FROM various experiments the writer concluded previously (1937, 1938) that

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1 [ 177 A HORMONE FOR CORRELATIVE INHIBITION BY R. SNOW Fellow of Magdalen College, Oxford (With I figure in the text) I. INTRODUCTION FROM various experiments the writer concluded previously (1937, 1938) that the inhibition of lateral buds and shoots by auxin, whether formed naturally or applied, is brought about indirectly and is due to a secondary inhibiting infiuence, which originates from some primary process promoted by the auxin in the main stem and travels upwards into lateral buds and shoots where the auxin cannot easily follow it. As to the nature of this inhibiting infiuence, an earlier experiment (Snow, 1929) indicated that it was probably an inhibiting substance, since inhibition could travel upwards through a dead zone with the transpiration stream. But for reasons given previously (1937, p. 298) it seemed desirable to obtain further evidence on this point, and an experiment providing such evidence will be reported here. This experiment, like others reported previously (1937, p. 292; 1938, p. 180) provides evidence against Went's "diversion" theory of inhibition also (1938, 1939), which will be discussed again in section EXPERIMENT Peas, of race "Thomas Laxton", were sown in pairs close together, and the main shoots were cut off as soon as they appeared. Later when the axillary shoots of the cotyledons, two from each plant, had grown out and had fully expanded two leaves, those pairs of plants were selected in which at least three of the four cotyledonary shoots were well developed, and the fourth shoot was removed, so that in each pair of plants there now remained one plant with two shoots and another with one (see Fig. i, for which I am indebted to my wife). Then one of the shoots of the "two-shoot" plant was tied with moist bast to the single shoot of the other plant, in such a way that the internodes second from the base were pressed fairly tightly into contact along most of their length. Before these internodes were tied together, thin strips of epidermis and a little cortex had been cut away from the sides which were to be brought into contact, and the cut surfaces had been washed. Care had been taken to select shoots in which these internodes would fit together easily and fairly exactly. Finally, the shoots which had been tied together were decapitated by cuts through the next internodes above those which had been tied. The object was now to note whether the intact shoot of the "two-shoot" plant would inhibit the lower axillary bud of the single shoot of the other plant beyond and below the region of contact. The controls were the corresponding buds of similar shoots which were decapitated at the same level but not bound to neighbouring plants. New Phytol. 39, 2 12

2 178 R. SNOW The buds to be observed were from 0-5 to i-o long at the start, and care was taken that those of experiments and controls were about equal in length. Shoots with larger buds were avoided. After the binding and final decapitation, the plants, grown in the summer in a green-house, were kept in the shade in rather Internodes of decapitated shoots tied together Position of measured bud Cotyledons of two-shoot plant Fig. I. Cotyledons of single-shoot plant damp air. The buds of experiments and controls were measured days after the final decapitation in the first batch of plants, and 5 days after it in the others. Table i shows the growth of the measured buds after these times. It can be seen that the mean growth of the buds at the lower nodes in the "single-shoot" experimental plants was less than half as much as in the controls.

3 A hormone for correlative inhibition 179 Moreover the greatest bud growth in the six experimental plants was less than the smallest bud growth in the sixteen controls. Clearly therefore the buds of the experimental plants were partially inhibited by the intact shoots of the other plants across the region of contact. The buds at the next higher node also, above the region of contact, grew distinctly less than the corresponding buds in the controls, but this will not be considered as evidence since it might be argued that they were retarded by the tightness of the ties round the stems below them. All the lateral buds on the "two-shoot" plants, including those on their decapitated shoots, were of course completely inhibited. Table i. Growth of measured buds after ^ or days Experimental plants Batch I Controls Experimental plants O Batch 2 Controls 8 9 Experimental plants Batch 3 Controls 8 7 II 14 1 Means: Experimental plants Controls After the experiment the bast ties were cut through and the stems fell apart at once, showing that they had not adhered nor grown together. So the inhibiting influence which travels up a side shoot can cross a moist protoplasmic discontinuity, and this result, together with the previous "dead-zone" experiment (1929), justifies the conclusion that the inhibiting influence is another hormone. Previous experiments (Snow, 1939 a) indicated that the inhibiting influence originates from the reaction or co-operation of auxin with some other factor or factors in the main stem. 3. THE "DIVERSION" THEORY OF INHIBITION Went (1939) has recently reported some experiments which he claims to support his "diversion" theory of inhibition by auxin, and these will be discussed below. According to this theory, the auxin, whether formed naturally by the apical bud or applied artificially, brings about the inhibition of lateral buds by somehow causing some substance necessary for the growth of buds to travel up the main stem to the apical bud instead of passing out into the lateral buds. Thus lateral buds (and presumably lateral shoots) remain cut off from their supplies at the base, which are diverted up the main stem. The writer has previously reported two experiments on correlative inhibition (1937, p. 292) and one on inhibition by applied auxin (1938, p. 180) which cannot be explained on this theory, besides another experiment (1937, p. 293) which would be difficult to explain on it; and Went has not attempted to answer these objections. The experiment reported in the previous section is also

