Plant Growth Regulation (2005) 46:133–141 DOI 10.1007/s10725-005-8379-5
Springer 2005
-1
Induction of heat stress tolerance in barley seedlings by pre-sowing seed treatment with glycinebetaine Abdul Wahid* and Asma Shabbir Department of Botany, University of Agriculture, Faisalabad-38040, Pakistan; *Author for correspondence (email:
[email protected]) Received 23 January 2005; accepted in revised form 6 June 2005
Key words: Correlations, GB, Heat stress, Ions, Leaf water status, Net photosynthetic rate, RMP
Abstract Heat stress adversely affects plant growth and development, while glycinebetaine (GB) plays a protective role under stressful conditions. The objective of this study was to assess the optimum level of GB for use as a presowing seed treatment and the subsequent effect on the heat tolerance of barley (Hordeum vulgare L. cv. Haider-93) seedlings. Among a range of GB levels, the 20 mM concentration emerged as the most effective in enhancing seed germination, shoot fresh and dry weight and shoot water content under heat stress, and this level was selected for further studies. Time course changes revealed that the seedlings developing from 20 mM GB treated seeds had greater shoot dry weight, net photosynthetic rate (PN), leaf water potential (ww) and reduced relative membrane permeability (RMP), compared to no-GB treated plants under heat stress. Correlations between dry weight and high PN (r = 0.881), low ww (r = 0.938) and RMP (r = 0.860) of shoots suggested the involvement of GB in heat stress tolerance. Leakage of Ca2+ and NO3 was the greatest followed by K+ and PO43 under no-GB seed treatment, and GB application under heat stress appreciably reduced the leakage of all these ions, particularly Ca2+, K+ and NO3. In conclusion GB absorbed by seeds, after translocation to the seedlings, enhanced their capacity to maintain greater water content, and higher seedling vigor by virtue of increased PN, reduced RMP and leakage of important ions under heat stress. These results have implications for final field stand under the conditions where the ambient temperature is supra-optimal for barley growth.
Introduction Heat stress is a major growth limiting factor for most crop plants. Prolonged or even a transitory exposure to high temperature leads to altered metabolic functions. Plant parts including leaves, flower buds and roots are all affected by it (Tsukaguchi et al. 2003; Iwaya-Inoue et al. 2004). The most conspicuous changes take place in the cellular membranes and enzymes. Enzymes from higher plants studied so far, with respect to heat
stress, include those from cytoplasm (Laurie and Stewart 1990), mitochondria (Nash et al. 1982) or chloroplasts (Dionisio-Sese et al. 1999). An in vivo study has shown that high temperature stress for prolonged periods inhibits Rubisco activity in heat sensitive cultivars of rice (Bose and Ghosh 1995). Elevated temperature results in rapid loss of water from the plant surface and causes a state of dehydration. This leads to the disruption of cellular membranes, making them more permeable to ions (Jiang and Huang 2001a, b) by increased
134 solubilization and peroxidation of membrane lipids under stressful conditions (Wen-yue et al. 2001; Iba 2002). Enhanced membrane stability determined in terms of changes in ion leakage has long been taken as an index of stress tolerance (Blum and Ebercon 1981; Rehman et al. 2004). Marcum (1998) noted a positive relationship between cell membrane thermo-stability and shoot dry weight for Kentucky bluegrass cultivars, and regarded it as a reliable screening tool for heat stress response. Photosynthetic systems in higher plants are the most sensitive to different stresses. Studies have revealed that specific effects of high temperature on photosynthetic