ON THE ROLE OF AUXIN IN PHOTOTROPISM AND LIGHT-GROWTHREACTIONS OF AVENA-COLEOPTILES

TER VERKRIJGING VAN DEN GRAAD VAN DOCTOR IN DE WIS- EN NATUURKUNDE AANnbsp;DE RIJKSUNIVERSITEIT TE UTRECHT, OPnbsp;GEZAG VAN DEN WAARNEMENDEN RECTOR-MAGNIFICUS L. VAN VUUREN, HOOGLEERAARnbsp;IN DE FACULTEIT DER LETTEREN EN WIJSBEGEERTE, VOLGENS BESLUIT VAN DENnbsp;SENAAT DER UNIVERSITEIT TE VERDEDIGENnbsp;TEGEN DE BEDENKINGEN VAN DE FACULTEITnbsp;DER WIS- EN Natuurkunde op maandagnbsp;15 DECEMBER 1941, DES NAMIDDAGS TE 3 UUR

WILLEM FREDERIK FLORUS OPPENOORTH Jr.

N.V. DRUKKERIJ v/h KOCH amp; KNUTTEL

Bij het afsluiten van mijn studietijd wil ik gaarne mijn dank betuigen aan allen, die tot mijn vorming hebben bijgedragen.

In de allereerste plaats dank ik mijn ouders, die deze studie hebben mogelijk gemaakt.

Hooggeleerde Koningsberger, Hooggeachte Promotor, U dank ik voor Uw hulp en Uw kritiek bij het bewerken van dit proefschrift. De tijd, waarin ik bij U assistent mocht zijn, is voor mij eennbsp;zeer belangrijke geweest. Daarnaast dank ik, ook namens mijnnbsp;vrouw. Mevrouw Koningsberger en U voor de gastvrijheid, dienbsp;wij van U beiden mochten ondervinden.

Hooggeleerde De Bussy, Honing, Jordan, Mohr, Pulle en Westerdijk, door mijn belangstelling hoop ik getoond te hebben,nbsp;hoe ik Uw colleges en Uw leiding bij het practisch werk gewaardeerd heb.

Zeergeleerde Katz, U dank ik voor de bijzonder plezierige wijze, waarop U mij geholpen heeft met het opbouwen van de filter-opstelling.

Het personeel van het Botanisch Laboratorium dank ik voor de aangename samenwerking en de prettige hulp gedurende de afge-loopen jaren; mijn collega�s assistenten tevens voor het vele werk,nbsp;dat zij van mij overnamen, teneinde mij in de gelegenheid te stellennbsp;dit proefschrift te bewerken.

Waarde Visser, U dank ik voor het nauwgezette testen van de vele extracten, die voor dit proefschrift noodig bleken te zijn.

Hans, zonder Jouw hulp had dit werk nog niet af kunnen zijn. Samen hebben we dit doel bereikt.

ON THE ROLE OF AUXIN IN PHOTOTROPISM AND LIGHT-GROWTHREACTIONS OF AVENA-COLEOPTILES

� I. The function of the coleoptile tip in phototropism . 295 � 2. Photo-effects, which could cause a lateral transport

c) nbsp;nbsp;nbsp;Photo-effectsnbsp;nbsp;nbsp;nbsp;onnbsp;nbsp;nbsp;nbsp;viscosity of the protoplasm . . 299

CHAPTER VII. General discussion and summary .... 348 � I. The types of phototropic, reactions, phototonus . . 348

INTRODUCTION AND STATEMENT OF THE PROBLEM.

� I. Introduction.

In 1909 Blaauw, discovering � simultaneously with Fr�schel (1908) � the stimulus quantity law, tried to explain phototropismnbsp;as well as changes in phototonus in terms of photochemical reactions.nbsp;Negative phototropism of normally positive phototropic organs,nbsp;occurring after the exposure to high amounts of light energy, werenbsp;compared to the solarization of the photographic plate. Blaauw thusnbsp;advocated a unity of the mechanism of both the positive and thenbsp;negative reaction.

A few years later (1914,1915,1918) Blaauw stated that all-round illumination causes light-growthreactions, their magnitude largelynbsp;depending on the light quantities. Since at unilateral illuminationnbsp;both halves of the phototropic organ are irradiated with differentnbsp;amounts of light energy, this illumination would cause an unequalnbsp;change in the growth rate of the light-(L) and of the dark-(D) sidenbsp;of the organ. Phototropism after unilateral illumination thereforenbsp;would represent a special case of the light-growthreactions afternbsp;all-round illumination, the latter being the primary phenomenonnbsp;(1915, page 187): ,,Die Lichtwachstumsreaktion ist die primare, dernbsp;Phototropismus die secundare Erscheinung, welche notwendig ausnbsp;ihr erfolgt, wenn durch �rtlich ungleiche Belichtung �rtlich ungleichenbsp;Wachstumsreaktionen entstehen.�

Meanwhile Arisz (1914) had extended the validity of the stimulus quantity law for Avena-colcoptiles. Up to a light quantity of lOOnbsp;M.C.S. the phototropic curvature proved to be proportional to thenbsp;light energy. Further the stimulus quantity law proved to be validnbsp;for the lower threshold value of the negative phototropic curvaturenbsp;of the Avena-coleoptile too, provided that the light energy (ca 4000nbsp;M.S.C.) be administered within a certain time limit (20 minutes).nbsp;Finally Arisz made it probable that changes in phototonus are notnbsp;due to changes in sensitivity of the organs, but to an interaction ofnbsp;different phototropic responses of the L- and D-side. The latternbsp;assumption urged Arisz to advocate that the photo tropic curvaturenbsp;is the primary reaction upon unilateral illumination, all-roundnbsp;illumination causing complicated secondary effects.

The apparently minor controverse between Blaauw�s and Arisz�s views in fact since has been the most disputed item in the problemnbsp;of phototropism and of light-growthreactions.

In a number of accurate investigations on growth and light-growthreactions Blaauw�s theory has been carefully checked, the majority of them endorsing its correctness a.o. V. J. Koningsbergernbsp;(1922), Brauner (1922), VAN Dillewijn (1927).

Especially VAN Dillewijn (1927) gave a thorough analysis of the light-growthreactions. In coleoptiles of Avena he found:

1) nbsp;nbsp;nbsp;a long lasting light-growthreaction after illumination of the tip,nbsp;even with light energies below 25 M.C.S. (tip reaction). This growth-reaction attains its peak after i�2 hours. At light quantities belownbsp;8000 M.C.S. a slowing down of the growth rate was found, fornbsp;light quantities above 8000 M.C.S. an acceleration.

2) nbsp;nbsp;nbsp;a short lasting light-growthreaction after illumination of thenbsp;base and � at large light quantities � also after that of the tip (basenbsp;reaction). This reaction always consists of a retardation of the growthnbsp;rate, which reaches its maximum after ca 30 minutes. For the basenbsp;reactions relatively high amounts of light energy (gt; 800 M.C.S.)nbsp;are required.

3) nbsp;nbsp;nbsp;a �dark� growthreaction after long lasting illumination. Thisnbsp;reaction, being a weak reflection of the base reaction, consists of anbsp;short lasting acceleration of the growth rate.

From these three types of responses, respectively the tip-, the base- and the dark reactions, the different types of phototropicnbsp;responses were derived. The so called first positive, the negativenbsp;and the second positive curvature would result from the interactionnbsp;between different competing light-growthreactions.

At the time, that Blaauw�s theory seemed so well corroborated, it was thoroughly shaken by the growth substance theory of Cho-LODNY-WENT (1928). Since Boysen Jensen (1910,1911,1913) ascribednbsp;phototropism to a phototropic stimulus substance, this view wasnbsp;held by a number of scientists (Purdy (1921), Stark amp; Drechselnbsp;(1922), Snow (1924), Boysen Jensen amp; Nielsen (1926) and Starknbsp;(1927) )�

Paal (1914, 1919) believed that this substance is always produced in the coleoptile tip as a growth promoting substance, that wouldnbsp;unequally be distributed by unilateral illumination. The definitenbsp;proof on the correctness of the latter conception of the substancenbsp;has been brought by Went (1928).

Trapping separately in agar blocks the auxin diffusing from L- and D-side of unilaterally illuminated coleoptile tips (looonbsp;M.C.S.), he found much more auxin delivered by the D-side (57 %)

than by the L-side (27 %), the total of diffused auxin amounting to 84 % of that of the dark controls. Originally Went attributed thesenbsp;results to:

2) nbsp;nbsp;nbsp;a �redistribution� of the auxin as a consequence of a lateralnbsp;shift of the auxin transport to the D-side by the illumination.

Later investigations, especially those of van Overbeek (1933), brought further evidence of a transversal diversion of the auxin bynbsp;the light. Since then this redistribution is the main item in thenbsp;Cholodny-Went theory on phototropism, as it had already beennbsp;formulated by Cholodny (1928 p. 134): ,,Die Ursache dieser Er-scheinung (the phototropic curvature) ist vielmehr darin zu suchen,nbsp;dass die aus der Spitze diffundierende Wuchshormone sich zwischennbsp;den verschiedenen Seiten des wachsendes Organs ungleichmassignbsp;ver teilen.�

The mechanism of the lateral transport still completely remaining obscure, the eventual effect of light upon the permeability of thenbsp;protoplasm, as earher advocated by Lepeschkin (1909) and Tr�ndlenbsp;(1910), got a new interest. Especially Brauner (1922, 1924) tried tonbsp;explain phototropism and light-growthreactions by light-inducednbsp;changes of the permeability and also van Dillewijn (1927) gavenbsp;some further evidence of the probability of such changes. If theynbsp;really occur, they could offer a means to explain the lateral transportnbsp;of auxin and also that of other substances.

According to the Cholodny-Went theory phototropism cannot directly depend on light-growthreactions. The latter are induced bynbsp;all-round illuminations, which, of course, cannot yield any transversal diversion of auxin. Therefore this theory is incompatible withnbsp;that of Blaauw. This hold true after the effort by van Overbeeknbsp;(1933) to bridge the gap by assuming that phototropism would benbsp;caused by a lateral transport of auxin and light-growthreactions bynbsp;a changed reactivity on auxin of the cell wall.

On the other hand Went (1928) himself had found also a consistent decrease of the auxin quantities diffusing from coleoptile tips thatnbsp;had been illuminated (1000 M.C.S. from the top side) table 20, p. 92:

haviour against light in decapitated coleoptiles provided with auxin and with indole-3-acetic acid. When illuminated all-sidedly duringnbsp;the time wanted for the auxin curvatures, these curvatures withnbsp;auxin proved to be much smaller than in the dark controls. Withnbsp;indole-3-acetic acid the difference was much smaller or almost zero.nbsp;He explains these results by a destruction of the auxin by the light,nbsp;whilst indole-3-acetic acid would almost not be affected by the light.

Meanwhile in further chemical investigations on the auto-inactivation of auxin-a K�gl, C. Koningsberger amp; Erxleben (1936) stated the photo-inactivation of auxin-a-lactone by ultra-violetnbsp;radiation.

C. Koningsberger (1936) showed that the absorption bands, immediately found with solutions of auxin-a-lactone in the ultraviolet spectrum, are not due to the lactone itself, but to its readynbsp;conversion by the radiation into a physiologically inactive product,nbsp;called lumi-auxin-a-lactone. In slightly acid solutions in vitro auxin-anbsp;is in equilibrium with its lactone. Accounting for the possibilitynbsp;that such an equilibrium would also occur within the living tissue,nbsp;K�gl c.s. (1936) wrote (p. 274): ,,Dieser Stoff (auxin-a-lactone)nbsp;wird �berraschenderweise auch dutch Bestrahlung physiolo-gisch inaktiv, eine Erscheinung, die vermutlich von grosser Wichtig-keit sein wird f�r die Deutung der phototropischen Kr�mmungen�.

This interest preliminarily has been shown by V. J. Koningsberger amp; Verkaaik (1938). They used deseeded and decapitated coleoptiles of Avena, according to Skoog�s method (1937). Suchnbsp;coleoptiles practically are free from auxin and ,,show phototropicnbsp;curvatures (base response) if auxin-a is supplied as growth substance,nbsp;and not with indole-3-acetic acid�. This base response (the 2ndnbsp;type of light-growthreactions after van Dillewijn) was ascribed by

auxin-a-lactone system; in their experiments there was no evidence of any �redistribution� in the base of the coleoptile.

Since the auto-inactivation of auxin-a-lactone in vitro occurs only by ultra-violet radiation, the authors assume the presence of a sensibilizer in the growing cells of the coleoptile. Referring to the statement of Wald amp; du Buy (1937) for Avena coleoptiles, and of B�n-NING (1937) for Phycomyces sporangiophores, that carotinoids occurnbsp;in these photo tropic organs, they linked up the auxin theory withnbsp;B�nning�s (1937) carotene theory of phototropism by assumingnbsp;that carotinoids may act as sensibilizers in the inactivation of auxin-a-lactone by light of visible wave lengths.

This hypothesis gained considerably in probability by the investigations of ScHURiNGA (1941), who stated that in vitro auxin-a-

lactone is readily inactivated indeed by light of visible wave lengths in the presence of a- and j3-carotene and lycopin and turns into thenbsp;inactive lumi-auxin-a-lactone. The maximum of the photo-inactivation in the presence of carotinoids shifts from the ultra-violetnbsp;(ca 3300 A) towards the maximum of the absorption band of thenbsp;carotinoid; i.c. for /S-carotene to about 4500 A, that is exactly thenbsp;peak of the light sensitivity of the Avena coleoptile.

There was therefore more than one reason to investigate once more the role of the auxin in phototropism and in the light-growth-reactions.

The photo-inactivation of auxin-a by the visible light in the presence of carotinoids can lead to a decrease of the auxin contentnbsp;after illumination according to the reaction;

The possibility remains open that this process actually plays a part in phototropism (i.c. also-in the tip reaction) and in the light-growth-reaction. In that case possibly the lateral shift of the auxin transport,nbsp;as postulated by the Cholodny-Went theory, has been shammednbsp;by an auxin-inactivation. If this proved to hold true, the explanationnbsp;of photo-tropism and light-growthreactions would greatly be linkednbsp;up again with Blaauw�s theory and this would match with a modifiednbsp;growth substance theory on phototropism.

First it had to be discriminated whether the inactivation of auxin-a-lactone actually plays a part in phototropism and in the light-growthreactions in general, apart from its probable effect in the s.c. base response.

On the other hand it might be possible, that the light also affects the synthesis of auxin in the tip. By using the diffusion method suchnbsp;a synthesis easily would be masked by a real or seeming lateral shiftnbsp;of the auxin transport.

Besides, the distribution of auxin after unilateral illumination had to be examined again. It was not to be excluded, that the earliernbsp;data on the auxin distribution at the L- and D-sides of the coleoptilenbsp;had erroneously been interpreted. In the first place because thenbsp;photo-inactivation had been ignored. Secondly, all data had beennbsp;gained by means of the diffusion method. This however, does not

294

give a right estimation of the auxin content at a certain moment, since the auxin needs a relative long time to diffuse from the tipnbsp;into the agar slice. During this diffusion time the production (respectively the inactivation) of auxin in the tip might be liable tonbsp;considerable alterations. This objection a fortiori holds true for thenbsp;efforts to explain still more complicated processes, e.g. the negativenbsp;phototropic curvature (Asana, 1938) by means of data obtained withnbsp;the diffusion method.

The only means correctly to estimate the auxin content of L- and D-sides at arbitrary moments is offered by the use of the extractionnbsp;method. The course of the auxin content only can be investigatednbsp;by extracting successive sets of equally treated coleoptiles at successive time intervals after the exposure to light.

Since photochemical reactions in the sense of Blaauw (1909) could be expected to occur in the coleoptile, in most experimentsnbsp;monochromatic light has been used to avoid possible complicationsnbsp;4ue to some interaction of different processes.

It was hoped that the data collected in this way would enable to elucidate the controverse between the theory of Blaauw on onenbsp;hand and those of Arisz and of Cholodny-Went on the other hand.nbsp;It will be discussed at the end of this paper how far this aim has beennbsp;approached at.

The experiments, reported in this paper, were started in the early spring 1938. It soon turned out that each auxin determination shouldnbsp;be repeated a number of times in order to get reliable mean values,nbsp;while each experiment costed a lot of time. It therefore was clearnbsp;that I had to restrict myself to a few selected light quantities, mainlynbsp;of the most promising short wave lengths. Unfortunately, this worknbsp;had to be interrupted for a long period by the mobilisation and thenbsp;subsequent war. This forced me to abandon a part of my program.nbsp;It is hoped to continue it in the near future.

CHAPTER II.

According to the statement of our problem, we will only discuss that part of the extensive literature, which deals with questionsnbsp;related to the auxin theory of phototropism. The earlier literaturenbsp;on this subject has been surveyed several times, e.g. by van Dille-wijN (i927),Went (1928), du Buy amp; Nuernbergk (1932,1934,1935).nbsp;In the first two paragraphs the function of the coleoptile tip and

factors, which may be involved in a lateral shift of the auxin transport, will be discussed. These factors can only play a part in phototropismnbsp;proper. The factors discussed in the other paragraphs are of interestnbsp;for phototropism as well as for light-growthreactions. It may benbsp;mentioned once for all that in the latter case one should read fornbsp;phototropism: phototropism and light-growthreactions.nbsp;A few own experiments, in which it had been tried to elucidatenbsp;discrepancies in the literature, are reported in this chapter too.

Since long, especially since the classic work of Rothert (1894), it is known that the coleoptile tip is many times more sensitive tonbsp;light than the lower zones. More recently this sensitivity has beennbsp;studied by Sierp amp; Seybold (1926) and by Lange (1927). As lightnbsp;cannot act unless it be absorbed, it often has been tried to estimatenbsp;the light absorption in the coleoptile. The gradient has been estimatednbsp;by a number of scientists, but the values found differ very much andnbsp;run from 10�50 at the L-side against i for the D-side. Curiouslynbsp;enough these estimations were done for the hollow sections of thenbsp;coleoptile only and never for the solid, light sensitive tip. On thenbsp;other hand the value of this absorption gradient does not seem sonbsp;very important, since Lundegardh (1922) did not find any differencenbsp;in the course of the phototropic curvature in hollow coleoptiles andnbsp;in those filled up with the primary leaf. Bergann (1930) filled isolatednbsp;coleoptiles with dye solutions (blue green, blue or black). He didnbsp;not find differences in their phototropic response as compared tonbsp;water filled coleoptiles.

The highest sensitivity is located in the very upper part of the solid tip. After Lange (1927) this part is only a section of 50 /� (seenbsp;his fig. 9). According to his fig. 10, were a longitudinal section otnbsp;the coleoptile has been reproduced, this means that the highestnbsp;phototropic sensitivity is confined to only three epidermic cells.nbsp;Here the lightabsorption and therefore the gradient must be muchnbsp;smaller than in the lower zones.

Apart from this, the solid tip must be greatly responsible for a redistribution of auxin. The solid region being not more thannbsp;200�300 high, the polarity of the cells of this tip region mustnbsp;be changed by light to make possible a lateral shift of the auxinnbsp;transport. It therefore is worth while to discuss the means by whichnbsp;such a shift might be initiated.

