Effects of Brassinolide on Growth and Chilling Resistance of Maize

Chapter 19 Effects of Brassinolide on Growth and Chilling Resistance of Maize Seedlings Ruo-yun He, Guan-jie Wang, and Xue-shu Wang

Downloaded by UNIV OF ARIZONA on January 9, 2013 | http://pubs.acs.org Publication Date: November 4, 1991 | doi: 10.1021/bk-1991-0474.ch019

Department of Basic Science, Shenyang Agricultural University, Shenyang 110161, China

Maize seedlings were cultured in plastic pots containing pearlite or on filter paper and treated with brassinolide (BR)in the dark or in the light. The results showed that at 10 ppm BR promoted the elongation of coleoptiles and mesocotyls but retarded the growth of leaves and roots, whether in the dark or in the light. The meso cotyls of etiolated seedlings showed either twining or transverse geotropism when treated by higher concentrations of BR. At 10 -10 ppm, BR improved the greening of etiolated leaves at different temperatures, especially at lower temperature in light. BR also promoted the growth recovery of maize seedlings following chilling treatment. The physiological effect of BR seems to be that of both auxin and cytokinin, and BR as a steroid may act on biological membrane systems. 0

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It has been reported that brassinolide (BR) has some actions similar to auxin and cytokinin (i). In order to compare the physiological effects of BR in these respects and to determine if this is so, we studied three aspects as follows: 1.

Characteristic responses to brassinolide in maize seedling organs

2.

Effect of brassinolide on greening of etiolated maize seedlings under light

3.

Effect of brassinolide on growth recovery of maize seedlings after chilling

Our experiments were carried out mainly with maize (Zea mays L.) hybrids 171 x 330A and Danyu 13, provided by Shenyang Agricultural University and Shenyang Seed Company. Brassinolide (2a,3a,22i?,23/?tetrahydroxy-245'-methyl-B-homo-oxa-5a-cholestan-6-one) (Figure 1), a natural type synthesized by Fugisawa Pharmaceutical Co., Ltd., was 0097-6156/91/0474-0220$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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provided by the National Federation of Agricultural Co-operative Association (Zen-Non) of Japan. It was dissolved in 85% alcohol to make a 200-ppm solution and then diluted into various concentrations with water. Maize seedlings were cultured in plastic pots containing pearlite, or on filter paper, and were treated with various concentrations of brassinolide that ranged from l O ^ - l O ppm in the dark (etiolated seedlings) or in the light (green seedlings). Details of experimental methods are as follows. 0


Characteristic Responses of Maize Seedling Organs to Brassinolide Maize seeds were soaked and germinated at 25 ° C. When the radicles were about 1 cm, the seedlings were treated with various concentrations of brassinolide that ranged from l O ^ - l O ppm in the light and in the dark for 4 days. Following treatment, the lengths and dry weights of various plant parts were measured for both green and etiolated seedlings. The following conclusions were drawn from the data obtained (Figure 2): 0

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• Concentrations of brassinolide from IO" ppm to 10~ ppm had no significant effects on the growth of coleoptile, mesocotyl, and the first true leaf. (In fact, the mesocotyl of etiolated seedling was slightly promoted and the first true leaf was slightly retarded.) At 10~~ ppm, brassinolide showed a tendency to promote the growth of the second leaf in the light and in the dark. These results paralleled those obtained for barley (2) and wheat (3, 4), which indicated that brassins and brassinosteroid promoted the elongation of true leaves in monocotyledons. 2

• At 10° ppm, brassinolide promoted the elongation of coleoptiles and mesocotyls but retarded the growths of true leaves and roots, whether in the light or in the dark. • At 10° ppm, brassinolide increased the dry weight of seedling shoots (including mesocotyls) and decreased the dry weight of roots; thus, the root/top ratio decreased remarkably. We suggest that the organic matter stored in the endosperm was distributed mainly to coleoptiles and mesocotyls as seeds germinated and developed into seedlings, because brassinolide at 10° ppm inhibited leaves from growing. • The effects of brassinolide were similar in the light and in the dark, but the coleoptile and mesocotyl were elongated more and leaf growth was retarded more in the dark than in the light by brassinolide at 10° ppm. These results were in sharp contrast to those experimental results obtained with Avena coleoptiles (J) and with soybean and mung bean tissues (1, 6). Etiolated maize seedling mesocotyls elicited two responses to higher concentrations of brassinolide ( 1 0 - 1 0 ° ppm). The first response was transverse geotropism and the second was twining (Figure 3). The seedlings in light culture treated with brassinolide at 10° ppm had the same _ 1

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

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4

0 10' 10 10' 10'

1

4

6

0 10 10* IO'* 10

PWD

Concentration of BR (ppm) 1-Mesocotyl 2-Coleoptile 3- First leaf

4-Second leaf 5-Root-top ratio

Figure 2. Effects of brassinolide on the growth of organs in maize seedlings.



