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Effects of endogenous abscisic acid, jasmonic acid, polyamines, and polyamine oxidase activity in tomato seedlings under drought stress
Scientia Horticulturae 159 (2013) 172–177
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Effects of endogenous abscisic acid, jasmonic acid, polyamines, and polyamine oxidase activity in tomato seedlings under drought stress Chunmei Zhang a,1 , Zhi Huang b,∗,1 a b
College of Agriculture and Biology Technology, Hexi University, ZhangYe, Gansu 734000, PR China College of Horticulture, Sichuan Agricultural University, Ya’an, Sichuan, 625014, PR China
a r t i c l e
i n f o
Article history: Received 13 February 2013 Received in revised form 25 April 2013 Accepted 16 May 2013 Keywords: Drought stress Abscisic acid Jasmonic acid Polyamines Polyamine oxidase activity Tomato
a b s t r a c t Drought stress causes various physiologic and biochemical effects in plants. The phytohormones in plant systems are closely involved in responses against drought stress. However, information on the regulatory effect of abscisic acid (ABA), and free polyamine (PA) metabolism on the accumulation of JA is limited. Elucidating the endogenous mechanisms that confer stress resistance is essential to providing insights into the potential of plants to adapt to environmental change. This study aims to determine the relationship between the concentrations of abscisic acid (ABA) and jasmonic acid (JA) and the accumulation of free PAs (putrescine, spermine, and spermidine), as well as with polyamine oxidase (PAO) activity, in tomato (Lycopersicon esculentum M.) seedlings grown hydroponically under polyethylene glycol – induced drought stress. The results indicate that the concentrations of endogenous ABA, JA, and free polyamines, and the PAO activity in the roots and leaves of tomato seedlings were generally higher in the treatment groups than in the untreated controls. A significantly positive correlation was observed between the concentrations of endogenous polyamines and PAO activity (R = 0.708**) in roots and leaves of tomato seedlings. The time course in the present experiment demonstrated that the ABA concentrations increase in the roots prior to that in the leaves. Therefore, under drought stress, the higher concentrations of endogenous spermine and spermidine in the roots and leaves stimulate the simultaneous accumulation of endogenous ABA and JA with increasing PAO activity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Plants encounter various stressful environmental conditions in their life cycle. About one-third of the global potentially viable land suffers from an inadequate water supply. Drought stress causes various physiologic and biochemical effects on plant populations (Farooq et al., 2009). Plants have systems for tolerating environmental stresses. Some phytohormones are closely associated with these systems. For instance, abscisic acid (ABA), as one of the major phytohormones linking plant responses to stress (Hirayama and Shinozaki, 2007), is ubiquitous in higher plants, and interacts with membrane phospholipids to stabilize plant cell membranes against stress conditions, and to enhance tolerance as a messenger in stress perception response pathways, such as in drought stress (Guschina
Abbreviations: ABA, Abscisic acid; JA, Jasmonic acid; PA, Polyamine; Pas, Putrescine/spermine and spermidine; PAO, Polyamine oxidase; PEG, Polyethylene glycol; Put, Putrescine; PVP, Poly vinyl pyrrolidone; Spd, Spermidine; Spm, Spermine. ∗ Corresponding author. Tel.: +86 835 2882515; fax: +86 835 2882515. E-mail address: [email protected] (Z. Huang). 1 These authors contributed equally to this work. 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.05.013
et al., 2002). Vegetative development is frequently influenced by environmental stress. Many studies have shown that ABA acclimatization occurs in plants exposed to drought, which indicates that ABA plays an important role in desiccation tolerance (Hirayama and Shinozaki, 2007). On the other hand, polyamines (PAs) are ubiquitous nitrogen-containing polycationic compounds found in all eukaryotic cells. The most abundant PAs in plants are putrescine (Put), spermidine (Spd) and spermine (Spm). As plant hormones, PAs are susceptible to environmental changes as indicated by the massive increase in PAs in plants for regulating plant growth and development under stress conditions, and for preventing stressinduced damage (Yamaguchi et al., 2007). PAs are closely associated with plant resistance to water stress (Groppa and Benavides, 2008). Environmental stress induces considerable increases or decreases in cellular PA concentrations, depending on the type of stress, plant species, and duration of stress application (Kasinathan and Wingler, 2004), which may mediate intracellular hormonal effects or act as a secondary hormonal messenger. Different stresses may influence PA metabolism in different manners and specific functions when exposed to stress conditions (Sharma and Dietz, 2006). PA oxidase (PAO) is one of the key enzymes that regulate the PA metabolic pathway in different model plant systems (Groppa and Benavides, 2008). Aside from PAs and ABA, jasmonic acid (JA) is also a key
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mediator in the signaling pathways in plant defense systems (Overmyer et al., 2003). Several studies provide ample evidence that JA concentrations increase rapidly and transiently in response to various types of stress, such as salt stress (Pedranzani et al., 2003), and desiccation stress (Shan and Liang, 2010), which both show that JA contributes to stress tolerance. PAs enhance the desiccation tolerance of plants by regulating endogenous ABA concentrations under water stress conditions. Under chill stress, the rice seedlings showed a remarkable ABA-increase and simultaneous enhanced PA synthesis (Gill and Tuteja, 2010). ABA treatment increases the PA concentrations in sugarcane as a response to desiccation, whereas JA treatment is ineffective under these processes (Nieves et al., 2001). ABA and JA generally increased in response to salinity, these phytohormones may have separate and interactive effects on how plants respond and adapt to stress in natural environments (Wang et al., 2001). There was also a remarkable JA-increase in two populations of Pinus pinaster Ait. under water stress, thus the JA-response was much more prominent under water stress than under cold stress(Pedranzani et al., 2007). Different patterns of JAs have been found in different varieties of the same species (e.g., tomato) related to the stress tolerance (Pedranzani et al., 2003). All these studies suggest that these phytohormones contribute to drought tolerance. Until now, the relationship between ABA, JA, and free PA metabolism in plant populations is unclear aside from the respective roles of each under drought stress. Information on the regulatory effect of ABA and free PA on JA accumulation in plants is limited. The mechanism by which endogenous substances modify stress tolerance in plants has to be further elucidated. To our knowledge, this is the first investigation to link responses to polyethylene glycol (PEG)-induced drought stress and the phytohormones ABA, JA, and PA in the same plant population. The findings would have important implications in revealing the mechanisms that confer stress resistance and provide insights into the potential of plants to adapt to environmental change. The objectives of this study are as follows: (1) to determine whether ABA and endogenous JA interact with PAs and to elucidate the underlying mechanisms in tomato seedlings (Lycopersicon esculentum M.) under PEG-induced drought stress using two cultivars with different degrees of drought resistance; (2) to find the relationship among endogenous ABA, JA, and PAs in plants under drought stress; (3) to identify differences in desiccation tolerance between the two tomato cultivars; and (4) to verify the roles of these hormones.
