Salicylic Acid Biosynthesis Inhibitor Research Articles

Salicylic Acid Is Involved in the Growth Inhibition Caused by Excessive Ammonium in Oilseed Rape (Brassica napus L.).

Rapeseed (Brassica napus L.) is extremely sensitive to excessive NH4+ toxicity. There remains incomplete knowledge of the causal factors behind the growth suppression in NH4+-nourished plants, with limited studies conducted specifically on field crop plants. In this study, we found that NH4+ toxicity significantly increased salicylic acid (SA) accumulation by accelerating the conversion of SA precursors. Moreover, exogenous SA application significantly aggravated NH4+ toxicity symptoms in the rapeseed shoots. Genome-wide differential transcriptomic analysis showed that NH4+ toxicity increased the expression of genes involved in the biosynthesis, transport, signaling transduction, and conversion of SA. SA treatment significantly increased shoot NH4+ concentrations by reducing the activities of glutamine synthase and glutamate synthase in NH4+-treated rapeseed plants. The application of an SA biosynthesis inhibitor, ABT, alleviated NH4+ toxicity symptoms. Furthermore, SA induced putrescine (Put) accumulation, resulting in an elevated ratio of Put to [spermidine (Spd) + spermine (Spm)] in the NH4+-treated plants, while the opposite was true for ABT. The application of exogenous Put and its biosynthesis inhibitor DFMA induced opposite effects on NH4+ toxicity in rapeseed shoots. These results indicated that the increased endogenous SA contributed noticeably to the toxicity caused by the sole NH4+-N supply in rapeseed shoots. This study provided fresh perspectives on the mechanism underlying excessive NH4+-induced toxicity and the corresponding alleviating strategies in plants.

NADPH oxidase-dependent H2O2 production mediates salicylic acid-induced salt tolerance in mangrove plant Kandelia obovata by regulating Na+/K+ and redox homeostasis.

Salicylic acid (SA) is known to enhance salt tolerance in plants. However, the mechanism of SA-mediated response to high salinity in halophyte remains unclear. Using electrophysiological and molecular biological methods, we investigated the role of SA in response to high salinity in mangrove species, Kandelia obovata, a typical halophyte. Exposure of K. obovata roots to high salinity resulted in a rapid increase in endogenous SA produced by phenylalanine ammonia lyase pathway. The application of exogenous SA improved the salt tolerance of K. obovata, which depended on the NADPH oxidase-mediated H2O2. Exogenous SA and H2O2 increased Na+ efflux and reduced K+ loss by regulating the transcription levels of Na+ and K+ transport-related genes, thus reducing the Na+/K+ ratio in the salt-treated K. obovata roots. In addition, exogenous SA-enhanced antioxidant enzyme activity and its transcripts, and the expressions of four genes related to AsA-GSH cycle as well, then alleviated oxidative damages in the salt-treated K. obovata roots. However, the above effects of SA could be reversed by diphenyleneiodonium chloride (the NADPH oxidase inhibitor) and paclobutrazol (a SA biosynthesis inhibitor). Collectively, our results demonstrated that SA-induced salt tolerance of K. obovata depends on NADPH oxidase-generated H2O2 that affects Na+/K+ and redox homeostasis in response to high salinity.

Endogenous salicylic acid mediates melatonin-induced chilling-and oxidative-stress tolerance in harvested kiwifruit

Phytohormone melatonin (MT) has shown promise in alleviating chilling injury (CI) in kiwifruit; however, the performance and mechanism remain largely unknown. In this study, the effect of pretreatment of MT at concentration of 0.05 mM on CI in ‘Xuxiang’ kiwifruit during storage at 1 °C for 90 d was investigated, as well as the implication of endogenous salicylic acid (SA) in MT-induced chilling-and oxidative-stress tolerance. MT pretreatment protected kiwifruit against CI, as figured out by lower CI index, firmness loss and soluble solids accumulation and better pulp appearance than those in control fruit. MT pretreatment stimulated endogenous SA generation by activating the gene expression of phenylalanine ammonia lyase (PAL) and activity of PAL and benzoic acid-2-hydroxylase, therefore up-regulated SA-responsive pathogenesis-related gene 1 expression, and provoked kiwifruit defense response to chilling stress. Moreover, reactive oxygen species in MT-pretreated fruit were neutralized by evaluating endogenous MT, ascorbic acid and glutathione contents and superoxide dismutase, catalase and ascorbate peroxidase activity, thus reducing membrane damage. However, such positive regulations of MT on kiwifruit were counteracted to varying extent by pretreatment of the combination of paclobutrazol PAC, a SA biosynthesis inhibitor, with MT. These findings indicated a mediating role of endogenous SA in chilling-and oxidative-stress tolerance in kiwifruit induced by MT pretreatment that could be operative by evoking defense response to chilling stress and enhancing antioxidative protection.

