AUXIN CONTROL OF ABSCISSION. I. EXPERI­ MENTS WITH ERVATAMIA DIVARICATA BURKILL., V AR. FLORE-PLENO

M. AcHARYYA CHOUDHURI and S. K. CHATTERJEE

Department if , Burdwan University, Burdwan, West Bengal

SUMMARY

The present study aims to analyse the effects of on abscission of of Ervatamia divaricata Burkill., var. flare-plena (Apocynaceae), with special reference to the structure of different auxins and their relation to abscission activity. Auxins promote and inhibit abscission of leaves, the effect is less pronounced in older leaves than in younger ones. The inhi­ bitory effect of A-stage of leaf cannot be traced in B or C stages of leaves of 3-node twigs. This indicates a lesser degree of control of abscission in older leaves. This is also confirmed in experiments with 2-node twigs which are of different physiological maturation. The occurrence of two distinct physiological steps in the process of abscission of A-stage of leaves has been established. Auxins inhibit the first step and promote the second step of this stage of leaf. Absence of promotion in older leaves (B and C stages) indicates that the ageing of leaves has decreased the sensitivity of the second step towards auxins. The increasing requirement of induction period to cause 50 per cent abscission of the debladed petioles in spring and summer months will suggest a possible correlation of the natural occurrence of two steps of abscission with the metabolic activities of the leaves. In winter months, weaker metabolic activities may lead to an earlier completion of the first step, whereas in summer months the first step of abscission of de bladed petioles is sufficiently prolonged. It appears, that the second step of abscission gradually loses sensitivity towards auxin when the leaves enter winter months of the year. An analysis of the results of the present study carried out in different seasons will point out an involvement of auxins in the abscission process during different seasons. ABSCISSION OF LEAVES 63

The promotive effects of IPA and NOA may be due to the slow transport rate; it is likely that these auxins reach the abscission zone sufficiently later and cause promotion by their effects on the second step. A distinct gradation in the activity of phenoxy auxins (e.g., 2, 4-D; d-2, 4-DP; l-2, 4-DP; and 2, 4, 5-T) having different spatial configurations, has been established by separately following their inhibition and promotion effects on two steps of abscission. An analysis of their effects points out that 2, 4-D is the strongest auxm in the series whereas PAA is the weakest auxin.

INTRODUCTION Abscission of leaves takes place by the formation of some specialized cells in the region of abscission zone. The physiologi­ cal basis of the formation of such cells is of special interest. The role of endogenous auxins in this phenomenon has been critically studied by a number of workers (Addicott and Lynch, 1951; Biggs and Leopold, 1958; Jacobs, 1958). Influence of amino acids on the abscission process has been studied by Rubinstein and Leopold ( 1962). Recently Chatterjee et al. (1965) and Chatterjee (1966) have stressed the significance of auxin/ balance on the natural ageing of leaves. ' - Attempts have also been made to isolate some abscission accelerating substances (Carns et al., 1959; Hall et al., 1961 ; Addicott, 1965). ' The auxin control of leaf abscission involves the Coleus occurrence of two distinct physiological steps (AcharyyaChaudhuri and Chatterjee, 1966). The present study aims to analyse the effect of various auxins on the abscission of Ervatamia divaricata Burkill., var. jlore-pleno and their abscission activities have been related to their structures. The abscission behaviour of leaves in different seasons has also been studied.