membranes result in loss of grana stacking or its swelling (Gounaris et al. 1984). The PSII oxygen evolution complex is considered as one of the most thermolabile components of the photosynthetic apparatus (Srivastava et al. 1997). Chlorophyll fluorescence is one of the few physiological parameters that have been shown to correlate with heat tolerance (Belkhodja et al. 1994; Yang et al. 1996). In response to heatinduced reduction in the tissue water content (Jiang and Huang 2001a), a decrease in net CO2 assimilation (Matos et al. 2002) and an increase in photorespiration (Sulpice et al. 2002) are generally observed. These effects can result from different events, such as inhibition of electron transport activity and limited generation of reducing powers for metabolic functions (Tardy and Havaux 1999; Allakhverdieva et al. 2001). Problems associated with seed germination and seedling survival of crops under a range of environmental stresses can variously affect crop establishment and final yield (Naidu 1995). There have been considerable research efforts regarding ways and means to alleviate the stress effects on laboratory or field grown plants. One approach has been the successful use of a quaternary ammonium compound (QAC), glycinebetaine (GB). It is non-toxic, highly water soluble and readily absorbed in various tissues (Diaz-Zorita et al. 2001). GB is naturally biosynthesized under stressful conditions (Jagendorf and Takabe 2001). Efforts are underway to metabolically engineer plants with enhanced capacity to accumulate GB (Alia 1998; Sakamoto and Murata 2002; Quan et al. 2004). Nevertheless, external use of GB was found to protect rice and sugarcane against salinity (Rahman et al. 2002; Wahid 2004), wheat
against drought (Wen-Yue et al. 2001), Arabidopsis against freezing stress (Xing and Rajashekar 2001) and to confer osmotic adaptation upon the prokaryotic and eukaryotic cells under heat stress (Caldas et al. 1999; Xing and Rajashekar 2001). It stabilized Rubisco when added to crude extract of rice seedlings that was subsequently heated to 50 C in vitro (Dionisio-Sese et al. 1999), protected numerous thylakoid membrane proteins and redox components, and stabilized higher order structures of PSI and PSII from high temperature induced inactivation (Allakhverdieva et al. 2001; Allakhverdiev et al. 2003). Germination and seedling vigor of crops such as wheat, cotton, sugarcane and pasture legumes were increased under stress by treating the seed with betaine (Agboma et al. 1997; Alia et al. 1998; Campbell et al. 1999). Foliar GB application increased seed set by 94 and 39% in Amaroo and Doongara rice varieties respectively (Naidu and Williams 2004). Endogenous GB biosynthesis profoundly affects various aspects of plant growth and development under stressful conditions (Wahid 2004; Quan et al. 2004). However, some plant species lack the ability to synthesize GB in sufficient amounts (Yang et al. 1996, 2003), and its exogenous application becomes imperative to induce stress tolerance. Some studies acclaim the beneficial role of GB as foliar application and others as seed treatment (Agboma et al. 1997; Sakamoto and Murata 2002; Naidu and Williams 2004). However, there is no consensus regarding appropriate levels of GB as foliar or presowing seed treatment to various crops in mitigating the stress effects. The objectives of this study were to (a) ascertain an optimum GB level for presowing seed treatment, (b) determine the effectiveness of selected levels in supporting barley (Hordeum vulgare L.) seedling growth under heat stress based on photosynthetic rate, water status and membrane permeability characteristics of shoots over time and (c) determine the alterations in the patterns of ion-leakage from shoots.
