Plant Physiology and Biochemistry 90 (2015) 50e57
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Research article
Priming effect of abscisic acid on alkaline stress tolerance in rice (Oryza sativa L.) seedlings Li-Xing Wei a, b, Bing-Sheng Lv a, b, Ming-Ming Wang a, c, Hong-Yuan Ma a, c, Hao-Yu Yang a, c, Xiao-Long Liu a, c, Chang-Jie Jiang e, *, Zheng-Wei Liang a, c, d, ** a
Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, Jilin, 130102, China University of Chinese Academy of Sciences, Beijing, 100049, China Da'an Sodic Land Experiment Station, Da'an, Jilin, 131317, China d Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin, Heilongjiang, 150081, China e National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, 305 8602, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 January 2015 Accepted 4 March 2015 Available online 12 March 2015
Salineealkaline stress is characterized by high salinity and high alkalinity (high pH); alkaline stress has been shown to be the primary factor inhibiting rice seedling growth. In this study, we investigated the potential priming effect of abscisic acid (ABA) on tolerance of rice seedlings to alkaline stress simulated by Na2CO3. Seedlings were pretreated with ABA at concentrations of 0 (control), 10, and 50 mM by rootdrench for 24 h and then transferred to a Na2CO3 solution that did not contain ABA. Compared to control treatment, pretreatment with ABA substantially improved the survival rate of rice seedlings and increased biomass accumulation after 7 days under the alkaline condition. ABA application at 10 mM also alleviated the inhibitory effects of alkaline stress on the total root length and root surface area. Physiologically, ABA increased relative water content (RWC) and decreased cell membrane injury degree (MI) and Naþ/Kþ ratios. In contrast, fluridone (an ABA biosynthesis inhibitor) decreased the RWC and increased MI in shoots under the alkaline conditions. These data suggest that ABA has a potent priming effect on the adaptive response to alkaline stress in rice and may be useful for improving rice growth in salineealkaline paddy fields. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Abscisic acid (ABA) Alkaline stress Priming Stress tolerance Rice (Oryza sativa)
1. Introduction Soil saline-alkalization is a major factor that limits crop growth and productivity worldwide (Islam et al., 2011; Uddin et al., 2011; Zhang et al., 2010; Zhang and Mu, 2009). Globally, there are more than 800 million hectares of land covered by saline and alkaline soil (Martinez-Beltran and Manzur, 2005). In northeastern China, the salineealkaline soil accounts for 6.2% of the land area (Li et al., 2002; Wang et al., 2004; Yang et al., 2008), in which only a few alkali-resistant plants can survive, causing a serious threat to crop
Abbreviations: ABA, abscisic acid; FW, fresh weight; RD, root diameter; RN, root numbers; RSA, root surface area; RV, root volume; SL, shoot length; TRL, total root length; RWC, relative water content; MI, membrane injury. * Corresponding author. ** Corresponding author. Da'an Sodic Land Experiment Station, Da'an, Jilin, 131317, China. E-mail addresses: [email protected] (C.-J. Jiang), [email protected] (Z.-W. Liang). http://dx.doi.org/10.1016/j.plaphy.2015.03.002 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.
production (Zhang et al., 2013). Soil alkalization is often accompanied by salinization and creates an inordinately high soil pH (ranging from 8.5 to 11) in addition to high salinity and high osmotic pressure (Ma and Liang, 2007). The salineealkaline soil is impregnated with Na2CO3 and NaHCO3, and the proportion of Naþ accounts for more than 70% of the total soil cations, and CO2 3 and HCO2 3 constituted the primary anions. Rice (Oryza sativa L.) is regarded as a desalinization crop because of its ability to grow well in standing water in alkaline soil (Van Asten et al., 2004; Wang et al., 2010). It has been proven that paddy rice cultivation can significantly increase the pace of reclamation of salineealkaline soils (Liu et al., 2010; Yu et al., 2010). However, rice is also known as a salt-sensitive crop (Munns and Tester, 2008), and rice seed germination rate was significantly reduced and seedling growth was delayed under salineealkaline conditions (Khan et al., 1997). Rice seedlings have shown decreases in dry weight, plant height, cell membrane stability and relative water content (RWC) in response to salineealkaline stress (Lv et al., 2013; Ruan and Xue, 2001; Wang et al., 2011). In addition,
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Fig. 1. Images of seeding growth under different treatments. Two-week-old rice seedlings were root-drenched with 0 mM ABA (control), 10 mM fluridone, 10 mM ABA (ABA-10) and 50 mM ABA (ABA-50) for 24 h, and then were subjected to unstressed or alkaline-stressed conditions. Photographs were taken at day 5 (5d) and day 7 (7d).
salineealkaline stress also decreased the main root length and lateral root number (Li et al., 2008). During the seedling stage, alkaline stress on rice can also cause damage to cell membranes, and even young plant death (Cheng et al., 2008; Sun et al., 2004; Wang et al., 2006). Moreover, in salineealkaline soils, the tillering ability of plants was decreased and the heading stage was significantly delayed (Liang et al., 2003). We have previously shown that alkaline stress has a more deleterious effect on rice seedlings than that of salt and osmotic stresses (Lv et al., 2013). Hence, improving the resistance of rice plants to alkaline condition is critical for increasing plant growth and productivity in salineealkaline paddy fields. The phytohormone abscisic acid (ABA) plays a vital role in the adaptive response of plants to various environmental stresses, such as cold (Chen et al., 1983), heat (Robertson et al., 1994), drought (Lu et al., 2009), and salt (Zhang et al., 2006). Recently, application of
Fig. 2. Survival rates of rice seedlings after growing for 7 days under alkaline stress condition. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.01) based on Duncan's test.