MATERIALS AND METHODS Healthy branches with fully expanded opposite decussate leaves of Ervatamia divaricata Burkill., var.jlore-pleno (Apocynaceae) were used in the abscission tests reported here. The physiological maturation of leaves as reported by the present authors, was also considered in the present study. 'A' represents the apical or the youngest pair of leaves, 5-8 days old; 'B' represents the middle pair of leaves, 15-18 days old; and 'C' represents the third pair of leaves, 30-35 days old. In 64 M. ACHARYYA CHOUDHURI AND S. K. CHATTERJEE addition, two-node twigs were also used in some of the experi­ ments. The physiological age of the youngest pair (A,) in such a twig varied from two to three days and that of the second pair (B,) varied from 10-12 days. The leaves were debladed and 1 em length of the petioles was left on the main stem. The abscission times (time required for 50 per cent of the debladed petioles to abscise) for the de bladed petioles of different nodes were separately recorded in each case. The following auxins were used Indole-acetic acid (IAA) Indole-propionic acid (IPA) a-Naphthalene acetic acid (NAA) a-Naphthoxy acetic acid (NOA) Phenoxy acetic acid (P AA) 2, 4-Dichlorophenoxy acetic acid (2, 4-D) 2, 4, 5-Trichlorophenoxy acetic acid (2, 4, 5-T) d-2, 4-Dichlorophenoxy-2-propionic acid (d-2, 4-DP) l-2, 4-Dichlorophenoxy-2-propionic acid (l-2, 4-DP) Different concentrations of IAA, IPA, NAA and NOA were applied in lanolin paste to the freshly cut ends of the de bladed petioles of both three-node and two-node twigs and the abscission times were recorded. In the experiments with phenoxy auxins, only A-stage of leaf of three-node twigs was considered. In the studies relating to the occurrence of two physio­ logical steps in the process of abscission, the method described by Rubinstein and Leopold (1963) and modified by the present authors ( 1966) had been followed. In seasonal studies, an initially inhibitory concentration of NAA (0·25 per cent) was applied distally to the petioles after 12, 14, 16 ...... 36, 38 and 40 hours of deblading. These experiments were conducted at different times of the year. In studies with phenoxy auxins, two sets of experiments were performed. In the first set, 0 · 25 per cent of each of the auxins was applied to the freshly cut ends ofthe petioles of A-stage of leaf and later the source of auxins was removed by trimming off 2 mm portion of the petioles after stipulated periods. In the second set of experiments, the debladed petioles were subject­ ed to a sufficiently prolonged induction period (36 hours) and later 0 · 25 per cent of each ofthe auxins was applied distally for 2, 4, 8 and 12 hours. The abscission times were noted as usual. The were maintained on a 12-hour photoperiod at a temperature of 25°-27°C and the relative humidity of l ABSCISSION OF LEAVES 65

70-80 per cent. Abscission readings, i.e. the detachment of the petioles from the main stem under slight pressure, were recorded at 12-hour intervals and the time taken for 50 per cent abscission was calculated. The experiments were replicated at least thrice with comparable results.

RESULTS

The effects of IAA, IPA, NAA and NOA on abscission of debladed petioles of three-node and two-node twigs are presen­ ted in Figs. la and 1b. Both IAA and NAA inhibited abscission of debladed petioles of A-stage of leaf markedly. Complete inhibition of abscission was obtained with IAA in concentrations of 0 · 25 per cent and 1 · 0 per cent and with 1·0 per cent NAA only. In case of two-node twigs, abscission of debladed petioles of both A, and B, stages of leaf was inhibi­ ted completely by IAA (0 · 25 per cent and 1 · 0 per cent) and by NAA (1·0 per cent). Partial inhibition was obtained with 0·25 per cent NAA, 0·062 per cent of both IAA and NAA and

• CEI • = 113111 -80 ' ..

. ,6Q .. 0 ...... +40 .. .. 1 ~~~ ABC A,B, .\BCA,B, ABC A,B, ABC A,B, -20 ""·'· •oc : ~ '' 'oc '" I -·40 IAA

-60

- -~._._____ - I ·O 0"25 o·062 O·OI5 I ·O 0•25 0·062 o·OI5 Concentration (per cent) Fra. Ia. Effects of different concentrations ofiAA and NAA on abscission times for 50 per cent of the debladed petioles of the two types of twigs. (Solid bars indicate the abscission times for the petioles of three-node twigs and dotted bars indicate the abscission times for two-node twigs.) >14 Control times for 50 per cent abscission of th(debladed petioles of A -stage of leaf - 96 hours B - ...... - 88 hours C - ...... - 78 hours A 1 - ••••••••••• - 98 hours B1 - .• , •••••••• - 90 hours 66 7\L ACHARYYA CHOUDHURI AND S. K. CHATTERJEE

IPA NOA

A,H ABC .~.ll, ·\BC A,ll, ABC .\,B. ABC .\,B, ABC .\,8,

1 ·U 0·25 0·062 I 0 l) l'• 0·062 0 015 Concentration (per cent) FIG. I b. Effects of different concentrations of IPA and NOA on abscission for 50 per cent ofthc debladed petioles of the two types of twigs. (Solid bars for three-node twigs and dotted bars for two-node twigs.)

