JXB Advance Access originally published online on August 14, 2006
Journal of Experimental Botany 2006 57(12):3337-3347; doi:10.1093/jxb/erl098
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Novel interrelationship between salicylic acid, abscisic acid, and PIP2-specific phospholipase C in heat acclimation-induced thermotolerance in pea leaves
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
*To whom correspondence should be addressed. E-mail: huanggwd{at}263.net
Received 16 May 2006; Accepted 27 June 2006
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Abstract |
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Increasing evidence suggests that heat acclimation and exogenous salicylic acid (SA) and abscisic acid (ABA) may lead to the enhancement of thermotolerance in plants. In this study, the roles that free SA, conjugated SA, ABA, and phosphatidylinositol-4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) play in thermotolerance development induced by heat acclimation (38 °C) were investigated. To evaluate their potential functions, three inhibitors of synthesis or activity were infiltrated into pea leaves prior to heat acclimation treatment. The results showed that the burst of free SA in response to heat acclimation could be attributed to the conversion of SA 2-O-D-glucose, the main conjugated form of SA, to free SA. Inhibition of ABA biosynthesis also resulted in a defect in the free SA peak during heat acclimation. In acquired thermotolerance assessment, the greatest weakness of antioxidant enzyme activity and the most severe heat injury (malondialdehyde content and degree of wilting) were found in pea leaves pre-treated with neomycin, a well-known inhibitor of PIP2-PLC activity. PsPLC gene expression was activated by exogenous ABA, SA treatments, and heat acclimation after pre-treatments with a SA biosynthesis inhibitor. From these results, PIP2-PLC appears to play a key role in free SA- and ABA-associated reinforcement of thermotolerance resulting from heat acclimation.
Key words: ABA, abscisic acid, heat acclimation, pea, PIP2-PLC, SA, salicylic acid, thermotolerance
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Introduction |
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Temperature levels beyond an organism's optimal life range are regarded as a major abiotic stress. Plants are often subjected to changes of temperature during growth. Thus, like other organisms, an integrated strategy for protecting plant cells from damage caused by rapid and/or violent changes in temperature is of particular interest for commercially significant plants. Heat stress typically results in increased membrane fluidization, whereas cold stress leads to deep membrane rigidity (Sangwan et al., 2002). This phenomenon begs the question as to whether plant cells are capable of sensing heat or cold. In addition to changes in membrane fluidity, a wide range of heat shock proteins (HSPs) are produced in response to high temperature (Howarth and Dugham, 1993; Schöffl et al., 1997). Several recent studies have suggested that salicylic acid (SA) is involved in thermotolerance, for instance in mustard (Dat et al., 1998) and potatoes (Lopez-Delgado et al., 1998). However, these results may be the result of a defence-like mechanism against more severe heat injury. If so, the question of whether or not SA is a necessary element in the signalling pathway resulting in thermotolerance is yet to be clarified. Larkindale and Knight (2002) reported that transgenic Arabidopsis seedlings expressing a bacterial SA-decomposing salicylate hydroxylase lost tolerance against heat stress in comparison with its background ecotype after 37 °C pre-treatment for 1 h. Results from Clarke (2004), whose conclusion suggested that SA signalling plays an important role in the acquisition of basal thermotolerance rather than in aquired thermotolerance, generally support the conclusions of Larkindale and Knight (2002).
SA, which derives from the shikimate–phenylpropanoid pathway (Zenk and Müller, 1964), is first converted to trans-cinnamic acid (t-CA) by phenylalanine ammonia lyase (PAL). t-CA is then either hydroxylated to o-coumaric acid before oxidation of the side chain, or the t-CA side chain is shortened to benzoic acid (BA), which is in turn hydroxylated to SA (Sticher et al., 1997). Based on these findings, several research groups have reported that BA hydroxylation to SA appears to be the final and rate-limiting step (León et al., 1995; Coquoz et al., 1998; Ribnicky et al., 1998). This is in contrast to earlier studies where the PAL enzyme was found to play a key role in SA biosynthesis. As a result, benzoic acid hydroxylase (BA2H), whose major biochemical function is catalysing the conversion of BA to SA, has received increased attention in the regulation of SA biosynthesis and physiological processes related to SA signalling.
Abscisic acid (ABA) shows a strong capacity in reinforcing thermotolerance in maize (Gong et al., 1998) and bromegrass (Robertson et al., 1994), where it is thought to act as a hormonal signalling device closely associated with water deficits. This novel role correlated with the heat stress response suggests that ABA may be a ubiquitous second messenger in a broad range of abiotic stresses. It appears that rapid increases in ABA content followed by its immediate decrease in response to various stresses can be attributed to de novo synthesis (Zeevaart and Creelman, 1988). 9-cis-Epoxycarotenoid dioxygenase (NCED), which catalyses the cleavage of 9-cis-epoxycarotenoids to apocarotenoid (C25) and xanthoxin (C15), has also been well documented as a key limiting enzyme in ABA synthesis via the oxidative cleavage of epoxycarotenoids (Schwartz et al., 1997; Taylor et al., 2000; Seo and Koshiba, 2002).