Materials and methods Experimental details and growth conditions Two experiments were conducted. In the first experiment, the optimum level of seed application of glycinebetaine (GB) was determined for the
135 alleviation of the heat stress effect on germination, seedling growth and shoot water content of barley (Hordeum vulgare L. cv. Haider-93). In the second experiment, optimum level of GB determined from first experiments was applied to seeds and effects were analyzed in terms of net photosynthetic rate (PN), leaf water potential (ww) and relative membrane permeability (RMP) of shoots. For both the experiments, selected healthy seeds were surface sterilized using 0.1% mercuric chloride for 3 min followed by repeated washing (four times) with sterile distilled water. Seeds were soaked for 24 h in 10, 20, 30, 40 and 50 mM levels of GB (Sigma Chemical Co. USA) or in sterilized distilled water. After soaking the seeds were washed in distilled water before sowing in pots containing 5 kg of loam soil. Analysis of the soil revealed its properties as: ECe, 1.23 dS m1; pH, 7.6; organic matter, 1.30%, and available K+ and P were 30.1 and 130 mg L1, respectively. After germination, 5 plants were maintained per pot. After seven days of growth, half of the plants raised from control or GB treated seeds were shifted to a growth chamber (Eyelatron, FLI-301N, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) set at 40 ± 1/32 ± 1 C day/ night temperature, 12 h day length, 45% RH and 550 mmol m2 s1 light intensity to apply heat stress, while the other similar set of plants was kept under control conditions in another chamber set at 25/22 ± 1 C day/night temperature, 12 h day length, 45% RH and 550 mmol m2 s1 light intensity. The plants under both conditions were grown for an additional 10 days.
Growth, leaf photosynthesis and water potential determinations Leaf area of the intact plants was determined as maximum leaf length · maximum leaf width · 0.70 (correction factor calibrated for all leaves). The PN was determined using an infra-red gas analyzer (LCA-4, Analytical Development Co., Hoddesdon, UK). The conditions for these determinations were: air flow per unit leaf area 344 mmol m2 s1, atmospheric pressure 99.4 kPa, PAR on the leaf surface 1280 lmol m2 s1, CO2 concentration 361 lmol mol1 and ambient temperature 23–25 C. The ww of leaves was measured using a pressure chamber (Scholander Bomb, Germany). Shoots were cut at ground level to
determine fresh weight and dried in an oven at 70 C for four days to take dry weight. RMP and pattern of ion-leakage The RMP and ion-leakage were determined as described by Yang et al. (1996). The leaves were excised and put in test tubes containing 20 ml of deionized distilled water. The test tubes were vortexed for 5 s and the solution was assayed for initial electrical conductivity (EC0). These tubes were kept at 4 C for 24 h and then assayed for EC1. Half of the leachate (10 ml) was saved from each tube and assayed for the pattern of ion leakage and the other half containing leaves was autoclaved to determine EC2. Percent RMP was calculated as; RMPð%Þ ¼ ½ðEC1 EC0Þ=ðEC2 EC0Þ 100 Concentrations of individual ions including K+, Ca2+, NO3 and PO43, from the leachate saved from EC1 samples, were determined. Concentrations of K+ and Ca2+ were estimated using a flame photometer (Jenway, PFP7, Essex, UK). Phosphate was determined with molybdate-vanadate reagent (Yoshida et al. 1976) and NO3 by its reaction with chromotropic acid (Kowalenko and Lowe 1973).
Statistical analysis Both the experiments were laid out in randomized complete block designs with four replications. The experiments were conducted twice and data were pooled for the analysis of variance and to determine the significant (p < 0.05) differences between treatments. Duncan’s Multiple Range test was applied to compare the treatment means. Correlation coefficients were computed and trend-lines were set in order to assess the relationships of various parameters and GB induced effects on heat stress tolerance of barley seedlings.
Results Optimization of glycinebetaine levels Figure 1 presents changes in the barley seed germination and early growth and water content of
136
Germination (%)
100 90 80 70
Fresh weight (mg shoot-1)
200
Dry weight (mg shoot-1)
60
60
a
ab
ab bcd
cd
160
ef ab
abc cd
120
de
f g
80
50 40 30
Shoot watercontent (%)
20
90 a 80
bc
70
ab
b
bc
c
bc bc
c
c
d
60 Leaf area (cm2 shoot-1)
a
20 15 10 5 0 0
10 20 30 40 Glycinebetaine levels (mM)
50
Figure 1. Interaction of glycinebetaine seed soaking and heat stress on seed germination and seedling growth characteristics of barley shoots. Open circles represent control and closed heat stress. Data points with same letters do not differ significantly. The interaction of glycinebetaine levels and high temperature is non-significant (p > 0.05) in case of germination percentage, shoot dry weight and leaf area per shoot.
