Functional validation of a water deficit stress responsive AP2/ERF family transcription factor-encoding gene in Oryza sativa

Cloning of drought responsive genes and validating their function are essential to crop improvement. In the present study, a drought responsive AP2/ERF family transcription factor was isolated from drought-tolerant Oryza sativa L. cv N-22 (AP2/ERF-‘N-22’). Embryogenic calli produced in vitro from dehusked mature seeds of rice were bombarded with a gene construct containing AP2/ERF-N22, driven under inducible promoter RD29A from Arabidopsis using the Biolistic method. The bombarded calli were selected on hygromycin-containing selection medium. Molecular analysis of regenerated plants confirmed the integration and enhanced expression of the gene under water deficit stress (WDS). Transgenics showed 1.4 fold more expression as compared to wild-type (WT) under control condition and up to 2 fold more expression of AP2/ERF-‘N-22’ under water deficit stress as compared to WT. Molecular analysis of regenerated plants confirmed the integration and enhanced expression of the gene under water deficit stress. The transformation efficiency was found to be nil, 0.97% and 3.11% for overstored seeds ≥ a year, ≥a year and fresh seeds respectively. Transgenics showed 1.4 fold more expression under control condition and up to 2 fold more expression under WDS as compared to wild-type (WT). About 90% of the plants reached maturity and showed no negative phenotypic effects or aberrations as observed earlier under a constitutive promoter from that of the WT. Physiobiochemical analysis of transgenics showed enhanced drought tolerance.

Expression analysis of bZIP transcription factor encoding genes in response to water deficit stress in rice.

In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate several developmental processes and activate genes in response to biotic and abiotic stresses. Role of stress responsive bZIP transcription factors was studied in paddy in relation to different stages of development and water deficit stress (WDS) in a drought tolerant cultivar N22 and susceptible IR 64. Further, relative water content (RWC), membrane stability index (MSI) and abscisic acid (ABA) content were measured as indices of WDS at different stages of development and levels of stress. Expression of stress responsive bZIP transcription factors was directly correlated to developmental stage and WDS and indirectly to RWC, MSI and ABA content.

Cloning and characterization of a water deficit stress responsive transcription factor gene from Oryza sativa L.

Understanding the biochemical and molecular basis of drought mechanism in rice is important as drought is one of the major causes affecting rice crop adversely. A 1017 bp gene sequence encoding AP2/ERF family TF was isolated from Oryza sativa sp. Indica cv N22 encoding a protein of 338 amino acid residues, with a molecular weight of 36.58 kDa, and no intron in the ORF. The gene was named as AP2/ERF-N22(2) different from the drought responsive gene AP2/ERF-N22 that we reported earlier. AP2/ERF-N22(2) has entirely different characteristics from that of AP2/ERF-N22. It has a single AP2 domain of 55 amino acid residues and a cluster of acidic amino acid residues at the C-terminal region, which could function as a trans-activation domain. Presence of NLS indicates that it is a nuclear localized transcription factor encoding gene. It falls in group VI L, sharing characteristic similarities. Arabidopsis members of group VI L have been shown to be involved in response to cytokinin under drought stress.

Role of γ-oryzanol in drought-tolerant and susceptible cultivars of rice (Oryza sativa L.).

Drought-tolerant cultivars and their phytochemical composition, which has a role in providing drought tolerance are gaining importance. In this study, rice bran oil and semi-purified oryzanol (SPO) obtained from five rice (Oryza sativa L.) cultivars, namely P1401 and PB1 (drought-susceptible) and N22, PNR381 and APO (drought-tolerant) were analyzed for the γ-oryzanol content, an antioxidant present in considerable amount in the rice bran. The higher level of γ-oryzanol and its antioxidant activity was observed in drought-tolerant cultivars (N22, PNR381 and APO) as compared to drought-susceptible (PB1 and P1401), suggesting the role of γ-oryzanol in drought tolerance, as antioxidants are known to play an important role by scavenging free radicals. The total antioxidant activity of γ-oryzanol might be attributed to 24-methylene cycloartanyl ferulate, a major component of γ-oryzanol. By enhancing the level of active oryzanol components identified in this study by genetic and molecular means could impart increased drought tolerance.

Cloning and characterization of a gene encoding novel zinc finger protein transcription factor induced under water deficit stress from rice (Oryza sativa) cv. N-22.

A gene OsZnI encoding Cys3/His1-type zinc finger protein was isolated from the water stress-induced cDNA library of rice (Oryza sativa) cv. N-22, an early maturing, deep-rooted, drought-tolerant genotype adapted to upland conditions. The in-silico analysis revealed an insert of 800 bp with an ORF of 663 nucleotides, encoding 221 amino acids. OsZnI had three distinct features — nuclear localization signal (NLS) present in Arg152-Arg168, Zn finger domain between 185-193 amino acids and 12 amino acids conserved domain in 71-82 amino acids homologous to LEA motif, and belonged to C-type family of Zn finger protein. OsZnI showed induced expression under water deficit stress.

Cloning and characterization of drought responsive partial gene sequence(s) from Oryza sativa L. subsp. Indica.

Differential display gels were run for the drought tolerant (N-22) and drought susceptible (Panidhan) genotypes of rice (Oryza sativa) to identify the genes showing differential expression with respect to moisture stress. Differential cDNA products were cloned in PCR-Trap vector and analyzed for differential expression by Northern hybridization. Two clones namely R4A and R7G were found to be associated with water deficit stress (WDS). Sequencing revealed an insert of 244 bp in the clone R4A. BLASTN and FASTA results showed that R4A had maximum homology with a full-length cDNA clone: 002-110-H10 and OSJNBa006109.12 protein. GO classification suggested that it had beta-glucosidase motif which had been implicated in ABA mobilization and thereby ABA dependent gene expression.

Isolation of stress responsive Psb A gene from rice (Oryza sativa l.) using differential display.

Differential display (DD) experiments were performed on drought-tolerant rice (Oryza sativa L.) genotype N22 to identify both upregulated and downregulated partial cDNAs with respect to moisture stress. DNA polymorphism was detected between drought-stressed and control leaf tissues on the DD gels. A partial cDNA showing differential expression, with respect to moisture stress was isolated from the gel. Northern blotting analysis was performed using this cDNA as a probe and it was observed that mRNA corresponding to this transcript was accumulated to high level in rice leaves under water deficit stress. At the DNA sequence level, the partial cDNA showed homology with psb A gene encoding for Dl protein.