Although its biochemical function is quite similar to that of the molecules mentioned above (SA and ABA), phosphatidylinositol-4,5-bisphosphate-specific-phospholipase C (PIP2-PLC) is a lipid-associated enzyme which employs PIP2 to produce inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These two messenger substances play crucial roles in amplifying extracellular stimuli and mediating various physiological processes caused by different abiotic stresses (Pical et al., 1999; Dewald et al., 2001; Takahashi et al., 2001; Ruelland et al., 2002; Zhao et al., 2004). It is generally believed that PIP2-PLC activation is both rapid and sensitive to changes in extracellular status, which suggests that PIP2-PLC is closely associated with an early response to environmental stress. The results obtained in an earlier study indicate that PIP2-PLC activation should relay the establishment of thermotolerance induced by moderate heat pre-treatment, i.e. heat acclimation at 37 °C, leading to a sudden increase in the free SA level in pea leaves (Liu et al., 2006). In this proposed pathway, PIP2-PLC stimulation is preceded by an increase in free SA content followed by an increase in IP3 production, which is triggered by the activation of PIP2-PLC. The burst in IP3 content induces a Ca2+ release from intracellular stores such as the tonoplast or endoplasmic reticulum. Thus far, however, and particularly with regard to the development of thermotolerance or the response of heat signal transduction, the nature of the relationship between SA, ABA, and the PIP2-PLC signalling pathways remains unclear. The present study represents an attempt to clarify the connection between these components in a temporal sequence.
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Plant materials, chemical pre-treatments, and heat treatments
Pea (Pisum sativum L. cv. Ningxia) seeds were grown for 8 d in a growth chamber containing pre-fertilized soil. Light intensity was maintained at 180 µmol m–2 s–1, with day/night temperatures controlled at 25/22 °C at 50% relative humidity. Heat treatments were conducted by transferring the chambers into an intelligent climate incubator at 38 °C and 48 °C to mimic heat acclimation and stress conditions. Chemical solutions [SA, ABA, abamine (ABM), paclobutrazol (PAC), and neomycin (Neo)] were sprayed on the pea seedling leaves until drips formed, for a 40 min infiltration period as the phytohormone or inhibitor pre-treatment. When heat treatments were completed, the pea leaves were harvested and immediately frozen in liquid nitrogen, and stored at –80 °C until further use.
Antioxidant enzyme activity assays
Superoxide dismutase (SOD) activity was assayed by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium according to the methods described in Beauchamp and Fridovich (1971). Catalase (CAT) activity was measured as described by Liang et al. (1982). Ascorbate peroxidase (APX) activity was assayed as described by Nakano and Asada (1981). Assays of glutathione reductase (GR) and peroxidase (POD) activities were carried out according to Cakmak and Marschner (1992). Dehydroascorbate reductase (DHAR) activity was assayed according to Nakano and Asada (1981).
Salicylic acid glucosyltransferase (SAGT) and BA2H activity assays
SAGT activity was assayed according to Yalpani et al. (1992). The standard incubation mixture consisted of 40 µl of protein solution containing 2–120 µg of total proteins, 5 mM NH4Cl, 10 mM UDP-glucose, and 0.4 mM SA in TM buffer in a final volume of 200 µl. Assays with radioactive SA also contained 1 kBq of [7-14C]SA (PerkinElmer, specific activity: 1.74 GBq mM–1). Incubations were performed at 30 °C and terminated by the addition of 200 µl of methanol after 30 min. SA was then separated from the SA 2-O-D-glucose (SAG) by chromatography on Polyamide 6 columns. For separations of [14C]SA from [14C] SAG on the Polyamide 6 columns, the entire 400 µl of the methanolic reaction mixture was loaded onto a 0.8x2 cm polyamide column equilibrated in 10 mM Tris-MES, pH 7.0. A 1 ml aliquot of H2O was passed through the column. SAG was then eluted with 5 ml of H2O, collected in scintillation vials, and 14.5 ml of scintillation liquid added. Radioactivity was determined using a scintillation counter (LKB 1217 Rackbeta).
BA2H activity was determined by quantifying the SA synthesized from BA according to León et al. (1995). The reaction mixture contained a final volume of 0.5 ml, 10 µM HEPES, pH 7.4, 1 µM NADPH, and 1 µM BA. Following 30 min incubation at 30 °C, the reaction was terminated by the addition of 0.5 ml of 15% (w/v) trichloroacetic acid (TCA). After the precipitated pellet was removed by centrifugation (5 min, 8000 g), the SA was partitioned three times with ethyl acetate:cyclopentane:isopropanol (100:99:1, by vol.). SA content was then quantified as described in the measurement of free SA content. The SA synthesized from BA was calculated from the SA present in the assay mixture by subtracting the SA present in blanks incubated under similar conditions.