4 i8o R. impossible to explain on Went's theory, as a glance at the illustration will soon show. For the auxin travelling down the main stem of the "two-shoot" plant cannot cut off the lateral buds of the other plant from their base of supplies, smce they have their own independent base which that auxin cannot reach. Indeed the arrangement is physiologically rather similar to that in the writer's previous experiment (1937, Figs. 2, 3), though differently reached. The earlier "dead-zone" experiment (Snow, 1929) is also quite inconsistent with the "diversion" theory. For it was found that if a zone of stem was killed near the base of a decapitated shoot of a "two-shoot" broad bean plant, the buds above the dead zone grew out a little, though they grew only very slowly since some inhibition from the other shoot travelled up to them through the dead zone. But still they did grow a httle, whereas in plants similarly prepared, but without a dead zone, the corresponding buds did not grow at all. Thus by kilhng a zone of stem on a decapitated shoot, one releases the buds above the zone partially, though not completely, from inhibition by another shoot. Yet one certainly does not improve their chances of drawing supplies from the base. Went (1939), by applying hetero-auxin in lanoline to decapitated dark-grown pea shoots, has confirmed the "increase of inhibition with distance" which was previously found by the writer (1931) using part of the shoot apex as the source of inhibition. This confirmation is welcome, and Went argues that it can be readily explained on his "diversion" theory since it may be supposed that for effective inhibition a sufficient length of stem is needed in which the factors for bud growth may accumulate above the lateral bud. This explanation is indeed plausible in itself, but Went leaves out of account the fact that he himself (1939, p. no) found that seven other chemicals ("auxinoids", as they may perhaps be called), which have some of the effects of auxin and hetero-auxin but are less rapidly transported, caused not an increase but a decrease of inhibition with distance. The conclusion should surely be that the "increase with distance", though still unexplained, is probably, when it occurs, somehow connected with the high mobility of natural auxin and hetero-auxin. It may be mentioned that Thimann (1937), using darkgrown pea shoots and hetero-auxin, found neither significant increase nor decrease with distances ranging from 2 to 150 Went also claims to show by some rather complicated experiments (1939, Tables 3, 4, 5) that in shoots of decapitated etiolated pea seedlings certain rather weak concentrations of hetero-auxin and of y-phenyl butyric acid in lanoline, applied on top of the stem, both inhibit lateral buds and also increase the accumulation of factors for bud growth in the stem after the decapitation. (Even when pure lanoline was applied, the factors for bud growth accumulated similarly in the stem, if Went's interpretation is correct, but to a somewhat less degree.) He claims that the results support his theory that inhibition of lateral buds is due to diversion of factors for bud growth to the top of the stem. But actually, as Thimann (1939, p. 337) has pointed out, in these experiments these concentrations of the applied chemicals did not inhibit the lateral buds, but promoted them, although they did inhibit the lateral buds in other conditions, when the shoots were left on the plants

