Salicylic Acid-binding Protein Research Articles

Overexpressing CsSABP2 enhances tolerance to Huanglongbing and citrus canker in C. sinensis.

Huanglongbing (HLB) and citrus canker, arising from Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. Citri (Xcc), respectively, have been imposing tremendous losses to the global citrus industry. Systemic acquired resistance (SAR) has been shown to be crucial for priming defense against pathogen in citrus. Salicylic acid (SA) binding protein 2 (SABP2), which is responsible for converting methyl salicylate (MeSA) to SA, is essential for full SAR establishment. Here, we characterized the functions of four citrus SABP2 genes (CsSABP2-1, CsSABP2-1V18A , CsSABP2-2 and CsSABP2-3) against HLB and citrus canker. In vitro enzymatic assay revealed that all four proteins had MeSA esterase activities, and CsSABP2-1 and CsSABP2-1V18A has the strongest activity. Their activities were inhibited by SA except for CsSABP2-1V18A. Four genes controlled by a strong promoter 35S were induced into Wanjincheng orange (Citrus sinensis Osbeck) to generate transgenic plants overexpressing CsSABP2. Overexpressing CsSABP2 increased SA and MeSA content and CsSABP2-1V18A had the strongest action on SA. Resistance evaluation demonstrated that only CsSABP2-1V18A had significantly enhanced tolerance to HLB, although all four CsSABP2s had increased tolerance to citrus canker. The data suggested the amino acid Val-18 in the active site of CsSABP2 plays a key role in protein function. Our study emphasized that balancing the levels of SA and MeSA is crucial for regulating SAR and conferring broad-spectrum resistance to HLB and citrus canker. This finding offers valuable insights for enhancing resistance through SAR engineering.

Molecular basis of methyl-salicylate-mediated plant airborne defence.

Aphids transmit viruses and are destructive crop pests1. Plants that have been attacked by aphids release volatile compounds to elicit airborne defence (AD) in neighbouring plants2-5. However, the mechanism underlying AD is unclear. Here we reveal that methyl-salicylate (MeSA), salicylicacid-binding protein-2 (SABP2), the transcription factor NAC2 and salicylicacid-carboxylmethyltransferase-1 (SAMT1) form a signalling circuit to mediate AD against aphids and viruses. Airborne MeSA is perceived and converted into salicylic acid by SABP2 in neighbouring plants. Salicylic acid then causes a signal transduction cascade to activate the NAC2-SAMT1 module for MeSA biosynthesis to induce plant anti-aphid immunity and reduce virus transmission. To counteract this, some aphid-transmitted viruses encode helicase-containing proteins to suppress AD by interacting with NAC2 to subcellularly relocalize and destabilize NAC2. As a consequence, plants become less repellent to aphids, and more suitable for aphid survival, infestation and viral transmission. Our findings uncover the mechanistic basis of AD and an aphid-virus co-evolutionary mutualism, demonstrating AD as a potential bioinspired strategy to control aphids and viruses.

Overexpression of salicylic acid methyltransferase reduces salicylic acid-mediated pathogen resistance in poplar

Salicylic acid (SA) is generally considered to be a critical signal transduction factor in plant defenses against pathogens. It could be converted to methyl salicylate (MeSA) for remote signals by salicylic acid methyltransferase (SAMT) and converted back to SA by SA-binding protein 2 (SABP2). In order to verify the function of SAMT in poplar plants, we isolated the full-length cDNA sequence of PagSAMT from 84K poplar and cultivated PagSAMT overexpression lines (OE-2 isolate) to test its role in SA-mediated defenses against the virulent fungal pathogen Botryosphaeria dothidea. Our results showed that after inoculation with B. dothidea, OE-2 significantly increased MeSA content and reduced SA content which is associated with increased expression of SAMT in both infected and uninfected leaves, when compared against the wild type (WT). Additionally, SAMT overexpression plant lines (OE-2) exhibited higher expression of pathogenesis-related genes PR-1 and PR-5, but were still susceptible to B. dothidea suggesting that in poplar SA might be responsible for resistance against this pathogen. This study expands the current understanding of joint regulation of SAMT and SABP2 and the balance between SA and MeSA in poplar responses to pathogen invasion.

