Salicylic Acid Glucosyltransferase Research Articles

OsTGAL1 suppresses the resistance of rice to bacterial blight disease by regulating the expression of salicylic acid glucosyltransferase OsSGT1.

Many TGA transcription factors participate in immune responses in the SA-mediated signaling pathway in Arabidopsis. This study identified a transcription factor OsTGAL1, which is induced upon infection by Xoo. Overexpression of OsTGAL1 increased the susceptibility of rice to Xoo. Plants overexpressing OsTGAL1 could affect the expression of many SA signaling-related genes. OsTGAL1 was able to interact with the promoter of OsSGT1, which encodes a key enzyme for SA metabolism. The transcript of OsSGT1 was induced by Xoo and this responsive expression was further increased in plants overexpressing OsTGAL1. OsSGT1 knockout lines had enhanced resistance to Xoo, and knocking out OsSGT1 in plants overexpressing OsTGAL1 blocked the susceptibility caused by OsTGAL1. Altered expression levels of several OsPRs in all the transgenic plants demonstrated that SA-mediated signaling had been affected. Furthermore, we identified an oxidoreductase of CC-type glutaredoxin, OsGRX17, which interacted with OsTGAL1. OsGRX17 reduced the regulation of OsTGAL1 on OsSGT1, and this may be due to its redox modulation. Thus, our results demonstrate that OsTGAL1 negatively regulates resistance to Xoo by its effects on SA metabolism via the activation of OsSGT1, which provides valuable targets for plant breeders in developing new cultivars that are resistant to Xoo.

ATG4 Mediated Psm ES4326/AvrRpt2-Induced Autophagy Dependent on Salicylic Acid in Arabidopsis Thaliana.

Psm ES4326/AvrRpt2 (AvrRpt2) was widely used as the reaction system of hypersensitive response (HR) in Arabidopsis. The study showed that in npr1 (GFP-ATG8a), AvrRpt2 was more effective at inducing the production of autophagosome and autophagy flux than that in GFP-ATG8a. The mRNA expression of ATG1, ATG6 and ATG8a were more in npr1 during the early HR. Based on transcriptome data analysis, enhanced disease susceptibility 1 (EDS1) was up-regulated in wild-type (WT) but was not induced in atg4a4b (ATG4 deletion mutant) during AvrRpt2 infection. Compared with WT, atg4a4b had higher expression of salicylic acid glucosyltransferase 1 (SGT1) and isochorismate synthase 1 (ICS1); but less salicylic acid (SA) in normal condition and the same level of free SA during AvrRpt2 infection. These results suggested that the consumption of free SA should be occurred in atg4a4b. AvrRpt2 may trigger the activation of Toll/Interleukin-1 receptor (TIR)-nucleotide binding site (NB)-leucine rich repeat (LRR)—TIR-NB-LRR—to induce autophagy via EDS1, which was inhibited by nonexpressor of PR genes 1 (NPR1). Moreover, high expression of NPR3 in atg4a4b may accelerate the degradation of NPR1 during AvrRpt2 infection.

Gene expression of salicylic acid and jasmonic acid pathways and photosynthesis parameters of sweet orange trees in response to acibenzolar-S-methyl

The use of resistance inducers may be a tool to assist in the management of citrus diseases. However, knowledge about how some inducers, such as the benzothiadiazoles, promote gene expression from the pathways involved in induced resistance and alter plant development are lacking. In this work, we evaluated the effect of the acibenzolar-S-methyl (ASM) on gene expression from the biosynthetic pathways of salicylic (SA) and jasmonic (JA) acids, as well as in physiological parameters of young sweet orange trees. After a single ASM application, gene expression assessed during six time points revealed no significant changes in the main pathway for SA synthesis: isochorismate synthases (ISC) 1 and ICS2 genes were up-regulated only at 6 days after treatment (dat). However, an increase in phenylalanine ammonia lyase (PAL) was observed. Salicylic acid carboxymethyl transferase (BSMT1) and salicylic acid glucosyltransferase I (SAGT1) were up-regulated, indicating expression of genes whose products modify SA. Pointedly up-regulation of lipoxygenase (LOX) and both up- and down-regulation of allene oxide synthase (AOS) and allene oxide cyclase (AOC) in biosynthetic pathways of JA was observed, while jasmonic acid methyl transferase (JMT), whose product modify JA, was consistently induced from three to 13 dat. Besides an increase in SA and JA modification enzymes, ASM reduced photosynthetic rate in treated plants, without affecting the stomatal conductance and the transpiration rate. Our results reveal how the SA and JA pathways are regulated soon after ASM treatment, in particular the up-regulation of BSTM1, SAGT1 and JMT.

