Thermotolerance and antioxidant systems in Agrostis stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethylene

This study investigated whether pre-treating plants with specific putative signaling components and heat acclimation would induce tolerance of a cool-season grass, creeping bentgrass (Agrostis stolonifera var. palustris), to subsequent heat stress and whether thermotolerance induction of those pretreatments was associated with the regulation of antioxidant regenerating enzymes. The treatments included foliar application of salicylic acid (SA), abscisic acid (ABA), calcium chloride (CaCl2), hydrogen peroxide (H2O2), 1-aminocyclopropane-1-carboxylic acid (ACC, a precursor of ethylene prior to the exposure of plants to heat stress (35 degrees C) in a growth chamber. Physiological measurements including turf quality, leaf photosynthetic rate, and levels of oxidative damage demonstrated that all treatments increased heat tolerance. The better heat tolerance for pre-treated plants as compared to controls was related to the protection of oxidative damage under heat stress. APX activity increased over the first 2 days and 5 days of heating for ACC and CaCl2 respectively, but for only 12 h for H2O2. SA and ABA pre-treatments had no effects on APX activity earlier, but maintained APX activity at a significantly higher level than in controls after 24 h of heating. SA and ABA pre-treatments had no effects on POX activity. ACC treatment significantly increased POX activity. Pre-treatment with CaCl2, H2O2, and HA reduced POX activity, particularly during the later phase of heating. Plants treated with SA, CaCl2, H2O2 and HA had lower CAT activity than their control plants prior to heating and within 48 h of heat stress. ABA and ACC pre-treatments maintained higher CAT activity than the controls after 48 h of heating. ACC, CaCl2, or HA pre-treatments increased SOD activity only before 5 days of heat stress. SA and ABA pre-treatments had less effect on APX activity earlier under heat stress. These results suggest that specific groups of potential signaling molecules may induce tolerance of creeping bentgrass to heat stress by reducing oxidative damage.     

Calcium-dependent signaling pathway in the heat-induced oxidative injury in <Emphasis Type="Italic">Amaranthus lividus</Emphasis>

Heat caused reduction in membrane protein thiol content, increased accumulation of thiobarbituric acid reactive substances and reduced germination rate and early growth in germinating Amaranthus lividus seeds. Imposition of heat stress during early germination also causes accumulation of reactive oxygen species like superoxide and hydrogen peroxide while activities of antioxidative enzymes catalase, ascorbate peroxidase, and glutathione reductase decreased. Calcium chelator (EGTA), calcium channel blocker (LaCl₃) and calmodulin inhibitor (trifluroperazine) aggravated these effects. Added calcium reversed the effect of heat, implying that protection against heat induced oxidative damage and improvement of germination requires calcium and calmodulin during the recovery phase of post-germination events in Amaranthus lividus.     

Effects of Abscisic Acid,Salicylic Acid,Ethylene and Hydrogen Peroxide in Thermotolerance and Recovery for Creeping Bentgrass

Abscisic acid (ABA), salicylic acid (SA), ethylene, and hydrogen peroxide (H₂O₂) may be involved in the regulation of plant responses to heat stress. The objective of this study was to determine whether these signaling molecules are involved in survival at high temperatures in creeping bentgrass (Agrostis stolonifera). We investigated the effects of treatment with ABA, SA, H₂O₂, and ACC (an ethylene precursor) on physiological damage occurring in creeping bentgrass during heat stress (35 °C for 1 month). We also compared the effects of chemical application and the induction of thermotolerance using moderate heat stress (30 °C for 24 h). All of the pre-treatments (heat or chemical) resulted in increased tolerance to prolonged heat stress (1 month) compared to control plants. All treated samples showed more green leaves, decreased membrane leakage and reduced oxidative damage compared to control plants. We then measured changes in the endogenous concentration of these chemical components during heat stress (35 °C) and during recovery after a stress treatment (cooling back to 20 °C). An oxidative burst was detected 5 min after the initiation of heat treatment, with the increase in H₂O₂ being detected primarily in the apoplast of the cells in both leaf and root tissues. Free SA was detected only an hour after the initiation of heat stress, and concentration remained low subsequently. Neither ABA nor ethylene concentrations rose during heat stress, but the concentration of both increased during subsequent cooling. These results suggest that the signaling components of interest are involved in thermotolerance in creeping bentgrass, but that the different chemicals are likely to be involved in separate signaling pathways. An oxidative burst and SA may be bona fide heat stress signals, but ABA and ethylene appear to be involved in signaling pathways in response to recovery from heat stress in this species.     

