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Different solute levels in two spring wheat cultivars induced by progressive field water stress at different developmental stages
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Journal of Arid Environments 62 (2005) 1–14 www.elsevier.com/locate/jnlabr/yjare
Different solute levels in two spring wheat cultivars induced by progressive field water stress at different developmental stages X. Zhu1, H. Gong, G. Chen, S. Wang, C. Zhang Institute of Botany and Plant Physiology, School of Life Science, Lanzhou University, Lanzhou 730000, Gansu, China Received 26 September 2002; received in revised form 28 May 2004; accepted 21 October 2004
Abstract Two cultivars of spring wheat (Triticum aestivum L.) with known drought-resistance rankings (cv. Dingxi 24 more tolerant than cv. Longchun 8139) were subjected to progressive field water stress throughout their growth. Except at the seedling stage, cv. Dingxi 24 showed a higher leaf water potential compared with cv. Longchun 8139 during the stress period. Leaf water potential, inorganic ions (Na+, K+, Ca2+, Mg2+, Cl), proline, betaine, soluble sugars and ATP content were studied in the two cultivars at various developmental stages. Contrary to the sensitive cv. Longchun 8139, consistent changes in K+ and Cl levels and selective accumulation in K+ and Na+ were observed in Dingxi 24 mesophyll and bundle sheath cells evaluated by X-ray microanalysis and in leaves measured by atomic absorption spectrophotometer at the seedling and jointing stages. The responses of the two wheat cultivars to a decline in leaf water at the seedling and jointing stages of development included a net accumulation of inorganic ions and soluble sugar, while at the heading and grain-filling stages, there was an accumulation of betaine and proline. These results suggest that the changes in solute levels in different cultivars were closely related to their drought tolerance at different developmental stages. Mechanisms involved in solute accumulation in different cultivars and
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E-mail address: [email protected] (S. Wang). Present address: School of Life Science, Xiamen University, Xiamen 361005, Fujian, China.
0140-1963/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2004.10.010
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the relationship between solute accumulation and ATP consumption at different developmental stages during water stress are also discussed. r 2004 Elsevier Ltd. All rights reserved. Keywords: Different developmental stage; Field drought; Solute response; Spring wheat; X-ray microanalysis
1. Introduction Environmental stress factors, such as salinity and drought, can result in osmotic stress by decreasing the chemical activity of water and causing loss of cell turgor (Serrano et al., 1999). As cell growth depends on turgor-driven stretching of the cell wall, it is important for plants to counter cell dehydration by selectively absorbing some inorganic ions and by synthesis of intracellular compatible solutes (McCue and Hanson, 1990; Holmberg and Bulow, 1998). It is well documented that plant species can adapt to water stress by changes in solute levels so that turgor and hence physiological activity is maintained at low leaf water potentials. All the data suggest that the accumulation of solutes in the stressed leaves contributes to plant tolerance of dehydration (Jones et al., 1980; Morgan 1984; Wood et al., 1996; Smienoff, 1998). Different species possess distinct characteristics of solute accumulation during water stress with the solutes involved varying between species (Jones et al., 1980). For example, under drought conditions, the main solutes in sorghum leaves were soluble sugar, K+ and Cl while in sunflower they were free amino acids, organic acids, K+, Ca2+, Mg2+ and NO 3, and in cotton leaves they were malic acid, soluble sugar, K+ and NO 3 (Cutler and Rains, 1978; Jones et al., 1980). However, in the vast majority of studies reporting solute accumulation during water stress, a single and abrupt artificial stress factor was imposed and the consequent responses were analysed. In contrast, field plants are subject to a more gradual stress since water availability in the soil does not change abruptly, thus the impacts of natural drought stress on plants in the field may differ from those prevailing in laboratory research. Under laboratory conditions, abrupt artificial stress can induce rapid cell damage so that many stress-responsive genes do not actually contribute to tolerance because their induction is likely to reflect, rather than precede stress damage (Bray, 1993; Xiong et al., 1999). Leone et al. (1994) showed that different sets of polypeptides were synthesized in potato cells depending upon whether they were submitted to abrupt or gradual osmotic stress. Furthermore, since quantitative trait loci (QTLs) that are linked to tolerance at one stage in plant development can differ from those linked to tolerance at other stages (Foolad, 1999), the responses of plants to water stress might also be different at different growth stages. No data are yet available on the extent to which there are common response characteristics of solute changes for different plant species or cultivars throughout
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growth and development in response to progressive water stress in the field. Little is known about in vivo compartmentation of inorganic ions, which are important osmolytes in leaves of many plant species during water stress. In order to better understand the effects and mechanisms of drought on plants, more information is needed on solute changes in leaves throughout growth under progressive field water stress. Comparative studies on the relationships between solute changes at different developmental stages during gradual water stress would contribute to our understanding of the effects of water stress on biochemical and physiological processes in plants, and lead to a sound scientific strategy for management of individual growth stages.
2. Materials and methods 2.1. Plant material Seeds of two spring wheat (Triticum aestivum) cultivars with different sensitivities to drought (cv. Dingxi 24 more tolerant than cv. Longchun 8139, hereafter referred to as dx24 and 8139, respectively) were provided by the Gansu Institute of Agriculture and Science, China. On March 17th, 2000, the seeds were sown in 16 plots (4 2.5 m each) in the plant garden of Lanzhou University (361040 N, 1031100 E; 1518 m elevation), a drought region of the north-west of China (mean annual precipitation 238 mm). All plots were fertilized and irrigated sufficiently before sowing. Water stress was imposed by termination of watering to the plants after sowing, and rainfall was excluded with a movable plastic shed, while the control plants were watered every 3–4 days. Four weeks after planting, four non-irrigated plots for each cultivar were randomly assigned to seedling, jointing, heading and grain-filling stages, respectively. On the sampling days for each of the four growth stages, between 10 AM and noon, the second fully expanded leaves from the apices were harvested and immediately frozen in liquid N2 until they were analysed. Leaf water potential ðcw Þ was measured using a pressure chamber on 5–8 different leaves on each sampling day. 2.2. Quantification of ion content One hundred milligram of dried and well-ground leaves were transferred to a 50 ml digestion flask to which 10 ml of a 60% TCA:HNO3:H2SO4 (1:5:0.5) mixture was added. The flasks were heated gently in a constant temperature water bath at 90 1C for 10 min. The digest was cooled and diluted to 25 ml with deionized water. Na+, K+, Ca2+, and Mg2+ were determined using an atomic absorption spectrophotometer (WFX-1D, Beijing, China). The tissue chloride content was extracted with 1% KNO3 and assayed by silver ion titration with 0.01 M AgNO3 (Jing and Ding, 1981, pp. 29–31).
