Sagarika Mishra

Arsenic stress in rice: Redox consequences and regulation by iron

Arsenic (As) contamination is a serious hazard to human health and agriculture. It has emerged as an important threat for rice cultivation mainly in South Asian countries. In this study, we investigated the effect of iron (Fe) supplementation on arsenic (As(V)) induced oxidative stress responses in rice (Oryza sativa L.). Rice seedlings treated with As(V) for 24 and 48 h in presence or absence of 2.5 mM Fe after which the root and shoot tissues were harvested for analysis. The results indicate significant (p ≤ 0.05) reduction in root and shoot length/dry biomass. Supplementation of Fe showed improved growth responses under stress as compared to As(V) alone. The scanning electron microscopy (SEM) analysis of roots under As(V) treatment for 48 h showed major alterations in root structure and integrity, although no noticeable changes were observed in Fe - supplemented seedlings. Significantly high (p ≤ 0.05) accumulation of As(V) was observed in root and shoot after 24 and 48 h of stress. However, under Fe - supplementation As accumulation in root and shoot were considerably low after 24 and 48 h of As(V) treatment. The hydrogen peroxide (H2O2) and malondialdehyde (MDA) content in both root and shoot increased significantly (p ≤ 0.05) after 24 and 48 h of As(V) treatment. In Fe - supplemented seedlings, the levels of H2O2 and MDA were considerably low as compared to As(V) alone. Ascorbate (AsA) and glutathione (GSH) levels also increased significantly (p ≤ 0.05) under As(V) stress as compared to control and Fe-supplemented seedlings. Activities of catalase (CAT) and superoxide dismutase (SOD) were significantly (p ≤ 0.05) high after 24 and 48 h of As(V) treatment as compared to Fe-supplemented seedlings. The gene expression analysis revealed up-regulation of metallothionein (MT1, MT2) and nodulin 26-like intrinsic protein (NIP2;1) genes after 5d of As treatment, while their expressions were repressed under Fe-supplementation. Our results indicate that Fe regulates oxidative stress and promotes growth under As stress.

Cloning and Functional Characterization of a Vacuolar Na+/H+ Antiporter Gene from Mungbean (VrNHX1) and Its Ectopic Expression Enhanced Salt Tolerance in Arabidopsis thaliana

Plant vacuolar NHX exchangers play a significant role in adaption to salt stress by compartmentalizing excess cytosolic Na+ into vacuoles and maintaining cellular homeostasis and ionic equilibrium. We cloned an orthologue of the vacuolar Na+/H+ antiporter gene, VrNHX1 from mungbean (Vigna radiata), an important Asiatic grain legume. The VrNHX1 (Genbank Accession number JN656211.1) contains 2095 nucleotides with an open reading frame of 1629 nucleotides encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. The consensus amiloride binding motif (84LFFIYLLPPI93) was observed in the third putative transmembrane domain of VrNHX1. Bioinformatic and phylogenetic analysis clearly suggested that VrNHX1 had high similarity to those of orthologs belonging to Class-I clade of plant NHX exchangers in leguminous crops. VrNHX1 could be strongly induced by salt stress in mungbean as the expression in roots significantly increased in presence of 200 mM NaCl with concomitant accumulation of total [Na+]. Induction of VrNHX1 was also observed under cold and dehydration stress, indicating a possible cross talk between various abiotic stresses. Heterologous expression in salt sensitive yeast mutant AXT3 complemented for the loss of yeast vacuolar NHX1 under NaCl, KCl and LiCl stress indicating that VrNHX1 was the orthologue of ScNHX1. Further, AXT3 cells expressing VrNHX1 survived under low pH environment and displayed vacuolar alkalinization analyzed using pH sensitive fluorescent dye BCECF-AM. The constitutive and stress inducible expression of VrNHX1 resulted in enhanced salt tolerance in transgenic Arabidopsis thaliana lines. Our work suggested that VrNHX1 was a salt tolerance determinant in mungbean.

Enhanced salinity tolerance in transgenic mungbean overexpressing Arabidopsis antiporter (NHX1) gene

Efficient compartmentalization of Na+ ions into the vacuole through heterologous overexpression of vacoular antiporter gene NHX1 is a promising approach to develop salt tolerance in plants. Mungbean (Vigna radiata L. Wilczek) is an important grain legume widely cultivated in Southeast Asia for its protein rich grains. Salinity affects growth and productivity of mungbean. In this paper, we report overexpression of an Arabidopsis NHX1 (AtNHX1) in transgenic mungbean plants conferred enhanced salt tolerance. Cotyledonary node explants were transformed via Agrobacterium tumefaciens mediated transformation using pCAMBIA2301 vector that harbours 35S::AtNHX1 in its T-DNA. Putative transformed plants were selected on kanamycin containing medium. Polymerase chain reaction and Southern blot analysis confirmed the presence, integration and copy number of transgenes in T1 transgenic lines. Reverse transcription-PCR analysis showed higher expression of AtNHX1 in transgenic plants as compared to wild type plants (WT). Under salt stress conditions, T2 transgenic lines displayed less damage and stronger growth phenotypes with concurrent physiological changes as compared to WT. In addition, T2 transgenic lines under salt stress accumulated higher K+/Na+ in the aerial parts and higher [Na+] in roots than WT. Moreover, the T2 transgenic lines showed under NaCl treatment reduced membrane lipid peroxidation and H2O2 and O2− accumulation, higher levels of antioxidant enzyme activity and increased accumulation of proline and ascorbate than WT. These results indicated that the activity of heterologous AtNHX1 protein contributing enhanced salt tolerance in transgenic mungbean.

Cowpea [Vigna unguiculata (L.) Walp].

Agrobacterium tumefaciens-mediated transformation is an efficient method for incorporating genes and recovering stable transgenic plants in cowpea because this method offers several advantages such as the defined integration of transgenes, potentially low copy number, and preferential integration into transcriptional active regions of the chromosome. Cotyledonary node explants of cowpea present an attractive target for T-DNA delivery followed by regeneration of shoots via axillary proliferation without involvement of a de novo regeneration pathway. In this chapter, we describe a detailed protocol for Agrobacterium-mediated transformation of the cowpea variety Pusa Komal. The seedling cotyledonary node explants are used for cocultivation with an Agrobacterium strain EHA105 harboring standard binary vector, pCAMBIA2301 or pNOV2819, and putative transformed plants are selected using aminoglycoside antibiotic or mannose as sole carbon source, respectively. The entire process includes explant infection to transgenic seed generation in greenhouse.