Soybean Senescence and Pod Development'. Received for publication September 11, 1984 and in revised form January 28, 1985. LARRY D. NOODEN* AND ...
Plant Physiol. (1985) 78, 263-266
0032-0889/85/78/0263/04/$01.00/0
Effects of Morphactin and Other Auxin Transport Inhibitors on Soybean Senescence and Pod Development' Received for publication September 11, 1984 and in revised form January 28, 1985
LARRY D. NOODEN* AND S. M. NOODEN Botany Department, University ofMichigan, Ann Arbor, Michigan 48109-1048 number in soybean. In addition, these inhibitors may provide a tool for probing the role of auxin transport in regulating pod Because trijodobenzoic acid increases pod number, albeit variably, in development and leaf senescence processes. soybean (Glycine max), we tested other auxin-transport inhibitors. MorIn this study, we have used auxin-transport inhibitors to exphactins, especially methylchlorflurenol (MCF), were found to be very amine (a) potential involvement of auxin in regulating pod active (optimal concentration 10 micromolar) when sprayed onto the development and (b) the effect of increased pod number on total foliage. Applications at 1 week after the start of flowering were most seed yield. effective, producing a 40% increase in pod number with little inhibition (12%) of stem elongation. MCF increased the number of pods initiated MATERIALS AND METHODS (reaching 1 cm length) at least partially by prologing the initiation period, while pod abortion (failure of pods > 1 cm long) remained low. Soybean plants (Glycine max [L.] Merr. var Anoka) were Generally, MCF did not increase seed yield (dry weight/plant) more, but grown indoors in pots ofsoil as described by Lindoo and Nooden smaller seeds, were formed by the treated plants. The promotive effect (16) and Derman et al. (5). After the second trifoliate leaf had of MCF on pod initiation seems to be independent of its inhibition of expanded (3-4 weeks), the plants were placed in environmental stem elongation, which is insignificant at 10 micromolar. MCF delayed control chambers (27°C day, 22°C night, 10-h day with lighting pod maturation by 3 to 4 days, while foliar yellowing, blade abscission, given in References 5 and 16). The visual procedures for scoring and petiole abscission were retarded by 2, 4, and 2 days, respectively. the yellowing of the leaves and pod development are discussed MCF has only a small effect on senescence and that could be indirect, by Lindoo and Nooden (16). due to a delay in pod development. Other auxin-transport inhibitors The treatment solutions containing 0.05% (v/v) Tween 80 tested, including N-1-naphthylphthalamic acid, produced little or no in- were sprayed onto the foliage almost to incipient runoff. Care crease in pod number, however, 0.1 millimolar 5-(2'-carboxyphenyl-3- was taken to avoid spraying the pods. Each treatment group phenylpyrazole caused a 27% increase. These results implicate auxin as contained five plants. a potential regulator of pod development, and they show that soybean CF and MCF were donated by Dr. H. Itzel of Celamerk Gmbh. seed yield is not simply sink limited. and Co., Ingelheim am Rhein, West Germany; CPD, CPP, and PBA by Dr. G. F. Katekar, CSIRO, Canberra, Australia; and NPA by Uniroyal Chemical, Naugatuck, CT. It should be noted that heating or prolonged storage of the morphactin solutions caused loss of activity. ABSTRACT
TIBA2, an inhibitor of auxin transport (22), can increase pod number in soybean; however, this effect and any consequent yield improvement are variable (10). DPX 1840, another auxin transport-inhibitor (2), can also raise pod number in soybean (1, 2). The discovery of a broad array of other auxin-transport inhibitors including morphactins (22), N-naphthylphthalamic acid (22), and others (8, 14, 15) has provided the means to further test the ability of auxin-transport inhibitors to increase pod ' Supported in part by a grant (901-15-35) from the United States Department of Agriculture Cooperative State Research Service under P.L. 89-106. We also acknowledge the University of Michigan Matthaei Botanical Gardens for maintaining these plants until they were 6 weeks old. 2Abbreviations: TIBA, 2,3,5-triiodobenzoic acid; Tween 80, polyoxethylene (20) sorbitan monooleate; CF, chlorflurenol or 2-chloro-9hydroxyflurene-9-carboxylic acid; CPD, 1-(2' carboxyphenyl)-3-phenylpropane-1,3-dione; CPP, 542'-carboxyphenyl)-3-phenylpyrazole; DPX 1840, 3,3a-dihydro-24p-methoxyphenyl)-8H-pyrazolo (5,la) isoindol8-one; MCF, methylchlorflurenol or methyl-2-chloro-9-hydroxyflurene9-carboxylate; NPA, N-l-naphthylphthalamic acid; PBA, 24 1-pyrenoyl)benzoic acid. 263