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2.3. Energy-dispersive X-ray microanalysis for ion distribution in the different leaf cells Leaf segments (2 mm) from the middle part of leaves were frozen in liquid nitrogen and inserted into a transfer device under liquid nitrogen and thence rapidly transferred to the cold stage of an JSM-5600 LV scanning electron microscope (Japan) fitted with a Kevex Energy-Dispersive X-ray detector (USA) as described by Harvey et al. (1985). Each segment was analysed without the application of a conductive coating under the following conditions: accelerating voltage, 15 kV; beam current, 150 pico Amps; solid angle beam current, 0.6; title angle, 01; take-off angle, 27.81; and working distance, 20 mm. The results were calculated by expressing the net counts for a particular element in a given cell (not always the same cell) as a percentage of the total net counts for all the elements measured in the cell ( Na, Mg P, S, Cl, K, Ca) using an automatic Quasar Pro software analysis system. E.g. Fractional Na% ¼ Na net counts/(Na+Mg+Ca+K+P+Cl+S) net counts 100.
2.4. Estimation of soluble sugar, proline and betaine in leaves Soluble sugars were extracted with 75% alcohol at 80 1C and assayed according to the Anthrone method (Yemm and Willis, 1954). For measurement of proline, 0.5 g fresh leaf tissue was ground in 20 ml 3% sulfosalicyclic acid. The homogenate was extracted in boiling water for 10 min. After cooling down, an aliquot of 2 ml was assayed for proline according to the method of Bates et al. (1973). Chromophores were extracted with 4 ml toluene then absorbance was read at 520 nm in a spectrophotometer. Leaves (0.5 g) were ground to a powder in liquid N2 and extracted to measure the content of betaine as reported by Pearce et al. (1976). Extracted periodides were dissolved in 1,2-dichloroethane and the absorbance was read at 365 nm.
2.5. Measurement of ATP content ATP contents were determined by the method of bioluminescence as previously described (Zhu et al., 2001).
2.6. Statistical analysis Least significant difference (LSD) of variance analysis of single factor was calculated to compare means (significance level was at 0.05 or 0.01).
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3. Results 3.1. Water status in leaves at different developmental stages Plants were submitted to progressive soil water deficits as indicated by a gradual decrease in soil moisture content (data not shown). Leaf water potentials ðcw Þ of the two stressed groups declined compared with their respective controls. Except at the seedling stage, the more tolerant dx24 showed a higher water potential compared with 8139 (po0:05) during the stress period (Fig. 1). In contrast to the abrupt laboratory stress, leaf water potential at the seedling stage remained relatively constant, but declined gradually at other stages. A slow decrease in cw in the control groups at heading and grain-filling stages could be the result of a larger leaf area and higher evaporation (Fig. 1). 3.2. Changes in contents of inorganic ions at different developmental stages during field drought The development of leaf water deficits in stressed leaves resulted in changes in each of the major inorganic ions of the two cultivars at different stages. Large differences existed between the two wheat cultivars, with dx24 stressed leaves showing marked changes in ion concentration, especially at the seedling and jointing stages. The levels of divalent cations, such as Ca2+ and Mg2+ in stressed leaves of both dx24 and 8139 Sampling days after sowing (days)
Seedling 35
45
Jointing 55
65
Heading 75
Grain filling 85
95
105
Leaf water potential (Mpa)
-0.5
-1
-1.5
-2
-2.5
dx24
ck dx24
8139
ck 8139
-3
Fig. 1. Changes in leaf water potential ðcw Þ of two spring wheat cultivars at different developmental stages subjected to gradual water deficits. Leaf water potential (pressure chamber) was conducted on each sampling day throughout the experiment. Values are the means of 5–8 measurements. LSD of single factor variance analysis (po0.05) was calculated. Bars represent standard errors. ck indicates control plants.
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were always higher than those of their respective controls, and were significantly increased (po0:01) in the dx24 stressed leaves. Changes in monocations such as Na+ were observed mainly at the seedling stage while changes in K+ were mainly at the jointing and heading stages. There was a concomitant increase in the Na+ content with the decrease in K+ level ðr2 ¼ 1Þ in stressed dx24 leaves. Changes in anion Cl
Fig. 2. Changes in ion contents of two wheat cultivars during an extended stress period. Values are the means of three replications. S: seedling stage; J: jointing stage; H: heading stage; GF: grain-filling stage. LSD of single factor variance analysis was calculated. Bars represent standard errors. Letters above bars indicate significant differences (po0:05).