Fig. 3. Shoots length of rice seedlings grown under unstressed (A) and alkalinestressed (B) conditions for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
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Fig. 4. Fresh weight of shoots (A) and roots (B), and dry weight of shoots (C) and roots (D) of rice seedlings grown under alkaline-stressed condition for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
exogenous ABA has been shown to improve drought tolerance of poplar (Popilus kangdingensis C.) (Yin et al., 2004) and salinity tolerance of tomato (Solanum lycopersicum L.) (Amjad et al., 2014), and common bean (Phaseolus vulgaris L.) (Khadri et al., 2007). In rice, pretreatment with ABA improved tolerance of rice calli to osmotic, saline, and freezing stresses (Martinez-Beltran and Manzur, 2005). Wang et al. (2013) suggested that exogenous ABA elevated rice chilling tolerance by inducing the antioxidant systems. It was also reported that pretreatment with ABA increased rice survival rate and growth under salt stress (Gurmani et al., 2013; Sripinyowanich et al., 2013). Bohra et al. (1995) suggested that the increased Kþ/Naþ ratio following exogenous ABA application is correlated with the increased salinity tolerance in rice. However, the effects of ABA on alkaline tolerance of rice remain unknown. Plants can acquire enhanced resistance to future stress exposure after experiencing a biotic or an abiotic stress; this is known as priming or hardening (Beckers and Conrath, 2007). Well-known examples include systemic acquired resistance (SAR) and seed preconditioning (seed priming) (Beckers and Conrath, 2007; Goellner and Conrath, 2008; Jisha et al., 2013). Priming can be triggered by a range of chemical compounds, including some phytohormones and their functional analogs (Aranega Bou et al., 2014). Under salt stress, ABA-primed Brassica napus seeds showed earlier germination and more radical protrusion than non-primed control seeds (Gao et al., 2002). Further, it was recently reported that presoaking of rice seeds with ABA increased grain yield by 21% in saline field conditions (Gurmani et al., 2011). In this study, we aimed to determine the potential priming effect of ABA on the physiological and biochemical adaptive responses of rice seedlings to alkaline stress.
2. Materials and methods 2.1. Plant material The rice cultivar Dongdao-4, a japonica type elite cultivar in the salineealkaline land area in China, was used throughout this study. Seed germination and seedling growth were carried out as described previously (Lv et al., 2013). Twenty-five uniformly germinated seeds were selected and pre-cultured for 14 d in a growth chamber (HPG-400HX; Hadonglian In., Beijing, China) at 25/20 C (light/dark), and 12 h photoperiods. 2.2. Alkaline-stress treatment and ABA application We used Na2CO3 at 15 mM, which had EC ¼ 2.76 ± 0.003 mS/cm and pH ¼ 10.87 ± 0.033, to simulate alkaline stress (Lv et al., 2013). The EC and pH of the solutions were measured using conductivity meter DDS-12 (Lida In.,Shanghai, China) and pH meter PHS-25 (Baiyuan In., Beijing, China) respectively. Abscisic acid (ABA) and fluridone (a carotenoid and ABA biosynthesis inhibitor; Sigma, Inc., St, Louis, MO, USA) were dissolved in a small amount of absolute ethanol and then diluted with deionized water to the desired concentrations. Seedlings at approximated three-leaf stage were pretreated with ABA at concentrations of 0 mM (control), 10 mM or 50 mM by root-drench for 24 h, and then transferred to deionized water (unstressed) or Na2CO3 solution that did not contain ABA. Fluridone at 10 mM was used as a negative control for ABA. Each treatment was arranged in a completely randomized design (CRD) with three replications, each of which contained 25 seedlings, and the solutions were changed every 2 days to maintain the stress conditions throughout the experiment.The seedlings were sampled for various
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Fig. 5. Total root lengths (A), root surface area (B), root diameters (C), root volume (D) and root numbers (E) of rice seedlings grown under alkaline-stressed condition for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
measurements after 7 days of stress treatment. Individual seedlings were classified as deadif all the leaves were dry and brown. 2.3. Measurement of seedling growth, sodium, potassium Ten rice seedlings were randomly selected from the 25 seedlings of each treatment group and scanned using the Epson Expression 10000XL (Epson America Inc., Long Beach, CA). The resulting images were digitized by using the WinRHIZO program according to the manufacturer's instruction (Regent Instruments Canada Inc., Quebec, Canada), and the average shoot length (SL, the length of the longest leaf), total root length (TRL), total root surface area (RSA), root diameter (RD), total root volume (RV), and root numbers (RN) were examined. After the scanning, the seedlings were cut and divided into shoots and roots, and measured their fresh weight. These samples were then dried at 105 C for 2 h, followed by 70 C until reaching a stable mass in a forced air-driven oven before measuring the dry mass. The roots were rinsed 4 times with deionized water before subjected to drying. After measuring the dry mass, the samples were cut with a scissor to 5e10 mm pieces and digested completely with an HNO3
