• 沒有找到結果。

NaCl stress in rice seedlings: starch mobilization and the influence of gibberellic acid on seedling growth

N/A
N/A
Protected

Academic year: 2021

Share "NaCl stress in rice seedlings: starch mobilization and the influence of gibberellic acid on seedling growth"

Copied!
5
0
0

加載中.... (立即查看全文)

全文

(1)

NaCl stress in rice seedlings: starch mobilization and the

influence of gibberellic acid on seedling growth

Chuan Chi Lin and Ching Huei Kao

1

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China

(Received January 6, 1995; Accepted April 12, 1995)

Abstract. The growth of shoots and roots of rice (Oryza sativa L., cv. Taichung Native 1) seedlings was significantly inhibited when the seeds were subjected to NaCl stress. NaCl markedly decreased the mobilization of starch in en-dosperm. Results also showed that α-amylase activities in endosperm were reduced when rice seeds were germinated in the presence of NaCl. NaCl-inhibition of α-amylase activities was counteracted by gibberellic acid (GA3). GA3

reduced NaCl-inhibition of shoot growth, but not of root growth. Sugars (sucrose, fructose, and glucose) were able to reduce NaCl-induced growth inhibition of shoots and roots. The possible mechanism by which shoot growth and root growth in NaCl media respond differently to GA3 is discussed.

Keywords: α-Amylase; Gibberellic acid; NaCl; Oryza sativa L.; Starch mobilization. Abbreviation: GA3, gibberellic acid.

1 Corresponding author.

Introduction

Mobilization of seed reserves, which occurs during early seed germination, is crucial because it supplies sub-strates for the proper functioning of different metabolic processes that are essential to growth of the embryonic axis (Mayer and Poljakoff-Mayber, 1975). Rice is a salt-sensi-tive crop species (Flowers and Yeo, 1981). The mecha-nism of NaCl inhibition of rice-seedling growth is unclear, but NaCl may inhibit mobilization of seed reserves (Prakash and Prathapasenan, 1988).

Gibberellic acid (GA3) is known to induce the

synthe-sis of α-amylase in embryo-less rice seeds (Palmiano and Juliano, 1972). It is not known whether GA3 can reduce

NaCl inhibition of rice-seedling growth, but GA3 has been

reported to promote the growth of cotton and some halo-phytes in saline condition (Agakishiev, 1964; Boucaud and Ungar, 1976a; 1976b; Zhao et al., 1986). Huber et al. (1974) also reported that GA3 counteracted the influence

of NaCl on the carbohydrate metabolism in leaves of Pennisetum typhoides. The present investigation was con-ducted to examine whether NaCl-inhibited growth of rice seedlings is mediated through diminishing mobilization of starch in endosperm, and to determine the influence of GA3

on NaCl-inhibited rice-seedling growth.

Materials and Methods

Rice (Oryza sativa L. cv. Taichung Native 1) seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed thoroughly with distilled water. These seeds were

then germinated for 1 day in petri dishes (20 cm) contain-ing distilled water at 37°C in the dark. Uniformly germi-nated seeds were selected and transferred to petri dishes (9 cm) containing two sheets of Whatman No. 1 filter pa-per moistened with 10 ml of distilled water or test solu-tion. Each petri dish contained 20 germinated seeds. Each treatment was performed 4 times. The germinated seeds were allowed to grow at 27°C in darkness, and 3 ml of distilled water or test solution was added to each petri dish on day 3.

Sugars and starch were extracted from the endosperm twice with hot ethanol (80%). The extract was evaporated to dryness and the residue was dissolved in 2 ml of dis-tilled water. A portion of this extract was used for the es-timation of total soluble sugars and reducing sugars using the methods of Yoshida et al. (1972) and Lindsay (1973), respectively. Total soluble sugars and reducing sugars are expressed as µg glucose equivalents per endosperm. The tissue residues were suspended in 2 ml of 20 mM sodium phosphate (pH 6.9) and 6 mM NaCl, and boiled for 15 min to gelatinize the starch. Crude boiled homogenates were then used to determine starch according to the method described previously (Hurng and Kao, 1993). Starch level is expressed as mg maltose equivalents per endosperm.

