| Histone H2B monoubiquitination (H2Bub) is being recognized as a regulatory mechanism that controls a range of cellular processes in Arabidopsis. H2Bub has been reported to affect leaf and root growth and seed dormancy, and H2Bub is also involved in regulating the expression of key flowering time genes for the control of flowering time and plant development. A recent study showed that the H2Bub-mediated regulation of gene expression played a role in the processes of photomorphogenesis and the circadian clock. Arabidopsis HUB1is a regulatory component of plant defense against necrotrophic fungal pathogens. H2Bub is involved in multiple developmental processes and responses to biotic stress in plants. Verticillium dahliae-toxins (Vd-toxins) purified from Verticillium dahliae secretions was pathogenic to cotton and Arabidopsis, Plant defense response including H2O2generation, activation of defense genes and rapid cell death arised in Arabidopsis treated with Vd-toxins. However, there was no report about the role of H2Bub in defense of Arabidopsis against Vd-toxins.The central features of the plant defense response are the generation of reactive oxygen species (ROS), activation of defense genes and rapid cell death. We analyzed the generation of H2O2, PR1and PDF1.2a expression and rapid cell death after treatment by Vd-toxins in the wild type and in the hubl-4, hub2-2, ubcl-1and ubcl-lubc2-1mutants of Arabidopsis. The H2O2was labeled with a permeable fluorescent probe,2’,7’-dichlorofluorescin diacetate (H2DCF-DA). We observed that the H2O2level increased significantly in the wild type, whereas the mutants showed a reduction of H2O2production. We used the trypan blue staining method to detect cell death. In agreement with the observations of H2O2content, relatively small areas of cell death were detected on the leaves of the hubl-4mutant after Vd-toxins treatment in comparison with the wild type. Furthermore, the relative expression levels of PR1and PDF1.2a were monitored by Real-time PCR. An increase in the expression level of PR1and PDF1.2a was strongly induced in the wild type after Vd-toxins treatment, whereas the expression level was reduced in the mutants. Taken together, the mutants showed a reduction of Vd-toxins-induced H2O2production, cell death and activation of defense genes, suggesting that H2Bub is an important modification with regulatory roles in the defense against Vd-toxins in Arabidopsis.H2O2marker gene GST1was investigated using Real-time PCR in Col-0and hubl-4treated with Vd-toxins, results showed that more significantly up-regulation of GST1in Col-0compared with hub1-4. To investigate the interaction between H2O2production and defence genes expression, the wild-type and hubl-4seedlings were co-treated with Vd-toxins plus DPI or DMTU. Results indicated that H2Bub is involved in modulating the defense responses, and H2O2is essential for salicylic acid but not essential for jasmonic acid signaling pathway in Arabidopsis defense response to Vd-toxins.Gene expression profiling in Col-0and the mutants were investigated with Vd-toxins treatment. It was found that genes of defense response, incompatible interaction defense response, response to bacterium, response to reactive oxygen species, cell redox homeostasis, toxin catabolic process, response to biotic stimulus, immune system process, response to fungus, response to salicylic acid stimulus were up-regulated or inhibited down-regulation in Col-0whereas none or less in the mutants. The result indicate that histone H2B monoubiquitination control many physiological processes associated with plant defense.The results will provide new insight into the mechanisms controlling the Arabidopsis defense response against Vd-toxins. |
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