and HClO4 (v/v ¼ 2:1) mixture and diluted to 50 mL. The Naþ and Kþ concentrations in the shoots and roots were determined by flame emission spectrometry (FP6410, Shanghai precision and scientific instrument Co., Ltd, China). Membrane injury (MI) was measured by means of electrolyte €rffling, 2006). Ten seedlings were randomly leakage (Tantau and Do selected from each treatment group and washed with deionized water in order to remove surface adhered electrolytes, and cut and divided into shoots and roots. Samples (2 g fresh weight) of the shoots and roots were submerged in 15 ml of deionized water in 50 ml conical tubes and kept at 20 C for 1 h, and the electrical conduction of effusion was measured (R1). The tissues of the samples were killed by heating tubes in a boiling bath for 40 min, cooled down to 20 C, and electrical conduction of the effusion was measured once again (R2). The MI was evaluated with the formula MI (%) ¼ R1/R2 100%. 2.4. Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) using the statistical software SPSS 21.0 (SPSS,
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Fig. 6. Relative water content (RWC) of shoots (A) and roots (B) of rice seedlings grown under alkaline-stressed condition for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
Chicago, IL). Based on the ANOVA results, a Duncan test for mean comparison was performed. All data were represented by an average of three biological replicate measurements and the standard error. The significance level was P < 0.05. 3. Results
Fig. 7. Membrane injury (MI) of shoots (A) and roots (B) of rice seedlings grown under alkaline-stressed conditions for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
that in the other treatments (P < 0.05) (Fig. 3B). High ABA concentration (50 mM) resulted in significant suppression in the shoot length as observed under unstressed condition (Fig. 3B). Compared to control and fluridone treatment, ABA pretreatment resulted in significantly higher shoot fresh weight (FW) under alkaline-stress (Fig. 4A). Moreover, pretreatment with ABA improved dry mass accumulation, as shown by higher shoot dry weight (DW) than the control and fluridone treatments (P < 0.05) (Fig. 4C). In contrast, no appreciable effect was observed for seedlings pretreated with fluridone (Fig. 4A and C).
3.1. Seedling survivals 3.3. Root growth Rice seedlings with ABA application had a significantly higher survival rate than those in the control and fluridone treatments under the alkaline stress after 7 days (P < 0.01) (Figs. 1 and 2). ABA application at 10 mM and 50 mM concentrations resulted in about 3fold and 2-fold as high survival rates as the control, respectively. The highest survival rate was observed with ABA treatment at 10 mM (Fig. 2). No seedling death was observed under unstressed conditions during the whole course of experiment (Fig. 1). 3.2. Shoot growth Under unstressed conditions (Fig. 3A), the shoot length of seedlings treated with fluridone was significantly higher than that of the other treatments (P < 0.05). In contrast, high ABA concentration (50 mM) resulted in significant reduction of shoot growth. However, under alkaline stress conditions, the shoot length of seedlings pretreated with 10 mM ABA was significantly higher than
After 7 days of stress treatment, seedlings pretreated with 10 mM ABA had significantly higher root FW and DW (P < 0.05) (Fig. 4B and D). In contrast, high concentration of ABA (50 mM) showed no significant positive effect on FW and DW (Fig. 4B and D). Seedlings pretreated with 10 mM ABA had significantly higher total root length (TRL) and root surface area (RSA) than those in the other treatments (P < 0.05) (Fig. 5A and B); however no appreciable changes were observed in root volume (RV), root diameter (RD) and root number (RN) (Fig. 5C and D). Further, there was no significant effect of ABA at 50 mM on the root growth indices, except for a higher RD (Fig. 5). 3.4. Relative water content and membrane injury The seedlings pretreated with ABA had significantly higher shoot relative water content (RWC) than those in the control and
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Fig. 8. Content of Naþ (A, B), Kþ (C, D) and Naþ/Kþ ratio (E, F) of rice seedlings grown under alkaline-stressed condition for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
fluridone treatments (Fig. 6A). Meanwhile, there was no significant difference in root RWC among different treatments, with the exception that seedlings pretreated with 10 mM ABA had a lower root RWC (Fig. 6B). Pretreatment with ABA significantly alleviated the membrane injury (MI, P < 0.05) (Fig. 7). In addition, the alkaline stress caused more MI (P < 0.05) to roots (38%e52%) than shoots (20%e32%) (Fig. 7). 3.5. Changes in sodium, potassium and their ratio ABA treatment resulted in a slightly, but significantly, lower Naþ content (Fig. 8A) and Naþ/Kþ ratio (Fig. 8E) in shoots compared with the control and fluridone treatments (P < 0.05). In roots, no difference in Naþ content was observed among the treatments, while compared to control and fluridone treatments, ABAtreatment resulted in higher Kþ content (Fig. 8D) and thus a lower Naþ/Kþ ratio (Fig. 8F). In addition, the alkaline stress caused a greater increase in the Naþ/Kþ ratio in roots than that in shoots (Fig. 8). The relatively high Na/K ratio shown in this study is probably due to no Kþ supplied in the stress solution.
