The combined use of photoaffinity labeling and surface plasmon resonance-based technology identifies multiple salicylic acid-binding proteins.

Salicylic acid (SA) is a small phenolic molecule that not only is the active ingredient in the multi-functional drug aspirin, but also serves as a plant hormone that affects diverse processes during growth, development, responses to abiotic stresses and disease resistance. Although a number of SA-binding proteins (SABPs) have been identified, the underlying mechanisms of action of SA remain largely unknown. Efforts to identify additional SA targets, and thereby elucidate the complex SA signaling network in plants, have been hindered by the lack of effective approaches. Here, we report two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SABPs from Arabidopsis. Using these approaches, multiple proteins, including the E2 subunit of α-ketoglutarate dehydrogenase and the glutathione S-transferases GSTF2, GSTF8, GSTF10 and GSTF11, were identified as SABPs. Their association with SA was further substantiated by the ability of SA to inhibit their enzymatic activity. The photoaffinity labeling and surface plasmon resonance-based approaches appear to be more sensitive than the traditional approach for identifying plant SABPs using size-exclusion chromatography with radiolabeled SA, as these proteins exhibited little to no SA-binding activity in such an assay. The development of these approaches therefore complements conventional techniques and helps dissect the SA signaling network in plants, and may also help elucidate the mechanisms through which SA acts as a multi-functional drug in mammalian systems.

The extent to which methyl salicylate is required for signaling systemic acquired resistance is dependent on exposure to light after infection.

Systemic acquired resistance (SAR) is a state of heightened defense to a broad spectrum of pathogens that is activated throughout a plant following local infection. Development of SAR requires the translocation of one or more mobile signals from the site of infection through the vascular system to distal (systemic) tissues. The first such signal identified was methyl salicylate (MeSA) in tobacco (Nicotiana tabacum). Subsequent studies demonstrated that MeSA also serves as a SAR signal in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum). By contrast, another study suggested that MeSA is not required for SAR in Arabidopsis and raised questions regarding its signaling role in tobacco. Differences in experimental design, including the developmental age of the plants, the light intensity, and/or the strain of bacterial pathogen, were proposed to explain these conflicting results. Here, we demonstrate that the length of light exposure that plants receive after the primary infection determines the extent to which MeSA is required for SAR signaling. When the primary infection occurred late in the day and as a result infected plants received very little light exposure before entering the night/dark period, MeSA and its metabolizing enzymes were essential for SAR development. In contrast, when infection was done in the morning followed by 3.5 h or more of exposure to light, SAR developed in the absence of MeSA. However, MeSA was generally required for optimal SAR development. In addition to resolving the conflicting results concerning MeSA and SAR, this study underscores the importance of environmental factors on the plant's response to infection.

Altering expression of benzoic acid/salicylic acid carboxyl methyltransferase 1 compromises systemic acquired resistance and PAMP-triggered immunity in arabidopsis.

Methyl salicylate (MeSA), which is synthesized in plants from salicylic acid (SA) by methyltransferases, has roles in defense against microbial and insect pests. Most of the MeSA that accumulates after pathogen attack is synthesized by benzoic acid/SA carboxyl methyltransferase 1 (AtBSMT1). To investigate the role of AtBSMT1 in plant defense, transgenic Arabidopsis with altered AtBSMT1 function or expression were assessed for their ability to resist pathogen infection. A knockout mutant (Atbsmt1) failed to accumulate MeSA following pathogen infection; these plants also failed to accumulate SA or its glucoside in the uninoculated leaves and did not develop systemic acquired resistance (SAR). However, the Atbsmt1 mutant exhibited normal levels of effector-triggered immunity and pathogen-associated molecular pattern (PAMP)-triggered immunity to Pseudomonas syringae and Hyaloperonospora arabidopsidis. Analyses of transgenic Arabidopsis plants overexpressing AtBSMT1 revealed that they accumulate elevated levels of MeSA in pathogen-infected leaves but fail to develop SAR. Since the levels of SA and its glucoside were reduced in uninoculated systemic leaves of these plants whereas MeSA levels were elevated, AtBSMT1-mediated conversion of SA to MeSA probably compromised SAR development by suppressing SA accumulation in uninoculated leaves. PAMP-triggered immunity also was compromised in the AtBSMT1 overexpressing plants, although effector-triggered immunity was not.

microRNA, seeds, and Darwin?: diverse function of miRNA in seed biology and plant responses to stress.

microRNAs (miRNAs) are small, single-stranded RNAs that down-regulate target genes at the post-transcriptional level. miRNAs regulate target genes by guiding mRNA cleavage or by repressing translation. miRNAs play crucial roles in a broad range of developmental processes in plants. Multiple miRNAs are present in germinating seeds and seedlings of Arabidopsis, some of which are involved in the regulation of germination and seedling growth by plant hormones such as abscisic acid (ABA) and auxin. The involvement of miRNAs in ABA responses is not limited to the early stages of plant development but seems to be important for general stress responses throughout the plant life cycle. This Darwin review summarizes recent progress in miRNA research focusing on seed and stress biology, two topics which were of interest to Charles Darwin.