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Table 1 The fractional elemental percentages analysed by the X-ray microanalysis from MCs and BSCs of two spring wheat cultivars at the jointing stage, showing significantly different ion levels in stressed leaves Cell type
Element Na
Mg
P
dx24 MC ck dx24 MC dx24 BSC ck dx24 BSC
**
**
8139 MC ck 8139 MC 8139 BSC ck 8139 BSC
60.0470.35** 13.4671.01 18.0274.93** 11.7371.54
S
Cl
K **
Ca
12.273.31 5.6871.52 1.0170.02 1.4370.18 12.871.17* 22.3871.75** 9.7872.01 14.0472.98
**
**
**
12.8871.92 11.3771.99 6.8970.63 3.2870.43 16.7272.54* 11.9070.53 13.3272.10 12.9571.63
9.3771.69 28.9072.77 8.4671.01** 21.3873.07
33.373.76 53.5070.99 16.5870.64 18.6074.34
16.972.54** 5.3370.72 11.2270.78 9.9771.52
1.2670.05** 8.2371.15 14.473.49** 5.4870.26
2.6670.21** 20.4072.59 13.9670.49** 21.6871.56
2.4370.42** 11.6871.53 9.6670.27* 14.4172.84
1.5870.37** 15.2973.81 10.8272.08* 15.1371.36
24.4172.45 9.6670.30* 23.4472.76 7.5070.99 22.9974.58* 10.1470.98 20.4871.63 10.7372.04
Values are the means7S.E. of 8–15 different cells of 4–5 leaf blades. The significance (LSD) between the stressed and control plants is indicated by: po0.01 (**) and po0.05 (*).
level, however, showed a positive correlation with K+ at several stages in stressed dx24 ðr2 ¼ 0:61Þ: These two relationships were not observed in 8139 (Fig. 2). Based on the above results of leaf ion contents, the distributions of ions in bundle sheath cells (BSCs) and mesophyll cells (MCs) of the two wheat cultivars at the jointing stage were further analyzed by X-ray microanalysis. As shown in Table 1, most element percentages in MCs of both dx24 and 8139 were lower than those in their corresponding BSCs with the exception of a relatively large percentage of K and Ca in MCs of dx24 and of Mg in MCs of 8139. This indicated a selective absorption when ions entered MCs from BSCs across the membrane. Stressed dx24 showed an increased percentage of Mg, Na, P, Ca in the MCs and BSCs but a declining Cl and K compared with the controls. In stressed 8139, however, only the cation elements Mg and Ca in MCs and Na, K, Mg increased in BSCs, suggesting that the effect of drought on ion accumulations and compartmentalization in different cells varied considerably between the cultivars. The drop in Na percentages in the MCs and their increase in the BSCs of stressed dx24 were correlated with the reverse changes in K levels, suggesting that selective absorbency for Na and K might have existed in dx24 (Table 1 and Fig. 3). 3.3. Compatible solute response to field drought at different developmental stages At the seedling stage, both stressed and control dx24 leaves showed an increased concentration in proline, while the reverse was observed in 8139. The content of proline in dx24 and 8139 remained relatively constant at the jointing and filling stages. At the end of the heading and beginning of grain-filling stages, the proline concentration in stressed dx24 plants and in stressed 8139 increased 2.4-fold and 1.3fold compared with their respective controls (Fig. 4).
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Fig. 3. X-ray spectra from cells of mesophyll and bundle sheath in stressed and control dx24 leaves at the jointing stage under progressive drought conditions. The vertical scale of the spectra is counts per seconds (CPS). Element line was defined as Ka.
Compared with the dx24, both stressed and control leaves of 8139 possessed higher soluble sugar level throughout most of the growth period, indicating that the content of soluble sugar per se was related to the cultivar. However, the difference in
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Proline content (umol g-1dw)
8 dx24
ck dx24
8139
ck 8139
6
4
2
0 35
45
55 Seedling
65
75 Jointing
85
95 Heading
105
Grain filling Fig. 4. Proline changes in leaves at different developmental stages during water stress. Values are the means of three replications. LSD of single factor at 0.05 level was calculated. Bars represent standard errors.
Souble sugar (mg g-1dw)
400 dx24 8139
ck dx24 ck 8139
300
200
100
0 35
45
Seedling
55
65
Jointing
75
85
95
105
Heading
Sampling days after sowing (days) Fig. 5. Changes in the content of soluble sugar of the two spring wheat cultivar at different developmental stages during progressive field water stress. Values are the means of 5 replications. LSD of single factor at 0.05 level was calculated. Bars represent standard errors.
soluble sugar concentration between stressed and control plants of dx24 was significantly greater than that in 8139, especially at the seedling (po0:01) and jointing stages (po0:01). The levels in dx24 reached 228.3% and 289.5% of their corresponding controls at the seedling and jointing stages, respectively, compared with 144.2% and 206.0% in 8139 (Fig. 5).
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Fig. 6. Measurements of betaine and ATP levels in the leaves of cv. dx24 and cv. 8139 at the four different developmental stages during progressive field water stress. Values are the means7S.E. (n ¼ 3 for betaine assay, n ¼ 5 to 7 for ATP estimation). LSD of single factor at 0.05 level was calculated. Bars represent standard error. Letters above bars indicate significant differences.
Both dx24 and 8139 showed similar changes in betaine levels at different stages of development: betaine contents of dx24 and 8139 stressed leaves were lower than, or close to, their respective controls at the seedling and jointing stages, while at the heading and grain-filling stages, they had a higher betaine content (Fig. 6). The maximum accumulation of betaine in the two stressed groups compared with their controls at the heading stage was 125% for 8139 and 187% for dx24. 3.4. ATP levels at different developmental stages under field water stress ATP contents in the leaves of dx24 and 8139 at the two earlier growth stages were lower than those in the two later stages, suggesting that the decrease in ATP content could be related to growth and development. A considerable decrease in the ATP content in stressed dx24 and 8139 at all four developmental stages might involve a large change in solute content, especially ion accumulation at the jointing and heading stages. The sensitive cultivar (8139) may have needed more ATP for its ion accumulation in comparison with the tolerant dx24 (Fig. 6).