Extraction and determination of free SA, SAG, and ABA content
Purification and quantification of free SA was carried out according to methods previously described by Rasmussen et al. (1991). A 2 g aliquot of pea leaves was ground in liquid nitrogen and extracted twice, once with 90% methanol and once with 100% methanol. After 5 min centrifugation at 10 000 g, the combined supernatants were condensed by rotary evaporation at 40 °C and ddH2O added to a total volume of 4 ml. The extract was then adjusted to 
pH 3.0 by metaphosphate and partitioned three times in succession with ethyl acetate. The organic phase containing free SA was evaporated under a stream of N2. The residue obtained after N2 drying was dissolved in 200 µl of 95% methanol. For purification of free SA, the methanol fraction containing the free SA was spotted onto silica gel 60 A chromatography plates (Whatman) and developed in toluene:dioxane:acetic acid (90:25:4, by vol.). Free SA was visualized on the plate under UV light (302 nm). The fluorescent band corresponding to SA was eluted from the silica gel with 4 ml of 95% methanol and the methanol removed under a stream of N2. The residue was dissolved in 200 µl of the mixture (methanol, 45%; phosphoric acid, 0.025%). Quantitative analysis was performed by high-performance liquid chromatography (HPLC) linked to fluorescence detectors (excitation wavelength=310 nm, emission wavelength=400 nm). SAG was indirectly quantified by acid hydrolys of the compounds remaining in a sodium acetate buffer after organic extraction and analysing the released SA by HPLC. Acid hydrolysis was performed by incubating the samples in a boiling water bath at pH 1–1.5 for 30 min and then extracting the free SA as above. ß-Glucosidase digestion was performed as described by Southerton and Deverall (1990). For each sample, the dried methanol extract was resuspended in 5 ml of water at 80 °C and the solution divided into two equal portions. To one portion, an equal volume of 0.2 M acetate buffer (pH 4.5) containing 0.1 mg ml–1 ß-glucosidase was added, while buffer alone was added to the other portion. Both portions were incubated at 37 °C overnight. After digestion, samples were acidified to pH 1–1.5 with HCl. SA was then extracted and back extracted from each half for quantification by HPLC.
ABA content was measured as described by López-Carbonell and Jáuregui (2005). Samples were extracted with acetone:water:acetic acid (80:19:1, by vol.) at –20 °C, after which they were vortexed and centrifuged at 13 000 g, 4 °C, for 10 min. The supernatants were then collected and the pellets re-extracted with 3 ml of the extraction solvent. The supernatants obtained after the second group of extracts was centrifuged were dried in a rotavapour until the aqueous fractions were obtained; these fractions were dried completely under a nitrogen stream. The dried samples were kept at –20 °C until analysis. The extracts were reconstituted in 200 µl of acetonitrile:water:acetic acid (90:10:0.05, by vol.), stirred, vortexed, centrifuged (7850 g, 10 min), filtered through a 0.45 µm PTFE filter (Waters, Milford, MA, USA), and 10 µl were injected into the liquid chromatograpy–tandem mass spectrometry (LC–MS/MS) system. Quantification was done by the standard addition method by spiking control plant samples with ABA solutions.
Malondialdehyde (MDA) content assay
MDA content was measured as described by Heath and Packer (1968). The pea leaves (0.3 g) were homogenized in 5 ml of 0.1% TCA and centrifuged at 10 000 g for 5 min. After centrifugation, 1 ml of supernatant was mixed with 4 ml of 0.5% thiobarbituric acid (TBA) in 20% TCA, and the mixture incubated in boiling water for 30 min, after which it was transferred to an ice bath to stop the reaction. The absorbance was read at 532 nm and adjusted for non-specific absorbance to 600 nm. MDA content was estimated by using an extinction coefficient of 155 mmol l–1 cm–1.
RNA isolation and PsPLC gene expression analysis by RT–PCR
Total RNA was extracted from pea leaf fragments according to Logemann et al. (1987). RNA yield was examined spectrophotometrically and its quality determined by electrophoresis in an agarose gel followed by ethidium bromide staining and UV light visualization. A 2 µg aliquot of RNA was used as a template for reverse transcription–polymerase chain reactions (RT–PCRs) using an AMV Reverse Transcription Kit (Promega, USA, A3500) according to procedures specified by the manufacturer. Gene-specific primers for PsPLC (forward, 5'-AGGAATGTTCAAAGCCAATG-3'; reverse, 5'-TTGCCCACCAAAGTCATC-3') and Actin (forward, 5'-TAACCCTAAGGCTAATCGG-3'; reverse, 5'-AACCACCACTCAAGACAAT-3') were designed for RT–PCR amplification according to published sequences for pea (GenBank accession nos Y15253 and X90378). Amplification of Actin cDNA was used as an internal control. The prospective sizes of the PCR products were 391 bp (PsPLC gene fragment) and 578 bp (Actin gene fragment), respectively. PCRs were carried out in 25 µl of PCR mixtures with 0.5 mM dNTPs, 2 U of Taq polymerase (Takara), and 0.1 nM of each gene-specific amplification primer. PCR conditions were as follows: 8 min initial heating at 94 °C, followed by 35/30 (PsPLC/Actin) three-step cycles of 1 min denaturation at 94 °C, 30 s annealing at 59 °C/53 °C (PsPLC/Actin), and 30 s elongation at 72 °C, followed by a final 8 min elongation step at 72 °C. The identity of all PCR products was confirmed by sequencing analysis at Invitrogen Co., Ltd. (Beijing, China).
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Change of SA content, BA2H, and SAGT activity during the heat acclimation period
A free SA content peak appeared 25 min after initiation of heat acclimation, i.e. sublethal temperature of 38 °C (Fig. 1A). Although the level of SAG, a major conjugated form of SA, decreased slightly at 20 min after the start of heat acclimation, it remained stable up to 60 min post-heat treatment (Fig. 1B). BA2H activity reached a maximum at the 50 min time point (Fig. 1C). However, no significant change in BA2H activity was observed during the 25 min prior to the peak. While SAGT activity experienced a significant drop at 20 min, a maximum occurred 60 min after the initiation of the heat acclimation treatment (Fig 1D).
