Takafumi Shimizu

Abscisic acid (ABA) is a plant hormone that induces seed dormancy during seed development and inhibits seed germination after imbibition. Although ABA is synthesized in the seed coat (testa), endosperm, and embryo, the physiological roles of the hormone derived from each tissue are not fully understood. We found that the gene encoding an Arabidopsis ABA importer, NPF5.1, was expressed in the seed coat during seed development. Dry seeds of loss-of-function npf5.1 mutants contained significantly higher levels of dihydrophaseic acid (DPA), an inactive ABA metabolite, than the wild type. The npf5.1 mutant also had a slight increase in ABA content. An increase in DPA was prominent in the fraction containing the seed coat and endosperm. Seed germination of the npf5.1 mutant was similar to the wild type in the presence of ABA or the gibberellin biosynthesis inhibitor paclobutrazol. However, a mutation in NPF5.1 suppressed the paclobutrazol-resistant germination of npf4.6, a mutant impaired in an ABA importer expressed in the embryo. These results suggest that ABA uptake into the seed coat mediated by NPF5.1 is important for ABA homeostasis during seed development and for regulating seed germination.

SUMMARY: Jasmonoyl‐isoleucine (JA‐Ile) is a key signaling molecule that activates jasmonate‐regulated flower development and the wound stress response. For years, JASMONATE RESISTANT1 (JAR1) has been the sole jasmonoyl‐amino acid synthetase known to conjugate jasmonic acid (JA) to isoleucine, and the source of persisting JA‐Ile in jar1 knockout mutants has remained elusive until now. Here we demonstrate through recombinant enzyme assays and loss‐of‐function mutant analyses that AtGH3.10 functions as a JA‐amido synthetase. Recombinant AtGH3.10 could conjugate JA to isoleucine, alanine, leucine, methionine, and valine. The JA‐Ile accumulation in the gh3.10–2 jar1‐11 double mutant was nearly eliminated in the leaves and flower buds while its catabolism derivative 12OH‐JA‐Ile was undetected in the flower buds and unwounded leaves. Residual levels of JA‐Ile, JA‐Ala, and JA‐Val were nonetheless detected in gh3.10–2 jar1‐11, suggesting the activities of similar promiscuous enzymes. Upon wounding, the accumulation of JA‐Ile and 12OH‐JA‐Ile and the expression of JA‐responsive genes OXOPHYTODIENOIC ACID REDUCTASE3 and JASMONATE ZIM‐DOMAIN1 observed in WT, gh3.10–1, and jar1‐11 leaves were effectively abolished in gh3.10–2 jar1‐11. Additionally, an increased proportion of undeveloped siliques associated with retarded stamen development was observed in gh3.10–2 jar1‐11. These findings conclusively show that AtGH3.10 contributes to JA‐amino acid biosynthesis and functions partially redundantly with AtJAR1 in sustaining flower development and the wound stress response in Arabidopsis.

The plant hormone abscisic acid (ABA) is actively synthesized in vascular tissues and transported to guard cells to promote stomatal closure. Although several transmembrane ABA transporters have been identified, how the movement of ABA within plants is regulated is not fully understood. In this study, we determined that Arabidopsis NPF4.6, previously identified as an ABA transporter expressed in vascular tissues, is also present in guard cells and positively regulates stomatal closure in leaves. We also found that mutants defective in NPF5.1 had a higher leaf surface temperature compared to the wild type. Additionally, NPF5.1 mediated cellular ABA uptake when expressed in a heterologous yeast system. Promoter activities of NPF5.1 were detected in several leaf cell types. Taken together, these observations indicate that NPF5.1 negatively regulates stomatal closure by regulating the amount of ABA that can be transported from vascular tissues to guard cells.

Transmembrane transport of plant hormones is required for plant growth and development. Despite reports of a number of proteins that can transport the plant hormone gibberellin (GA), the mechanistic basis for GA transport and the identities of the transporters involved remain incomplete. Here, we provide evidence that Arabidopsis SWEET proteins, AtSWEET13 and AtSWEET14, which are members of a family that had previously been linked to sugar transport, are able to mediate cellular GA uptake when expressed in yeast and oocytes. A double sweet13 sweet14 mutant has a defect in anther dehiscence and this phenotype can be reversed by exogenous GA treatment. In addition, sweet13 sweet14 exhibits altered long distant transport of exogenously applied GA and altered responses to GA during germination and seedling stages. These results suggest that AtSWEET13 and AtSWEET14 may be involved in modulating GA response in Arabidopsis.

The root hemiparasite witchweed (Striga spp.) is a devastating agricultural pest that causes losses of up to $1 billion US annually in sub-Saharan Africa. Development of resistant crops is one of the cost-effective ways to address this problem. However, the molecular mechanisms underlying resistance are not well understood. To understand molecular events upon Striga spp. infection, we conducted genome-scale RNA sequencing expression analysis using Strigahermonthica-infected rice (Oryza sativa) roots. We found that transcripts grouped under the Gene Ontology term defense response were significantly enriched in up-regulated differentially expressed genes. In particular, we found that both jasmonic acid (JA) and salicylic acid (SA) pathways were induced, but the induction of the JA pathway preceded that of the SA pathway. Foliar application of JA resulted in higher resistance. The hebiba mutant plants, which lack the JA biosynthesis gene ALLENE OXIDE CYCLASE, exhibited severe S. hermonthica susceptibility. The resistant phenotype was recovered by application of JA. By contrast, the SA-deficient NahG rice plants were resistant against S. hermonthica, indicating that endogenous SA is not required for resistance. However, knocking down WRKY45, a regulator of the SA/benzothiadiazole pathway, resulted in enhanced susceptibility. Interestingly, NahG plants induced the JA pathway, which was down-regulated in WRKY45-knockdown plants, linking the resistant and susceptible phenotypes to the JA pathway. Consistently, the susceptibility phenotype in the WRKY45-knockdown plants was recovered by foliar JA application. These results point to a model in which WRKY45 modulates a cross talk in resistance against S. hermonthica by positively regulating both SA/benzothiadiazole and JA pathways.

NRT1/PTR FAMILY (NPF) proteins were originally identified as nitrate or di/tri-peptide transporters. Recent studies revealed that this transporter family also transports the plant hormones auxin (indole-3-acetic acid), abscisic acid (ABA), and gibberellin (GA), as well as secondary metabolites (glucosinolates). We developed modified yeast two-hybrid systems with receptor complexes for GA and jasmonoyl-isoleucine (JA-Ile), to detect GA and JA-Ile transport activities of proteins expressed in the yeast cells. Using these GA and JA-Ile systems as well as the ABA system that we had introduced previously, we determined the capacities of Arabidopsis NPFs to transport these hormones. Several NPFs induced the formation of receptor complexes under relatively low hormone concentrations. Hormone transport activities were confirmed for some NPFs by direct analysis of hormone uptake of yeast cells by liquid chromatography–tandem mass spectrometry. Our results suggest that at least some NPFs could function as hormone transporters.

Studies have indicated that endogenous concentrations of plant hormones are regulated very locally within plants. To understand the mechanisms underlying hormone mediated physiological processes, it is indispensable to know the exact hormone concentrations at cellular levels. In the present study, we established a system to determine levels of abscisic acid (ABA) and jasmonoyl-isoleucine (JA-Ile) from single cells. Samples taken from a cell of Vicia faba leaves using nano-electrospray ionization (ESI) tips under a microscope were directly introduced into mass spectrometers by infusion and subjected to tandem-mass spectrometry (MS/MS) analysis. Stable isotope labeled D₆-ABA or ¹³C₆-JA-Ile was used as an internal standard to compensate ionization efficiencies, which determine the amount of ions introduced into mass spectrometers. We detected ABA and JA-Ile from single cells of water- and wound-stressed leaves, whereas they were almost undetectable in non-stressed single cells. The levels of ABA and JA-Ile found in the single cell analysis were compared with levels found by analysis of purified extracts with liquid-chromatography tandem-mass spectrometry (LC-MS/MS). These results demonstrated that stress-induced accumulation of ABA and JA-Ile could be monitored from living single cells.

WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

Key message Endogenous JA production is not necessary for wound-induced expression of JA-biosynthetic lipase genes such as DAD1 in Arabidopsis. However, the JA-Ile receptor COI1 is often required for their JA-independent induction. Abstract Wounding is a serious event in plants that may result from insect feeding and increase the risk of pathogen infection. Wounded plants produce high amounts of jasmonic acid (JA), which triggers the expression of insect and pathogen resistance genes. We focused on the transcriptional regulation of DEFECTIVE IN ANTHER DEHISCENCE1 and six of its homologs including DONGLE (DGL) in Arabidopsis, which encode lipases involved in JA biosynthesis. Plants constitutively expressing DAD1 accumulated a higher amount of JA than control plants after wounding, indicating that the expression of these lipase genes contributes to determining JA levels. We found that the expression of DAD1, DGL, and other DAD1-LIKE LIPASE (DALL) genes is induced upon wounding. Some DALLs were also expressed in unwounded leaves. Further experiments using JA-biosynthetic and JA-response mutants revealed that the wound induction of these genes is regulated by several distinct pathways. DAD1 and most of its homologs other than DALL4 were fully induced without relying on endogenous JA-Ile production and were only partly affected by JA deficiency, indicating that positive feedback by JA is not necessary for induction of these genes. However, DAD1 and DGL required CORONATINE INSENSITIVE1 (COI1) for their expression, suggesting that a molecule other than JA might act as a regulator of COI1. Wound induction of DALL1, DALL2, and DALL3 did not require COI1. This differential regulation of DAD1 and its homologs might explain their functions at different time points after wounding.