2. Materials and methods 2.1. Plant materials and treatments The study was conducted from October to December of 2009 and repeated in May to July of 2010 in an environmentally controlled greenhouse at the Horticultural College in Northwest A & F University, Yangling, Shanxi Province, China (34◦ 20 N, 108◦ 7 E). The two tomato cultivars HuangGuan (drought-susceptible), which originated from the southwestern climatic region (Guangzhou, 23◦ 8 N, 113◦ 17 E), and MaoFen802 (drought-resistant), which originated from northwestern climatic region (Yangling), were selected for this study. Both tomato cultivars were selected among eight cultivars that originated from different climatic regions of China. The seeds of the two tomato cultivars were surface-sterilized with 50% NaClO (8% active Cl2 ) for 10 min, thoroughly rinsed in distilled water, and germinated at 25 ◦ C in moistened vermiculite for 3–4 days. The germinated seeds were then sown individually in 10 cm-diameter plastic pots filled with homogenized soil that contained 4 g of a slow-release fertilizer (Osmocote 17-6-12 with micronutrients), and were placed in a naturally lit greenhouse
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under semicontrolled environmental condition. The temperatures in the greenhouse ranged from 20 ◦ C to 30 ◦ C in the day and from 15 ◦ C to 18 ◦ C at night, with a 12 h photoperiod under a photosynthetic photon flux density of 130 mol m−2 s−1 , and relative aerial humidity fluctuating between 60% and 75%. The plants were watered with half strength Hoagland’s solution. After the 4th leaf of the seedlings was fully expanded, seedlings were selected for uniformity and transplanted to troughs containing 7 L of full-strength Hoagland’s solution (pH 6.5 ± 0.1; EC from 2.2 mS cm−1 to 2.5 mS cm−1 ). The nutrient solution was aerated using an air pump to supply oxygen intermittently (40 min h−1 ) to maintain the dissolved oxygen at 7.8 mg L−1 ± 0.2 mg L−1 . The solutions were renewed every 5 d and pre-culturing was conducted at the sixth leaf stage. Experimental treatments were started 7 days after preculturing. The treatments were as follows: (a) control, full-strength Hoagland solution; and (b) polyethylene glycol 6000 (PEG6000) treatment, 10% PEG, which is equivalent to an osmotic potential of −0.40 MPa. The troughs were arranged in a completely randomized block design with three replicates, providing 12 troughs with 54 plants per treatment. At the beginning (0 h), and after 6, 12, 24, 36, and 48 h of treatment, roots and leaves were sampled from each treatment, rinsed in distilled water, flash frozen in liquid nitrogen, and stored at −72 ◦ C for further use. The content of ABA and JA, as well as the three free PAs (Put, Spm, and Spd) were simultaneously measured and recorded with three replicates. 2.2. Determination of the ABA content ABA was extracted from plant tissues using the method by Kondo et al. (2001) with minor modifications, depending on the plant material. During extraction for each cultivar and treatment, ABA content was determined for each sample. Then, 50 mg of sample plant material were kept on a shaker at 4 ◦ C in the dark to prevent ABA isomerization. A series of organic solvent mixtures were used to remove interfering substances. After the filtrate was dried, it was re-dissolved in 10 mL of 0.1 M phosphate buffer at pH 8, and the pH adjusted to 2.5. ABA was extracted with ethyl acetate. The extracts were vacuum-dried, dissolved in 1 mL of absolute methanol, and passed through 0.45 m filters. ABA was quantified using high-performance liquid chromatography (HPLC; Waters 600E system, Waters Corporation, USA) equipped with two pumps and a diode array UV2487 detector. The separation was carried out on a 4.5 mm × 250 mm C–18 column (Spherisorb ODS 2–3 mm) using an isocratic system of solvent (water/methanol/acetic acid, 40:60:1). The mobile phase solutions were filtered through at 0.45 m membrane filter and degassed before use. The highperformance liquid chromatography was operated at a flow rate of 0.8 mL min−1 and was adjusted to pH 2.8; detection was performed at 254 nm. Identification and quantification were performed using pure ABA (cis–trans isomer, Sigma) as the external standard. The ABA concentration was calculated as ng g−1 fresh weight. 2.3. Determination of the JA content The JA content was determined via enzyme-linked immunosorbent assay (ELISA), using polyclonal anti-JA antibodies prepared using the method by Albrechet et al. (Albrecht et al., 1993) with slight modifications. Then, 0.5 g of samples were ground in liquid nitrogen with 3 mL of 80% methanol (1% PVP), allowed to stand overnight at 4 ◦ C, ultrasonicated, and centrifuged at 10,000 × g for 20 min at 4 ◦ C. The supernates were collected, and the precipitates were extracted once more with 80% methanol. The supernatant liquid was pooled, dried with N2 , and redissolved in 1.5 mL of the extraction solution before the ELISA. According to ELISA process,
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microtitration plates were coated with 100 L of JA coating buffer for 3 h at 37 ◦ C, and then the plates were washed with washing buffer. Then, 50 L of a series of JA standards and samples in different diluents were added into each well in triplicate, and 50 L of diluted anti-rabbit serum were added into each well. The plates were homogenized and incubated for 2 h at 37 ◦ C. Anti-JA antibodies were added into each well, and the plates were incubated for 1 h at 37 ◦ C. The absorbance at OD490nm was read after applying 50 L of stopping solution (2 mol L−1 H2 SO4 ).