5 A hormone for correlative inhibition instead of being used as cuttings (Went, 1939, p. 114 and Table 7). But then in those conditions there was no evidence of increased accumulation of bud growth factors in the main stem. Even apart from this objection, and even if Went's interpretation of these experiments were entirely granted, it would only have been shown that, after the substances mentioned have been applied, two results follow; the factors for bud growth accumulate in the stem, and the lateral buds are inhibited. Erom this it would not follow that one of these two results is the cause of the other. Went also reports (p. 11) that some low concentration of hetero-auxin inhibits the lateral buds if applied continuously after decapitation, but promotes them if applied for the first 2 days only: and he claims that this fact is "completely unexplainable on the basis of Snow's theory". But this is not so, for at least one possible explanation can readily be suggested, which is not inconsistent with that theory. Thus application of hetero-auxin for 2 days only may perhaps be equivalent to application of a very small continuous dose. If so, then the explanation may be that a very small dose of hetero-auxin, leading, according to the writer's theory, to the formation of a very small amount of inhibitor, may act on the lateral buds in the opposite way to a larger dose; just as hetero-auxin promotes root growth in very low concentration, but inhibits it in higher concentration. However, the true explanation may of course really be quite different. i8i 4. THE DIRECT AND INDIRECT THEORIES OE INHIBITION The indirect theory would be much strengthened if an inhibitor could be extracted from those parts, and only those, in which it would be expected. Recently, several workers have obtained extracts containing inhibitors of coleoptile growth. Thus Viehmann (1939) has obtained by diffusion from sunflower hypocotyls a growth inhibitor which, as he concludes, is formed either in the hght, or else in the dark, but then only under the influence of applied acetic or formic acid. Stewart and others (1939) and Stewart (1939) have extracted with ether from radish cotyledons an inhibitor which is transported in both directions. Larsen (1939) has extracted inhibitors with ether from tomato fruits and from meal of maize seeds, and Goodwin (1939) has concluded from indirect evidence that ether extracts of maize meal and of broad bean shoots contain an inhibitor. The writer (1939), following Stewart and others, has extracted a rather different inhibitor with wet ether from pea leaves, but this inhibitor was found sometimes to persist for several days at least in the leaves of decapitated disbudded shoots, and not to disappear as the correlative inhibitor would be expected to do. The same test would need to be applied to the other inhibitors also, before they could be accepted as likely candidates for the position of correlative inhibitor. In a valuable review Thimann (1939) considers the "indirect" theory of inhibition as still remaining open, but raises some difficulties for it, which to the writer do not seem insuperable. Thus in referring (p. 327) to his experiments in which he inhibited buds by applying auxin in lanoline to their outer sides (1937),

i82 R. SNOW he does not take into account the fact that in such experiments the auxin travels down the outer leaves and enters the axis of the bud from its base.

6 i82 R. SNOW he does not take into account the fact that in such experiments the auxin travels down the outer leaves and enters the axis of the bud from its base. Consequently the inhibition can be understood on the indirect theory. Thimann objects also (p. 332) that the indirect theory does not explain the great decrease of auxin in inhibited parts. But it is to be expected that when these parts are inhibited in growth, they will cease to form auxin, as seems probably to have happened in the seedlings of Avery et al. (1937) when grown with shortage of nitrogen. Or again, if the suppression of auxin formation in inhibited buds and shoots is primary and the cessation of growth secondary, then the correlative inhibitor may act by suppressing the auxin formation. In spite of what Thimann writes on this point, the essential difference from his "direct" theory would still remain, that on that theory the auxin from the inhibiting region must travel into all inhibited lateral buds and shoots, whereas on the "indirect" theory it does not do so, or not in any effective concentration. Indeed on the "indirect" theory those parts of the shoot system which are so situated and orientated that the main stream of auxin, travelling chiefly downwards, cannot easily enter them are just those which become inhibited. Evidence that this is indeed the rule was offered previously (Le Fanu, 193; Snow, 1937, 1938). It is not necessary on this theory to suppose that absolutely no auxin enters the lateral buds and shoots; for it is enough if the ratio of auxin to inhibitor is much lower in them than in the main stem. Indeed van Overbeek (1938, p. 10) has obtained evidence of some slight upward transport of hetero-auxin into lateral buds growing out in the dark. It would, however, undoubtedly be difficult to explain, on the indirect theory, the inhibition of the formation of adventitious buds in flax hypocotyls by heteroauxin, or by ether extracts of shoots, applied in lanoline on top (Link & Eggers, 1938), or again, as Thimann has pointed out, to explain the inhibition of regeneration in fern prothalli by hetero-auxin in lanoline applied to the apical cut surface (Albaum, 1938). For in these examples the inhibited centres of regeneration are not inserted laterally, so that the downward-moving auxin probably passes directly through them. Contrasting indeed with these two examples are several others in which the formation of adventitious buds from stems or leaves in various species has actually been provoked by hetero-auxin in lanoline applied from above (Greenleaf, 1937; Beal, 1937; Goldberg, 1938; Prevot, 1938). Nevertheless it may be pointed out that it would be quite consistent to explain on the basis of Thimann's "direct" theory such examples as the inhibition of regeneration in flax hypocotyls and fern prothalli, while still explaining on the "indirect" theory the inhibition of lateral buds and shoots, for which alone it was intended. On this combined view the fate of any part of the shoot system depends on two variables, the relative amounts of auxin and inhibitor which it receives, and its inherent sensitivity. Thus some parts, such as lateral buds and shoots, are so situated and orientated that the main stream of auxin, travelling chiefly downwards, cannot enter them or only in small amount; and these parts are regularly inhibited by the inhibitor whatever their stage of development, in accordance with the "indirect" theory. But those parts through which the descending stream of auxin