The most direct evidence for a lateral transport in the tip has been given by Boysen Jensen (1928). He placed a piece of a covernbsp;glass in a longitudinally split tip of the coleoptile of Avena. Illumi-

nation perpendicular to the cover glass practically did not yield any curvature, while a normal curvature occurred after illumination innbsp;the plane of the glass plate.

Normally the transport of auxin is strictly polarized; it only moves from the tip to the base. Van der Wey (1932), who especiallynbsp;studied this transport, confirmed Went�s (1928) statement that itnbsp;is a vital phenomenon. It cannot be a mere diffusion, since its ratenbsp;is too high (ca. 8 mm/h) and it shows an optimum curve in itsnbsp;dependence on temperature. Went (1932) tried to explain the movingnbsp;agent in terms of a cataphoretic transport, but this idea has beennbsp;rejected for different reasons (see Hellinga 1937, Thomas 1938).nbsp;Vapour of ethylene causes an abnormal growth. Borgstr�m (1939)nbsp;pretends that under the influence of ethylene the auxin would movenbsp;transversally. In fact van der Laan (1934) found that geotropismnbsp;in Vida Faba was disturbed by ethylene vapour since the lateralnbsp;transport of auxin was hampered. It is, however, not clear how thisnbsp;statement can be applied upon a vertically growing plant surroundednbsp;by ethylene vapour.

The change in the direction of transport must be preceded by a change in polarization. I therefore will briefly discuss those factors,nbsp;which might initiate a change of the polarization under influencenbsp;of unilateral illumination.

The data in the literature on the effect of light on the permeability are incomplete and contradictory. Even the results obtained withnbsp;experiments on artificial membranes are far from uniform (cf.nbsp;Becking amp; Gregerson (1924), Calabek (1927), Pincussen (1930),nbsp;Pyrkosch (1936), L. amp; M. Brauner (1937)). A number of authors,nbsp;working with the most different biological objects, pleads in favournbsp;of a change of the permeability of the protoplasm by light (f.i.nbsp;Blackman amp; Paine (1918), Packard (1925), Brooks (1926), Hoffmann (1927), Lepeschkin (1908,1909 a and b, 1930), Meindl (1934),nbsp;Heilbrunn amp; Daugherti (1933), Kaho (1937))? but even theirnbsp;results are often contradictory. On the other hand such an effectnbsp;is denied by many others.

Only a few investigations on this subject had a direct bearing on phototropism. Working with leaf-cells of Tilia and Buxus, Tr�ndlenbsp;(1910) stated a decrease of the permeability by illumination. Withnbsp;increasing light quantities, however, the permeability increased, to

decrease again at still higher light quantities. Tr�ndle wrote in his theoretical considerations (p. 223): �Die Erklarung des Umschlagensnbsp;von positiver in negativer Reaktion ist auch von Blaauw (1909 p. 81)nbsp;versucht worden durch die Annahme einer positiven und einer nega-tiven Reaktion ebenso von der Zufuhr einer bestimmten Lichtmengenbsp;abhangt, wie der positiven. Ich sehe deshalb die Bedeutung meinernbsp;obigen theoretischen Ausfiihrungen nicht darin, aufs neue auf dasnbsp;Vorhandensein zweier entgegengesetzter ungleich schnell verlau-fender Erregungen hingewiesen zu haben, sondern ich wollte haupt-sachlich zeigen, wie man in einem speziellen Fall die Wirkung dernbsp;beiden Erregungen im einzeln verstehen kann�. Tr�ndle therefore,nbsp;applying his results upon phototropism, believes that the decreasenbsp;of the permeability (positive reaction) gradually reaches its maximum, which causes an increase of the permeability (negativenbsp;reaction).

L. Brauner (1922, 1924), working with coleoptiles of Avena found changes in the permeability after illumination. The appliednbsp;light quantities were large; 50.000 M.C.S. or permanent illuminationnbsp;with too M.C. His conclusion (p. 131, 1924) was: �Bei gleichernbsp;Lichtmenge verlauft die Kurve dieser Permeabilitatszunahme an-naherend parallel mit der phototropische Kr�mmungsgeschwindig-keit. Dies liegt den Gedanken an einer Zusammenhang dieser beidennbsp;Reaktionen nahe�. Unfortunately Brauner later abandoned thisnbsp;object to study the parenchyma-cells of Daucus carota. In light thenbsp;permeability for sugars of the parenchyma-cells is decreased andnbsp;that for water is increased (L. amp; M. Brauner, 1936). They explainednbsp;this as follows: The electric charge of the surface layer of the plasmnbsp;would be decreased by the light and so the swelling. By decreasednbsp;swelling the permeability for water would be increased, but that fornbsp;sugar decreased. In 1937 they gave the results of experiments withnbsp;E/odea-leaves and models. The light would cause a double effectnbsp;on the permeability:

1. nbsp;nbsp;nbsp;a loss of potential of negatively charged membranes and consequently a decrease of the motility of the cations and an increasenbsp;of that of the anions (,,primarer Photoeffekt�).

2. nbsp;nbsp;nbsp;a condensation of the disperse system, due to a decrease of thenbsp;E.M.F., the pores becoming smaller, which does not hamper thenbsp;cations but strongly inhibits the motility of the anions (,,sekun-darer Photoeffekt�).

They used vellum paper as model of the membrane. If this was coloured a potential difference was found after illumination. Thenbsp;strongest photo-electric effect was obtained, when it was blackened

The scanty data on this subject, still interwoven with many hypothetical elements, are not conclusive. One must, however, account for the possibility that light affects the permeability of the protoplasm and that this effect decreases with increasing wave length.

Many investigators take such a change of permeability for changes of the electric charge of the protoplasm particles. If this potentialnbsp;changes, it is attended with a change in hydratation. A further effectnbsp;of it is, that the viscosity of the protoplasm must change too.

Although there is no definite indication that transport of substances in the tissue is linked with protoplasmic streaming, many scientists believe that the transport largely depends on the latternbsp;process. In this connection it is important that Bottelier (1933)nbsp;stated that the rate of protoplasmic streaming is decreased by illumination. Further he stated that the spectral sensitivity curves fornbsp;phototropic curvature and for protoplasmic streaming reactions ofnbsp;oat coleoptiles are similar. In both cases, there is no reaction to wavenbsp;lengths longer than about 5400 A. Both curves show a maximumnbsp;between 4800�4300 A. The quantity of light needed for the firstnbsp;phototropic curvature (tip illumination) and for a marked decreasenbsp;in protoplasmic streaming is about the same. The relation betweennbsp;the two phenomena still being obscure, the parallelism in theirnbsp;behaviour against light seems too great to be merely incidental. Itnbsp;may be possible that both effects primarily are due to some photochemical �master�-reaction.

In this connection it is of interest to note that Thimann amp; Sweeney (1937, 1938) found an acceleration by indole-3-acetic acid (in physiological concentrations lt; 0,5 mg/l) on the protoplasmic streaming.nbsp;This effect, however, lasts only for a short time (30 minutes). Innbsp;the presence of a suitable sugar, however, this effect was not transient,nbsp;but is maintained for at least two hours (1938).

On the other hand Clark (1938) brought some evidence that the longitudinal transport of auxin does not depend on the protoplasmicnbsp;streaming. A 0,05 % solution of saponin stops the protoplasmicnbsp;streaming in Avena without affecting the transport of indole-3-aceticnbsp;acid, while sodium glycocholate, in non toxic concentrations, abolishes the transport of indole-3-acetic acid without affecting the ratenbsp;of protoplasmic streaming, permeability, respiration, potential etc.

In the mean time, however, Clark found that coleoptiles infiltrated with Na-glycocholate solutions show normal phototropism

and geotropism. He concludes therefore that the lateral transport of auxin depends on a mechanism, quite different from that of thenbsp;longitudinal transport.

It therefore still is impossible to value the parallellism between the spectral photo-sensitivity of phototropism and of protoplasmicnbsp;streaming.

In literature many data are given on the influence of light upon the viscosity of the protoplasm and of the cell wall, as well for zoological as for botanical objects. The results, however, can hardly benbsp;compared, as they greatly will depend on the method used fornbsp;determining the viscosity. For our purpose it is of interest thatnbsp;Gibbs (1926) stated for Spirogyra that a short exposure to ultraviolet light induced a liquefaction and a longer exposure rather anbsp;stiffening of the protoplasm. Here again a typical analogy is foundnbsp;that the result of a short exposure has an effect opposite to that ofnbsp;a long one. According to Strugger (1934) the plasm of growing cellsnbsp;(stated with Helianthus hypocotyls) is of much higher viscosity thannbsp;that of full-grown cells. Though this could not be affirmed bynbsp;Borriss (1937), it is possible that light has another, i.c. a stronger,nbsp;effect upon young, still growing cells than upon older ones.

In connection with our problem it is of interest briefly to survey the literature on the effect of P.D. on auxin and reversely. Brauner amp;nbsp;B�nning (1930) claim that roots of Vida Fata seedlings, placed innbsp;an electric field of 640 Volt/cm^, grow towards the negative platenbsp;and the coleoptiles of Avena towards the positive plate; in the rootsnbsp;the deviation got clearly visible after one hour, in the coleoptilenbsp;already after 20 minutes. This is explained by a lateral shift of thenbsp;auxin. The positive plate induces a negative potential in the side ofnbsp;the plant facing it; the auxin molecules (anions) should consequentlynbsp;migrate by cataphoresis towards the other side. This would causenbsp;a growth acceleration in this side of the coleoptile, in the root anbsp;growth inhibition. After some time a recovery takes place; the curvature of the coleoptile decreases after two to three hours and entirely disappears after five hours. This would indicate a re-establishment of the potential balance in the plant, which, however, is hardnbsp;to be understood.

Ramshorn (1934) found a potential increase with decapitated Avena, when growth substance was applied (p. 750): �Die Potential-differenzen anderen sich entsprechend der Wuchsstoffzugabe� and

on p. 765: �Wachstumsvariationen durch Zugabe von Wuchsstoff rufen ebenfalls eine Aenderung der Potentialdifferenzen hervor. Dienbsp;starker wachsende Zone erweisst sich als electropositiv. Angelegtenbsp;Potentialdifferenzen bedingen Wachstumsanderungen am Hypocotylnbsp;von Helianthus. Liegt der Pluspol an der Zuwachszone, erfoigt einenbsp;kurze Steigerung der Zuwachsgeschwindigkeit, liegt der Minuspolnbsp;an ihr, vermindert sich die Zuwachsgeschwindigkeit�.

A high potential is attended with an intensive respiration. Though Ramshorn does not state it distinctly, he does apparently not believenbsp;that the P.D. is a primary effect, but might be caused by a changednbsp;permeability. Subsequently the migration of ions would be facilitatednbsp;and P.D. changes could be brought about.

Koch (1934) let a current pass (tension: 4,5V) through auxin agar and stated that after short time the auxin had gathered at the positivenbsp;pole. According Koch in the hypocotyl of Helianthus by a P. D.nbsp;of 4,5 V the auxin was shifted to the positive pole. In this way henbsp;succeeded in preventing photo- and geotropic reactions of hypocotylsnbsp;by an electric current (a tension of 1,5�2 V was sufficient). Is thisnbsp;cataphoresis or electro-osmosis? The latter possibility was excludednbsp;by Koch, p. 218: ,,Dass der Wuchsstoff durch einen elektro-osmo-tisch bewirkten Wasserstr�m zum positiven Pol befordert wird, istnbsp;ausgeschlossen. In Agar wenigstens, der selbst negativ geladen ist,nbsp;m�sste die Elektro-osmose des Wassers zum negativen Pol hinnbsp;stattf inden�.

PoHL (1936) later carried out a nice experiment. Two glass tubes filled with oat meal and water and fitted with platinum electrodes,nbsp;were connected in such a way that an electric current could pass.nbsp;After applying a P.D. of 15 volts for 24 hours, the two halves werenbsp;separated and the oat meal extracted apart. It now appeared that allnbsp;auxin present in the meal had assembled in the anode half and innbsp;this way even a greater output was obtained than with the normalnbsp;extraction method, without a preceding electric current (see table i).

The behaviour of auxin, resp. indole-3-acetic acid in agar gels against a P.D., however, seems to be different from that in living

tissues. Clark (1937 a, b; 1938) in a large number of experiments stated, that the longitudinal transport of indole-3-acetic acid in thenbsp;plant cannot be affected by a P.D. applied from outside. Accordingnbsp;to him there is no relation between direction of transport and potentialnbsp;gradient in the plant. The same was found for auxin by K�gl amp;nbsp;Haagen Smit amp; van Hulssen (1936), who did not succeed innbsp;influencing the transport in the plant by means of a P. D. By anbsp;P.D., however, the dislocation of the growth substance in agar-agarnbsp;is inHuenced. The increased sensitivity against auxin (K�gl 1933)nbsp;cannot be ascribed to an increased transport within the plant, butnbsp;only to an accumulation of the growth substance near the woundnbsp;surface of the coleoptile. Since indole-3-acetic acid and auxin verynbsp;easily migrate in an agar gel under the influence of a potential difference and this does not happen in the plant, experiments on modelsnbsp;have no physiological interest.

Speaking generally, it seems that one should be very cautious in interpreting results obtained on bio-potentials. Ramshorn (1937)nbsp;found the highest positive potentials in the zones of the fastestnbsp;growth; according to him the tip is often positive against the base.

Clark (1935, 1937 ^ b, 1938) regularly states the reverse. In a number of thorough experiments the tip was negative againstnbsp;the base. Even in cylinders of coleoptiles the top end proved to benbsp;negative with regard to the base.

There are also a few data on the influence of a P.D. on growth. Cholodny amp; Sankewitsch (1937) measured the growth of intactnbsp;coleoptiles of Avena when a current of 10-'^�lO-c Amp. passed fromnbsp;the base to the tip. This current induced a short growth acceleration,nbsp;usually followed by a growth inhibition. A current from the tip tonbsp;the base caused a growth inhibition, that still lasted for a long timenbsp;after switching off the current.

As for the effect of light on bio-potentials, I may refer to Glass (1933). He stated that apical and basal leaves of Elodea respondnbsp;to illumination of the apex by a strong increase of the potentialnbsp;(H- 100 mV) of the apex with respect to that of the base. The decreasenbsp;in the magnitudes of the E.M.F.�s along the leaf from apex to basenbsp;was uniform. In the roots of onions after illumination no rise of thenbsp;P.D. was found. Glass supposed that the P.D. effect is not a directnbsp;photo-electrical effect, but that some action of the chloroplast is thenbsp;primary effect. He thus advocates the much referred opinion thatnbsp;to get a photo-effect on the P.D. the cell should contain chlorophyll.nbsp;It seems, however, probable that this effect is not confined to chlorophyll only; perhaps other pigments, f.i. carotinoids, too may accountnbsp;for it, for etiolated plants can it show too.

Finally we owe some data on the effect of illumination on the P.D. in the ^dwraa-coleoptile to Clark (1935). After an all-round exposurenbsp;to 600.000 M.C.S. changes in the P.D. were found, which ran aboutnbsp;parallel to those of the growth. The fluctuations in the growth rate,nbsp;however, were about half an hour ahead to those in the P.D. Retardations of the growth corresponded with positivation, accelerationsnbsp;with negativation of the tip.

It seemed worth while to investigate the P.D. between L- and D-side after an unilateral illumination of my subject, the Avenanbsp;coleoptile.

By the courteous help of Dr. J. B. Thomas I was able to obtain some measurements of the potential differences in the .^vena-coleoptile after illumination. I thank him sincerely for his help and for the use of the equipmentnbsp;(Thomas 1939). Table 2 and fig. i represent the data of one of some similarnbsp;experiments on the P.D. after an exposure to 500 M.C.S. (390 erg/cm^) withnbsp;unfiltered mercury light. These experiments had to be stopped when I wasnbsp;mobilized and had to join the army.

303

Fig. I. After illumination with 500 M.C.S. (390 ergs/cm^), white light, in the Avena coleoptile a P.D. arises. O = relative potential of the D-against the L-sidej L becomes positive against D. Readings each 2 minutes.

The plants were cultivated in an usual air-conditioned dark room in the usual glassholders. The seedlings had the normal age of 90 hours. For thenbsp;experiment they were placed with their glassholders in a small moist chambernbsp;with one glass side for the illumination. The opposite wall was of paraffinnbsp;through which the electrodes were introduced. The glassholder was fixednbsp;in a paraffin block of a special shape which contained a vial of water, allowingnbsp;the water-uptake by the roots during the experiment. The electrodes werenbsp;long flexible gelatine-threads sticked on the epidermis without wounding it.nbsp;This kind of electrodes allows the coleoptile to curve after the exposure. Theynbsp;were melted on the L- and D-side just below the tip. A third electrode wasnbsp;fixed at the basal region perpendiculary to the other two. This one was thenbsp;zero^ the measurements of L and D thus were done against the basal electrode,nbsp;by substraction the difference between L and D could be found.

It appeared that L- and D-side had the same P.D. with respect to the base. Shortly after the exposure, however, the L-side became positive and maintained this level during about 100 minutes. Here the experiments were stopped.nbsp;The coleoptiles in these experiments showed only a slight phototropic response;nbsp;probably the gelatine electrodes still were too stiff to allow a normal curvature.

Waller (1900), Bose (1907) and Brauner (1927) did similar experiments but obtained opposite results. They probably used

an other light quantity than was applied in my experiments; On the other hand, with the controverse between Ramshorn and Clark innbsp;mind, this discrepancy is not amazing.

In Thomas� (1939) thesis a review of the different theories on the origin of bio-potentials is given. Not feeling myself an expert in thisnbsp;field, I will not choose between these theories, but only point to thenbsp;fact that the rising P.D. at the L- and D-side matches the transversalnbsp;transport as supported by me lateron (Chapter VII).

Gessner (1938) and F�ckler (1939) investigated the influence of light upon the respiration of cells free of chlorophyll. They believenbsp;that light stimulates the respiration. On the other hand a numbernbsp;of other authors could not state an influence of light on the respirationnbsp;(see f.i. Gaffron, 1939).

On this subject only few data are available; it is, however, an important question since respiration masters a number of othernbsp;processes.

Bonner (1933, 1936) investigated the influence of respiratory poisons on growth. He found that transport and activity of auxinnbsp;is blocked by HCN and phenylurethane. Pure auxin has no influencenbsp;on respiration or on the respiratory quotient. With increasing agenbsp;the growth rate decreases much more than the respiration. Becausenbsp;HCN and phenylurethane inhibits the respiration as well as the growthnbsp;at the same rate, and since in the absence of oxygen no growth takesnbsp;place, he concluded that the respiration was the essential conditionnbsp;for growth.

Skoog (1934) reported that in plants, treated with X-rays, the auxin content strongly decreased as compared to that of non-treatednbsp;controls. The modified appearance of X-radiated plants was ascribednbsp;to an oxydative inactivation of the auxin. In 1935 the same inactivation could be stated with white light, when the plants previouslynbsp;had been stained with eosin. The yIz;e�a-coleoptiles grew normallynbsp;in the dark after treatment with an i in lO� solution of eosin, butnbsp;they had lost their phototropism.

The oxydative nature of the inactivation was derived from the fact, that also in vitro ,,auxin� proved to be inactivated by whitenbsp;light in presence of eosin. When, however, a water solution of growthnbsp;substance was irradiated in an atmosphere of nitrogen, there was nonbsp;inactivation, indicating that the reaction was an oxydation (p. 256).

