Immunocompromisation of wheat host by L-BSO and 2,4-DPA induces susceptibility to the fungal pathogen Fusarium oxysporum.

Susceptibility is defined as the disruption of host defence systems that promotes infection or limits pathogenicity. Glutathione (GSH) is a major component of defence signalling pathways that maintain redox status and is synthesised by γ-glutamyl cysteine synthetase (γ-ECS). On the other hand, lignin acts as a barrier in the primary cell wall of vascular bundles (VBs) synthesised by phenylalanine ammonia-lyase (PAL) in the intracellular system of plants. In this study, we used two inhibitors, such as L-Buthionine-sulfoximine (BSO), which irreversibly inhibits γ-ECS, and 2,4-dichlorophenoxyacetic acid (DPA), which reduces PAL activity and leads to the induction of oxidative stress in wheat (Triticum aestivum) seedlings after exposure to Fusarium oxysporum. Seedlings treated with 1 mM L-BSO and 2,4-DPA showed high levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), carbonyl (CO) content, and low activity of antioxidative enzymes [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR)] as compared to wild-type (WT) seedlings under F. oxysporum infection. Further, the content of reduced glutathione (RGSH), ascorbate (ASC), and lignin was decreased in BSO and DPA treated seedlings as compared to WT seedlings during Fusarium infection. Moreover, treatment with BSO and DPA significantly inhibited the relative activity of γ-ECS and PAL (P ≤ 0.001) in WT seedlings during Fusarium infection, which led to disintegrated VBs and, finally, cell death. Our results demonstrate that inhibition of γ-ECS and PAL by BSO and DPA, respectively, disrupts the defence mechanisms of wheat seedlings and induces susceptibility to F. oxysporum.

Expression analysis of chitinase upon challenge inoculation to Alternaria wounding and defense inducers in Brassica juncea.

Chitinases are the hydrolytic enzymes which belong to the pathogenesis-related (PR) protein family and play an important role not only in plant defense but also in various abiotic stresses. However, only a limited number of chitinase genes have been characterised in B. juncea. In this study, we have characterised B. juncea class IV chitinase gene (accession no EF586206) in response to fungal infection, salicylic acid (SA), jasmonic acid (JA) treatments and wounding. Gene expression studies revealed that the transcript levels of Bjchitinase (BjChp) gene increases significantly both in local and distal tissues after Alternaria infection. Bjchitinase gene was also induced by jasmonic acid and wounding but moderately by salicylic acid. A 2.5 kb class IV chitinase promoter of this gene was isolated from B. juncea by Genome walking (accession no KF055403.1). In-silico analysis of this promoter revealed a number of conserved cis-regulatory elements related to defense, wounding and signalling molecules like SA, and JA. For validation, chitinase promoter was fused to the GUS gene, and the resultant construct was then introduced into Arabidopsis plants. Histochemical analysis of T2 transgenic Arabidopsis plants showed that higher GUS activity in leaves after fungal infection, wounding and JA treatment but weakly by SA. GUS activity was seen in meristematic tissues, young leaves, seeds and siliques. Finally investigation has led to the identification of a pathogen-inducible, developmentally regulated and organ-specific promoter. Present study revealed that Bjchitinase (BjChp) promoter is induced during biotic and environmental stress and it can be used in developing finely tuned transgenics.

Protein glutathionylation protects wheat (Triticum aestivum Var. Sonalika) against Fusarium induced oxidative stress.

