Dose-dependent response of Trichoderma harzianum in improving drought tolerance in rice genotypes.

MAIN CONCLUSION: This study demonstrates a dose-dependent response of Trichoderma harzianum Th-56 in improving drought tolerance in rice by modulating proline, SOD, lipid peroxidation product and DHN / AQU transcript level, and the growth attributes. In the present study, the effect of colonization of different doses of T. harzianum Th-56 strain in rice genotypes were evaluated under drought stress. The rice genotypes treated with increasing dose of T. harzianum strain Th-56 showed better drought tolerance as compared with untreated control plant. There was significant change in malondialdehyde, proline, higher superoxide dismutase level, plant height, total dry matter, relative chlorophyll content, leaf rolling, leaf tip burn, and the number of scorched/senesced leaves in T. harzianum Th-56 treated rice genotypes under drought stress. This was corroborated with altered expression of aquaporin and dehydrin genes in T. harzianum Th-56 treated rice genotypes. The present findings suggest that a dose of 30 g/L was the most effective in improving drought tolerance in rice, and its potential exploitation will contribute to the advancement of rice genotypes to sustain crop productivity under drought stress. Interaction studies of T. harzianum with three aromatic rice genotypes suggested that PSD-17 was highly benefitted from T. harzianum colonization under drought stress.

PDH45 transgenic rice maintain cell viability through lower accumulation of Na(+), ROS and calcium homeostasis in roots under salinity stress.

Salinity severely affects the growth/productivity of rice, which is utilized as major staple food crop worldwide. PDH45 (pea DNA helicase 45), a member of the DEAD-box helicase family, actively provides salinity stress tolerance, but the mechanism behind this is not well known. Therefore, in order to understand the mechanism of stress tolerance, sodium ion (Na(+)), reactive oxygen species (ROS), cytosolic calcium [Ca(2+)]cyt and cell viability were analyzed in roots of PDH45 transgenic-IR64 rice lines along with wild-type (WT) IR64 rice under salinity stress (100mM and 200 mM NaCl). In addition, the roots of salinity-tolerant (FL478) and susceptible (Pusa-44) rice varieties were also analyzed under salinity stress for comparative analysis. The results reveal that, under salinity stress (100mM and 200 mM NaCl), roots of PDH45 transgenic lines accumulate lower levels of Na(+), ROS and maintain [Ca(2+)]cyt and exhibit higher cell viability as compared with roots of WT (IR64) plants. Similar results were also obtained in the salinity-tolerant FL478 rice. However, the roots of WT and salinity-susceptible Pusa-44 rice accumulated higher levels of Na(+), ROS and [Ca(2+)]cyt imbalance and lower cell viability during salinity stress, which is in contrast to the overexpressing PDH45 transgenic lines and salinity-tolerant FL478 rice. Further, to understand the mechanism of PDH45 at molecular level, comparative expression profiling of 12 cation transporters/genes was also conducted in roots of WT (IR64) and overexpressing PDH45 transgenic lines (L1 and L2) under salt stress (24h of 200 mM NaCl). The expression analysis results show altered and differential gene expression of cation transporters/genes in salt-stressed roots of WT (IR64) and overexpressing transgenic lines (L1 and L2). These observations collectively suggest that, under salinity stress conditions, PDH45 is involved in the regulation of Na(+) level, ROS production, [Ca(2+)]cyt homeostasis, cell viability and cation transporters in roots of PDH45 transgenic-IR64 rice and consequently provide salinity tolerance. Elucidating the detailed regulatory mechanism of PDH45 will provide a better understanding of salinity stress tolerance and further open new ways to manipulate genome to achieve higher agricultural production under stress.

Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum.

