Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases, which play key roles in the regulation of eukaryotic gene expression. Previously, we have identified a yeast histone deacetylase REDUCED POTASSIUM DEPENDENCY3 (RPD3) homolog, HISTONE DEACETYLASE19 (HDA19) (AtRPD3A), in Arabidopsis thaliana. Here, we report further study of the expression and function of HDA19. Analysis of Arabidopsis plants containing the HDA19:b-glucuronidase fusion gene revealed that HDA19 was expressed throughout the life of the plant and in most plant organs examined. In addition, the expression of HDA19 was induced by wounding, the pathogen Alternaria brassicicola, and the plant hormones jasmonic acid and ethylene. Using green fluorescent protein fusion, we demonstrated that HDA19 accumulated in the nuclei of Arabidopsis cells. Overexpression of HDA19 in 35S:HDA19 plants decreased histone acetylation levels, whereas downregulation of HDA19 in HDA19-RNA interference (RNAi) plants increased histone acetylation levels. In comparison with wild-type plants, 35S:HDA19 transgenic plants had increased expression of ETHYLENE RESPONSE FACTOR1 and were more resistant to the pathogen A. brassicicola. The expression of jasmonic acid and ethylene regulated PATHOGENESIS-RELATED genes, Basic Chitinase and b-1,3-Glucanase, was upregulated in 35S:HDA19 plants but downregulated in HDA19-RNAi plants. Our studies provide evidence that HDA19 may regulate gene expression involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.

Post-translational modifications of histones, including acetylation, play a key role in modulating dynamic changes in chromatin structure and gene activity. Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases. HDA6 is a RPD3-type histone deacetylase in Arabidopsis. The Arabidopsis HDA6 mutant, axe1-5, and HDA6 RNA-interfering (HDA6-RNAi) plants displayed higher levels of acetylated H3 compared with wild-type, suggesting that HDA6 affects histone acetylation levels globally. The expression of the jasmonate responsive genes, PDF1.2, VSP2, JIN1, and ERF1, was down-regulated in axe1-5 and HDA6-RNAi plants. Furthermore, axe1-5 and HDA6-RNAi plants displayed increased leaf longevity compared with the wild type. The expression of the senescence-associated genes, SAG12 and SEN4, was down-regulated in the axe1-5 and HDA6-RNAi plants. In addition, axe1-5 and HDA6-RNAi plants displayed late-flowering. The expression of FLC was up-regulated and hyperacetylated in axe1-5 and HDA6-RNAi plants, suggesting that HDA6 is required to deacetylate FLC chromatin and thereby repress its expression. Our results suggest that HDA6 is involved in jasmonate response, senescence, and flowering in Arabidopsis.

SummaryHD2 (histone deacetylase) proteins are plant-speci®c histone deacetylases (HDACs). The Arabidopsis genome contains four HD2 genes, namely HD2A, HD2B, HD2C, and HD2D. We have previously demonstrated that HD2A, HD2B, and HD2C can repress transcription directly by targeting to promoters in planta. Here, we show that the N-terminal conserved motif (EFWG) and histidine 25 (H25), a potential catalytic residue, were important for the gene repression activity of HD2A. In situ hybridization indicated that HD2A, HD2B, and HD2C were expressed in ovules, embryos, shoot apical meristems, and primary leaves. Furthermore, all three genes were strongly induced during the process of somatic embryogenesis. HD2D mRNA was only detected in the stems and¯owers with young siliques and may have adopted different functions. Using green¯uorescent protein (GFP) fusions, we demonstrated that HD2A, HD2B, and HD2C accumulated in the nuclei of Arabidopsis cells. Overexpression of 35S::GFP±HD2A in transgenic Arabidopsis plants generated pleiotropic developmental abnormalities, including abnormal leaves, delayed¯owering, and aborted seed development. The data showed that normal pattern of HD2 expression was essential for normal plant development and that HD2A, HD2B, and HD2C may be needed for embryogenesis and embryo development. Reverse transcriptase (RT)-PCR analysis revealed that a number of genes involved in seed development and maturation were repressed in the 35S::GFP±HD2A plants, supporting a role of HD2A in the regulation of gene expression during seed development.

Three beta-glucosidases (At1g66270-BGLU21, At1g66280-BGLU22, and At3g09260-BGLU23) were purified from the roots of Arabidopsis and their cDNAs were expressed in insect cells. In addition, two beta-glucosidase binding protein cDNAs (At3g16420; PBPI and At3g16430; PBPII) were expressed in Escherichia coli and their protein products purified. These binding proteins interact with beta-glucosidases and activate them. BGLU21, 22 and 23 hydrolyzed the natural substrate scopolin specifically and also hydrolyzed to some extent substrates whose aglycone moiety is similar to scopolin (e.g. esculin and 4-MU-glucoside). In contrast, they hydrolyzed poorly DIMBOA-glucoside and did not hydrolyze pNP- and oNP-glucosides. We determined the physicochemical properties of native and recombinant BGLUs, and found no differences between them. They were stable in a narrow pH range (5-7.5) and had temperature and pH optima for activity at 35 degrees C and pH 5.5, respectively. As for thermostability, >95% of their activity was retained at 40 degrees C but dramatically decreased at >50 degrees C. The apparent K(m) of native and recombinant enzymes for scopolin was 0.73 and 0.81 mM, respectively, and it was 5.8 and 9.7 mM, respectively, for esculin. Western blot analysis showed that all three enzymes were exclusively expressed in roots of seedlings but not in any other plant part or organ under normal conditions. Furthermore, spatial expression patterns of all eight genes belonging to subfamily 3 were investigated at the transcription level by RT-PCR.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) disease on rice plants. Xoc delivers a type III effector AvrRxo1-ORF1 into rice plant cells that can be recognized by disease resistance (R) protein Rxo1, and triggers resistance to BLS disease. However, the mechanism and virulence role of AvrRxo1 is not known. In the genome of Xoc, AvrRxo1-ORF1 is adjacent to another gene AvrRxo1-ORF2, which was predicted to encode a molecular chaperone of AvrRxo1-ORF1. We report the co-purification and crystallization of the AvrRxo1-ORF1:AvrRxo1-ORF2 tetramer complex at 1.64 Å resolution. AvrRxo1-ORF1 has a T4 polynucleotide kinase domain, and expression of AvrRxo1-ORF1 suppresses bacterial growth in a manner dependent on the kinase motif. Although AvrRxo1-ORF2 binds AvrRxo1-ORF1, it is structurally different from typical effector-binding chaperones, in that it has a distinct fold containing a novel kinase-binding domain. AvrRxo1-ORF2 functions to suppress the bacteriostatic activity of AvrRxo1-ORF1 in bacterial cells.