reactions AS>BY ¢ . The temperature b coefficient of ais 2, that of bis 1l. For a, & = an k,8 at B . Boke ac _ _ Ty and at KB at T,- For b at K,8 at qT and = “ KB at t,- In comparison with its rate of production, B is used more rapidly at To and therefore the concentration of B at the steady state will be less. Therefore the final rate of preduction of CO will be less at To, and, depending upon the relative values of RB, and 4, the apparent coefficient of the entire process will assume values intermediate between 1 and & for a longer or shorter time . ‘afte a change from.T, to Te. -136- this effect could he duplicated with other unsaturated acids such as erotonic and ricinolkic which have no growth substance—like activity upon cell extension. It was conceivable that growth substance could be the heat stable, dializacle, coenzyme of carboxylase (Wetzel, 1932) but an extended series of manometric experiments showed that this is not the case. Growth substance also apparently cannot replace the catechol component of the coleoptile oxidase system. This type of in-vitro experiment, seems nevertheless, a possible fruitful method of future attack. @._ Discussion The significant results of this part may be summarized as follows: 1) Over the grand period of the coleoptile, total respiration and total elongation run qualitatively parallel. Over the lenzth of a given coleoptile, respiration per unit length is almost constant, altho elongation per unit length varies. Neither over the grand period or in a given coleoptile does respiration per unit protein or per unit dry weight parallel elongation, 2) Cyanidég decreases growth and respiration to about the same extent. Lower temperature decreases respiration more than elongation. Neutral buffers decrease elongation without exhibiting any effect upon the magnitude of respiration. From 1 and 2 it is necessary to conclude that decreases in respiration decrease elongation but not the reverse, i.e. no con— siderable portion of the respiration is a result of elongation pro— cesses. The dependance of growth upon the existence of a large respiration may be viewed in two ways ~137- | . re a) the entire respiration is a "formal praquisite of growth" (Pfeffer); b} one of the processes of growth is itself a respiratory process but one of small oxygen uptake. It has been shown in.the introduction that the entire respiration cannot be needed for the supoly of energy needed in growth. @) can give therefore no clear idea of why a reduction of respiration by HCN for example should decrease elongation to even approximately the same extent. This brings up the question as to in what way respiration might be a "formal prequisite for growth. It is clear that it can be necessary only if it performs functions which are essential to growth. The principal function it performs is the pro- auction of heat, which it is difficult to visualize as useful for @lonzation. It is likely therefore that only a portion of the res— piration is needed for actual growth, and a) would become identical with b). Experimentally, the distinction between a) and b) is also difficult if not impossible. Upon a), growth is cut when respiration is cut because the whole respiration is essential. Upon b), elon gation is cut iz respiration is cut because the two kinds of res- piration have similar properties. Uoon a) elongation can cease and respiration continue because the latter is a prerequisite of the former. Upon b) elongation can cease and respiration continue because either of somewhat different properties of the two respirations or because of the inhibition of other portions of the elongation process. Concept a) cannot then be either theoretically or experi- mentally strictly differentiated from concept b). It would seem quite desirable then to avoid the ambiguity of