Genome-wide Analysis of bZIP Transcription Factors in wheat and Functional Characterization of a TabZIP under Abiotic Stress

Abstract

The basic leucine zipper (bZIP) represents one of the largest as well as most diverse transcription factor (TFs) families. They are known to play role in both stress as well as in various plant developmental processes. In the present study, a total of 191 bZIP transcription factors have been identified from Triticum aestivum. Expression analysis during various stress conditions, developmental stages, different varieties and gene ontology enrichment analysis suggest their possible roles in abiotic stress as well as in developmental responses. In the current analysis, one of the members named as TabZIP (Traes_7AL_25850F96F.1) was selected for detailed analysis to understand its role under different abiotic stress conditions. Gene expression studies revealed differential expression of TabZIP in various abiotic stress conditions like heat, salinity and dehydration suggesting the possible role of bZIP in various stress mitigation mechanism. Arabidopsis transgenics overexpressing TabZIP showed enhanced tolerance to salinity, drought, heat and oxidative stress. Thus TabZIP (Traes_7AL_25850F96F.1) can serve as a candidate gene for improving heat as well as other abiotic stress tolerance and can be helpful in enhancing the crop productivity under stress conditions.

Figure 1

Expression profiling of TabZIP transcription factor using real-time PCR in response to various stresses. Change in the transcript level of TabZIP in (a) Ten days grown wheat seedlings of PBW343 in response to heat, cold, salt, mannitol and ABA treatment. (b) In shoot and reproductive tissues (from mature plant) of PBW343 after heat stress. The expression level of control tissue (0 h of treatment) was normalized as 1.0. Results obtained are the mean ± standard deviations of minimum three independent experiments. CT-Control, HS-Heat Stress, DAA (Days After Anthesis)-D3, D5, D7, D10, D20). *Asterick represents the significant difference i.e. Student’s t-test, P- value of ≤0.05.

Figure 2

Expression analysis of TabZIP transcription factor in response to heat stress in five varieties of Triticum aestivum. (a) PBW343, (b) HD2329, (c) CPAN1676, (d) K7903 and (e) C306. Seedlings were given heat stress (37 °C and 42 °C) for two hrs, followed by four hrs of recovery. The expression level of TabZIP of control sample (0 h of treatment) was normalized to 1.0. Results obtained are mean ± standard deviations of minimum three independent experiments. *Asterick represents the significant difference i.e. Student’s t-test, P- value of ≤ 0.05.

Figure 3

Transcription activation assay of TabZIP in yeast. Growth of yeast AH109 strain harbouring full length CDS of TabZIP::pGBKT7 constructs along with both positive and negative controls on (a) SD-H and (b) SD-HW media. Growth of yeast AH109 strains containing various deletions construct of TabZIP::pGBKT7 on (c) SD-W and (d) SD-HW media.

Figure 4

TabZIP over expressing Arabidopsis plants showing increased tolerance to salt stress. (a) Effect of salt stress on TabZIP overexpression Arabidopsis transgenic lines subjected to stress by supplementing media with 150 mM NaCl and phenotype was observed after twenty days. (b) Effect of salt stress on 14-day-old TabZIP over-expressing transgenic lines of Arabidopsis subjected to salt solution treatments i.e. 150 mM NaCl for four days followed by 200 mM NaCl for another four days and then subjected to 300 mM NaCl for sixteen days. (c) Relative abundance of SOS1, SOS2, SOS3 and (d) ATF1. The expression level of wild type (0 h of treatment) was normalized to 1.0. Results depicted are mean ± standard deviations of minimum three independent experiments. *Asterick represents the significant difference i.e. Student’s t-test, P- value of ≤ 0.05.

Figure 5

TabZIP overexpression plants showed enhanced tolerance to dehydration stress. (a) Effect of dehydration on TabZIP overexpression Arabidopsis transgenic lines subjected to stress by supplementing media with 4% PEG and phenotype was observed after twenty days. (b) Effect of drought stress by subjecting 22-days-old wild type and transgenic seedlings to drought treatment for fourteen days and then followed by recovery i.e. rewatering the plants for five days.

Figure 6

TabZIP overexpression plants showed enhanced tolerance to drought stress. Effect of drought stress on transgenic lines (one month old) subjected to drought stress for almost one month. (a) Phenotype of wild type and transgenic plants observed after stress, (b) size of siliques, (c) leaf morphology and (d) relative transcript abundance of drought stress marker gene in wild type and TabZIP overexpression transgenics. Expression level in wild type was normalized to 1.0. Results obtained are mean value ± standard deviations of minimum three independent experiments. *Asterick represents the significant difference i.e. Student’s t-test, P- value of ≤0.05.

Figure 7

TabZIP overexpressing Arabidopsis plants enhances thermotolerance. Arabidopsis transgenic harboring TabZIP under constitutive promoter showed better response to heat stress treatment (42 °C) for two hrs in comparison to the wild type. (a) Phenotype of plants post recovery at 20 days. (b) Silique size. (c) Seed morphology assay of WT and TabZIP overexpressing transgenics of Arabidopsis. (d) Yield per plant. (e) Relative transcript abundance estimation of heat stress marker gene (HSF) in wild type and TabZIP overexpression transgenic plants. The expression level of wild type was normalized to 1.0. Results obtained are mean value ± standard deviations of minimum three independent experiments. *Asterick indicates the significant difference i.e. Student’s t-test, P- value of ≤0.05.

Figure 8

Oxidative stress response of TabZIP overexpressing plants. Oxidative stress response of wild type and TabZIP overexpressing Arabidopsis seedlings examined for comparative accumulation of ROS i.e. O₂⁻ under heat stress i.e. 37 °C for two hrs by (a) NBT for O₂⁻ species, (b) DAB for H₂O₂ species and (c) under oxidative stress treatment by methyl viologen i.e. 50 µM for four hrs by DAB staining. (d) Relative transcript abundance of APXs and (e) CATs in wild type and TabZIP overexpressing Arabidopsis plants. The expression level of wild type was normalized to 1.0. Results obtained are mean value ± standard deviations of minimum three independent experiments. *Asterick represents the significant difference i.e. Student’s t-test, P- value of ≤0.05.