4. Discussion Although the responsiveness of solute concentration to water stress in plant species has been studied for many years, research on solute changes at different developmental stages under natural drought lag far behind. The present study showed that stress triggered some solute accumulation, but that this accumulation of different solutes was related to differences between cultivars and in developmental stages. These findings differ from those of laboratory studies, in which several of the major solutes, such as proline, betaine, soluble sugar, inorganic ions were together responsible for seedling responses to water stress. We have shown that different solutes respond individually to gradual water deficit at different developmental stages. Xu and Yu (1990) reported that synthesis and accumulation of organic solutes consumes more energy than uptake of inorganic ions. It seems to be of significance
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that plants respond to water stress by selectively absorbing and accumulating inorganic ions at the seedling and jointing stages, when more ATP is needed for growth and development of the young seedlings. Although the present study cannot elucidate the relationships between ionic regulation and detailed molecular reactions, both ion content analysis of leaves and X-ray microanalysis of cells indicated that synergistic changes in Cl and K+ and selective uptake of Na+ and K+ exist in drought tolerant dx24 (Fig. 2 and Table 1). When fully expanded sorghum leaves at the heading-to-anthesis stage were subjected to moderate water stress, they also accumulated K+ and Cl (Jones et al., 1980). This implies that K+ and Cl have certain common or similar actions and to a certain degree, Cl probably plays an important role in the regulation of cation and anion equilibrium, at least for K+. Contrary to reports of little change in the levels of Ca2+ and Mg2+ in water-stressed leaves (Ford and Wilson, 1981), in the current work, both stressed dx24 and 8139 leaves showed a higher concentration of these two divalent cations in comparison with their respective controls (Fig. 2). It is difficult to interpret why K+ accumulated in the leaves of stressed dx24, while it declined in the MCs and the BSCs of stressed dx24 at the jointing stage (Fig. 2 and Table 1). However, our recent study on the distribution of K+ in the different tissues and cells indicated that a large amount of potassium accumulated in the epidermal cells of dx24 (data not shown). The accumulation of free proline in water-stressed leaves was first described in perennial ryegrass and this phenomenon has been subsequently observed in various plant species (Hanson et al., 1977; Jones et al., 1980; Corcuera et al., 1989; Gao et al., 1999). However, conflicting results have been reported from different plant species (Blum and Ebercon, 1976; Tan and Halloran, 1982; Xue, 1998, pp. 78–95). For example, enhanced proline accumulation in different soybean cultivars subjected to water stress was not related to drought tolerance but to the damage index (Chen, 1998, pp. 18–34). Some plant species with higher proline levels have been cultured successfully, but they did not show increased osmotic regulation under water deficit (Xue, 1998). Under the present experimental conditions, a higher accumulation of proline in the stressed leaves of dx24 at the grain-filling stage clearly resulted from a moderate water deficit ðcw ¼ 2:1 to 2:3Þ (Figs. 1 and 4), suggesting that the role of proline is related to a protective action in the case of moderate stress. However, proline accumulation in dx24 leaves at the seedlings stage was not triggered by water deficit, since the proline concentration in both stressed and control dx24 plants showed similar changes at this stage of growth. In agreement with its reported role as an osmotic regulator in a wide range of species, soluble sugar accumulated significantly in the stressed leaves of dx24 and to lesser extent in stressed 8139 at the seedling and jointing stages (Fig. 5). This indicates it is common for both the drought-tolerant dx24 and the drought-sensitive 8139 to accumulate soluble sugar as a response to soil-water deficit. The active accumulation of solutes such as betaine is thought to play a central role in plant response to desiccation since the ability to synthesize and accumulate betaine is found in plants subjected to drought stress (Ladyman et al., 1980; Grumet and Hanson, 1986; Rhodes and Hanson, 1993). The present results showed that along with the continued decrease in leaf water potential, betaine levels in
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drought-tolerant dx24 were markedly enhanced at the two later growth stages compared with the unstressed control. Because betaine is not appreciably catabolized in higher plants (Ladyman et al., 1980), its accumulation at the two later stages is probably the result of increased biosynthesis, though another possibility is that an altered transportation system is possibly involved. The changes in ATP content at the two earlier growth stages were mainly related to the growth and development of seedlings. Although higher accumulation of solutes clearly used some of the ATP (Fig. 6), the possibility that other metabolisms could affect ATP levels at the same stage could not be excluded. Under progressive drought conditions, the stressed, sensitive 8139 accumulated more solutes compared to its control but at the expense of more consumption of ATP compared to the stressed, tolerant dx24. In summary, under progressive field drought, changes in the various solute levels in different cultivars were closely related not only to their drought tolerance but also to the different developmental stages. Dx24, the drought-tolerant cultivar, showed a higher degree of accumulation of various solutes than the sensitive 8139. A significant accumulation of soluble sugar and inorganic ions seems to contribute much to the response of stressed dx24 to water deficit at the seedling and jointing stages. At these stages, soil water content was still sufficient for plants to increase ion uptake and absorb water. Betaine and proline might play important roles at the two later developmental stages, when soil in the root zone has dried and accumulated solutes might be more important for protective action against moderate water stress (Figs. 5 and 6). Inorganic ion content and compatible solutes in the leaves of dx24 are better integrated at the different developmental stages, and this probably explains the better performance of dx24 than 8139 under drought condition. Besides, compared with abrupt, artificial stress, the accumulation of solutes in leaves in response to gradual field stress was markedly lower. The phenomenon of solute contents enhanced dozens or even hundreds of times in stressed leaves in response to abrupt stress was not observed in the present study.
Acknowledgements We thank Professor Peter Long, Massey University, New Zealand for correcting the English version of the manuscript. The author also wishes to thank the anonymous reviewers of the original manuscript for making important suggestions. This research was supported by the National Key Basic Research Special Funds, China, No. G 1999011700. References Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Blum, A., Ebercon, A., 1976. Genotypic responses in sorghum to drought stress 3. Free proline accumulation and drought resistance. Crop Science 16, 28–431.