with 0·015 per cent IAA and NAA in A., A, and B, stages of leaves. The petioles of B and C stages of leaves of three-node twigs showed little or no change in their abscission behaviour in different treatments of these t\VO auxins. Both IPA and NOA were effective in promoting abscission. The promotion of abscission was obtained with all the concen­ trations used and the extent of promotion was maximum with 1·0 per cent IPA and NOA and minimum with 0·015 per cent of these auxins in A, B and C stages of leaves of three-node twigs and A, and B, stages of leaves of two-node twigs. Table I shows the effects of phenoxy auxins having different spatial configurations on the abscission of debladed petioles of

Table 1. Effects of dijferent concentrations ofphenoxy auxins of varying configurations on abscission times for 50 per cent of the deb laded petioles of.first node (A-stage of leaf)

Chang<' in abscission time in hours over controls at concentration,; Phenoxy auxins ···-·-- ···•-'""' --··-·------·------... l·O·;~ ..______, ... ·-----·------.. - ..------···----- '"·-··---- PAA 40 38 28 --18 2, 4-D 2, +.:i-T 1-0 30 2!i d-2, 4-Dl' -70 ')6 1-_; 4-DP ___ --~T:. 7'2 __ . -:-60 H7 ..,-32 Control time for 50 per cent abscission of the debladed petioles: 96 hr. >+=Complete inhibition L =Inhibition in hours over control .....-Promotion in hours over control I' ~~ ,,

tfll ~r ABSCISSION OF LEAVES 67 I. t A-stage of leaves. 2, 4-D caused total inhibition of abscission it with all the concentrations tried, whereas d-2, 4-DP produced l total inhibition in 1 · 0 per cent and 0 · 25 per cent concentrations only. Abscission was partially inhibited by l-2, 4-DP and 2, j 4, 5-T at all the concentrations and was promoted by PAA. The requirements of varying induction periods for 50 per cent promotion of abscission in A-stage ofleaves of three-node I twigs in different seasons are presented in Fig. 2a. The induc­ tion period was maximum in late summer (36 hours) and was minimum in winter (18 hours) and was medium during spring JI r

45 '/" 1-;"' 6 "0 .8 v1-; 0.. :: ·t 30 g I,. - .a"0 '-0

";:l ~ 15 t :: ~ ~

Season Fra. 2a. Seasonal variations in the requirement of induction period to promote 50 per cent abscission of the debladed petioles (A-stage) when an initially inhibitory cone. of NAA (0· 25 per cent) was applied distally.

S, = Mean value of induction period during winter (Dec.-Feb.) S2 = spring (Mar.-May) Ss = late summer (June-Aug.). (26 hours). When a constant induction period of 40 hours was applied to the explants in different seasons, the extent of promotion was maximum during late summer (66 hours) and minimum during winter months-42 hours (Fig. 2b). The effects of 0 · 25 per cent of different phenoxy auxins when applied for different durations just after deblading, J ' 68 !\1. ACHARYYA CHOt'DHURl AND S, K. CHATTERJEE

0 U'l 'c 75

60 .g ~ 45 ::...\..,

30

15

~ ·• Season FIG. 2b. Seasonal variations in the extent of promotion of 50 per cent abscission of the debladed petioles (A-stage) when an initially inhibitory cone. of NA.\ (0· 25 per cent) was applied distally after a constant induction period of 40 hours.

S ~ ;vit-an Yalue of the extent of promotion during winter s21 ~' . . spring s3 = .. late summer. on abscission of A-stage of leaves are shown in Table II. 2, 4-D showed a gradual increase in the extent of inhibition of abscission which was complete in 12 and 16 hours of treatments. d-2, 4-DP; l-2, 4-DP; and 2, 4, 5-T induced partial inhibition of abscission in 8,12, and 16 hours of treatments and the extent of inhibition successively decreased. P AA clearly promoted abscission which increased with the duration of treatment. Table III shows the effects of phenoxy auxins when applied after 36 hours of induction period for different durations. Abscission of A-stage ofleaves was promoted in all the treatments and the effect was maximum with 2, 4-D and minimum with PAA, the response to other auxins falling in between. d-2, 4-DP; l-2, 4-DP; and 2,4, 5-T also produced promotion of abscission in different treatments but the extent of promotion was successively reduced. ABSCISSION O'F LEAVES G9

Table II. Effects of different phenoxy auxins on inhibition ( +) or promotion ( - ) of 50 per cent abscission of the debladed petioles (A-stage) over control during varying induction periods

Extent of inhibition ( +) or promotion ( --) after different Phenoxy auxins durations (hr) of auxin application (hr)

2 4 8 12 16

PAA 4 --20 -28 30 35 2, 4-D -r8 +30 +44 -T -)- '-+ 2, 4, 5-T -2 0 +24 +26 +30 d-2, 4-DP 0 0 +34 +54 +60 l-2, 4-DP 0 0 -t-26 +40 +48

·--·~-~------·--·------Control times for 50% abscision of the debladed petioles of A-stage of leaf: 98 hr. ( + + indicates complete inhibition of abscission.)