BACKGROUND: CuZn-Superoxide dismutase (SOD) is a unique enzyme, which can catalyzes the dismutation of inevitable metabolic product i.e.; superoxide anion into molecular oxygen and hydrogen peroxide. The enzyme has gained wide interest in pharmaceutical industries due to its highly acclaimed antioxidative properties. The recombinant expression of this protein in its enzymatically active and stable form is highly desired and hence optimization of culture conditions and characterization of the related biochemical properties are essential to explore the significance of the enzyme in physiological, therapeutic, structural and transgenic research. RESULTS: High-level expression of the chloroplastic isoform of Pisum sativum CuZn-SOD was achieved at 18°C, upon isopropyl ß-D-1-thiogalactopyranoside induction and the process was optimized for maximum recovery of the protein in its soluble (enzymatically active) form. Both crude and purified protein fractions display significant increase in activity following supplementation of defined concentration Cu (CuSO4) and Zn (ZnSO4). Yield of the purified recombinant protein was ~ 4 mg L(-1) of culture volume and the bacterial biomass was ~ 4.5 g L(-1). The recombinant pea chloroplastic SOD was found to possess nearly 6 fold higher superoxide dismutase activity and the peroxidase activity was also 5 fold higher as compared to commercially available CuZn-superoxide dismutase. The computational, spectroscopic and biochemical characterization reveals that the protein harbors all the characteristics features of this class of enzyme. The enzyme was found to be exceptionally stable as evident from pH and temperature incubation studies and maintenance of SOD activity upon prolonged storage. CONCLUSIONS: Overexpression and purification strategy presented here describes an efficient protocol for the production of a highly active and stable CuZn-superoxide dismutase in its recombinant form in E. coli system. The strategy can be utilized for the large-scale preparation of active CuZn-superoxide dismutase and thus it has wide application in pharmaceutical industries and also for elucidating the potential of this protein endowed with exceptional stability and activity.

Salinity and drought tolerant OsACA6 enhances cold tolerance in transgenic tobacco by interacting with stress-inducible proteins.

Plant Ca(2+)ATPases regulate many signalling pathways which are important for plant growth, development and abiotic stress responses. Our previous work identified that overexpression of OsACA6 confers salinity and drought tolerance in tobacco. In the present work we report, the function of OsACA6 in cold stress tolerance in transgenic tobacco plants. The expression of OsACA6 was induced by cold stress. The promoter-GUS fusion analyses in the different tissues of transgenic tobacoco confirmed that OsACA6 promoter is cold stress-inducible. Transgenic tobacco plants overexpressing OsACA6 exhibited cold tolerance compared to the wild type (WT) controls. The enhanced tolerance was confirmed by phenotypic analyses as well as by measuring germination, survival rate, chlorophyll content, cell membrane stability, malondialdehyde and proline content. Compared to the WT, the expression of catalase, ascorbate peroxidase and superoxide dismutase increased in the OsACA6 overexpressing plants, which was inversely correlated with the levels of H2O2 in the transgenic lines. We also identified interacting proteins of OsACA6 by using yeast two-hybrid screening assay. Most of the interacting partners of OsACA6 are associated with the widespread biological processes including plant growth, development, signalling and stress adaptation. Furthermore, we also confirmed that OsACA6 is able to self-interact. Overall, these results suggest that OsACA6 plays an important role in cold tolerance at least in part, by regulating antioxidants-mediated removal of reactive oxygen species or by interacting with different calcium signal decoders including calmodulin-like proteins (CaM) calcium/calmodulin dependent protein kinases (CDPKs) and receptor-like protein kinases (RLKs).

Genome-wide analysis of plant-type II Ca(2+)ATPases gene family from rice and Arabidopsis: potential role in abiotic stresses.