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Journal of Arid Environments 62 (2005) 1–14 www.elsevier.com/locate/jnlabr/yjare
Different solute levels in two spring wheat cultivars induced by progressive field water stress at different developmental stages X. Zhu1, H. Gong, G. Chen, S. Wang, C. Zhang Institute of Botany and Plant Physiology, School of Life Science, Lanzhou University, Lanzhou 730000, Gansu, China Received 26 September 2002; received in revised form 28 May 2004; accepted 21 October 2004
Abstract Two cultivars of spring wheat (Triticum aestivum L.) with known drought-resistance rankings (cv. Dingxi 24 more tolerant than cv. Longchun 8139) were subjected to progressive field water stress throughout their growth. Except at the seedling stage, cv. Dingxi 24 showed a higher leaf water potential compared with cv. Longchun 8139 during the stress period. Leaf water potential, inorganic ions (Na+, K+, Ca2+, Mg2+, Cl), proline, betaine, soluble sugars and ATP content were studied in the two cultivars at various developmental stages. Contrary to the sensitive cv. Longchun 8139, consistent changes in K+ and Cl levels and selective accumulation in K+ and Na+ were observed in Dingxi 24 mesophyll and bundle sheath cells evaluated by X-ray microanalysis and in leaves measured by atomic absorption spectrophotometer at the seedling and jointing stages. The responses of the two wheat cultivars to a decline in leaf water at the seedling and jointing stages of development included a net accumulation of inorganic ions and soluble sugar, while at the heading and grain-filling stages, there was an accumulation of betaine and proline. These results suggest that the changes in solute levels in different cultivars were closely related to their drought tolerance at different developmental stages. Mechanisms involved in solute accumulation in different cultivars and
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E-mail address: [email protected] (S. Wang). Present address: School of Life Science, Xiamen University, Xiamen 361005, Fujian, China.
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the relationship between solute accumulation and ATP consumption at different developmental stages during water stress are also discussed. r 2004 Elsevier Ltd. All rights reserved. Keywords: Different developmental stage; Field drought; Solute response; Spring wheat; X-ray microanalysis
1. Introduction Environmental stress factors, such as salinity and drought, can result in osmotic stress by decreasing the chemical activity of water and causing loss of cell turgor (Serrano et al., 1999). As cell growth depends on turgor-driven stretching of the cell wall, it is important for plants to counter cell dehydration by selectively absorbing some inorganic ions and by synthesis of intracellular compatible solutes (McCue and Hanson, 1990; Holmberg and Bulow, 1998). It is well documented that plant species can adapt to water stress by changes in solute levels so that turgor and hence physiological activity is maintained at low leaf water potentials. All the data suggest that the accumulation of solutes in the stressed leaves contributes to plant tolerance of dehydration (Jones et al., 1980; Morgan 1984; Wood et al., 1996; Smienoff, 1998). Different species possess distinct characteristics of solute accumulation during water stress with the solutes involved varying between species (Jones et al., 1980). For example, under drought conditions, the main solutes in sorghum leaves were soluble sugar, K+ and Cl while in sunflower they were free amino acids, organic acids, K+, Ca2+, Mg2+ and NO 3, and in cotton leaves they were malic acid, soluble sugar, K+ and NO 3 (Cutler and Rains, 1978; Jones et al., 1980). However, in the vast majority of studies reporting solute accumulation during water stress, a single and abrupt artificial stress factor was imposed and the consequent responses were analysed. In contrast, field plants are subject to a more gradual stress since water availability in the soil does not change abruptly, thus the impacts of natural drought stress on plants in the field may differ from those prevailing in laboratory research. Under laboratory conditions, abrupt artificial stress can induce rapid cell damage so that many stress-responsive genes do not actually contribute to tolerance because their induction is likely to reflect, rather than precede stress damage (Bray, 1993; Xiong et al., 1999). Leone et al. (1994) showed that different sets of polypeptides were synthesized in potato cells depending upon whether they were submitted to abrupt or gradual osmotic stress. Furthermore, since quantitative trait loci (QTLs) that are linked to tolerance at one stage in plant development can differ from those linked to tolerance at other stages (Foolad, 1999), the responses of plants to water stress might also be different at different growth stages. No data are yet available on the extent to which there are common response characteristics of solute changes for different plant species or cultivars throughout
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growth and development in response to progressive water stress in the field. Little is known about in vivo compartmentation of inorganic ions, which are important osmolytes in leaves of many plant species during water stress. In order to better understand the effects and mechanisms of drought on plants, more information is needed on solute changes in leaves throughout growth under progressive field water stress. Comparative studies on the relationships between solute changes at different developmental stages during gradual water stress would contribute to our understanding of the effects of water stress on biochemical and physiological processes in plants, and lead to a sound scientific strategy for management of individual growth stages.
2. Materials and methods 2.1. Plant material Seeds of two spring wheat (Triticum aestivum) cultivars with different sensitivities to drought (cv. Dingxi 24 more tolerant than cv. Longchun 8139, hereafter referred to as dx24 and 8139, respectively) were provided by the Gansu Institute of Agriculture and Science, China. On March 17th, 2000, the seeds were sown in 16 plots (4 2.5 m each) in the plant garden of Lanzhou University (361040 N, 1031100 E; 1518 m elevation), a drought region of the north-west of China (mean annual precipitation 238 mm). All plots were fertilized and irrigated sufficiently before sowing. Water stress was imposed by termination of watering to the plants after sowing, and rainfall was excluded with a movable plastic shed, while the control plants were watered every 3–4 days. Four weeks after planting, four non-irrigated plots for each cultivar were randomly assigned to seedling, jointing, heading and grain-filling stages, respectively. On the sampling days for each of the four growth stages, between 10 AM and noon, the second fully expanded leaves from the apices were harvested and immediately frozen in liquid N2 until they were analysed. Leaf water potential ðcw Þ was measured using a pressure chamber on 5–8 different leaves on each sampling day. 2.2. Quantification of ion content One hundred milligram of dried and well-ground leaves were transferred to a 50 ml digestion flask to which 10 ml of a 60% TCA:HNO3:H2SO4 (1:5:0.5) mixture was added. The flasks were heated gently in a constant temperature water bath at 90 1C for 10 min. The digest was cooled and diluted to 25 ml with deionized water. Na+, K+, Ca2+, and Mg2+ were determined using an atomic absorption spectrophotometer (WFX-1D, Beijing, China). The tissue chloride content was extracted with 1% KNO3 and assayed by silver ion titration with 0.01 M AgNO3 (Jing and Ding, 1981, pp. 29–31).