Fusarium induced oxidative stress could be recovered by reversible protein oxidative modification through the process of glutathionylation in co-stressed (low-dose (50 µM) Cd2+ pre-treatment followed by Fusarium inoculation) wheat seedlings. Co-stressed seedlings showed low disease severity index as compared to Fusarium infected seedlings. A reduced level of hydrogen peroxide (H2O2) and carbonyl contents due to irreversible protein oxidation were observed in co-stressed seedlings as compared to Fusarium infected seedlings. Further, a comparative biochemical assay showed an enhanced glutathione content in co-stressed tissues as compared to Fusarium infected tissues. In an investigation, reduced glutathione pre-coated agarose gel beads were used to pull down proteins having affinity with GSH. Fructose-1, 6-bisphosphate aldolase and 3-Phosphoglycerate kinase were observed to be co-existed in co-stressed seedlings when analysed by LC-MS/MS after being processed through protein-pull assay. Co-stressed tissues showed an enhanced free protein thiol content as compared to Fusarium infected tissues. The ratio of free thiol to thiol disulfides was also observed to be increased in co-stressed tissues as compared to Fusarium infected tissues. In contrast, the quantitative assay by Ellman's reagent and qualitative analysis by diagonal gel electrophoresis showed enhanced protein thiol disulfides in Fusarium infected tissues as compared to co-stressed tissues. Further, glutaredoxin, responsible for the reverse reduction of proteins was observed to be enhanced in co-stressed tissues as compared to Fusarium infected tissues. Thus, a low dose Cd2+ triggered glutathionylation is suggestive of offering tolerance against Fusarium induced oxidative stress and protects target proteins from irreversible modification and permanent damage in wheat.

3D cell growth and proliferation on a RGD functionalized nanofibrillar hydrogel based on a conformationally restricted residue containing dipeptide.

Three-dimensional (3D) hydrogels incorporating a compendium of bioactive molecules can allow efficient proliferation and differentiation of cells and can thus act as successful tissue engineering scaffolds. Self-assembled peptide-based hydrogels can be worthy candidates for such applications as peptides are biocompatible, biodegradable and can be easily functionalized with desired moieties. Here, we report 3D growth and proliferation of mammalian cells (HeLa and L929) on a dipeptide hydrogel chemically functionalized with a pentapeptide containing Arg-Gly-Asp (RGD) motif. The method of functionalization is simple, direct and can be adapted to other functional moieties as well. The functionalized gel was noncytotoxic, exhibited enhanced cell growth promoting properties, and promoted 3D growth and proliferation of cells for almost 2 weeks, with simultaneous preservation of their metabolic activities. The presence of effective cell growth supporting properties in a simple and easy to functionalize dipeptide hydrogel is unique and makes it a promising candidate for tissue engineering and cell biological applications.

High salinity reduces the content of a highly abundant 23-kDa protein of the mangrove Bruguiera parviflora.

A significant decrease in the amount of a protein, whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 23 kDa and pI = 6.5, was observed in leaves of NaCl-treated Bruguiera parviflora (Roxb.) Wt. & Arn. ex Griff. seedlings. This particular salt-sensitive protein, designated as SSP-23, almost disappeared after 45 days of treatment in 400 mM NaCl as compared to untreated seedlings (0 mM NaCl) where the presence of the protein was significant. A polyclonal antibody raised against the 23-kDa protein was used to determine the subcellular localization of this protein in leaves by cross-reaction with proteins from isolated chloroplasts, mitochondria, peroxisomes and cytosol fractions on Western blots. SSP-23 was confirmed to be localized in the cytosol by immunoblotting. The disappearance of SSP-23 as a result of high NaCl treatment suggests that this protein is salt-sensitive and has a possible role in salt adaptation.

Salt-stress induced alterations in protein profile and protease activity in the mangrove Bruguiera parviflora.

Two-month-old seedlings of Bruguiera parvifora were treated with varying levels of NaCl (100, 200 and 400 mM) under hydroponic culture. Total proteins were extracted from leaves of control and NaCl treated plants after 7, 14, 30 and 45 d of treatment and analysed by SDS-PAGE. As visualized from SDS-PAGE, the intensity of several protein bands of molecular weight 17, 23, 32, 33 and 34 kDa decreased as a result of NaCl treatment. The degree of decrease of these protein bands seemed to be roughly proportional to the external NaCl concentration. The most obvious change concerned a 23 kDa-polypeptide (SSP-23), which disappeared after 45 d treatment in 400 mM NaCl. Moreover, the SSP-23 protein, which disappeared in B. parviflora under salinity stress, reappeared when these salinized seedlings were desalinized. These observations suggest the possible involvement of these polypeptides for osmotic adjustment under salt stress. NaCl stress also caused an increase in the activity of both acid and alkaline protease. The increasing activity of proteases functions as a signal of salt stress in B. parviflora, which induces the reduction of protein level.