Arabidopsis plants overexpressing additional copies of heat shock protein Hsp101 showed high heat tolerance and endo-gene silencing.

Hsp101 chaperone is vital for survival of plants under heat stress. We generated transgenic Arabidopsis thaliana (Arabidopsis) lines with extra copies of Hsp101 gene using diverse approaches. Arabidopsis plants transformed with rice Hsp101 cDNA driven by Arabidopsis Hsp101 promoter (IN lines) showed high heat tolerance while the plants transformed with rice Hsp101 cDNA driven by CaMV35S promoter (C lines) were like wild type plants in heat stress response. Transformation of Col-0 plants with 4633 bp Hsp101 genomic fragment (GF lines) from A. thaliana containing both its coding and the regulatory sequence resulted in mostly over-expressor (OX) lines and a few under-expressor (UX) lines of Hsp101. OX lines showed enhanced heat tolerance while the UX lines were overly heat sensitive. In UX lines, silencing of not only Hsp101 endo-gene was noted but also transcript of choline kinase (CK2) was silenced. Previous work established that in Arabidopsis, CK2 and Hsp101 are convergent gene pairs sharing a bidirectional promoter. The elevated AtHsp101 protein amount in most GF and IN lines was accompanied by lowered CK2 transcript levels under HS. We observed increased methylation of the promoter and gene sequence region in UX lines; however, methylation was lacking in OX lines.

Sida cordifolia accelerates wound healing process delayed by dexamethasone in rats: Effect on ROS and probable mechanism of action.

ETHNO PHARMACOLOGICAL RELEVANCE: Sida cordifolia is used commonly in traditional systems of medicine (TSM) and as folk remedies for treating the wounds (both external and internal), infected area, rheumatic disorders, muscular weakness, tuberculosis, heart problems, bronchitis, neurological problems etc. Therefore, in order to authenticate the claims, a mechanism-oriented investigation of the wound healing properties of this plant is essential. AIM OF THE STUDY: The overall aim of the present research is to understand the precise unknown cellular and molecular mechanism by which S. cordifolia accelerates wound healing delay caused by the steroidal drug dexamethasone. Here, we have also tried to quantify intracellular superoxide with the help of a unique fluoroprobe MitoSOX based on fluorescence measurements in yeast MATERIALS AND METHODS: Wound healing property of successive extracts (ethyl acetate, methanol and aqueous) of S. cordifolia against dexamethasone-induced retardation of wound healing in rats was studied. The various extracts of S. cordifolia were characterised by determining the various phytochemicals and quantifying the total phenolic content and flavonoidal content by High throughput assays. In order to know the probable mechanism of action of the successive fractionates, assessed the antioxidant activity both by in-vitro (DPPH-assay) and in-vivo methods in wild-type Saccharomyces cerevisiae BY 4743 (WT) and knock-out strain (Δtrx2) against H2O2-induced stress mediated damages. The cell survival was evaluated after exposure to the oxidizing reagent (4 mM H2O2) by two methods which included the ability of cells to proliferate on solid or liquid medium. The cell membrane integrity/amount of mitochondrial ROS was determined by treating the strains with extract/standard in presence of H2O2 and propidium iodide (PI)/MitoSOX Red RESULTS: During the preliminary in-vivo wound healing study, the period for complete re-epithelialization of the wound tissue was reduced significantly (pin the treatment groups as compared to the negative control group. The formulation HF3 containing aqueous extract of S. cordifolia (SCA) showed highest wound healing potential against dexamethasone-retarded wounds in rats which justifies its traditional use. In the growth curve assay, the H2O2-induced growth arrest was restored by aqueous extract of S. cordifolia (SCA) in a concentration-dependent(pmanner both in the WT and Δtrx2 strains similar to the standard (ascorbic acid), H2O2 after 24 hours incubation which was also confirmed by the findings of CFU method. We got almost similar results of cell viability when stained with PI. The lower level of mitochondrial superoxide was indicated by a significant (preduction in the amount of MitoSOX stained cells, in the extract-treated group in contrast to the H2O2-stressed group. CONCLUSION: It was concluded that HF3 can be applied topically in hydrogel form in the case of delayed wound healing caused by the steroidal drug-dexamethasone, aptly justifying its traditional use. Regarding its mechanism of action, our findings report that the potent adaptive response of SCA-treated WT and Δtrx2 strains towards intracellular ROS specifically mitochondrial-ROS confirms its antioxidant potential. Moreover, as SCA was able to rescue the Δtrx2 strains from stress, it can be inferred that it might be able to induce the enzyme thioredoxin-II to restore redox homeostasis. The findings with the conditional mutant ∆trx2 are the first proof linking SCA action related to particular cellular pathways which may be because of the phenols and flavonoids and their synergistic effect.

Chymotrypsin protease inhibitor gene family in rice: Genomic organization and evidence for the presence of a bidirectional promoter shared between two chymotrypsin protease inhibitor genes.

Protease inhibitors play important roles in stress and developmental responses of plants. Rice genome contains 17 putative members in chymotrypsin protease inhibitor (ranging in size from 7.21 to 11.9 kDa) gene family with different predicted localization sites. Full-length cDNA encoding for a putative subtilisin-chymotrypsin protease inhibitor (OCPI2) was obtained from Pusa basmati 1 (indica) rice seedlings. 620 bp-long OCPI2 cDNA contained 219 bp-long ORF, coding for 72 amino acid-long 7.7 kDa subtilisin-chymotrypsin protease inhibitor (CPI) cytoplasmic protein. Expression analysis by semi-quantitative RT-PCR analysis showed that OCPI2 transcript is induced by varied stresses including salt, ABA, low temperature and mechanical injury in both root and shoot tissues of the seedlings. Transgenic rice plants produced with OCPI2 promoter-gus reporter gene showed that this promoter directs high salt- and ABA-regulated expression of the GUS gene. Another CPI gene (OCPI1) upstream to OCPI2 (with 1126 bp distance between the transcription initiation sites of the two genes; transcription in the reverse orientation) was noted in genome sequence of rice genome. A vector that had GFP and GUS reporter genes in opposite orientations driven by 1881 bp intergenic sequence between the OCPI2 and OCPI1 (encompassing the region between the translation initiation sites of the two genes) was constructed and shot in onion epidermal cells by particle bombardment. Expression of both GFP and GUS from the same epidermal cell showed that this sequence represents a bidirectional promoter. Examples illustrating gene pairs showing co-expression of two divergent neighboring genes sharing a bidirectional promoter have recently been extensively worked out in yeast and human systems. We provide an example of a gene pair constituted of two homologous genes showing co-expression governed by a bidirectional promoter in rice.