Arabidopsis SMALL DEFENSE-ASSOCIATED PROTEIN 1 Modulates Pathogen Defense and Tolerance to Oxidative Stress.

Salicylic acid (SA) and reactive oxygen species (ROS) are known to be key modulators of plant defense. However, mechanisms of molecular signal perception and appropriate physiological responses to SA and ROS during biotic or abiotic stress are poorly understood. Here we report characterization of SMALL DEFENSE-ASSOCIATED PROTEIN 1 (SDA1), which modulates defense against bacterial pathogens and tolerance to oxidative stress. sda1 mutants are compromised in defense gene expression, SA accumulation, and defense against bacterial pathogens. External application of SA rescues compromised defense in sda1 mutants. sda1 mutants are also compromised in tolerance to ROS-generating chemicals. Overexpression of SDA1 leads to enhanced resistance against bacterial pathogens and tolerance to oxidative stress. These results suggest that SDA1 regulates plant immunity via the SA-mediated defense pathway and tolerance to oxidative stress. SDA1 encodes a novel small plant-specific protein containing a highly conserved seven amino acid (S/G)WA(D/E)QWD domain at the N-terminus that is critical for SDA1 function in pathogen defense and tolerance to oxidative stress. Taken together, our studies suggest that SDA1 plays a critical role in modulating both biotic and abiotic stresses in Arabidopsis (Arabidopsis thaliana) and appears to be a plant-specific stress responsive protein.

Identification of likely orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana.

Salicylic acid-binding protein 2 (SABP2) is essential for the establishment of systemic acquired resistance (SAR) in tobacco; SABP2's methyl salicylate (MeSA) esterase activity is required in healthy systemic tissues of infected plants to release the active defense phytohormone SA from MeSA, which serves as a long-distance signal for SAR. In the current study, we characterize a new gene family from Arabidopsis thaliana encoding 18 potentially active alpha/beta fold hydrolases that share 32-57% identity with SABP2. Of 14 recombinant AtMES (MES for methyl esterase) proteins tested, five showed preference for MeSA as a substrate and displayed SA inhibition of MeSA esterase activity in vitro (AtMES1, -2, -4, -7, and -9). The two genes encoding MeSA esterases with the greatest activity, AtMES1 and -9, as well as AtMES7 were transcriptionally upregulated during infection of Arabidopsis with avirulent Pseudomonas syringae. In addition, conditional expression of AtMES1, -7, or -9 complemented SAR deficiency in SABP2-silenced tobacco, suggesting that these three members of the AtMES family are SABP2 functional homologs (orthologs). Underexpression by knockout mutation and/or RNAi-mediated silencing of multiple AtMES genes, including AtMES1, -2, -7, and -9, compromised SAR in Arabidopsis and correlated with enhanced accumulation of MeSA in the systemic tissue of SAR-induced plants. Together, the data show that several members of the AtMES gene family are functionally homologous to SABP2 and redundant for MeSA hydrolysis and probably SAR. These data suggest that MeSA is a conserved SAR signal in Arabidopsis and tobacco.

Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation.

Phytopathogens can manipulate plant hormone signaling to access nutrients and counteract defense responses. Pseudomonas syringae produces coronatine, a toxin that mimics the plant hormone jasmonic acid isoleucine and promotes opening of stomata for bacterial entry, bacterial growth in the apoplast, systemic susceptibility, and disease symptoms. We examined the mechanisms underlying coronatine-mediated virulence and show that coronatine activates three homologous NAC transcription factor (TF) genes, ANAC019, ANAC055, and ANAC072, through direct activity of the TF, MYC2. Genetic characterization of NAC TF mutants demonstrates that these TFs mediate coronatine-induced stomatal reopening and bacterial propagation in both local and systemic tissues by inhibiting the accumulation of the key plant immune signal salicylic acid (SA). These NAC TFs exert this inhibitory effect by repressing ICS1 and activating BSMT1, genes involved in SA biosynthesis and metabolism, respectively. Thus, a signaling cascade by which coronatine confers its multiple virulence activities has been elucidated.

CRT1 is a nuclear-translocated MORC endonuclease that participates in multiple levels of plant immunity.