To extract α-amylase, endosperm was homogenized in a chilled (4°C) mortar and pestle with 0.2 M sodium ac-etate (pH 5.4) containing 3 mM CaCl2. Crude extract was

used to determine α-amylase activities by the method de-veloped by Rinderknecht et al. (1967), which uses starch azure as substrate. The change in A595 was used to

calcu-late the α-amylase activity. One unit of enzyme activity is defined as an increase of 1 A595 min-1.

(2)

For all measurements, each treatment was performed four times, and all experiments described here were per-formed three times. Similar results and identical trends were obtained each time. The data reported here are from a single experiment.

Results

The influence of NaCl concentrations on the levels of starch, total soluble sugars, and reducing sugars in en-dosperm of germinating seeds is shown in Figure 1. En-dosperm of germinating rice seeds treated with NaCl

contained higher levels of starch than that treated with dis-tilled water—the higher the NaCl concentration, the higher the level of starch in endosperm. Figure 1 also shows that the levels of total soluble and reducing sugars were lower in endosperm of seeds treated with NaCl than in that of the non-stressed control. The starch level in endosperm decreased and the levels of total soluble and reducing sug-ars increased as germination progressed. This process was greatly inhibited by NaCl (Figure 2). In the endosperm of both non-stressed and NaCl-stressed seeds, starch level decreased with a parallel increase in the levels of total soluble and reducing sugars (Figure 2). The fresh weight

Figure 1. Influence of NaCl on the levels of starch, total soluble sugars, and reducing sugars in endosperm of germinating rice seeds. Starch, total soluble sugars, and reducing sugars were determined after 5 days of treatment. Vertical bars represent stan-dard errors. Only those stanstan-dard errors larger than the symbol size are shown.

Figure 2. Changes in the levels of starch, total soluble sugars, and reducing sugars in endosperm of germinating rice seeds treated with 150-mM NaCl. Vertical bars represent standard er-rors. Only those standard errors larger than the symbol size are shown. mg endosperm -1 µ g endosperm -1 4 3 2 1 0 Starch 700 550 400 250 0 Total sugars µ g endosperm -1 600 450 300 150 0 Reducing sugars 0 50 100 150 NaCl, mM Time, days 0 1 2 3 4 5 Reducing sugars 600 400 200 0 µ g endosperm -1 600 400 200 0 µ g endosperm -1 mg endosperm -1 Total sugars Starch 5.0 4.5 3.0 1.5 0 H2O NaCl

(3)

per endosperm (23 mg) treated with NaCl (150 mM) for 5 days was found to be higher than that of the control (18 mg). Our results were similar to those reported by Prakash and Prathapasenan (1988).

Comparative studies of α-amylase activity in en-dosperm revealed a continuous increase in enzyme activity with duration of germination in both the control and NaCl treatments (Figure 3). NaCl inhibited α-amylase activity in endosperm in a concentration-dependent manner (Figure 3). Dubey (1982), however, demonstrated that 150-mM NaCl increased the activities of α-amylase in rice endosperm.

Figure 5. Counteraction by GA3 of the NaCl-induced inhibition

of α-amylase activities (units/endosperm) in endosperm. The concentration of GA3 was 0.1 µM. α-Amylase was assayed

af-ter 5 days of treatment. Vertical bars represent standard errors. Only those standard errors larger than the symbol size are shown. Figure 4. Counteraction by GA3 of the NaCl-induced inhibition

of seedling growth. The concentration of GA3 was 0.1 µM.

Seed-ling growth was measured after 5 days of treatment. Vertical bars represent standard errors. Only those standard errors larger than the symbol size are shown.

Figure 3. Influence of NaCl on the activity of α-amylase in en-dosperm of germinating rice seeds. For the dose-response study,

α-amylase in the endosperm was assayed after 5 days of treat-ment. The concentration of NaCl used in the time-course study was 150 mM. Vertical bars represent standard errors. Only those standard errors larger than the symbol size are shown.