His figures (summarized on p. 238, table 5) on this inactivation are convincing.

It is, however, doubtful which growth substance has been used by Skoog. He speaks of �auxin obtained in highly active purifiednbsp;preparations from Rhizopus suinus and from urin� and of indole-3-acetic acid.

In order to study the eventual part of pH in this inactivation, I did some experiments with eosin myself with solutions of auxin-a,nbsp;auxin-b and indole-3-acetic acid. Curiously enough, I never couldnbsp;find an inactivation in the presence of eosin after irradiating thenbsp;solutions for 15�30 minutes with unfiltered mercury light (3300nbsp;ergs/cm^/sec) nor with blue light (A = 4360 A, 70 ergs/cm^/sec.).

The solutions were brought on pH == 4 by means of a 5 times diluted Me. Ilvaine�s standard buffer solution. Each solution was tested on 12 Avenanbsp;test plants. For each test fresh solutions were prepared and irradiated. Eosinnbsp;was added in a concentration i in lo'^.The auxin-b was obtained by extractingnbsp;rice-bran. As a control blanc buffer solution-agar was tested too. The resultsnbsp;are summarized in table 3.

auxin-b of the

same activity

dark		ilium.		dark		ilium.		dark		ilium.
no	with	no	with	no	with	no	with	no	with	no	with
eo-	eo-	eo-	eo-	eo-	eo-	eo-	eo-	eo-	eo-	eo-	eo-
sin	sin	sin	sin	sin	sin	sin	sin	sin	sin	sin	sin
0	0	0	0	15,0	7gt;6	IL5	2lt;7
0	0	0	0	18,0	�gt;7	18,5	8,4
				18,2	14,7	19,6	14,3	I4gt;5	10,2	14,0	12,2
								18,7	13,8	17,6	i4j7
0	0	0	0	15,0	13,5	13,2	IL5	13^0	11,2	15.9	9,0
								13,3	9,0	12,7	7,7
0	0	0	0	16,6	14,1	16,4	12,9	12,9	9,2	12,9	9,2

Curvatures in degrees of the test plants. pH = 4, eosin concentration i in 10�. Illumination during 15�30 minutes with unfiltered mercury light (3300nbsp;ergs/cm^/sec.). The activity of the auxin-b solution was the same as that ofnbsp;the solution containing hetero-auxin. See text.

The blanc buffer-agar did never give curvatures. On the other hand eosin strongly reduced the curvature, in the control dark solution as well as in thenbsp;irradiated ones. There is, however, no question of any photo-inactivation ofnbsp;auxin-b or of indole-3-acetic acid in the presence of eosin. To avoid the

3o6

inhibition of the curvature by eosin, its concentration was reduced in the next set of experiments.

Accordingly the prescriptions by K�gl, Haagen Smit amp; Erxleben (1933) auxin-a was prepared from urin and purified up to the extraction with petrol-ether. The auxin-a was taken up in a buffer solution of the desired pH andnbsp;eosin (in controls water) was added. The eosin concentration finally was

in to�; these solutions still were distinctly coloured. As a control physiologically aequivalent solutions of indole-3-acetic acid were prepared in a buffer solution of pH=4, with and without eosin. The concentration of thenbsp;growth substances in these solutions was aequivalent to indole-3-acetic acidnbsp;I in 10^. In order to abolish the strong effect of pH of the agar blocks onnbsp;the curvatures (see Chapter HI � 5 b) all solutions were �neutralized� bynbsp;diluting them 1000 times with a buffer solution of pH = 4 (that is about thenbsp;optimal pH for the test) before bringing them in agar for the test. The illuminated preparations were irradiated with unfiltered mercury light (3300 ergs/nbsp;cm^/sec) for half an hour.

The mean curvatures, obtained with the indole-3-acetic acid preparations, tested on 29 plants were;

Curvatures in degrees of the test plants. Auxin-a in solutions of pH = 4, = 6 and = 8 during the illumination brought to pH = 4 for the test. Eosinnbsp;concentration during the illumination 4 in 10^, for the test brought tonbsp;in 10�. Illumination during 15�30 minutes with unfiltered mercury lightnbsp;(3300 ergs/cm�/sec.).

The inhibiting effect of eosin on the curvature has disappeared in this lower concentration. No indication of any photo-inactivation in the presencenbsp;of eosin can be stated. On the contrary at pH = 6 and = 8 the curvaturesnbsp;are slightly higher after illumination with eosin than without. It might benbsp;that a slight inactivation occurred in the latter preparations.

From all this we may conclude that at none of the investigated pH eosin can act as a sensibilizer for the photo-inactivation of the growthnbsp;substance as reported by Skoog.

On the other hand I briefly surveyed in Chapter I the more recent work on the photo-inactivation of the auxin-a-lactone and its possiblenbsp;physiological interest. It is known from the work of C. Koningsberger (1936) and ScHURiNGA (1941) that in solutions auxin-a is innbsp;equilibrium with its lactone. At a low pH (about 4,5) a considerablenbsp;amount (up to 60 %) of the auxin-a is present as auxin-a-lactone. �

Apart from Koningsberger amp; Verkaaik (1938), whose work has already been discussed, also Larsen (1939) gave evidence, that innbsp;the plant auxin-a is in equilibrium with its lactone. From, etiolatednbsp;pea seedlings he isolated two growth substances, an acid one and anbsp;neutral one (�Skototenin�). The latter amounts to about 30�50 %nbsp;of the total amount of growth substance and would be inactivatednbsp;by illumination. Larsen himself mentioned the possibility that hisnbsp;�Skototenin� could be identical with the auxin-a-lactone.

Besides the photo-inactivation of the auxin-a-lactone fraction, there are still two other ways in which the auxin-a and the auxin-bnbsp;can be inactivated: the so called auto-inactivation and the oxydativenbsp;inactivation. The auto-inactivation only has been studied in vitronbsp;(K�GL, C. Koningsberger amp; Erxleben, 1936) and then occursnbsp;slowly in the lapse of several weeks. It is unknown, whether this typenbsp;of inactivation has any physiological interest. To an oxydativenbsp;inactivation many difficulties of the extraction method are ascribed,nbsp;although the chemical details of this process are still unknown. Vannbsp;OvERBEEK (1935) has given some evidence that such an inactivationnbsp;may have a physiological meaning. Also van Raalte (1937) found anbsp;relation between the auxin content of root tips and the state of thenbsp;redox system. The possibility may not be excluded that the redoxnbsp;system of living tissues is affected by illumination. A shift of thenbsp;redox potential towards a higher rH in that case might cause annbsp;oxydative inactivation of auxin too. This could happen in thenbsp;presence of certain dyes, discussed in � 5.

Finally I must mention that in the literature several indications are given of changes of the plasticity of the cell wall after illuminationnbsp;with various wave lengths, the shorter ones of which being claimednbsp;to be the most active in this regard. For the moment, however, itnbsp;cannot be discriminated whether these changes are caused directlynbsp;by changes of the physico-chemical properties of the cell wall, ornbsp;indirectly via a photo-inactivation of auxin-a-lactone or by both.nbsp;Such changes probably would result in a changed reactivity of thenbsp;cell wall on illumination. Since in my own experiments (Chapter IV

� 2) nothing could be stated on such changes in reactivity in the range of the first positive and the first negative curvature, this question will not be discussed further. It remains possible, however, thatnbsp;such phenomena are implicite with the second positive curvature,nbsp;which has not been , studied by me.

In most of the older experiments on phototropism white light has been used. Since long it is known that positive and negative curvaturesnbsp;alternate. This so called phototonus has been most thoroughlynbsp;studied by Arisz (1914), who found, that with ever increasing lightnbsp;quantities the first positive curvature is replaced by the first negativenbsp;one and this on its turn by the second positive curvature. Du Buynbsp;(1933) claims that there is also a range of light quantities, wherenbsp;second negative and third positive curvatures would occur. Thenbsp;second positive curvature of Arisz would be identical with the thirdnbsp;one of DU Buy. These results have all been obtained with white light.nbsp;Since it is possible that the action of the different spectral regionsnbsp;in phototropism would be different, it seemed worth while to studynbsp;these phenomena with monochromatic light.

With monochromatic light only threshold values (of the first positive curvature) have been investigated. Blaauw (1909) has givennbsp;the first curves of the distribution of the light sensitivity in thenbsp;spectrum, in which the light quantities have been accounted for.nbsp;He found a maximum of this sensitivity at A = 4670 A for Avenanbsp;and of A == 4950 A for Phycomyces. This sensitivity slowly decreasesnbsp;towards the sWter wave lengths and much more steeply towardsnbsp;the longer wave lengths. Koningsberger (1922) studied the light-growthreactions of Avena in monochromatic light and found thenbsp;same distribution of the activity of spectral light on the growth, thusnbsp;greatly endorsing Blaauw�s theory on phototropism. Later investigators, a.o. DU Buy (1933) repeated Blaauw�s experiments.nbsp;Du Buy critisizes the purity of the monochromatic light of othernbsp;investigators, but he found the maximum very close to that ofnbsp;Blaauw; for Avena this was at A = 4600 A. According to DU Buynbsp;the sensitivity very steeply drops towards the longer wave lengths.nbsp;With A = 5460 A he still could get photo tropic curvatures, but withnbsp;A = 5780 A this proved to be impossible.

As stated above, thus far only threshold values have been studied. Only in du Buy�s experiments there is an indication that A = 4360 Anbsp;can induce negative curvatures. With other wave lengths, however,nbsp;negative responses have not yet been obtained. This possibly mightnbsp;be due to the low intensity of the monochromatic light used, since

negative responses only occur, if the light quantity used is administered within a certain time. It is therefore still to discriminate whether negative curvatures can be induced by wave lengths other thannbsp;I = 4360 A.

B�NNING (1937) and Wald amp; DU Buy (1936) already suggested that carotinoids play a part in phototropism. B�nning comparednbsp;the spectral distribution of the phototropic sensitivity of Phycomycesnbsp;to the absorption spectrum of the carotinoids extracted from thenbsp;sporangiophores. From the striking parallelism between these twonbsp;B�nning concluded upon an important part of carotinoids in thenbsp;perception of light. He and Wald amp; du Buy showed the presence ofnbsp;carotene and carotinoids in the etiolated Avena coleoptiles and discussed the resemblance of the absorption spectrum of these pigmentsnbsp;and the spectral sensitivity of Avena. The later investigations referrednbsp;to in Chapter I made it probable that carotinoids act indeed as sensibilizers of the photo-inactivation of auxin-a-lactone. Especiallynbsp;Schuringa�s work (1941) made this probability almost to a certainty.

Since in my own experiments only tips of Avena-coleoptiles of 3 mm were extracted, I tried to ascertain the statement by Wald amp; DU Buy thatnbsp;carotinoids are also present in these tips. After the extraction of auxin with ether,nbsp;in which perhaps the major part of the carotinoids will have been dissolvednbsp;too, the tips were gathered in acetone and kept in the refrigerator until severalnbsp;thousands of tips were gathered. The yellow solution then was decanted andnbsp;centrifuged. By the kindness of the staffs) of the Biophysical Research of thenbsp;Rockefeller Foundation, Utrecht, the absorption spectrum of this solutionnbsp;was measured (fig. 2).

The strong absorption in the ultra-violet being due to other substances (probably proteins), the maximum at A = 4300 A still indicates the presencenbsp;of carotinoids in the tips extracted with ether.

It certainly would be worth while to check whether carotinoids are also present in phototropic roots and absent in non-phototropicnbsp;organs.

Several authors succeeded in sensibilizing non-phototropic roots by treating them with dye solutions, so that they curved phototropicallynbsp;after this treatment. Metzner (1923) for instance found that roots,nbsp;grown in a solution containing fluorescein, were sensitive to lightnbsp;and showed phototropism. The same was stated for wheat roots bynbsp;Blum amp; Scott (1933) in solutions of erythrosin i in 10�. Theynbsp;concluded (p. 535) �the wave length of the light producing the phototropic bending corresponds to the absorption spectrum of the dyenbsp;^ To the members of which I tender my best thanks.

indicating that the dye acts as a photosensibilizer�. Some scientists reported a detrimental effect of the dyes apphed and did not get thenbsp;same results. They observed an abnormal growth and a stronglynbsp;decreased auxin content (Boas amp; Merckenschlager 1925, Boasnbsp;19333 SCHWEIGHART 1935). It is likely that in these cases the dyesnbsp;were applied in too high concentrations. In fact it would not benbsp;strange if further investigations would prove that phototropismnbsp;of roots is attended with the presence of suitable photosensibilizingnbsp;pigments.

In the above discussion special attention was paid to carotinoids. It remains possible, however, that also other pigments are involvednbsp;in phototropism, not only in the photo-inactivation of the auxin-a-lactone fraction, but also in other processes. It will appear later thatnbsp;in phototropism also an increase of the auxin content plays a part.nbsp;The nature of this increase still is unknown, but it seems likely thatnbsp;this process too is sensibilized by pigments, either by carotinoids ornbsp;by other light absorbing substances.

Finally pigments possibly could play an indirect part in phototropism. At the end of � 3 I discussed the possibility of an oxydative inactivation of auxin by shifts of the redox system. Positivation ofnbsp;the redox potential (a raise of the rH) most probably would leadnbsp;towards a decrease of the auxin content. Also in this process pigmentsnbsp;might act as sensibilizers. In this connection I refer the conceptionnbsp;of Lazar (1935) and of Beck (1937) ^I^^t carotinoids might act asnbsp;oxygen vectors and to the statement by Syre (1938) that erythrosinnbsp;makes the redox system shift towards a higher rH. Such photochemical properties of pigments and dyes possibly largely depend onnbsp;illumination and on light absorption. The present knowledge onnbsp;bio-sensibilizers in physiological processes mastered by light stillnbsp;being very scanty, the urgency of further investigations in this fieldnbsp;may be stressed.

MATERIAL AND METHODS.

� I. The plants used.

In all experiments coleoptiles of Avena were used. The plants used for photo tropic reactions, as well as those from which the tips werenbsp;taken for auxin extraction, always were of the same age as the testnbsp;plants in the auxin test, that is 90 hours. They were grown in the

usual type of air conditioned dark room (relative humidity 96 %, temperature 23� C.) as described by Nuernbergk amp; du Buy (1930);nbsp;where the experiments were taken too. Eventual illuminations tooknbsp;place in an adjacent room, equally conditioned, but with blackenednbsp;walls and ceiling. By placing the racks almost parallel to the axisnbsp;of the light beam, the light passed through the coleoptiles in thenbsp;direction of the longer diameter of their elliptic cross section. Eithernbsp;immediately after the illumination, or after certain time intervals, thenbsp;tips of the coleoptiles had to be removed for the extraction of auxin.nbsp;Since the auxin content of the light- and shade-side had to be determined separately, the isolated tips had to be longitudinally splitnbsp;before being put into ether. This proved to be a delicate work, whichnbsp;demanded special precautions.

After cutting the about 10 mm long tips in the usual way with the decapitation scissors, the tips were only loosened a little, painstakingly taking care not to turn them along their longitudinal axis.nbsp;As soon as a dozen of coleoptiles (one rack) was treated in this way,nbsp;the tips were carefully removed from the primary leaf by means ofnbsp;tweezers and placed in an ebonite mould. This mould consists ofnbsp;two halfs; in each half twelve small furrows make a dozen of holes,nbsp;when the halves are clasped together. In each hole fits exactly anbsp;coleoptile tip of 3 mm length. As soon as a dozen of tips is put intonbsp;the holes in the correct orientation, they are cut to the same lengthnbsp;of 3 mm with one stroke of a razor blade alongside the mould. Thennbsp;the blade is pulled through the split between the two halves of thenbsp;mould, so that the coleoptiles are split longitudinally as precisely asnbsp;possible, in the plane of the short axis of their cross section. Onenbsp;set of the halves of the tips thus represent the light side (L in thenbsp;tables and the graphs), the other those of the shade-side (D). Thenbsp;sets of the halves are picked up with tweezers and put into ether.

In order to control the reactivity of the test plants, with each test parallels ran with two to four different concentrations of indole-3-acetic acid ^). By doing so, one can rule out the daily fluctuations innbsp;the reactivity of the test plants and the amounts of auxin in thenbsp;coleoptile extracts can be expressed in aequivalents of indole-3-aceticnbsp;acid.

The procedure of splitting and extracting illuminated tips was not applied until its reliability had been checked in a larger number ofnbsp;blank experiments with not-illuminated, �dark� coleoptile tips.nbsp;Since it proved to be very difficult to obtain constant and reproducible results with the ether extraction of coleoptile tips, it seems

For the experiments as a light source a high pressure mercury bulb from Philips, Eindhoven, of the commercial �Philora� typenbsp;(H.P. 300) was used, which � after a few minutes of preheating �nbsp;gives a light of a fairly constant intensity and rich in blue, violetnbsp;and ultra-violet. This bulb had been mounted vertically in a lightproof copper tube with a hole on one side; the desired light quantitynbsp;was administrated by means of a photographic shutter.

At first unfiltered white light was applied (Chapter II). Lateron monochromatic light was used (Chapter III and IV).

To obtain monochromatic light of an intensity as high as possible a filter device was arranged. The main apparatus of the set is thenbsp;�Dispersionsfilter� according to Christiansen amp; Weigert, suppliednbsp;by C. Zeiss, Jena. The transmitted wave length depends on the temperature of the filter, therefore it is placed in a thermostat, the temperature of which may vary 0,1� C. at most.

Fig. 3 shows the arrangement. L.S. is the H.P. 300 mounted in the copper tube. Its light, collected by lense L i (diameter 4 cm,nbsp;focus 6 cm), has to pass diaphragm D i. Lense L 2 (diameter 10 cm,nbsp;focus 36 cm) makes a slightly diverging beam to obtain a correctnbsp;dispersion by the filter (diameter 9,4 cm, thickness 5 cm). Lensenbsp;L 3 (identical with L 2) collects the rays again, diaphragm D 2nbsp;(identical with D i), connected with a photographic shutter, standsnbsp;in its focus. Two large wooden compartments cut off all of thenbsp;diffuse light. Behind D 2 a liquid filter (L.F.) is placed. By meansnbsp;of lense L 3 (diameter 7 cm, focus 36 cm) a parallel light beam isnbsp;obtained. This had the advantage that, if wanted, several racks withnbsp;plants could be placed behind each other, meanwhile taking carenbsp;that the successive coleoptiles were not shaded by each other. In thenbsp;parallel beam the distance between plants and light source does notnbsp;affect the light intensity.

The lenses L 2, L 3 and L 4 are biconvex; the ratio of the radii of the two sides being 9:1. The most convex sides of L 2 and L 3nbsp;face each other.

Light, obtained in this way, proved to be fairly monochromatic i). Controlling it with a spectrograph only a weak trace of the neighbouring wave-lengths could be detected. The emission spectrum of

^ In the arrangement and calibration of this equipment I had the unvaluable assistance of Dr. E. Katz. I feel much indebted towards him for his courteousnbsp;help and precious advices.

w 6

� the mercury bulb, being composed of lines or narrow bands, isnbsp;especially adapted to this purpose.

suitable light source is used. Intending to study the influence of X = 4360 A and of A = 5460 A, the mercury bulb was chosen because of its great intensity in these wave lengths.

