Physiological Modes of Brassinolide Action in Cucumber Hypocotyl

Chapter 21 Physiological Modes of Brassinolide Action in Cucumber Hypocotyl Growth Masayuki Katsumi

Downloaded by UNIV OF ARIZONA on January 8, 2013 | http://pubs.acs.org Publication Date: November 4, 1991 | doi: 10.1021/bk-1991-0474.ch021

Biology Department, International Christian University, Osawa, Mitaka-shi, Tokyo 181, Japan To show that the modes of brassinolide (BR) action in the control of seedling growth are unique and different from those of auxin (IAA) and gibberellin (GA4), experimental results obtained with hypocotyls of light-grown cucumber seedlings are described The stimulating effect of BR on the elon gation of hypocotyl sections is characteristic in terms of its very low effec tive concentration range, pH independence, the modes of interactions with other hormones, the effects of inhibitors, etc. The most interesting action of BR is to reinforce seedlings against cold stress. Since the first isolation and identification of brassinolide (BR) from rape pollen by Grove et al in 1978 (1), a wide occurrence in the plant kingdom of related steroidal compounds which are now collectively called brassinosteroids, as well as BR itself, has been confirmed (2). Brassinosteroids have been demonstrated to elicit various pronounced effects on plant growth and development (3), and may now be considered as comprising an entirely new class of plant hormones in terms of their physiological modes of action and chemical structures as well. Brassinosteroids stimulate the growth of seedlings and the elongation of stem segments (4-9), the expansion of etiolated cotyledons (6), the enlargement of cultured cells (10), the growth of callus (11), pollen tube elongation (12), lamina joint bending(73,14), epinasty (75), leaf senescence (6), proton secretion (16-20), ethylene formation (21), photosynthetic activity (22) and both nucleic acid and protein syntheses (23). Thus, they show activities similar to those of auxin, gibberrellin and cytokinin. However, their physiological modes of action are unique and distinctly different from those of the other hormones, as will be explained later. In this Chapter, results of experiments on the analysis of the modes of brassinolide (BR) action in growth stimulation are described, particularly, as compared with those of auxin (IAA) and gibberellin (GA4). The experiments were carried out in most cases by using hypocotyl sections of light-grown cucumber (Cucumis sativus L . cv.Aonagajibai and cv, Spacemaster) seedlings which have been studied in detail for their physiological responses to auxin and gibberellin (24). Effects of BR on Intact Seedlings Hypocotyl Elongation. Although BR applied to the apex has been reported to have no effect on the hypocotyl growth of intact mung bean seedlings (3), it can elicit a distinct growth response in cucumber seedlings. BR is almost a hundred times as active as IAA which has also been shown to have the same effect in the same material (26). The activity of BR at the dosage levels of 10 -100 ng/plant is approximately the 0097-6156/91/0474-0246$06.00/0 © 1991 American Chemical Society

In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Physiological Modes of Action in Cucumbers

same as that of G A 4 which is the native physiological gibberellin of cucumber and which very actively stimulates hypocotyl elongation (27), but at higher dosages G A 4 is much more active than BR. Hypocotyls responding to a high dosage of BR are slightly twisted and partly thickened, which resemble those responding to higher dosages of IAA. Such abnormal morphology due to BR treatment has also been reported for other plant materials (5). This morphological effect of BR may be due to ethylene formation induced by BR as is known for IAA. In this case BR acts similarly to IAA. Cotyledon Expansion. Cotyledon expansion is markedly stimulated by G A 4 and by BR as well, but to a lesser extent: the activity is about 1/100 that of G A 4 . On the other hand, IAA has practically no effect Dark-grown cucumber cotyledons have also been shown to expand when subjected to BR treatment (6). Since expansion of cotyledons and leaves of intact seedlings is a normal effect of gibberellin, in this case BR acts like gibberellin.


)

Adventitious Root Formation. IAA stimulates adventitious root formation in cucumber hypocotyl cuttings (28). On the other hand, BR has either no effect or is slightly inhibitory, while it stimulates hypocotyl elongation. Gibberellin is usually inhibitory. However, it has been reported that in mung bean cuttings, the formation of root primordia was not inhibited (29). Growth under Chilling Stress. In recent years, brassinosteroids have been evaluated for practical use in agriculture, especially in Japan and China. Of great interest is the effect of BR in reinforcing the resistance of plants against various external stresses such as high concentrations of salts, low temperatures, pathogens, agricultural chemicals, etc.(30). When cucumber seedlings, germinated and grown in the light at 25 C for 4 days, are subjected to cold treatment at 5 C for 3 days, the growth rate of the seedlings as measured by dry weight increase is markedly reduced during the cold period, and the recovery of the growth rate after transferring back to 25 C is very small for at least several days. However, when seeds are soaked with BR solution during imbibition, the growth of seedlings exposed to cold temperature is significantly greater than in controls (without BR treatment) (Figure 1 A). Since BR has practically no effect on the growth of seedlings which have not been exposed to cold temperature, this growth stimulation effect of BR can be attributed to its reinforcement of seedlings against chilling stress. Another parameter of the chilling effect is the decrease in chlorophyll content which is more sensitive than the reduction of dry weight increase. In seedlings continuously grown at a constant 25 C the chlorophyll content in the cotyledons increases as growth proceeds. On the other hand, in seedlings subjected to cold temperature, the content decreases distinctly, even after transfer back to 25 C. With BR treatment, however, the chlorophyll level before cold treatment is retained, and it increases after transfer back to 25*C (Figure IB). These results indicate that the growth stimulating effect of BR is more significant under conditions where normal growth is reduced, and suggest that BR may act to help seedlings maintain their normal cell activities under unfavorable temperature conditions. e