RESULTS We attempted to increase pod set by spraying with 4 x 10-1 M TIBA in 0.05% Tween 80 at 1 week before flowering, at flowering, at 1 week after flowering started and when the most advanced pods were 1.0 cm long (data not shown). The earliest treatment produced stunted plants (fewer nodes) with more pods per node but not more pods per plant. These applications also delayed pod development and thereby, indirectly, senescence. The treatments given at flowering or 1 week later produced a small (015%) but variable increase in pod number, and this basically confirms the extensive published data (see 10). The morphactin, chlorflurenol, at 101 M gave similar though quantitatively magnified results. CF was most effective at 1 week after flowering began, but the magnitude was variable. At a higher concentration, 10-1 M, CF produced a greater increase in pod number, but total seed yield (dry weight) did not increase, because the seeds were smaller. CF delayed foliar senescence only to the extent that it retarded pod development. MCF was more effective and more consistent than CF. The optimum concentration was about I0-' M and the optimum time for application about 1 week after the start of flowering. Higher concentrations did not produce more pods. At 10-5 M MCF did not significantly reduce stem elongation (Table I), while higher
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Table I. Effect of-MCF, NPA, and Other Auxin-Transport Inhibitors on Pod Development and Stem Elongation In 0.05% (v/v) Tween 80. Applied to the leaves 1 week after the start of flowering. The data for MCF and the control are from the same plants as in Figures I and 2. SE are shown. Stem Length Pods/Plant Seeds/Plant Seed Dry Wt Seed Dry Wt Treatment
glplant
glseed
Control
116±4
26±2
57±2
13.0±0.4
0.23
MCF1O-5M
102±6
37±2
78±5
13.6±0.5
0.17
Control NPA 10-8M NPA 106M
116 ± 4 125±5 102±2
26 ± 1 29±2 24±2
57 ± 2 61 ±3 55±4
13.0 ± 0.4 12.9±0.5 11.7±0.8
0.23 0.21 0.22
Control CPD 104M CPP 104 M PBA 10-4M
103 ± 4 103±2 74 ± 4 96±4
30 ± 2 26± 1 38 ± 2 30± 1
72 ± 3 65±3 66 ± 6 72±2
14.1 ± 0.7 12.7±0.5 11.5± 0.2
0.20 0.20 0.17 0.19
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14.0±0.7
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parameters (Fig. 2), the same plants and time axes are shown in Figures 1 and 2. MCF did not alter the sequence of leaf changes which start with yellowing, followed by blade abscission and finally petiole abscission. MCF caused a brief delay in leaf yellowing (Fig. 2A), about 2 d (at 50% of leaves 2 1/2 yellow). Comparing these curves at the 50% point, blade abscission (Fig. 2B) is delayed about 4 d and petiole abscission (data not shown) about 2 d. A higher concentration (101 M) applied at 1 week after the start of flowering delayed pod development by 5 d, leaf yellowing by 4 d, blade abscission by 5 d, and petiole abscission by 20+ d. Of the other transport inhibitors, NPA was tested at 10-9 to 101 M and CPD, CPP, and PBA at 10-1 and 10' M, but only 10' M CPP produced a large increase in pod initiation (Table I).These treatments with other transport inhibitors, except 10' M CPP, did not significantly suppress stem elongation. Thus, we have no assurance that the chemicals were exerting any substantial physiological effect. cence
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32
40
48 56 64 SHORT DAYS
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FIG. 1. Effect of MCF on pod initation, pod abortion, and pod maturation (fruit maturity index). The foliage was sprayed once with IO-S M MCF in water plus 0.05% (v/v) Tween 80 at I week after the start of flowering. SE are shown as bars.
concentrations did. MCF (10-5 M) applied earlier also inhibited stem elongation and decreased pod number; application at 1 week before flowering decreased stem length by 69%, pod number by 74%, and seed yield by 56%. MCF at 10-5 M induced a substantial (40%) increase in pod number (Fig. 1) when applied at week after the start of flowering. This increase seems to be due mainly to prolongation of the period of pod initiation (Fig. 1). The fruit maturity index (which is a measure of the average developmental stage of the pods) indicates that MCF treatment caused some delay (3-4 d) during pod maturation. Since MCF treatment caused a similar delay in the fruit maturity index calculated for the 10 most advanced pods on each plant (data not shown), MCF actually retarded pod development rather than just shifting the average by adding younger pods. Pod abortion (failure of pods > I cm in length) did not offset the increases in pod load but remained at a fairly low level (about one pod per plant) in both the MCF-treated and control plants. Overall seed yield (dry weight) was not increased in the MCF-treated plants (Table I). Thus, the treated plants produced more but correspondingly smaller seeds.