In the first experiments with X = 4360 A it turned out that no negative response could be obtained. The intensity then was 22nbsp;erg/cm^/sec. Increasing the intensity up to 198 erg/cm^/sec. bynbsp;changing the diaphragms of i mm by such of 10 mm and L 3 bynbsp;a lense of shorter focus, negative reponses were obtained. Thesenbsp;changes hardly did affect the purity of the light. Still in this casenbsp;special filters were added to the equipment. To obtain:

X = 4360 A, a solution of Malachite green as a liquid filter was used; temperature of the thermostat was 46,3� C., andnbsp;X = 5460 A, an alcoholic solution of Victoria blue B; temperaturenbsp;of the thermostat 25,6� C.

The thickness of the filter was 5 cm. The filter solutions were prepared according to data from �The International Critical Tables�nbsp;VII (1930).

The H.P. 300 was fed by the A.C. net of the town, the tension of which is not constant. In measuring the radiation intensity, itnbsp;appeared that the fluctuations of the tension were rather small, andnbsp;did not affect radiation for more than 5 %. This is, however, enoughnbsp;to affect the results if the exposure times are short, for instancenbsp;i/io sec. For each extraction about 9 dozens of entire tips or 18nbsp;dozens of halves were needed. By exposing each dozen of plantsnbsp;apart the effect of these fluctuations greatly will neutralize eachnbsp;other. Further each point in the graph is the mean value of 8 or morenbsp;extractions. Finally, the variability of the auxin content as foundnbsp;with the extraction method (see dark controls) is so great, that slightnbsp;differences in the amounts of applied light energy cannot be of greatnbsp;importance. Therefore no further attemps were made to reduce thisnbsp;source of error.

The light intensity was measured with a thermopile after Moll, constructed by Kipp amp; Zoon, Delft, or with a barrier-layer cell fromnbsp;Tungsram.

The thermopile was chosen because of its non-selectivity in the different regions of the spectrum, but is not very sensitive. Thenbsp;photo-electric ceil on the other hand is much more sensitive, butnbsp;it has a selective spectral sensitivity. So the photoelectric cellnbsp;had to be calibrated with the thermopile.

In the beginning in the filter arrangement diaphragms of i mm were used. The obtained monochromatic green A = 5460 A andnbsp;blue A == 4360 A was too weak to measure it with the thermopile,nbsp;therefore the photo-electric cell was used. It was necessary to recalibrate the photo-electric cell after some time.

Lateron, when using diaphragms of 10 mm, the intensity allowed the use of the thermopile.

The electric currents, delivered by these instruments were estimated with a bifilar-galvanometer from C. Zeiss, Jena ^). Its highest sensitivity amounts to 7,5 x lo-^ Amp/scale unit. This means thatnbsp;with the photo-electric cell one scale unit corresponds to 0,216nbsp;erg/cm^/sec. at A = 4360 A and with the thermopile to 33 erg/cm^/sec.nbsp;at the same wave-length. This accuracy matches with my purpose.

In the beginning the prescription for auxin extraction, given by VAN Raalte (1937) was followed. As a matter of precaution allnbsp;manipulations for the extracting and evaporation of the ether werenbsp;done in a dark room in the same orange light as used in the air conditioned rooms. Immediately before the extraction the ether wasnbsp;freed from peroxides by redistilling it over CaO and FeS04 (ethernbsp;400 cm�, FeS04 10 gr, CaO i gr, H^O 40 cm�).

The coleoptile tips were thoroughly ground with washed quartz sand under ether and a few drops of a 0,1 n H2SO4 solution. Thennbsp;the ether was decanted and the residue washed twice more with ether.nbsp;After this, the extract was shaken with slightly accidified distillednbsp;water to remove the acid. The ether fraction subsequently wasnbsp;evaporated to a volume of 0,5 cm� and brought into a small test tubenbsp;with 0,1 cm� of a buffer solution of pH=4,5 and an agar slice. Thenbsp;rest of the ether was evaporated on a water bath by means of an airnbsp;current.

Several authors (Thimann 1934, van Overbeek 1936, Went amp; Thimann 1937) report an inactivation of auxin during the extractionnbsp;by enzymatic processes; for that reason they are also afraid ofnbsp;peroxides in the ether.

Following the prescription mentioned above, it was first tried to find out how many tips, or halves of tips were needed to obtainnbsp;measurable curvatures in the test. The results proved to be verynbsp;uncertain and variable. Four different extracts from 24 coleoptilenbsp;tips, made on the same day, gave the results of table 5.

^ I have to thank Dr. E. Nuernbergk for leaving this instrument at my disposal and Mr. J. G. Hagedoorn for calibrating of the thermopile.

Sometimes the same variability was found when diluting the extract. Three different extracts of 84 coleoptile tips were tested innbsp;3 different concentrations. Concentration i : i means that one agarnbsp;slice, divided into 12 blocks and tested on 12 plants, contains thenbsp;extract of 12 tips; concentration 5 : i contains 5 times as much,nbsp;that is the extract of 60 tips etc. The results are represented in table 6;nbsp;each experiment is done on a different day and consists of threenbsp;parallels (a, b and c).

In the mean values, however, there is a certain proportionality. It can easily be understood that the results obtained with split coleop-tiles also were uncertain.

Since the halves of the not illuminated tips ought to have equal auxin contents, we will indicate them arbitrarily as F(ront) andnbsp;B(ack). In each experiment 28 dozens of tips were extracted; table 7nbsp;gives the results.

Since in spite of all fluctuations in the individual experiments, the means match reasonably well and in most cases the differentnbsp;concentrations show a fair proportionality, the source of the errornbsp;had to'be found in the operations preceding the final evaporationnbsp;of the ether. It turned out, that the principal source of the variabilitynbsp;lies in grinding the tips; here the most serious destruction of auxinnbsp;was found. The auxin in the ether solution was relatively stable.

In many cases the amount of auxin, extracted from the coleoptiles, was extremely low too. Therefore a number of modifications of thenbsp;extraction method, described in literature, were tested. The sulphuricnbsp;acid was replaced by hydrochloric and by acetic acid; both gavenbsp;worse results. Also the use of chloroform instead of ether, as practisednbsp;by Thimann (1934) and Boysen Jensen (1936), with hydrochloricnbsp;and acetic acid failed to improve the yield.

Alcohol 96 % and cold or hot water proved to be unsuitable media for extraction. The destruction of the auxin, caused by enzymaticnbsp;processes, perhaps could be prevented by boiling the living tips;nbsp;therefore hot water was used. (Table 8A).

Since the water, with which the sulphuric acid was washed away, contained some ether, which was therefore lost together with eventualnbsp;auxin dissolved in it, it was tried to wash with a saturated solutionnbsp;of ammonium sulphate, but this meant no improvement either.

When VAN Overbeek (1938) reported that the acid could be omitted in the extraction of Avena, I had arrived at the same conclusionnbsp;myself. Also the grinding of the tissue proved to be superfluousnbsp;with coleoptile tips (table 8B).

With regard to indications in literature, it was tested whether auxin is inactivated by oxidation during the evaporation of the ether.nbsp;To that purpose the evaporation was done on a hot waterbath by

means of a nitrogen current. The yield of auxin was only a little increased but it is a great advantage, that the agar slice with the auxinnbsp;preparation can be kept much longer in a nitrogen atmosphere thannbsp;in the air. The activity is preserved for at least 48 hours, which hasnbsp;a special advantage, when the test plants have been spoiled for somenbsp;reason.

The possibility of enzymatic oxidation was considered too. To eliminate the eventually responsible enzymes, the tips were dippednbsp;into boiling water or the action of enzymes was checked with H2S.nbsp;The yield of auxin, however, did not increase. Extreme low temperature did not help either.The dry-ice extraction after DU Buy (1938)nbsp;too gave largely varying results. Since, however, the evaporation ofnbsp;the ether is much less at a lower temperature, and also to inhibitnbsp;eventual enzymatic effects, the ether further was cooled with icenbsp;during the treatment of the coleoptile tips in the dark room and thenbsp;extracts were kept in the refrigerator.

Finally it was tried to omit the buffer solution and to soak the agar slice directly with the extract during the evaporation of thenbsp;ether. To that purpose the 0,5 cm� of ether extract was brought innbsp;the small test tube with an agar slice only. It was hoped that, during

the evaporation of the ether with nitrogen, the auxin would enter the agar. This method in eight experiments gave the same, resultsnbsp;as with the buffer solution as intermedium, but it has at least thenbsp;advantage that the material can be reduced to one third (table 9).

After all these modifications the variations in the individual experiments were not yet eliminated, but a fairly reliable mean value was obtained. In table 10 five experiments are resumed, in each ofnbsp;which two parallel sets of 60 coleoptile tips were extracted and testednbsp;in the concentration 5:3.

With this method also a number of blank experiments with split tips was taken. The tips were not illuminated and therefore the twonbsp;sets of F- and B-halves should give the same results. In each experiment of table II 240 halves of tips (in the controls 120 entirenbsp;tips) have been extracted and tested on 24 plants, the concentrationnbsp;thus being 5:1.

The means of four replications in table 11 fairly match each other. Since it proved to be impossible to get reliable figures from onenbsp;experiment (mean of 24 test plants), it was decided to use statisticalnbsp;values and to take into account only the means of at least four replications of each experiment. This means a lot of more work, butnbsp;it was absolutely necessary, since the kind of processes, which destroynbsp;the auxin or are responsible for the fluctuations, could not be detected.

The procedure of the extraction practised for the experiments, described in this paper, briefly can be resumed as follows.

The halves of the coleoptile tips, obtained in the way described above, were picked up with tweezers and put into 20 cm� ether innbsp;Erlemeyer flasks of 25 cm�, cooled in a beaker with shredded ice.nbsp;For each extract normally nine dozens of entire tips or 18 dozensnbsp;of half tips were used; the volume of ether thus being relativelynbsp;very large. Immediately before using it the ether was freed fromnbsp;peroxides by redistillation. As soon as the operations in the airnbsp;conditioned dark room were finished, the bottles with ether werenbsp;placed in a light-tight box and transported into the refrigerator,nbsp;where they remained for at least five hours. Then the ether wasnbsp;decanted and evaporated to a volume of 0,5 cm�. This volume wasnbsp;brought together with an agar slice into a small type of test tube.nbsp;Then the rest of the ether was evaporated on a hot water-bath bynbsp;means of a current of nitrogen. The tightly closed tubes with thenbsp;agar slice in an atmosphere of nitrogen were left for one night in thenbsp;refrigerator and the preparations were tested the next day.

Two parallel sets of 60 coleoptile tips (not illuminated) were extracted and tested on 36 plants, the concentration thus being 5:3. This was repeatednbsp;5 times. The variation of the individual experiments were not yet eliminated,nbsp;but a fairly reliable mean value was obtained.

Recently seveial times (Fr�schel 1940, Linser 1940, Ruge 1939, Voss 1939) the presence of growth inhibiting substances in plantnbsp;extracts has been mentioned. With my own extracts never positivenbsp;curvatures were obtained in the Avena test. Since no indicationnbsp;of the interaction of such growth inhibitors was found in mynbsp;experiments, they are left out of consideration.

For the estimation of the auxin content of the agar blocks the Avena-test of Went (1928) in its later modification (van der Weynbsp;1931) was used. For the test the pure line of oats �Segrehafer� wasnbsp;used from Sval�v, kindly supplied by Prof. A. Akerman, directornbsp;of the Experiment Station of the �Svensk Utsades F�rening�. Thenbsp;coleoptiles were decapitated twice, with an interval of hours.nbsp;Immediately after the second decapitation the agar blocks werenbsp;placed upon the stumps. These agar blocks were obtained by devidingnbsp;up an agar slice of 8 X 6 X 0,9 mm into 12 equal parts. In this papernbsp;an agar slice means a sheet of agar of the given dimensions, an agarnbsp;block is V12 part of it and therefore has a volume of 3,6 mm^.

Shadowgraphs of the test plants were taken 2 hours after placing the agar blocks upon the stumps. The curvatures were measured bynbsp;means of a protractor. In the tables the auxin quantities always arenbsp;expressed by the curvatures (in degrees) of the test plants. Thesenbsp;figures are the average of 10�36 plants, the mean error being calculated from the formula:

From the, first experiments it appeared that for each extraction a large number of tips was needed to get measurable curvatures in thenbsp;test. Therefore a more sensitive test method would be desirable.nbsp;The deseeded test according to Skoog (1937) did not give reliablenbsp;results. The sensitivity one time was much higher, another timenbsp;the same as that of the standard test of WENT.Thereforethe deseedednbsp;test was not used.

Funke (1939) propagated a new test. The coleoptiles were cut off near above the mesocotyl and then prepared in the usual way. Thisnbsp;method did not give an important increase in the sensitivity. Therefore at last the old standard test was maintained.

Failing to find a better test method, I tried to increase the auxin content of the tips according to Gorter amp; Funke (1937). Theynbsp;found that Raphanus hypocotyls contained more auxin in a drynbsp;atmosphere (50 %) than when cultivated at a higher air humiditynbsp;(too %). Some experiments in this direction were taken. One partnbsp;of the plants was cultivated as normally in the air-conditioned darknbsp;room but at an air humidity of 50 %, the other part in another roomnbsp;at an air-humidity of 96 %.

It appeared that there is some variation in auxin content, but the mean of these experiments almost yields the same result. The airnbsp;humidity therefore practically has no influence on the auxin contentnbsp;of the tip of the Avena coleoptile.

It is a well known fact that the presence of electrolytes affects the auxin curvature. K�gl amp; Haagen Smit (1931) already used a solution of 160 mg/1 KCl 0,2 cm^/l of glacial acetic acid to dilute theirnbsp;hetero-auxin standards. I found that the pH of the agar blocks toonbsp;is very important. As already is shown in table 3 and 4, referringnbsp;to experiments on inactivation, and in table 12, the concentrationnbsp;of indole-3-acetic acid I in 10'^ at pH = 4 induces a curvature ofnbsp;about 13�, while the same concentration at pH = 8 did not yield anbsp;curvature at all.

Therefore the most suitable pH of the agar blocks was tested. These experiments were carried out with indole-3-acetic acid andnbsp;with auxin-a, extracted from oats. Plotting the auxin curvature

324

against pH an optimum curve is obtained with a peak at pH = 4. So the agar slices, used in the test, were soaked in a buffer of pH= 4nbsp;during one night. The next day they were brought in the test tubesnbsp;as described above.

CHAPTER IV

PRELIMINARY EXPERIMENTS.

� I. The first orientation.

Since I intended to study the eventual part of photo-inactivation of auxin in phototropism, it had first to be determined which othernbsp;factors participate in this phenomenon and to what extent.

In view of the Cholodny-Went theory the so called redistribution, that is a lateral shifting of the auxin towards the shade side, shouldnbsp;be expected to be the most important factor. The evidence, givennbsp;by Went (1928) and van Overbeek (1933) for this redistribution,nbsp;will be discussed first. It may be mentioned ahead, that in manynbsp;cases it will be very difficult or even impossible to discriminatenbsp;between a lateral transport of auxin and an increase of the synthesisnbsp;of auxin at the shade side of the coleoptile. The latter possibihtynbsp;will be discussed later, but should be kept in mind and criticallynbsp;judged whenever accounting for the results of experiments on redistribution of auxin.

Went first exposed his plants vertically from the top side and found an inactivation as read from his tables XX and XXI, reprintednbsp;in my table 13. He studied also the delivery of auxin after unilateralnbsp;illumination in experiments, where the auxin from the light- andnbsp;the shade side was trapped separately, by placing tips over a razornbsp;blade after exposure to light. He states that the amount of growthnbsp;substance obtained from the shade side of illuminated coleoptilesnbsp;is higher than it would be of halves of the dark controls. In his tablenbsp;XXH (my table 14) he only gives relative figures, but an inactivationnbsp;of 16 % is detectable. In his table XXHI (my table 15) he does notnbsp;give dark controls, but it is the most conclusive one in favour of anbsp;transversal transport, especially the figures of the second interval.nbsp;I decided to repeat experiments of this type.

The most direct evidence, however, for a transversal transport has been given by van Overbeek (1933) in experiments with sections ofnbsp;hypocotyls of Raphanus, supplied with agar blocks containing growthnbsp;substance and unilaterally illuminated. From his tables XII, XIIInbsp;and XIV (my tables 16, 17 and 18) it can also be concluded that in

325

TABLE 17 (Van Overbeek�s table XIII, 19333 modified). Curvatures in degrees of the test plants.

TABLE J18 (Van Overbeek�s table XIV, 1933, modified). Curvatures in degrees of the test plants.

the light a part of the growth substance has been inactivated. In his table XII, where no auxin was supplied but the auxin from thenbsp;cotyledon-petioles was collected, this inactivation is very pronounced.nbsp;At that time a photo-inactivation of auxin in vitro had not yet beennbsp;shown and so its possibility too was mistaken (du Buy 1934).

Since in Went�s table XXIII no dark control is recorded as a standard, I have repeated experiments on this subject, but in anbsp;distinctly different way. Immediately after illumination with 500nbsp;M.C.S., the coleoptile tips were cut off and split; the halves, thosenbsp;of the light- and of the shade side apart, were placed on an agar slice.

Table 19 gives the means of 5 experiments; sets of 36 half tips, 3 mm long, were placed on one agar slice each. One and two hoursnbsp;after illumination the same sets of half tips were transferred to anbsp;new agar slice.

A redistribution in my experiments was prevented by splitting the tips directly after exposure, which fact explains the differencenbsp;between Went�s figures and mine for the later periods. In my

Delivery of auxin after illumination with 500 M.C.S. (diffusion method). Immediately after exposure the tips are split and put on agar.nbsp;Curvatures in degrees of the test plants.

experiment a long lasting decrease of the auxin delivery both by the light- and by the shade halves is visible. Another experiment, however, in which the coleoptile tips were cut and split partly immediately after illumination and partly resp. one and two hours afterwards,nbsp;pleads in favour of a transversal transport. In this experiment thenbsp;split tips were directly placed unilaterally on the decapitated testnbsp;plants (no diffusion into agar, table 20).

Although it is not possible in the last experiment to discriminate between transversal transport and changed synthesis of auxin in thenbsp;split coleoptile tip, it seems well established that redistributionnbsp;actually plays a certain part in the phototropic response after illumination with 500 M.C.S.

Wilden (1939) yet applied another method. She placed the entire illuminated tip on the test plants and found 17 : 83 as the comparativenbsp;concentrations of auxin of the light- and the shade side after 130�140nbsp;minutes (first phototropic curvature). Also in these experimentsnbsp;there were no dark controls as a standard.

Now it has been pointed out that no definite conclusion can be drawn from experiments by means of the diffusion method, via agar

Illumination of the test plants with 500�960.000 M.C.S. just before putting on the auxin agar. The arrows indicate the direction of the light beam.nbsp;Curvatures in degrees of the test plants.

