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ABSTRACTSmall interfering RNAs (siRNAs) are involved in the regulation of plant development and response to stress. We have previously shown that mutants impaired in Dicer-like 2(DCL2), DCL3and DCL4, RDR2, RDR6and NPRD1are partially impaired in their response to stress and dcl2and dcl3plants are also impaired in transgenerational response to stress, including changes in homologous recombination frequency (HRF). Here, we have analyzed genome stability of dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl3 dcl4and rdr6mutants by measuring the non-induced and the stress-induced recombination frequency. We found that all mutants had the lower spontaneous HRF. The analysis of strand breaks showed that all tested Arabidopsis mutants had a higher level of spontaneous strand breaks, suggesting that the lower HRF is not due to the unusually low level of breaks. Exposure to methyl methane sulfonate (MMS) resulted in an increase in the level of strand breaks in wild-type plants and a decrease in mutants. All mutants had the higher methylation of cytosines at CpG sites under non-induced conditions. Exposure to MMS resulted in a decrease in methylation level in wild-type plants and an increase in methylation in all dclmutants. The expression of several DNA repair genes was altered in dcl4plants under non-induced and induced conditions. Our data suggest that siRNA biogenesis may be essential for the maintenance of the genome stability and stress response in Arabidopsis.

Exposure to heat stress causes physiological and epigenetic changes in plants, which may also be altered in the progeny. We compared the progeny of stressed and control Arabidopsis thalianawild type and Dicer-like mutant dcl2, dcl3, and dcl4plants for variations in physiology and molecular profile, including global genome methylation, mRNA levels, and histone modifications in the subset of differentially expressed genes at normal conditions and in response to heat stress. We found that the immediate progeny of heat-stressed plants had fewer, but larger leaves, and tended to bolt earlier. Transposon expression was elevated in the progeny of heat-stressed plants, and heat stress in the same generation tended to decrease global genome methylation. Progeny of stressed plants had increased expression of HSFA2, and reduction in MSH2, ROS1, and several SUVHgenes. Gene expression positively correlated with permissive histone marks and negatively correlated with repressive marks. Overall, the progeny of heat stressed plants varied in both their physiology and epigenome and dcl2and dcl3mutants were partially deficient for these changes.

The role of resistance (R) genes in plant pathogen interaction has been studied extensively due to its economical impact on agriculture. Interaction between tobacco mosaic virus(TMV) and the N protein from tobacco is one of the most widely used models to understand various aspects of pathogen resistance. The transcription activity governed by Ngene promoter is one of the least understood elements of the model. In this study, the Ngene promoter was cloned and fused with two different reporter genes, one encoding β-glucuronidase (N::GUS) and another, luciferase (N::LUC). Tobacco plants transformed with the N::GUS or N::LUC reporter constructs were screened for homozygosity and stable expression. Histochemical analysis of N::GUS tobacco plants revealed that the expression is organ specific and developmentally regulated. Whereas two week old plants expressed GUS in midveins only, 6-wk-old plants also expressed GUS in leaf lamella. Roots did not show GUS expression at any time during development. Experiments to address effects of external stress were performed using N::LUC tobacco plants. These experiments showed that Ngene promoter expression was suppressed when plants were exposed to high but not low temperatures. Expression was also upregulated in response to TMV, but no changes were observed in plants treated with SA.