Arabidopsis thaliana CRT1 (compromised for recognition of Turnip Crinkle Virus) was previously shown to be required for effector-triggered immunity. Sequence analyses previously revealed that CRT1 contains the ATPase and S5 domains characteristic of Microchidia (MORC) proteins; these proteins are associated with DNA modification and repair. Here we show that CRT1 and its closest homologue, CRH1, are also required for pathogen-associated molecular pattern (PAMP)-triggered immunity, basal resistance, non-host resistance and systemic acquired resistance. Consistent with its role in PAMP-triggered immunity, CRT1 interacted with the PAMP recognition receptor FLS2. Subcellular fractionation and transmission electron microscopy detected a subpopulation of CRT1 in the nucleus, whose levels increased following PAMP treatment or infection with an avirulent pathogen. These results, combined with the demonstration that CRT1 binds DNA, exhibits endonuclease activity, and affects tolerance to the DNA-damaging agent mitomycin C, argue that this prototypic eukaryotic member of the MORC superfamily has important nuclear functions during immune response activation.

Systemic acquired resistance is induced by R gene-mediated responses independent of cell death.

On infection by pathogens, plants initiate defence responses that are able to curtail infection locally. These responses are mediated either by receptor-like proteins that recognize pathogen-associated molecular patterns or by the protein products of disease resistance (R) genes. At the same time, primary defence responses often result in the generation of signals that induce what is known as systemic acquired resistance (SAR), such that defence responses are enhanced on secondary pathogen challenge in distal tissues. R protein-mediated SAR induction is normally accompanied by a type of programmed cell death known as the hypersensitive response (HR) and, in some instances, cell death alone has been implicated in the induction of SAR. This has raised the question of whether R protein-mediated signalling per se induces SAR or whether SAR is an indirect result of the induction of HR. Using the Rx gene of potato, which confers resistance to Potato Virus X in the absence of cell death, we have shown that the HR is dispensable for R protein-mediated induction of SAR and that Rx-induced SAR is mediated by the same salicylate-dependent pathway induced by other R proteins.

Use of a synthetic salicylic acid analog to investigate the roles of methyl salicylate and its esterases in plant disease resistance.

We previously demonstrated that salicylic acid-binding protein 2 (SABP2) of tobacco is an integral component of systemic acquired resistance (SAR). SABP2 is a methyl salicylate (MeSA) esterase that has high affinity for SA, which feedback inhibits its esterase activity. MeSA esterase activity is required in distal, healthy tissue of pathogen-infected plants to hydrolyze MeSA, which functions as a long-distance, phloem-mobile SAR signal; this hydrolysis releases the biologically active defense hormone SA. In this study, we examined the inhibitory interaction of SA with SABP2, and identified a synthetic SA analog, 2,2,2,2'-tetra-f luoroacetophenone (tetraFA) that, like SA, competitively inhibits the activity of SABP2 and targets esterases, which utilize MeSA as a substrate. However, in contrast to SA, tetraFA does not induce downstream defense responses and, therefore, is effective in planta at blocking SAR development in tobacco mosaic virus (TMV)-infected tobacco and Pseudomonas syringae-infected Arabidopsis. These results confirm the importance of SABP2 and MeSA for SAR development in tobacco and establish similar roles for MeSA and the orthologs of SABP2 in Arabidopsis. Moreover, they demonstrate that tetraFA can be used to determine whether MeSA and its corresponding esterase(s) play a role in SAR signaling in other plant species. In planta analyses using tetraFA, in conjunction with leaf detachment assays and MeSA quantification, were used to assess the kinetics with which MeSA is generated in pathogen-infected leaves, transmitted through the phloem, and processed in the distal healthy leaves. In TMV-infected tobacco, these studies revealed that critical amounts of MeSA are generated, transmitted, and processed between 48 and 72 h post primary infection.

Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages.

AUXIN RESPONSE FACTORS (ARFs) are transcription factors involved in auxin signal transduction during many stages of plant growth development. ARF10, ARF16 and ARF17 are targeted by microRNA160 (miR160) in Arabidopsis thaliana. Here, we show that negative regulation of ARF10 by miR160 plays important roles in seed germination and post-germination. Transgenic plants expressing an miR160-resistant form of ARF10, which has silent mutations in the miRNA target site (termed mARF10), exhibited developmental defects such as serrated leaves, curled stems, contorted flowers and twisted siliques. These phenotypes were not observed in wild-type plants or plants transformed with the targeted ARF10 gene. During sensu stricto germination and post-germination, mARF10 mutant seeds and plants were hypersensitive to ABA in a dose-dependent manner. ABA hypersensitivity was mimicked in wild-type plants by exogenous auxin. In contrast, overexpression of MIR160 (35S:MIR160) resulted in reduced sensitivity to ABA during germination. Transcriptome analysis of germinating ARF10 and mARF10 seeds indicated that typical ABA-responsive genes expressed during seed maturation were overexpressed in germinating mARF10 seeds. These results indicate that negative regulation of ARF10 by miR160 plays a critical role in seed germination and post-embryonic developmental programs, at least in part by mechanisms involving interactions between ARF10-dependent auxin and ABA pathways.