Overexpression of the salicylic acid binding protein 2 (SABP2) from tobacco enhances tolerance against Huanglongbing in transgenic citrus.

Overexpression of the salicylic acid binding protein 2 (SABP2) gene from Tobacco results in enhanced tolerance to Huanglongbing (HLB; citrus greening disease) in transgenic sweet oranges. Huanglongbing (HLB), the most destructive citrus disease, is caused by Candidatus Liberibacter asiaticus (CaLas). Currently, no cure for this disease exists, and all commercially planted cultivars are highly susceptible. Salicylic Acid Binding Protein 2 (SABP2) is a well-characterized protein essential for establishing systemic acquired resistance (SAR) in tobacco.The constitutive over expression of SABP2 from tobacco (NtSABP2) in 'Hamlin' sweet orangeresulted in the production of several transgenic lines with variable transcript levels. Transient expression ofthe NtSABP2-EGFP fusion protein in Nicotiana benthamiana plants demonstrated thatNtSABP2was cytosolicin its subcellular localization. In a long-term fieldstudy, we identified a SABP2 transgenic linewithsignificantly reduced HLB symptoms that maintained aconsistently low CaLas titer. Transcriptome analysis of this selectedtransgenic line demonstrated upregulation of several genes related to plant defense and SAR pathways. Genes, such as NPR family genes and those coding for monooxygenases and lipoxygenases, were upregulated in the35S-NtSABP2 overexpressing line and might be candidates for incorporation into our citrus improvement program.

Comparative Transcriptome Analysis Reveals Differential Gene Expression in Resistant and Susceptible Watermelon Varieties in Response to Meloidogyne incognita.

M. incognita is a major parasitic plant disease in watermelon production, causing serious economic losses. Although there are many studies on root-knot nematode, the resistance mechanism is still unclear. In this study, in order to fully understand the mechanism of watermelon resistance to root-knot nematode, the relatively strongly resistant ‘Hongzi watermelon’ variety and the susceptible ‘M16’ watermelon variety were used as materials, combined with RNA sequencing (RNA-seq), to analyze the expression abundance of resistant and susceptible varieties at 0, 2, 8 and 15 days post-infection (DPI) by M. incognita. The number of differentially expressed genes (DEGs) in the four comparison groups (A0_B0, A1_B1, A2_B2 and A3_B3) was 3645, 2306, 4449 and 2362, respectively, and there were 835 shared DEGs among them. GO annotation and KEGG pathway enrichment analysis showed that 835 DEGs were mainly involved in phenylpropane biosynthesis and carbon metabolism. Furthermore, lignin-biosynthesis-related genes (4CL (4-coumaric acid-CoA ligase), C3H (coumaric acid 3-hydroxylase), CSE (caffeoyl shikimate esterase), COMT (caffeic acid-O-methyltransferase), CCR (cinnamyl CoA reductase) and PRX (peroxidase)), defense-related proteins (UDP-glucoronosyl/UDP-glucosyl transferase, UGT84A13; salicylic acid binding protein, SABP2) and some transcription factors (TFs) were highlighted, which may be potential candidate genes for further analysis in the infection process of M. incognita. These results suggest that watermelon can achieve resistance to M. incognita by increasing the content of lignin and phenols in root or improving ROS level. These RNA-seq data provide new knowledge for future functional studies and will be helpful to further elucidate the molecular mechanism of resistance to M. incognita in watermelon.

Ellman's method-based determination of acibenzolar-S-methyl in tobacco by transesterification with methanol and SABP2-catalyzed hydrolysis.