DISCUSSION The observations that both TIBA and DPX 1840, which are inhibitors of auxin transport (22), can increase pod number on soybean, albeit variably, suggests that auxin flux may play a role in regulating pod number. Thus, it seemed worthwhile to determine whether or not other inhibitors of auxin transport can promote pod initiation in soybean. Ofthe auxin-transport inhibitors tested (CF, CPD, CPP, MCF, NPA, PBA, and TIBA) by application to the foliage of soybeans, MCF is clearly the most effective in increasing pod number. The promotive effect of morphactin on fruit development seems not to be unique to soybean; however, morphactin can also be inhibitory (3, 6, 17, 24). MCF apparently increases the number of pods initiated (reaching cm length) on soybean plants by prolonging the period of pod initiation. With or without the MCF treatment, abortion of pods-, 1 cm in length remains low; thus MCF does not act through prevention of pod abortion. Seed number is also increased, though not so much as pod number. Seed yield (dry weight/plant) does not increase with the number of pods and seeds; the treated plants produce more but smaller seeds. Thus, simply increasing pod number will not increase seed yield. Using a wide range of doses including some about equal to our maximum, Clapp (4) reported no increase in pod number (and possibly a decrease at the highest dose); however, his conditions and formulation were very different. Likewise, Dybing and Lay (6) tested several morphactins but found no yield
AUXIN TRANSPORT INHIBITORS AND POD DEVELOPMENT
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increase. TIBA inhibits soybean stem elongation and may alter apical dominance (10, 25). While this reduction in stem length can increase soybean yields through a decrease in lodging (1 1), the promotion of axillary outgrowth producing more flowers may also be a factor leading to increased pod number (25). Like TIBA, MCF and the other auxin-transport inhibitors studied here can interfere with stem elongation, but as with TIBA, that effect is not necessarily correlated with increased pod number. Although MCF (0.36-3.6 x 10' M) may inhibit Chl breakdown in Rumex leaf discs (7), a variety of effects, including promotion of Chl loss by CF and other morphactins, has been reported recently (7). Several morphactins may cause a slight inhibition of soybean leaf yellowing under field conditions (6), and we have observed that MCF (10-0 M) causes only a small delay in the yellowing and abscission of soybean leaves in environmental control chambers. Since any retardation of pod development could also be expected to retard leaf senescence (19, 20), the effect of MCF on senescence could be an indirect result of its action on pod development. At a high concentration (10' M), MCF caused a similar delay in pod development, leaf yellowing, and blade abscission. Petiole abscission was, however, inhibited much more, which seems contrary to the idea that abscission is promoted by reduced auxin flux (21). Still, foliar applications of auxin delay leaf yellowing in soybean and some other species (19, 21) suggesting that endogenous auxin levels may participate in regulation of foliar senescence and auxin flux from the leaf blade may control leaf abscission. MCF does not, however, seem to alter these processes beyond its effect on pod development except at high concentrations I0O M and then only petiole abscission, not blade abscission, shows pronounced delay. In this respect, the soybean seems to differ from cotton where auxin transport inhibitors promote leaf abscission ( 18) as would be expected. TIBA, DPX 1840, MCF, etc., are very different chemically, and no doubt, they have diverse physiological effects (nonspecificity), yet they share (a) an ability to inhibit auxin transport and (b) a capacity to increase pod number. These results imply, but do not prove, that auxin (possibly auxin from the leaves) may play a role in regulating pod number (apparently acting at an early stage). The apparent inability of NPA, CPD, and PBA to elevate pod numbers seems contrary to this idea; however, these compounds may not be inhibiting auxin transport here for any of several reasons including penetration. Quite a different line of evidence based on the ability of auxin applications to duplicate the repressive interactions between young reproductive structures on the same raceme (12) also implicates auxin, but this could be a different phenomenon. It is important to note that proposed auxin-mediated effects of Phaseolus vulgaris fruit (23) seem different and may reflect yet another difference between soybean and other species (19, 20). In any case, other studies on the involvement of auxin in the complex web of correlative controls operating in the reproductive soybean (20) seem warranted. LITERATURE CITIED 1. ANONYMOUS 1971. Experimental growth regulant DPX1840. Du Pont Information Bulletin. E. I. duPont de Nemours and Company, Inc., Industrial and Biochemicals Department, Wilmington, DE 2. BEYER EM 1972 Auxin transport: A new synthetic inhibitor. Plant Physiol 50:
0
32
40
64 56 48 SHORT DAYS
72
80
FIG. 2. Effect of MCF on leaf yellowing (A) and blade abscission (B). Same plants as in Figure 1. The vertical arrows indicate the time at which the 50% point (along the vertical axis) is reached for the control and MCF-treated plants, respectively.