meaning in statement -138- ads and to express the necessity of respiration for. growth in the etatement that a process of respiratory nature is one of the com ponent processes of elongation. One of oxe most significant resultsof the present part is the demonstration that growth substance action probably takes place continuously during wall extension. This continuous action cannot be due to intussusception of new material (Part ¥). The remaining possibilities are a) that it affects orientation of wall micelles b) that it causes production of substances havingconsiderable influence upon the colloidal nature of the cell wail. The first possibility may be studied by X-ray methods. The second possibility may be studied only by investigation of the effects of growth substance upon metabolism and the relation of this to the cell wall. The present part has succeeded only in outlining the general nature of the metabolic processes which should be studied. —138a- Summary The results of the present part may be summarized as follows: 1,Over the grand period of the coleoptile,respiration parallels qual— itatively rate of slongation. Respiration per unit dry weight,per unit ’ protein,or per unit length exhibits no clear correlation with growth rate. ; 2, Respiration falls steadily from tip to base of the coleoptile,in contrast to thse growth rate which passes through a maximum,Over the length of the coleoptile, then the two processes exhibit no clear correlation, 3.Concerning the relation of growth and respiration in the excised rapidly growing zone,the following facts have been established; a.The growth of the section falls to about 2 per cent of its initial value,under the experimental conditions, while the res— piration falls only about 17 per cent. b. Inhibition of respiration by HON is accompanied by a corres ponding inhibition of growth. c.Decreass of temperature, however, causes a larger per centage decrease in respiration than in growth. d,Changes in growth rate due to changes in the growth substance supply are not accompanied by any detectable change in the rate of respiration . From a.,b.,c.,and d.git is clear that decreases in respiration are always accompanied by decreases in growth rate.It is suggested that this due to the fact that one or more of the processes of growth are of a respiratory nature,although processes of small oxygen up— take, e.Growth substance has no detectable effect upon the respira- tory quotient,although changes of the pH of the buffer in which the coleoptiles are suspended cause large variations of this quantity. f,The growth of sections under the present conditions is lim- ited by the action of growth substance after its entry into the section and not by the rate of this entry.It seems probable that the action of growth substance is an aerobic process which must go on during the actual plastic deformation of the cell wall. TABLE 45, EFFECT OF CUTTING UPON THE RATE OF RESPIRATION OF COLEOPTILE SECTIONS? , No. cut surfaces Q5 per No. cut surfaces Qoo BEL Experiment per pection sedtion per section gestion 1 2 3.08 4 3.06 2 2 2.94 S 2.80 °In this and in all subsequent tables 99, = ma? oxygen consumed - per hour and measured at standard condifions of temperature and pressure. TABLE 6. COMPARABILITY OF DUPLICATE SAMPLES OF COLEOPTILE SECTIONS Mean variation Qoo per section. Mean Expt. Vessel 1 2 mean siation 28 of ~— — _— nee mean I 1 y 1.40 1.43 1.42 1.30 1.40 0.037 2.7 2 1.68 1.72 1.56 1.641.65 0.050 3.0 1.35 1.32 1.29 1.33 1.32 0.018 1.3 ~140- TABLE 47. SUMMARIZED DATA OF GRAND puaropy OFr GROWTH AND RESPIR.TION Age . Height Gon ver Dry wght. Protein per 5 Piant per plant plant nours mm mgs. mgs. 12 (embryo) 2.35 0.50 0.254 0.08 24 (embryo) 2.80 1.49 0.304 0.12 36 (embryo) 3.95 2.51 0.475 0.18 36 (shoot) 1.34 — 0.161 — 4 (embryo) 13.80 