4. Discussion Salineealkaline stress is characterized by high salinity and high alkalinity, and can damage plants not only through ion toxicity from high salinity but also from a high pH (Lv et al., 2013). Recently, it was shown that exogenous application of ABA enhanced salt stress tolerance in rice (Bohra et al., 1995; Gao et al., 2002; Gurmani et al., 2013; Sripinyowanich et al., 2013) However, the effect of ABA on tolerance of rice to alkaline stress remains unknown. In this study, we have shown for the first time that pretreatment with exogenous ABA significantly improved the survival rate and growth of rice seedlings under alkaline stress conditions (Figs. 1e5). In addition, there was some evidence that ABA biosynthesis inhibitor fluridone may slightly compromised tolerance of the seedlings to alkaline stress (Figs. 1e5). These data demonstrate that ABA has a potent priming effect on the adaptive response to alkaline stress in rice plants. It has been shown that young rice seedlings after transplanting are particularly vulnerable to salineealkaline stress, and the returning green phase of rice seedlings is delayed for 3e7 days in salineealkaline paddy fields (Liang et al., 2004). Moreover, alkaline
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stress causes a series of damages to rice seedlings, such as cell membrane damage, resulting in growth retardation, and even young plant death (Qi et al., 2007; Shan et al., 2006). In this study, we showed that pretreatment with ABA significantly increased the survival rate of rice seedlings under alkaline stress conditions (Figs. 1 and 2). The ABA application also promoted the growth of shoots and roots under alkaline stress (Figs. 1 and 3e5). These results suggest that activating the ABA signaling pathway may provide a new approach for improving rice seedling survival and growth in salineealkaline soil. Under unstressed condition, ABA application, particularly at the high concentration (50 mM), suppressed shoot growth; conversely, fluridone treatment promoted it (Fig. 3). This phenomenon is in accordance with the inhibitory effect of ABA on plant growth and development, as opposed to enhancing tolerance to various environmental stresses (Tuteja, 2007). Moreover, a high concentration of ABA was less beneficial to seedling survival than a low concentration (10 mM) under alkaline stress condition (Fig. 2), and in some instances caused inhibition of seedling growth (Figs. 3e5). This is also in accordance with the findings that phytohormones function only within a threshold range of concentration levels (Khan et al., 2012). These results indicate that further optimization of ABA concentration is required to maximize the beneficial effects of ABA on the alkaline stress tolerance in rice seedlings. The Naþ and Kþ transport in plants have been extensively studied, and the Naþ/Kþ ratio is often regarded as an indicator of plant tolerance to various stresses (Ren et al., 2005; RodríguezNavarro and Rubio, 2006). It has been shown that plant tolerance to salt stress depends on the ability to maintain an ion balance, including lower Naþ and Naþ/Kþ ratio and high Kþ concentration (Inan et al., 2004), It was reported that exogenous ABA application enhanced salt tolerance by reducing Naþ transport and thus the Naþ/Kþ ratio in rice (Gurmani et al., 2013). In this study, the Naþ/Kþ ratio in both the shoots (Fig. 8E) and roots (Fig. 8F) under the exogenous ABA treatment was significantly lower than that in the control. In addition, exogenous ABA application increased RWC in shoots (Fig. 6A) and alleviated membrane injury of both shoots and roots (Fig. 7) under alkaline stress. These data together suggest that the priming effect of ABA on the tolerance of rice seedlings to alkaline stress is attributable to the improvement of multiple physiological factors. However, the molecular mechanism for this occurrence remains to be further elucidated. 5. Conclusions In this study, we demonstrated that ABA-pretreatment significantly enhanced tolerance of rice seedlings to alkaline stress as observed by increases in survival rate and seedling growth. This priming effect of ABA was attributed at least in part to decrease in Naþ/Kþ ratio, water loss, and cell membrane injury under alkaline stress. Our results suggest that ABA positively regulates alkaline stress tolerance and an adequate application of ABA can provide a potential approach to improve rice growth in salineealkaline paddy fields. A field study that applies our present findings is in progress to determine methods for improving rice growth and productivity in salineealkaline paddy fields. Contribution Zheng-Wei Liang, Chang-Jie Jiang and Li-Xing Wei designed the research, interpreted results and wrote the manuscript; Li-Xing Wei, Bing-Sheng Lv, Ming-Ming Wang, Hong-Yuan Ma, Hao-Yu Yang and Xiao-Long Liu performed laboratory experiments, data collection and statistical analysis.
