Publications

Protein kinases are major regulatory components in almost all cellular processes in eukaryotic cells. By adding phosphate groups, protein kinases regulate the activity, localization, protein–protein interactions, and other features of their target proteins. It is known that protein kinases are central components in plant responses to environmental stresses such as drought, high salinity, cold, and pathogen attack. However, only a few targets of these protein kinases have been identified. Moreover, how these protein kinases regulate downstream biological processes and mediate stress responses is still largely unknown. In this study, we introduce a strategy based on isotope-labeled in vitro phosphorylation reactions using in vivo phosphorylated peptides as substrate pools and apply this strategy to identify putative substrates of nine protein kinases that function in plant abiotic and biotic stress responses. As a result, we identified more than 5,000 putative target sites of osmotic stress-activated SnRK2.4 and SnRK2.6, abscisic acid-activated protein kinases SnRK2.6 and casein kinase 1-like 2 (CKL2), elicitor-activated protein kinase CDPK11 and MPK6, cold-activated protein kinase MPK6, H 2 O 2 -activated protein kinase OXI1 and MPK6, and salt-induced protein kinase SOS1 and MPK6, as well as the low-potassium-activated protein kinase CIPK23. These results provide comprehensive information on the role of these protein kinases in the control of cellular activities and could be a valuable resource for further studies on the mechanisms underlying plant responses to environmental stresses.

Abstract Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

Abstract DNA methylation is an epigenetic mark important for genome stability and gene expression. In Arabidopsis thaliana , the 5‐methylcytosine DNA glycosylase/demethylase DEMETER (DME) controls active DNA demethylation during the reproductive stage; however, the lethality of loss‐of‐function dme mutations has made it difficult to assess DME function in vegetative tissues. Here, we edited DME using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR‐associated protein 9 and created three weak dme mutants that produced a few viable seeds. We also performed central cell‐specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3 ( drdd ) quadruple mutant. A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues. A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues. Despite the limited role of DME in regulating DNA methylation in vegetative tissues, the dme mutants showed increased susceptibility to bacterial and fungal pathogens. Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants.

Paramutation is an epigenetic phenomenon that has been observed in a number of multicellular organisms. The epigenetically silenced state of paramutated alleles is not only meiotically stable but also "infectious" to active homologous alleles. The molecular mechanism of paramutation remains unclear, but components involved in RNA-directed DNA methylation (RdDM) are required. Here, we report a multi-copy pRD29A-LUC transgene in Arabidopsis thaliana that behaves like a paramutation locus. The silent state of LUC is induced by mutations in the DNA glycosylase gene ROS1. The silent alleles of LUC are not only meiotically stable but also able to transform active LUC alleles into silent ones, in the absence of ros1 mutations. Maintaining silencing at the LUC gene requires action of multiple pathways besides RdDM. Our study identified specific factors that are involved in the paramutation-like phenomenon and established a model system for the study of paramutation in Arabidopsis.

To identify the genetic loci that control salt tolerance in higher plants, a large-scale screen was conducted with a bialaphos marker-based T-DNA insertional collection of Arabidopsis ecotype C24 mutants. One line, osm1 (for osmotic stress-sensitive mutant), exhibited increased sensitivity to both ionic (NaCl) and nonionic (mannitol) osmotic stress in a root-bending assay. The osm1 mutant displayed a more branched root pattern with or without stress and was hypersensitive to inhibition by Na(+), K(+), and Li(+) but not Cs(+). Plants of the osm1 mutant also were more prone to wilting when grown with limited soil moisture compared with wild-type plants. The stomata of osm1 plants were insensitive to both ABA-induced closing and inhibition of opening compared with wild-type plants. The T-DNA insertion appeared in the first exon of an open reading frame on chromosome 1 (F3M18.7, which is the same as AtSYP61). This insertion mutation cosegregated closely with the osm1 phenotype and was the only functional T-DNA in the mutant genome. Expression of the OSM1 gene was disrupted in mutant plants, and abnormal transcripts accumulated. Gene complementation with the native gene from the wild-type genome completely restored the mutant phenotype to the wild type. Analysis of the deduced amino acid sequence of the affected gene revealed that OSM1 is related most closely to mammalian syntaxins 6 and 10, which are members of the SNARE superfamily of proteins required for vesicular/target membrane fusions. Expression of the OSM1 promoter::beta-glucuronidase gene in transformants indicated that OSM1 is expressed in all tissues except hypocotyls and young leaves and is hyperexpressed in epidermal guard cells. Together, our results demonstrate important roles of OSM1/SYP61 in osmotic stress tolerance and in the ABA regulation of stomatal responses.

Summary Forty‐one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP‐tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety‐five percent of the TAP‐tagged kinases were recovered. Fifty‐six percent of the TAP‐tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP‐tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14‐3‐3 proteins or cyclins.