e

e

e

e

Hypocotyl Sections The Activity of BR. Five mm long hypocotyl sections excised below the cotyledonary node respond to both IAA and gibberellin, with the younger hypocotyl tissue being more sensitive to gibberellin, while older tissue is more sensitive to IAA (31). BR also stimulates the elongation of hypocotyl sections, and it is almost 100 times as active as IAA. The lowest concentration eliciting activity is O.lnM (Figure 2). Younger tissue is much more sensitive to BR (8). The activity of BR is not D H deoendent exceut at a verv acidic D H of 3.5. while that In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


248

BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS

400 314

c o " 300 o

307

| 200 |§

100

130

100 B

0.1

10 BR,

Figure 1. Effect of brassinolide on the growth (A) and cotyledon-chlorophyll content (B) of cucumber seedlings subjected to chilling stress, measured 5 days after chilling treatment. (Ochi, N . and Katsumi, M . unpublished)


21. KATSUMI


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of IAA is significantly affected by pH change. The activity declines at a pH lower than 7.0. Since IAA is a weak electrolyte and is transported into the cytosol through the plasma membrane in its dissociated form, the activity of IAA must be pH-dependent. On the other hand, BR is lipophilic and is a non-electrolyte. Therefore, its activity may not be affected by the pH of the medium. BR-induced elongation also differs from that induced by IAA in that the former is not affected but rather enhanced in the presence of 50mM sucrose in the light, while the latter is inhibited. A similar sucrose-enhancement has been shown for gibberellininduced elongation (24). In this case the mode of BR action is similar to that of gibberellin. Time-Course Response. The chronological effect of BR is characteristic. As shown in Figure 3, BR also shows a biphasic response pattern similar to that of IAA (32). Thefirstphase is presumably ascribed to acid growth. The unique pattern of BRinduced response is that the second phase lags behind that of the IAA-induced second phase by 6 h. This may indicate that the action of BR is not immediate as far as elongation is concerned. Effects of Inhibitors. BR-induced elongation of sections is inhibited in the presence of /?-chlorophenoxyisobutyric acid (PCIB), an antiauxin (8). The same effect has also been demonstrated in other plant materials (5,14). Kinetin is also an inhibitor of IAA-induced elongation (33), and inhibits BR-induced elongation (#).These facts suggest that the presence of endogenous auxin is necessary for BR-induced elongation.The fact that BR is more active in younger tissue may reflect a higher endogenous level of auxin in this tissue. As to whether or not BR affects the endogenous level of auxin is not clear and conflicting results have been reported (34,35). N,N'-Dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane bound ATPase, has been shown to strongly inhibit IAA-induced elongation of cucumber hypocotyl sections, while it has no effect on GA-induced elongation (36). DCCD markedly inhibits BR-induced elongation (8), suggesting that BR acts differently from GA, but similarly to IAA in this particular case. Interaction with Auxin and Gibberellin. Simultaneous Interaction. In the literature, the interaction of BR with auxin has been reported to be additive or synergistic depending on experimental systems and conditions (3). In cucumber hypocotyl sections, the interaction is definitely synergistic (8). The synergism is especially significant when the concentration of one of the two is suboptimal. The interaction at their optimal concentrations is synergistic only during the early period of incubation and becomes additive finally. A synthetic auxin, 2,4-D also interacts with BR similarly (8). On the other hand, the interaction of BR with GA is entirely additive (8). This has been confirmed in other plant materials (3). Interaction of BR with IAA in a Curvature Test. Cucumber hypocotyl sections with the epidermis of the one side of the square pillar peeled, respond to IAA and low pH very quickly by curvature (32). Curvature toward the non-peeled side (+) occurs within a few minutes and this represents acid growth, while curvature toward the peeled side (-) in response to IAA occurs within 30 minutes, and the latter reflects epidermis-dependent growth (Figure 4). The maximum point of plus curvature indicates that the growth rates of both peeled and non-peeled sides have equilibrated, and the point where a given curve crosses the line zero line of curvature is the point at which negative curvature starts. As shown in Figure 4, BR alone induced a slightly positive curvature but has no further effect until 6 h, corresponding to the lag period of


250


9.0-


BR

Figure 2. Comparison of the activities of brassinolide, IAA and G A 4 in the stimulation of hypocotyl sections during 24 hr treatment

0

2

4

6

8 10 12 14 16 18 20 TIME, h

Figure 3. Time-course responses of hypocotyl sections to brassinolide and IAA.