Role of salicylic acid glucosyltransferase in balancing growth and defence for optimum plant fitness.

Salicylic acid (SA), an essential secondary messenger for plant defence responses, plays a role in maintaining a balance (trade‐off) between plant growth and resistance induction, but the detailed mechanism has not been explored. Because the SA mimic benzothiadiazole (BTH) is a more stable inducer of plant defence than SA after exogenous application, we analysed expression profiles of defence genes after BTH treatment to better understand SA‐mediated immune induction. Transcript levels of the salicylic acid glucosyltransferase (SAGT) gene were significantly lower in BTH‐treated Nicotiana tabacum (Nt) plants than in SA‐treated Nt control plants, suggesting that SAGT may play an important role in SA‐related host defence responses. Treatment with BTH followed by SA suppressed SAGT transcription, indicating that the inhibitory effect of BTH is not reversible. In addition, in BTH‐treated Nt and Nicotiana benthamiana (Nb) plants, an early high accumulation of SA and SA 2‐O‐β‐d‐glucoside was only transient compared to the control. This observation agreed well with the finding that SAGT‐overexpressing (OE) Nb lines contained less SA and jasmonic acid (JA) than in the Nb plants. When inoculated with a virus, the OE Nb plants showed more severe symptoms and accumulated higher levels of virus, while resistance increased in SAGT‐silenced (IR) Nb plants. In addition, the IR plants restricted bacterial spread to the inoculated leaves. After the BTH treatment, OE Nb plants were slightly larger than the Nb plants. These results together indicate that SAGT has a pivotal role in the balance between plant growth and SA/JA‐mediated defence for optimum plant fitness.

Analysis of salicylic acid-dependent pathways in Arabidopsis thaliana following infection with Plasmodiophora brassicae and the influence of salicylic acid on disease.

Salicylic acid (SA) biosynthesis, the expression of SA-related genes and the effect of SA on the Arabidopsis-Plasmodiophora brassicae interaction were examined. Biochemical analyses revealed that, in P. brassicae-infected Arabidopsis, the majority of SA is synthesized from chorismate. Real-time monitored expression of a gene for isochorismate synthase was induced on infection. SA can be modified after accumulation, either by methylation, improving its mobility, or by glycosylation, as one possible reaction for inactivation. Quantitative reverse transcription-polymerase chain reaction (qPCR) confirmed the induction of an SA methyltransferase gene, whereas SA glucosyltransferase expression was not changed after infection. Col-0 wild-type (wt) did not provide a visible phenotypic resistance response, whereas the Arabidopsis mutant dnd1, which constitutively activates the immune system, showed reduced gall scores. As dnd1 showed control of the pathogen, exogenous SA was applied to Arabidopsis in order to test whether it could suppress clubroot. In wt, sid2 (SA biosynthesis), NahG (SA-deficient) and npr1 (SA signalling-impaired) mutants, SA treatment did not alter the gall score, but positively affected the shoot weight. This suggests that SA alone is not sufficient for Arabidopsis resistance against P. brassicae. Semi-quantitative PCR revealed that wt, cpr1, dnd1 and sid2 showed elevated PR-1 expression on P. brassicae and SA + P. brassicae inoculation at 2 and 3 weeks post-inoculation (wpi), whereas NahG and npr1 showed no expression. This work contributes to the understanding of SA involvement in the Arabidopsis-P. brassicae interaction.