shoots raised from seeds soaked in solutions containing various GB concentrations for 24 h, and determinations made following 10 days of heat
stress. There was a difference among GB (p < 0.05) and temperature (p < 0.001) levels for percent seed germination but no interaction (p > 0.05) between these factors was detected. GB application did not remarkably promote seed germination under non-stressed conditions. However, it was increased under heat stress up to 20 mM GB and became steady thereafter (Figure 1). Shoot fresh weight was greatly increased by GB treatment under both the temperatures. This resulted in differences (p < 0.001) among GB treatments, temperatures and an interaction of these factors. GB applied at 20 mM was the most effective in improving the shoot fresh weight. Fresh weight declined at concentration above 20 mM under heat stress. Effective levels of GB under control conditions were 30 and 40 mM (Figure 1). Despite differences among the GB levels (p < 0.05) and temperature treatments (p < 0.001), increase in dry weight was lower than fresh weight under either condition of temperature resulting in no interaction (p > 0.05) of GB and temperature treatments. Although there was no difference among GB levels for shoot water content under the control condition, they were effective in improving this attribute under heat stress. Among the various GB levels, 20 mM was, again, the most effective concentration (Figure 1). These changes led to differences (p < 0.01) among the GB levels, prevailing temperatures and their interaction (p < 0.001). Leaf area per plant improved slightly with the GB levels under control conditions. There was a difference (p < 0.001) among GB levels and temperature treatments under heat stress, although there was no interaction (p > 0.05) between these factors. In this case too, 20 mM GB was the most effective (Figure 1). Being the most effective, 20 mM GB was used to note time course changes in various plant attributes.
Time course changes in barley under GB and heat stress treatments Determinations made for 10 days, at a 2 day intervals, indicated that while heat stress reduced (p < 0.001) dry weight, GB used as seed treatment alleviated (p < 0.001) the heat stress effect, as was noted from the enhanced shoot dry weight (Figure 2). There was no difference in the PN of
137 GB treated or untreated seedlings under control conditions. Under heat stress and no-GB application, PN declined drastically (68.45%), but at 20 mM GB application, this attribute was declined by just 27.41% on the second day and attained a steady state afterwards (Figure 2). This resulted in the differences (p < 0.001) of these factors individually as well as interactions (p < 0.05) of stress period, temperatures and GB levels. The shoot ww did not differ much in the presence or absence of presowing GB seed treatment
Shootdry weight (g)
60 50 40 30 20 10
Relative permeability (%)
Leaf water potential (-MPa)
A (µmol m -2 s-1 )
20 16 12
a
a
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8
a
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Pattern of ion-leakage
a b
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1 g
0.8
h
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0.6
c
d b
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c
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ab
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g
0.4 0.2
ab
ab
ab
b
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0.0
60
j i h
40
g e
20
c bc
bc c
bc
bc
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c
4
6
8
10
0 0
f
d
d
2
No GB seed treatment
under the control temperature. However, there was sharp decline (88.09%) in ww under heat stress with the no-GB application, but a minimal reduction (37.84%) at 20 mM seed GB treatment under heat stress (Figure 2). This resulted in differences (p < 0.001) among stress period, prevailing temperature and GB levels together with an interaction (p < 0.001) of all these factors. The RMP determined in terms of solute leakage from shoots, revealed differences (p < 0.001) amongst GB levels, prevailing temperatures and exposure time with an interaction (p < 0.001) among these factors. GB seed treatment markedly reduced the RMP compared to those receiving no-GB under the control temperature. However, RMP was greatly enhanced (68.54%) in the shoots receiving no-GB, compared to those receiving GB as seed treatment (49.54%), under heat stress, and became steady towards the end of the stress period (Figure 2).
ab
a
a
a
a
a
0
2
4
6
8
10
20 mM GB seed treatment
Days after heat stress
Figure 2. Changes in dry weight, photosynthetic rate, leaf water potential and membrane permeability characteristics of barley shoots raised from seeds soaked in water or glycinebetaine (20 mM) solution. Open circles represent the control condition and closed heat stress. Data points with same letters do not differ significantly. In the case of shoot dry weight the interaction of glycinebetaine levels, days after heat stress and high temperature is non-significant (p > 0.05).