Other possible factors, involved in phototropism, are a change of the reactivity of the illuminated tissue or a change in the rate ofnbsp;transport of auxin. In order to investigate whether these factors,nbsp;which can hardly be separated from each other, did play a part innbsp;my experiments, the decapitated test plants were illuminated withnbsp;500 M.C.S. before the application of the auxin agar, in the waynbsp;indicated in the head of table 21 by means of arrows. This experimentnbsp;further was modified as indicated in table 22. When the light energynbsp;was raised from 500 M.C.S. to 960.000 M.C.S. no curvature couldnbsp;be obtained (table 22), neither with auxin agar nor with blank agarnbsp;(as a control). From these data we may conclude, that after an

illumination with 500 M.C.S. no essential change of the reactivity of the tissue could be detected. Du Buy (1933) reported the samenbsp;results.

The only factor therefore, that can be expected to play a part in my experiments by means of the auxin extraction method is, besidesnbsp;an eventual photo-inactivation, the lateral shifting of the auxinnbsp;eventually attended with a change in its synthesis.

� 2. The auxin cont ent of coleoptile tips after illumination with 500 M.C.S. (390 ergs/cm^), unfiltered mercury light.

In this paragraph the auxin content of the tips of Avena coleoptiles after radiation with white mercury light will be reported. Sincenbsp;about the maximum of the first curvature is obtained with a lightnbsp;quantity of about 500 M.C.S., this amount of light energy has beennbsp;applied for the first orientation. In all experiments, reported in thisnbsp;paragraph, the plants were irradiated with 500 M.C.S. (= 390nbsp;ergs/cm^ for the mercury bulb). Each experiment is an extractionnbsp;either immediately after illumination, or after 15 or 30 minutes,nbsp;I, 2, 3, 4, 5 or 6 hours after the illumination. It could not be avoidednbsp;that decapitation, splitting of the coleoptile tips and putting thenbsp;halves into the ether took about 5 minutes. In this way at 9 differentnbsp;moments after illumination determinations of the auxin content ofnbsp;the previous light- (L) and shade-side (D) were made. As a controlnbsp;and comparison in each experiment an equal number of plants, butnbsp;not illuminated, were extracted; the curvature obtained with thenbsp;extract of their split tips is used as a standard (lOO %); in eachnbsp;experiment the values of the halves of the illuminated tips are calculated as per cent of those of the �dark� split tips. Further in eachnbsp;experiment control sets of test plants were treated with mdole-3-aceticnbsp;acid in different concentrations and finally at least one set of coleoptiles was used in each experiment to control the phototropic curvature.nbsp;Several of the latter sets have been recorded photographically andnbsp;the average course of the phototropic curvature is represented innbsp;fig. 4. These plants were not rotating on a clinostat; especially in thenbsp;later hours the phototropic response will have been decreased bynbsp;geotropic interaction; according to Arisz (1915), on the clinostat thenbsp;phototropic curvature would have increased for many hours. Fornbsp;each experiment (on one day) 50 racks with 12 coleoptiles each werenbsp;needed. It appeared impossible to clinostat such a large number ofnbsp;water-grown plants, therefore never a clinostat was used.

The results of the extraction experiments have been summarized in table 23 and fig. 5.

330

4. The average course of the phototropic curvature after illumination with 500 M.C.S. (390 ergs/cm^), unfiltered mercury light.

The auxin content of illuminated coleoptile tips (3 mm) at different times after exposure to 500 M.C.S. white light (390 ergs/cm^), exposure timenbsp;= 1 second.

As a control each day the phototropic curvature was determined too (2 hours after exposure).

The most striking result is that, immediately after the illumination (that is after about 5 minutes), the auxin content proves to be decreasednbsp;for about 30 %, as well at the light as at the shade side. There isnbsp;no doubt that this decrease must be due to a photo-inactivation ofnbsp;auxin, that is of auxin-a-lactone. It is evident, however, that thisnbsp;inactivation, occurring equally strong at both sides of the coleoptilenbsp;tip with a light quantity of 500 M.C.S. cannot be related with anynbsp;phototropic response, since it cannot induce a different growth ratenbsp;at both sides. On the other hand this sudden decrease of the auxinnbsp;content may be responsible for the so called �long� light-growth-reaction after all-round illumination. According to the ideas ofnbsp;Went (1928), it seems demonstrated once more that phototropism isnbsp;not merely depending on a light-growthreaction. Cholodny (1931)nbsp;too has endorsed this idea.

A difference of the auxin content of the light- and shade side sets in later and reaches its maximum not until one or two hoursnbsp;have passed after the illumination. At that time the auxin contentnbsp;at the shade side begins to surpass that of the �dark� controls.

The long lasting decrease of the auxin content of the light side, coinciding with a gradual increase at the shade side, speaks in favournbsp;of a lateral transport. After two hours, however, the total amount of

auxin in the entire coleoptile tip begins to surpass the auxin content immediately after the illumination. This can only mean that alsonbsp;the synthesis of auxin is changed by or after the illumination. Tonbsp;this point special attention will be paid in the next chapters. Thenbsp;values found after 3 hours are even higher than those of the �dark�nbsp;plants, but it is questionable whether this difference is consistent; thenbsp;same holds true for the figures found after four hours, where thenbsp;auxin content of the light-side is somewhat higher than that of thenbsp;shade-side. After 3 hours the L and D curves show slight fluctuations;nbsp;these, however, are probable due to an interaction of gravity (phototropic curvature = 22�) and will be left out of consideration.

Koningsberger amp; Verkaaik (1938) explained the photo tropic base response and also the �short� light-growthreactions, inducednbsp;by illumination of the base by a photo-inactivation of auxin-a-lactone. This view is supported by my own observations on decapitated coleoptiles that had regenerated their �physiological tip� andnbsp;were illuminated with about 500 M.C.S. In these coleoptiles thenbsp;phototropic curvatures were only weak and never did surpass 5�nbsp;(see table 24, fig. 6).

According to Koch (1934) a lateral transport seems hardly possible in the hollow base of the cylindrical coleoptile and chiefly must benbsp;governed in the extreme solid tip. This too pleads in favour of thenbsp;conclusion of Koningsberger amp; Verkaaik. In this regard one maynbsp;speak of a special phototropic function of the tip of Avena (seenbsp;Paal, 1919; and Boysen Jensen amp; Niels Nielsen, 1925).

I found another strong indication in this direction. It is known that when the primary leaf has peared through the coleoptile nonbsp;longer a phototropic response can be obtained. By means of thenbsp;extraction method however, it appeared to me that in the first timenbsp;the auxin content of the tip has not yet decreased. We may thereforenbsp;conclude that no phototropic curvature occurs while no lateralnbsp;shifting of the auxin is possible. So the tip is not only the auxinnbsp;producer.

Koningsberger amp; Verkaaik (1938 p. 12) concluded: �The base response in decapitated coleoptile tips is to be ascribed only to thenbsp;partial photo-inactivation of the auxin-a; no evidence of a �redistribution� (lateral transport) of the growth substance in the base of thenbsp;coleoptile, as postulated by the Cholodny-Went theory, could benbsp;obtained�.

At first sight the reader will feel an apparent controversy between my experiments on the auxin content of illuminated tips and those

7 nbsp;nbsp;nbsp;2^,3nbsp;nbsp;nbsp;nbsp;4nbsp;nbsp;nbsp;nbsp;5nbsp;nbsp;nbsp;nbsp;6

Fig. 6. The phototropic curvature of decapitated coleoptiles in relation to different regeneration times after an illumination with 500 M.C.S.

334

of Koningsberger amp; Verkaaik. In fact, however, there is no discrepancy, but our investigations are complementary to each other.

Koningsberger amp; Verkaaik studied the phototropic base response, caused by a long lasting illumination of lOo M.C.S., that is an amount of light energy far within the range of the second positivenbsp;phototropic curvature, when the entire coleoptiles are illuminated.

I examined the auxin content of the coleoptile tips at different intervals after illumination with a light quantity causing about thenbsp;maximum of the first positive phototropic curvature.

In both cases a certain photo-inactivation of auxin has been found, which can be responsible for the light-growthreactions. In thenbsp;experiments of Koningsberger amp; Verkaaik auxin present in thenbsp;base was inactivated, which may correspond with the �short� light-growthreaction of the base. In my case 30 % of the auxin in the tipnbsp;was inactivated, which certainly also would have happened after annbsp;all-round illumination. This fact probably may be responsible fornbsp;the �long� light-growthreaction, since it must take time for thenbsp;auxin in the tip to be transported to the region of actual growthnbsp;(5�7 mm below the tip).

Phototropism, however, in both cases should be interpreted in quite a different way. According to Koningsberger amp; Verkaaik,nbsp;the base response can be explained entirely by a photo-inactivationnbsp;of auxin. It had to be investigated whether the same holds true fornbsp;the second positive phototropic curvature of the intact coleoptile too.

As for the first positive curvature (my own experiments) this explanation is impossible since with 500 M.C.S. the degree of inactivation at L- and D-side is equal. We have seen, that the firstnbsp;experiments strongly are in favour of a redistribution of auxin. Butnbsp;further there are indications that also the synthesis of auxin isnbsp;changed by radiation. If this would be confirmed, it certainly wouldnbsp;have to be accounted for in the light-growthreactions too. In thisnbsp;respect Wents�s fig. 3, 4 and 5 (1925) are very interesting. Afternbsp;illumination with 500 M.C.S. first a decrease of the growth rate wasnbsp;recorded, there upon an increase so that the growth rate grew evennbsp;larger than that in the dark, finally followed by a decrease till thenbsp;original value had been reached. This is quite in keeping with thenbsp;auxin content curve of the entire coleoptile tip in my fig. 5 (p. 331).

The interaction between �redistribution� and a possible change in synthesis of auxin still is obscuring our problem. I hoped that itnbsp;would be possible to disentangle these two factors by repeating thenbsp;experiments with different light quantities, not only with suchnbsp;causing first positive curvatures, but also with those in the regionnbsp;of negative and second positive curvatures. It was thought to be

335

possible that light quantities, smaller or greater than 500 M.C.S., would have a different effect on the photo-inactivation and wouldnbsp;also affect the redistribution and an eventual auxin synthesis in anbsp;different way.

EXPERIMENTS WITH MONOCHROMATIC LIGHT.

� I. Introduction.

Many indications are found in literature that the different wave lengths have a different effect in phototropism and on the growthnbsp;processes. Blaauw (1909) a.o. have analyzed the long known factnbsp;(M�ller, 1872) that the phototropic sensitivity is not equal for thenbsp;different wave lengths. Koningsberger (1922) showed that the wavenbsp;lengths lt; A = 4800 A cause a long lasting reaction with a smallnbsp;energy quantity (i�5 ergs/cm^), wave lengths gt; /I = 4800 A causenbsp;short lasting reactions, for which much larger light quantities arenbsp;needed. It seems possible that the difference in activity of thenbsp;different wave lengths is not only quantitative but also qualitative.nbsp;With white light in that case phototropism and light-growthreactionsnbsp;would be complicated by the quality of the light and the simplestnbsp;reactions would occur on monochromatic illumination. For thatnbsp;reason I used monochromatic light, since this seemed to offer anbsp;possibility to discriminate between photo-inactivation, transversalnbsp;transport and changed synthesis of auxin.

The monochromatic light, used in these experiments was obtained as described in Chapter III � 3.

The auxin from the illuminated coleoptile tips was extracted by means of the described method and estimated by means of thenbsp;Avena test as in the preceding experiments. Each figure in the tablesnbsp;represents the mean curvature of 12�24 testplants on the same day.nbsp;From these values the means of parallel series (different days) werenbsp;calculated and the auxin content of the exposed tips was expressednbsp;in percent of the dark values. They were plotted in a curve, auxinnbsp;percent against the time elapsed after exposure to light. Since eachnbsp;experiment takes a lot of time a limited number of curves could benbsp;obtained.

1st. X = 4360 A; 330 ergs/cm^; this quantity induced about a maximal first positive curvature of � in my case � 15�.

The light quantity, used in the experiments with white light (fig. 8) induces a maximal response too though thenbsp;absolute light quantities slightly differ. The curves of thenbsp;auxin content proved to differ much too.

2nd. 7. = 4360 A; 3.000 ergs/cm^; this quantity induces a first negative curvature. When the intensity per time unit isnbsp;large enough, negative curvatures with monochromaticnbsp;light were obtained.

3rd. / = 5460 A; 26.400 ergs/cm^; this quantity was the smallest one which with this wave length induced a positive curvature of 15�.

It was planned to compare the fate of auxin after an exposure with the light quantities of 7. = 4360 A and I = 5460 A which inducednbsp;equal curvatures. To obtain this quantity of energy with / = 5460 Anbsp;an exposure of 20 minutes proved to be necessary.lt is possible thatnbsp;this long lasting exposure complicates the result. With the highestnbsp;intensity used a negative response could not be obtained withnbsp;A = 5460 A.

This energy quantity induces the maximal response in the region of the first positive phototropic curvature. In this case one maynbsp;expect a higher auxin content in the relatively fastergrowing D-side.nbsp;This is shown indeed by the curves.

As after the exposure to white light (fig. 5, p. 331) here too the auxin content in the L- as well as in the D-side decreases withnbsp;30�40 %. The auxin content of the L-side continues to decreasenbsp;during the first hour after the exposure, then it increases slowlynbsp;again. In the D-side the auxin content starts to increase during thenbsp;first hour, then it gradually falls off until 3 hours after the exposurenbsp;the contents of L- and D-side are about equal, namely 75 % of thatnbsp;of non-radiated controls.

After the inactivation the auxin content of the L- side only slowly increases; after 3 hours this increase is not more than � 10 % (ofnbsp;the content of the controls).

So the bending, induced by 330 ergs/cm^, A = 4360 A, probably must chiefly result from a transversal transport of auxin from thenbsp;L- to the D-side in the (solid) coleoptile tip. The increase of thenbsp;auxin content, which must be due to some change in the rate ofnbsp;auxin synthesis, is only slight, as the auxin content of the entire

337

exposed tip indicates (broken line in the figure; these values are obtained by halving the sum of L- and D-values).

The decrease of the auxin content of the total coleoptile, undoubtedly due to a photo-inactivation, can entirely account for the long lasting light-growthreaction (retardation) as registered by Koningsberger (1922) for the same spectral region (4200�4400 A andnbsp;4400�4600 A) for much smaller light quantities (2 ergs/cm^).

A comparison of fig. 5, obtained with an exposure to white light and fig. 7 teaches that the synthesis of auxin is not or almost notnbsp;increased until 2 hours after the exposure. Then in the white lightnbsp;curve a consistent increase is shown, but equal in both sides of thenbsp;coleoptile. We did not find this large increase in fig. 7, here at mostnbsp;a slight increase is visible. Three hours after exposure the auxinnbsp;content of the controls is not yet attained.

3rd .this inactivation can account for the long lasting growth retardation after ail-round illumination (Koningsberger 1922),nbsp;4th. a probable transversal transport during the first hour after thenbsp;exposure,

This quantity of energy causes a negative bending of the coleoptile. It is in the beginning of the range of the first negative phototropicnbsp;curvature. According to the expectation the concave L-side of thenbsp;negatively curved coleoptile contains most of the auxin.

In fig. 8 it is shown that no inactivation could be stated. On the contrary, in fact the determinations immediately (5 min.) afternbsp;exposure turned out on L = iio %, D = 105 % of the controls.nbsp;The increase of these values, being small and perhaps not real, isnbsp;neglected when drawing the curves. The increase of the auxin

339

content in the D-side shows a linear course, in contrast to that of the L-side, where the increase is the largest during the first hour afternbsp;the exposure, then it follows a same linear course as in the D-side.

The course of the mean auxin content of the entire tip becomes linear after the first hour. Up to the end of the fourth hour thenbsp;increase proceeds steadily and is still important. Experiments duringnbsp;a longer time fail (owing to gravity), so we cannot say whether andnbsp;when the auxin content of the tip grows normal again. The afternbsp;effect of 3.000 ergs therefore is much greater than that of 330 ergsnbsp;(of the same wave length).

The degree of photo-inactivation of the auxin-a iizr? auxin-a-lactone system certainly will depend upon the light quantity, but it is still unknown how. It seems, however, impossible from a photochemical point of view, that it would not occur within a certain rangenbsp;of light energy. We therefore must assume, that also here a part ofnbsp;the auxin is inactivated, but that this inactivation immediately isnbsp;overbalanced by an increase of the auxin content. This increasenbsp;cannot be but a synthesis either a production or an activation or anbsp;liberation of auxin. The nature of this phenomenon is still completelynbsp;obscure. It is, however, consistent and will be discussed later (p. 347).

3rd. in stead of it an almost immediately starting, long lasting and steady increase of the auxin content is found, for at leastnbsp;during 4 hours after the exposure.

4th. under the prevailing conditions nothing can be concluded on an eventual lateral transport of auxin.

This light quantity administered in 20 minutes induces a strong positive curvature. In contrast to the previous experiments no shortnbsp;exposure times could be applied to obtain the large light quantity;nbsp;the time of 20 minutes approaches more or less that of continuousnbsp;illumination.

The reaction time of the phototropic curvature and the time of cutting the tips is counted from the end of the exposure time andnbsp;therefore the abscissa of fig. 9 actually should be shifted for � 20nbsp;minutes to the left for a comparison with the other figures.

Firstly fig. 9 shows that no inactivation can be stated. An increase of the auxin content is found, it is strongest in the D-side and mostnbsp;prominent during the first hour after exposure. The maximum isnbsp;attained after about two hours, then a rather steep decrease sets innbsp;after about 3 hours and after about 4 hours the dark value is attainednbsp;again. Longer lasting experiments being impracticable, we cannotnbsp;say whether the dark value further is maintained or not.

A typical difference with the preceding experiments is, that the divergency between the curves of the L- and D-side lasts for suchnbsp;a long time. In the other cases after i�2 hours the curves eithernbsp;converged or ran parallel to each other. Here the increase of thenbsp;auxin content of the D-side remains for about three hours ahead asnbsp;compared to that of the L-side.

4th. an increase of the auxin content of L- and D-side during about 2�3 hours, followed by a rather steep decrease to the original

At the end of the preceding chapter we expressed the hope that we would be enabled to discriminate between the three phenomenanbsp;which come into play in the phototropic response by using monochromatic light. These three phenomena were: a) photo-inactivationnbsp;of auxin, b) a transversal transport of auxin and c) a changednbsp;synthesis of auxin.

Illumination with 330 ergs/cm^, X -- 4360 A, causes a photo-inactivatioi^ of auxin of 30�40 % at both sides and probably also a transversal transport. The auxin content later only slightly increases; this must be due to an auxin synthesis, increased in somenbsp;way. When increasing the light quantity up to 3.000 ergs/cm^,nbsp;1 == 4360 A, however, no photo-inactivation of auxin can be found,nbsp;but only a distinct increase in the auxin content in both sides, in thenbsp;L-side, however, to a much larger extent. No conclusions can benbsp;drawn on a lateral transport of auxin.

With illumination with 26.400 ergs/cm^, X = 5460 A, no photoinactivation of auxin could be detected either. Also here a pronounced, long lasting increase of auxin content was found, but that in the D-side is more pronounced. A conclusion on lateral transportnbsp;of auxin cannot be drawn.

From these series it appears that photo-inactivation of auxin cannot always be stated. In the next chapter we will investigate bynbsp;which light quantities photo-inactivation of auxin can be shownnbsp;and by which not. There this process will be treated more in detail.

