Characterization of wheat homeodomain-leucine zipper family genes and functional analysis of TaHDZ5-6A in drought tolerance in transgenic Arabidopsis

Abstract

Background: Many studies in Arabidopsis and rice have demonstrated that HD-Zip transcription factors play important roles in plant development and responses to abiotic stresses. Although common wheat (Triticum aestivum L.) is one of the most widely cultivated and consumed food crops in the world, the function of the HD-Zip proteins in wheat is still largely unknown.

Results: To explore the potential biological functions of HD-Zip genes in wheat, we performed a bioinformatics and gene expression analysis of the HD-Zip family. We identified 113 HD-Zip members from wheat and classified them into four subfamilies (I-IV) based on phylogenic analysis against proteins from Arabidopsis, rice, and maize. Most HD-Zip genes are represented by two to three homeoalleles in wheat, which are named as TaHDZX_ZA, TaHDZX_ZB, or TaHDZX_ZD, where X denotes the gene number and Z the wheat chromosome on which it is located. TaHDZs in the same subfamily have similar protein motifs and intron/exon structures. The expression profiles of TaHDZ genes were analysed in different tissues, at different stages of vegetative growth, during seed development, and under drought stress. We found that most TaHDZ genes, especially those in subfamilies I and II, were induced by drought stress, suggesting the potential importance of subfamily I and II TaHDZ members in the responses to abiotic stress. Compared with wild-type (WT) plants, transgenic Arabidopsis plants overexpressing TaHDZ5-6A displayed enhanced drought tolerance, lower water loss rates, higher survival rates, and higher proline content under drought conditions. Additionally, the transcriptome analysis identified a number of differentially expressed genes between 35S::TaHDZ5-6A transgenic and wild-type plants, many of which are involved in stress response.

Conclusions: Our results will facilitate further functional analysis of wheat HD-Zip genes, and also indicate that TaHDZ5-6A may participate in regulating the plant response to drought stress. Our experiments show that TaHDZ5-6A holds great potential for genetic improvement of abiotic stress tolerance in crops.

Keywords: Drought tolerance; Expression profiles; HD-zip gene family; Phylogenetic relationships; TaHDZ5-6A; Wheat.

Fig. 1

Phylogeny and distribution of HD-Zip proteins from eight plant species. a Phylogenetic tree of HD-Zip proteins from Arabidopsis, Populus, Vitis, wheat, rice, maize, Brachypodium, and moss. Phylogeny was constructed by PhyML using maximum likelihood analysis. Bootstrap support values as percentage, are shown on selected major branches. The bar indicates substitutions per site; b Percentage representation of HD-Zips across the eight plant species within each subfamily; c Percentage representation of distributions for HD-Zips within each plant species

Fig. 2

The phylogenetic tree of HD-Zip proteins from wheat, Arabidopsis, maize and rice. Members of the HD-zip genes from wheat are marked in red. Two-letter prefixes for sequence identifiers indicate species of origin. Ta, Triticum aestivum; At, Arabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays. The tree was constructed using the Neighbor-Joining algorithm with 1000 bootstrap based on the full length sequences of HD-Zip proteins. The HD-Zip proteins are grouped into four distinct groups

Fig. 3

Phylogenetic relationships and gene structures of wheat HD-Zip genes. a Phylogenetic tree of 113 full length HD-Zip proteins from wheat were constructed by MEGA 6.0 using the Neighbour-Joining (NJ) method with 1000 bootstrap values. b Exon/intron structures of wheat HD-Zip genes. Exons and introns are represented by purple boxes and grey lines, respectively. c The distribution of intron numbers between four distinct HD-Zip subfamily of wheat

Fig. 4

Expression profiles of TaHDZ genes in ten different organs or tissues. The heat map was drawn in Log₁₀-transformed expression values. The red or green colors represent the higher or lower expression level of each transcript in each sample. R, root of wheat seedling at five-leaf stage; S, stem of wheat seedling at five-leaf stage; L, leaf of wheat seedling at five-leaf stage; FL, flag leaf at heading stage; YS5, young spike at early booting stage; YS15, spike at heading stage; GR5, grain of 5 days post-anthesis; GR10, grain of 10 days post-anthesis; GR15, grain of 15 days post-anthesis; GR20, grain of 20 days post-anthesis

Fig. 5

Expression profiles of TaHDZ genes in seedling leaves under drought stress treatment. a hierarchical clustering of the relative expression level of TaHDZ genes under drought stress treatment. The heat map was drawn in Log₁₀-transformed expression values. The red or green colors represent the higher or lower relative abundance of each transcript in each sample. b Expression patterns of TaHDZ genes under drought stress treatment. c The numbers of up-regulated and down-regulated TaHDZ genes in four HD-Zip subfamilies. d The ratios of up-regulated and down-regulated TaHDZ genes in four HD-Zip subfamilies

Fig. 6

Expression profiles of TaHDZ genes in seedling roots under drought stress treatment. a Hierarchical clustering of the relative expression level of TaHDZ genes under drought stress treatment. The heat map was drawn in Log₁₀-transformed expression values. The red or green colors represent the higher or lower relative abundance of each transcript in each sample. b Expression patterns of TaHDZ genes under drought stress treatment. c The numbers of up-regulated and down-regulated TaHDZ genes in four HD-Zip subfamilies. d The ratios of up-regulated and down-regulated TaHDZ genes in four HD-Zip subfamilies

Fig. 7

Phenotype of the 35S:TaHDZ5-6A transgenic Arabidopsis. a RT-PCR analysis of TaHDZ5-6A transcript levels in the three transgenic lines. b Statistical analysis of survival rates after the drought-stress treatment. The average survival rates and standard errors were calculated based on data obtained from three independent experiments. Significant differences were determined by a t-test. *P < 0.05, **P < 0.01. c Drought tolerance of 35S:TaHDZ5-6A transgenic Arabidopsis. Photographs were taken before and after the drought treatment, and followed by a six-day period of re-watering. D Stomatal aperture of WT and 35S::TaHDZ5-6A transgenic plants under normal and drought conditions. e Statistical analysis of stomatal aperture of WT and 35S::TaHDZ5-6A transgenic plants. Values are mean ratios of width to length. Error bars represent standard errors of three independent experiments (n = 60). Bars, 10 μm. f Water loss from detached rosettes of WT and 35S::TaHDZ5-6A transgenic plants. Water loss was expressed as the percentage of initial fresh weight. Values are means from eight plants for each of three independent experiments. Significant differences were determined by a t-test. *P < 0.05, **P < 0.01. g Free proline content of WT and 35S::TaHDZ5-6A transgenic plants under normal and drought stress treatment

Fig. 8

Transcriptomic analyses of the 35S::TaHDZ5-6A transgenic Arabidopsis under normal condition. a venn diagrams of up- or down-regulated genes in transgenic plants relative to WT plants using a significance cutoff of P < 0.001, and a fold-change (FC) > 2. b Hierarchical clustering of up- or down-regulated genes in 35S::TaHDZ5-6A transgenic Arabidopsis lines relative to WT plants. The indicated scale is the log₂ value of the normalized level of gene expression. c Gene ontology of biological pathways (GOBPs) enriched in TaHDZ5-6A transgenic plants based on up or downregulated genes. d qRT-PCR analysis of drought induced genes in the transgenic and WT plants under normal and drought conditions. The error bars indicate standard deviations derived from three independent biological experiments. Significant differences were determined by a t-test. *P < 0.05, **P < 0.01