2.6. Statistical analysis Data are presented as the mean and standard deviation of three replicates in each treatment and in the control groups. The data are expressed as mean ± standard error (±S.E.) in every treatment. All statistical analyses were performed using Microsoft Excel and the SPSS statistical package, version 12.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results
2.4. Determination of the free PA content
3.1. Endogenous JA and ABA content
Free PA was determined according to the method by Silveira et al. (2004) with some modifications. The root and leaf samples (0.5 g) were extracted, homogenized in 5% perchloric acid (v/v), and placed on ice for 1 h. After centrifugation at 12,000 × g for 30 min at 4 ◦ C, the PAs were purified using a 4.5 mm × 250 mm reverse-phase C–18 column (5 m particle size). The eluent consisted of acetronitrile in water (pH 3.5). The acetonitrile gradient was maintained at 65% over the first 10 min, increased from 65% to 100% for 10 min to 15 min, and then maintained at 100% for 15 min to 22 min. The column was washed with 100% methanol for 5 min, re-equilibrated with 65% methanol before the next sample was injected. The samples were run isocratically at a flow rate of 0.5 mL min−1 to 0.7 mL min−1 at 37 ◦ C for 20 min to 25 min, or at 30 ◦ C for 30 min 40 min. The reaction was terminated by adding 4 mL of saturated NaCl solution, and 20 L of the sample was collected for HPLC analysis on a programmable HPLC (Model Waters 600E, Waters Inc., USA). Standards were processed simultaneously and 20 nmol for each standard PA (Sigma, U.S.A.) was dansylated. The detector was set to 254 nm the UV absorption was measured. The eluent peaks, with their retention time and area, were recorded using an attached integrator. The reagents (Put, Spd, and Spm) were purchased from Sigma Chemical Co. (P.O. Box 14508, St. Louis, MO 63178, USA).)
Time course of changes in JA and ABA concentrations were monitored for 48 h in the leaves and roots of tomato seedlings exposed to 10% PEG-induced drought treatment. The endogenous JA and ABA contents, in both cultivars, increased steadily under PEGinduced drought-stress compared with the control groups (Fig. 1). Small quantities of JA were detected in normal leaves of two cultivars. The JA concentrations in the root segments were higher compared with the control groups (Fig. 1A), increased rapidly at 6 h after treatment, and reached a maximum after 12 h. The JA concentrations in the leaves increased during the successive sampling periods, and reached its maximum after 24 h (Fig. 1B). Significant differences in ABA concentration were observed between the two cultivars after drought stress. The ABA concentrations in the roots of both cultivars also increased and peaked after 24 h for MaoFen802 and after 12 h for HuangGuan (Fig. 1C). However, the ABA concentrations in the leaves peaked after 36 h for MaoFen802 and after 24 h for HuangGuan (Fig. 1D). The time course of the experiment demonstrated that the ABA concentrations in the roots increased prior to that in the leaves. The same pattern was noted for both cultivars. The 10% PEG treatments significantly increased the endogenous ABA concentrations, although the JA concentrations were higher in MaoFen802 than in HuangGuan. The results suggest that the synergistic interaction between ABA and JA mediates drought resistance in seedlings subjected to drought stress. Furthermore, the time course of the experiment demonstrated that the ABA concentrations in the roots increased prior to that in the leaves. Therefore, the ABA and JA concentrations in tomato seedlings are correlated with an early response to drought stress.
2.5. Determination of the polyamine oxidase Polyamine oxidase (PAO) activity was determined according to the method by Smith (1972) with key modifications depending on the plant materials. Potassium phosphate buffer (1.6 mL; 0.1 M), adjusted to pH 6.5 using NaOH, was added to the samples. The mixture was homogenized in an ice bath, transferred into a centrifuge tube, and centrifuged at 4 ◦ C for 20 min at 10,000 × g. Then, the supernatant solution was used for enzyme assays, with 200 L of the remaining enzyme extract set aside for the next procedure. The reaction was carried out at 25 ◦ C in a reaction mixture (3.0 mL) containing 2.5 mL of 0.1 M potassium phosphate buffer (pH 6.5), 0.2 mL of 0.1 M staining solution containing phosphatebuffered saline (pH 6.5), 4-aminoantipyrine/N,N-dimethylaniline solution, and 0.1 mL of horseradish peroxidase solution (specific activity 250 units/mL of protein; Sigma), and added to 200 L of the enzyme extract. The reaction was initiated with the addition of 100 L of 20 mM spermine. The reaction mixture was incubated at 25 ◦ C for 30 min, and then terminated with the addition of 0.5 mL of 10% trichloroacetic acid (TCA). The optical density (OD) of the reaction mixture was then read at 550 nm using a UV–1700 spectrophotometer. All procedures were carried out between 0 ◦ C and 4 ◦ C. One unit of enzyme activity is defined as 0.001 absorbance unit of change in the optical density at 550 nm min−1 . Enzyme activity was determined in three independent experiments with three replicates each. Assessment of activity was based on measurements obtained within the first 3 min of the assay.
3.2. The endogenous PA content and PAO activity In the following, PEG-induced drought stress triggered organspecific changes in PA concentrations and PAO activity. Short-term stress (6 h) caused a significant accumulation of the three free PAs in the roots and leaves (Fig. 2). In the control group, the concentrations of the three free PAs in the roots were relatively stable. The drought treatment caused a continuous increase during the successive sampling time points and the concentrations of the three free PAs increased slightly within the first 6 h. The increase started upon stress initiation for both cultivars, and then reached their maximum at 12 h after treatment. Put increased 2.67fold, Spd increased 6.9-fold, and Spm increased 6.21-fold in the roots of MaoFen802 seedlings, whereas in the roots of HuangGuan seedlings, Put increased 1.87-fold, Spd increased 3.34-fold, and Spm increased 3.82-fold (Fig. 2A, C, and E). After a longer exposure time (12 h), the PA concentrations in the roots decreased, but remained significantly higher than those in the control groups. Similar changes were observed in the drought-stressed leaves. The PA concentrations peaked at 12 h after treatment. Put increased 2.77fold, Spd increased 4.51-fold, and Spm increased 3.69-fold in the leaves of MaoFen802 seedlings, whereas Put increased 2.08-fold, Spd increased 2.95-fold, and Spm increased 3.02-fold in the leaves