Leakage of some important ions was determined at the end of the stress period in order to find differences in their leakage as affected by high temperature and 20 mM GB levels as seed treatment. The leakage of K+, NO3 and Ca2+ indicated no difference (p < 0.01) under 20 mM or no-GB applied under the control temperatures, but PO43 leakage was slightly higher under no-GB treatment (Table 1). Under heat stress, shoots raised from no-GB treated seeds had the greatest leakage of all the ions, but those arising from seeds treated with 20 mM GB showed substantially reduced ion-leakage under heat stress. Differences (p < 0.05) in the leakage of these ions were noted under heat stress, except PO43. Notwithstanding the lack of difference among GB levels for leakage of ions under the control temperature, seed GB treatment improved the membrane structure and this improvement was much more pronounced under heat stress, as was evident from the substantially reduced leakage of Ca2+, NO3 and K+.
Trend analysis The effectiveness of presowing seed treatment with GB on shoot dry matter production was
138 Table 1. Heat shock induced leakage of some ions from barley shoots from seed treated overnight with or without 20 mM glycinebetaine solution. The data were recorded after 10 days of heat stress. Temperature (C)
25
Glycinebetaine levels (mM)
0 20 0 20
40
Concentration (mg l1) K+
NO3
PO43
Ca2+
25.33 ± 2.52a 17.67 ± 1.53a 56.33 ± 5.08c 34.33 ± 2.08b
33.30 ± 3.06a 30.90 ± 3.04a 70.17 ± 5.00c 40.10 ± 4.26b
22.37 ± 2.06b 16.03 ± 2.48a 52.50 ± 5.00d 35.63 ± 3.91c
14.37 ± 2.10a 11.53 ± 1.10a 41.90 ± 5.23c 18.53 ± 1.90b
Means with different letters within a column differ significantly (p < 0.05).
substantiated by establishing correlations and drawing trend-lines between various parameters. The application of GB indicated no relationship (p > 0.05) of dry weight with PN, ww or RMP of shoots under the control temperature, but under heat
stress these relationships were present (p < 0.01), although the trend did not differ from control shoots (Figure 3). This indicated that seed GB application has a profound role on the heat stress tolerance as determined in terms of shoot dry weight.
Water potential(-Mpa)
0.40
1.20
18
A (µmol m-2 s-1)
14
16 14 12 10 8 6 4 2 0
Relative permeability(%)
17 r = 0.540ns 16 15
r = -0.263ns
r = 0.881**
r = -0.938**
0.90
0.36
0.60 0.32
0.30 0.00
0.28
60 r = -0.860**
r =-0.460ns
16
40
14 12
20
10 8
0
20
30
40
50
Shoot dry weight (g)
60
20
25
30
35
40
45
Shoot dry weight (g)
Figure 3. Interrelationships of dry matter with photosynthetic rate, water potential and relative membrane permeability of barley shoots rose from seeds treated with 0 or 20 mM glycinebetaine and exposed to heat stress. The left panel is data from control plants and the right panel from plants exposed to heat stress (n = 12).