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Decrease in free SA content was triggered by pre-treatment with an ABA biosynthesis inhibitor during the heat acclimation period
When PAC, an effective SA biosynthesis-related BA2H enzyme inhibitor (León et al., 1995), was sprayed on the pea leaves, the free SA level still reached a maximum 20 min after the heat treatment (Fig. 2A). However, the SAG content remained at the control level and no peak was observed following heat acclimation, with the exception of the slight decrease that occurred at 20 min (Fig. 2B). Thus, PAC operated more as a chelator of conjugated SA production rather than free SA in this study. Interestingly, and at least in part similar to the PAC pre-treatment, pre-treatment with the 100 µM ABA biosynthesis inhibitor ABM resulted in the disappearance of the free SA peak compared with no-inhibitor pre-treatment (Fig. 2C). However, a free SA peak at 25 min was still triggered by pre-treatment with 100 µM Neo, which is a representative PIP2-PLC activity inhibitor (Fig. 2D).
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ABA content change is rapidly responsive to heat acclimation with pre-treatment with SA biosynthesis and PIP2-PLC activity inhibitors rather than an ABA biosynthesis inhibitor
When there was no inhibitor pre-treatment, the ABA peak appeared to be very sensitive to heat acclimation at 38 °C (Fig. 3A). At 10 min after beginning the heat acclimation treatment, the ABA content increased rapidly to 3.51-fold relative to the control (0 min), then declined to the control level during the following 110 min. To examine whether ABA was correlated with SA and PIP2-PLC signalling, the pea leaves were treated with 100 µM PAC and Neo, effective inhibitors of BA2H and PIP2-PLC activity, respectively. As shown in Fig. 3C and D, ABA content changes were compatible with the no-inhibitor pre-treatment (Fig. 3A). In comparison with the inhibitors mentioned above, pre-treatment with 100 µM ABM, which was found by Han et al. (2004) to function as a novel ABA synthesis inhibitor, did not result in a significant change in ABA level. Further, no peak was observed within a 2 h acclimation period (Fig. 3B).
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Significant thermotolerance losses resulted from pre-treatments with neomycin, a well-known PIP2-PLC activity inhibitor, and could not be reversed by ABA or SA
In order to determine which signal molecule (ABA, SA, or PIP2-PLC) plays the most important role in establishing heat acclimation-induced thermotolerance, PAC (or ABM) and Neo were employed to evaluate the loss of thermotolerance. As shown in Fig. 4A, pre-treatment with the ABA biosynthesis inhibitor ABM did not cause a profound difference in either the MDA level or the degree of leaf wilting compared with the control (H2O pre-treatment). However, a significant difference was triggered by pre-treatment with PAC, an SA biosynthesis inhibitor. PAC combined with an ABM pre-treatment also resulted in a significant difference in thermotolerance compared with the control. In previous experiments, Neo was thought to be a major PIP2-PLC activity inhibitor (Kashem et al., 2000; Liu et al., 2006). Here, a stronger effect on weakness of thermotolerance was found with Neo pre-treatment relative to PAC and ABM (Fig. 4B, D). To clarify whether spraying with SA or ABA could protect pea leaves from heat injury with Neo pre-treatments, combined pre-treatments of Neo with 100 µM SA, 20 µM ABA, and 100 µM SA plus 20 µM ABA were performed. As shown in Fig, 4C, pre-treatments with SA, ABA, or SA plus ABA did not reverse the thermotolerance loss caused by Neo pre-treatment.
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0.05 among treatments according to Duncan's multiple range test.Changes of activities of reactive-oxygen scavenging antioxidant enzymes after applications of different inhibitor pre-treatments followed by heat acclimation prior to heat stress
Compared with the control, SOD activity decreased significantly (reductions of 37% and 45.8% over the control) with PAC and Neo pre-treatments. However, no significant reduction was observed following ABM pre-treatment (Fig. 5A). Interestingly, changes in POD activity in all pre-treatments including ABM, PAC, and Neo were similar to the control (Fig. 5B). As shown in Fig. 5C and D, PAC and Neo pre-treatments reduced CAT by 27% and 39.2%, respectively, and APX activity by 26.3% and 36.4%, respectively, whereas no profound difference was observed in CAT or APX activities with ABM pre-treatment compared with the control. However, pre-treatments with PAC and Neo did cause a significant decrease in GR activity (Fig. 5F). In contrast to GR activity, only the Neo pre-treatment resulted in a decrease in DHAR (Fig. 5E).
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ABA regulates SAGT activity in a negative manner not only in vivo but also in vitro
As shown in Fig. 2C, the free SA peak disappeared with pre-treatment with the ABA biosynthesis inhibitor ABM during heat acclimation (Fig. 1A), although it is unclear why inhibition of ABA synthesis led to the disappearance of the free SA peak. Given the rapid reduction in SAGT activity 20 min after heat acclimation, we believe it is possible that ABA might cause SAGT activity to stop the elevation of free SA in response to heat acclimation. In order to evaluate the feasibility of this hypothesis, we tested whether or not different concentrations of ABA affected SAGT activities in vivo and in vitro. In Fig. 6A, a negative proportional change of SAGT activity was observed as the concentration of ABA increased in vitro. However, spraying pea leaves with ABA in vivo produced only a slight modulation of SAGT activity (Fig. 6B).