Transgenic pearl millet male fertility restorer line (ICMP451) and hybrid (ICMH451) expressing Brassica juncea Nonexpressor of pathogenesis related genes 1 (BjNPR1) exhibit resistance to downy mildew disease.

Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into pearl millet male fertility restorer line ICMP451 by Agrobacterium tumefaciens-mediated genetic transformation. Transgenic pearl millet plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with A. tumefaciens strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in transgenic pearl millet lines. Transgenes BjNPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Transgenic pearl millet hybrid ICMH451-BjNPR1 was developed by crossing male-sterile line 81A X homozygous transgenic line ICMP451-BjNPR1. T3 and T4 homozygous lines of ICMP451-BjNPR1 and hybrid ICMH451-BjNPR1 exhibited resistance to three strains of downy mildew pathogen, while the untransformed ICMP451 and the isogenic hybrid ICMH451 plants were found susceptible. Following infection with S. graminicola, differential expression of systemic acquired resistance pathway genes, UDP-glucose salicylic acid glucosyl transferase and pathogenesis related gene 1 was observed in transgenic ICMP451-BjNPR1 and untransformed plants indicating the activation of systemic acquired resistance pathway contributing to the transgene-mediated resistance against downy mildew. The transgenic pearl millet expressing BjNPR1 showed resistance to multiple strains of S. graminicola and, as such, seems promising for the development of durable downy mildew resistant hybrids.

Up-Regulation of Antioxidants in Tobacco by Low Concentrations of H2O2 Suppresses Necrotic Disease Symptoms

Pretreatment of tobacco leaves with low concentrations (5 to 10 mM) of H₂O₂ suppressed hypersensitive-type necrosis associated with resistance to Tobacco mosaic virus (TMV) or Pseudomonas syringae pv. phaseolicola. The same pretreatment resulted in suppression of normosensitive necrosis associated with susceptibility to Botrytis cinerea. This type of H₂O₂-mediated, induced disease symptom resistance correlated with enhanced host antioxidant capacity, i.e., elevated enzymatic activities of catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POX) after viral and bacterial infections. Induction of genes that encode the antioxidants superoxide dismutase (SOD), CAT, and APX was also enhanced early after TMV infection. Artificial application of SOD and CAT suppressed necroses caused by viral, bacterial, or fungal pathogens similarly as H₂O₂ pretreatment, implying that H₂O₂-mediated symptom resistance operates through enhancement of plant antioxidant capacity. Pathogen multiplication was not significantly affected in H₂O₂-pretreated plants. Salicylic acid (SA), a central component of plant defense, does not seem to function in this type of H₂O₂-mediated symptom resistance, indicated by unchanged levels of free and bound SA and a lack of early up-regulation of an SA glucosyltransferase gene in TMV-infected H₂O₂-pretreated tobacco. Taken together, H₂O₂-mediated, induced resistance to necrotic symptoms in tobacco seems to depend on enhanced antioxidant capacity.

Tobacco Salicylic Acid Glucosyltransferase Is Active toward Tuberonic Acid (12-Hydroxyjasmonic Acid) and Is Induced by Mechanical Wounding Stress

Recently we reported that rice salicylic acid (SA) glucosyltransferase (OsSGT) is active toward 12-hydroxyjasmonic acid (tuberonic acid, TA) and that OsSGT gene expression is induced by wounding stress. Here we report that tobacco SA glucosyltransferase (NtSGT), which is thought to be an ortholog of OsSGT, is also active toward TA. Although NtSGT expression is known to be induced by biotrophic stress, it was also induced by wounding stress in the same manner as OsSGT. These results indicate that this glucosyltransferase is important not only in biotrophic stress but also for wounding stress. It was found that this enzyme is dually functional, with activity both toward TA and SA.

The expression patterns of AtBSMT1 and AtSAGT1 encoding a salicylic acid (SA) methyltransferase and a SA glucosyltransferase, respectively, in Arabidopsis plants with altered defense responses.