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2.3. Energy-dispersive X-ray microanalysis for ion distribution in the different leaf cells Leaf segments (2 mm) from the middle part of leaves were frozen in liquid nitrogen and inserted into a transfer device under liquid nitrogen and thence rapidly transferred to the cold stage of an JSM-5600 LV scanning electron microscope (Japan) fitted with a Kevex Energy-Dispersive X-ray detector (USA) as described by Harvey et al. (1985). Each segment was analysed without the application of a conductive coating under the following conditions: accelerating voltage, 15 kV; beam current, 150 pico Amps; solid angle beam current, 0.6; title angle, 01; take-off angle, 27.81; and working distance, 20 mm. The results were calculated by expressing the net counts for a particular element in a given cell (not always the same cell) as a percentage of the total net counts for all the elements measured in the cell ( Na, Mg P, S, Cl, K, Ca) using an automatic Quasar Pro software analysis system. E.g. Fractional Na% ¼ Na net counts/(Na+Mg+Ca+K+P+Cl+S) net counts 100.
2.4. Estimation of soluble sugar, proline and betaine in leaves Soluble sugars were extracted with 75% alcohol at 80 1C and assayed according to the Anthrone method (Yemm and Willis, 1954). For measurement of proline, 0.5 g fresh leaf tissue was ground in 20 ml 3% sulfosalicyclic acid. The homogenate was extracted in boiling water for 10 min. After cooling down, an aliquot of 2 ml was assayed for proline according to the method of Bates et al. (1973). Chromophores were extracted with 4 ml toluene then absorbance was read at 520 nm in a spectrophotometer. Leaves (0.5 g) were ground to a powder in liquid N2 and extracted to measure the content of betaine as reported by Pearce et al. (1976). Extracted periodides were dissolved in 1,2-dichloroethane and the absorbance was read at 365 nm.
2.5. Measurement of ATP content ATP contents were determined by the method of bioluminescence as previously described (Zhu et al., 2001).
2.6. Statistical analysis Least significant difference (LSD) of variance analysis of single factor was calculated to compare means (significance level was at 0.05 or 0.01).
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3. Results 3.1. Water status in leaves at different developmental stages Plants were submitted to progressive soil water deficits as indicated by a gradual decrease in soil moisture content (data not shown). Leaf water potentials ðcw Þ of the two stressed groups declined compared with their respective controls. Except at the seedling stage, the more tolerant dx24 showed a higher water potential compared with 8139 (po0:05) during the stress period (Fig. 1). In contrast to the abrupt laboratory stress, leaf water potential at the seedling stage remained relatively constant, but declined gradually at other stages. A slow decrease in cw in the control groups at heading and grain-filling stages could be the result of a larger leaf area and higher evaporation (Fig. 1). 3.2. Changes in contents of inorganic ions at different developmental stages during field drought The development of leaf water deficits in stressed leaves resulted in changes in each of the major inorganic ions of the two cultivars at different stages. Large differences existed between the two wheat cultivars, with dx24 stressed leaves showing marked changes in ion concentration, especially at the seedling and jointing stages. The levels of divalent cations, such as Ca2+ and Mg2+ in stressed leaves of both dx24 and 8139 Sampling days after sowing (days)
Seedling 35
45
Jointing 55
65
Heading 75
Grain filling 85
95
105
Leaf water potential (Mpa)
-0.5
-1
-1.5
-2
-2.5
dx24
ck dx24
8139
ck 8139
-3
Fig. 1. Changes in leaf water potential ðcw Þ of two spring wheat cultivars at different developmental stages subjected to gradual water deficits. Leaf water potential (pressure chamber) was conducted on each sampling day throughout the experiment. Values are the means of 5–8 measurements. LSD of single factor variance analysis (po0.05) was calculated. Bars represent standard errors. ck indicates control plants.
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were always higher than those of their respective controls, and were significantly increased (po0:01) in the dx24 stressed leaves. Changes in monocations such as Na+ were observed mainly at the seedling stage while changes in K+ were mainly at the jointing and heading stages. There was a concomitant increase in the Na+ content with the decrease in K+ level ðr2 ¼ 1Þ in stressed dx24 leaves. Changes in anion Cl
Fig. 2. Changes in ion contents of two wheat cultivars during an extended stress period. Values are the means of three replications. S: seedling stage; J: jointing stage; H: heading stage; GF: grain-filling stage. LSD of single factor variance analysis was calculated. Bars represent standard errors. Letters above bars indicate significant differences (po0:05).