One only can conclude upon a lateral transport of auxin when a decrease of the auxin content of one side runs parallel with annbsp;increase of that in the other side. Such a parallelism only was foundnbsp;for the auxin curves of L- and D-side in the experiments with 500nbsp;M.C.S. (white light) and with 330 ergs/cm^, X = 4360 A. In thesenbsp;cases it seems justified to argue in favour of a lateral transport. Alsonbsp;here, however, such a transport is attended with, or followed by anbsp;change in the auxin content, which only can be due to a change innbsp;�synthesis� of auxin (in the broadest sense). It is, therefore, anbsp;question of personal feeling whether one wants to explain the opposite

parallel changes of the auxin content of L- and D-sides by a lateral transport or by a different change of the auxin synthesis at bothnbsp;sides. The same holds true for the other curves with 3.000 ergs/cm^,nbsp;1 = 4360 A (negative curvature) and with 26.400 ergs/cm^,

X = 5460 A, (positive curvature). One may argue the differences between the L- and D-side by a lateral transport attended with anbsp;homogenously increased auxin synthesis, or by an increased auxinnbsp;synthesis different in both sides of the coleoptile. This question willnbsp;be discussed more thoroughly at the end of this paper.

In all four series of experiments an increase of the auxin content could be shown, which must be due to some increased synthesisnbsp;of auxin.

In the experiments with small light quantities i.c. 500 M.C.S. (white light) and 330 ergs/cm^, X = 4360 A, this increased synthesisnbsp;was only slight and preceded by a marked photo-inactivation ofnbsp;auxin. With 330 ergs/cm^, X = 4360 A, the inactivated amount ofnbsp;auxin was 30�40 % and thus the main auxin content of the tipnbsp;decreased to � 65 % of the dark value. Three hours later it hadnbsp;slowly increased up to � 75 %. About the same was found for 500nbsp;M.C.S. (white light). During the second hour the increase amounts

the dark value. Then, however, a markedly quick increase occurs so that one hour later, 3 hours after the exposure, the auxin level ofnbsp;the dark controls has been surpassed. This last feature fails in thenbsp;curve of 330 ergs/cm^, X = 4360 A. A longer continuation of thesenbsp;experiments is impossible since geotropism interacts with the photo-tropism and obscures the results. The later fluctuations found withnbsp;500 M.C.S. therefore will not be valued.

Far more prominent is the increased auxin content in the experiments with 3.000 ergs/cm^, X = 4360 A, and with 26.400 ergs/cm^, X = 5460 A. With 3.000 ergs/cm^, X = 4360 A (negative curvature)nbsp;the increase immediately appears and it lasts for 4 hours at least. Itnbsp;is most prominent in the L-side. Due to the long exposure timenbsp;things are a little different in the experiments with 26.400 ergs/cm^,nbsp;X == 5460 A. Here after about half an hour (since the radiation began)nbsp;an increased auxin content is found, being strongest at the D-side.nbsp;It lasts not so long; after 4 hours the surplus production practicallynbsp;has disappeared from the tip.

We thus have to conclude that light induces either immediately or later an increase of the auxin synthesis in the coleoptile tip. Thenbsp;nature of this synthesis still being completely obscure, we cannot

discriminate between a real synthesis or a photo-activation or a liberation of auxin from a bound state. Also this question, which fornbsp;the present is a completely unknown item of our problem will benbsp;discussed at the end of this paper.

THE PHOTOTROPIC CURVATURE AS A FUNCTION OF THE LIGHT ENERGY (A = 4360 A)

� I. Introduction.

In the preceding chapter it turned out, that photo-inactivation of auxin cannot always be stated. It had to be investigated how thisnbsp;phenomenon is related with the amount of light energy. For it cannotnbsp;be understood why by a certain amount of light energy auxin isnbsp;inactivated, and why by increasing this energy no inactivation isnbsp;detectable.

In the beginning, working with monochromatic blue light, A == 4360 A, no negative phototropic curvature could be obtained.nbsp;Fig. 10 shows the curve (broken line) when the phototropic reactionnbsp;is plotted against the log. of the light energy. The light intensitynbsp;was 22 ergs/cm^/sec. The reason why no negative response could benbsp;obtained was the rather low intensity. This turned out when, innbsp;order to get more suitable exposure times, the intensity was raisednbsp;up to 198 ergs/cm^sec., by making the light beam narrower and bynbsp;opening the diaphragms. Now negative curvatures could be obtainednbsp;indeed. So it seems that the curvature (see the graph) obtained withnbsp;unfiltered white light presumably does not differ from that inducednbsp;by monochromatic blue A == 4360 A. Only if the light intensity isnbsp;sufficiently high � i.c. the time of exposure short enough (cf.nbsp;Arisz 1914) � negative curvatures will appear. When using annbsp;intensity, i.c. an energy-quantity per time unit � not large enoughnbsp;to produce a negative curvature � the positive curvature does notnbsp;show the rapid decrease with increasing light quantity. There is onlynbsp;a slow decrease which is converted into an increase again withoutnbsp;entering the domain of negative curvatures or even reaching zero.nbsp;So the curve does show a flat sinking slope, but a slight one only.nbsp;It should be realised that in these cases the exposure-times amountednbsp;to 20�60 minutes and therefore were very long. For the present

344

I wil) confine myself to the short-exposure effect, since with the long one complicating time effects may be involved. Therefore I havenbsp;studied the short-exposure effect only.

The next series of experiments was carried out with monochromatic blue light A = 4360 A. with varying light quantities. The auxin extraction took place immediately after illumination. Innbsp;Chapter V I accounted for the course of the auxin content duringnbsp;several hours after an illumination with 330 ergs/cm^ and withnbsp;3.000 ergs/cm^ of the same wave length. The former quantity causesnbsp;an inactivation of the auxin for about 30�40 %; with the latter lightnbsp;quantity no inactivation could be stated. It was strange not to findnbsp;any inactivation by increasing the light energy. So it seemed worthnbsp;while to test other light quantities to see whether inactivation takesnbsp;place or not. With monochromatic blue light the smallest energynbsp;used was 35 ergs/cm�^; in this case no inactivation was detectable.nbsp;Exposure to 150 ergs/cm^ had an equal negative result. So the phototropic curvature in this region cannot be induced by a partly photoinactivation of auxin, but must be due to either a transversal transportnbsp;or an altered auxin synthesis in the tip or to both. The appliednbsp;amounts of light energy fall within the range, where the phototropicnbsp;curvature is proportional with log. light energy. When the lightnbsp;energy surmounts about 330 ergs/cm^ this proportionality disappearsnbsp;and here the first positive phototropic curvature reaches its maximum. In this region the photo-inactivation of auxin occurs, but thisnbsp;cannot have a share in the curving process, since it is practicallynbsp;equal in both sides. On the other hand the auxin content immediatelynbsp;after the application of 150 ergs/cm^, ?. = 4360 A, namely L = 85%,nbsp;D = 115 % as compared to the dark controls may be explained innbsp;favour of a lateral transport of auxin. One should remind, however,nbsp;that it is hard to understand, how such a quick lateral move of auxinnbsp;can be realized. It is true, that van der Wey (1932) estimated thenbsp;rate of the longitudinal auxin transport on 2 mm in 10�12 minutes.nbsp;There is, however, no indication that a lateral transport is based onnbsp;the same mechanism (see Clark 1938).

When the light quantity is increased further the phototropic response decreases. In this range of energy no inactivation is detectable either. I used 700, 1000 and 1400 ergs/cm^, A = 4360 A. Sonbsp;it turned out that only in a narrow region at about 330 ergs/cm^,nbsp;that is in the region of the maximum of the first positive phototropicnbsp;curvature, inactivation can be stated. Moreover this inactivation

The first negative curvature can be obtained with a light quantity of 3.000 ergs/cm^, A = 4360 A. Here also no photo-inactivation ofnbsp;auxin could be found. Finally an auxin determination was madenbsp;after an exposure to 30.000 ergs/cm^, A = 4360 A. The phototropicnbsp;curvature is about zero, here the region of the second positive curvature begins. Here again a photo-inactivation of auxin is obvious. Nonbsp;experiments were taken with still larger light quantities because thenbsp;exposure time then would be too long. The time needed for 30.000nbsp;ergs/cm^ amounted to 100 seconds for this wave length. To getnbsp;100.000 ergs/cm^ an exposure of about 5 minutes would be required.nbsp;Intending to study the effect after short exposures, to avoid eventualnbsp;complications due to a time effect, experiments with larger energynbsp;quantities were omitted, because light of greater intensity could notnbsp;be obtained. To ascertain that the photo-inactivation of auxin,nbsp;obtained with 330 ergs/cm^, A = 4360 A, was not an illusive incident,nbsp;at the end of this series a few experiments with 350 ergs/cm^ werenbsp;taken. They showed a photo-inactivation of auxin too.

To induce a phototropic curvature a certain quantity of light energy is needed. When increasing the light energy the curvaturenbsp;proves to be proportional to the log. of the energy until the curvenbsp;flattens and a maximal first positive curvature is reached (fig. 10).

In the region where the phototropic curvature is proportional to log. light quantity no photo-inactivation of auxin by the appliednbsp;doses of radiation (35 and 150 ergs/cm^) could be stated. Phototropism here must be due to a lateral transport of auxin or by anbsp;change in the auxin synthesis or by both.

It is strange that a photo-inactivation was only apparent in the region of the maximal first positive phototropic curvature (330,350nbsp;ergs/cm^). This is the more strange, since Koningsberger (1922)nbsp;recorded light-growthreactions after illumination with very muchnbsp;smaller light quantities (2 ergs/cm^). But it is consistent and it wasnbsp;regularly found in three different series of experiments, one withnbsp;white light and two with blue light, A = 4360 A. This inactivation,nbsp;however, cannot cause the phototropic curvature, since it is equalnbsp;at both sides. On the other hand it can fully account for the longnbsp;lasting light-growthreaction.

Still stranger is, that this photo-inactivation could not be found with larger light quantities (700, 1000, 1400 and 3.000 ergs/cm^).nbsp;As we pointed out on page 339 it seems impossible that the inactivation

347

would not occur in this range of light energy, that cannot be attained without passing the critical range of inactivating light quantities.nbsp;The most probable explanation is offered by the assumption thatnbsp;also the auxin synthesis is changed by these amounts of light energynbsp;so quickly that the inactivation is immediately overbalanced.

The inactivation found with still higher amounts of light energy (30.000 ergs/cm^) then only can mean that there also the lactonenbsp;fraction of the newly produced auxin-a is inactivated. That wouldnbsp;mean, that the equilibrium auxin-a ^ auxin-a-lactone estabhshesnbsp;itself within 100 seconds (exposure time). On its turn this suggestionnbsp;offers a possibility to account for the time-effect (Arisz 1914), butnbsp;we will discuss this question later.

Meanwhile the data collected on the photo-inactivation are completely enigmatic. I therefore tried to find an explanation in the following direction. Schuringa (1941) found that the equilibriumnbsp;auxin-a: auxin-a-lactone is catalyzed by H-ions and that the photoinactivation auxin-a-lactone ^^ lumi-auxin-a-lactone by ultraviolet radiation is readily achieved in an ethanolic solution atnbsp;pH == 4. It would be possible that the internal pH of the coleoptilenbsp;tip shifts towards a lower value under the influence of the light. Sonbsp;I tried to alter this internal pH artificially. Van Santen (1940)nbsp;showed that the internal pH of coleoptile sections equals that of thenbsp;medium when they have been soaked for 12�24 hours in buffernbsp;solutions. In my experiments the entire plants were soaked for 12nbsp;hours in buffer solutions of different pH. They then were exposednbsp;to light in the air and placed back again in the solution. As controlsnbsp;plants were used that had been soaked in tap water. I first comparednbsp;such water soaked coleoptiles to normal �air-grown� coleoptiles.nbsp;The phototropic curvature was smaller in the �tap water coleoptiles�.nbsp;The curvatures shown by coleoptiles from the buffer solutions pH = 4,nbsp;=6 and=8 (see table 29) were different indeed.The growth rate, however, proved to be different too; the plants in pH = 8 grew morenbsp;slowly than the others. The differences in phototropism thereforenbsp;may be considered primarily due to differences in growth rate.nbsp;These experiments therefore are not conclusive. Though they failnbsp;to elucidate the curious relation between light energy and photoinactivation of auxin, it still remains possible that the internal pH isnbsp;affected by light in such a way that the energy / inactivation relationnbsp;depends on it. I only did not succeed in giving experimental supportnbsp;to this possibility.

Another possibility is that this relation depends on specific properties of the photo-sensibilizer. According to several authors and more especially Schuringa (1941) carotinoids are the physiological sensi-

bilizers in the photo-inactivation of auxin-a-lactone. The possibility that the action of the sensibilizer could be affected by light-absorption in the medium, had not to be excluded. Skoog (1935) reportednbsp;an auxin inactivation by light in the presence of eosin. Since thisnbsp;dye is water soluble and carotinoids not, I treated auxin solutionsnbsp;with eosin and then irradiated them. I could, however, not confirmnbsp;the results of Skoog. Neither by white light, nor by monochromaticnbsp;blue light, tested at different pH�s an inactivation of auxin couldnbsp;be stated (see Chapter II � 3).

GENERAL DISCUSSION AND SUMMARY.

� I. The types of phototropic reactions; Phototonus.

From the preceding experimental data can be concluded upon a threefold part played by auxin in phototropism:

The last item only has a certain degree of probability. The present technique does not enable to discriminate with certainty between anbsp;change of the auxin synthesis and a lateral transport. Only in thosenbsp;cases, where the total auxin content of the illuminated tips is too %nbsp;of-or less than-that of the dark controls and where a decrease of thenbsp;auxin content of one side is attended with a simultaneous parallelnbsp;increase of that of the other side, a lateral transport seems evident.nbsp;When, however, the total amount of auxin in the tip increases and anbsp;change in the auxin synthesis is apparent, one cannot conclude

whether the differences between L- and D-side are due to a lateral transport or to a different synthesis or to both. For the light-growth-reactions the lateral transport can be left out of consideration.

Before considering these three processes from a theoretical point of view, I will survey the different types of phototropic responsesnbsp;studied with monochromatic light. With X = 4360 A I succeeded innbsp;inducing the first positive, the first negative and the threshold of thenbsp;second positive curvature. I stated a photo-inactivation of the extractable auxin for resp. 30 � 40 % and 20 � 25 % in the regionsnbsp;of the maximum of the first positive reaction and in that of thenbsp;threshold value of the second positive reaction. Although this inactivation, being about equal at both L- and D-side, cannot in itselfnbsp;explain the phototropic response, it certainly complicates the phototropic phenomena, known as phototonus, in this spectral region.

This follows from the fact that I could not obtain other than positive reactions with X = 5460 A; all light quantities of this wavenbsp;length yielded positive curvatures. I only studied the course of thenbsp;auxin content after a radiation with 26.400 ergs/cm^ of X ~ 5460 A.nbsp;No auxin inactivation could be stated, but the auxin synthesis provednbsp;to be increased, the auxin content of the D-side being more increasednbsp;than that of the L-side. The first extraction being started 25 minutesnbsp;after the beginning of the illumination (which lasted 20 minutes), thenbsp;possibility remains, that a photo-inactivation escaped from ournbsp;observation and had been balanced by an increased synthesis. This,nbsp;however, seems not probable. Although with this wave length I onlynbsp;studied the effect of one light quantity and I did not investigate thenbsp;curvatures due to the less frangible wave lengths of the spectrum,nbsp;it seems justified to conclude that phototropism, as far as it occursnbsp;in this spectral region, can only be positive and chiefly is due to annbsp;increased auxin synthesis; in the tip this increase being stronger innbsp;the D-side than in the L-side.

The only data available on light-growthreactions in this part of the spectrum (Koningsberger 1922) show short lasting and verynbsp;weak growth retardations with 6 ergs/cm^, X = 5300�5700 A andnbsp;8 ergs/cm^, X = 5700�6200 A; with 60 ergs/cm^, X = 6200�7000 Anbsp;a much stronger, short lasting growth retardation was found. Thesenbsp;reactions, which are typical base responses, can be left out of consideration, since my experiments exclusively deal with changes ofnbsp;the auxin content in the tip. Nowhere, however. Koningsbergernbsp;has reported an acceleration of the growth rate, as could be expectednbsp;from my own data. Perhaps his observation time (2 hours) has beennbsp;too short to this purpose.

an increased auxin synthesis, which in its turn is caused by the radiation, it seems impossible to explain the different auxin contentsnbsp;of L- and D-side by a higher auxin synthesis in the D-side, thatnbsp;received less light energy. Most probably this difference must benbsp;ascribed to a lateral transport.

The uniform phototropic behaviour of Avena against light of A = 5460 A suggests that the complicated phenomena of phototonusnbsp;must be due to the action of the short wave lengths (i.c. A = 4360 A),nbsp;which consist of photo-inactivation and increased auxin synthesis.nbsp;These two factors, eventually combined with a lateral transport,nbsp;compete and interact with each other and the result, observed in thenbsp;phototropic response, is called �phototonus�.

The strangest result of my experiments with light quantities A = 4360 A in the range of the first positive curvature is that anbsp;photo-inactivation of auxin only could be stated in the region ofnbsp;the maximal curvature (330�350 ergs/cm^). It is possible that somenbsp;inactivation occurred indeed with smaller amounts of light energy,nbsp;but that they were only too slight to be detected with our method.nbsp;On the other hand Schuringa (1941) found a practically completenbsp;inactivation of auxin-a-lactone by ultra-violet light A = 3340 A,nbsp;with 11,4 ergs/cm^, and, in the presence of j3-carotene, by violetnbsp;A = 4360 A with 6,9 ergs/cm^. It is not clear why such an inactivationnbsp;could not be found under physiological conditions. As I pointed outnbsp;on p. 346 also Koningsberger (1922) found consistent light-growth-reactions after illumination with 2 ergs/cm^, A = 4200�4400 A andnbsp;A = 4400�6400 A, which indicates that a photo-inactivation mustnbsp;have occurred. Perhaps another statement by Schuringa (1941) cannbsp;account for my curious negative result. Estimating the quantumnbsp;output of the photo-chemical inactivation of auxin-a-lactone, thisnbsp;author reasoned that one light quantum v = 9.101* (;i = 3340 A)nbsp;inactivates at least more than 3.10� molecules of auxin-a-lactone. Henbsp;suggests that each auto-inactivation of auxin originates by �germs�nbsp;of lumi-auxin-a-lactone and that these germs catalyze or sensibilizenbsp;the further inactivation. When applying this idea upon our case onenbsp;can admit that a very slight inactivation initiated by radiation, later-on automatically could increase in the dark. This hypothesis dealsnbsp;with a kind of �photo-mechanical induction� advocated in plantnbsp;physiology as early as 1878 by Julius Wiesner. Since my own auxinnbsp;determinations with low light quantities were done only immediatelynbsp;(i.c. � 5 minutes) after the illumination, it must be left to furthernbsp;investigations to check this possibility. In favour of it pleads the fact

that after ail-round illumination with light quantities within the range of the first positive curvature always long lasting light-growth-reactions have been found as retardations of the growth rate (Koningsberger, 1922; VAN Dillewiin, 1927). Further the auxinnbsp;content of the L-side, in the region of the maximum of the firstnbsp;positive curvature, continues to decrease for about one hour afternbsp;the illumination.