Table III. Effects of different phenoxy auxins on promotion ( - ) of 50 per cent abscission of the debladed petioles (A-stage) over control when applied for different duration after 36 hours of induction period

Extent of promotion H when applied for different Phenoxy auxins durations (hr)

2 4 8 12 PAA 0 4 12 -30 2, 4-D 10 --36 56 -66 2, 4, 5-T 0 10 -20 -48 d-2, 4-DP -6 -25 31 - 36 1-2, 4-DP -4 -17 -20 -33

Control times as indicated in Table II. l DISCUSSION Both promotion and inhibition of abscission of leaves by auxins were reported in bean by Gaur and Leopold ( 1955) and Biggs and Leopold ( 1958), and in cotton by Addicott et al. ( 1955). Chatterjee et al. ( 1955) analysed the effects of auxins on abscis­ sion of bean leaves of varying ages and established that auxin effects were less pronounced in older leaves than in younger ones. In the present study with three-node twigs, the inhibition of abscission of A-stage of leaf could not be traced in B or C stages of the same. This indicated the possibility of a lesser degree of auxin control of abscission in older leaves. Experiments with two-node twigs confirm this observation. 70 M. ACHARYYA CHOUDHURI AND S. K. CHATTERJEE

The presence of two distinct physiological steps in the process of bean leaf abscission was shown by Rubinstein and Leopold (1963). Similar studies extended by the present authors in Coleus leaf abscission (1966) also indicated that the abscission process involved two distinct physiological steps of which the first step could be inhibited and the second step could be promoted by auxins. In the present study with Ervatamia the occurrence of similar steps in the process of abscission of debladed petioles has been followed with NAA and other phenoxy auxins. There was no inhibition of abscission of older leaves (B or C stages) when auxins were applied on the cut ends of the petioles. This will point out that the leaves are no longer in the first step. On the contrary, the absence of distinct promotion in these leaves will indicate that the leaves which were supposed to be in the second step, have become less sensitive towards auxins. It is likely that the natural process of ageing in these leaves has decreased the sensitivity of the second step of abscission towards auxins. This is in complete agreement with the observa­ tions of Chatterjee and Leopold ( 1955) with bean leaves where a decrease in overall sensitivity of abscission process towards auxins with increase in leaf age has been shown. The inhibition of abscission of leaves was erased and there was promotion when NAA was applied after a suitable induc­ tion period. The increasing requirement of this induction period to cause 50 per cent abscission of the debladed petioles in spring and summer months will suggest a possible correlation of the natural occurrence of two steps of abscission with the meta­ bolic activities of the leaves. In winter months, weaker metabolic activities may lead to an earlier completion of the first step whereas in summer months the first step of abscission of de bladed petioles is sufficiently prolonged. By examining the extent of promotion caused by the application of NAA after 40 hours of induction period in different seasons, it appears that the second step of abscission gradually loses sensitivity towards auxin when the leaves enter in winter months of the year. Different authors (Myers, 1940 and Kaushik, 1965) found that IAA was more effective in inhibiting abscission in summer than in winter. Their results might be interpreted in terms of weaker sensitivity of the abscission process towards auxin in winter months. An analysis of the results of the present study carried out in different seasons will point out a similar involvement of auxins in the abscission process during different seasons. .\.BSCISSION OF LEA\'ES i I