Sulfur dioxide improves the thermotolerance of maize seedlings by regulating salicylic acid biosynthesis

Heat stress (HS) has become a serious threat to crop growth and yield. Sulfur dioxide (SO2) is being verified as a signal molecule in regulating the plant stress response. However, it is unknown whether SO2 plays a significant role in the plant heat stress response (HSR). Herein, maize seedlings were pretreated with various concentrations of SO2 and then kept at 45 °C for heat stress treatment, aiming to study the effect of SO2 pretreatment on HSR in maize by phenotypic, physiological, and biochemical analyses. It was found that SO2 pretreatment greatly improved the thermotolerance of maize seedlings. The SO2-pretreated seedlings showed 30–40% lower ROS accumulation and membrane peroxidation, but 55–110% higher activities of antioxidant enzymes than the distilled water-pretreated seedlings under heat stress. Interestingly, endogenous salicylic acid (SA) levels were increased by ∼85% in SO2-pretreated seedlings, as revealed by phytohormone analyses. Furthermore, the SA biosynthesis inhibitor paclobutrazol markedly reduced SA levels and attenuated SO2-triggered thermotolerance of maize seedlings. Meanwhile, transcripts of several SA biosynthesis and signaling, and heat stress-responsive genes in SO2-pretreated seedlings were significantly elevated under HS. These data have demonstrated that SO2 pretreatment increased endogenous SA levels, which activated the antioxidant machinery and strengthened the stress defense system, thereby improving the thermotolerance of maize seedlings under HS. Our current study provides a new strategy for mitigating heat stress damage for safe crop production.

Role of Endogenous Salicylic Acid as a Hormonal Intermediate in the Bacterial Endophyte Bacillus subtilis-Induced Protection of Wheat Genotypes Contrasting in Drought Susceptibility under Dehydration.

Endophytic Bacillus subtilis is a non-pathogenic beneficial bacterium which promotes plant growth and tolerance to abiotic stresses, including drought. However, the underlying physiological mechanisms are not well understood. In this study, the potential role that endogenous salicylic acid (SA) plays in regulating endophytic B. subtilis-mediated drought tolerance in wheat (Triticum aestivum L.) was examined. The study was conducted on genotypes with contrasting levels of intrinsic drought tolerance (drought-tolerant (DT) cv. Ekada70; drought-susceptible (DS) cv. Salavat Yulaev). It was revealed that B. subtilis 10-4 promoted endogenous SA accumulation and increased the relative level of transcripts of the PR-1 gene, a marker of the SA-dependent defense pathway, but two wheat cultivars responded differently, with the highest levels exhibited in DT wheat seedlings. These had a positive correlation with the ability of strain 10-4 to effectively protect DT wheat seedlings against drought injury by decreasing osmotic and oxidative damages (i.e., proline, water holding capacity (WHC), and malondialdehyde (MDA)). However, the use of the SA biosynthesis inhibitor 1-aminobenzotriazole prevented endogenous SA accumulation under normal conditions and the maintenance of its increased level under stress as well as abolished the effects of B. subtilis treatment. Particularly, the suppression of strain 10-4-induced effects on proline and WHC, which are both contributing factors to dehydration tolerance, was found. Moreover, the prevention of strain 10-4-induced wheat tolerance to the adverse impacts of drought, as judged by the degree of membrane lipid peroxidation (MDA) and plant growth (length, biomass), was revealed. Thus, these data provide an argument in favor of a key role of endogenous SA as a hormone intermediate in triggering the defense responses by B. subtilis 10-4, which also afford the foundation for the development of the bacterial-induced tolerance of these two different wheat genotypes under dehydration.