Acibenzolar-S-methyl (ASM) is the most commercially successful biological antibacterial agent used for crop through systemic acquired resistance (SAR). In this study, a reproducible and accurate procedure, based on the spectrophotometric/microplate reader analysis, has been developed to detect ASM in tobacco leaves. This method involves oxidation of methyl mercaptan by the Ellman's reagent 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB2-), measurable at 412nm. Methyl mercaptan can be produced by either the ASM transesterification with methanol or the SA-binding protein 2 (SABP2)-catalyzed ASM hydrolysis. The proportions of methanol, reaction time, temperature, the concentrations of EDTA and DTNB were optimized in a 96-well plate. The calibration curve of ASM was linear over the range of 25.2-315μgg-1. The results of the intra- and inter-day accuracy and precision data were within the FDA acceptance criteria. With ASM as substrate, the turnover number of SABP2 was determined, with the kcat value of 31.1min-1 using the Michaelis-Menten equation. In tobacco plants treated with 100μM ASM, it was decreased as time elapsed in treated tobacco, reaching negligible values 72h after treatment. The optimized method was applied for the determination of ASM transesterification with methanol and the kinetic data determination of SABP2-catalyzed ASM hydrolysis.

Identification of yield-related genes through genome-wide association: case study of weeping forsythia, an emerging medicinal crop.

Key messageThis study identified candidate genes related to fruit yield for an emerging medicinal crop, weeping forsythia.BackgroundThe genetic basis of crop yield is an agricultural research hotspot. Identifying the genes related to yield traits is the key to increase the yield. Weeping forsythia is an emerging medicinal crop that currently lacks excellent varieties. The genes related to fruit yield in weeping forsythia have not been identified.ObjectiveThus, we aimed to screen the candidate genes related to fruit yield of weeping forsythia by using genome-wide association analysis.MethodsHere, 60 samples from the same field and source of weeping forsythia were collected to identify its yield-related candidate genes. Association analysis was performed on the variant loci and the traits related to yield, i.e., fruit length, width, thickness, and weight.ResultsResults from admixture, neighbor-joining, and kinship matrix analyses supported the non-significant genetic differentiation of these samples. Significant association was found between 2 variant loci and fruit length, 8 loci and fruit width, 24 loci and fruit thickness, and 13 loci and fruit weight. Further search on the 20 kb up/downstream of these variant loci revealed 1 gene related to fruit length, 16 genes related to fruit width, 12 genes related to fruit thickness, and 13 genes related to fruit weight. Among which, 4 genes, namely, WRKY transcription factor 35, salicylic acid-binding protein, auxin response factor 6, and alpha-mannosidase were highly related to the fruit development of weeping forsythia.ConclusionThis study identify four candidate genes related to fruit development, which will provide useful information for the subsequent molecular-assisted and genetic breeding of weeping forsythia.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13258-021-01186-w.

Benzoic Acid and Its Hydroxylated Derivatives Suppress Early Blight of Tomato (Alternaria solani) via the Induction of Salicylic Acid Biosynthesis and Enzymatic and Nonenzymatic Antioxidant Defense Machinery.

Tomato early blight, caused by Alternaria solani, is a destructive foliar fungal disease. Herein, the potential defensive roles of benzoic acid (BA) and two of its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA), and protocatechuic acid (PCA) against A. solani were investigated. All tested compounds showed strong dose-dependent fungistatic activity against A. solani and significantly reduced the disease development. Benzoic acid, and its hydroxylated derivatives, enhanced vegetative growth and yield traits. Moreover, BA and its derivatives induce the activation of enzymatic (POX, PPO, CAT, SlAPXs, and SlSODs) and non-enzymatic (phenolics, flavonoids, and carotenoids) antioxidant defense machinery to maintain reactive oxygen species (ROS) homeostasis within infected leaves. Additionally, BA and its hydroxylated derivatives induce the accumulation of salicylic acid (SA) and its biosynthetic genes including isochorismate synthase (SlICS), aldehyde oxidases (SlAO1 and SlAO2), and phenylalanine ammonia-lyases (SlPAL1, SlPAL2, SlPAL3, SlPAL5, and SlPAL6). Higher SA levels were associated with upregulation of pathogenesis-related proteins (SlPR-1, SlPR1a2, SlPRB1-2, SlPR4, SlPR5, SlPR6), nonexpressor of pathogenesis-related protein 1 (SlNPR1), and salicylic acid-binding protein (SlSABP2). These findings outline the potential application of BA and its hydroxylated derivatives as a sustainable alternative control strategy for early blight disease and also deciphering the physiological and biochemical mechanisms behind their protective role.