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of HuangGuan seedlings (Fig. 2B, D, and F). In particular, Spd and Spm increased much faster than Put both in the drought-stressed leaves and roots. In general, all three PAs remarkably increased during the early drought period in the MaoFen802 seedlings. However, the increase in PAs was less than that in the MaoFen802 seedling in the HuangGuan seedlings. In the control groups, the PAO activity in the droughtsusceptible seedlings was higher than that of drought-resistant ones. The PAO activity increased rapidly and peaked after 12 h of treatment in both the roots and the leaves, and followed a similar trend in both cultivars (Fig. 3A and B). The time course of the changes suggests that the PAO activity increased with increasing PA accumulation. A strong increase in PAO activity was observed in the HuangGuan seedlings. The change in PAO activity in the droughtsensitive HuangGuan seedlings was much greater than that in the drought-resistant MaoFen802 seedlings. The results show a significantly positive correlation between endogenous PA content and PAO activity (R = 0.708**) in the roots and leaves of the tomato seedlings. At the end of treatment, the PAO activity in both cultivars decreased to that in the control groups. 4. Discussion 4.1. Effect of the content of endogenous JA and ABA in tomato seedlings under PEG-induced drought stress The response of plants to unfavorable stress involves complex physiologic responses, including changes in the concentrations and ratios of endogenous hormones. As a generic stress hormone with multiple functions, ABA plays an important role in drought adaptation. Previous studies have shown that endogenous ABA content increases rapidly under water stress, which enhances drought tolerance in woody plants (Zhang et al., 2004). Increased ABA concentration have been reported in wheat exposed to drought stress (Farooq et al., 2009). In the present study, the relationship between phytohormones and drought tolerance has been demonstrated in
tomato cultivars. After short-term exposure of seedlings of both cultivars to PEG-induced drought stress, the ABA and JA accumulated in the roots and leaves. As shown in Fig. 1, the ABA concentrations in the two tomato cultivars changed after drought treatment, which is consistent with previously reported data in rice spikelets (Yang et al., 2007) wherein the ABA concentrations increased against water stress. No significant difference in ABA concentrations was observed between the two cultivars with different drought resistance. One possible explanation is that the susceptibility to water stress varied with the species of plants. Adaptation to drought stress resulted in different responses in different plant species when exposed to stress conditions. Moreover, the results indicate that the 10% PEG treatments significantly increased endogenous ABA and JA in MaoFen802 more than in HuangGuan, which confirms that the former is more drought-tolerant than the latter. ABA concentrations are higher in the drought-tolerant cultivars than in drought-sensitive cultivars (Gunes et al., 2008). The current study presents evidence that tomato cultivars with different drought tolerance also differ in JA basal content. JA rapidly accumulated in both tomato cultivars after they were subjected to drought stress. The increase in JA and ABA concentrations in the tomato seedlings confirms that the phytohormones play important roles in the adaptation of plant growth and development to drought stress (Farooq et al., 2009). Similar results were also observed by Pedranzani et al. (2003) who found that exposure of tomato cultivars to salt stress increases the accumulation of endogenous JA, and that tomato cultivars with different concentrations of tolerance have different basal JA content and different levels of JA responses to salt stress. Horváth et al. (2007) found that JA and ABA concentrations increase in vegetative organs and the two hormones may act synergistically to increase salt tolerance. These results show adaptive differentiation between different L. esculentum populations, and support the relationship between environmental heterogeneity and the magnitude of responses in plants. Genetic differences in drought tolerance provide the unique opportunity of comparing physiologic responses to drought stress.
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4.2. Effect of the endogenous PA content and PAO activity in tomato seedlings under PEG-induced drought stress
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Plant species and cultivars with high stress tolerance have greater capacities to enhance PA biosynthesis for regulating environmental stresses, including drought (Groppa and Benavides, Huang guan 10%PEG Mao fen 802 10%PEG Huang guan CK Mao fen 802 CK
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2008). As shown in Fig. 2, drought stress induced a continuous increase in the content of Put, Spd and Spm. Among the three major PAs, Spd and Spm accumulated at higher concentrations than Put in both cultivars, which indicates that maintaining Spm and Spd are required for drought tolerance. These results agree with previous reports that demonstrated that increases in Spd and Spm
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concentrations are regulated more closely than those of Put under drought stress (Urano et al., 2003). Basu et al. (1988) reported that the salt-tolerant cultivars showed a slight increase in Put and higher increases in Spd and Spm. Moreover, salt-tolerant cultivars have higher Spd and Spm concentrations (Chattopadhayay et al., 2002). Similarly, Spd and Spm are greatly involved in ABA-induced sugarcane maturation combined with JA (Nieves et al., 2001). In the current study, prolonged stress conditions (24 h to 48 h) resulted in a decline in the three PAs in the roots and in the leaves, which may indicate that the PA biosynthetic pathway is sensitive to prolonged exposure to high water deficits. The PA biosynthesis in wheat varieties with different drought and salt tolerance show genotypedependent responses (Kusano et al., 2008). As shown in Fig. 2 and Fig. 3, a parallel increase in the concentrations of the three free PAs and the increase in PAO activity under drought stress suggest that PAO plays an important role in regulating drought-induced PA accumulation in tomato seedlings. On the other hand, PAO activity increased considerably after short-term exposure to stress, similar to the results of Duan et al. (2008). The significant increase in PAO activity in HuangGuan under drought stress could account for the lesser increase in Spd and Spm in HuangGuan compared with Maofen802. The positive correlation between endogenous PA content and PAO activity (R = 0.708**) in the roots and leaves of tomato suggest that PAO regulates the free PA content. In the current study, higher PA concentrations were detected in the roots and leaves of stressed plants, which suggest that PA metabolism in drought-tolerant tomato plants is an adaptive mechanism to drought stress. 5. Conclusions Elucidating the endogenous mechanisms that confer stress resistance is essential to providing insights into the potential of plants to adapt to environmental change. In summary, the present study shows that PEG-induced drought stress has significant and different effects on endogenous ABA and JA. The increase in free PAs in response to desiccation stress increases these endogenous substances are important mechanisms for initiating a corresponding defense in plants. The increase in ABA concentrations in the roots occurred prior to that in the leaves. The time course analysis of the changes in the concentrations of ABA, JA, and PAs indicated that the increase in PA content occurred prior to the increase in ABA and JA content. Consequently, the PAs may promote the accumulation of ABA and JA in plants under PEG-induced drought stress. The results also show a significant positive correlation between endogenous PA content and PAO activity (R = 0.708**) in the roots and the leaves of the seedlings. The concentrations of ABA, JA, and PAs increased in the drought-stressed plants, which suggest that these phytohormones play critical roles in plant responses to drought stress. The endogenous ABA and JA content exhibited an extremely close correlation with PA accumulation. Our results confirm that hormonal responses in plants regulate their metabolism to facilitate their survival and adaptation to stressful environments. Acknowledgments This work was supported by the Department of Education of Sichuan (Project 12ZB090). The authors are very grateful to Prof. Q.C. Wang for critical reading and comments of the manuscript.