The growth of shoots and roots was tracked by mea-suring their fresh weight. Figure 4 shows the influence of NaCl on the growth of seedlings with and without GA3

treatment. In the absence of GA3, NaCl significantly

sup-pressed the growth of shoots and roots. The growth of shoots and roots was reduced to 15 and 30% of the con-trol values (without NaCl), respectively, by 150 mM NaCl. These results are generally consistent with those reported by Flowers and Yeo (1981). NaCl-induced inhibition of shoot growth was significantly reduced by GA3, but GA3

did nothing to counteract NaCl inhibition of root growth. Figure 5 shows the influence of GA3 on the activities

of α-amylase in endosperm of seedlings treated with vari-ous concentrations of NaCl. In the absence of GA3, NaCl

inhibited α-amylase activities in endosperm of seedlings. GA3 effectively reduced NaCl-induced inhibition of α

-amylase activities in endosperm.

H2O NaCl 1.8 1.5 1.2 0.9 0.5 0.3 0 α

-Amylase, units endosperm

-1 1.8 1.5 1.2 0.9 0.5 0.3 0 α

-Amylase, units endosperm

-1 0 50 100 150 0 1 2 3 4 5 0 50 100 150 NaCl, mM 30 25 20 15 10 5 0

Fresh weight, mg organ

-1 -GA +GA Shoot 12 8 4 0 0 50 100 150 NaCl, mM -GA +GA

NaCl, mM Time, Days

α

-Amylase, units endosperm

-1 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0

(4)

enous sugars, other factors responsible for growth inhibi-tion by NaCl can not be ruled out.

Sucrose is considered to be a sole transport sugar in the majority of plants (Giaquinta, 1980). It seems that the up-take of glucose and fructose by rice seedlings is less ef-fective than that of sucrose. This may explain why glucose and fructose have little influence on the growth of shoots or roots.

GA3 is an important factor in enhancing the α-amylase

activities in germinating rice seeds (Palmiano and Juliano, 1972). It is conceivable that the mechanism by which NaCl-induced inhibition of α-amylase activities is coun-teracted by GA3 is related to a deficiency of GA3 in

NaCl-stressed endosperm.

GA3 has been reported to reduce NaCl-induced growth

inhibition in some plant species (Agakishiev, 1964; Boucaud and Ungar, 1976a; 1976b; Zhao et al., 1986). We found, unexpectedly, that in rice seedlings, GA3

counter-acted NaCl-inhibited shoot growth, but not root growth. This is the first evidence that under NaCl stress, shoot growth and root growth respond differently to GA3.

The results of our analysis of α-amylase activities show that the reduction of NaCl-inhibition of shoot growth by GA3 results from an enhancement of hydrolysis of starch

in endosperm. Because the addition of sugars improved the root growth of seedlings in NaCl medium, and because GA3 did not reduce NaCl-inhibition of root growth, the

translocation of sugars from endosperm into roots is most likely inhibited by NaCl. We cannot, however, exclude the possibility that in the presence of NaCl, rice-seedling shoots draw more sugars from the endosperm. A deeper knowledge of NaCl-inhibition of the export of starch-mobilization products from the endosperm to roots is nec-essary for a better understanding of the mechanism of NaCl-inhibition of root growth.

Literature Cited

Agakishiev, D. 1964. Effect of gibberellin on cotton under con-ditions of salinization. Fiziol. Rast. 11: 210–205.

Boucaud, J. and I. A. Ungar. 1976a. Influence of hormonal treat-ments on the growth of two halophytic species. Am. J. Bot. 63: 694–699.

Boucaud, J. and I. A. Ungar. 1976b. Hormonal control of ger-mination under saline conditions of three halophytic taxa in the genus Suaeda. Physiol. Plant. 37: 143–148. Dubey, R. S. 1982. Biochemical changes in germinating rice

seeds under saline stress. Biochem. Physiol. Pflanzen 177: 523–535.

Flowers, T. J. and A. R. Yeo. 1981. Variability in the resistance of sodium chloride salinity within rice (Oryza sativa L.) va-rieties. New Phytol. 88: 363–373.

Giaquinta, R. T. 1980. Translocation of sucrose and oligosac-charides. In J. Preiss (ed.), The Biochemistry of Plants. A Comprehensive Treatise, vol. 3. Academic Press, New York, London, Toronto, Sydney, San Francisco, pp. 271–320. Huber, W., P. N. Rustagi, and N. Sankhla. 1974.