Regulations of m6A methylation on tomato fruit chilling injury

Tomato fruit are sensitive to chilling injury (CI) during cold storage. Several factors have been discovered to be involved in chilling injury of tomato fruit. Plant hormones play an important regulatory role, however, the relationship between chilling injury and N6-methyladenosine (m6A) methylation of transcripts in plant hormone pathways has not been reported yet. In order to clarify the complex regulatory mechanism of m6A methylation on chilling injury in tomato fruit, Nanopore direct RNA sequencing was employed. A large number of enzymes and transcription factors were found to be involved in the regulation process of fruit chilling injury, which were associated with plant hormone, such as 1-aminocyclopropane 1-carboxylate synthase (ACS), aspartate aminotransferase (AST), auxin response factor (ARF2), ethylene response factor 2 (ERF2), gibberellin 20-oxidase-3 (GA20ox) and jasmonic acid (JA). By conjoint analysis of the differential expression transcripts related to chilling injury and m6A methylation differential expression transcripts 41 differential expression transcripts were identified involved in chilling injury including 1-aminocyclopropane-1-carboxylate oxidase (ACO) and pectinesterase (PE) were down-regulated and heat shock cognate 70 kD protein 2 (cpHSC70), HSP70-binding protein (HspBP) and salicylic acid-binding protein 2 (SABP2) were up-regulated. Our results will provide a deeper understanding for chilling injury regulatory mechanism and post-harvest cold storage of tomato fruit.

Mechanism of growth amelioration of triclosan-stressed tobacco (Nicotiana tabacum) by endogenous salicylic acid

Among emerging organic contaminants (EOCs), triclosan (TCS) is an antibacterial agent and frequently detected in sludge. In this study, RNA sequencing (RNA-seq) was used to obtain the first transcriptomic profile of tobacco with TCS treatment in comparison with control. The results of transcriptome profiling indicated that salicylic acid (SA) signalling pathway actively participated in the tobacco’s response to TCS treatment. The accumulation of endogenous SA in transgene tobacco lines transformed with a homologous gene of SA binding protein (LcSABP) was significantly enhanced. The resistance of transgenic tobacco lines to TCS was markedly enhanced revealed by morphological and physiological indexes while the total Chl level and Pn of transgenic individuals showed about 180% and 250% higher than that of WT on average, and the accumulation of H2O2 and O2− induced by TCS in SABP overexpressing tobacco was 35.3%–37.3% and 53.0%–56.0% lower than that of WT. In order to further explore the mechanism of TCS tolerance in transgenic plants, RNA-seq was then performed to obtain the second transcriptomic profile between wild type and transgenic samples with TCS exposure. The results indicated that differentially expressed genes (DEGs) were most highly enriched in MAPK signalling pathway, amino acid synthesis pathway and plant hormone transduction pathway. Especially, genes encoding key proteins such as cytochrome P450, laccase, peroxidase, glycosyl transferase, glutathione S-transferase and ATP-binding cassette were considered to be related to the increased tolerance ability of transgenic tobacco to the treatment of TCS stress. This research will likely provide novel insights into the molecular mechanism of SA-mediated amelioration of TCS stress on tobacco.