Calcium, Calmodulin and the Control of Respiration in Protoplasts Isolated from Meristematic Tissues8

Owen, J. H., Hetherington, A. M. and Wellburn, A. R. 1987. Calcium,calmodulin and the control of respiration in protoplasts isolatedfrom meristematic tissues by abscisic acid.—J. exp. Bot.38: 1356–1361. A study was made of the possible involvement of calcium channelsand calmodulin during the calcium-dependent inhibition of mitochondrialrespiration by abscisic acid (ABA) in meristematic protoplastsobtained from light-grown barley (Hordeum vulgare L. cv. Patty)seedlings. The calcium channel blockers lanthanum, verapamiland nifedipine were all found to reduce the Ca²⁺-dependent inhibitionof protoplast respiration by ABA. The ionophore A23187 [GenBank] itselfcaused an inhibition of protoplast respiration, possibly becauseit mimicked the action of ABA by increasing plasmalemma permeabilityto extracellular calcium. By contrast, calmodulin antagoniststrifluoperazine and compound 48/80 both caused a partial decreasein the Ca²⁺-dependent inhibition of protoplast respiration byABA. In contrast to the action of ABA, gibberellic acid markedlyincreased the rates of protoplast respiration but this did notappear to require the presence of extracellular calcium ions.These results support the hypothesis that ABA increases plasmalemmapermeability to extracellular calcium which might then directlyor indirectly act as a second messenger, possibly in conjunctionwith calmodulin, to regulate mitochondrial dark respirationwhich is an important part of early meristematic cell development. Key words: Abscisic acid, calcium, calmodulin, meristematic respiration     

Regulation of Arabidopsis thaliana 14-3-3 gene expression by gamma-aminobutyric acid

The function in plants of the non-protein amino acid, gamma-aminobutyric acid (GABA) is poorly understood. In this study, we show that GABA down-regulates the expression of a large subset of 14-3-3 gene family members in Arabidopsis thaliana seedlings in a calcium, ethylene and abscisic acid (ABA)-dependent manner. Gene expression is not affected when seedlings are supplied with glutamate (GLU), a precursor of GABA. The repression of 14-3-3 gene expression by GABA is dependent on functional ethylene and ABA signalling pathways, because the response is lost in the etr1-1, abi1-1 and abi2-1 mutants. Calcium measurements show that in contrast to GLU, GABA does not elicit a cytoplasmic calcium elevation, suggesting that the GABA response is unlikely to be mediated by GLU receptors (GLRs), as has been suggested previously. We suggest that in addition to its role as a stress-related metabolite, GABA may regulate gene expression in A. thaliana, including members of the 14-3-3 gene family.     

Interaction of salicylic acid and ethylene and their effects on some physiological and biochemical parameters in canola plants (Brassica napus L.)

Environmental stresses, such as cold, heat, salinity, and drought, induce ethylene production and oxidative stress and cause damage in plants. On the other hand, studies have shown that salicylic acid (SA) induced resistance to environmental stresses in plants. In this research, the effects of ethylene on chlorophyll (Chl), carotenoid (Car), anthocyanin, flavonoids, ascorbic acid, dehydroascorbic acid, total ascorbate, lipid peroxidation, and ethylene production in leaves of canola pretreated with SA were studied. The plants were grown in pots until they have four leaves. Leaves were sprayed for two days with three different concentrations of SA (0, 0.5, and 1 mM). The plants were treated for three days with three concentrations of ethylene (0, 50, and 100 ppm). At the end of the ethylene treatments, all examined parameters were measured. The results showed that the ethylene treatments induced lipid peroxidation, while SA mitigated this effect. The ethylene treatment lowered significantly Chl and Car contents and anthocyanin accumulation, but SA alleviated these effects. SA induced an increase in ascorbic acid content in canola plants after the ethylene treatments. Therefore, we concluded that SA played an important role in the alleviation of damages caused by stress conditions.     

Regulation of Arabidopsis thaliana Ku genes at different developmental stages under heat stress

Ku, a heterodimeric protein consisting of 70- and 80-kDa subunits, is involved in many cellular processes, such as DNA replication, cell cycle regulation and heat shock response. Moreover, the expression of Arabidopsis thaliana Ku genes (AtKu) is modulated by certain plant hormones through several signal transduction pathways. This study investigated how AtKu are regulated by heat stress. AtKu expression in 3-week-old young seedlings was down-regulated by heat stress in a time-dependent manner, as examined using real-time quantitative PCR, GUS reporter systems, and western blotting analysis. Additionally, the heat-induced repression of AtKu was mediated through the abscisic acid (ABA) biosynthetic pathway, as shown by the reversal of AtKu suppression in the ABA biosynthesis mutant, aba3, and by an increase in the ABA level as analyzed by reverse-phase high performance liquid chromatography. Heat stress-induced regulation of AtKu repression also involved ethylene signaling, DNA repair pathways, and fatty acid synthesis. Furthermore, AtKu expression was repressed in stems, rosette leaves, and cauline leaves in 4-5-week-old plants under heat stress, whereas it remained unchanged in roots and primary inflorescence, indicating that heat differentially modulated AtKu expression in distinct tissues of Arabidopsis.     

