Fig 1.
Analysis of natural polymorphism in the coding DNA sequence of OsbZIP23 gene.
(A) PCR amplification of OsbZIP23 CDS from selected genotypes using gene-specific primers. Lanes 1–15 represents O. rufipogon, O. nivara, Vandana, Swarna, Nagina22, Manipuri, HRC300, IR20, IR36, IR64, IR72, sorghum, maize, Brachypodium and bajra respectively. Lane M- HinfI digested pUC18 plasmid as a standard molecular weight marker. (B) Allelic polymorphism in the CDS of OsbZIP23 gene across selected rice genotypes is represented by multiple sequence alignment in Jalview software.
Fig 2.
Analysis of the transcript expression level and copy number of the endogenous OsbZIP23 gene.
(A) Relative expression level of OsbZIP23 gene in different tissues of an upland indica rice genotype, Vandana. Real time PCR analysis of OsbZIP23 transcript in vegetative (B) and reproductive (grain filling) stage (C), in 11 selected rice genotypes grown under before stress (BS), after stress (AS) and after recovery (AR) condition as described in method section. Rice polyubiquitin1 (OsUbi1) gene was taken as internal reference. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis of Student’s t test indicated significant differences (* P<0.05, **P<0.01). (D) Southern blot showing the single copy of endogenous OsbZIP23 gene in 11 selected rice genotypes. Lanes 1–11 represent O. rufipogon, O. nivara, Vandana, IR20, Swarna, IR36, IR64, IR72, Nagina22, Manipuri, HRC300 rice genotypes, respectively.
Fig 3.
Cloning and in silico analysis of OsbZIP23 promoter sequences.
(A) PCR amplification of the OsbZIP23 gene promoter region from two selected rice genotypes using gene-specific primers. Lanes 1–2 represent O. rufipogon and IR20, respectively. Lane M- HinfI digested pUC18 plasmid as a standard molecular weight marker. (B) Alignment of nucleotide sequences and distribution of major cis-regulatory elements in the OsbZIP23 promoter of O. rufipogon and IR20.
Fig 4.
Southern blot and GFP fluorescence analyses of transgenic and NT plants for studying the promoter activity of OsbZIP23 gene.
(A) Southern blot analysis of rice T0 transformants of RuP and 20P lines developed with the O. rufipogon promoter-GFP construct (S1 Fig) and O. sativa IR20 promoter-GFP construct (S2 Fig), respectively. The genomic DNA of plant sample was digested with HindIII, and the CDS of GFP gene was used as hybridization probe. Lane NT- non-transgenic, Lane M- molecular weight marker. (B) GFP expression in finely cross-sectioned leaves of drought stressed single integration transgenic rice lines under the confocal microscope (Olympus FV1200). Scale bar 50 μm. (C) Real time PCR analysis showing relative expression level of GFP in leaf samples of RuP and 20P lines under drought stress condition, where rice polyubiquitin1 (OsUbi1) gene was taken as internal reference. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis of Student’s t test indicated significant differences (**P<0.01).
Fig 5.
Molecular analyses of transgenic and NT plants for studying the CDS activity of OsbZIP23 gene.
(A) Southern blot analysis of T0 transformants of OEN and OER developed with the genetic construct of OsbZIP23 CDS from O. rufipogon (S3 Fig) and O. nivara (S4 Fig). The genomic DNA was digested with HindIII and probed with the 739 bp OsbZIP23 CDS. (B) Southern blot analysis of T0 transformants of RNAi developed with gene silencing construct (S5 Fig) using HindIII digested genomic DNA and probed with 739 bp OsbZIP23 CDS. Lane NT- non-transgenic, Lane M- molecular weight marker. (C) Real time PCR analysis showing relative expression level of OsbZIP23 in three OE lines, two RNAi lines and non-transgenic (NT) plants in vegetative stage, where rice polyubiquitin1 (OsUbi1) gene was taken as internal reference. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis of Student’s t test indicated significant differences (* P<0.05, **P<0.01). (D) Western blot analysis showing the expression level of OsbZIP23 protein in three OE lines, two RNAi lines and NT plant (upper panel). Equal loading of protein was confirmed by using β-actin antibody (lower panel).