Effects of Combined Application of Potassium Silicate and Salicylic Acid on the Defense Response of Hydroponically Grown Tomato Plants to Ralstonia solanacearum Infection

Bacterial wilt, caused by soilborne pathogenic bacterium Ralstonia solanacearum, is a serious and widespread disease that affects global tomato production. Both silicon (Si) and salicylic acid (SA) play important roles in enhancing tomato resistance against bacterial wilt, however, their combined effects on the defense responses of infected tomato plants remain unknown. Hence, the combined effects of Si and SA on physiological and biochemical parameters of R. solanacearum-infected tomato plants were investigated. The combination treatment of Si and SA significantly decreased disease incidences, lipoxygenase (LOX) activity and ethylene (ET) production. The combined treatments were more prominent in improving the morphological traits of root systems, such as root length, root surface area, average root diameter and root volume. The activities of polyphenol oxidase (PPO) and peroxidase (POD) and the concentrations of total soluble phenolics (TSPs) and lignin-thioglycolic acid (LTGA) derivatives were significantly increased in the plants with combined treatments. Si in combination with SA could significantly enhance neutral invertase (NI) and acid invertases (AI) activities in the leaves of tomato plants at 3 days post-infection (dpi) compared with application of Si alone. Three defense-related genes, PAL, POD and pathogenesis-related protein 1 (PR1), were significantly induced in Si+SA treatment at 7 dpi when compared with individual application of Si or SA. The expression level of salicylic acid-binding protein 2 (SABP2) was significantly higher for combination treatment when compared with treatment of Si or SA alone. The possible mechanisms involved in the synergistic effects of Si and SA on the control of tomato bacterial wilt were proposed. This study indicates that under hypertonic conditions, the combined application of 2.0 mM potassium silicate (K2SiO3) and 0.5 mM SA had a synergistic effect on the control of tomato bacterial wilt.

Identification and analysis of key genes involved in methyl salicylate biosynthesis in different birch species.

Species of the perennial woody plant genus Betula dominate subalpine forests and play a significant role in preserving biological diversity. In addition to their conventional benefits, birches synthesize a wide range of secondary metabolites having pharmacological significance. Methyl salicylate (MeSA) is one of these naturally occurring compounds constitutively produced by different birch species. MeSA is therapeutically important in human medicine for muscle injuries and joint pain. However, MeSA is now mainly produced synthetically due to a lack of information relating to MeSA biosynthesis and regulation. In this study, we performed a comprehensive bioinformatics analysis of two candidate genes mediating MeSA biosynthesis, SALICYLIC ACID METHYLTRANSFERASE (SAMT) and SALICYLIC ACID-BINDING PROTEIN 2 (SABP2), of high (B. lenta, B. alleghaniensis, B. medwediewii, and B. grossa) and low (B. pendula, B. utilis, B. alnoides, and B. nana) MeSA-producing birch species. Phylogenetic analyses of SAMT and SABP2 genes and homologous genes from other plant species confirmed their evolutionary relationships. Multiple sequence alignments of the amino acid revealed the occurrence of important residues for substrate specificity in SAMT and SABP2. The analysis of cis elements in different birches indicated a functional multiplicity of SAMT and SABP2 and provided insights into the regulation of both genes. We successfully developed six prominent single nucleotide substitution markers that were validated with 38 additional birch individuals to differentiate high and low MeSA-producing birch species. Relative tissue-specific expression analysis of SAMT in leaf and bark tissue of two high and two low MeSA-synthesizing birches revealed a high expression in the bark of both high MeSA-synthesizing birches. In contrast, SABP2 expression in tissues revealed indifferent levels of expression between species belonging to the two groups. The comparative expression and bioinformatics analyses provided vital information that could be used to apply plant genetic engineering technology in the mass production of organic MeSA.

One- and Two-Proton Transfer Mechanisms Coexist in One Active Site.

Acibenzolar-S-methyl (ASM) is one of the most successfully commercialized plant activators of the systemic acquired resistance (SAR). However, its activation (hydrolysis) mechanism catalyzed by the salicylic acid binding protein 2 (SABP2) remains elusive. The fundamental catalytic mechanism of the SABP2-catalyzed hydrolysis of the ASM had been investigated by extensive computational and experimental studies, including QM/MM simulations, charge transfer analysis, small-molecule synthesis, and biochemical assays. Here we report that the promiscuous SABP2 shows different catalytic mechanisms toward different substrates. To catalyze the ASM hydrolysis, the SABP2 uses a two-proton transfer mechanism, and the key intermediate is stabilized by the charge transfer effect; to catalyze the ethyl 1,2,3-benzothiadiazole-7-carboxylate (BTM, an ASM analogue) hydrolysis, the SABP2 applies the one-proton transfer mechanism, and the classic tetrahedral intermediate is stabilized by the electrostatic effect. The HPLC analyses of the SABP2 esterase activities toward the ASM and the BTM show comparable results with our computaional results, suggesting that the obtained computational mechanism insights are reasonable. The obtained mechanism is not only an important supplement to the theory of enzymes' catalytic promiscuity, but it also contributes a possible strategy for the design of next generation plant SAR activators.

Species determination and phylogenetic relationships of the genus Betula inferred from multiple chloroplast and nuclear regions reveal the high methyl salicylate-producing ability of the ancestor

Key messageThe investigation provides initial knowledge on the distribution and evolution of the high and low methyl salicylate-producing trait in the Betula genus. Mislabelled birch species could be identified and removed.The genus Betula is the largest group of ecologically and economically dominant perennial woody plants in subalpine forests. The taxonomy of Betula is complex due to an extensive history of hybridization and periodic introgression events among the species. Although almost all land plants including birches produce methyl salicylate (MeSA) as a signaling molecule and in response to stress (“low MeSA producer”), some birch species produce high amounts of MeSA in the leaves and bark (“high MeSA producer”). Unfortunately, the evolution of high levels of MeSA production in the genus Betula remains unclear. The salicylic acid-binding protein 2 (SABP2) and salicylic acid methyltransferase (SAMT) genes involved in MeSA biosynthesis were incorporated into this study to examine the interspecific relationship of high and low MeSA-producing birches. Additionally, eight chloroplast and three nuclear regions were included to evaluate their potential application in species determination. The analysis resulted in 25 and 61 nucleotide variations, respectively, which allowed for a visualization of the genetic architecture in the 18 Betula species investigated. The high MeSA-producing B. lenta, B. grossa, and B. alleghaniensis formed the basal clade in the phylogenetic analysis, thus revealing their ancestral status, and the network analysis postulates that the diploid B. lenta is one of the ancestors of the genus Betula. The results indicate that the ability to produce high levels of MeSA that were initially present in the genus has been lost several times during its evolution. Placing species of the subgenus Acuminata alongside the subgenus Betula, together with a fragrance analysis, questions their ability to produce high levels of MeSA.

Tobacco SABP2-interacting protein SIP428 is a SIR2 type deacetylase

Salicylic acid is widely studied for its role in biotic stress signaling in plants. Several SA-binding proteins, including SABP2 (salicylic acid-binding protein 2) has been identified and characterized for their role in plant disease resistance. SABP2 is a 29 kDA tobacco protein that binds to salicylic acid with high affinity. It is a methylesterase enzyme that catalyzes the conversion of methyl salicylate into salicylic acid required for inducing a robust systemic acquired resistance (SAR) in plants. Methyl salicylic acid is one of the several mobile SAR signals identified in plants. SABP2-interacting protein 428 (SIP428) was identified in a yeast two-hybrid screen using tobacco SABP2 as a bait. In silico analysis shows that SIP428 possesses the SIR2 (silent information regulatory 2)-like conserved motifs. SIR2 enzymes are orthologs of sirtuin proteins that catalyze the NAD+-dependent deacetylation of Nε lysine-acetylated proteins. The recombinant SIP428 expressed in E. coli exhibits SIR2-like deacetylase activity. SIP428 shows homology to Arabidopsis AtSRT2 (67% identity), which is implicated in SA-mediated basal defenses. Immunoblot analysis using anti-acetylated lysine antibodies showed that the recombinant SIP428 is lysine acetylated. The expression of SIP428 transcripts was moderately downregulated upon infection by TMV. In the presence of SIP428, the esterase activity of SABP2 increased modestly. The interaction of SIP428 with SABP2, it's regulation upon pathogen infection, and similarity with AtSRT2 suggests that SIP428 is likely to play a role in stress signaling in plants.