Eco-physiologi-cal studies on Indian arid zone plants. III. Effect of sodium

Experiments were conducted to determine whether sug-ars can counter the growth inhibition induced by NaCl. Shoot growth and root growth in NaCl medium were both found to be enhanced by the addition of sucrose, fructose, or glucose (Figure 6).

Discussion

Our results are generally consistent with the hypoth-esis that NaCl-induced inhibition of early seedling growth is mediated through mobilization of endosperm reserves (Prakash and Prathapasenan, 1988). Since NaCl-inhibited seedling growth was not completely reversed by

exog-Figure 6. Counteraction by sugars of the NaCl-induced inhibi-tion of seedling growth. The concentrainhibi-tion of NaCl was 150 mM. For all treatments, 0.25 mg/l chloramphenicol was added to pre-vent bacterial growth. Seedling growth was measured 5 days af-ter treatment. Vertical bars represent standard errors. Only those standard errors larger than the symbol size are shown.

7 5 3 0 9 7 5 3 0 Root Shoot Sucrose Glucose Fructose

Fresh weight, mg organ

-1

0 15 30 45

(5)

chloride and gibberellin on the activity of the enzymes of carbohydrate metabolism in leaves of Pennisetum typhoides. Oecologia 15: 77–86.

Hurng, W. P. and C. H. Kao. 1993. Loss of starch and increase of α-amylase activity in leaves of flooded tobacco plants. Plant Cell Physiol. 34: 531–534.

Lindsay, H. 1973. A colorimetric estimation of reducing sugars in potatoes with 3,5-dinitrosalicylic acid. Potato Res. 16: 176–179.

Mayer, A. M. and A. Poljakoff-Mayber. 1975. The germination of seeds, 2nd edn. Pergamon Press, New York.

Palmiano, E. P. and B. O. Juliano. 1972. Biochemical changes in the rice grain during germination. Plant Physiol. 49: 751–756.

Prakash, L. and G. Prathapasenan. 1988. Putrescine reduces NaCl-induced inhibition of germination and early seedling growth of rice (Oryza sativa L.). Aust. J. Plant Physiol. 15: 761–767.

Rinderknecht, H., P. Wilding, and B. J. Haverback. 1967. A new method for the determination of α-amylase. Experientia 23: 805.

Yoshida, S., D. A. Forno, J. H. Cock, and K. A. Gomoz. 1972. Laboratory Manual for Physiological Studies of Rice, 2nd edn. International Rice Research Institute, Loss Banos, Phil-ippines.

Zhao, K.-F., M.-L. Li, and J.-Y. Liu. 1986. Reduction by GA3

of NaCl-induced inhibition of growth and development in Suaeda ussuriensis. Aust. J. Plant Physiol. 13: 547–551.

數據

Figure 1. Influence of NaCl on the levels of starch, total soluble sugars, and reducing sugars in endosperm of germinating rice seeds
Figure 5. Counteraction by GA 3  of the NaCl-induced inhibition of α-amylase activities (units/endosperm) in endosperm

參考文獻

相關文件

6 《中論·觀因緣品》,《佛藏要籍選刊》第 9 冊,上海古籍出版社 1994 年版,第 1

The first row shows the eyespot with white inner ring, black middle ring, and yellow outer ring in Bicyclus anynana.. The second row provides the eyespot with black inner ring

In particular, we present a linear-time algorithm for the k-tuple total domination problem for graphs in which each block is a clique, a cycle or a complete bipartite graph,

You are given the wavelength and total energy of a light pulse and asked to find the number of photons it

Wang, Solving pseudomonotone variational inequalities and pseudocon- vex optimization problems using the projection neural network, IEEE Transactions on Neural Networks 17

Hope theory: A member of the positive psychology family. Lopez (Eds.), Handbook of positive

Define instead the imaginary.. potential, magnetic field, lattice…) Dirac-BdG Hamiltonian:. with small, and matrix

• elearning pilot scheme (Four True Light Schools): WIFI construction, iPad procurement, elearning school visit and teacher training, English starts the elearning lesson.. 2012 •