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the time course curve (Figure 3). In thisfigure,the curve starts to bend toward the negative side at 6 h. In fact, if incubation is continued beyond 6 h, BR-induced negative curvature is observed. Simultaneous application of BR and IAA results clearly and drastically in synergism. The turning point of BR+IAA is much earlier than that with IAA alone, and the degree of curvature is also larger for BR+IAA. Sequential Interaction. Synergism between two hormones is sometimes more clearly observed in sequential treatments of the two hormones. Thus, GA pretreatment always results in synergistic enhancement of IAA-induced elongation of hypocotyl sections (57). Sequential interaction between BR and IAA is very similar to that between GA and IAA (8). Sections pretreated with BR for 2 h respond to IAA synergistically. The reverse order treatment is rather inhibitory as is the case for GA. Therefore, both BR and GA seem to sensitize hypocotyl sections to a later response by IAA. These facts suggest that cells which have already been initiated to elongation by IAA are not the target cells of BR/GA. Although BR and GA behave very similarly in their relationship to IAA, the mechanisms of how the two hormones act are probably differentfromeach other. First, the GA-pretreatment effect can be reduced by a high concentration of mannitol, while the BR pretreatment effect is not much affected by mannitol. Second, the GApretreatment effect is diminished by washing pretreated sections before IAA treatment, while the BR pretreatment effect is not (8). Third, the interaction of BR and GA in their pretreatment effects is simply additive (8).

Proton Secretion. Auxin is known to stimulate proton secretionfromthe cytosol to the cell wall matrix. BR also does the same in cucumber hypocotyl sections as has been reported for other plant materials (16-20). When sections with the epidermis peeled off are incubated in a weakly alkaline buffer, the pH of the buffer drops considerably, indicating that protons are secretedfromthe tissue to the medium. BR at 10 nM and IAA at 10 |iM are almost equally effective (Figure 5). The interaction of BR and IAA is rather inhibitory at the early period of incubation. However, proton secretion continues longer in the presence of both BR and IAA, and finally exceeds those by BR or IAA alone. BR behaves similarly to IAA in this effect.

Conclusion The experimental results on BR behavior described above are summarized in Table I in comparison with those of IAA and GA. BR elicits physiological effects that are very similar to those of other plant hormones, in particular, auxin and GA. However, the behavior of BR is unique and different from those of the others. The facts that BR modifies plant growth by itself or in association with other hormones, that its effective concentration is very low (lOnM), that it has different modes of action, and that it occurs widely in the plant kingdom strongly indicate that BR is a new plant hormone which is probably essential for plant growth and development.



252


0

1

2

3

4

5

6

Time, h Figure 4. Time-course responses of one-side peeled hypocotyl sections to brassinolide, IAA, IAA+brassinolide and acidic pH.

Figure 5. Effects of Brassinolide and IAA on proton secretion from peeled hypocotyl sections.



young

inhibitory inhibitory

promotive inhibitory

Sucrose (50mM) in the light

promotive promotive inhibitory

Hypocotyl growth

Cotyledon expansion

Advent, root formation

promotive

slightly inhibitory

promotive

inhibitory

DCCD

antagonistic

inhibitory inhibitory

Kinetin

Intact Seedlings

old

inhibitory

highly promotive

highly promotive

no effect

promotive

inhibitory

inhibitory

synergistic slightly inhibitory

^

additive

young

synergistic

synergistic

synergistic

old

>100|iM

>100nM

GA4

synergistic

old

10(1 M

lOOnM - 100|aM

IAA

Antiauxin

Pretreatment for BR/GA

Pretreatment for IAA

Interaction of IAA with

Interaction of BR with

young >•

IOO11M

Optimal concentration

Tissue age-Response

O.lnM - lOnM

Effective cone, range

hypocotyl Sections

BR

Table I. Comparison of the Modes of Actions of BR, IAA and G A 4


254


Acknowledgment The author wishes to thank ZEN-NOH Agricultural Technical Center, Kanagawa for their kind supply of brassinolide and for providing us with a research grant.