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Reactive oxygen species and hormonal control of cell death. Trends Plant Sci. 8, 335–342. ˜ Pedranzani, H., Racagni, G., Alemano, S., Miersch, O., Ramírez, I., Pena-Cortés, H., Taleisnik, E., Machado-Domenech, E., Abdala, G., 2003. Salt tolerant tomato plants show increased levels of jasmonic acid. Plant Growth Regul. 41, 149–158. Pedranzani, H., Sierra-de-Grado, R., Vigliocco, A., Miersch, O., Abdala, G., 2007. Cold and water stresses produce changes in endogenous jasmonates in two populations of Pinus pinaster Ait. Plant Growth Regul. 52, 111–116. Shan, C., Liang, Z., 2010. Jasmonic acid regulates ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stress. Plant Sci 178, 130–139. Sharma, S.S., Dietz, K.J., 2006. The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress. J. Exp. Bot. 57, 711–726. Silveira, V., Iochevet Segal Floh, E., Handro, W., Pedro Guerra, M., 2004. 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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Effects of endogenous abscisic acid, jasmonic acid, polyamines, and polyamine oxidase activity in tomato seedlings under drought stress Chunmei Zhang a,1 , Zhi Huang b,∗,1 a b
College of Agriculture and Biology Technology, Hexi University, ZhangYe, Gansu 734000, PR China College of Horticulture, Sichuan Agricultural University, Ya’an, Sichuan, 625014, PR China
a r t i c l e
i n f o
Article history: Received 13 February 2013 Received in revised form 25 April 2013 Accepted 16 May 2013 Keywords: Drought stress Abscisic acid Jasmonic acid Polyamines Polyamine oxidase activity Tomato
a b s t r a c t Drought stress causes various physiologic and biochemical effects in plants. The phytohormones in plant systems are closely involved in responses against drought stress. However, information on the regulatory effect of abscisic acid (ABA), and free polyamine (PA) metabolism on the accumulation of JA is limited. Elucidating the endogenous mechanisms that confer stress resistance is essential to providing insights into the potential of plants to adapt to environmental change. This study aims to determine the relationship between the concentrations of abscisic acid (ABA) and jasmonic acid (JA) and the accumulation of free PAs (putrescine, spermine, and spermidine), as well as with polyamine oxidase (PAO) activity, in tomato (Lycopersicon esculentum M.) seedlings grown hydroponically under polyethylene glycol – induced drought stress. The results indicate that the concentrations of endogenous ABA, JA, and free polyamines, and the PAO activity in the roots and leaves of tomato seedlings were generally higher in the treatment groups than in the untreated controls. A significantly positive correlation was observed between the concentrations of endogenous polyamines and PAO activity (R = 0.708**) in roots and leaves of tomato seedlings. The time course in the present experiment demonstrated that the ABA concentrations increase in the roots prior to that in the leaves. Therefore, under drought stress, the higher concentrations of endogenous spermine and spermidine in the roots and leaves stimulate the simultaneous accumulation of endogenous ABA and JA with increasing PAO activity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Plants encounter various stressful environmental conditions in their life cycle. About one-third of the global potentially viable land suffers from an inadequate water supply. Drought stress causes various physiologic and biochemical effects on plant populations (Farooq et al., 2009). Plants have systems for tolerating environmental stresses. Some phytohormones are closely associated with these systems. For instance, abscisic acid (ABA), as one of the major phytohormones linking plant responses to stress (Hirayama and Shinozaki, 2007), is ubiquitous in higher plants, and interacts with membrane phospholipids to stabilize plant cell membranes against stress conditions, and to enhance tolerance as a messenger in stress perception response pathways, such as in drought stress (Guschina
Abbreviations: ABA, Abscisic acid; JA, Jasmonic acid; PA, Polyamine; Pas, Putrescine/spermine and spermidine; PAO, Polyamine oxidase; PEG, Polyethylene glycol; Put, Putrescine; PVP, Poly vinyl pyrrolidone; Spd, Spermidine; Spm, Spermine. ∗ Corresponding author. Tel.: +86 835 2882515; fax: +86 835 2882515. E-mail address: [email protected] (Z. Huang). 1 These authors contributed equally to this work. 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.05.013
et al., 2002). Vegetative development is frequently influenced by environmental stress. Many studies have shown that ABA acclimatization occurs in plants exposed to drought, which indicates that ABA plays an important role in desiccation tolerance (Hirayama and Shinozaki, 2007). On the other hand, polyamines (PAs) are ubiquitous nitrogen-containing polycationic compounds found in all eukaryotic cells. The most abundant PAs in plants are putrescine (Put), spermidine (Spd) and spermine (Spm). As plant hormones, PAs are susceptible to environmental changes as indicated by the massive increase in PAs in plants for regulating plant growth and development under stress conditions, and for preventing stressinduced damage (Yamaguchi et al., 2007). PAs are closely associated with plant resistance to water stress (Groppa and Benavides, 2008). Environmental stress induces considerable increases or decreases in cellular PA concentrations, depending on the type of stress, plant species, and duration of stress application (Kasinathan and Wingler, 2004), which may mediate intracellular hormonal effects or act as a secondary hormonal messenger. Different stresses may influence PA metabolism in different manners and specific functions when exposed to stress conditions (Sharma and Dietz, 2006). PA oxidase (PAO) is one of the key enzymes that regulate the PA metabolic pathway in different model plant systems (Groppa and Benavides, 2008). Aside from PAs and ABA, jasmonic acid (JA) is also a key
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mediator in the signaling pathways in plant defense systems (Overmyer et al., 2003). Several studies provide ample evidence that JA concentrations increase rapidly and transiently in response to various types of stress, such as salt stress (Pedranzani et al., 2003), and desiccation stress (Shan and Liang, 2010), which both show that JA contributes to stress tolerance. PAs enhance the desiccation tolerance of plants by regulating endogenous ABA concentrations under water stress conditions. Under chill stress, the rice seedlings showed a remarkable ABA-increase and simultaneous enhanced PA synthesis (Gill and Tuteja, 2010). ABA treatment increases the PA concentrations in sugarcane as a response to desiccation, whereas JA treatment is ineffective under these processes (Nieves et al., 2001). ABA and JA generally increased in response to salinity, these phytohormones may have separate and interactive effects on how plants respond and adapt to stress in natural environments (Wang et al., 2001). There was also a remarkable JA-increase in two populations of Pinus pinaster Ait. under water stress, thus the JA-response was much more prominent under water stress than under cold stress(Pedranzani et al., 2007). Different patterns of JAs have been found in different varieties of the same species (e.g., tomato) related to the stress tolerance (Pedranzani et al., 2003). All these studies suggest that these phytohormones contribute to drought tolerance. Until now, the relationship between ABA, JA, and free PA metabolism in plant populations is unclear aside from the respective roles of each under drought stress. Information on the regulatory effect of ABA and free PA on JA accumulation in plants is limited. The mechanism by which endogenous substances modify stress tolerance in plants has to be further elucidated. To our knowledge, this is the first investigation to link responses to polyethylene glycol (PEG)-induced drought stress and the phytohormones ABA, JA, and PA in the same plant population. The findings would have important implications in revealing the mechanisms that confer stress resistance and provide insights into the potential of plants to adapt to environmental change. The objectives of this study are as follows: (1) to determine whether ABA and endogenous JA interact with PAs and to elucidate the underlying mechanisms in tomato seedlings (Lycopersicon esculentum M.) under PEG-induced drought stress using two cultivars with different degrees of drought resistance; (2) to find the relationship among endogenous ABA, JA, and PAs in plants under drought stress; (3) to identify differences in desiccation tolerance between the two tomato cultivars; and (4) to verify the roles of these hormones.