322-327 3. CANTLIFFE DJ, RW ROBINSON, S SHANNON 1972 Promotion of cucumber fruit set and development by chlorflurenol. HortScience 7: 416-418 4. CLAPP JG JR 1975 Response of soybeans to a morphactin. Crop Sci 15: 157158 5. DERMAN BD, DC Rupp, LD NOODbN 1978 Mineral distribution in relation to fruit development and monocarpic senescence in Anoka soybean. Am J Bot 65: 205-213 6. DYBING CD, C LAY 1981 Yields and yield components of flax, soybean, wheat and oats treated with morphactins and other growth regulators for senescence delay. Crop Sci 21: 904-908
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7. DYBING CD, GL YARROW 1984 Morphactin effects on soybean leaf anatomy and chlorophyll content. J Plant Growth Regul 3: 9-21 8. GEISSLER AE, JL HUPPATz, GF KATEKAR 1975 Effect of substituted pyrazoles and related compounds on geotropism in cress seedlings. Pestic Sci 6: 441450 9. HARADA H 1967 Effects of morphactin on the negative geotropic response and leaf senescence, Naturwissenschaften 54: 95 10. HicKs DR 1978 Growth and development. In AG Norman, ed, Soybean Physiology Agronomy and Utilization. Academic Press, New York, pp 2744 11. HicKs DR, JW PENDLETON, WO SCoTT 1967 Response of soybeans to TIBA (2,3,5-triiodobenzoic acid) and high fertility levels. Crop Sci 7: 397-398 12. HUFF A, CD DYBING 1980 Factors affecting shedding of flowers in soybean [Glycine max (L.) Merrill]. J Exp Bot 31: 751-762 13. HUMPHRIES EC, U PETHIYAGODA 1969 Effect of morphactin on growth of potatoes. Ber Dtsch Bot Ges 3: 139-148 14. KATEKAR GF, AE GEISSLER 1977 Auxin transport inhibitors. II. 2-(1-pyrenoyl)Benzoic acid, a potent inhibitor of polar auxin transport. Aust J Plant Physiol 4: 321-325 15. KATEKAR GF, AE GEISSLER 1977 Auxin transport inhibitors. III. Chemical requirements of a class of auxin transport inhibitors. Plant Physiol 60: 826829 16. LINDOO SJ, LD NOODEN 1976 The interrelation of fruit development and leaf senescence in 'Anoka' soybeans. Bot Gaz 137: 218-223
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17. LUKASIK S 1975 Some observations upon the influence of morphactin IT3456 applied into the soil on the yield of tomatoes. Acta Agrobot 28: 43-55 18. MORGAN PW, Jl DURHAM 1972 Abscission: Potentiating action of auxin transport inhibitors. Plant Physiol 50: 313-318 19. NOODiN LD 1980 Senesence in the whole plant. In KV Thimann, ed, Senescence in Plants. CRC Press, Boca Raton, FL, pp 219-258 20. NooDoN LD 1984 Integration of soybean pod development and monocarpic senescence. Physiol Plant 62: 273-284 21. NOODEN LD, AC LEOPOLD 1978 Phytohormones and the endogenous regulation of senescence and abscission. In DS Letham, PB Goodwin, TJV Higgins, eds, Phytohormones and Related Compounds-A Comprehensive Treatise, Vol II. Elsevier/North-Holland Biomedical Press, Amsterdam, pp 329-369 22. SCHNEIDER EA, F WIGHTMAN 1978 Auxins. In DS Letham, PB Goodwin, and TJV Higgins, eds, Phytohormones and Related Compounds-A Comprehensive Treatise, Vol I. Elsevier/North-Holland Biomedical Press, Amsterdam, pp 29-105 23. TAMAS IA, CJ ENGELS, SL KAPLAN, JL OZBUN, DH WALLACE 1981 Role of indoleacetic acid and abscisic acid in the correlative control by fruits of axillary bud development and leaf senescence. Plant Physiol 68: 476-481 24. WEAVER RJ, RM POOL 1969 Effect of ethrel, abscisic acid, and a morphactin on flower and berry abscission and shoot growth in Vitis vinifera. J Am Soc Hortic Sci 94: 474-478 25. ZIMMERMAN PW, AE HITCHCOCK 1942 Flowering habit and correlation of organs modified by triiodobenzoic acid. Contr Boyce Thompson Inst 12: 491-496