19. HE ET AL.


Figure 3. The characteristic responses of mesocotyls of etiolated maize seedlings to high concentrations of BR.


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transverse geotropism, but no twining growth response was observed. However, with brassinolide treatment at K T ppm and exposure to light, mesocotyls were not twined, nor was transverse geotropism noted. Transverse geotropism in stems is a characteristic response to ethylene. It has been reported that treatments with epibrassinolide at concentrations of 1—10 ppm accelerated ethylene production in etiolated mung bean hypocotyl segments by increasing A C C (1-aminocyclopropane-l-carboxylic acid) (7). Thus it is possible that the transverse geotropism and even the twining growth response evoked by brassinolide at 10"" -10° ppm were related to the production of ethylene. We cultured maize seedlings with various concentrations (200-4000 ppm) of Ethrel (2-chloroethylphosphoric acid) in the dark to ascertain if ethylene induced the same responses in corn. The results showed that mesocotyls had neither a transverse geotropism response nor a twining response. Earlier, we stated that both transverse geotropism and twining growth were the characteristic responses of maize mesocotyls to high concentrations of brassinolide. We now suggest that the transverse geotropism and the twining response are specific phenomena of brassinolide treatment and are not induced by ethylene in corn. In addition, the degree of mesocotyl twining response induced by brassinolide is different in different maize hybrids and is concentration dependent. For example, hybrid 171 x 330A is more sensitive than hybrid Danyu 13, with twining mesocotyls of 87.5% and 52.6%, respectively, upon treatment with 10° ppm of brassinolide, and the probability of twining was about 25% for hybrid 117 x 330A when treated with 10~~* ppm of brassinolide. Dong Jiao-wang (Beijing Agricultural University, in Proceedings of the 2nd National Plant Growth Substance Seminar, Shanghai, China, 1987; unpublished data) found that the primary roots of rice had greater twining when the brassinolide concentration increased from 10~ ppm to 10~"* ppm, but IAA, GA, and ABA (1-10 ppm) did not cause the rice roots to twine. Wang Yu-qin (Shanghai Plant Physiology Institute, in Proceedings of a National Plant Growth Substance Seminar, Nanjing, China, 1989; unpublished data) also found that rice primary roots twined with epibrassinolide treatment at 1 ppm. GA, auxin, and cytokinin did not induce twining; with four BR derivatives, however, the twining response was seen in primary roots of rice. We also found that young roots of wheat cultured in brassinolide also had twining. All the data presented so far indicate that the twining effect in roots or mesocotyls is a characteristic response to brassinolide or brassinosteroids. The mechanism whereby twining is induced by brassinolide and the significance of this phenomenon in bioassays have yet to be elucidated. 1


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Effect of Brassinolide on Greening of Etiolated Maize Seedlings under Light It has been reported (8-12) that brassinolide increases the chlorophyll content of several crops in the field, but the effect of brassinolide on the


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synthesis of chlorophyll in etiolated leaves has not yet been elucidated. Therefore, we studied the effect of brassinolide on the greening of etiolated maize leaves at different temperatures in light and compared the response of maize with the response of cucumber. Etiolated seedlings were grown in brassinolide solution and in water as control. The first true leaves (sometimes also the second leaves) were detached and greened in the light, and then the chlorophyll content was determined. The results are shown in Tables I and II.


Table I. Effect of BR on Greening of Etiolated Maize Leaves Exposed to Light (Hybrid Danyu 13; 2 klux; 30 ° C; 12 h) First Leaf Chi (mg/gfw)

Treatment Water (CK) BR 10" ppm BR 1 0 ppm BR 10° ppm

0.0702 0.1244 0.1348 0.0334

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Second Leaf

%

%

Chi (mg/gfw)

100 177.2 192 47

0.1797 0.3061 0.4253 0.2278

100 170.3 236.7 126.8

a

Approaching senescence Not yet unfolding

b

Table II. Effects of BR on Greening of Etiolated Maize Leaves Exposed to Light at Various Temperatures (Hybrid 171 x 330A; 5 klux) 30 °C, 24 h Treatment Water (CK) BR 10~ ppm BR 1 0 ppm 3

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Chi (mg/gfw) 0.472 0.598 0.641

% 100 126.7 135.8

18'C, 24 h Chi (mg/gfw) 0.151 0.379 0.356

% 100 251 236

15 °C, 48 h Chi (mg/gfw) 0.060 0.077 0.130

% 100 124 209

NOTE: Figures are average values from three experiments. From data in Tables I and II, the following conclusions can be made: 3

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• Brassinolide concentrations ranging from 10~ ppm to 1 0 ppm improved the greening of etiolated leaves, although the degree of greening was different for the two hybrids. • Maize is a hot-weather crop. Table II shows that low temperature depressed the synthesis of chlorophyll in etiolated leaves exposed to light, and brassinolide relaxed the depression of chlorophyll synthesis, especially at low temperature. For example, the chlorophyll content of the control at 18 ° C was 68% less than that of control at 30 ° C. However, the chlorophyll content of brassinolide-treated leaves at 18 ° C was only 36-44.5% less than that of brassinolide-treated leaves at 30 ° C. A In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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temperature of 15 ° C is the critical point of growth in thermophilic plants. Chlorophyll content of the control at 15 ° C was 94% less than that of the the control at 30 °C. For plants treated with BR (10 ppm), the chlorophyll content at 15 ° C was 90% less than at 30 ° C.