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Spraying pea leaves with conjugated SA in vivo promotes tolerance to heat stress
The negative impact of ABA on SAGT activity (Fig. 6), together with the fact that inhibition of ABA biosynthesis did not lead to thermotolerance loss as much as inhibition of SA biosynthesis (Fig. 4A), suggests that conjugated SA is probably involved in the development of thermotolerance induced by heat acclimation. To evaluate whether SAG (the major form of conjugated SA) could reinforce partial thermotolerance, a simple and direct experiment was performed. After spraying 100 µM conjugated SA, it was found that the extent of injury in terms of MDA content and apparent wilting caused by heat stress was markedly less than that of the control (Fig. 7). Accordingly, it is concluded that the importance of SAG in the development of thermotolerance is analogous to SA or heat acclimation (Fig. 7).
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PIP2-PLC gene expression is stimulated by pre-treatments with SA, ABA, and an SA biosynthesis inhibitor prior to heat acclimation
In this study, Neo, an inhibitor of the activity of PIP2-PLC, resulted in maximum weakness in anti-thermal indicators including lipid peroxidation and degree of wilting, and antioxidant enzyme activity (Figs 4, 5). To evaluate PIP2-PLC gene expression under conditions of heat acclimation and infiltration with biosynthesis inhibitors, a semi-quantitive RT–PCR analysis was performed. As shown in Fig. 8A–D, PsPLC transcription was stimulated by heat acclimation (38 °C), 100 µM SA, 20 µM ABA, and 100 µM SA combined with 20 µM ABA, with the highest levels reached 45, 45, 45, and 30 min post-heat acclimation, respectively. However, neither 100 µM ABM nor 100 µM PAC combined with 100 µM ABM had an effect on PsPLC mRNA accumulation (Fig. 8F, G). Interestingly, the maximum accumulation of PsPLC occurred after 45 min in leaves infiltrated with 100 µM PAC followed by heat acclimation (Fig. 8E).
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Historically, the conversion of phenylalanine to cinnamic acid catalysed by PAL has been acknowledged as the rate-limiting step in the de novo biosynthesis of SA (Coquoz et al., 1998). Recently, special attention has been paid to the BA2H enzyme, which displays its biochemical function in the hydroxylation of BA to SA. However, whether or not the abrupt elevation in the free SA level corresponding to a heat stimulus is a result of biosynthesis or conversion of conjugated SA to free SA remains a matter of debate. Dat et al. (1998) found that both free SA and total SA levels were elevated during the first 30 min following heat acclimation. Their results indicated that the rapid increase in free SA content, for the most part, could be attributed to the biosynthesis of SA, with the final step catalysed by BA2H. However, the present findings regarding the source of the free SA burst triggered by heat acclimation (Fig. 1A) are not in agreement with those of Dat et al. (1998). The difference, to some extent, might be attributed to endogenous basal SA levels of different biomaterials. In mustard, for example, there is as much endogenous free SA as conjugated SA. At the same time, the basal level of SAG is
5-fold more than that of free SA in pea leaves. The rapid SAG detoxification 20 min after beginning heat acclimation (Fig. 1B) might simply be an emergency conversion of SAG to free SA under stress. This could be a significant improvement in adaptation to sudden heat damage during the initial periods, since the hydrolysis of SAG to free SA may require as much as 20 min, whereas in vivo SA biosynthesis requires at least 1 h. Although subsequent BA2H activity increased 3-fold over the control at 50 min (Fig. 1C), the elevation in free SA may be due to SAG rather than newly synthesized SA. Glucosylation is a common modification of plant secondary metabolites (Jones and Vogt, 2001; Claire et al., 2005). In tobacco, exogenously sprayed SA is primarily metabolized to SAG (Lee and Raskin, 1998). Therefore, in most cases, SAG is the major SA metabolite (Silverman et al., 1995; Dean et al., 2003). Dean et al. (2003) found that SA was converted to SAG in cytoplasm catalysed by the SAGT enzyme, with the resulting newly formed SAG stored in the vacuole. On the basis of effective inhibition of NCED activity and modification of nordihydroguaiaretic acid (NDGA) structure, ABM represents a novel specific inhibitor of ABA biosynthesis (Han et al., 2004). However, its inhibitory effect on ABA biosynthesis is stronger than that of NDGA, which has traditionally been used as an ABA biosynthesis inhibitor in plant physiology. In this study, 100 µM ABM not only prevented a burst in the ABA content but also inhibited drastic free SA formation during the period following heat acclimation (Figs 2C, 3A). The double effect of the ABA inhibitor on both ABA and free SA levels implies that rapid ABA elevation corresponding to heat acclimation should precede a free SA peak. Despite a number of studies on the roles of ABA in physiological processes including dehydration (Zeevaart and Creelman, 1988; Thompson et al., 2000), seed maturation and dormancy (Yoshioka et al., 1998; Frey et al., 1999; Grappin et al., 2000), as well as sugar response (Arenas-Huertero et al., 2000; Laby et al., 2000; Rook et al., 2001), the relationship between ABA and the heat signal pathway remains unclear. In the present study, inhibition of ABA biosynthesis had almost no effect on thermotolerance weakening, which was evaluated by the MDA level and the degree of wilting (Fig. 4). ABA seems not to be as important as SA and PIP2-PLC in the heat signal-related relay. Larkindale et al. (2005) employed several Arabidopsis mutants of signalling substances including ABA, ethylene, and oxidative burst to assess their importance in development of thermotolerance. In the experiment, the ABA signalling mutant showed the strongest defect in acquired thermotolerance for seedling survival. The disagreement between the two conclusions may be explained by the distinct hormone thermotolerance regulation mechanism in different biomaterials. For instance, in tobacco and Arabidopsis, the basal SA levels are very low. As such, a small increase in the SA level is sufficient for the development of system acquired resistance (SAR) (Enyedi et al., 1992; Vernooij et al., 1994). In other words, while these plant species contain low levels of SA, their levels are still effective in SA perception and transduction. In some plant species, such as potato, rice, and tomato, SA is abundant for the establishment of a defence system against biotic and/or abiotic stress, but active signal perception and transduction is poor (Raskin et al., 1990; Coquoz et al., 1995; Chen et al., 1997). Whether the ABA-related heat signal pathway and regulation mechanism is strictly determined by the ABA level remains unclear and will need further investigation before any consensus can be reached.