We reported previously that overexpression of a salicylic acid (SA) methyltransferase1 gene from rice (OsBSMT1) or a SA glucosyltransferase1 gene from Arabidopsis thaliana (AtSAGT1) leads to increased susceptibility to Pseudomonas syringae due to reduced SA levels. To further examine their roles in the defense responses, we assayed the transcript levels of AtBSMT1 or AtSAGT1 in plants with altered levels of SA and/or other defense components. These data showed that AtSAGT1 expression is regulated partially by SA, or non-expressor of pathogenesis related protein1, whereas AtBSMT1 expression was induced in SA-deficient mutant plants. In addition, we produced the transgenic Arabidopsis plants with RNAi-mediated inhibition of AtSAGT1 and isolated a null mutant of AtBSMT1 and then analyzed their phenotypes. A T-DNA insertion mutation in the AtBSMT1 resulted in reduced methyl salicylate (MeSA) levels upon P. syringae infection. However, accumulation of SA and glucosyl SA was similar in both the atbsmt1 and wild-type plants, indicating the presence of another SA methyltransferase or an alternative pathway for MeSA production. The AtSAGT1-RNAi line exhibited no altered phenotypes upon pathogen infection, compared to wild-type plants, suggesting that (an)other SA glucosyltransferase(s) in Arabidopsis plants may be important for the pathogenesis of P. syringae.

Purification and cDNA cloning of a wound inducible glucosyltransferase active toward 12-hydroxy jasmonic acid

Tuberonic acid (12-hydroxy epi-jasmonic acid, TA) and its glucoside (TAG) were isolated from potato leaflets ( Solanum tuberosum L.) and shown to have tuber-inducing properties. The metabolism of jasmonic acid (JA) to TAG in plant leaflets, and translocation of the resulting TAG to the distal parts, was demonstrated in a previous study. It is thought that TAG generated from JA transmits a signal from the damaged parts to the undamaged parts by this mechanism. In this report, the metabolism of TA in higher plants was demonstrated using [12- 3H]TA, and a glucosyltransferase active toward TA was purified from the rice cell cultures. The purified protein was shown to be a putative salicylic acid (SA) glucosyltransferase (OsSGT) by MALDI-TOF-MS analysis. Recombinant OsSGT obtained by overexpression in Escherichia coli was active not only toward TA but also toward SA. The OsSGT characterized in this research was not specific, but this is the first report of a glucosyltransferase active toward TA. mRNA expressional analysis of OsSGT and quantification of TA, TAG, SA and SAG after mechanical wounding indicated that OsSGT is involved in the wounding response. These results demonstrated a crucial role for TAG not only in potato tuber formation, but also in the stress response in plants and that the SA glucosyltransferase can work for TA glucosylation.

Contribution of salicylic acid glucosyltransferase, OsSGT1, to chemically induced disease resistance in rice plants

Systemic acquired resistance (SAR), a natural disease response in plants, can be induced chemically. Salicylic acid (SA) acts as a key endogenous signaling molecule that mediates SAR in dicotyledonous plants. However, the role of SA in monocotyledonous plants has yet to be elucidated. In this study, the mode of action of the agrochemical protectant chemical probenazole was assessed by microarray-based determination of gene expression. Cloning and characterization of the most highly activated probenazole-responsive gene revealed that it encodes UDP-glucose:SA glucosyltransferase (OsSGT1), which catalyzes the conversion of free SA into SA O-beta-glucoside (SAG). We found that SAG accumulated in rice leaf tissue following treatment with probenazole or 2,6-dichloroisonicotinic acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and the gene product expressed in Escherichia coli was characterized, and the results suggested that probenazole-responsive OsSGT1 is involved in the production of SAG. Furthermore, RNAi-mediated silencing of the OsSGT1 gene significantly reduced the probenazole-dependent development of resistance against blast disease, further supporting the suggestion that OsSGT1 is a key mediator of development of chemically induced disease resistance. The OsSGT1 gene may contribute to the SA signaling mechanism by inducing up-regulation of SAG in rice plants.