Fig 6.
Assessing drought stress tolerance and grain yield of OsbZIP23 overexpression (OE) and down-regulated (RNAi) transgenic lines.
(A) Photographs of OE lines, RNAi lines and non-transgenic (NT) plants in the vegetative stage- before and after drought stress and their subsequent recovery after the drought treatment. (B) Survival rates of transgenic lines and NT plants, calculated in percentage (%). (C) Photographs of OE lines, RNAi lines and NT plants in early reproductive (panicle initiation) stage- before and after drought stress and their subsequent recovery after the drought treatment. (D) Survival rates of transgenic lines and NT plants, calculated in %. (E) Drought stress in PVC pipes in flowering stage and subsequent recovery till seed maturation stage of OE lines, RNAi lines and NT plants. (F) Mature panicle of OE lines, RNAi lines and NT plant. (G) Measurement of panicle weight in OE lines, RNAi lines and NT plant. (H) Spikelet fertility measurement in OE lines, RNAi lines and NT plants, calculated in %. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis by Student’s t-test indicated significant differences (*P<0.05, ** P<0.01). All the results were based on three independent experiments.
Fig 7.
Comparison of leaf water retention capacity and reactive oxygen species (ROS) activity in OsbZIP23 OE and RNAi lines.
(A) Water loss rates and (B) relative water contents of detached leaves, at the five-leaf stage amongst OsbZIP23 OE lines, RNAi lines and NT plants. Measurement of (C) proline and (D) soluble sugar contents in OsbZIP23 OE lines, RNAi lines and NT plants, after extraction from leaf tissues before and after water stress. All the results were based on three independent experiments. Data bars represent the mean ±SD of triplicate measurement. A statistical analysis by Student’s t-test indicated significant differences (*P<0.05, **P<0.01). (E) Measurement of MDA content in OsbZIP23 OE, RNAi lines and NT plants, after extraction from leaf tissue of rice plants before and after water stress. Data bars represent the mean ±SD of triplicate measurement. A statistical analysis by Student’s t-test indicated significant differences (*P<0.05, **P<0.01). (F) Detection of ROS by monitoring H2O2 production in leaves of OsbZIP23 OE, RNAi lines and NT plants were visualized by staining with 3, 3׳–diaminobenzidine (DAB) under well-watered (normal) and drought stress condition. (G) Production of O2− ions in leaves of OsbZIP23 OE, RNAi lines and NT plants were visualized by staining with nitro blue tetrazolium (NBT) under normal and drought stress condition. The results were based on three independent experiments; one set of result is represented here.
Fig 8.
Real time PCR analysis in vegetative stage showing relative expression level of three selected representative drought inducible genes working downstream of OsbZIP23 in the ABA signaling pathway.
(A) OsRab16B, (B) OsRab21 and (C) OsLEA3-1 in OsbZIP23 OE lines, RNAi lines and NT plants, where rice polyubiquitin1 (OsUbi1) gene was taken as internal reference. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis by Student’s t-test indicated significant differences (**P<0.01).
Fig 9.
Evaluating ABA sensitivity of OsbZIP23 OE and RNAi lines at germination and post-germination stages.
Germination performance of seeds from (A) OsbZIP23 OE lines (OER#5, OEN#9), and (B) OsbZIP23 RNAi lines (RNAi#1, RNAi#4) in comparison to NT plants on MS agar medium containing 0, 1, 3 and 6 μM ABA at 10th day. (C) Calculation of the germination rates (%) of OsbZIP23 OE, RNAi and NT seeds. (D and E) Performance of OE, RNAi and NT seedlings in ½ MS liquid medium containing 0, 1, 3 and 6 μM of ABA. Measurement of (F) shoot length and (G) root length of OE, RNAi and NT seedlings grown on different concentrations of ABA after 14 days. Data bars represent the mean ±SD of triplicate measurement. Statistical analysis by Student’s t-test indicated significant differences (*P<0.05, ** P<0.01). All the results were based on three independent experiments.