In this study, the negative regulation by ABA biosynthesis on SAGT activity only decreased free SA and did not affect SAG levels (Fig. 2A). In addition, the fact that PAC pre-treatment led to more loss of thermotolerance suggests that newly synthesized SA plays a crucial role in heat acclimation-induced thermotolerance. Based on evidence presented in Fig. 1B and C, we deduce that the primary newly synthesized SA is SAG, a glusosylated form of conjugated SA. However, it is still unclear whether or not SAG involves signal transduction in relation to the development of thermotolerance. Hennig et al. (1993) reported that SAG was as active as free SA in the induction of PR-1 gene expression and establishment of SAR in tobacco. These results indicate that conjugated SA could promote thermotolerance as effectively as free SA and heat acclimation at sublethal temperatures (Fig. 7). Consequently, it seems unwise to consider conjugated SA, especially SAG, as a final product of SA metabolism in response to abiotic stress.
Abiotic stresses, such as cold, salt, and drought, can all induce the accumulation of reactive oxygen species including 
, H2O2, and hydroxyl radicals (Hasegawa et al., 2000). As is well known, SOD is responsible for scavenging and dismutating to H2O2. There are two systems whose capacities for decomposing H2O2 are most prominent in the cells of higher plants. One is co-operation of POD and CAT located in the cytoplasm. Another is the AsA–GSH redox cycle consisting of APX, GR, and DHAR residing in the chloroplast. Except for POD, Neo pre-treatment led to the highest reduction of antioxidant enzyme activity compared with the control and other inhibitor pre-treatments (Fig. 5A, C–F). This implies that collaboration between antioxidant enzymes involves enhancement of thermotolerance induced by heat acclimation and that PIP2-PLC plays a very important role in the development of acquired thermotolerance. A large body of evidence indicates that SA and ABA are involved in the establishment of thermotolerance (Robertson et al., 1994; Larkindale and Knight, 2002; Clarke et al., 2004; Larkindale et al., 2005). Here, more attention has been paid to PIP2-PLC as a messenger and membrane-associated enzyme. In order to assess the effects of pre-infiltration with SA, ABA, and their biosynthesis inhibitors prior to heat acclimation on the expression of the PIP2-PLC gene, a PsPLC gene fragment was cloned. Obvious and rapid increases in the PsPLC transcription level were found in the leaves infiltrated with SA, ABA, and SA plus ABA, and that pre-infiltrated with H2O and the SA biosynthesis inhibitor PAC prior to heat acclimation (Fig. 8). However, the ABA biosynthesis inhibitor and its combination with the SA synthesis inhibitor did not result in quantitative changes of PsPLC transcription. Together, the RT–PCR analysis with the SA and ABA assay results suggest that the response of PIP2-PLC to heat acclimation should be preceded by the peak in ABA and free SA rather than conjugated SA, whose level was shown to be controlled by SA biosynthesis during heat acclimation (Fig. 2B). The conclusions drawn in this study are in agreement with our previous work where free SA was acknowledged to function as an upstream element in the stimulation of PIP2-PLC in response to heat treatment (Liu et al., 2006). Meanwhile, PAC did not stimulate expression of PsPLC mRNA during the heat acclimation period, indicating that conjugated SA as an effective signal molecule to induce an increase in tolerance to heat stress (Fig. 7) is independent of reception and transduction by membrane-associated PIP2-PLC.
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Acknowledgements |
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This study was financially supported by the National Natural Science Foundation of China (No. 30270918, 30471192) and Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20050019015).
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Abbreviations |
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ABA, abscisic acid; ABM, abamine; APX, ascorbate peroxidase; BA, benzoic acid; BA2H, benzoic acid-2-hydroxylase; CAT, catalase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; MDA, malondialdehyde; Neo, neomycin; PAC, paclobutrazol; PAL, phenylalanine ammonia lyase; PIP2-PLC, PIP2-specific-phospholipase C; POD, peroxidase; RT–PCR, reverse transcription–polymerase chain reaction; SA, salicylic acid; SAG, salicylic acid 2-O-ß-D-glucose; SAGT, salicylic acid glucosyltransferase; SAR, systemic acquired resistance; SOD, superoxide dismutase; t-CA, trans-cinnamic acid; TCA, trichloroacetic acid.
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Arenas-Huertero F, Arroyo A, Zhou L, Sheen J, León P. (2000) Analysis of Arabidopsis glucose-insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar. Genes and Development 14:2085–2096.
Beauchamp C and Fridovich I. (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry 44:276–287.[CrossRef][ISI][Medline]
Cakmak J and Marschner H. (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiology 98:1222–1227.