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO2, abscisic acid,darkness, vapor pressure deficit and ozone

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO₂, light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO₂, light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO₂ induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5–2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO₂ and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).     

高温胁迫及其信号转导

高温是影响当前农业生产重要的不利环境因素之一。本文综述了4种信号分子脱落酸(ABA),Ca2+,水杨酸(SA),茉莉酸(JA)对高温胁迫的响应以及它们的相互关系,高温胁迫能够诱导ABA,Ca2+,SA的含量升高,并且通过外施ABA,Ca2+,SA,JA都能提高植物的抗热性。作为胞内第二信使,外源Ca2+能够提高植物超氧化物歧化酶(SOD),过氧化氢酶(CAT)以及抗坏血酸氧化酶(APX)的活性,且能提高钙调蛋白水平。ABA诱导的抗热性受胞质游离的Ca2+介导。SA被认为是对胁迫反应所必需的信号分子,H2O2很可能是信号转导链的一部分。JA和ABA在生理功能上有很多相似之处,JA独自或通过提高ABA含量来起作用,JA和SA有不同的生理功能,也有相同的(不过它们的信号转导途径可能不同),最后,提出了今后高温胁迫信号转导研究的一些思路。     

Effect of salicylic and abscisic acid administered through detached tillers on antioxidant system in developing wheat grains under heat stress

The mechanism imparting thermotolerance by salicylic acid (SA) and abscisic acid (ABA) is still unresolved using either spraying technique or in vitro conditions. Alternative way of studying these effects under near in vivo conditions is through the use of liquid culturing technique. Effects of SA and ABA (100 μM) on antioxidative enzymes, antioxidants and lipid peroxidation were studied in detached tillers of three wheat (Triticum aestivum L.) cultivars PBW 343, C 306 (heat tolerant) and WH 542 (heat susceptible) cultured in a liquid medium. Ears were subjected to heat shock treatment (45°C for 2 h) and then maintained at 25°C for 5 days. Heat shock treatment resulted in increased peroxidase (POD) activity, while superoxide dismutase (SOD) and catalase (CAT) activities were reduced compared to control. The decrease in CAT activity was more significant in susceptible cultivar WH 542. Concomitantly, content of α-tocopherol and lipid peroxides increased in heat-treated wheat ears, whereas contents of total ascorbate level were reduced. Following treatment with SA and ABA, activities of all three antioxidative enzymes increased in correspondence with an increase in ascorbate and α-tocopherol content. Apparently, lipid peroxide content was reduced by SA in heat tolerant cultivars (PBW 343 and C 306) whereas in susceptible cultivar it was decreased by ABA. The up-regulation of the antioxidant system by SA and ABA possibly contributes to better tolerance against heat shock-induced oxidative damage in wheat grains.     

Novel interrelationship between salicylic acid, abscisic acid, and PIP2-specific phospholipase C in heat acclimation-induced thermotolerance in pea leaves

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 (PIP(2))-specific phospholipase C (PLC) play in thermotolerance development induced by heat acclimation (38 degrees 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 PIP(2)-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, PIP(2)-PLC appears to play a key role in free SA- and ABA-associated reinforcement of thermotolerance resulting from heat acclimation.     

高温胁迫及其信号转导

高温是影响当前农业生产重要的不得环境因素之一。本文综述了４种信号分子脱落酸（ＡＢＡ）,Ｃa^2 ,水杨酸（ＳＡ）,茉莉酸（ＪＡ）对高温胁迫的响应以及它们的相互关系,高温胁迫能够诱导ＡＢＡ,Ｃa^2 ,ＳＡ的含量升高,并且通过外施ＡＢＡ,Ｃa^2 ,ＳＡ,ＪＡ都能提高植物的抗热性。作为胞内第二信使,外源Ｃa^2 Ｃ能够提高植物超氧化物歧化mei（ＳＯＤ）,过氧化氢mei（ＣＡＴ）以及抗杯血酸氧化mei（ＡＰＸ）的活性,且能     

Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. A family of seven related proteins named AtGPX1- AtGPX7 in Arabidopsis was identified, and the genomic organization of this family was reported. The putative subcellular localizations of the encoded proteins are the cytosol, chloroplast, mitochondria, and endoplasmic reticulum. Expressed sequence tags (ESTs) for all the genes except AtGPX7 were identified. Expression analysis of AtGPX genes in Arabidopsis tissues was performed, and different patterns were detected. Interestingly, several genes were up-regulated coordinately in response to abiotic stresses. AtGPX6, like human phospholipid hydroperoxide GPX (PHGPX), possibly encodes mitochondrial and cytosolic isoforms by alternative initiation. In addition, this gene showed the strongest responses under most abiotic stresses tested. AtGPX6::GUS analysis in transgenic Arabidopsis showed that AtGPX6 is highly expressed throughout development in most tissues, thus supporting an important role for this gene in protection against oxidative damage. The different effects of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and auxin on the expression of the genes indicate that the AtGPX family is regulated by multiple signaling pathways. Analysis of the upstream region of the AtGPX genes revealed the presence of multiple conserved motifs, and some of them resembled antioxidant-responsive elements found in plant and human promoters. The potential regulatory role of specific sequences is discussed.     

Changes in antioxidant enzymes activity and oxidative stress by abscisic acid and salicylic acid in wheat genotypes

Abscisic acid (ABA) and salicylic acid (SA) were sprayed on leaves of wheat genotypes C 306 and Hira at 25 and 40 d after sowing under moderate water stress (−0.8 MPa) imposed by adding PEG-6000 in nutrient solution. ABA and SA increased the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in comparison to unsprayed control plants. Both ABA and SA treatments decreased the contents of hydrogen peroxide and thiobarbituric acid reactive substances, a measure of lipid peroxidation, compared to unsprayed plants. The beneficial effect of increase in antioxidant enzymes activity and decrease in oxidative stress was reflected in increase in chlorophyll and carotenoid contents, relative water content, membrane stability index, leaf area and total biomass over control plants. The lower concentrations of ABA (0.5 mM) and SA (1.0 mM) were generally more effective than higher concentrations.     

Uniconazole-induced tolerance of rape plants to heat stress in relation to changes in hormonal levels, enzyme activities and lipid peroxidation

Winter rape (Brassica napus L. cv. 601) seedlings were treated with 50 mg.l-1of foliar-applied uniconazole and then exposed to heat stress with a light/dark temperature regime of 43 °C/38 °C for 3 days at the stem elongation stage. Heat stressed plants contained lower endogenous GA3, IAA and zeatin contents than the controls, while ABA content and ethylene level were increased significantly. Uniconazole-treated plants had lower endogenous GA3 and IAA contents, and higher zeatin and ABA contents and ethylene levels. Leaf chlorophyll content and respiratory capacity of roots were reduced markedly after plants were subjected to heat stress, and foliar sprays of uniconazole retarded the degradation of chlorophyll and increased respiratory capacity of roots. Following exposure to heat, the activities of superoxide dismutase and peroxidase were significantly reduced. Uniconazole-induced heat tolerance was accompanied by increased activities of various antioxidant enzymes. Foliar applications of uniconazole reduced electrolyte leakage and malondialdehyde accumulation caused by heat stress, suggesting that uniconazole may have decreased heat-induced lipid peroxidation and membrane damage. Foliar sprays of uniconazole increased the tolerance of rape plants to heat stress.     

The calcium sensor calcineurin B-like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis

Calcium plays a pivotal role in plant responses to several stimuli, including pathogens, abiotic stresses, and hormones. However, the molecular mechanisms underlying calcium functions are poorly understood. It is hypothesized that calcium serves as second messenger and, in many cases, requires intracellular protein sensors to transduce the signal further downstream in the pathways. The calcineurin B-like proteins (CBLs) represent a unique family of calcium sensors in plant cells. Here, we report our analysis of the CBL9 member of this gene family. Expression of CBL9 was inducible by multiple stress signals and abscisic acid (ABA) in young seedlings. When CBL9 gene function was disrupted in Arabidopsis thaliana plants, the responses to ABA were drastically altered. The mutant plants became hypersensitive to ABA in the early developmental stages, including seed germination and post-germination seedling growth. In addition, seed germination in the mutant also showed increased sensitivity to inhibition by osmotic stress conditions produced by high concentrations of salt and mannitol. Further analyses indicated that increased stress sensitivity in the mutant may be a result of both ABA hypersensitivity and increased accumulation of ABA under the stress conditions. The cbl9 mutant plants showed enhanced expression of genes involved in ABA signaling, such as ABA-INSENSITIVE 4 and 5. This study has identified a calcium sensor as a common element in the ABA signaling and stress-induced ABA biosynthesis pathways.     

Disruption of a guard cell-expressed protein phosphatase 2A regulatory subunit,RCN1, confers abscisic acid insensitivity in Arabidopsis

Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell-expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca(2+) increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling.