OsHsfB4b Confers Enhanced Drought Tolerance in Transgenic Arabidopsis and Rice

Abstract

Heat shock factors (Hsfs) play pivotal roles in plant stress responses and confer stress tolerance. However, the functions of several Hsfs in rice (Oryza sativa L.) are not yet known. In this study, genome-wide analysis of the Hsf gene family in rice was performed. A total of 25 OsHsf genes were identified, which could be clearly clustered into three major groups, A, B, and C, based on the characteristics of the sequences. Bioinformatics analysis showed that tandem duplication and fragment replication were two important driving forces in the process of evolution and expansion of the OsHsf family genes. Both OsHsfB4b and OsHsfB4d showed strong responses to the stress treatment. The results of subcellular localization showed that the OsHsfB4b protein was in the nucleus whereas the OsHsfB4d protein was located in both the nucleus and cytoplasm. Over-expression of the OsHsfB4b gene in Arabidopsis and rice can increase the resistance to drought stress. This study provides a basis for understanding the function and evolutionary history of the OsHsf gene family, enriching our knowledge of understanding the biological functions of OsHsfB4b and OsHsfB4d genes involved in the stress response in rice, and also reveals the potential value of OsHsfB4b in rice environmental adaptation improvement.

Keywords: Hsf; drought stress; expression profiles; genome-wide analysis; rice.

Figure 1

Complex phylogenetic tree of Hsf proteins in rice (Oryza sativa L. Os), Arabidopsis (Arabidopsis thaliana L. At), and maize (Zea mays L. Zm). The tree was generated using the protein sequences of rice (pink triangle), Arabidopsis (red circle), and maize (blue square). The tree shows three major groups (A–C) and 15 subgroups with different colored backgrounds.

Figure 2

Schematic representations for chromosomal localization and gene duplication events of OsHsf genes. Twenty-five OsHsf genes were unevenly mapped on the 10 chromosomes. The lines represent the gene density in each chromosome. * indicated the tandem duplication events of OsHsfs.

Figure 3

Synteny analysis of OsHsf genes. (a) Interchromosomal relationships of OsHsf genes. Grey lines indicate all synteny blocks of rice, and red lines indicate links beween OsHsf syntenic genes; (b) Synteny analysis of OsHsf genes between maize, rice, and wheat. Red triangles indicate the positions of OsHsfs. All collinearity blocks and collinearity blocks of Hsf gene pairs within rice, maize, and wheat are indicated by grey lines and blue lines, respectively.

Figure 4

Expression profiles of the OsHsf genes. (a) Hierarchical clustering of expression profiles of OsHsf genes in 48 samples including developmental stages of different tissues. DAF: day after fertilization; (b) Expression analysis of some OsHsf genes in different tissues (leaf, root, and stem) by qRT-PCR. Data were normalized to OsActin1 and OsActin2. Different letters indicate significant differences at p < 0.05 according to one-way ANOVA and post-hoc Tukey’s test.

Figure 5

Expression levels of OsHsfB4d and OsHsfB4d in response to cold, heat, NaCl, ABA, and PEG treatments. Relative expression levels of OsHsfB4b and OsHsfB4d in response to drought for 1 h to 24 h in the leaves at the three-leaf stage of rice. Data were normalized to OsActin1 and OsActin2. Values were the mean ± standard deviation of three biological replicates. ANOVA and Tukey’s test (ns inditated non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

Subcellular localization of OsHsfB4b and OsHsfB4d proteins in rice protoplast. OsHsfB4b-GFP and OsHsfB4d-GFP fusion protein driven by the 35S promoter were transformed into the rice protoplast. GFP and mCherry signals are represented by a green and red color, respectively. Scale bars = 5 μM.

Figure 7

Over-expression of the OsHsB4b gene in Arabidopsis and rice enhances plants resistance to drought stress. Photographs of 7-day-old WT and OsHsB4b-over-expressed Arabidopsis seedlings grown on 1/2 MS (a) and 1/2 MS containing 300 mM Mannitol (b); The germination rates (c,d) and root lengths (e) of seedlings in (a,b); (f) Percentage of B (big), M (moderate), and S (small) seedlings in (a,b). According to the one-way ANOVA and post-hoc Tukey’s test, the significant differences were indicated by different letters in (e), n ≥ 40. Statistical comparisons were performed by two-tailed independent sample t-test (*** p < 0.001) in (f); (g) Photographs of WT and OE rice plants exposed to 20% PEG treatment. Fourteen-day-old rice seedlings were treated with 20% PEG 6000 for 8 days followed by recovering for 7 days. Thirty plants per line were used per replicate; (h,i) The fresh weight (h) and the chlorophyll content (i) of rice seedlings in (g); (j–l) The expression levels of APX2, CAT2, and DREB in leaves of WT or OE rice lines upon PEG treatment. Different letters indicate significant differences at p < 0.05 according to one-way ANOVA and post-hoc Tukey’s test.