Melatonin induces improved protection against Botrytis cinerea in cherry tomato fruit by activating salicylic acid signaling pathway

Melatonin modulated immune responses to various stresses in plants. However, the crosstalk between melatonin and the other signal molecules, especially with salicylic acid (SA), is largely unclear in fruit. The objective of this research was to investigate the mechanism of melatonin induced disease resistance in cherry tomato fruit. The results indicated that melatonin treatment significantly inhibited gray mold development, induced reactive oxygen species (ROS) burst, enhanced SA accumulation accompanied by increasing its synthesis-related enzymes activity, up-regulated expression of defense genes, including SlWRKY70, SlNPR1, SlTGA5, SlPR1, SlPR2 and SlGLU, but decreased nitric oxide (NO) content during storage. Moreover, melatonin treatment increased endogenous melatonin content by up-regulating the expressions of SlTDC, SlSNAT and SlASMT in fruit during storage. However, the melatonin-enhanced disease resistance in fruit was completely abolished by a paclobutrazol (PAC) (SA biosynthesis inhibitor) treatment. Thus, the results suggested that hydrogen peroxide (H2O2) and SA signaling pathway would be collectively involved in melatonin induced-disease resistance in cherry tomato fruit during storage.

Acetic acid enhances the tolerance of grapevines to NaHCO3 stress by increasing SA production

To demonstrate the role of acetic acid in increasing the tolerance of grapevines to NaHCO3 and reveal the underlying mechanism, the alkali-tolerant rootstock AB11, planted in a split-root system, was used in this study. The content of acetic acid was largely increased in the NaHCO3-treated half and nontreated half of split-root grapevines. When 25 mM acetic acid was applied to half the roots and the other half was treated with NaHCO3, acetic acid decreased the injury of the NaHCO3-treated roots and increased the grapevine tolerance to NaHCO3. Both acetic acid and NaHCO3 treatments largely increased the content of salicylic acid (SA) in roots, and additionally, the two treatments generated additive effects on SA accumulation. The application of the SA biosynthesis inhibitor 1-aminobenzotriazole decreased the grapevine tolerance to NaHCO3 induced by acetic acid. Taken together, the synthesis of acetic acid was induced by NaHCO3, and acetic acid treatment enhanced grapevine tolerance to NaHCO3 via SA.

Hydrogen sulfide acted as a downstream signal was involved in the regulation of salicylic acid on photosynthesis of cucumber seedlings under low temperature and low light intensity

Both salicylic acid (SA) and hydrogen sulfide (H2S) play an important role in regulating plant growth and development and physiological metabolism under abiotic stresses. As signal molecules, the interaction between them in regulating cucumber photosynthesis under low temperature and low light is still unclear. Here, we examined the regulation and interaction of SA and H2S on photosynthesis in cucumber seedlings under low temperature (8 ℃/5 ℃, day/night) and low light (100 μmol·m-2·s-1). Seedlings were foliar-sprayed with SA, sodium sulfide (NaHS, H2S donor), and their scavenger or biosynthesis inhibitors, respectively. Seedlings treated with deionized water at suitable temperature and light condition were used as the control. The results showed that SA increased the L-/D-cysteine desulfhydrase (LCD, DCD) activities and relative mRNA expression, and consequently promoted the endogenous H2S production. However, NaHS did not affect the activities and gene expressions of phenylalnine ammonialyase and isochorismate and endogenous SA level. Compared with the H2O-treated seedlings under low temperature and low light, SA- and NaHS-treated seedlings showed an increase in the photosynthetic rate, stomatal conductance and transpiration rate, while a decrease in intercellular CO2 concentration. SA and NaHS increased the CO2 assimilation, which mainly attributed to the increases in the activities of the ribulose-1, 5-bisphosphate carboxylase, rubisco activase, sedoheptulose-1, 7-bisphosphatase and fructose-1, 6-bisphosphatase, as well as their mRNA expression. Meanwhile, SA and NaHS improved the actual photochemical efficiency and maximal photochemical efficiency of PSII, and therefore alleviated the damage in photosynthetic apparatus and negative effect on growth from low temperature and low light stress. The SA-induced higher photosynthesis and growth in stressed seedlings were suppressed by addition of H2S scavenger hypotaurine. However, the H2S-induced tolerance of photosynthetic apparatus to low temperature and low light was not affected by SA biosynthesis inhibitor paclobutrazol and 2-aminoindan-2-phosphonic acid. Our results suggested that H2S, as a downstream signal of SA, was involved in regulating photosynthesis in cucumber seedlings under low temperature and low light.