The Plant Ca(2+)ATPases are members of the P-type ATPase superfamily and play essential roles in pollen tube growth, vegetative development, inflorescence architecture, stomatal opening or closing as well as transport of Ca(2+), Mn(2+) and Zn(2+). Their role in abiotic stress adaptation by activation of different signaling pathways is emerging. In Arabidopsis, the P-type Ca(2+)ATPases can be classified in two distinct groups: type IIA (ECA) and type IIB (ACA). The availability of rice genome sequence allowed performing a genome-wide search for P-type Ca(2+)ATPases proteins, and the comparison of the identified proteins with their homologs in Arabidopsis model plant. In the present study, we identified the P-type II Ca(2+)ATPases from rice by analyzing their phylogenetic relationship, multiple alignment, cis-regulatory elements, protein domains, motifs and homology percentage. The phylogenetic analysis revealed that rice type IIA Ca(2+)ATPases clustered with Arabidopsis type IIA Ca(2+)ATPases and showed high sequence similarity within the group, whereas rice type IIB Ca(2+)ATPases presented variable sequence similarities with Arabidopsis type IIB members. The protein homology modeling, identification of putative transmembrane domains and conserved motifs of rice P-type II Ca(2+)ATPases provided information on their functions and structural architecture. The analysis of P-type II Ca(2+)ATPases promoter regions in rice showed multiple stress-induced cis-acting elements. The expression profile analysis indicated vital roles of P-type II Ca(2+)ATPases in stress signaling, plant development and abiotic stress responses. The comprehensive analysis and expression profiling provided a critical platform for functional characterization of P-type II Ca(2+)ATPase genes that could be applied in engineering crop plants with modified calcium signaling and homeostatic pathways.

Genome-wide analysis of glutathione reductase (GR) genes from rice and Arabidopsis.

Plant cells and tissues remain always on risk under abiotic and biotic stresses due to increased production of reactive oxygen species (ROS). Plants protect themselves against ROS induced oxidative damage by the upregulation of antioxidant machinery. Out of many components of antioxidant machinery, glutathione reductase (GR, EC 1.6.4.2) and glutathione (GSH, γ-Glu-Cys-Gly) play important role in the protection of cell against oxidative damage. In stress condition, the GR helps in maintaining the reduced glutathione pool for strengthening the antioxidative processes in plants. Present study investigates genome wide analysis of GR from rice and Arabidopsis. We were able to identify 3 rice GR genes (LOC_Os02 g56850, LOC_Os03 g06740, LOC_Os10 g28000) and 2 Arabidopsis GR genes (AT3G54660, AT3G24170) from their respective genomes on the basis of their annotation as well as the presence of pyridine nucleotide-disulphide oxidoreductases class-I active site. The evolutionary relationship of the GR genes from rice and Arabidopsis genomes was analyzed using the multiple sequence alignment and phylogenetic tree. This revealed evolutionary conserved pyridine nucleotide-disulphide oxidoreductases class-I active site among the GR protein in rice and Arabidopsis. This study should make an important contribution to our better understanding of the GR under normal and stress condition in plants.

Genome wide analysis of Cyclophilin gene family from rice and Arabidopsis and its comparison with yeast.

Cyclophilin proteins are the members of immunophillin group of proteins, known for their property of binding to the immune-suppressant drug cyclosporin A, hence named as cyclophilins. These proteins are characterized by the presence of peptidyl prolyl isomerase (PPIase) domain which catalyzes the cis-trans isomerisation process of proline residues. In the present study, an in-silico based approach was followed to identify and characterize the cyclophilin family from rice, Arabidopsis and yeast. We were able to identify 28 rice, 35 Arabidopsis and 8 yeast cyclophilin genes from their respective genomes on the basis of their annotation as well as the presence of highly conserved PPIase domain. The evolutionary relationship of the cyclophilin genes from the three genomes was analyzed using the phylogenetic tree. We have also classified the rice cyclophilin genes on the basis of localization of the protein in cell. The structural similarity of the cyclophilins was also analyzed on the basis of their homology model. The expression analysis performed using Genevestigator revealed a very strong stress responsive behavior of the gene family which was more prominent in later stages of stress. The study indicates the importance of the gene family in stress response as well as several developmental stages thus opening up many avenues for future study on the cyclophilin proteins.