Publications

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf.

RNA interference, mediated by small interfering RNAs (siRNAs), is a conserved regulatory process that has evolved as an antiviral defense mechanism in plants and animals. It is not known whether host cells also use siRNAs as an antibacterial defense mechanism in eukaryotes. Here, we report the discovery of an endogenous siRNA, nat-siRNAATGB2, that is specifically induced by the bacterial pathogen Pseudomonas syringae carrying effector avrRpt2. We demonstrate that the biogenesis of this siRNA requires DCL1, HYL1, HEN1, RDR6, NRPD1A, and SGS3. Its induction also depends on the cognate host disease resistance gene RPS2 and the NDR1 gene that is required for RPS2-specified resistance. This siRNA contributes to RPS2-mediated race-specific disease resistance by repressing PPRL, a putative negative regulator of the RPS2 resistance pathway.

Summary Osmotic stress activates the expression of many plant genes through ABA‐dependent as well as ABA‐independent signaling pathways. We report here the characterization of a novel mutant of Arabidopsis thaliana , hos5‐1 , which exhibits increased expression of the osmotic stress responsive RD29A gene. The expression of several other stress genes are also enhanced by the hos5‐1 mutation. The enhanced expression is specific to ABA and osmotic stress because low temperature regulation of these genes is not altered in the mutant. Genetic analysis indicated that hos5‐1 is a recessive mutation in a single nuclear gene on chromosome III. Double mutant analysis of hos5‐1 and the ABA‐deficient aba1‐1 as well as the ABA‐insensitive abi1‐1 mutant indicated that the osmotic stress hypersensitivity of hos5‐1 is not affected by ABA deficiency or insensitivity. Furthermore, combined treatments of hos5‐1 with ABA and osmotic stress had an additive effect on RD29A‐LUC expression. These results suggest that the osmotic stress hypersensitivity in hos5‐1 may be ABA‐independent. The germination of hos5‐1 seeds was more resistant to ABA. However, the hos5‐1 mutation did not influence stomatal control and only slightly affected the regulation of growth and proline accumulation by ABA. The hos5‐1 mutation reveals a negative regulator of osmotic stress‐responsive gene expression shared by ABA‐dependent and ABA‐independent osmotic stress signaling pathways.

Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation.

Protein phosphorylation is one of the most important and widespread molecular regulatory mechanisms that controls almost all aspects of cellular functions in animals and plants. Here, we introduce a novel chemically functionalized reverse phase phosphoprotein array (RP3A) to capture and measure phosphoproteomes. RP3A uses polyamidoamine (PAMAM) dendrimer immobilized with Ti(IV) ions to functionalize nitrocellulose membrane, facilitating specific chelation of phosphoproteins from complex protein samples on the array. Globular, water-soluble Ti(IV)-dendrimer allows the RP3A surface to be highly accessible to phosphoproteins multidimensionally, and the captured phosphoproteins were subsequently detected using the same validated antibodies as in regular reverse-phase protein arrays. The novel chemical strategy demonstrated superior specificity (1:10 000), high sensitivity (fg level), and good quantitative nature (R2 = 0.99) for measuring phosphoproteins. We further applied quantitative phosphoproteomics followed by RP3A to validate the phosphorylation status of a panel of phosphoproteins in response to environmental stresses in Arabidopsis.

The adverse effects of high salt on plants include Na(+) toxicity and hyperosmotic and oxidative stresses. The plasma membrane-localized Na(+)/H(+) antiporter SOS1 functions in the extrusion of toxic Na(+) from cells and is essential for plant salt tolerance. We report here that, under salt or oxidative stress, SOS1 interacts through its predicted cytoplasmic tail with RCD1, a regulator of oxidative-stress responses. Without stress treatment, RCD1 is localized in the nucleus. Under high salt or oxidative stress, RCD1 is found not only in the nucleus but also in the cytoplasm. Like rcd1 mutants, sos1 mutant plants show an altered sensitivity to oxidative stresses. The rcd1mutation causes a decrease in salt tolerance and enhances the salt-stress sensitivity of sos1 mutant plants. Several genes related to oxidative-stress tolerance were found to be regulated by both RCD1 and SOS1. These results reveal a previously uncharacterized function of a plasma membrane Na(+)/H(+) antiporter in oxidative-stress tolerance and shed light on the cross-talk between the ion-homeostasis and oxidative-stress detoxification pathways involved in plant salt tolerance.

Abstract Background Cowpea ( Vigna unguiculata ) is an important crop in arid and semi-arid regions and is a good model for studying drought tolerance. MicroRNAs (miRNAs) are known to play critical roles in plant stress responses, but drought-associated miRNAs have not been identified in cowpea. In addition, it is not understood how miRNAs might contribute to different capacities of drought tolerance in different cowpea genotypes. Results We generated deep sequencing small RNA reads from two cowpea genotypes (CB46, drought-sensitive, and IT93K503-1, drought-tolerant) that grew under well-watered and drought stress conditions. We mapped small RNA reads to cowpea genomic sequences and identified 157 miRNA genes that belong to 89 families. Among 44 drought-associated miRNAs, 30 were upregulated in drought condition and 14 were downregulated. Although miRNA expression was in general consistent in two genotypes, we found that nine miRNAs were predominantly or exclusively expressed in one of the two genotypes and that 11 miRNAs were drought-regulated in only one genotype, but not the other. Conclusions These results suggest that miRNAs may play important roles in drought tolerance in cowpea and may be a key factor in determining the level of drought tolerance in different cowpea genotypes.