Acknowledgment This study was supported by the National science & Technology pillar Program of China (No. 2012BAD20B03-05), the Chinese Academy of Sciences Action-plan for West Development (KZCX2XB3-16), the Key Research Program of the Chinese Academy of Sciences (KZZD-EW-TZ-07-08). References Amjad, M., Akhtar, J., Anwar-ul-Haq, M., Yang, A., Akhtar, S.S., Jacobsen, S.-E., 2014. Integrating role of ethylene and ABA in tomato plants adaptation to salt stress. Sci. Hortic. 172, 109e116. http://dx.doi.org/10.1016/j.scienta.2014.03.024. Aranega Bou, P., Leyva, M.D.L.O., Finiti, I., García-Agustín, P., Gonz alez-Bosch, C., 2014. Priming of plant resistance by natural compounds. Hexanoic acid as a model. Front. Plant Sci. 5, 488. http://dx.doi.org/10.3389/fpls.2014.00488. Beckers, G.J., Conrath, U., 2007. Priming for stress resistance: from the lab to the field. Curr. Opin. Plant Biol. 10, 425e431. http://dx.doi.org/10.1016./ j.pbi.2007.06.002. €rffling, H., Do € rffling, K., 1995. 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Research article
Priming effect of abscisic acid on alkaline stress tolerance in rice (Oryza sativa L.) seedlings Li-Xing Wei a, b, Bing-Sheng Lv a, b, Ming-Ming Wang a, c, Hong-Yuan Ma a, c, Hao-Yu Yang a, c, Xiao-Long Liu a, c, Chang-Jie Jiang e, *, Zheng-Wei Liang a, c, d, ** a
Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, Jilin, 130102, China University of Chinese Academy of Sciences, Beijing, 100049, China Da'an Sodic Land Experiment Station, Da'an, Jilin, 131317, China d Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin, Heilongjiang, 150081, China e National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, 305 8602, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 January 2015 Accepted 4 March 2015 Available online 12 March 2015
Salineealkaline stress is characterized by high salinity and high alkalinity (high pH); alkaline stress has been shown to be the primary factor inhibiting rice seedling growth. In this study, we investigated the potential priming effect of abscisic acid (ABA) on tolerance of rice seedlings to alkaline stress simulated by Na2CO3. Seedlings were pretreated with ABA at concentrations of 0 (control), 10, and 50 mM by rootdrench for 24 h and then transferred to a Na2CO3 solution that did not contain ABA. Compared to control treatment, pretreatment with ABA substantially improved the survival rate of rice seedlings and increased biomass accumulation after 7 days under the alkaline condition. ABA application at 10 mM also alleviated the inhibitory effects of alkaline stress on the total root length and root surface area. Physiologically, ABA increased relative water content (RWC) and decreased cell membrane injury degree (MI) and Naþ/Kþ ratios. In contrast, fluridone (an ABA biosynthesis inhibitor) decreased the RWC and increased MI in shoots under the alkaline conditions. These data suggest that ABA has a potent priming effect on the adaptive response to alkaline stress in rice and may be useful for improving rice growth in salineealkaline paddy fields. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Abscisic acid (ABA) Alkaline stress Priming Stress tolerance Rice (Oryza sativa)
1. Introduction Soil saline-alkalization is a major factor that limits crop growth and productivity worldwide (Islam et al., 2011; Uddin et al., 2011; Zhang et al., 2010; Zhang and Mu, 2009). Globally, there are more than 800 million hectares of land covered by saline and alkaline soil (Martinez-Beltran and Manzur, 2005). In northeastern China, the salineealkaline soil accounts for 6.2% of the land area (Li et al., 2002; Wang et al., 2004; Yang et al., 2008), in which only a few alkali-resistant plants can survive, causing a serious threat to crop
Abbreviations: ABA, abscisic acid; FW, fresh weight; RD, root diameter; RN, root numbers; RSA, root surface area; RV, root volume; SL, shoot length; TRL, total root length; RWC, relative water content; MI, membrane injury. * Corresponding author. ** Corresponding author. Da'an Sodic Land Experiment Station, Da'an, Jilin, 131317, China. E-mail addresses: [email protected] (C.-J. Jiang), [email protected] (Z.-W. Liang). http://dx.doi.org/10.1016/j.plaphy.2015.03.002 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.
production (Zhang et al., 2013). Soil alkalization is often accompanied by salinization and creates an inordinately high soil pH (ranging from 8.5 to 11) in addition to high salinity and high osmotic pressure (Ma and Liang, 2007). The salineealkaline soil is impregnated with Na2CO3 and NaHCO3, and the proportion of Naþ accounts for more than 70% of the total soil cations, and CO2 3 and HCO2 3 constituted the primary anions. Rice (Oryza sativa L.) is regarded as a desalinization crop because of its ability to grow well in standing water in alkaline soil (Van Asten et al., 2004; Wang et al., 2010). It has been proven that paddy rice cultivation can significantly increase the pace of reclamation of salineealkaline soils (Liu et al., 2010; Yu et al., 2010). However, rice is also known as a salt-sensitive crop (Munns and Tester, 2008), and rice seed germination rate was significantly reduced and seedling growth was delayed under salineealkaline conditions (Khan et al., 1997). Rice seedlings have shown decreases in dry weight, plant height, cell membrane stability and relative water content (RWC) in response to salineealkaline stress (Lv et al., 2013; Ruan and Xue, 2001; Wang et al., 2011). In addition,
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Fig. 1. Images of seeding growth under different treatments. Two-week-old rice seedlings were root-drenched with 0 mM ABA (control), 10 mM fluridone, 10 mM ABA (ABA-10) and 50 mM ABA (ABA-50) for 24 h, and then were subjected to unstressed or alkaline-stressed conditions. Photographs were taken at day 5 (5d) and day 7 (7d).