Exogenous application and endogenous elevation of salicylic acid levels by overexpressing a salicylic acid-binding protein 2 gene enhance nZnO tolerance of tobacco plants

Salicylic acid (SA) plays vital roles in the stress response of plants. However, the relationship between SA signaling and plant tolerance to nZnO stress has not been reported. The effects of different sizes and concentrations of nZnO on tobacco plants grown in MS medium or soil were evaluated. The effects of exogenous and endogenous SA on the morphological and biochemical parameters of tobacco plants by exogenous application of SA and overexpression of LcSABP2 gene were also investigated in this study. The endogenous SA content and the expression of salicylic acid-binding protein 2 (SABP2) gene increased in tobacco individuals with nZnO treatment. Exogenously applied SA increased the germination rate of tobacco seeds, and enhanced the root length and chlorophyll content of tobacco seedlings. Overexpression of LcSABP2 gene in transgenic tobacco significantly increased SA content in plants with nZnO treatment. Compared to the wild type plants, transgenic tobacco with nZnO treatment showed higher chlorophyll content, net photosynthetic rate, related gene expression, activities of SOD, POD and CAT, and less contents of H2O2, O2− and MDA, which suggested that LcSABP2 might play important regulatory roles in plant resistance to nZnO toxicity by elevating the level of endogenous SA, promoting the action of photosynthetic system, and enhancing the activities of antioxidant enzymes. This study illustrated that exogenous and endogenous SA might all be involved in alleviating the toxicity of nZnO on plants.

Extracellular alpha/beta-hydrolase from Paenibacillus species shares structural and functional homology to tobacco salicylic acid binding protein 2

An alpha/ beta hydrolase annotated as a putative salicylate esterase within the genome of a species of Paenibacillus previously identified from differential and selective growth on Kraft lignin was structurally and functionally characterised. Feruloyl esterases are key to the degradation of lignin in several bacterial species and although this activity was investigated, no such activity was observed. The crystal structure of the Paenibacillus esterase, here denoted as PnbE, was determined at 1.32 Å resolution, showing high similarity to Nicotiana tabacum salicylic acid binding protein 2 from the protein database. Structural similarities between these two structures across the core domains and key catalytic residues were observed, with superposition of catalytic residues giving an RMSD of 0.5 Å across equivalent Cα atoms. Conversely, the cap domains of PnbE and Nicotiana tabacum SABP2 showed greater divergence with decreased flexibility in the PnbE cap structure. Activity of PnbE as a putative methyl salicylate esterase was supported with binding studies showing affinity for salicylic acid and functional studies showing methyl salicylate esterase activity. We hypothesise that this activity could enrich Paenibacillus sp. within the rhizosphere by increasing salicylic acid concentrations within the soil.

Enhancement of endogenous SA accumulation improves poor-nutrition stress tolerance in transgenic tobacco plants overexpressing a SA-binding protein gene

Salicylic acid (SA) plays an important role in the response of plants to abiotic stresses. Starvation stress affects plant cell metabolic activities, which further limits the normal growth and development of plants. It was reported that SA might play a regulatory role in the process of plant against starvation stress, but the mechanism involved in this process is still unclear. Thus, in this study, the transgenic plants overexpressing a SA binding protein 2 (SABP2) gene were exposed to starvation stress and the transgenic lines showed starvation-tolerant phenotype. Compared with wild-type (WT) plants, transgenic plants showed better growth status under poor-nutrition stress. Transgenic plants also showed more vigorous roots than WT plants. Physiological tests indicated that the transgenic plants showed higher relative water content (RWC), chlorophyll content, photosynthetic capacity, endogenous SA content, and lower ROS level compared to WT plants. Transcriptome analysis of tobacco plants identified 3, 748 differentially expressed genes (DEGs) between transgenic and WT plants under starvation stress. These DEGs are mainly involved in glycolysis/gluconeogenesis pathway group, MAPK signaling pathway group and plant hormone signal transduction pathway group. As determined by qPCR, up-regulated expression of fifteen genes such as abscisic acid receptor PYR1-like gene (NtPYR1-like), bidirectional sugar transporter N3-like gene (NtSWEETN3-like) and superoxide dismutase [Fe] chloroplastic-like gene (NtFeSOD-like), etc., was observed in transgenic plants under poor-nutrition stress which was in accordance with RNA-sequencing results. The modified pathways involved in plant hormone signaling are thought to be at least one of the main causes of the increased starvation tolerance of transgenic tobacco plants with altered SA homeostasis.