GmSnRK1.1, a Sucrose Non-fermenting-1(SNF1)-Related Protein Kinase, Promotes Soybean Resistance to Phytophthora sojae.

Phytophthora root and stem rot, a destructive disease of soybean [Glycine max (L.) Merr.], is caused by the oomycete Phytophthora sojae. However, how the disease resistance mechanisms of soybean respond to P. sojae infection remains unclear. Previously, we showed that GmWRKY31, which interacts with a sucrose non-fermenting-1(SNF1)-related protein kinase (SnRK), enhances resistance to P. sojae in soybean. Here, we report that the membrane-localized SnRK GmSnRK1.1 is involved in the soybean host response to P. sojae. The overexpression of GmSnRK1.1 (GmSnRK1.1-OE) increased soybean resistance to P. sojae, and the RNA interference (RNAi)-mediated silencing of GmSnRK1.1 (GmSnRK1.1-R) reduced resistance to P. sojae. Moreover, the activities and transcript levels of the antioxidant enzymes superoxide dismutase and peroxidase were markedly higher in the GmSnRK1.1-OE transgenic soybean plants than in the wild type (WT), but were reduced in the GmSnRK1.1-R plants. Several isoflavonoid phytoalexins related genes GmPAL, GmIFR, Gm4CL and GmCHS were significantly higher in “Suinong 10” and GmSnRK1.1-OE lines than these in “Dongnong 50,” and were significantly lower in GmSnRK1.1-R lines. In addition, the accumulation of salicylic acid (SA) and the expression level of the SA biosynthesis-related gene were significantly higher in the GmSnRK1.1-OE plants than in the WT and GmSnRK1.1-R plants, moreover, SA biosynthesis inhibitor treated GmSnRK1.1-R lines plants displayed clearly increased pathogen biomass compared with H2O-treated plants after 24 h post-inoculation. These results showed that GmSnRK1.1 positively regulates soybean resistance to P. sojae, potentially functioning via effects on the expression of SA-related genes and increased accumulation of SA.

A unique aluminum resistance mechanism conferred by aluminum and salicylic-acid-activated root efflux of benzoxazinoids in maize

Although aluminum (Al) exclusion via root exudation of organic matters is a common resistance mechanism adopted by many plant species, whether root exudation of benzoxazinoids, such as hydroxamic acids (HAs), confers Al resistance remains unclear. We performed physiological characterization for an Al-resistant maize cultivar TY and a sensitive maize cultivar ZD. First, Al exposure induced HA exudation from the root tip of TY, but not from ZD. Second, HAs formed non-toxic Al chelation complexes in vitro and exogenous HAs alleviated root damage and improved root growth under Al stresses. Third, both Al and exogenous salicylic acid (SA) treatments induced accumulation of endogenous SAs in the root apices of TY, which in turn enhanced root HA exudation and Al resistance in TY. Furthermore, an SA biosynthesis inhibitor significantly decreased Al resistance in TY and abolished the beneficial effects of exogenous SA on Al resistance, suggesting a key role of the endogenous SAs in induction of Al resistance. Finally, it was the root-tip HA exudation but not the root-tip HA contents that determined Al resistance in maize. We have revealed a unique Al exclusion mechanism underlying Al resistance via Al and SA-mediated root HA efflux in maize.

Salicylic acid biosynthesis inhibitors increase chilling injury to maize (Zea mays L.) seedlings

Salicylic acid (SA) is involved in plant response to many abiotic stresses. This study investigated the potential role of SA in regulating chilling tolerance of maize (Zea mays L.) seedlings. The results showed that chilling stress induced transient accumulation of free SA in maize plants, and stimulated the enzymatic activities of phenylalanine ammonia-lyase (PAL) and benzoic acid-2-hydroxylase. However, seedlings pretreated with SA biosynthesis inhibitors (i.e., 2-aminoindane-2-phosphonic acid or 1-aminobenzotriazole) accumulated significantly less free and bound SA and suffered more severe cold injury. Together, these results indicated that the chilling-induced SA accumulation in maize seedlings was mainly attributed to the PAL pathway. SA application also induced chilling tolerance of maize seedlings; whereas, inhibition of SA biosynthesis significantly increased membrane peroxidation, decreased antioxidant enzymes activities, and resulted in growth retardation. Exogenous SA treatments reversed the effects of the SA inhibitors on chilling tolerance, indicating the important endogenous role of SA in inducing chilling tolerance of maize plants.

