The rice ortholog of DREB1, OsDREB1b, is transcriptionally induced by cold stress and over-expression of OsDREB1b results in increase tolerance towards high salt and freezing stress. This spatio-temporal expression of OsDREB1b is preceded by the change in chromatin structure at the promoter and the upstream region for gene activation. The promoter and the upstream region of OsDREB1b genes appear to be arranged into a nucleosome array. Nucleosome mapping of ∼700bp upstream region of OsDREB1b shows two positioned nucleosomes between −610 to −258 and a weakly positioned nucleosome at the core promoter and the TSS. Upon cold stress, there is a significant change in the nucleosome arrangement at the upstream region with increase in DNaseI hypersensitivity or MNase digestion in the vicinity of cis elements and TATA box at the core promoter. ChIP assays shows hyper-acetylation of histone H3K9 throughout the locus whereas region specific increase was observed in H3K14ac and H3K27ac. Moreover, there is an enrichment of RNA PolII occupancy at the promoter region during transcription activation. There is no significant change in the H3 occupancy in OsDREB1b locus negating the possibility of nucleosome loss during cold stress. Interestingly, cold induced enhanced transcript level of OsDREB1b as well as histone H3 acetylation at the upstream region was found to diminish when stressed plants were returned to normal temperature. The result indicates absolute necessity of changes in chromatin conformation for the transcription up-regulation of OsDREB1b gene in response to cold stress. The combined results show the existence of closed chromatin conformation at the upstream and promoter region of OsDREB1b in the transcription “off” state. During cold stress, changes in region specific histone modification marks promote the alteration of chromatin structure to facilitate the binding of transcription machinery for proper gene expression.

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ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (Kd = 475 ± 17.9 nM) compared to Arabidopsis HMGB1 protein (Kd = 39.8 ± 2.68 nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys(85) from the ARID domain and Arg(199) & Lys(202) from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.

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In flowering plants, jasmonic acid (JA) signalling regulates the complex process of male gametophyte development. JA signalling initiates with the activation of MYC2 transcription factor, for the expression of several JA responsive genes throughout stamen development and pollen maturation. However, the regulation of JA signalling during different developmental stages of male gametophytes is still less understood. In this study we have characterized T-DNA insertion line of AtHMGB15. Phenotypic characterization of athmgb15-4 mutant plants showed delayed bolting, shorter siliques and reduced seed set compared to wildtype. Moreover, deletion of AtHMGB15 resulted in defective pollen morphology, delayed pollen germination, abberant pollen tube growth and a higher percentage of non-viable pollen population in athmgb15-4 compared to wildtype. Molecular analysis indicated down-regulation of JA-biosynthesis and JA-signalling genes viz MYC2, MYB21 and MYB24 in athmgb15-4 mutant. Furthermore, jasmonic acid and its derivatives were found almost ten-fold lower in athmgb15-4 flowers. However, exogenous application of jasmonate could restore pollen morphology and pollen germination, suggesting that impaired JA signalling is responsible for the pollen phenotype in athmgb15 mutant. AtHMGB15 physically interacts with MYC2 protein to form the transcription activation complex for promoting transcription of genes responsible for JA signalling during stamen and pollen development. Collectively, our findings indicate that AtHMGB15, a plant specific DNA binding protein of the ARID-HMG group, acts as a positive regulator of JA signalling to control the spatiotemporal expression of key regulators responsible for stamen and pollen development.