LcSABP2, a salicylic acid binding protein 2 gene from Lycium chinense, confers resistance to triclosan stress in Nicotiana tabacum

The triclosan (TCS) is one of the most commonly detected organic pollutants in the sewage sludge. TCS could induce phytotoxicity in plants. Salicylic acid (SA) is a phenolic compound capable of enhancing plant growth and development. It is well documented that abiotic stress tolerance could be enhanced by exogenous application of SA. However, the regulatory mechanisms for functions of endogenous SA in plants' responses to xenobiotics stress remains unclear. Our results indicated that TCS suppressed plant growth by restricting photosynthesis, decreasing chlorophyll contents and inducing over production of reactive oxygen species (ROS). Interestingly, SA or glutathione (GSH) application could significantly improve plant tolerance to TCS. Moreover, endogenous SA and the expression of a SA binding protein 2 (SABP2) gene were found to be elevated in tobacco under TCS treatment. The overexpression of LcSABP, a SABP2-like gene cloned from the leaves of Lycium chinense, markedly enhanced the SA content in the transgenic plants under TCS stress. The LcSABP-overexpressing plants presented higher photosynthesis rate, chlorophyll content, glutathione reductase (GR) and glutathione-S-transferase (GST) enzymes activities, GSH content and lower O2−•, H2O2 and malondialdehyde (MDA) content in comparison with WT tobacco with TCS treatment. One of the GSH synthesis-related gene, NtGSHS, also showed higher expression level in the transgenic tobacco in comparison with control plants with TCS stress treatment. These results indicated that SABP2 played a positive regulatory role in plant response to TCS stress via increasing the endogenous SA levels. The increased SA content might then increase the GSH content, probably through an increase in GR activity and GSHS gene expression, thus inducing the antioxidant and xenobiotics detoxification systems, which promoted TCS stress tolerance in tobacco plants.

Overexpression of LcSABP, an Orthologous Gene for Salicylic Acid Binding Protein 2, Enhances Drought Stress Tolerance in Transgenic Tobacco.

Salicylic acid (SA) plays an essential role in the growth and development of plants, and in their response to abiotic stress. Previous studies have mostly focused on the effects of exogenously applied SA on the physiological response of plants to abiotic stresses; however, the underlying genetic mechanisms for the regulatory functions of endogenous SA in the defense response of plants remain unclear. In plants, SA binding protein 2 (SABP2), possessing methyl salicylate (MeSA) esterase activity, catalyzes the conversion of MeSA to SA. Herein, a SABP2-like gene, LcSABP, was cloned from Lycium chinense, which contained a complete open reading frame of 795 bp and encoded a protein of 264 amino acids that shared high sequence similarities with SABP2 orthologs from other plants. Overexpression of LcSABP enhanced the drought tolerance of transgenic tobacco plants. The results indicated that increased levels of LcSABP transcripts and endogenous SA content were involved in the enhanced drought tolerance. Physiological and biochemical studies further demonstrated that higher chlorophyll content, increased photosynthetic capacity, lower malondialdehyde content, and higher activities of superoxide dismutase, peroxidase, and catalase enhanced the drought tolerance of transgenic plants. Moreover, overexpression of LcSABP also increased the expression of reactive oxygen species (ROS)- and stress-responsive genes under drought stress. Overall, our results demonstrate that LcSABP plays a positive regulatory role in drought stress response by enhancing the endogenous SA content, promoting the scavenging of ROS, and regulating of the expression of stress-related transcription factor genes. Our findings indicate that LcSABP functions as a major regulator of the plant’s response to drought stress through a SA-dependent defense pathway.