On the other hand the almost parallel course of the curves of the auxin content of the L- and D-side strongly suggest a lateral transport.

The main auxin content of the entire tip, however, immediately after the illumination decreased to 65 %, soon begins slightly tonbsp;increase which points in the direction of a slight change of the auxinnbsp;synthesis by the radiation. One can also explain the differences ofnbsp;the L- and D-side by different changes of the auxin synthesis.nbsp;Perhaps this question would have found a more conclusive answernbsp;if only the extreme solid tips had been extracted ''200�300 m).nbsp;From a technical point of view this would have been too difficult,nbsp;so that I always extracted tips of 3 mm.

The increase of the auxin synthesis, only slight in the range of the first positive curvature, entirely prevails in the region of the firstnbsp;negative reaction. As we discussed on p. 339 it cannot be understoodnbsp;that with these amounts �f light energy no photo-inactivation wouldnbsp;occur, so that, for the moment, the only possible explanation is,nbsp;that a decrease of the auxin content by photo-inactivation is overbalanced by an immediate and strong increase of the auxin synthesis.nbsp;The possible nature of this increase will be discussed later in � 5.nbsp;We here will mention that negative curvatures only can be obtainednbsp;� as already stated by Arisz (1914) � when the amount of lightnbsp;energy required is administered within a certain time. This timenbsp;effect most probably must be due to the light-intensity, the latternbsp;determining the slope of the curve of the change of the auxin synthesis. It is in agreement with this hypothesis that the auxin content ofnbsp;the L-side shows a steeper increase than that of the D-side. On thenbsp;other hand this difference also can be explained by a lateral transport,nbsp;in this case from the D- towards the L-side. When comparing thenbsp;curves of the auxin contents with the light-growthreactions as studiednbsp;by VAN Dillewijn (1927) we have to choose those experiments innbsp;which only the extreme tip (J mm) had been illuminated. Theynbsp;show either a growth acceleration or practically no change. Sincenbsp;van Dillewijn applied white light, the amounts of light energy

cannot be compared. In the experiments, in which the entire coleop-tile was illuminated, the light-growthreactions are complicated by strong base responses, which obscure the comparison.

We still have to mention that negative curvatures could not regulary be obtained with 3.000 ergs/cm^, A = 4360 A. On severalnbsp;days the plants don�t curve at all, they are indifferent. This variability, perhaps due to the same factors as that in the Avena-test,nbsp;can be understood from the complicated nature of the processes,nbsp;described above, involved in the negative phototropic response.

At about the threshold value of amounts of light energy, inducing a second positive curvature, I found a distinct inactivation of auxinnbsp;again. Although I did not further study the course of the auxinnbsp;content in the range of the second positive curvature, it is possiblenbsp;to predict the factors on which the auxin content of the tip mustnbsp;depend: an increase of the auxin synthesis depending upon the lightnbsp;intensity and a photo-inactivation of the auxin-a-lactonenbsp;already present, and of that newly delivered by the increased synthesis. If the increase of the auxin synthesis is so steep (highnbsp;light intensity) that the establishment of the equilibriumnbsp;auxin-a ^ auxin-a-lactone cannot keep pace with it, negativenbsp;curvatures will result. If the light intensity is lower, thenbsp;increase of the auxin synthesis will be too slow to overrun the photoinactivation of the auxin-a-lactone fraction. In this case only secondnbsp;positive curvatures will result. This seems the most probable explanation of the time effect as stated by Arisz (1914), many othernbsp;authors and myself. It remains possible that also a lateral transport isnbsp;involved in the distribution of the auxin in the second positive curvature.

Further, in all cases, also a photo-inactivation of auxin present in the sub-apical regions of the coleoptile occurs (Koningsberger amp;nbsp;Verkaaik, 1938).

The latter phenomenon accounts for the short lasting base response of the light-growthreactions, which always is a retardation of thenbsp;growth rate. The later course of the light-growthreaction depends onnbsp;the interaction of the increased synthesis in the tip and the photoinactivation of auxin as discussed above.

In all cases, where the establishment of the equilibrium auxin-a T ^ auxin-a-lactone in newly synthezised auxin can keep pacenbsp;with the rate of photo-inactivation, second positive curvatures willnbsp;occur. This always will be the case with continuous illuminations.nbsp;The reasoning, given here, matches the statement by Konings-

353

BERGER (1922), that in continuous light the �sensitivity� of the light-growthreaction for a higher light intensity is fully preserved. This can be explained by the fact that to each light intensity a specificnbsp;increase in the auxin synthesis belongs and consequently an increased amount of auxin-a-lactone, liable to photo-inactivation.

Further the conclusion of Haig (1935) that in Avena �there should be two photo-receptor systems, which have separate loci� matchesnbsp;my view that photo-inactivation of auxin in the entire coleoptilenbsp;interacts with changes of the auxin synthesis in the tip, perhapsnbsp;attended with a lateral transport in the tip too.

The photo-inactivation of a part of the auxin, as stated in the more basal regions of the coleoptile by Koningsberger amp; Verkaaiknbsp;(1938), certainly also occurs in the tip of the coleoptiles. The coursenbsp;of this inactivation in its dependence on the light energy still isnbsp;enigmatic. The inactivation only could be stated in the region of thenbsp;maximum of the first positive curvature [light energy = 500 M.C.S.nbsp;(390 ergs/cm^), white light; or 330�350 ergs/cm^, X = 4360 A] andnbsp;at the threshold of the second positive curvature (light energy =nbsp;30.000 ergs/cm^, X = 4360 A). On p. 347 I tried to account for thenbsp;fact that no photo-inactivation could be stated for amounts of lightnbsp;energy below 330 ergs/cm^, X = 4360 A, by referring to the datanbsp;collected by Schuringa (1941) on the photo-inactivation of auxin-a-lactone in vitro and the �photo-mechanical induction� assumed bynbsp;him. As most probable explanation for the fact that no photoinactivation was found in the region of the decreasing first positivenbsp;and in that of the negative curvatures I assumed that the inactivationnbsp;is masked and overbalanced by a steeply increasing auxin synthesis.

Since in vitro pure solutions of auxin-a-lactone are only inactivated by ultra-violet radiation, in the coleoptile the inactivation by visible light must be sensibilized. According to B�nning�s (1937)nbsp;and DU Buy amp; Olson�s (1938) statement that carotinoids play a partnbsp;in phototropism. Koningsberger amp; Verkaaik (1938) suggested thatnbsp;these substances would act as sensibilizers in the photo-inactivationnbsp;of auxin in the coleoptile. This view is strongly supported by thenbsp;work of Schuringa (1941). The rate of sensibilization being directlynbsp;proportional to the light absorption i.c. the light gradient, theie isnbsp;� as far as the photo-inactivation is concerned � no reason to joinnbsp;Atkins� view (1936): �a relationship is suggested between the phototropic curvature and the light gradient of the photosensitive area,nbsp;rather than a direct photochemical effect of some growth regulator�.nbsp;Further Atkins (1936) leaves open the possibility that also chloro-

354

phyll and anthocyanids could act as photo-sensibilizers. For the present there is no indication that this view holds true.

It remains possible, however, that in the increase of the auxin synthesis by light (� 5) also photo-sensibilizers are involved, othersnbsp;than carotinoids. My results, inclusively those obtained with light ofnbsp;2 = 5460 A, suggest that these sensibilizers absorb a broader regionnbsp;of the spectrum than carotinoids do.

Earlier or later in all experiments with different light quantities an increase of the auxin content was apparent. After exposure tonbsp;small light quantities (500 M.C.S., white light and 330 ergs/cm^,nbsp;A = 4360 A) the increase of the auxin content is only slow andnbsp;shght but with larger light quantities this increase is the only prominent feature of the auxin curvature not only with short wavenbsp;lengths, but also with /I = 5460 A. This increase of the auxin contentnbsp;of the tip can only be identical with an increase of the auxin synthesis, if this term be used in its broadest sense. The nature of thisnbsp;synthesis still is completely unknown. We only tried to argue thatnbsp;the rate of the increase of the auxin synthesis depends on the lightnbsp;quantity administered per time unit, i.c. on the light intensity. Thisnbsp;dependency seems logical for each kind of photo-catalyzed synthesis,nbsp;either for an inversion of an inactive precursor into auxin, or for anbsp;liberation of free auxin from a bound state. The last mentionednbsp;possibility gets a special interest in connection with Went�s (1938)nbsp;opinion on the occurrance of auxin in the tissue. He believes thatnbsp;auxin in the cell occurs in a �free� and in a �bound� state.The formernbsp;is diffusable and can be transported, the latter is bound somewherenbsp;to the plasm and cannot diffuse nor be transported. With the diffusionnbsp;method only the free moving auxin is determined, with the extractionnbsp;method both. In the statement by Stewart amp; Went (1940, p. 710);nbsp;�light apparently does not decrease the amount of free moving auxinnbsp;in the Avena coleoptiles, in contrast to its effect on bound auxin�nbsp;should be read in photo-chemical terms: �auxin-a� for �free movingnbsp;auxin� and ,,auxin-a-lactone� for �bound auxin�. It is, however,nbsp;hard to be understood how the polar acid itself could be free andnbsp;would not be adsorbed to the protoplasm, while the neutral lactonenbsp;would be. Moreover this does not agree with the fact that the auxin-a-lactone is transported in the plant: in the Avena-tt^X it has the samenbsp;activity as auxin-a. It seems much more likely that the free auxinnbsp;consists of the auxin-a :�� auxin-a-lactone system and the boundnbsp;auxin of some adsorbed and therefore inactive precursor, that is

355

liberated by light ^). Instead of the quoted lines we would prefer to read: �light apparently can decrease the amount of free movingnbsp;auxin in the tip of the Avena coleoptiles and liberate the boundnbsp;auxin�. Since the latter most probably is adsorbed to some boundarynbsp;layer of the protoplasm this version of Went�s hypothesis wouldnbsp;include a change in the permeability. This in its turn could accountnbsp;for changes in the normal basipetal transport, known as lateralnbsp;transport.

The course of the auxin content of L- and D-sides in all experiments can be explained by factors other than a lateral transport. There is not one experiment which gives evident proof of a lateralnbsp;transport. As we stated earlier (p. 342) it is more or less a questionnbsp;of personal feeling, whether one accepts a lateral transport or not.nbsp;Personally, however. I�m convinced that this lateral transport existsnbsp;on the following reasons:

2nd. the distribution of auxin after exposure to light quantities inducing the maximum of the first positive phototropic curvature,

3rd. the fact that after exposure to 26.400 ergs/cm^, X = 5460 A the D-side continuously has a higher auxin content than the L-side,nbsp;4th. the fact that all changes in the auxin content found innbsp;the L- and D-sides are too small entirely to account for thenbsp;phototropic response. Presumably they coincide with changesnbsp;in the physico-chemical properties of the protoplasm and/or ofnbsp;the cell wall, f.i. of the permeability of the protoplasm asnbsp;explained in the end of the preceding paragraph,

5th. the different results of Went�s experiments (1928) on the auxin delivery by diffusion of coleoptile tips split only at theirnbsp;base and my experiments on the auxin diffusion from entirelynbsp;split tips (p. 324). Also my experiments resumed in table 19nbsp;as compared to those of table 20 show consistent differences.nbsp;6th. the fact that geotropism entirely is ascribed to such a lateralnbsp;transport.

Since the data on geotropism of the earlier authors (Went, 1928; Cholodny, 1929, 1930; Dolk, 1930 and Dijkman, 1934) were all obtained by meansnbsp;of the diffusion method, I decided to check them by means of the extractionnbsp;method. In two replications sets of coleoptiles were either decapitated andnbsp;extracted or placed horizontal and then decapitated and extracted after i,

Voss (1939) suggests that auxin, inactivated by the scutellum, can be stored in this inactive form in the cells of the seedlings.

356

Many authors believe that geptropism and phototropism are of a different nature. In my opinion they have at least one importantnbsp;factor in common; the lateral transport of auxin, for Avena in thenbsp;extreme solid tip of the coleoptile.

Still less than proving the existence of a lateral transport in phototropism, I succeeded in elucidating the mechanism of such a change of the polarity. In the end of � 3 only a hypothetical possibility hasnbsp;been mentioned; the light would liberate an auxin precursor (�boundnbsp;auxin�) adsorbed to the boundery layer of the protoplasm in thenbsp;tip cells of the coleoptile. Consequently the properties of that layernbsp;and also its permeability would change and this change would involvenbsp;all still deficiently investigated phenomena referred to in Chapter II,nbsp;such as changes in viscosity of the cell wall, of bio-potentials, ofnbsp;respiration, of the protoplasmic streaming and also of the turgor etc.

This merely hypothetical explanation can also account for long distance effects of illumination. Proest (1927) for instance stated annbsp;increase of the growth rate of .iluena-coleoptiles when the roots arenbsp;illuminated. He ascribed this to an increased permeability of thenbsp;root cells by which the water supply of the coleoptile would benbsp;promoted.

As far as the photo-inactivation of auxin is concerned, it has turned out with certainty that phototropism is not a special case of

357

a light-growthreaction after all-round illumination. In those cases were this photo-inactivation was stated, it was equal at the D- andnbsp;the L-side and therefore cannot account for the phototropic response.nbsp;On the other hand this inactivation fully can be responsible for thenbsp;�tip-reaction� as far as it consists of a long lasting growth retardation.nbsp;Blaauw�s theory therefore cannot be entirely valid.

If one attributes a part to lateral transport in phototropism, as I do, the discrepancy with Blaauw�s theory is largest in the range ofnbsp;the first positive curvature. In this connection it must be mentionednbsp;that the course and the shape of the first and the second positivenbsp;curvature are different. In the first positive reaction the curvaturenbsp;starts in a narrow section just under the tip; it then gradually shiftsnbsp;towards the base, while the tip region straightens out again or, undernbsp;the influence of gravity, curves in the opposite direction, the cole-optile becoming S-shaped. In the second positive curvature thenbsp;entire coleoptile curves simultaneously. Furthermore it lasts muchnbsp;longer; the first positive curvature disappears after 2�3 hours, thenbsp;second one, according to DU Buy (1933), lasts for 5 hours.

This feature of the second positive curvature agrees with an inactivation of the auxin-a-lactone fraction present all along the coleoptile. The short base response of the light-growthreaction will be due to this inactivation. With an unilateral illumination this inactivation in basal zones will � according to the strong light gradient innbsp;this region � be less at the D- than at the L-side, so that at leastnbsp;part of the second positive curvature can be explained in Blaauw�snbsp;terms. That presumably is not the case with the reactions inducednbsp;by the tip, which, however, in this range of the light quantities havenbsp;not yet been thoroughly studied.

By means of the extraction method data were collected on the course of the auxin content of the light- and shade-side of coleoptilenbsp;tips of Avena after unilateral illumination. As light quantities werenbsp;applied:

a) nbsp;nbsp;nbsp;500 M.C.S. (390 ergs/cm^), white light and 330 ergs/cm^,nbsp;monochromatic blue light of 2. = 4360 A, both inducing aboutnbsp;the maximum of the first positive curvature,

Further a number of preliminary experiments were carried out with other amounts of monochromatic light (A = 4360 A). Thenbsp;results can be resumed as follows:

358

2. nbsp;nbsp;nbsp;Although no proof could be given for a lateral transport, there isnbsp;evidence that it actually plays an important part in phototropism.

3. nbsp;nbsp;nbsp;With the longer wave lengths (il = 5460 A) only positive curvatures could be obtained; negative curvatures occurred afternbsp;exposure to adequate amounts of light X = 4360 A of highnbsp;intensity.

4. nbsp;nbsp;nbsp;This difference is reduced to a different interaction of the threenbsp;factors, mentioned under i (see p. 341).

5. nbsp;nbsp;nbsp;Photo-inactivation of auxin was stated in the region of thenbsp;maximum of the first positive curvature (light energy: 500 M.C.Snbsp;(390 erg/cm^), white light; 330 ergs/cm^, X = 4360 A) and atnbsp;the threshold of the second positive curvature (light energy:nbsp;30.000 ergs/cm^, X =- 4360 A).

6. nbsp;nbsp;nbsp;This photo-inactivation can account for the long lasting growth-retardation of the so-called �tip� light-growthreaction, but notnbsp;for the phototropic response since it is equal at the light- andnbsp;the shade-side of the tip.

7. nbsp;nbsp;nbsp;Blaauw�s theory, reducing phototropism to special cases of light-growthreactions, can therefore not be entirely valid, especiallynbsp;not for the first positive curvature.

8. nbsp;nbsp;nbsp;Always, and especially after exposure to larger light, quantities,nbsp;the synthesis of auxin is increased. The unknown nature of thisnbsp;increased synthesis has been discussed (� 3, p. 354) and thenbsp;rate of the increase has been ascribed to the light quantum pernbsp;time unit, i.c. the light intensity.

9. nbsp;nbsp;nbsp;It was tried to explain phototonus in terms of an interactionnbsp;between photo-inactivation and rate of the increase of auxinnbsp;synthesis (� i, p. 350).

lO. Theories are given to explain the different phototropic responsesnbsp;(� I, a, b, c, p. 350). Especially in the first positive reaction anbsp;prominent part is ascribed to the lateral transport of auxin.nbsp;In the second positive reaction the photo-inactivation of auxin-a-lactone all alongside of the coleoptile greatly attributes to thenbsp;curvature.

The investigations were carried out in the Botanical Laboratory of the State University, Utrecht. I owe much to Prof. Dr. V. J.nbsp;Koningsberger for his interest in my work and his valuable criticism.

359

The auxiHoContent of illuminated tips after illumination with 330 ergs/cm-, A = 4360 A at different times after exposure.

360

3 hours after exposure.

29-io-�40	6.5	7-4	7-9	� 4.0	7-2	5-1	0
	2.0	5-4	5-1
30-io-�40	7-7	10.0	6.6	� 6.0	8.0	6.0	0
	2.2	3.0	2-3
i2-ri-�40	I.O	7.6	6.5	-lo.o )	�	�	7-3
	1.2	3-3	2.5
i3-ii-�40	9-9	8.4	4.8	� 5.0)
	4-5	10.3	6.7
mean:	4.6	6.9	5-3	� 6.2	7.6	5-5	�

4 hours after exposure.

2I-I0-�40	5-2	7.0	8.5	� 8.0	8.2	5-5	3-3
	4-5	6.0	7.6
	2.0	4.0	2.1	� 1-3	0	0	0
22-I0-�40	3-4	6.0	3-4
	4-3	7.6	4-3	� 9.0	8.0	5-7	2.5
4-11-�40	3-7	53	2.0
mean:	3-9	6.0	4-7	� 6.1	8.1	5.6	2.9

Summary of the mean values.

Extraction after exposure:	In degrees			Phototropic curvature	In .%
Extraction after exposure:	C	L	D	Phototropic curvature	C	L	D
Immediately:	5-9	6.5	6.2	� 7.2	100	no	105
After I hour:	5.8	7-9	6.2	� 6.0	100	136	107
After 2 hours:	2.5	3-7	2.8	�9.1	100	148	II2
After 3 hours:	4.6	6.9	5.3	� 6,2	100	150	115
After 4 hours:	3-9	6.0	4-7	� 6.1	100	154	120

The auxin�content of illuminated tips after illumination with 26.400 ergs/cm^, A = 5460 A at different times after exposure.