Salicylic Acid Alleviates Aluminum Toxicity in Soybean Roots through Modulation of Reactive Oxygen Species Metabolism.

As an important signal molecule, salicylic acid (SA) improves plant tolerance to aluminum (Al) stress. The objective of this study was to investigate the effects of exogenous SA application on the dynamics of endogenous SA and reactive oxygen species in soybean (Glycine max L.) exposed to Al stress. The roots of soybean seedlings were exposed to a combination of AlCl3 (30 μM) and SA (10 μM)/PAC (100 μM, paclobutrazol, SA biosynthesis inhibitor) for 3, 6, 9, and 12 h. Al stress induced an increase in endogenous SA concentration in a time-dependent manner, also verified by the up-regulated expression of GmNPR1, an SA-responsive gene. Al stress increased the activities of phenylalanine ammonia-lyase (PAL) and benzoic acid 2-hydroxylase (BA2H), and the contents of SA, and malondialdehyde (MDA) in the root apex. The application of exogenous SA increased PAL and BA2H, and reduced and MDA contents in soybean roots under Al stress. PAC inhibited the SA induced increase in BA2H activity. In addition, the SA application resulted in a rapid increase in hydrogen peroxide (H2O2) concentration under Al stress, followed by a sharp decrease. Compared with the plants exposed to Al alone, Al+SA plants possessed higher activities of superoxide dismutase, peroxidase, and ascorbate peroxidase, and lower catalase activity, indicating that SA alleviated Al-induced oxidative damage. These results suggested that PAL and BA2H were involved in Al-induced SA production and showed that SA alleviated the adverse effects of Al toxicity by modulating the cellular H2O2 level and the antioxidant enzyme activities in the soybean root apex.

Fungal endophyte protects Atractylodes lancea from root rot caused by Fusarium oxysporum

Endophytic fungi, which stimulate a variety of defence reactions in host plants without causing visible disease symptoms, have been isolated from almost every plant. However, beneficial interactions between fungal endophytes and pathogens from the same habitat remain largely unknown. An inoculation of Atractylodes lancea plantlets with Gilmaniella sp. AL12 (AL12) prior to infection with Fusarium oxysporum prevented the necrotization of root tissues and plant growth retardation commonly associated with fusarium root rot. Quantification of F. oxysporum infections using real‐time PCR revealed a correlation between root rot symptoms and the relative amount of fungal DNA. Pretreatment with AL12 reduced the accumulation of reactive oxygen species stimulated by F. oxysporum. An in vitro analysis of their interactions under axenic culture conditions showed AL12 could inhibit F. oxysporum growth. Additionally, F. oxysporum infections were shown to decrease salicylic acid (SA) production compared with control plantlets. SA biosynthesis inhibitors, 2‐aminoindan‐2‐phosphonic acid and paclobutrazol, abolished the inhibition of F. oxysporum growth in A. lancea even after inoculation with AL12. The results indicated that the fungal endophyte protected A. lancea not only by direct antibiosis, but also by reversing the F. oxysporum‐mediated suppression of SA production.

The interaction of salicylic acid and Ca2+ alleviates aluminum toxicity in soybean (Glycine max L.)