Chen Z, Iyer S, Caplan A, Klessig DF, Fan B. (1997) Differential accumulation of salicylic acid and salicylic acid-sensitive catalase in different rice tissues. Plant Physiology 114:193–201.[Abstract]
Claire MMG, Mathilde L-M, Patrick S. (2005) Plant secondary metabolism glycosyltransferases: the emerging functional analysis. Trends in Plant Science 10:542–549.[CrossRef][ISI][Medline]
Clarke SM, Mur LAJ, Wood JE, Scott ZM. (2004) Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana. The Plant Journal 38:432–447.[CrossRef][ISI][Medline]
Coquoz JL, Buchala A, Métraux JP. (1998) The biosynthesis of salicylic acid in potato plants. Plant Physiology 117:1095–1101.
Coquoz JL, Buchala AJ, Meuwly PH, Métraux JP. (1995) Arachidonic acid treatment of potato plants induces local synthesis of salicylic acid and confers systemic resistance to Phytophthora infestans and Alternaria solani. Phytopathology 85:1219–1225.
Dat JF, Lopez-Delgado H, Foyer CH, Scott IM. (1998) Parallel change in H2O2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedlings. Plant Physiology 116:1351–1357.
Dean JV, Shah RP, Mohammed LA. (2003) Formation and vacuolar localization of salicylic acid glucose conjugates in soybean cell suspension cultures. Physiologia Plantarum 118:328–336.[CrossRef]
Dewald DB, Torabinejad J, Jones CA, Shope JC, Cangelosi AR, Thompson JE, Prestwich GD, Hama H. (2001) Rapid accumulation of phosphatidylinositol 4,5-bisphosphate correlates with calcium mobilization in salt-stressed arabidopsis. Plant Physiology 126:759–769.
Enyedi A, Yalpani N, Silverman P, Raskin I. (1992) Localization, conjugation and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco mosaic virus. Proceedings of the National Academy of Sciences, USA 89:2480–2484.
Frey A, Audran C, Marin E, Sotta B, Marion-Poll A. (1999) Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression. Plant Molecular Biology 39:1267–1274.[CrossRef][ISI][Medline]
Grappin P, Bouinot D, Sotta B, Miginiac E, Jullien M. (2000) Control of seed dormancy in Nicotiana plumbaginifolia: post-imbibition abscisic acid synthesis imposes dormancy maintenance. Planta 210:279–285.[CrossRef][ISI][Medline]
Gong M, Li Y-J, Chen SZ. (1998) Abscisic acid induced thermotolerance in maize seedlings is mediated by Ca2+ and associated with antioxidant systems. Journal of Plant Physiology 153:488–496.
Han SY, Kitahata N, Sekimata K, Saito T, Kobayashi M, Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K, Yoshida S, Asami T. (2004) A novel inhibitor of 9-cis-epoxycarotenoid dioxygenase in abscisic acid biosynthesis in higher plants. Plant Physiology 135:1574–1582.
Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ. (2000) Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51:463–499.[CrossRef][ISI]
Heath RL and Packer L. (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics 125:189–198.[CrossRef][ISI][Medline]
Hennig J, Malamy J, Grynkiewicz G, Indulski J, Klessig DF. (1993) Interconversion of the salicylic acid signal and its glucoside in tobacco. The Plant Journal 4:593–600.[CrossRef][ISI][Medline]
Howarth CJ and Dugham HJ. (1993) Gene expression under temperature stress. New Phytologist 125:1–26.
Jones P and Vogt T. (2001) Glycosyltransferases in secondary plant metabolism: tranquilizers and stimulant controllers. Planta 213:164–174.[CrossRef][ISI][Medline]
Kashem MA, Itoh K, Iwabuchi S, Hori H, Mitsui T. (2000) Possible involvement of phosphoinositide–Ca2+ signaling in the regulation of alpha-amylase expression and germination of rice seed (Oryza sativa L.). Plant and Cell Physiology 41:399–407.[ISI][Medline]
Laby RJ, Kincaid MS, Kim D, Gibson SI. (2000) The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response. The Plant Journal 23:587–596.[CrossRef][ISI][Medline]
Larkindale J and Knight MR. (2002) Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiology 128:682–695.
Lee H-I and Raskin I. (1998) Glucosylation of salicylic acid in Nicotiana tabacum cv. Xanthi-nc. Phytopathology 88:692–697.
León J, Shulaev V, Yalpani N, Lawton MA, Raskin I. (1995) Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis. Proceedings of the National Academy of Sciences, USA 92:10413–10417.
Liang Z, Yu C, Huang AHC. (1982) Isolation of spinach leaf peroxisomes in 0.25 molar sucrose solution by percoll density gradient centrifugation. Plant Physiology 70:1210–1222.
Liu H-T, Huang W-D, Pan Q-H, Weng F-H, Zhan J-C, Liu Y, Wan S-B, Liu Y-Y. (2006) Contributions of PIP2-specific-phospholipase C and free salicylic acid to heat acclimation-induced thermotolerance in pea leaves. Journal of Plant Physiology 163:405–416.[CrossRef][ISI][Medline]
Logemann J, Schell J, Willmitzer L. (1987) Improved method for the isolation of RNA from plant tissues. Analytical Biochemistry 163:16–20.[CrossRef][ISI][Medline]
López-Carbonell M and Jáuregui O. (2005) A rapid method for analysis of abscisic acid (ABA) in crude extracts of water-stressed Arabidopsis thaliana plants by liquid chromatography–mass spectrometry in tandem mode. Plant Physiology and Biochemistry 43:407–411.[ISI][Medline]
Lopez-Delgado H, Dat JF, Foyer CH, Scott IM. (1998) Induction of thermotolerance in potato microplants by acetylsalicylic acid and H2O2. Journal of Experimental Botany 49:713–720.