363

364

The auxin content of illuminated �tips immediately after exposure with different light quantities, A =4360 A.

Continuation Table 28.

366

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370

KahOj H. 1937, �ber den Einfluss der Kohlensaure auf die Exosmose von Elektrolyten aus Stengelzellen. Protoplasma 27 p. 502�522.

KocHj K.j 1934, Untersuchungen �ber den Quer- und Langstransport des Wuchsstoffes in Pflanzenorganen. Planta 22 p. 190�220.

K�GL5 F. amp; A. J. Haagen Smit, 1931, �ber die Chemie des Wuchsstoffs. Proc. Kon. Akad. Wet. Amsterdam 34 p. 1411�1416.

K�gl, F., a. J. Haagen Smit amp; H. Erxleben, 1933, �ber ein Phytohormon der Zellstreckung. Reindarstellung des Auxins aus menschhchem Ham.nbsp;IV Mitteilung. Z. physiol. Chem. 214 p. 241�261.

K�gl, F., A. J. Haagen Smit amp; C. J. van Hulssen, 1936, �ber den Einfluss unbekannter ausserer Faktoren bei Versuchen mit Avena saliva. Z.nbsp;physiol. Chem. 241 p. 17�33.

K�gl, F., C. Koningsberger amp; H. Erxleben, 1936, �ber die Selbstinak-tivierung der Auxine-a und -b. Z. physiol. Chem. 244 p. 266�278.

Koningsberger, V. J., 1922, Tropismus und Wachstum. Rec. trav. bot. n�erl. 19 p. i�136.

Koningsberger, V. J. amp; B. Verkaaik, 1938, On phototropic curvatures in Avena, caused by photochemical inactivation of auxin-a via its lactone.nbsp;Rec. trav. bot. n�erl. 35 p. i�13.

Laan, P. A. van der, 1934, Der Einfluss von Aethylen auf die Wuchsstoff-bildung bei Avena und Vida. Rec. trav. bot. n�erl. 31 p. 691�742.

Lange, S., 1937, Die Verteilung der Lichtempfindhchkeit in der Spitze der Haferkoleoptile. Jhr. wiss. Bot. 67 p. i�51.

Larsen, P., 1939, Skototenin, ein neuer Streckungswuchsstoff in h�heren Pflanzen. Die Naturwissenschaften 27, p. 549�550.

Lazar, O., 1935, L�influence du carot�ne sur la n�oformation des racines et Ie d�veloppement de la gemmule chez Impatiens balsamina L. Compt.nbsp;Rend. soc. biol. Paris 120 p. 1374�1376.

Lepeschkin, W. W., 1908, Zur Kenntnis des Mechanismus der Variations-bewegungen. Ber. bot. Ges. 26 p. 724�735.

bewegungen und der Einwirkimg des Beleuchtungswechsels auf die Plasmamembran. B.B.C. 24 I p. 308�356.

Linser, Hans, 1940, Ueber das Vorkommen von Hemmstoff in Pflanzen-extrakten, sowie �ber das Verhaltnis von Wuchsstoffgehalt und Wuchs-stoffabgabe bei Pflanzen oder Pflanzenteilen. Planta 31 p. 32�60.

Lundegardh, H., 1922, Ein Beitrag zur quantitativen Analyse des Photo-tropismus. Arkiv f�r Eotanik 18 no. 3.

Meindl, T., 1934, Weitere Beitrage zur protoplasmatischen Anatomie des He/odea-Blattes. Protoplasma 21 p. 362�393.

Metzner, P., 1923, �ber induzierten Phototropismus. Ber. bot. Ges. 41 p. 268�274.

M�ller, N. J. C., 1872/77, Untersuchungen �ber die Ki�mmungen der Pflanzen gegen das Sonnenlicht. Bot. Untersuchungen I Heidelberg.

Nuernbergk, E. amp; H. G. du Buy, 1930, �ber Methoden zur Analyse von Wachstumserscheinungen. Rec. trav. bot. n�erl. 27 p. 417�520.

Oppenoorth, W. F. F., 1939, Photo-inactivation of auxin in the coleoptile of Avena and its bearing on phototropism. Proc. Kon. Akad. Wet.Am-sterdam 42 p. 3�16.

371

OvERBEEK, J. VAN, 1933, Wuchsstoff, Lichtwachstumreaktion und Photo-tropismus bei Raphanus. Ree. trav. bot. n�erl. 30 p. 537�627.

Paal, A., 1914, �ber phototropische Reizleitungen. Ber. bot. Ges. 32 p. 499�502.

PoHL, R., 1936, Die Abhangigkeit des Wachstums der yitiewa-Koleoptile und ihrer sogenannten Wuchsstoffproduction vom Auxingehalt des Endosperms. Planta 25 p. 720�750.

Proest, S., 1927. �ber den Einfluss einer Sprossbelichtung auf das Wurzel-wachstum und denjenigen einer Wurzelbelichtung auf das Sprosswach-stum. Planta 4 p. 651�710.

Purdy, H. A., 1921, Studies on the path of transmission of phototropic and geotropic stimuli in the coleoptile of Avena. Det Kgl. Danske Videnskabnbsp;Selskab, Biol. Meddel. 3 p. 3�29.

Pyrkosch, G., 1936, Licht und Transpiradonswiderstand. I. Die transpira-tionswiderstande im monochromatischen Licht. Protoplasma 26 p.

Raalte, M. H. van, 1937, On factors determining the auxin content of the root tip. Rec. trav. bot. n�erl. 34 p. 278�333.

Ramshorn, K., 1934, Experimentelle Beitrage zur elektrophysiologischen Wachstumtheorie, Planta 22 p. 737�766.

Rothert, W., 1894. �ber Heliotropismus. Cohns Beitr. Biol. Pfl. 7 p. i�212. Ruge, U., 1939, Zur Physiologic der genuinen keimungshemmenden undnbsp;keimungsbeschleunigenden Stoffen von Helianthus annuus. Z. Bot.nbsp;33 P- 529�571-

Santen, A. M. A. van, 194O3 Groei, groeistof en pH. Thesis Utrecht. Schweighart, O., 1935, Eosin und Keimpflanzen. B.B.C. 53A p. 217�292.nbsp;SCHURINGA, G. J., 194I3 De foto-inactiveering van auxine-a-lacton. Thesisnbsp;Utrecht.

SiERP, H. amp; A. Seybold, 1926, Untersuchungen �ber die Lichtempfindlich-keit der Spitze und des Stumpfes in der Koleoptile von Avena saliva. Jhr. wiss. Bot. 65 p. 592�610.

Skoog, F., 1934, The effect of x-rays on growth substance and plant growth. Science 79 p. 256.

372

Skoog F., 1937, A deseeded Avena test method for small amounts of auxin and auxin precursors. J. Gen. Physiol. 20 p. 311�334.

Snow, R., 1924, Further experiments on the conduction of tropic excitation. Ann. Bot. 38 p. 163�174.

Stark, P., 1927, Das Reizleitungsproblem bei dem Pflanzenim Lichte neuerer Erfahrungen. Ergeb. Biol. 2 p. i�94.

Stark, P. amp; O. Drechsel, 1922, Phototropische Reizleitungsvorgange bei Unterbrechung des organischen Zusammenhangs. Jhr. wiss. Bot. 61nbsp;P- 339�371-

Stewart, W. S. amp; F. W. Went, 1940, Light stability of auxin in Avena coleoptiles. The bot. Gaz. loi p. 706�714.

Strugger, S., 1934, Beitrage zur Physiologie des Wachstums. I. Zur proto-plasma-physiologischen Kausalanalyse des Streckungswachstums. Jhr. wiss. Bot. 79 p. 406�471.

Sweeney, B. M. amp; K. V. Thimann, 1937, The effect of auxins on protoplasmic streaming. II. J. Gen. Physiol. 21 p. 439�461.

Syre, H., 1938, Untersuchungen �ber Statolithenstarke und Wuchsstoff an vorbehandelten Wurzeln. Z.f. Bot. 33 p. 129�182.

Thimann, K. V., 1934, Studies on the growth hormone of plants. VI. The distribution of the growth substance in the plant tissues. J. Gen. Physiol.nbsp;18 p. 23�34.

Thimann, K. V. amp; B. M. Sweeney, 1937, The effect of auxins upon protoplasmic streaming. J. Gen. Physiol. 21 p. 123�135.

Thomas, J. B., 1939, Electric control of polarity in plants. Rec. trav. bot. n�erl. 36 p. 374�438.

Tr�ndle, a., 1910, Der Einfluss des Lichtes auf die Permeabilitat der Plasma-haut. Jhr. wiss. Bot. 48 p. 171�285.

Voss, H., 1939, Nachweis des inaktieven Wuchsstoffes, einer Wuchsstoff-antagonisten und deren wachstumsregulatorische Bedeutung. Planta 30 p. 252�286.

Wald, G. amp; H. G. du Buy, 1936, Pigments of the oat coleoptile. Science 84 p. 247.

Waller, A. D., 1900, The electrical effects of light upon green leaves. Proc. Roy. Soc. B 67 p. 129�137.

Wey, H. G. van der, 1931, Die quantitatieve Arbeitsmethode mit Wuchsstoff. Proc. Kon. Acad. Wet. Amsterdam 34 p. 875�892.

Went, F. W. amp; K. V. Thimann, 1937, Phytohormones. New York. WiESNER, J., 1878, Die heliotropischen Erscheinungen im Pflanzenreich I.

WiESNER, J., 1880, Die heliotropischen Erscheinungen im Pflanzenreich II Denkschr. k. Akad. Wiss. Wien. 43 p. i�92.

Wilden, M., 1939, Zur Analyse der positiven und negativen phototropischen Kr�mmungen. Planta 30 p. 286�289.

STELLINGEN

Op physiologische gronden moet aangenomen worden, dat in de plant het evenwicht auxine-a = auxine-a-lacton zich binnen drienbsp;minuten instelt.

Het is zeer waarschijnlijk, dat het vermogen om phototropisch te reageer en berust op de aanwezigheid van carotino�den.

III

Bij Chlorella pyrenoidosa gaat de basale ademhaling tijdens de oxydatieve verwerking van toegevoerde organische stoffen onveranderd door.

Uit de proeven van Melchers moet de conclusie getrokken worden, dat voor het in bloei komen van planten de samenwerking van florigeen en vernaline noodig is.

De pollenanalytische methode mag, met eenige voorzichtigheid, ook op aeolische afzettingen toegepast worden.

De bouw van de angiosperme embryozak, ook wanneer deze van het normale type afwijkt, kan verklaard worden door aan te nemen,nbsp;dat in principe twee gereduceerde archegoni�n aanwezig zijn en eennbsp;vegetatief prothallium ontbreekt.

VII

De tegengestelde reactie ten gevolge van exstirpatie van de pedaal-ganglia, wanneer de slakkenvoet (Helix pomatia) in ,,Muskei-� dan

wel in �Zentraltonuskonstanz� verkeert, moet verklaard worden door de wisselwerking tusschen de periferie en de twee antagonistische tonuscentra in de pedaalganglia in verband met hun specifiekenbsp;reactie op de grootte van den rekkingslast.

VIII

Met behulp van gistingsproeven kan worden bepaald of het aneurine-disulfide een physiologische beteekenis heeft.

Het heidepodsolprofiel is genetisch ��n geheel en is ontstaan door uitlooging der bovenliggende en inspoeling in de daaronder liggendenbsp;lagen. Dit profiel kan ook in den huldigen tijd nog ontstaan.

Door uitwendige omstandigheden is de stofwisseling der planten zoo te be�nvloeden, dat zoowel de aantasting door als de verspreidingnbsp;van parasitaire plantenziekten zeer bemoeilijkt wordt.

Actieve resistentie tegen virusziekten berust waarschijnlijk niet op een verandering van het virus zelf, maar op een veranderingnbsp;in de plant.

XII

Het uiteengaan der chromosomen bij de kerndeeling geschiedt door middel van trekdraden.

XIH

Het chromosoom in het leptoteen-stadium der reductiedeeling is niet uit ��n enkelvoudige draad opgebouwd.

i5-2-�40	4.6	2.5	3.0	9.0	10.1	5-1	2.0
	4-5	6.3
i-7-�40	5-7	6.3	5-9
		8.4	9.8	9-4	12.4	7.2	5-4
2-7-�40	7-7	5.8	6.6
		6.2	6.1	9-9	5-7	4.6	2.2
3-7-�40	8.4	II-3	12.2	12.2	8.7	7-1	4-9
I2-8-�40	5-7	9-5	7-1	15.0	�	�	�
	8.0	3-3	8.0
mean;	6.4	6.7	7.3	II.I	9.2	6.0	3-7
	30 minutes after			exposure.
20-6-�40	�	4-7	7.0	9-1	9.6	8.3	6.2
	5.8	4-7	7.8
27-6-�40	5.8	3.0	2.5	10.3	11.4	5-2	3-r
		6.1	2.5
28-6-�40	7-7	�	6.0	8.3	12.7	8.6	5-3
	5.2	10.3	�
i6-8-�4o	10.5	9.8	lO.I	23-3	lO.O	5.0	2.6
	5-7	9-5	10.4
I9-8-�40	8.0	10,7	14.6	9-3	II.3	6.1	2.6
	5.2
	9-7	10.0	12.3
mean:	7-2	7.6	8.1	12,0	10.7	7-1	4.0
I hour after exposure.
4-7-�4D	7.2	10.8	8.4	13.9	10.5	3.8	0
5-7-�40	4.6	5.0	8.6	9-9	13-3	8.0	3-3
		6.4	9.0
8-7-�40	6.2	8.3	9-7	10.4	12.2	8.6	4.8
i3-8-�4o	8.3	7-9	4.2
i3-8-�4o	8.3	7.0	II.O	21.0	�	�	�
	5-7	7.2	6.0
mean:	6.4 *9�	7.5	8.1	14.0	12.0	6.8	4.0

i7-3-�4i	18.6	134	8.5	20.0		19.0	8.0
	7-5	10.8	8.5
i8-3-�4I	29.4	25.5	27.4	17.4		lO.O	4.2
	26.2	23.0	25.7
i9-3-�4i	14.0	21.1	8.1	27.0	25.4	18.4	lO.O
	6.3	13.6	13-0
20-3-�4i	12.0	8.4	�	17.6	22.0	14.8	9-3
	7-4	4.6	4-1
mean:	15.2	15.1	13.6	21,0	23.7	15-5	7-9

ii-3-�4r	9.0	9.0	12.0	15.0	16.0	8.6
	8.0	44	12.0
i2-3-�4i	6.0	� 5-0	4.0	12.5	lO.O	4.0
	6.4	6.2	6.2
i3-3-�4i	15.0	18.5	13-5	17.0	17.0	6.0
	lO.O	12.0	lO.O
i4-3-�4i	10.5	9.0	8.0	19.0	9-7
	6.7	9.0	9-5
mean:	9.0	9-1	94	16.0	13.2	6.2

2-4-�41	25.7 6.6	22,0 20.0	13.0 19.0	-I.O	14.0	7-7	2.2
3-4-�4i	20,7 15.0	10.7 12.3	19.3	-I.O	15.2	7-7	0
4-4-�4i	15.0 8.0	8.6	5.8 II.0	3.0	10.7	8.0	0
7-4-�4i	13.1 20.6	12.2 11.5	9.7 10.7	4-3-0	(7-0)	(20.0)	(II.8)
mean:	16.9	13-9	12.9	1.0	13.3	7.8	0

	Control		Photo-
Exp.	not	Illuminated	tropic
on:	ilium.		curva-
	C	L 1 D	ture

16-9-�40 24-9-�40	17.0 9.2	14.4 4.8 6.6 6.3	14.0 7.0 5-7	10.4 21.3	I3.I 8.0	8.1 3-1	3.3 0.6
mean:	13.1	8,0	8.9
I hour after exposure.
17-9-�40	6.0	3.2	5.0	13.8	�	7.0	4.8
	7-5	3-5	6.7
i8-9-�40	5-4	4.5	5-3	11.3	II.6	5-4	0
	5-2	2.8	4.2
	�	2.6	4.6
mean:	6.0	3-3	5.2			1
		2 hours	after exposure.
I9-9-�40	6.0	2.8	3.2	8.0	�	9.2	3.4
	4-4	4-7	4-5
		�	4.0
2o-9-�40	6.0	4.0	4-3	10,0	�	�	�
	7-4	5-1	7-4
		3-3	7.0
mean:	6.0	4.0	5-1

24-io-�40	5.8	5.8	6.5	�II.0	7-3	3.6	1-5
	5-3	5.6	5.6		8.5
25-io-�4o	8.7	9-5	7-3	� 9.0	8.5	4.0	0
	8.6	7.0	5-7	� i.6\
3i-io-�4o	4-3	7.0	7.0	� i.6\
	7.6	7.0	8.0		8.9	4-5	I.O
i-ii-�40	3-1	5-1	3-7
	4.0	5-2	6.1
mean:	5-9	6.5	6.2	� 7-2	8.2	4.0	1.2

Energy in ergs/cm^ � = 4360 A	Curvature in degrees			Phototropic curvature	Curvature in % of the control
Energy in ergs/cm^ � = 4360 A	C	L	D	Phototropic curvature	C	L	D
35	II.I	II.2	II.0	15-3	100	lOI	99
150	4.0	34	4.6	28.0	100	85	115
330	13.1	8.0	8.9	15.0	100	61	68
350	5.8	3-7	3-5	20.5	100	64	61
700	5-1	5-5	4-9	27.0	100	108	96
1.000	15.2	15.1	13.6	21.0	100	100	90
1.400	9.0	9-1	94	16.0	100	lOI	104
3.000	5-9	6.5	6.2	-6.0	roo	no	105
30.000	16.9	13.9	12.9	-f i.o	100	82	76

tc'

^1'

ï-

miX

0^.

WILLEM FREDERIK FLORUS OPPENOORTH Jr.

N.V. DRUKKERIJ v/h KOCH amp; KNUTTEL

INTRODUCTION AND STATEMENT OF THE PROBLEM.

� I. Introduction.

88%

294

CHAPTER II.

303

Waller (1900), Bose (1907) and Brauner (1927) did similar experiments but obtained opposite results. They probably used

3o6

MATERIAL AND METHODS.

� I. The plants used.

w 6

� the mercury bulb, being composed of lines or narrow bands, isnbsp;especially adapted to this purpose.

324

CHAPTER IV

PRELIMINARY EXPERIMENTS.

� I. The first orientation.

325

57 (68 %)

8,8�

330

334

335

EXPERIMENTS WITH MONOCHROMATIC LIGHT.

� I. Introduction.

337

339

THE PHOTOTROPIC CURVATURE AS A FUNCTION OF THE LIGHT ENERGY (A = 4360 A)

� I. Introduction.

344

347

GENERAL DISCUSSION AND SUMMARY.

� I. The types of phototropic reactions; Phototonus.

353

354

355

356

357

358

359

360

363

364

365

Continuation Table 28.

366

LITERATURE.

368

370

371

372

STELLINGEN

II

III

IV

VI

VII

ti

VIII

IX

X

XI

XII

XIH