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The greening of etiolated cucumber cotyledons exposed to light was also accelerated by brassinolide. We found that the effect of brassinolide on accelerating chlorophyll synthesis in etiolated cucumber cotyledons was similar to that of cytokinin, which deleted the lag phase induced by light during greening( He et al., Shenyang Agricultural University, unpublished data; 13, 14). Otherwise, the accumulation of chlorophyll, especially at low temperature, was promoted by brassinolide, and it is possible that brassinolide protects the biological membrane system and protects chlorophyll from photooxidation (15). We (He et al., unpublished data) studied the activity of superoxide dismutase (SOD) in etiolated and greened cucumber cotyledons that were cultured with brassinolide and benzyladenine (BA) for 6 days. The results showed that BR and B A promoted the activity of SOD in both etiolated and greened cotyledons (Table III).

Table m. Effects of BR and BA on SOD Activity in Etiolated and Greened Cucumber Cotyledons 0

Etiolated cotyledon Treatment Water (CK) BR 10~ ppm BR 10" ppm BA I O ppm BA 10~ ppm 3

1

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2

SOD (units/gdw) 708.83 892.17 923.00 747.82 789.26

b

Greened cotyledon SOD (units/gdw)

% 100 125.9 130.2 105.5 111.3

1100.22 1652.97 2366.19 1543.22 1767.52

% 100 150.2 215.1 140.3 160.7

Chf (mg/gdw) 1.166 2.676 3.420 1.580 2.727

a

Detached and cultured for 6 days in the dark Detached and cultured for 12 h in the light and 5.5 days in the dark Measured after greening in the light for 12 h

b

c

SOD played an important role in protecting the stability of the membrane system because it scavenged 0 ~ radicals (superoxide free radicals). The effect of SOD is to protect chlorophyll from photooxidation at low temperature (16), thus it causes accumulation of chlorophyll in leaves under light. 2

Effect of Brassinolide on Growth Recovery of Maize Seedlings after Chill Stress Organs of maize seedlings are very sensitive to chilling stress during germination and the early growth stage. Chilling temperature not only affects


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organ growth at that time but also prevents growth recovery after rewarming, which is unfavourable to the development of roots and the emergence of seedlings in early spring. It was reported (17—19) that brassinolide induced resistance to chilling in rice, cucumber, and corn. We have examined the effect of brassinolide on the growth recovery of maize seedlings following chilling treatment to confirm the role of BR in chilling resistance. Maize (Zea mays L. hybrid 171 x 330A) seeds were soaked in BR, calcium chloride (CaCl ), and water at 25 ° C for 24 h and cultured in the same solutions for 2 days. Water treatment was used as control. The germinating seeds were subjected to chilling (0-3 ° C) in the dark when their coleoptiles were about 1 cm and radicles were 2 cm in length. The seedlings were rewarmed at 25 ° C after chilling for 2, 4, 6, 8, and 10 days and cultured at the same temperature for 2 days. The lengths of organs were measured before and after culture. The daily growth rate (cm/day) was used as an index of growth recovery. The growth recoveries in three organs of maize seedlings were as follows: Coleoptile. The effect of brassinolide on the average daily growth rate of coleoptiles following seedlings chilled for different days is shown in Figure 4A, which shows that the growth rate decreased as the number of days of chilling increased. The growth of controls (water treatment) ceased after 8 days of chilling, which indicated irreversible damage. Both 10~ and 10 ppm concentrations of brassinolide unequivocally promoted the growth recovery of coleoptile. The effect of CaCl on growth recovery was approximately equal to that of BR for 2 days of chilling. For over 2 days of chilling, however, the growth recovery of coleoptiles with CaCl treatment decreased sharply and finally looked like that of the controls (water treatment). Mesocotyl. The effect of brassinolide on the average daily growth rate of mesocotyls following chilling for different days is shown in Figure 4B. The growth trend for mesocotyls paralleled that for coleoptiles (Figure 4A), but a higher concentration (10 ppm) of BR was more favourable to the growth recovery of mesocotyls. Radicle. Maize radicles were very sensitive to chilling, and the growth of radicles was nearly zero after more than 2 days of chilling. The different effects of the agents appeared only for 2 days of chilling. Figure 5 shows the effect of agents (BR 10~ and 10"" ppm and CaCl 0.25%) on the daily growth rate of maize radicles chilled for 2 days. From Figure 5 we conclude that both BR and CaCL, especially CaCl , have obvious effects on the growth recovery of maize radicles. On the first day of rewarming, the BR 10~ -ppm treatment yielded values close to that of the CaCl treatment, but on the second day, the growth rate was lower than that of the first day. This result shows that the ability for growth recovery was lower. The control and BR 10 -ppm treatment exhibited the same phenomenon. However, the daily growth rate on the second day of rewarming was higher than that of the first day with CaCl treatment. It appeared that growth recovery by CaCl treatment was higher than that by BR treatment. Brassinolide obviously affected the growth recovery of maize seedlings