Metabolism of salicylic acid in wild‐type, ugt74f1 and ugt74f2 glucosyltransferase mutants of Arabidopsis thaliana

Arabidopsis thaliana contains two salicylic acid (SA) glucosyltransferase enzymes designated UGT74F1 and UGT74F2. UGT74F1 forms only SA 2-O-beta-D-glucose (SAG), while UGT74F2 forms both SAG and the SA glucose ester (SGE). In an attempt to determine the in vivo role of each SA glucosyltransferase (SAGT), the metabolism of SA in ugt74f1 and ugt74f2 mutants was examined and compared with that of the wild-type. The three major metabolites formed in wild-type Arabidopsis included SAG, SGE, and 2,5-dihydroxbenzoic acid 2-O-beta-D-glucose (DHB2G). This is the first description of DHB2G as a major metabolite of SA in plants. The major metabolites of SA formed in ugt74f1 mutants were SGE, SAG and 2,5-dihydroxybenzoic acid 5-O-beta-D-glucose (DHB5G). DHB5G was not formed in the wild-type plants. SAG and DHB2G were the main metabolites of SA in ugt74f2 mutants. The ugt74f2 mutant was unable to form SGE. Only SGE could be detected during in vitro SAGT assays of untreated wild-type and ugt74f1 mutants. This activity was because of constitutive UGT74F2 activity. Both SGE and SAG could be formed during in vitro assays of SA-pretreated wild-type and ugt74f1 leaves. Neither SAG nor SGE could be detected during the in vitro SAGT assays of untreated ugt74f2 leaves. Only SAG was formed during the in vitro SAGT assays of SA-pretreated ugt74f2 leaves. The SAG formation was a result of the UGT74F1 activity. This work demonstrates that changes in the activity of either SAGT enzyme can have a dramatic effect on the metabolism of exogenously supplied SA in Arabidopsis.

Overexpression of AtSGT1, an Arabidopsis salicylic acid glucosyltransferase, leads to increased susceptibility to Pseudomonas syringae

We reported previously that a recombinant salicylic acid (SA) glucosyltransferase1 (AtSGT1) from Arabidopsis thaliana catalyzes the formation of both SA 2- O-β- d-glucoside (SAG) and the glucose ester of SA (SGE). Here, transgenic Arabidopsis plants overexpressing AtSGT1 have been constructed, and their phenotypes analyzed. Compared to wild-type plants, transgenic plants showed an increased susceptibility to Pseudomonas syringae and reduced the accumulation levels of both free SA and its glucosylated forms (SAG and SGE). On the other hand, the overexpression increased the levels of methyl salicylate (MeSA) and methyl salicylate 2- O-β- d-glucoside (MeSAG), and also induced SA carboxyl methyltransferase1 ( AtBSMT1) expression, whose products catalyze the conversion of SA to MeSA. Our data indicate that reduced resistance by AtSGT1 overexpression results from a reduction in SA content, which is at least in part caused by increases in MeSAG and MeSA levels at the expense of SA. Our study also suggests that genetic manipulation of AtSGT1 can be utilized as an important regulatory tool for pathogen control.

Induction of a Salicylic Acid Glucosyltransferase, AtSGT1, Is an Early Disease Response in Arabidopsis thaliana

Induction of a Salicylic Acid Glucosyltransferase, AtSGT1, Is an Early Disease Response in Arabidopsis thaliana