Literature Cited 1. Grove, M.D.; Spencer, G. F.; Rohwedder, W.K.; Mandava, N.B.; Worley, J.F.; Warthen, J.D., Jr.; Steffens, G.L.; Flippen-Anderson, J.L.; Cook, J., Jr. Nature 1979, 281, 216. 2. Yokota, T.; Takahashi, N. In Plant Growth Substance 1985; Bopp, M., Ed.; Springer- Verlag: Berlin/Heidelberg, 1986; pp.124-38. 3. Mandava, N.B. Ann. Rev. Plant Physiol. 1989, 39, 23. 4. Meudt, W.J. In Ecology and Metabolism of Plant Lipids ; Fuller, G.; Nes, W.D., Ed.; ACS Symposium Ser. No. 325 ; ACS : Washington, DC, 1987, pp.53-75. 5. Yopp., J.H.; Mandava, N.B.; Sasse, J.M. Physiol.Plant. 1981, 53, 445. 6. Mandava, N.B.; Sasse, J.M.; Yopp, J.H. Physiol. Plant. 1981, 53, 453. 7. Gregory, L.E.; Mandava., N.B. Physiol. Plant. 1982, 54, 239. 8. Katsumi, M. Plant Cell Physiol, 1985, 26, 615. 9. Sasse, J.M. Physiol. Plant, 1985, 63, 303. 10. Sala, C.; Sala, F. Plant Cell Rep, 1985, 41, 144. 11. Takematsu, T.; Takeuchi, Y.; Koguchi, M. Chem. Regul. Plants, 1983, 18, 2. (in Japanese) 12. Hewitt, F.R.; Hough, T.; O'Neill, P.; Sasse, J.M.; Williams, E.G.; Rowan, K.S. Aust. J. Plant Physiol. 1985, 12, 201. 13. Wada, K.; Marumo, S., Ikekawa, N.; Morisaki, M.; Mori, K. Plant Cell Physiol. 1981, 22, 323. 14. Takeno, K.; Pharis, R.P. Plant Cell Physiol, 1982, 23, 1275. 15. Schlagnhaufer,C.D.;Arteca, R.N. Plant Physiol. 1985, 78, 300. 16. Cerana, R.; Bonetti; A.; Marre, M.T.; Romani. G.; Lado, P.; Marre, E. Physiol. Plant. 1983, 59, 23. 17. Romani, G,; Marre, M.T.; Bonetti, A.; Cerana, R.; Lado, P.; Marre, E. Physiol. Plant, 1983, 59, 528. 18. Cerana, R.; Colombo, R.; Lado, P. Physiol. Veg. 1983, 21, 875. 19. Cerana, R.; Lado, P.; Anastasia, M.; Ciuffreda, P.; Allevi:, P. J. Plant Physiol. 1984, 114, 221. 20. Cerana, R.; Spelta, M.; Bonetti, A.; Lado, P. Plant Sci. 1985, 38, 99. 21. Mandava, N.B. Physiol. Plant. 1983, 59, 539. 22. Braun, P.; Wild, A. J. Plant Physiol. 1984, 116, 189. 23. Kaeinich, J.F.; Mandava, N.B.; Todhunter, J.A. J.Plant Physiol. 1985, 120, 207. 24. Katsumi, M.; Kazama, H. Bot. Mag. Tokyo 1978, Special Issue 1, 141. 25. Kazama, M.; Katsumi, M. Plant Cell Physiol. 1973, 14, 449. 26. Katsumi, M.; Purves, W.K.; Phinney, B.O. Physiol. Plant. 1964, 18, 550. 27. Nakayama, M; Yamane, H.; Yamaguchi, I.; Murofushi, N.; Takahashi, N,; Katsumi, M. J. Plant Growth Regul. 1989, 8, 237. 28. Katsumi. M.; Chiba, Y.; Fukuyama, M. Physiol. Plant. 1969, 22, 993. 29. Gregory, L.E.; Mandava, N.B. Physiol. Plant. 1982, 54, 239. 30. Takematsu, T.; Takeuchi, Y.; Choi, C - D. Shokucho 1986, 20, 2. (in Japanese ) 31. Kazama, H.; Katsumi, M. Plant Cell Physiol. 1973, 14, 449. 32. Kazama, H.; Katsumi, M. Plant Cell Physiol. 1976, 17, 467. 33. Katsumi, M.; Kazama, H. Plant Cell Physiol. 1978, 19, 107. 34. Cohen, J.D.; Meudt, W.J. Plant Physiol. 1983, 72, 691. 35. Eun, J.-S.; Kuraishi, S.; Sakurai, N. Plant Cell Physiol. 1989, 30, 807. 36. Katsumi, M. Plant Cell Physiol. 1976, 17, 139. 37. Kazama, H.; Katsumi, M. Plant Cell Physiol. 1974, 15, 1307. R E C E I V E D May 14, 1991


Physiological Modes of Brassinolide Action in Cucumber Hypocotyl

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