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Table 1 The fractional elemental percentages analysed by the X-ray microanalysis from MCs and BSCs of two spring wheat cultivars at the jointing stage, showing significantly different ion levels in stressed leaves Cell type
Element Na
Mg
P
dx24 MC ck dx24 MC dx24 BSC ck dx24 BSC
**
**
8139 MC ck 8139 MC 8139 BSC ck 8139 BSC
60.0470.35** 13.4671.01 18.0274.93** 11.7371.54
S
Cl
K **
Ca
12.273.31 5.6871.52 1.0170.02 1.4370.18 12.871.17* 22.3871.75** 9.7872.01 14.0472.98
**
**
**
12.8871.92 11.3771.99 6.8970.63 3.2870.43 16.7272.54* 11.9070.53 13.3272.10 12.9571.63
9.3771.69 28.9072.77 8.4671.01** 21.3873.07
33.373.76 53.5070.99 16.5870.64 18.6074.34
16.972.54** 5.3370.72 11.2270.78 9.9771.52
1.2670.05** 8.2371.15 14.473.49** 5.4870.26
2.6670.21** 20.4072.59 13.9670.49** 21.6871.56
2.4370.42** 11.6871.53 9.6670.27* 14.4172.84
1.5870.37** 15.2973.81 10.8272.08* 15.1371.36
24.4172.45 9.6670.30* 23.4472.76 7.5070.99 22.9974.58* 10.1470.98 20.4871.63 10.7372.04
Values are the means7S.E. of 8–15 different cells of 4–5 leaf blades. The significance (LSD) between the stressed and control plants is indicated by: po0.01 (**) and po0.05 (*).
level, however, showed a positive correlation with K+ at several stages in stressed dx24 ðr2 ¼ 0:61Þ: These two relationships were not observed in 8139 (Fig. 2). Based on the above results of leaf ion contents, the distributions of ions in bundle sheath cells (BSCs) and mesophyll cells (MCs) of the two wheat cultivars at the jointing stage were further analyzed by X-ray microanalysis. As shown in Table 1, most element percentages in MCs of both dx24 and 8139 were lower than those in their corresponding BSCs with the exception of a relatively large percentage of K and Ca in MCs of dx24 and of Mg in MCs of 8139. This indicated a selective absorption when ions entered MCs from BSCs across the membrane. Stressed dx24 showed an increased percentage of Mg, Na, P, Ca in the MCs and BSCs but a declining Cl and K compared with the controls. In stressed 8139, however, only the cation elements Mg and Ca in MCs and Na, K, Mg increased in BSCs, suggesting that the effect of drought on ion accumulations and compartmentalization in different cells varied considerably between the cultivars. The drop in Na percentages in the MCs and their increase in the BSCs of stressed dx24 were correlated with the reverse changes in K levels, suggesting that selective absorbency for Na and K might have existed in dx24 (Table 1 and Fig. 3). 3.3. Compatible solute response to field drought at different developmental stages At the seedling stage, both stressed and control dx24 leaves showed an increased concentration in proline, while the reverse was observed in 8139. The content of proline in dx24 and 8139 remained relatively constant at the jointing and filling stages. At the end of the heading and beginning of grain-filling stages, the proline concentration in stressed dx24 plants and in stressed 8139 increased 2.4-fold and 1.3fold compared with their respective controls (Fig. 4).
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Fig. 3. X-ray spectra from cells of mesophyll and bundle sheath in stressed and control dx24 leaves at the jointing stage under progressive drought conditions. The vertical scale of the spectra is counts per seconds (CPS). Element line was defined as Ka.
Compared with the dx24, both stressed and control leaves of 8139 possessed higher soluble sugar level throughout most of the growth period, indicating that the content of soluble sugar per se was related to the cultivar. However, the difference in
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Proline content (umol g-1dw)
8 dx24
ck dx24
8139
ck 8139
6
4
2
0 35
45
55 Seedling
65
75 Jointing
85
95 Heading
105
Grain filling Fig. 4. Proline changes in leaves at different developmental stages during water stress. Values are the means of three replications. LSD of single factor at 0.05 level was calculated. Bars represent standard errors.
Souble sugar (mg g-1dw)
400 dx24 8139
ck dx24 ck 8139
300
200
100
0 35
45
Seedling
55
65
Jointing
75
85
95
105
Heading
Sampling days after sowing (days) Fig. 5. Changes in the content of soluble sugar of the two spring wheat cultivar at different developmental stages during progressive field water stress. Values are the means of 5 replications. LSD of single factor at 0.05 level was calculated. Bars represent standard errors.
soluble sugar concentration between stressed and control plants of dx24 was significantly greater than that in 8139, especially at the seedling (po0:01) and jointing stages (po0:01). The levels in dx24 reached 228.3% and 289.5% of their corresponding controls at the seedling and jointing stages, respectively, compared with 144.2% and 206.0% in 8139 (Fig. 5).
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Fig. 6. Measurements of betaine and ATP levels in the leaves of cv. dx24 and cv. 8139 at the four different developmental stages during progressive field water stress. Values are the means7S.E. (n ¼ 3 for betaine assay, n ¼ 5 to 7 for ATP estimation). LSD of single factor at 0.05 level was calculated. Bars represent standard error. Letters above bars indicate significant differences.
Both dx24 and 8139 showed similar changes in betaine levels at different stages of development: betaine contents of dx24 and 8139 stressed leaves were lower than, or close to, their respective controls at the seedling and jointing stages, while at the heading and grain-filling stages, they had a higher betaine content (Fig. 6). The maximum accumulation of betaine in the two stressed groups compared with their controls at the heading stage was 125% for 8139 and 187% for dx24. 3.4. ATP levels at different developmental stages under field water stress ATP contents in the leaves of dx24 and 8139 at the two earlier growth stages were lower than those in the two later stages, suggesting that the decrease in ATP content could be related to growth and development. A considerable decrease in the ATP content in stressed dx24 and 8139 at all four developmental stages might involve a large change in solute content, especially ion accumulation at the jointing and heading stages. The sensitive cultivar (8139) may have needed more ATP for its ion accumulation in comparison with the tolerant dx24 (Fig. 6).