Th(' promotive effects of auxins, like IPA and NOA on abscission of debladcd petioles and the complete absence of inhibition in these cases lead to some interesting speculation regarding the two step hypothesis of abscission. It can be argued that due to the slow transport rate, these auxins reach the abscission zone sufficiently later and cause promotion by their dfects on the second step. Among the five phenoxy compounds used in the present investigation the effects caused by 2, 4-D; d-2, 4-DP; l-2, 4-DP; and 2, 4, 5-T will suggest that all these substances possess consi­ derable auxin activity and 2, 4-D is the strongest in the series. A somewhat similar indication has been put forward by Chatterjee et al. (1963) in their studies with bean leaf explants. Abscission effects caused by P AA are, however, contradictory to their observation in the sense that this weak auxin fails to cause even slightest inhibition when applied during the first step. On the other hand, PAA induces promotion and it can be argued that a variety of other chemical agents (Gaur and Leopold, 1955 and Biggs, 1957) which are not at all auxins cause promotion of abscission. Weak auxin activity of PAA can, however, be tested if it is applied in the second step of abscission. When different phenoxy auxins are applied after 36 hours of deblading 2, 4-D appears to have maximum auxin activity and PAA the minimum. A distinct gradation in the activity of phenoxy auxins having diflerent structural configurations ha:; been, thus, established by separately following their inhibition and promotion effects on two steps of abscission. An analysis of their effects will point out that auxins are playin~ a very significant role in the control of abscission of leaves.

ACKNOWLEDGEMENT The authors wish to express their deep sense of gratitude to Professor A. C. Leopold for going through the manuscript and for his valuable suggestions. Sincere thanks are also due to Professor P. N. Bhaduri, Head of thr. Department of Botany, University of Burdwan.. for providing laboratory and other facilities and to Dr. S. C. Dutta, Lecturer in Botany, University of Calcutta, for kindly supplying a small quantity nf 2,4,5-T used in the present investigation.

REFERENCES

Acharyya Choudhuri, :VI. and Chatte~jee, S.K. (1966). Abscission of lea\-cs. l'ror. 53rd Ind. Sc. Gong. Asscn. I Abstract\. Part III, 252. 72 M. ACHARYYA CHOVDHVRI AND S. K. CHATTERJEE

Acharyya Choudhuri, M. and Chatterjee, S. K. ( 1966). Studies on the occurrence of two physiological steps in the abscission process of Coleus leaves. Scienc< and Culture. (in Press). Addicott, F.T. (1965). Physiology of abscission. Handbuch der Pflanzenphysio­ logie, XV, Part II, 1094-1122. ----and Lynch, R.S. (1951). Acceleration and retardation of abscission by indole acetic acid. Science, 114: 688-89. Biggs, R.H. (1957). Physiological basis of abscission in plants. Doct. Thesis, Purdue Univ., Lafayette, Indiana. ---- and Leopold, A.C. (1958). Two-phase action of auxin on abscission. Amer. Jr. Bot., 45: 547-51. Carns, H.R., McMeans, J.C. and Addicott, F.T. (1959). An abscission accelerating hormone in cotton and some of its interactions with auxins and gibberellic acid. (Abstract), Proc. 9th Internal, Bot. Gong., 2: 60. Chatterjee, S.K. (1966). Analysis of the effects of extracts of ageing leaves on the abscission process. Ind. Jr. Physiol. 9( l): 52-58. ---- and Leopold, A. C., (1965). Changes in abscission process with ageing. Plant Physiol., 40: 96-101. ---- and ---- (1963). Auxin structure and abscission activity. Plant Physiol., 38(3): 268-73. Gaur, B.K. and Leopold, A. C. (1955). The promotion of abscission by auxin. Plant Physiol., 30: 487-90. Gawadi, A.G. and Avery, G.S. Jr. (1950). Leaf abscission and the so-called 'abs­ cission layer'. Amer. ]r. Bot., 37: 172-80. Hall, W.C., Herrero, F.A., and Katterman, F.R.H. (1961). Leaf abscission in cotton. IV. Effects of a natural promoter and amino-acids on abscission in coty­ ledonary node explants. Bot. Gaz., 123: 29-34. Jacobs, \,Y.P. ( 1958). Further studies of the relation between auxin and abscission of Coleus leaves. Amer. Jr. Bot., 45: 673-75. Kaushik, M.P. (1965). Exact substitution of leaf blade by £-indolyl acetic acid with respect to the retardation of abscission of debladed petioles of Coleus Blumei Benth. Ind. Jr. Plant Physiol., 8( 1): 23-35. Myers, R.M. (1940). Effects of growth substances on the abscission layer in leaves of Coleus. Bot. Gaz., 102: 323-38. Rubinstein, B. and Leopold, A.C. (1962). Effects of amino-acids on bean leaf abs­ cission. Plant Physiol., 37: 398-401. ---- and (1963). An analysis of auxin control of abscission. Plant Physiol., 38: 262-67.