salineealkaline stress also decreased the main root length and lateral root number (Li et al., 2008). During the seedling stage, alkaline stress on rice can also cause damage to cell membranes, and even young plant death (Cheng et al., 2008; Sun et al., 2004; Wang et al., 2006). Moreover, in salineealkaline soils, the tillering ability of plants was decreased and the heading stage was significantly delayed (Liang et al., 2003). We have previously shown that alkaline stress has a more deleterious effect on rice seedlings than that of salt and osmotic stresses (Lv et al., 2013). Hence, improving the resistance of rice plants to alkaline condition is critical for increasing plant growth and productivity in salineealkaline paddy fields. The phytohormone abscisic acid (ABA) plays a vital role in the adaptive response of plants to various environmental stresses, such as cold (Chen et al., 1983), heat (Robertson et al., 1994), drought (Lu et al., 2009), and salt (Zhang et al., 2006). Recently, application of
Fig. 2. Survival rates of rice seedlings after growing for 7 days under alkaline stress condition. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.01) based on Duncan's test.
Fig. 3. Shoots length of rice seedlings grown under unstressed (A) and alkalinestressed (B) conditions for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
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Fig. 4. Fresh weight of shoots (A) and roots (B), and dry weight of shoots (C) and roots (D) of rice seedlings grown under alkaline-stressed condition for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
exogenous ABA has been shown to improve drought tolerance of poplar (Popilus kangdingensis C.) (Yin et al., 2004) and salinity tolerance of tomato (Solanum lycopersicum L.) (Amjad et al., 2014), and common bean (Phaseolus vulgaris L.) (Khadri et al., 2007). In rice, pretreatment with ABA improved tolerance of rice calli to osmotic, saline, and freezing stresses (Martinez-Beltran and Manzur, 2005). Wang et al. (2013) suggested that exogenous ABA elevated rice chilling tolerance by inducing the antioxidant systems. It was also reported that pretreatment with ABA increased rice survival rate and growth under salt stress (Gurmani et al., 2013; Sripinyowanich et al., 2013). Bohra et al. (1995) suggested that the increased Kþ/Naþ ratio following exogenous ABA application is correlated with the increased salinity tolerance in rice. However, the effects of ABA on alkaline tolerance of rice remain unknown. Plants can acquire enhanced resistance to future stress exposure after experiencing a biotic or an abiotic stress; this is known as priming or hardening (Beckers and Conrath, 2007). Well-known examples include systemic acquired resistance (SAR) and seed preconditioning (seed priming) (Beckers and Conrath, 2007; Goellner and Conrath, 2008; Jisha et al., 2013). Priming can be triggered by a range of chemical compounds, including some phytohormones and their functional analogs (Aranega Bou et al., 2014). Under salt stress, ABA-primed Brassica napus seeds showed earlier germination and more radical protrusion than non-primed control seeds (Gao et al., 2002). Further, it was recently reported that presoaking of rice seeds with ABA increased grain yield by 21% in saline field conditions (Gurmani et al., 2011). In this study, we aimed to determine the potential priming effect of ABA on the physiological and biochemical adaptive responses of rice seedlings to alkaline stress.
2. Materials and methods 2.1. Plant material The rice cultivar Dongdao-4, a japonica type elite cultivar in the salineealkaline land area in China, was used throughout this study. Seed germination and seedling growth were carried out as described previously (Lv et al., 2013). Twenty-five uniformly germinated seeds were selected and pre-cultured for 14 d in a growth chamber (HPG-400HX; Hadonglian In., Beijing, China) at 25/20 C (light/dark), and 12 h photoperiods. 2.2. Alkaline-stress treatment and ABA application We used Na2CO3 at 15 mM, which had EC ¼ 2.76 ± 0.003 mS/cm and pH ¼ 10.87 ± 0.033, to simulate alkaline stress (Lv et al., 2013). The EC and pH of the solutions were measured using conductivity meter DDS-12 (Lida In.,Shanghai, China) and pH meter PHS-25 (Baiyuan In., Beijing, China) respectively. Abscisic acid (ABA) and fluridone (a carotenoid and ABA biosynthesis inhibitor; Sigma, Inc., St, Louis, MO, USA) were dissolved in a small amount of absolute ethanol and then diluted with deionized water to the desired concentrations. Seedlings at approximated three-leaf stage were pretreated with ABA at concentrations of 0 mM (control), 10 mM or 50 mM by root-drench for 24 h, and then transferred to deionized water (unstressed) or Na2CO3 solution that did not contain ABA. Fluridone at 10 mM was used as a negative control for ABA. Each treatment was arranged in a completely randomized design (CRD) with three replications, each of which contained 25 seedlings, and the solutions were changed every 2 days to maintain the stress conditions throughout the experiment.The seedlings were sampled for various
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Fig. 5. Total root lengths (A), root surface area (B), root diameters (C), root volume (D) and root numbers (E) of rice seedlings grown under alkaline-stressed condition for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
measurements after 7 days of stress treatment. Individual seedlings were classified as deadif all the leaves were dry and brown. 2.3. Measurement of seedling growth, sodium, potassium Ten rice seedlings were randomly selected from the 25 seedlings of each treatment group and scanned using the Epson Expression 10000XL (Epson America Inc., Long Beach, CA). The resulting images were digitized by using the WinRHIZO program according to the manufacturer's instruction (Regent Instruments Canada Inc., Quebec, Canada), and the average shoot length (SL, the length of the longest leaf), total root length (TRL), total root surface area (RSA), root diameter (RD), total root volume (RV), and root numbers (RN) were examined. After the scanning, the seedlings were cut and divided into shoots and roots, and measured their fresh weight. These samples were then dried at 105 C for 2 h, followed by 70 C until reaching a stable mass in a forced air-driven oven before measuring the dry mass. The roots were rinsed 4 times with deionized water before subjected to drying. After measuring the dry mass, the samples were cut with a scissor to 5e10 mm pieces and digested completely with an HNO3