139 Discussion Increased ambient temperature is emerging as a great threat to the growth and development of most crop plants. Among the various non-toxic compatible cytosolutes, the induction of GB accumulation plays an important role in stress tolerance because of its property of improving cell water balance (Jagendorf and Takabe 2001). Various studies highlight profound effects of exogenous GB application as foliar or presowing seed treatment. Naidu and William (2004) have reported that seed application of GB at 8 and 2 mM as foliar spray were sufficient for enhanced rice seed germination and economic yield, respectively. The present study on the presowing seed GB treatment revealed that 20 mM GB was most effective in promoting various parameters. There was a great improvement in the shoot fresh weight and its water content under heat stress (Figure 1). Interaction of GB levels with heat stress revealed that improved shoot water content is important for heat tolerance. Quan et al. (2004) reported that seed GB content of transgenic lines of maize in the range of 4.1–5.8 lmol g1 dry weight was sufficient to induce drought tolerance. Chen and Murata (2002) reported that as a result of metabolic engineering of the cox gene 13 lmol GB g1, dry weight, was sufficient to induce drought tolerance in Brassica napus. This accumulation, as a result of seed soaking in 25 mM GB solution, was up to 35 lmol g1 fresh weight in Morex barley seeds (A. Wahid unpublished results). This implies that enhancing GB levels of seedlings by using 20 mM GB as a seed soaking treatment is practicable for induction of heat tolerance in barley seedlings. Time dependent changes involving 20 mM GB as seed treatment revealed that among the various parameters, shoot PN and its ww were improved while RMP was markedly improved under heat stress. However, these parameters were adversely affected in the shoots receiving no-GB as presowing seed treatment (Figure 2). We believe that GB absorbed by the seed was most likely distributed in the seedlings, which maintained the shoot water status and stabilized the cellular membranes. Earlier studies indicate that heat stress has a pronounced effect on the gas exchange properties of leaves (Karim et al. 2000; Matos et al. 2002), and endogenous biosynthesis or exogenous application of GB has a stabilizing effect on the photosynthetic
membranes under heat stress (Yang et al. 1996; Agboma et al. 1997; Allakhverdiev et al. 2003). Tsukaguchi et al. (2003) reported that exposure to heat stress induced a sharp increase in the leaf transpiration rate and deterioration of leaf water status of Snap bean. In the present case barley seedlings receiving no-GB as presowing seed treatment had a marked reduction in shoot water potential under heat stress, which was offset by the GB present in the seedlings (Figure 2). This finding is further supported by tight correlations between increased shoot dry weight and improved shoot PN and its ww but reduced RMP under heat stress (Figure 3). Hence the improved leaf water status as a result of GB treatment is a plausible reason for improved photosynthesis and enhanced dry matter yield of barley shoots. Many studies indicate that heat induced loss of membrane stability is a major reason for reduced growth of various plant species (Blum and Ebercon 1981; Bajji et al. 2002; Iba 2002), and has been used as selection criteria for heat tolerance (Marcum 1998; Rehman et al. 2004). Studies showing the pattern of solute leakage under heat stress are relatively scanty (Jiang and Huang 2001b) and there are none showing the altered pattern of solute leakage with GB application. In this study we have determined the leakage pattern of NO3, PO43, Ca2+ and K+ ions (Table 1). Exposure to heat stress indicated a greater leakage of NO3 and Ca2+ followed by K+ and PO43. The GB application reversed this pattern and the leakage of NO3, Ca2+ and K+ was reduced substantially. Such a change in the ion-leakage pattern is important in view of the structural integrity of membranes (Ca2+), osmotic adjustment (K+), nutritional balance and energy transfer processes (NO3 and PO43) under stressful conditions (Marschner 1995; Taiz and Zeiger 2002). In conclusion, the GB absorbed by the seed was highly effective in improving shoot water status, which enhanced the shoot PN, stabilized the cellular membrane and produced vigorous shoots with greater dry mass under heat stress. The pattern of leakage of individual ions indicated the involvement of GB in the stabilization of cellular membranes. These findings have great implications for barley, in particular, and other crops, in general, in the seedling establishment and accomplishment of requisite crop stand in the fields where the temperature becomes supra-optimal for plant growth.
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