A hormone for correlative inhibition 183 directly passes react to it in a manner depending on their inherent sensitivity, as proposed by Thimann (1937).

7 A hormone for correlative inhibition 183 directly passes react to it in a manner depending on their inherent sensitivity, as proposed by Thimann (1937). For instance stems generally have their growth in thickness promoted by moderate concentrations of auxin passing down them, and tbeir growth in length eitber increased or, if tbey bave enougb auxin already, not mucb affected by it. Also growtb in buds is apparently promoted by moderate amounts of auxin wben it is really applied to tbeir morpbological apices, and not to tbeir outer leaves: for betero-auxin in lanoline applied to part of tbe stem apex was found to promote tbe formation and growtb of members from tbat part (Snow & Snow, 1937). (Tbimann (1939) bas a little misunderstood tbese experiments: tbe auxin was applied to tbe stem apex or to part of it, not to tbe existing leaf primordia.) But otber parts, sucb as tbe centres of incipient regeneration in flax bypocotyls and fern protballi, may be more sensitive to auxin passing tbrougb tbem, so tbat unless tbe concentration of auxin is low, it may be too mucb for tbem and inbibit tbem. Tbis explanation agrees well witb experiments of Stougbton & Plant (1938) also, wbicb indicate tbat in sea-kale cuttings a bigb content of auxin tends to inbibit formation of adventitious buds. Tbe very earliest stages of formation of axillary buds can also be sbown to be liable to correlative inbibition, in peas at least, if tbe plants are given tbe opportunity to form new buds by regeneration in one of tbe lower axils (Snow, 1931); and for tbe present it must be left an open question wbetber tbis inbibition is direct or indirect. But furtber evidence tbat from quite an early stage of development tbe axillary buds are inbibited indirectly is tbe fact tbat tbey can quite early be sbown to be negatively geotropic, even wbile partially inbibited. To test tbis point, tbe writer bent down long sboots of pea seedlings borizontally and reduced tbe inbibiting power of tbe apex eitber by removing tbe older developing leaves, or else by decapitating bigb up and leaving a single developing leaf (see Snow, 1931). Controls were decapitated and deprived of all developing leaves. Tbe buds of tbe plants witb apex or single young leaf remaining grew out mucb tbe more slowly, being partially inbibited; but yet tbey sbowed clear geo-negative curves in tbeir basal internodes wben tbese internodes were only from 4 to 8 long, and tbe wbole buds from 8 to 13 long. Tbeir negative geotropism indicated tbat auxin was promoting tbeir growth, wbile at tbe same time tbey were being correlatively inbibited. Tbe inbibition tberefore can bardly bave been directly due to auxin. In general, if axillary buds were inbibited directly by auxin, one would expect often to notice tbem making geo-positive curves in nature. SUMMARY 1. An experiment illustrated in Fig. i sbows tbat in pea plants correlative inbibition from a growing sboot apex can travel down one sboot and up anotber decapitated sboot, and can tben enter a decapitated sboot of anotber plant, crossing a protoplasmic discontinuity wbere tbe tissues are only in moist contact. 2. Since previous experiments bad sbown tbat tbe inbibiting influence wbicb travels up a lateral sboot is not ordinary auxin (Snow, 1937), it is concluded