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Coleoptile (A)

Mesocotyl (B) o

Water o 5

BRIO" * 1

*

BR1CT*

«

CaCl 0.25%^

*


t

8

10

2

4

6

8

10 days

Chilling treatment Figure 4. The effect of BR on the growth recovery of maize organs after chilling.

Figure 5. The effect of BR on the growth recovery of maize radicles on the 1st and 2nd days of rewarming after chilling for 2 days.


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after chilling. The damage induced by chilling stress to thermophilic crops is generally realized to be brought about by imbalance of the endogenous growth-regulating substances (20, 21). The effect of brassinolide on the growth recovery of seedlings following chilling was most probably due to the role that brassinolide plays in adjusting or compensating the equilibrium of plant growth regulators in the plant (22, 23). In other words, exo genous brassinolide acted as a growth regulator and most probably as a steroid. The latter favoured growth recovery after chilling probably through a protective effect brought about by a change in the membrane system.


Summary The three experiments with brassinolide showed that the physiological effect of brassinolide seems to be that of both an auxin and cytokinin. The twin ing growth effect, however, is the characteristic response evoked by brassi nosteroids, and it was thought that the twining movement of the tendril and the twiner (climber) reported for auxin, or abscisic acid (ABA), or ethylene earlier is doubtful. In other words, brassinolide as a steroid may act on membrane systems. Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Mandava, N. B. Ann. Rev. Plant Physiol. Plant Mol. 1988, 39:23-52. Gregory, L. E . Am. J. Bot. 1981, 65:585-588. Braun, P.; Wild, A. In Advances in Photosynthesis Research Proc. 6th Congress on Photosynthesis; Sybesma, C., Ed.; Nijhoff: The Hague, 1984; 3:461-464. Braun, P.; Wild, A. J. Plant Physiol. 1984, 116:189-196. Yokota, T.; Takahashi, N. In Plant Growth Substances; 1985. Bopp, M., Ed.; Springer: Berlin, 1986; pp 129-138. Gregory, L. E.; Mandava, N. B. Physiol. Plant. 1982, 54:239-243. Wu, Y. M.; Bao, Y. W.; Liu, Y. Acta Phytophysiologica Sinica 1987, 13(1):107-111. Chen, F. Y.; Bin, Y. Q; He, R. Y. Lioning Agric. Sciences (China) 1988, 5:37-41. Yu, S., et al. ZHIWU ZAZHI (Plants) 1990, 17(5):24-25. Hao, J. J.; Xuan, Y. S.; He, R. Y. J. Shenyang Agric. University 1990, 21(1):43-47. Zhang, X. M., et al. Acta Agri. Universitatis Henanensis 1987, 21(1):1-7. Luo, B. S. Plant Physiol. Commun. 1986, 2:14-17. Beevers, L., et al. Planta 1970, 90:256. Fletcher, R. A., et al. Can. J. Bot. 1971, 49:2197. Hasselt, P. R. V. Acta Bot. Neerl. 1972, 21:539-548. Wang, Y. R., et al. Acta Phytophysiologica Sinica 1986, 12(3):244-251. Fujita, F. Farming and Technique (Japan) 1988, 43(1):19-24. Zhou, A. Q.; Luo, B. S.; Ren, X. B. J. Hwazhong Agric. University 1987, 6(1):8-13. Luo, B. S. Plant Physiol. Commun. 1986, 1:11-14.


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20. Luo, Z . R. Plant Physiol. Commun. 1989, 3:1-5. 21. Guo, Q.; Pan, R. C. Acta Phytophysiologica Sinica 1984, 10(4):295-303. 22. Eun J. S.; Kuraishi, S.; Sakurai, N. Plant Cell Physiol. 1989, 30(6):807-810. 23. Luo, B. S.; Yu, D. Q.; Zhou, D. Y. Plant Physiol. Commun. 1988, 5:31-34.


R E C E I V E D June 14, 1991


Effects of Brassinolide on Growth and Chilling Resistance of Maize

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