and HClO4 (v/v ¼ 2:1) mixture and diluted to 50 mL. The Naþ and Kþ concentrations in the shoots and roots were determined by flame emission spectrometry (FP6410, Shanghai precision and scientific instrument Co., Ltd, China). Membrane injury (MI) was measured by means of electrolyte €rffling, 2006). Ten seedlings were randomly leakage (Tantau and Do selected from each treatment group and washed with deionized water in order to remove surface adhered electrolytes, and cut and divided into shoots and roots. Samples (2 g fresh weight) of the shoots and roots were submerged in 15 ml of deionized water in 50 ml conical tubes and kept at 20 C for 1 h, and the electrical conduction of effusion was measured (R1). The tissues of the samples were killed by heating tubes in a boiling bath for 40 min, cooled down to 20 C, and electrical conduction of the effusion was measured once again (R2). The MI was evaluated with the formula MI (%) ¼ R1/R2 100%. 2.4. Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) using the statistical software SPSS 21.0 (SPSS,
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Fig. 6. Relative water content (RWC) of shoots (A) and roots (B) of rice seedlings grown under alkaline-stressed condition for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
Chicago, IL). Based on the ANOVA results, a Duncan test for mean comparison was performed. All data were represented by an average of three biological replicate measurements and the standard error. The significance level was P < 0.05. 3. Results
Fig. 7. Membrane injury (MI) of shoots (A) and roots (B) of rice seedlings grown under alkaline-stressed conditions for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
that in the other treatments (P < 0.05) (Fig. 3B). High ABA concentration (50 mM) resulted in significant suppression in the shoot length as observed under unstressed condition (Fig. 3B). Compared to control and fluridone treatment, ABA pretreatment resulted in significantly higher shoot fresh weight (FW) under alkaline-stress (Fig. 4A). Moreover, pretreatment with ABA improved dry mass accumulation, as shown by higher shoot dry weight (DW) than the control and fluridone treatments (P < 0.05) (Fig. 4C). In contrast, no appreciable effect was observed for seedlings pretreated with fluridone (Fig. 4A and C).
3.1. Seedling survivals 3.3. Root growth Rice seedlings with ABA application had a significantly higher survival rate than those in the control and fluridone treatments under the alkaline stress after 7 days (P < 0.01) (Figs. 1 and 2). ABA application at 10 mM and 50 mM concentrations resulted in about 3fold and 2-fold as high survival rates as the control, respectively. The highest survival rate was observed with ABA treatment at 10 mM (Fig. 2). No seedling death was observed under unstressed conditions during the whole course of experiment (Fig. 1). 3.2. Shoot growth Under unstressed conditions (Fig. 3A), the shoot length of seedlings treated with fluridone was significantly higher than that of the other treatments (P < 0.05). In contrast, high ABA concentration (50 mM) resulted in significant reduction of shoot growth. However, under alkaline stress conditions, the shoot length of seedlings pretreated with 10 mM ABA was significantly higher than
After 7 days of stress treatment, seedlings pretreated with 10 mM ABA had significantly higher root FW and DW (P < 0.05) (Fig. 4B and D). In contrast, high concentration of ABA (50 mM) showed no significant positive effect on FW and DW (Fig. 4B and D). Seedlings pretreated with 10 mM ABA had significantly higher total root length (TRL) and root surface area (RSA) than those in the other treatments (P < 0.05) (Fig. 5A and B); however no appreciable changes were observed in root volume (RV), root diameter (RD) and root number (RN) (Fig. 5C and D). Further, there was no significant effect of ABA at 50 mM on the root growth indices, except for a higher RD (Fig. 5). 3.4. Relative water content and membrane injury The seedlings pretreated with ABA had significantly higher shoot relative water content (RWC) than those in the control and
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Fig. 8. Content of Naþ (A, B), Kþ (C, D) and Naþ/Kþ ratio (E, F) of rice seedlings grown under alkaline-stressed condition for 7 days. Values are means ± standard errors of three biological replicates. Different letters on the column represent significant difference (P < 0.05) based on Duncan's test.
fluridone treatments (Fig. 6A). Meanwhile, there was no significant difference in root RWC among different treatments, with the exception that seedlings pretreated with 10 mM ABA had a lower root RWC (Fig. 6B). Pretreatment with ABA significantly alleviated the membrane injury (MI, P < 0.05) (Fig. 7). In addition, the alkaline stress caused more MI (P < 0.05) to roots (38%e52%) than shoots (20%e32%) (Fig. 7). 3.5. Changes in sodium, potassium and their ratio ABA treatment resulted in a slightly, but significantly, lower Naþ content (Fig. 8A) and Naþ/Kþ ratio (Fig. 8E) in shoots compared with the control and fluridone treatments (P < 0.05). In roots, no difference in Naþ content was observed among the treatments, while compared to control and fluridone treatments, ABAtreatment resulted in higher Kþ content (Fig. 8D) and thus a lower Naþ/Kþ ratio (Fig. 8F). In addition, the alkaline stress caused a greater increase in the Naþ/Kþ ratio in roots than that in shoots (Fig. 8). The relatively high Na/K ratio shown in this study is probably due to no Kþ supplied in the stress solution.