Both calcium ion (Ca2+) and salicylic acid (SA) influence various stress responses in plants. In acidic soils, aluminum (Al) toxicity adversely affects crop yield. In this study, we determined the influences of Ca2+ and SA on root elongation, Al accumulation, and citrate secretion in soybean plant. We also investigated the activity of antioxidative enzymes in Al-exposed soybean roots. Root elongation was severally inhibited when the roots were exposed to 30 μM Al. The Al-induced inhibition of root elongation was ameliorated by Ca2+ and SA but aggravated by Ca2+ channel inhibitor (VP), CaM antagonists (TFP), Ca2+ chelator (EGTA), and SA biosynthesis inhibitor (PAC). Furthermore, 1.0 mM CaCl2 and 10 μM SA reduced the accumulation of Al in roots, but their inhibitors stimulated the accumulation of Al in roots. Citrate secretion from these roots increased with the addition of either 1.0 mM CaCl2 or 10 μM SA but did not increase significantly when treated with higher Ca2+ concentration. Enzymatic analysis showed that Ca2+ and SA stimulated the activities of superoxidase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) in Al-treated roots. In addition, SA restored the inhibition of Ca2+ inhibitors on root elongation and Al content. Thus, both Ca2+ and SA contribute to Al tolerance in soybean. Furthermore, Ca2+ supplements rapidly increased Al-induced accumulation of free-SA or conjugated SA (SAG), while Ca2+ inhibitors delayed the accumulation of SA for more than 8 h. Within 4 h of treatment, SA increased cytosolic Ca2+ concentration in Al-treated roots, and upregulated the expression of four genes that possibly encode calmodulin-like (CML) proteins. These findings indicate that SA is involved in Ca2+-mediated signal transduction pathways in Al tolerance.

Synergistic effect of antioxidant system and osmolyte in hydrogen sulfide and salicylic acid crosstalk-induced heat tolerance in maize (Zea mays L.) seedlings

Salicylic acid (SA), is a plant hormone with multifunction that is involved in plant growth, development and the acquisition of stress tolerance. Hydrogen sulfide (H2S) is emerging similar functions, but crosstalk between SA and H2S in the acquisition of heat tolerance is not clear. Our recent study firstly reported that SA treatment enhanced the activity of L-cysteine desulfhydrase (L-DES), a key enzyme in H2S biosynthesis, followed by induced endogenous H2S accumulation, which in turn improved the heat tolerance of maize seedlings.1 In addition, NaHS, a H2S donor, enhanced SA-induced heat tolerance, while its biosynthesis inhibitor DL-propargylglycine (PAG) and scavenger hydroxylamine (HT) weakened SA-induced heat tolerance. Also, NaHS had no significant effect on SA accumulation and its biosynthesis enzymes phenylalanine ammonia lyase (PAL) and benzoic-acid-2-hydroxylase (BA2H) activities, as well as significant difference was not observed in NaHS-induced heat tolerance of maize seedlings by SA biosynthesis inhibitors paclobutrazol (PAC) and 2-aminoindan-2-phosph- onic acid (AIP) treatment.1 Further study displayed that SA induced osmolytes (proline, betaine and trehalose) accumulation and enhancement in activity of antioxidant system in maize seedlings. These results showed that antioxidant system and osmolyte play a synergistic role in SA and H2S crosstalk-induced heat tolerance of maize seedlings.

Nitric oxide mediates hypocrellin accumulation induced by fungal elicitor in submerged cultures of Shiraia bambusicola.

Multiple responses of Shiraia bambusicola, including nitric oxide (NO) generation, hypocrellins production and salicylic acid (SA) biosynthesis, were induced by a fungal elicitor prepared from the mycelium of Aspergillum niger. Both the NO donator, sodium nitroprusside, and SA enhanced hypocrellin production without the fungal elicitor. However, the NO scavenger, 2,4-carboxyphenyl-4,4, 5,5- tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and the SA biosynthesis inhibitor, cinnamic acid (CA), inhibited hypocrellin accumulation in the presence of the elicitor. cPTIO also inhibited SA production induced by the A. niger elicitor. CA failed to inhibit NO production but it significantly inhibited hypocrellin accumulation. Aspergillum niger elicitor induced an NO burst, SA accumulation, and hypocrellin production in S. bambusicola. Therefore, the fungal elicitor was involved in the signaling pathway, which is a mechanism different from that of higher plants.

Endogenous salicylic acid accumulation is required for chilling tolerance in cucumber (Cucumis sativus L.) seedlings.