Nakano Y and Asada K. (1981) Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant and Cell Physiology 22:867–880.
Pical C, Westergren T, Dove SK, Larsson C, Sommarin M. (1999) Salinity and hyperosmotic stress induce rapid increase in phosphatidylinositol 4,5-bisphosphate, diacylglycerol pyrophosphate, and phosphatidylcholine in Arabidopsis thaliana cells. Journal of Biological Chemistry 274:38232–38240.
Raskin I, Skubatz H, Tang W, Meeuse BJD. (1990) Salicylic acid levels in thermogenic and nonthermogenic plants. Annals of Botany 66:369–373.
Rasmussen JB, Hammerschmidt R, Zook MN. (1991) Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv. syringae. Plant Physiology 97:1342–1347.
Ribnicky D, Shulaev V, Raskin I. (1998) Intermediates of salicylic acid biosynthesis in tobacco. Plant Physiology 118:565–572.
Robertson AJ, Ishikawa M, Gusta LV, MacKenzie SL. (1994) Abscisic acid induced heat tolerance in Bromus inermis Leyss cell-suspension cultures. Plant Physiology 105:181–190.[Abstract]
Rook F, Corke F, Card R, Munz G, Smith C, Bevan MW. (2001) Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signaling. The Plant Journal 26:421–433.[CrossRef][ISI][Medline]
Ruelland E, Cantrel C, Grawer M, Kader JC, Zachowski A. (2002) Activation of phospholipase C and D is an early response to a cold exposure in Arabidopsis suspension cells. Plant Physiology 130:999–1007.
Sangwan V, Örvar BL, Beyerly J, Hirt H, Dhindsa RS. (2002) Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways. The Plant Journal 31:629–638.[CrossRef][ISI][Medline]
Schöffl F, Rossol I, Angermuller S. (1997) Regulation of the transcription of heat shock genes in nuclei from soybean (Glycine max) seedlings. Plant, Cell and Environment 10:113–119.
Schwartz SH, Tan BC, Gage DA, Zeevaart JAD, McCarty DR. (1997) Specific oxidative cleavage of carotenoids by VP14 of maize. Science 276:1872–1874.
Seo M and Koshiba J. (2002) Complex regulation of ABA biosynthesis in plants. Trends in Plant Science 7:41–48.[CrossRef][ISI][Medline]
Silverman P, Seskar M, Kanter D, Schweizer P, Métraux J-P, Raskin I. (1995) Salicylic acid in rice: biosynthesis, conjugation, and possible role. Plant Physiology 108:633–639.[Abstract]
Southerton SG and Deverall BJ. (1990) Changes in phenolic acid levels in wheat leaves expressing resistance to Puccinia recondite f. sp. tritici. Physiological and Molecular Plant Pathology 37:437–450.[CrossRef]
Sticher L, Mauch-mani B, Métraux JP. (1997) Systemic acquired resistance. Annual Reviews of Phytopathology 35:235–270.
Takahashi S, Katagiri T, Hirayama T, Yamaguchi-Shinozaki K, Shinozaki K. (2001) Hyperosmotic stress induces a rapid and transient increase in inositol 1,4,5-trisphosphate independent of abscisic acid in Arabidopsis cell culture. Plant and Cell Physiology 42:214–222.
Taylor IB, Burbidge A, Thompson AJ. (2000) Control of abscisic acid synthesis. Journal of Experimental Botany 51:1563–1574.
Thompson AJ, Jackson AC, Parker RA, Morpeth DR, Burbidge A, Taylor IB. (2000) Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles, water stress and abscisic acid. Plant Molecular Biology 42:833–845.[CrossRef][ISI][Medline]
Vernooij B, Friedrich L, Morse A, Reist R, Kolditz-Jawhar R, Ward E, Uknes S, Kessmann H, Ryals J. (1994) Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction. The Plant Cell 6:959–969.[Abstract]
Yalpani N, Schulz M, Davis MP, Balke NE. (1992) Partial purification and properties of an inducible uridine 5'-diphosphate-glucose: salicylic acid glucosyltrasferase from oat roots. Plant Physiology 100:457–463.
Yoshioka T, Endo T, Satoh S. (1998) Restoration of seed germination at supraoptimal temperatures by uridone, an inhibitor of abscisic acid biosynthesis. Plant and Cell Physiology 39:307–312.
Zhao J, Guo YQ, Kosaihira A, Sakai K. (2004) Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated Cupressus lusitanica cell cultures. Planta 219:121–131.[CrossRef][ISI][Medline]
Zeevaart JAD and Creelman RA. (1988) Metabolism and physiology of abscisic acid. Annual Review of Plant Physiology and Plant Molecular Biology 39:439–473.[CrossRef][ISI]
Zenk MHM and Müller G. (1964) Biosynthesis von p-hydroxyben-Zoesäure und anderer Benzoesäuren in höheren Pflanzen. Zeitschrift für Naturforschung 19B:398–405.
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