2. Materials and methods 2.1. Plant materials and treatments The study was conducted from October to December of 2009 and repeated in May to July of 2010 in an environmentally controlled greenhouse at the Horticultural College in Northwest A & F University, Yangling, Shanxi Province, China (34◦ 20 N, 108◦ 7 E). The two tomato cultivars HuangGuan (drought-susceptible), which originated from the southwestern climatic region (Guangzhou, 23◦ 8 N, 113◦ 17 E), and MaoFen802 (drought-resistant), which originated from northwestern climatic region (Yangling), were selected for this study. Both tomato cultivars were selected among eight cultivars that originated from different climatic regions of China. The seeds of the two tomato cultivars were surface-sterilized with 50% NaClO (8% active Cl2 ) for 10 min, thoroughly rinsed in distilled water, and germinated at 25 ◦ C in moistened vermiculite for 3–4 days. The germinated seeds were then sown individually in 10 cm-diameter plastic pots filled with homogenized soil that contained 4 g of a slow-release fertilizer (Osmocote 17-6-12 with micronutrients), and were placed in a naturally lit greenhouse
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under semicontrolled environmental condition. The temperatures in the greenhouse ranged from 20 ◦ C to 30 ◦ C in the day and from 15 ◦ C to 18 ◦ C at night, with a 12 h photoperiod under a photosynthetic photon flux density of 130 mol m−2 s−1 , and relative aerial humidity fluctuating between 60% and 75%. The plants were watered with half strength Hoagland’s solution. After the 4th leaf of the seedlings was fully expanded, seedlings were selected for uniformity and transplanted to troughs containing 7 L of full-strength Hoagland’s solution (pH 6.5 ± 0.1; EC from 2.2 mS cm−1 to 2.5 mS cm−1 ). The nutrient solution was aerated using an air pump to supply oxygen intermittently (40 min h−1 ) to maintain the dissolved oxygen at 7.8 mg L−1 ± 0.2 mg L−1 . The solutions were renewed every 5 d and pre-culturing was conducted at the sixth leaf stage. Experimental treatments were started 7 days after preculturing. The treatments were as follows: (a) control, full-strength Hoagland solution; and (b) polyethylene glycol 6000 (PEG6000) treatment, 10% PEG, which is equivalent to an osmotic potential of −0.40 MPa. The troughs were arranged in a completely randomized block design with three replicates, providing 12 troughs with 54 plants per treatment. At the beginning (0 h), and after 6, 12, 24, 36, and 48 h of treatment, roots and leaves were sampled from each treatment, rinsed in distilled water, flash frozen in liquid nitrogen, and stored at −72 ◦ C for further use. The content of ABA and JA, as well as the three free PAs (Put, Spm, and Spd) were simultaneously measured and recorded with three replicates. 2.2. Determination of the ABA content ABA was extracted from plant tissues using the method by Kondo et al. (2001) with minor modifications, depending on the plant material. During extraction for each cultivar and treatment, ABA content was determined for each sample. Then, 50 mg of sample plant material were kept on a shaker at 4 ◦ C in the dark to prevent ABA isomerization. A series of organic solvent mixtures were used to remove interfering substances. After the filtrate was dried, it was re-dissolved in 10 mL of 0.1 M phosphate buffer at pH 8, and the pH adjusted to 2.5. ABA was extracted with ethyl acetate. The extracts were vacuum-dried, dissolved in 1 mL of absolute methanol, and passed through 0.45 m filters. ABA was quantified using high-performance liquid chromatography (HPLC; Waters 600E system, Waters Corporation, USA) equipped with two pumps and a diode array UV2487 detector. The separation was carried out on a 4.5 mm × 250 mm C–18 column (Spherisorb ODS 2–3 mm) using an isocratic system of solvent (water/methanol/acetic acid, 40:60:1). The mobile phase solutions were filtered through at 0.45 m membrane filter and degassed before use. The highperformance liquid chromatography was operated at a flow rate of 0.8 mL min−1 and was adjusted to pH 2.8; detection was performed at 254 nm. Identification and quantification were performed using pure ABA (cis–trans isomer, Sigma) as the external standard. The ABA concentration was calculated as ng g−1 fresh weight. 2.3. Determination of the JA content The JA content was determined via enzyme-linked immunosorbent assay (ELISA), using polyclonal anti-JA antibodies prepared using the method by Albrechet et al. (Albrecht et al., 1993) with slight modifications. Then, 0.5 g of samples were ground in liquid nitrogen with 3 mL of 80% methanol (1% PVP), allowed to stand overnight at 4 ◦ C, ultrasonicated, and centrifuged at 10,000 × g for 20 min at 4 ◦ C. The supernates were collected, and the precipitates were extracted once more with 80% methanol. The supernatant liquid was pooled, dried with N2 , and redissolved in 1.5 mL of the extraction solution before the ELISA. According to ELISA process,
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microtitration plates were coated with 100 L of JA coating buffer for 3 h at 37 ◦ C, and then the plates were washed with washing buffer. Then, 50 L of a series of JA standards and samples in different diluents were added into each well in triplicate, and 50 L of diluted anti-rabbit serum were added into each well. The plates were homogenized and incubated for 2 h at 37 ◦ C. Anti-JA antibodies were added into each well, and the plates were incubated for 1 h at 37 ◦ C. The absorbance at OD490nm was read after applying 50 L of stopping solution (2 mol L−1 H2 SO4 ).