Salicylic acid (SA) is an important plant hormone, and its exogenous application can induce tolerance to multiple environmental stresses in plants. In this study, we examine the potential involvement of endogenous SA in response to chilling in cucumber (Cucumis sativus L.) seedlings. A low temperature of 8 °C induces a moderate increase in endogenous SA levels. Chilling stimulates the enzymatic activities and the expression of genes for phenylalanine ammonia-lyase (PAL) and benzoic acid-2-hydroxylase rather than isochorismate synthase. This indicates that the PAL enzymatic pathway contributes to chilling-induced SA production. Cucumber seedlings pretreated with SA biosynthesis inhibitors accumulate less endogenous SA and suffer more from chilling damage. The expression of cold-responsive genes is also repressed by SA inhibitors. The reduction in stress tolerance and in gene expression can be restored by the exogenous application of SA, confirming the critical roles of SA in chilling responses in cucumber seedlings. Furthermore, the inhibition of SA biosynthesis under chilling stress results in a prolonged and enhanced hydrogen peroxide (H2O2) accumulation. The application of exogenous SA and the chemical scavenger of H2O2 reduces the excess H2O2 and alleviates chilling injury. In contrast, the protective effects of SA are negated by foliar spraying with high concentrations of H2O2 and an inhibitor of the antioxidant enzyme. These results suggest that endogenous SA is required in response to chilling stress in cucumber seedlings, by modulating the expression of cold-responsive genes and the precise induction of cellular H2O2 levels.

Signaling Effects of Nitric Oxide, Salicylic Acid, and Reactive Oxygen Species on Isoeuphpekinensin Accumulation in Euphorbia pekinensis Suspension Cells Induced by an Endophytic Fungal Elicitor

Nitric oxide (NO), salicylic acid (SA), and reactive oxygen species (ROS) are important signal molecules that mediate plant resistance reactions and play important roles in secondary metabolism. To research the signal transduction pathway of the endophytic fungal elicitor from Fusarium sp. E5 promoting secondary metabolism in Euphorbia pekinensis suspension cells, the changes in NO, SA, ROS, and isoeuphpekinensin contents in the cells were investigated after elicitor addition to the cell suspension culture. The elicitor did not change H2O2 or O2− contents notably, whereas NO and SA contents were enhanced. Both the NO donator sodium nitroprusside (SNP) and SA enhanced isoeuphpekinensin content in the absence of the fungal elicitor, whereas the NO scavenger cPTIO and SA biosynthesis inhibitor cinnamic acid (CA) inhibited isoeuphpekinensin accumulation in the presence of the elicitor. In addition, cPTIO inhibited SA production induced by the fungal elicitor. CA did not inhibit NO production, but it significantly inhibited isoeuphpekinensin accumulation. The results demonstrated that in Euphorbia pekinensis suspension cells the endophytic fungal elicitor induced increased NO content and SA production, which promoted isoeuphpekinensin accumulation. ROS are clearly not involved in the endophytic fungus–host interaction signaling pathway.

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.

Salicylic acid synthesized by benzoic acid 2-hydroxylase participates in the development of thermotolerance in pea plants

To investigate importance of salicylic acid (SA) and SA biosynthesis pathway in the development of plant thermotolerance, the research tested the effects of heat acclimation on pea plants ( Pisum sativun L.) at 37 °C for 2 h and examined in vivo pretreatments of the plants in a medium containing either SA or SA biosynthesis inhibitors before heat treatment. The results showed that both heat acclimation and SA application could reduce leaf injury caused by subsequent heat stress and induce the synthesis of heat shock proteins (Hsp 70 and Hsp 17.6). Further, membrane lipid peroxidation caused by the heat stress was found to decrease, suggesting that plant's thermotolerance developed as a result of heat acclimation and SA application. A rapid transient increase of endogenous free SA and a subsequent enrichment in Hsp 70 were both elevated by heat acclimation. Changes of phenylalanine ammonia lyase (PAL) and benzoic acid 2-hydroxylase (BA2H) activities roughly paralleled those of free SA. In addition, pretreatment with the inhibitors of either PAL or BA2H effectively blocked SA accumulation and Hsp 70 enrichment in the plants subjected to heat acclimation, thus reduced plant tolerance to heat stress. This finding indicates that inhibition of SA biosynthesis during heat acclimation can annul the development of plant thermotolerance and that SA synthesized by BA2H participates in the acquisition of heat acclimation-induced thermotolerance.