7.18 1.000 0.38 4s (shoot) 2.82 1.34 0.257 0.12 60 (shoot) 6.6 2.95 0.70 0.24 72 (shoot) 18.7 6.82 1.81 0.54 72 (coleoptild 16.0 5.62 0.915 0.25 64 (coleoptild 27.3 6.79 1.32 0.335 96 (coleoptild 46.2 9.25 2.22 QO. 108 (coleoptild 46.0 9.19 2.42 0.51 120 (coleoptild 46.4 8.05 2.49 0.49 -li- LTO 02°0 T2°0 G2°0 GE°O "0 Lr°o 4g°0 me €2°0 “Uo G2"0 92°0 92 O£°O te 22°0 62 G2°0 ‘um qusteq = “um /#OB quetd ‘ay aseg +°9T 2°s ) O°8T gee 0 6°6T us 1° TS G*02 rit n°2S ned T°9 3°16 0°22 6°9 0°23 - 0°02 H°S G*al — 1°3 g°ee _— ni L°6T —_ *¢ “goad "Su ‘qm 4ap 9-4. Mo zed $0 ged “Om -Su zad 20% % 62°0 92°0 0£°O 92°0 22°0 orm [20% ‘mm g 4xeq 62°0 of °0 Lz°0 12°0 1£°O 12°0 92°0 12°00 42°O 92°0 “unt [COR “um /“O% ‘uml g 9XeN dtq4 ‘mu G esojonty YT at CO usoy eu0Z HIILdOMION FHL NI NOLLVAIdSaY iO NOTLNEMLSIC G°OT 2°¢ qe°T TZ°O 4t*O “Ua Y4Mor? ZT 02 T2 a2 G2 Le ae on $ 6h HIEVL 6n°0 1S°0 Bo $2°0 TT°O GS0°O ———_- —_we ufegord “sat uyeyorg 6n°2 en’? zd*2 ek °T G16°0 G°O 2T°0 g0°0 Go°o 70°O *sFul GO°s 61°6 G)°6 6L°9 29° ene? OT°T ol °O 270 KT°O . quertd “qu faq dod 0% ANUIV TIIGAOTTON FO FCOIMAd GNVED AO ViVd CAZINVMNNS 4 ; Bh TIA. use ¢ res T quent cedxy a 85139 942 TeH -142. TABLE 50. DISTRIBUTION OF PROTEIN AND DRY WEIGHT IN THE COLEOPTILE Experiment Zone 45 mm. tip Next 6 mn. Next 6 mm. mgs. orotein per mm. 1 0.96x107° 1.21x107° 1. 30x10 2 1.03 — 1.43 3 1.28 1.16 1.23 1.13 1.27 1.42 Wean ~.toxi0o™ = 121x107 1.36x107° dry weight mgs. per ma, 1 92105 , 81075 5 liom 2 0x10", 4. 8x10—5 5.2x10 5 3 %,7x10 5.1x10 5,2x10 ~ Mean 3.9x10~° .gx107~ 5. 3x10° Mean % protein 2g 25 25.5 Base 1.39x107* 1.39 1.53 - 1.63 L.47xl 07 5. 5x10, 56x10 » 5. 5x10 5.5x107= 27 -143- TABLE 51 INHIBITION OF RESPIRATION BY HCN - % Inhibition Expt. HON conc. | molal sel0™? so xio7* = ato? =o"? Sgt 1 22 28 60 79 100 2 7 51 63 76 80 3 22 43 60 15 au 28 35 68 89 90 5 23 61 76 83 90 12 71 oy 18) &S Mean 1943 ysts5 674 gite agts TABLE 52. INHIBITION OF ELONGATION BY HCN % Inhibition t. HCN conc. ie molal sxi0™ = izio™ = amo =o = ato 1 30 51 67 Tl 87 2 34 ye 57 67 BL é 30 38 58 69 — 31 — —_ 69 — Mean | 31 4h. 61 69 34 Total plants | 30 18 18 30 12 Ly TABLE 53. AVERAGE GROWTH RALES IN VARIOUS BUFFER SOLUTIONS % Elongation during hour Solution 1 2 3 oH WL 5.9 3.5 2.3 pH 4.1 + g.s 7.4 6.9 Wed pH 7.2 2.1 0.6 0.5 pH 7.2 + 2.8. 2.6 2.2 2.1 TABLE 54. EYFEOT OF oH OF EXTERNAL SOLUTION UPON RESPIRATION . pH 4.1 pH 7.2 Expt. Qop/ section Qgo/ section 1 1.89 1.76 2 1.38 1.47 2 1.60 1.49 1.45 1.57 5 1.68 1.67 6 1.62 1.46 7 1.59 - 1,38 9 1.49 1.57 10 1.48 1.36 11 1.37 1.32 Mean 1.534.095 1.492.05 Mean variation g g as % of mean, —145- TABLE 55 EFFECTS OF TWO DIFFERENT GROWTH SUBSTANCE PREPARATIONS UPON RESPIRATION Experiment Without growth substance With growth substance 22 802 Lahr. 2 hr. L hr. 2 hr. Activity 2x107° ngs. unit 1 1.05 1.12 1.13 1.33 2 1.68 1.60 1.78 2.08 3 1.52 1.43 2.05 1.81 Activity $a07’ mes. /onit 1 1.65 1.49 1.74 1s 2 1.62 1.4 1.53 1.38 TABLE 56. EFFECTS OF ALANINE AND HISTIDWEON RESPIRATION Time hr. Rate of oxygen uptake in % 1 100 100 100 100 Solution vothing 1% fractose 0.05% 0.05% | added alanine Histidine 1 95 107 gu 77 2 gy 98 91 vis) 3 86 ge 87 15 -146- OL ‘UzMOrs Te{04 Ge “UWMOIs Teqoy G°GT ge°e nie LT eonmeene g°e HT care G6*2 G°2T g°e tt LE°T T°e tI G2e°T GG°s 2° 09°0 oe 6 13°0 . 9°0 Leg L be-0 g°t G £9°O % toy 4088 / cum o0T '°O THIF U4 MOI O T8404 oge "CO Tey0g eOT G‘t2 Ze t2 G'02 6T ec G°Ht Gt 9T ot % Y4MO.10) ea res oe » 00°6 00°6 98°C HB * a3°' Bl°2 g¢°2 ot’2 G6°T HZ*2 NOILVULdSHA NOdN CNV HEMOWD NO PAMLVATIAAL KOT AO SLOALIT LG gieve een et *4cx uson ol weeny weex ‘say ‘potsed Jo yqeueT —L47- TABLE 58. SULMARY OF RESPIRATION RATES AT 10° and at 25° + ment 25° 10° Experi O,/section/2hrs. 0,/section/2hrs. 1 2.04 0.56 2 2.35 0.58 3 2.36 0.64 4 2.09 0.57 5 1.98 0.62 6 1.92 0.62 1 1.80 0.63 8 2.24 0.50 9 2.13 0,66 Mean 2.10 0.60 Respiration Ger B5#OL Y= 28 Elongation _ . (Téstns.) Up =ZP*OL Youre TABLE 59. EFFECT OF HCN UPON RESPIRATION OF SECTIONS FROM YOUNG AND FROM OLD COLEOPTILES 8% hour coleoptile | 108 hour coleoptile Expt. | now cone.| 0 1x10 g x10 ¢ o | ixlog Uxlos molal { 2 | 2 aahipip 92 inhin.| %2| %2 inhin| 2 innin. 1 1.56 [0.66 58 ]0.37 76 }0.92|0.74 20 | 0.63 69 2 1.42 [0.85 4o {0.34 76 | 1.04) 0.83 21 10.65 63 —lig— TABLE 60 INHIBITION OF RESPIRATION BY HCN IN SOLUTIONS OF pH 4.1 and pH 7.2 - % inhibition EGN conc. pH 4.2 pH 7.2 Experiment = nolal ixi07* “x07 1x10 “elo7* 1 pt 73 16 55 2 7 76 30 58 3 76 85 20 5S 4 76 82 20 57 5 65 89 30 62 Means 59+8 gate exth = 8 58h. TABLE 61 HON SENSITIVITY GF ELONGATION AT pH 4 and pH 7. z HCN conc. b Inhibition P molal 1x10 “xO 4 (Table 52) yy 69 __ T 50 71 2 —i Total plants -149- TABLE 62 RESPIRATORY QUOTIENTS IN SOLUTIONS OF pH 4.1 and 7.2 pH UL DH 7.2 Expt. B.Q. Expt. RQ. 1 1.05 L 0.76 2 1.07 2 0.99 H 1.09 H 0.97 1.08 0.62 5 1.04 5 0.74 6 1.04 6 0.77 7 1.16 1 0.85 & 1.05 9 1.07 10 1.06 11 1.05 Mean 1.07 = .01 0.81 + .06 -150- 74.Mo.2H 96°0 0°82 £3°0 o*cne 62°t O°SLt gum ‘oa exegdn % gu ssueyoxe see Te{o] (su0tyoes prepueys Of) ‘gwarya pet £°t+ 6°T + qi Ler a Tet O°St+ £°2t+ gua pyoe SuTppe uo esuvyoxe ses 1°N 43°49 2°9- n°6 - 6°0l- 2°stt 0° 6f+ fre sutanp eSueyore og peuzoserd ses eq =~) aso road of, ] ‘s°3 4 "ab a PeTITISTP “at OH POTITISTP *s°2 + g°l Hd 2°) Hd 6) f°) ‘O08 *8°2 + T*h Hd 0 Tt Hd BOTIATOS INATZON AHOLVAIGSHE BHD NOdN HONVESENS HIMOE) 40 LORIAT “Ch wave | *qdoor -151- TABLE 64 EFFECT OF TEMPERATURE UPON GROWTH SUBSTANCE ENTRY Pratreatment : Plants Rigpexinoand 2 hrs. with 1000 u/ce F Elongation at 25° in H,0 blocks in 2 hours "2 Yo at 10° 20 from 10° 7.5 ab 25° 20 from 25° 69 2.0 2 hrs. with 1000 ufos Bending at 25°, Ll hr. 250 mg. riders solution Elastic bending Plastic bending Qo at 10° 2h = from 10° 4.3 11.0 1.0 - ab 25° 24 from 25° 3.2 11.2 ° TABLE 65 EFFECT OF pH UPON ELONGATION AFTER ENTRY OF GROWTH SUBSTANCE Elongation in % at 25° C. Pretreatment 2 hr. h hr. g hr. 2 hr. with 1000 u/ec pH 441 904 2G 3.0 blacks at 25° pH 7.2 4.7 1.4 1.2 TABLE 66 EFFECT OF TEMPERATURE UPON ELONGATION AFTER GROWTH SUBSTANCE ENTRY AT 25° Pretreatment ‘Plante Blongation in % per 2 hr. ho . o 2hr. with 1000 u/ec 2° at 10 2.8 1.4 blocks at 25° 10 at 25° 4,3 “152- Conclusions Detailed summaries have been given at the ends of each of the sections and it will neccessary to outline only the general imortance of the present work for the knowledge of cell elongation. 1.The growth substance controlling cell elongation is a trues phyto—hormone and not a structural component of the cell wall. 2,It is impossible that the elongation of the Avena coleop~ tile is by the intussusception of new material.It is due then,to plastic stretching. 3.The action of growth substance is not as has been suggested directly upon the properties of the cell wall.It is indin- ect and through the functions of the protoplasm. fhe. action of acids upon elongation is due to the depression of the ionization of the weak acid,growth substance, which is principaly active in causing cell elongation when in the non—dissociated form,Growth substance itself has no effect upon the acid metaboliem of the coleoptile.Effecta of acid wpen hydration and imbibition pressure of the protoplasm, long disseussed in the literature,have in the present case only minor importance, 5.Both transport and action of growth substance depend upon respiration—Like processes,and this is probably the reason for the well known dependance of the growth of aerobic plants upon the presence of respiration.The existance of the entire aerobic metabolism for the supply of growth energy is unnece cessery. -153- 6. 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