2.6. Statistical analysis Data are presented as the mean and standard deviation of three replicates in each treatment and in the control groups. The data are expressed as mean ± standard error (±S.E.) in every treatment. All statistical analyses were performed using Microsoft Excel and the SPSS statistical package, version 12.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results
2.4. Determination of the free PA content
3.1. Endogenous JA and ABA content
Free PA was determined according to the method by Silveira et al. (2004) with some modifications. The root and leaf samples (0.5 g) were extracted, homogenized in 5% perchloric acid (v/v), and placed on ice for 1 h. After centrifugation at 12,000 × g for 30 min at 4 ◦ C, the PAs were purified using a 4.5 mm × 250 mm reverse-phase C–18 column (5 m particle size). The eluent consisted of acetronitrile in water (pH 3.5). The acetonitrile gradient was maintained at 65% over the first 10 min, increased from 65% to 100% for 10 min to 15 min, and then maintained at 100% for 15 min to 22 min. The column was washed with 100% methanol for 5 min, re-equilibrated with 65% methanol before the next sample was injected. The samples were run isocratically at a flow rate of 0.5 mL min−1 to 0.7 mL min−1 at 37 ◦ C for 20 min to 25 min, or at 30 ◦ C for 30 min 40 min. The reaction was terminated by adding 4 mL of saturated NaCl solution, and 20 L of the sample was collected for HPLC analysis on a programmable HPLC (Model Waters 600E, Waters Inc., USA). Standards were processed simultaneously and 20 nmol for each standard PA (Sigma, U.S.A.) was dansylated. The detector was set to 254 nm the UV absorption was measured. The eluent peaks, with their retention time and area, were recorded using an attached integrator. The reagents (Put, Spd, and Spm) were purchased from Sigma Chemical Co. (P.O. Box 14508, St. Louis, MO 63178, USA).)
Time course of changes in JA and ABA concentrations were monitored for 48 h in the leaves and roots of tomato seedlings exposed to 10% PEG-induced drought treatment. The endogenous JA and ABA contents, in both cultivars, increased steadily under PEGinduced drought-stress compared with the control groups (Fig. 1). Small quantities of JA were detected in normal leaves of two cultivars. The JA concentrations in the root segments were higher compared with the control groups (Fig. 1A), increased rapidly at 6 h after treatment, and reached a maximum after 12 h. The JA concentrations in the leaves increased during the successive sampling periods, and reached its maximum after 24 h (Fig. 1B). Significant differences in ABA concentration were observed between the two cultivars after drought stress. The ABA concentrations in the roots of both cultivars also increased and peaked after 24 h for MaoFen802 and after 12 h for HuangGuan (Fig. 1C). However, the ABA concentrations in the leaves peaked after 36 h for MaoFen802 and after 24 h for HuangGuan (Fig. 1D). The time course of the experiment demonstrated that the ABA concentrations in the roots increased prior to that in the leaves. The same pattern was noted for both cultivars. The 10% PEG treatments significantly increased the endogenous ABA concentrations, although the JA concentrations were higher in MaoFen802 than in HuangGuan. The results suggest that the synergistic interaction between ABA and JA mediates drought resistance in seedlings subjected to drought stress. Furthermore, the time course of the experiment demonstrated that the ABA concentrations in the roots increased prior to that in the leaves. Therefore, the ABA and JA concentrations in tomato seedlings are correlated with an early response to drought stress.
2.5. Determination of the polyamine oxidase Polyamine oxidase (PAO) activity was determined according to the method by Smith (1972) with key modifications depending on the plant materials. Potassium phosphate buffer (1.6 mL; 0.1 M), adjusted to pH 6.5 using NaOH, was added to the samples. The mixture was homogenized in an ice bath, transferred into a centrifuge tube, and centrifuged at 4 ◦ C for 20 min at 10,000 × g. Then, the supernatant solution was used for enzyme assays, with 200 L of the remaining enzyme extract set aside for the next procedure. The reaction was carried out at 25 ◦ C in a reaction mixture (3.0 mL) containing 2.5 mL of 0.1 M potassium phosphate buffer (pH 6.5), 0.2 mL of 0.1 M staining solution containing phosphatebuffered saline (pH 6.5), 4-aminoantipyrine/N,N-dimethylaniline solution, and 0.1 mL of horseradish peroxidase solution (specific activity 250 units/mL of protein; Sigma), and added to 200 L of the enzyme extract. The reaction was initiated with the addition of 100 L of 20 mM spermine. The reaction mixture was incubated at 25 ◦ C for 30 min, and then terminated with the addition of 0.5 mL of 10% trichloroacetic acid (TCA). The optical density (OD) of the reaction mixture was then read at 550 nm using a UV–1700 spectrophotometer. All procedures were carried out between 0 ◦ C and 4 ◦ C. One unit of enzyme activity is defined as 0.001 absorbance unit of change in the optical density at 550 nm min−1 . Enzyme activity was determined in three independent experiments with three replicates each. Assessment of activity was based on measurements obtained within the first 3 min of the assay.