4. Discussion Although the responsiveness of solute concentration to water stress in plant species has been studied for many years, research on solute changes at different developmental stages under natural drought lag far behind. The present study showed that stress triggered some solute accumulation, but that this accumulation of different solutes was related to differences between cultivars and in developmental stages. These findings differ from those of laboratory studies, in which several of the major solutes, such as proline, betaine, soluble sugar, inorganic ions were together responsible for seedling responses to water stress. We have shown that different solutes respond individually to gradual water deficit at different developmental stages. Xu and Yu (1990) reported that synthesis and accumulation of organic solutes consumes more energy than uptake of inorganic ions. It seems to be of significance
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that plants respond to water stress by selectively absorbing and accumulating inorganic ions at the seedling and jointing stages, when more ATP is needed for growth and development of the young seedlings. Although the present study cannot elucidate the relationships between ionic regulation and detailed molecular reactions, both ion content analysis of leaves and X-ray microanalysis of cells indicated that synergistic changes in Cl and K+ and selective uptake of Na+ and K+ exist in drought tolerant dx24 (Fig. 2 and Table 1). When fully expanded sorghum leaves at the heading-to-anthesis stage were subjected to moderate water stress, they also accumulated K+ and Cl (Jones et al., 1980). This implies that K+ and Cl have certain common or similar actions and to a certain degree, Cl probably plays an important role in the regulation of cation and anion equilibrium, at least for K+. Contrary to reports of little change in the levels of Ca2+ and Mg2+ in water-stressed leaves (Ford and Wilson, 1981), in the current work, both stressed dx24 and 8139 leaves showed a higher concentration of these two divalent cations in comparison with their respective controls (Fig. 2). It is difficult to interpret why K+ accumulated in the leaves of stressed dx24, while it declined in the MCs and the BSCs of stressed dx24 at the jointing stage (Fig. 2 and Table 1). However, our recent study on the distribution of K+ in the different tissues and cells indicated that a large amount of potassium accumulated in the epidermal cells of dx24 (data not shown). The accumulation of free proline in water-stressed leaves was first described in perennial ryegrass and this phenomenon has been subsequently observed in various plant species (Hanson et al., 1977; Jones et al., 1980; Corcuera et al., 1989; Gao et al., 1999). However, conflicting results have been reported from different plant species (Blum and Ebercon, 1976; Tan and Halloran, 1982; Xue, 1998, pp. 78–95). For example, enhanced proline accumulation in different soybean cultivars subjected to water stress was not related to drought tolerance but to the damage index (Chen, 1998, pp. 18–34). Some plant species with higher proline levels have been cultured successfully, but they did not show increased osmotic regulation under water deficit (Xue, 1998). Under the present experimental conditions, a higher accumulation of proline in the stressed leaves of dx24 at the grain-filling stage clearly resulted from a moderate water deficit ðcw ¼ 2:1 to 2:3Þ (Figs. 1 and 4), suggesting that the role of proline is related to a protective action in the case of moderate stress. However, proline accumulation in dx24 leaves at the seedlings stage was not triggered by water deficit, since the proline concentration in both stressed and control dx24 plants showed similar changes at this stage of growth. In agreement with its reported role as an osmotic regulator in a wide range of species, soluble sugar accumulated significantly in the stressed leaves of dx24 and to lesser extent in stressed 8139 at the seedling and jointing stages (Fig. 5). This indicates it is common for both the drought-tolerant dx24 and the drought-sensitive 8139 to accumulate soluble sugar as a response to soil-water deficit. The active accumulation of solutes such as betaine is thought to play a central role in plant response to desiccation since the ability to synthesize and accumulate betaine is found in plants subjected to drought stress (Ladyman et al., 1980; Grumet and Hanson, 1986; Rhodes and Hanson, 1993). The present results showed that along with the continued decrease in leaf water potential, betaine levels in
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drought-tolerant dx24 were markedly enhanced at the two later growth stages compared with the unstressed control. Because betaine is not appreciably catabolized in higher plants (Ladyman et al., 1980), its accumulation at the two later stages is probably the result of increased biosynthesis, though another possibility is that an altered transportation system is possibly involved. The changes in ATP content at the two earlier growth stages were mainly related to the growth and development of seedlings. Although higher accumulation of solutes clearly used some of the ATP (Fig. 6), the possibility that other metabolisms could affect ATP levels at the same stage could not be excluded. Under progressive drought conditions, the stressed, sensitive 8139 accumulated more solutes compared to its control but at the expense of more consumption of ATP compared to the stressed, tolerant dx24. In summary, under progressive field drought, changes in the various solute levels in different cultivars were closely related not only to their drought tolerance but also to the different developmental stages. Dx24, the drought-tolerant cultivar, showed a higher degree of accumulation of various solutes than the sensitive 8139. A significant accumulation of soluble sugar and inorganic ions seems to contribute much to the response of stressed dx24 to water deficit at the seedling and jointing stages. At these stages, soil water content was still sufficient for plants to increase ion uptake and absorb water. Betaine and proline might play important roles at the two later developmental stages, when soil in the root zone has dried and accumulated solutes might be more important for protective action against moderate water stress (Figs. 5 and 6). Inorganic ion content and compatible solutes in the leaves of dx24 are better integrated at the different developmental stages, and this probably explains the better performance of dx24 than 8139 under drought condition. Besides, compared with abrupt, artificial stress, the accumulation of solutes in leaves in response to gradual field stress was markedly lower. The phenomenon of solute contents enhanced dozens or even hundreds of times in stressed leaves in response to abrupt stress was not observed in the present study.
Acknowledgements We thank Professor Peter Long, Massey University, New Zealand for correcting the English version of the manuscript. The author also wishes to thank the anonymous reviewers of the original manuscript for making important suggestions. This research was supported by the National Key Basic Research Special Funds, China, No. G 1999011700. References Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Blum, A., Ebercon, A., 1976. Genotypic responses in sorghum to drought stress 3. Free proline accumulation and drought resistance. Crop Science 16, 28–431.
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