The BME3 (Blue Micropylar End 3) GATA zinc finger transcription factor is a positive regulator of Arabidopsis seed germination.

In many plant species, seed dormancy is broken by cold stratification, a pre-chilling treatment of fully imbibed seeds. Although the ecological importance of seed response to cold temperature is well appreciated, the mechanisms underlying the physiological changes during cold stratification is unknown. Here we show that the GATA zinc finger protein expressed in Arabidopsis seeds during cold stratification plays a critical role in germination. Characterization of an enhancer-trap population identified multiple lines that exhibited beta-glucuronidase (GUS) expression in the micropylar end of the seed (named Blue Micropylar End, BME lines). One of these lines, BME3, had a T-DNA insertion site in the 5' upstream region of a GATA-type zinc finger transcription factor gene (termed BME3-ZF). The BME3-ZF mRNA accumulated in seeds during cold stratification. Characterization of the BME3-ZF promoter indicated that this gene was activated specifically in the embryonic axis, which was still enclosed by the endosperm. The zinc finger gene knockout plants produced seeds exhibiting deeper dormancy, which showed reduced response to cold stratification. The ungerminated knockout seeds exhibited testa rupture, but failed to penetrate the endosperm layer. Application of gibberellic acid (GA3) rescued impaired germination of knockout seeds without cold stratification, indicating that the normal GA signal transduction pathway is present in the knockout mutants. Expression of GA20-oxidase and GA3-oxidase genes was greatly reduced in the knockout seeds, suggesting the potential involvement of the zinc finger protein in GA biosynthesis. These results suggest that the GATA zinc finger protein is a positive regulator of seed germination.

Large-scale screening of Arabidopsis enhancer-trap lines for seed germination-associated genes.

Enhancer trap is a powerful approach for identifying tissue- and stage-specific gene expression in plants and animals. For Arabidopsis research, beta-glucuronidase (GUS) enhancer-trap lines have been created and successfully used to identify tissue-specific gene expression in many plant organs. However, limited applications of these lines for seed germination research have been reported. This is probably due to the impermeability of the testa to the GUS substrate. By focusing on the stages between testa and endosperm rupture, we were able to circumvent the testa barrier to the GUS substrate and observe diverse tissue-specific gene expression during germination sensu stricto. One hundred and twenty-one positive subpools of 10 lines out of 1130 were isolated. Approximately 4500 plants from these subpools were grown in a greenhouse and one to seven individual plants exhibiting GUS expression in seeds were isolated for each subpool. This library of the Arabidopsis seed enhancer-trap lines is an efficient tool for identifying seed germination-associated genes. The individual lines from this library will be provided to the international seed biology research community. International collaboration to identify the trapped genes using genome-walking PCR and to characterize the gene functions using knockout plants will significantly enhance our understanding of the molecular mechanisms of seed germination.

A novel endo-beta-mannanase gene in tomato LeMAN5 is associated with anther and pollen development.

Endo-beta-mannanase (EC 3.2.1.78) is involved in cell wall disassembly and the weakening of plant tissues by degrading mannan polymers in the cell walls. Endo-beta-mannanase genes are expressed in tomato (Lycopersicon esculentum) seeds (LeMAN1 and LeMAN2) and fruits (LeMAN3 and LeMAN4). A novel endo-beta-mannanase gene (termed LeMAN5) was found in the tomato genome by genome-walking PCR and bacterial artificial chromosome library screening. The 5'-upstream region of this endo-beta-mannanase gene contained four copies of the pollen-specific cis-acting elements POLLEN1LELAT52 (AGAAA). A GUS-reporter gene driven with the putative LeMAN5 promoter (-543 to +38) was activated in anthers and pollen of transgenic Arabidopsis, with the highest beta-glucuronidase activity detected in pollen. beta-Glucuronidase expression was detected in mature pollen retained in sporangia, discharged pollen, and elongating pollen tubes in transgenic Arabidopsis. Consistently, expression of LeMAN5 mRNA and endo-beta-mannnanase activity was detected in tomato anthers and pollen. In anthers, the highest mRNA expression and endo-beta-mannanase activity were detected during late stages of anther development, when pollen maturation occurred. Endo-beta-mannanase activity was present in discharged pollen, which was easily eluted in a buffer, indicating that the enzyme proteins are probably secreted from, and deposited on, the surface of pollen. These data suggest that the LeMAN5 endo-beta-mannanase is associated with anther and pollen development.