3.2. The endogenous PA content and PAO activity In the following, PEG-induced drought stress triggered organspecific changes in PA concentrations and PAO activity. Short-term stress (6 h) caused a significant accumulation of the three free PAs in the roots and leaves (Fig. 2). In the control group, the concentrations of the three free PAs in the roots were relatively stable. The drought treatment caused a continuous increase during the successive sampling time points and the concentrations of the three free PAs increased slightly within the first 6 h. The increase started upon stress initiation for both cultivars, and then reached their maximum at 12 h after treatment. Put increased 2.67fold, Spd increased 6.9-fold, and Spm increased 6.21-fold in the roots of MaoFen802 seedlings, whereas in the roots of HuangGuan seedlings, Put increased 1.87-fold, Spd increased 3.34-fold, and Spm increased 3.82-fold (Fig. 2A, C, and E). After a longer exposure time (12 h), the PA concentrations in the roots decreased, but remained significantly higher than those in the control groups. Similar changes were observed in the drought-stressed leaves. The PA concentrations peaked at 12 h after treatment. Put increased 2.77fold, Spd increased 4.51-fold, and Spm increased 3.69-fold in the leaves of MaoFen802 seedlings, whereas Put increased 2.08-fold, Spd increased 2.95-fold, and Spm increased 3.02-fold in the leaves
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of HuangGuan seedlings (Fig. 2B, D, and F). In particular, Spd and Spm increased much faster than Put both in the drought-stressed leaves and roots. In general, all three PAs remarkably increased during the early drought period in the MaoFen802 seedlings. However, the increase in PAs was less than that in the MaoFen802 seedling in the HuangGuan seedlings. In the control groups, the PAO activity in the droughtsusceptible seedlings was higher than that of drought-resistant ones. The PAO activity increased rapidly and peaked after 12 h of treatment in both the roots and the leaves, and followed a similar trend in both cultivars (Fig. 3A and B). The time course of the changes suggests that the PAO activity increased with increasing PA accumulation. A strong increase in PAO activity was observed in the HuangGuan seedlings. The change in PAO activity in the droughtsensitive HuangGuan seedlings was much greater than that in the drought-resistant MaoFen802 seedlings. The results show a significantly positive correlation between endogenous PA content and PAO activity (R = 0.708**) in the roots and leaves of the tomato seedlings. At the end of treatment, the PAO activity in both cultivars decreased to that in the control groups. 4. Discussion 4.1. Effect of the content of endogenous JA and ABA in tomato seedlings under PEG-induced drought stress The response of plants to unfavorable stress involves complex physiologic responses, including changes in the concentrations and ratios of endogenous hormones. As a generic stress hormone with multiple functions, ABA plays an important role in drought adaptation. Previous studies have shown that endogenous ABA content increases rapidly under water stress, which enhances drought tolerance in woody plants (Zhang et al., 2004). Increased ABA concentration have been reported in wheat exposed to drought stress (Farooq et al., 2009). In the present study, the relationship between phytohormones and drought tolerance has been demonstrated in
tomato cultivars. After short-term exposure of seedlings of both cultivars to PEG-induced drought stress, the ABA and JA accumulated in the roots and leaves. As shown in Fig. 1, the ABA concentrations in the two tomato cultivars changed after drought treatment, which is consistent with previously reported data in rice spikelets (Yang et al., 2007) wherein the ABA concentrations increased against water stress. No significant difference in ABA concentrations was observed between the two cultivars with different drought resistance. One possible explanation is that the susceptibility to water stress varied with the species of plants. Adaptation to drought stress resulted in different responses in different plant species when exposed to stress conditions. Moreover, the results indicate that the 10% PEG treatments significantly increased endogenous ABA and JA in MaoFen802 more than in HuangGuan, which confirms that the former is more drought-tolerant than the latter. ABA concentrations are higher in the drought-tolerant cultivars than in drought-sensitive cultivars (Gunes et al., 2008). The current study presents evidence that tomato cultivars with different drought tolerance also differ in JA basal content. JA rapidly accumulated in both tomato cultivars after they were subjected to drought stress. The increase in JA and ABA concentrations in the tomato seedlings confirms that the phytohormones play important roles in the adaptation of plant growth and development to drought stress (Farooq et al., 2009). Similar results were also observed by Pedranzani et al. (2003) who found that exposure of tomato cultivars to salt stress increases the accumulation of endogenous JA, and that tomato cultivars with different concentrations of tolerance have different basal JA content and different levels of JA responses to salt stress. Horváth et al. (2007) found that JA and ABA concentrations increase in vegetative organs and the two hormones may act synergistically to increase salt tolerance. These results show adaptive differentiation between different L. esculentum populations, and support the relationship between environmental heterogeneity and the magnitude of responses in plants. Genetic differences in drought tolerance provide the unique opportunity of comparing physiologic responses to drought stress.
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2008). As shown in Fig. 2, drought stress induced a continuous increase in the content of Put, Spd and Spm. Among the three major PAs, Spd and Spm accumulated at higher concentrations than Put in both cultivars, which indicates that maintaining Spm and Spd are required for drought tolerance. These results agree with previous reports that demonstrated that increases in Spd and Spm
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Fig. 3. Changes in PAO activity in the roots and leaves of two tomato cultivars seedlings under drought stress.
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concentrations are regulated more closely than those of Put under drought stress (Urano et al., 2003). Basu et al. (1988) reported that the salt-tolerant cultivars showed a slight increase in Put and higher increases in Spd and Spm. Moreover, salt-tolerant cultivars have higher Spd and Spm concentrations (Chattopadhayay et al., 2002). Similarly, Spd and Spm are greatly involved in ABA-induced sugarcane maturation combined with JA (Nieves et al., 2001). In the current study, prolonged stress conditions (24 h to 48 h) resulted in a decline in the three PAs in the roots and in the leaves, which may indicate that the PA biosynthetic pathway is sensitive to prolonged exposure to high water deficits. The PA biosynthesis in wheat varieties with different drought and salt tolerance show genotypedependent responses (Kusano et al., 2008). As shown in Fig. 2 and Fig. 3, a parallel increase in the concentrations of the three free PAs and the increase in PAO activity under drought stress suggest that PAO plays an important role in regulating drought-induced PA accumulation in tomato seedlings. On the other hand, PAO activity increased considerably after short-term exposure to stress, similar to the results of Duan et al. (2008). The significant increase in PAO activity in HuangGuan under drought stress could account for the lesser increase in Spd and Spm in HuangGuan compared with Maofen802. The positive correlation between endogenous PA content and PAO activity (R = 0.708**) in the roots and leaves of tomato suggest that PAO regulates the free PA content. In the current study, higher PA concentrations were detected in the roots and leaves of stressed plants, which suggest that PA metabolism in drought-tolerant tomato plants is an adaptive mechanism to drought stress. 5. Conclusions Elucidating the endogenous mechanisms that confer stress resistance is essential to providing insights into the potential of plants to adapt to environmental change. In summary, the present study shows that PEG-induced drought stress has significant and different effects on endogenous ABA and JA. The increase in free PAs in response to desiccation stress increases these endogenous substances are important mechanisms for initiating a corresponding defense in plants. The increase in ABA concentrations in the roots occurred prior to that in the leaves. The time course analysis of the changes in the concentrations of ABA, JA, and PAs indicated that the increase in PA content occurred prior to the increase in ABA and JA content. Consequently, the PAs may promote the accumulation of ABA and JA in plants under PEG-induced drought stress. The results also show a significant positive correlation between endogenous PA content and PAO activity (R = 0.708**) in the roots and the leaves of the seedlings. The concentrations of ABA, JA, and PAs increased in the drought-stressed plants, which suggest that these phytohormones play critical roles in plant responses to drought stress. The endogenous ABA and JA content exhibited an extremely close correlation with PA accumulation. Our results confirm that hormonal responses in plants regulate their metabolism to facilitate their survival and adaptation to stressful environments. Acknowledgments This work was supported by the Department of Education of Sichuan (Project 12ZB090). The authors are very grateful to Prof. Q.C. Wang for critical reading and comments of the manuscript.
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