8 184 R- SNOW from this experiment together with an earlier one (Snow, 1929) that the inhibiting influence is another hormone, 3, The above experiment, like others reported previously (Snow, 1937, 1938), cannot possibly be explained by Went's "diversion" theory of inhibition (1938), Some recent experiments, which Went (1939) claims to support his theory, are discussed and considered inconclusive, 4, Some difliculties raised by Thimann (1939), against the "indirect" theory of inhibition in shoots, are discussed, and it is pointed out that the inhibition by auxin of the formation of adventitious buds in the main axis may be direct, even if the inhibition of lateral buds and shoots is indirect. Further evidence that the inhibition of lateral buds is not directly due to auxin is provided by an experiment showing that in peas they are negatively geotropic even while partially inhibited, REFERENCES ALBAUM, H, G, (1938), Inhibitions due to growth hormones in fern prothallia and sporophytes, Amer. J. Bot. 2,$, 124, AvERY, G, S,, BuRKHOLDER, P, R. & CREIGHTON, H, B, (I937). Nutrient deficiencies and growth hormone concentration, etc, Amer. J. Bot. 34, 553, BEAL, J, M, (1937), Bud development in Lilium Harrisii, etc, Proe. nat. Aead. Sci., Wash., 23, 304, GOLDBERG, E, (1938), Root and shoot production induced in cabbage by beta-3-indoie-acetic acid. Science, N,S, 87, 511, GOODWIN, R, H, (1939), Evidence for the presence in certain ether extracts of substances, etc, Amer. jf. Bot. 2, 130, GREENLEAF, W, H, (1937), Induction of polyploidy in Nicotiana. Science, N,S, 8, 55, LARSEN, P, (1939), Uber Hemmung des Streckungswachstum durch naturlich vorkotnmende atherlosliche Stoffe, Planta, 30, 10, LE FANU, B, (193), Auxin and correlative inhibition, Nezu Phytol. 35, 205. LINK, G, K, & EGGERS, V, (1938), Inhibition of adventitious bud initiation in hypocotyls of Flax, etc. Nature, Lond., 142, 39S, PREVOT, P, C, (1938), Infiuence de l'h^t^ro-auxine sur la n^oformation des bourgeons chez Begonia Rex Putz, Bull. Soc. roy. Sci., Liege, no, 3, 284, SNOW, R, (1929), The transmission of inhibition through dead stretches of stem, Ann. Bot., Lond., 43. 2(>i. (1931), Experiments on growth and inhibition. Part i, Proc. roy. Soc. B, 108, 209, (1937), On the nature of correlative inhibition. New Phytol. 3, 283, (1938), On the upward inhibiting effect of auxin in shoots. New Phytol. 37, 173, (1939a), A second factor involved in inhibition by auxin in shoots. New Phytol. 38, 210, (i939i). An inhibitor of growth extracted from pea leaves. Nature, Lond., 144, 90, SNOW, M, & SNOW, R, (1937), Auxin and leaf formation. New Phytol. 3, i, STEWART, W, S, (1939), A plant growth inhibitor and plant growth inhibition, Bot. Gaz. ioi, 91, STEWART, W, S,, BERGREN, W, & REDEMANN, C, E, (1939), A plant growth inhibitor. Science, N,S, 89, 185, STOUGHTON, R, H, & PLANT, W, (1938), Regeneration of root cuttings as influenced by plant hormones. Nature, Lond., 142, 293, THIMANN, K, V, (1937), On the nature of inhibitions caused by auxin, Amer. J. Bot. 24, 407, (1939), Auxin and the inhibition of plant growth, Biol. Rev. 14, 319, VAN OVERBEEK, J, (1938), Auxin distribution in seedlings, etc, Bot. Gaz. 100, 133, ViEHMANN, H, (1939), Untersuchungen (iber die chemotropische Wirkung organischer Sauren, y. wiss. Bot. 87, 408, WENT, F, W, (1938), Specific factors other than auxin affecting growth and root formation. Plant Physiol. 13, 55, (1939)- Some experiments on bud growth, Amer. J. Bot. 2, 109,

FURTHER EXPERIMENTS ON PLAGIOTROPISM AND CORRELATIVE INHIBITION

[254] FURTHER EXPERIMENTS ON PLAGIOTROPISM AND CORRELATIVE INHIBITION BY R. SNOW Fellozv of Magdalen College, Oxford From his excellent study of correlative inhibition in Araucaria excelsa Massart (1924)