The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures

The metabolism of salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv. KY 14) cell suspension cultures was examined by adding [7-14C]SA to the cell cultures for 24 h and identifying the metabolites through high performance liquid chromatography analysis. The three major metabolites of SA were SA 2-O-beta-D: -glucose (SAG), methylsalicylate 2-O-beta-D: -glucose (MeSAG) and methylsalicylate. Studies on the intracellular localization of the metabolites revealed that all of the SAG associated with tobacco protoplasts was localized in the vacuole. However, the majority of the MeSAG was located outside the vacuole. The tobacco cells contained an SA inducible SA glucosyltransferase (SAGT) enzyme that formed SAG. The SAGT enzyme was not associated with the vacuole and appeared to be a cytoplasmic enzyme. The vacuolar transport of SAG was characterized by measuring the uptake of [14C]SAG into tonoplast vesicles isolated from tobacco cell cultures. SAG uptake was stimulated eightfold by the addition of MgATP. The ATP-dependent uptake of SAG was inhibited by bafilomycin A1 (a specific inhibitor of the vacuolar H(+)-ATPase) and dissipation of the transtonoplast H(+)-electrochemical gradient. Vanadate was not an inhibitor of SAG uptake. Several beta-glucose conjugates were strong inhibitors of SAG uptake, whereas glutathione and glucuronide conjugates were only marginally inhibitory. The SAG uptake exhibited Michaelis-Menten type saturation kinetics with a K(m) and V(max) value of 11 microM and 205 pmol min-1 mg-1, respectively, for SAG. Based on the transport characteristics it appears as if the vacuolar uptake of SAG in tobacco cells occurs through an H(+)-antiport-type mechanism.

Formation and vacuolar localization of salicylic acid glucose conjugates in soybean cell suspension cultures

The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean (Glycine max[L.] cv Williams 82) cell suspension cultures. [7–14C]SA was added to the cell cultures, the metabolites were extracted from the cells at various time points and analysed by TLC and HPLC. The [7–14C]SA was taken up rapidly from the culture media and converted primarily to SA 2‐O‐β‐d‐glucose (SAG). Lower levels of glucosylated 2,5‐dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2‐O‐β‐d‐glucose were also formed. Examination of the intracellular localization of the glucose conjugates revealed that all of the conjugates associated with the protoplasts were found in the vacuoles. An SA glucosyltransferase (SAGT) that could catalyse the formation of SAG from SA and UDP‐glucose could be extracted from soybean cells and assayed in vitro. Increasing concentrations of SA added to the culture media induced the SAGT activity. The highest levels of SAGT activity were observed in cells treated with 0.5 mM SA. The SAGT activity in these cells was 88‐fold greater than the SAGT activity in the untreated cells. The intracellular localization of the SAGT activity was also examined and it was determined that the majority of the SAGT activity in the protoplasts was located outside the vacuole. Therefore, it appears as if SAG is formed from SA outside the vacuole, presumably in the cytoplasm, and then subsequently transported into the vacuole where it accumulates.

Purification, Cloning, and Expression of a Pathogen Inducible UDP-glucose:Salicylic Acid Glucosyltransferase from Tobacco

Salicylic acid (SA) plays an important role in plant disease resistance. Inoculation of tobacco leaves with incompatible pathogens triggers the biosynthesis of SA which accumulates primarily as the SA 2-O-beta-D-glucoside (SAG) and glucosyl salicylate (GS). The tobacco UDP-glucose:salicylic acid glucosyltransferase (SA GTase) capable of forming both SAG and GS was purified, characterized, and partially sequenced. It has an apparent molecular mass of 48 kDa, a pH optimum of 7.0, and an isoelectric point at pH 4.4. UDP-glucose was the sole sugar donor for the enzyme. However, SA and several phenolics served as glucose acceptors. The apparent K(m) values for UDP-glucose and SA were 0.27 and 1-2 mM, respectively. Zn(2+) and UDP inhibited its activity. The corresponding cDNA clone which encoded a protein of 459 amino acids was isolated from an SA-induced tobacco cDNA library and overexpressed in Escherichia coli. The recombinant protein catalyzed the formation of SAG and GS, and exhibited a broad specificity to simple phenolics, similar to that of the purified enzyme. Northern blot analysis showed that the SA GTase mRNA was induced both by SA and incompatible pathogens. The rapid induction timing of the mRNA by SA indicates that it belongs to the early SA response genes.

Biosynthesis and metabolism of salicylic acid.

Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-beta-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance.