Responses to water stress are thought to be mediated by transcriptional regulation of gene expression via reversible protein phosphorylation events. Previously, we reported that bZIP (basic-domain leucine zipper)-type AREB/ABF (ABA-responsive element-binding protein/factor) transcription factors are involved in ABA signaling under water stress conditions in Arabidopsis. The AREB1 protein is phosphorylated in vitro by ABA-activated SNF1-related protein kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). Consistent with this, we now show that SRK2D/E/I and AREB1 co-localize and interact in nuclei in planta. Our results show that unlike srk2d, srk2e and srk2i single and double mutants, srk2d srk2e srk2i (srk2d/e/i) triple mutants exhibit greatly reduced tolerance to drought stress and highly enhanced insensitivity to ABA. Under water stress conditions, ABA- and water stress-dependent gene expression, including that of transcription factors, is globally and drastically impaired, and jasmonic acid (JA)-responsive and flowering genes are up-regulated in srk2d/e/i triple mutants, but not in other single and double mutants. The down-regulated genes in srk2d/e/i and areb/abf triple mutants largely overlap in ABA-dependent expression, supporting the view that SRK2D/E/I regulate AREB/ABFs in ABA signaling in response to water stress. Almost all dehydration-responsive LEA (late embryogenesis abundant) protein genes and group-A PP2C (protein phosphatase 2C) genes are strongly down-regulated in the srk2d/e/i triple mutants. Further, our data show that these group-A PP2Cs, such as HAI1 and ABI1, interact with SRK2D. Together, our results indicate that SRK2D/E/I function as main positive regulators, and suggest that ABA signaling is controlled by the dual modulation of SRK2D/E/I and group-A PP2Cs.

DREB1A/CBF3 and DREB2A are transcription factors that specifically interact with a cis-acting dehydration-responsive element (DRE), which is involved in cold-and dehydration-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Overexpression of DREB1A improves stress tolerance to both freezing and dehydration in transgenic plants. In contrast, overexpression of an active form of DREB2A results in significant stress tolerance to dehydration but only slight tolerance to freezing in transgenic plants. The downstream gene products for DREB1A and DREB2A are reported to have similar putative functions, but downstream genes encoding enzymes for carbohydrate metabolism are very different between DREB1A and DREB2A. We demonstrate that under cold and dehydration conditions, the expression of many genes encoding starchdegrading enzymes, sucrose metabolism enzymes, and sugar alcohol synthases changes dynamically; consequently, many kinds of monosaccharides, disaccharides, trisaccharides, and sugar alcohols accumulate in Arabidopsis. We also show that DREB1A overexpression can cause almost the same changes in these metabolic processes and that these changes seem to improve freezing and dehydration stress tolerance in transgenic plants. In contrast, DREB2A overexpression did not increase the level of any of these metabolites in transgenic plants. Strong freezing stress tolerance of the transgenic plants overexpressing DREB1A may depend on accumulation of these metabolites.

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K + uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K + channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using 86 radioactive rubidium ion ( 86 Rb + ) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABAmediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.

Microbial pathogens strategically acquire metabolites from their hosts during infection. Here we show that the host can intervene to prevent such metabolite loss to pathogens. Phosphorylation-dependent regulation of sugar transport protein 13 (STP13) is required for antibacterial defense in the plant Arabidopsis thaliana STP13 physically associates with the flagellin receptor flagellin-sensitive 2 (FLS2) and its co-receptor BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1). BAK1 phosphorylates STP13 at threonine 485, which enhances its monosaccharide uptake activity to compete with bacteria for extracellular sugars. Limiting the availability of extracellular sugar deprives bacteria of an energy source and restricts virulence factor delivery. Our results reveal that control of sugar uptake, managed by regulation of a host sugar transporter, is a defense strategy deployed against microbial infection. Competition for sugar thus shapes host-pathogen interactions.

Recognition of molecular patterns characteristic of microbes or altered-self leads to immune activation in multicellular eukaryotes. In Arabidopsis thaliana, the leucine-rich-repeat receptor kinases FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) recognize bacterial flagellin and elongation factor EF-Tu (and their elicitor-active epitopes flg22 and elf18), respectively. Likewise, PEP1 RECEP-TOR1 (PEPR1) and PEPR2 recognize the elicitor-active Pep epitopes conserved in Arabidopsis ELICITOR PEPTIDE PRECURSORs (PROPEPs). Here we reveal that loss of ETHYLENE-INSENSITIVE2 (EIN2), a master signaling regulator of the phytohormone ethylene (ET), lowers sensitivity to both elf18 and flg22 in different defense-related outputs. Remarkably, in contrast to a large decrease in FLS2 expression, EFR expression and receptor accumulation remain unaffected in ein2 plants. Genome-wide transcriptome profiling has uncovered an inventory of EIN2-dependent and EFR-regulated genes. This dataset highlights important aspects of how ET modulates EFR-triggered immunity: the potentiation of salicylate-based immunity and the repression of a jasmonate-related branch. EFR requires ET signaling components for PROPEP2 activation but not for PROPEP3 activation, pointing to both ET-dependent and -independent engagement of the PEPR pathway during EFR-triggered immunity. Moreover, PEPR activation compensates the ein2 defects for a subset of EFR-regulated genes. Accordingly, ein2 pepr1 pepr2 plants exhibit additive defects in EFR-triggered antibacterial immunity, compared with ein2 or pepr1 pepr2 plants. Our findings suggest that the PEPR pathway not only mediates ET signaling but also compensates for its absence in enhancing plant immunity.microbe-associated molecular pattern | danger-associated molecular pattern | disease resistance | plant hormone | defense-related genes

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using ␤-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K m for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.

SignificanceMultiple effectors of bacterial pathogens target immune kinases such as BAK1 and BIK1, but it is unclear whether this strategy is employed by fungal pathogens. We reveal here that a fungal effector named NIS1 is broadly conserved in filamentous fungi in the Ascomycota and Basidiomycota, thus being regarded as a core effector, and has the ability to suppress PAMP-triggered immunity. Importantly, NIS1 targets BAK1 and BIK1, interfering with their essential functions for immune activation upon pathogen recognition. Multifaceted analyses including the knockout of NIS1 revealed that it plays a critical role in fungal infection. These findings demonstrate that to infect host plants, filamentous fungi deploy a core effector that attacks conserved immune kinases critical for the ancestral defense system.

Polyamines (PAs) are ubiquitous, polycationic compounds that are essential for the growth and survival of all organisms. Although the PA-uptake system plays a key role in mammalian cancer and in plant survival, the underlying molecular mechanisms are not well understood. Here, we identified an Arabidopsis L-type amino acid transporter (LAT) family transporter, named RMV1 (resistant to methyl viologen 1), responsible for uptake of PA and its analog paraquat (PQ). The natural variation in PQ tolerance was determined in 22 Arabidopsis thaliana accessions based on the polymorphic variation of RMV1. An RMV1-GFP fusion protein localized to the plasma membrane in transformed cells. The Arabidopsis rmv1 mutant was highly resistant to PQ because of the reduction of PQ uptake activity. Uptake studies indicated that RMV1 mediates proton gradient-driven PQ transport. RMV1 overexpressing plants were hypersensitive to PA and PQ and showed elevated PA/PQ uptake activity, supporting the notion that PQ enters plant cells via a carrier system that inherently functions in PA transport. Furthermore, we demonstrated that polymorphic variation in RMV1 controls PA/PQ uptake activity. Our identification of a molecular entity for PA/PQ uptake and sensitivity provides an important clue for our understanding of the mechanism and biological significance of PA uptake.