4. Discussion Salineealkaline stress is characterized by high salinity and high alkalinity, and can damage plants not only through ion toxicity from high salinity but also from a high pH (Lv et al., 2013). Recently, it was shown that exogenous application of ABA enhanced salt stress tolerance in rice (Bohra et al., 1995; Gao et al., 2002; Gurmani et al., 2013; Sripinyowanich et al., 2013) However, the effect of ABA on tolerance of rice to alkaline stress remains unknown. In this study, we have shown for the first time that pretreatment with exogenous ABA significantly improved the survival rate and growth of rice seedlings under alkaline stress conditions (Figs. 1e5). In addition, there was some evidence that ABA biosynthesis inhibitor fluridone may slightly compromised tolerance of the seedlings to alkaline stress (Figs. 1e5). These data demonstrate that ABA has a potent priming effect on the adaptive response to alkaline stress in rice plants. It has been shown that young rice seedlings after transplanting are particularly vulnerable to salineealkaline stress, and the returning green phase of rice seedlings is delayed for 3e7 days in salineealkaline paddy fields (Liang et al., 2004). Moreover, alkaline
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stress causes a series of damages to rice seedlings, such as cell membrane damage, resulting in growth retardation, and even young plant death (Qi et al., 2007; Shan et al., 2006). In this study, we showed that pretreatment with ABA significantly increased the survival rate of rice seedlings under alkaline stress conditions (Figs. 1 and 2). The ABA application also promoted the growth of shoots and roots under alkaline stress (Figs. 1 and 3e5). These results suggest that activating the ABA signaling pathway may provide a new approach for improving rice seedling survival and growth in salineealkaline soil. Under unstressed condition, ABA application, particularly at the high concentration (50 mM), suppressed shoot growth; conversely, fluridone treatment promoted it (Fig. 3). This phenomenon is in accordance with the inhibitory effect of ABA on plant growth and development, as opposed to enhancing tolerance to various environmental stresses (Tuteja, 2007). Moreover, a high concentration of ABA was less beneficial to seedling survival than a low concentration (10 mM) under alkaline stress condition (Fig. 2), and in some instances caused inhibition of seedling growth (Figs. 3e5). This is also in accordance with the findings that phytohormones function only within a threshold range of concentration levels (Khan et al., 2012). These results indicate that further optimization of ABA concentration is required to maximize the beneficial effects of ABA on the alkaline stress tolerance in rice seedlings. The Naþ and Kþ transport in plants have been extensively studied, and the Naþ/Kþ ratio is often regarded as an indicator of plant tolerance to various stresses (Ren et al., 2005; RodríguezNavarro and Rubio, 2006). It has been shown that plant tolerance to salt stress depends on the ability to maintain an ion balance, including lower Naþ and Naþ/Kþ ratio and high Kþ concentration (Inan et al., 2004), It was reported that exogenous ABA application enhanced salt tolerance by reducing Naþ transport and thus the Naþ/Kþ ratio in rice (Gurmani et al., 2013). In this study, the Naþ/Kþ ratio in both the shoots (Fig. 8E) and roots (Fig. 8F) under the exogenous ABA treatment was significantly lower than that in the control. In addition, exogenous ABA application increased RWC in shoots (Fig. 6A) and alleviated membrane injury of both shoots and roots (Fig. 7) under alkaline stress. These data together suggest that the priming effect of ABA on the tolerance of rice seedlings to alkaline stress is attributable to the improvement of multiple physiological factors. However, the molecular mechanism for this occurrence remains to be further elucidated. 5. Conclusions In this study, we demonstrated that ABA-pretreatment significantly enhanced tolerance of rice seedlings to alkaline stress as observed by increases in survival rate and seedling growth. This priming effect of ABA was attributed at least in part to decrease in Naþ/Kþ ratio, water loss, and cell membrane injury under alkaline stress. Our results suggest that ABA positively regulates alkaline stress tolerance and an adequate application of ABA can provide a potential approach to improve rice growth in salineealkaline paddy fields. A field study that applies our present findings is in progress to determine methods for improving rice growth and productivity in salineealkaline paddy fields. Contribution Zheng-Wei Liang, Chang-Jie Jiang and Li-Xing Wei designed the research, interpreted results and wrote the manuscript; Li-Xing Wei, Bing-Sheng Lv, Ming-Ming Wang, Hong-Yuan Ma, Hao-Yu Yang and Xiao-Long Liu performed laboratory experiments, data collection and statistical analysis.
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