Overexpression of the class I homeodomain transcription factor TaHDZipI-5 increases drought and frost tolerance in transgenic wheat

Abstract

Characterization of the function of stress-related genes helps to understand the mechanisms of plant responses to environmental conditions. The findings of this work defined the role of the wheat TaHDZipI-5 gene, encoding a stress-responsive homeodomain-leucine zipper class I (HD-Zip I) transcription factor, during the development of plant tolerance to frost and drought. Strong induction of TaHDZipI-5 expression by low temperatures, and the elevated TaHDZipI-5 levels of expression in flowers and early developing grains in the absence of stress, suggests that TaHDZipI-5 is involved in the regulation of frost tolerance at flowering. The TaHDZipI-5 protein behaved as an activator in a yeast transactivation assay, and the TaHDZipI-5 activation domain was localized to its C-terminus. The TaHDZipI-5 protein homo- and hetero-dimerizes with related TaHDZipI-3, and differences between DNA interactions in both dimers were specified at 3D molecular levels. The constitutive overexpression of TaHDZipI-5 in bread wheat significantly enhanced frost and drought tolerance of transgenic wheat lines with the appearance of undesired phenotypic features, which included a reduced plant size and biomass, delayed flowering and a grain yield decrease. An attempt to improve the phenotype of transgenic wheat by the application of stress-inducible promoters with contrasting properties did not lead to the elimination of undesired phenotype, apparently due to strict spatial requirements for TaHDZipI-5 overexpression.

Keywords: 3D protein modelling; abiotic stress; activation domain; phenotypic features; protein homo- and hetero-dimerization; stress-inducible promoters.

Figure 1

A rectangular phylogenetic tree displaying the evolutionary relationships of HD‐Zip I γ‐clade TFs from Arabidopsis and selected monocots. Abbreviations of species: At, Arabidopsis thaliana; Os, Oryza sativa; Sb, Sorghum bicolor; Ta, Triticum aestivum; Zm, Zea mays.

Figure 2

Characterization of the TaHDZipI‐5 gene. (a) Transcript numbers of the TaHDZipI‐5 gene in wheat tissues were estimated by Q‐PCR. (b) Mapping of cis‐elements responsible for the abscisic acid (ABA)‐dependent activation of the TdHDZipI‐5A promoter, using a transient expression assay in wheat cell culture. Depicted is a schematic representation of ABA‐responsive element (ABRE) and MYB responsive (MYBR) cis‐elements in four promoter deletions (D1–D4) of the TdHDZipI‐5A promoter, and the graph shows activation of GUS expression by the deletions detected in a transient expression assay, in the presence (black bars) or absence (control; grey bars) of 0.5 mm ABA in the culture medium. Error bars were calculated from three technical replicates.

Figure 3

Identification of the TaHDZipI‐5 activation domain (AD) using an in‐yeast activation assay. (a) Transcription activity determined through the in‐yeast activation assay, where homeodomain (HD), Zip and AD designate putative homeodomain, leucine zipper and AD, respectively. Amino acid residues at the beginning and end of each truncated protein are indicated with numbers. (b) Conserved sequences of the identified AD in TaHDZipI‐5 and close homologues from other monocots. Ta, Triticum aestivum; Zm, Zea mays; Os, Oryza sativa; Sb, Sorghum bicolor. Amino acid residues, which are the same in more than the half of the investigated sequences, are in bold.

Figure 4

Molecular features of homeodomains (HDs) of TaHDZipI‐3 and TaHDZipI‐5 in homo‐ and hetero‐dimeric forms in complex with the HDZ1 cis‐element. (a) A sequence alignment of TaHDZipI‐3 and TaHDZipI‐5 HDs and of even‐skipped HD from Drosophila melanogaster (PDB: 1JGG). Identical amino acid residues are coloured based on their properties. α‐Helical secondary structural elements are indicated with ‘h’ below the sequences. HD residues that interact with DNA cis‐elements are indicated by inverted triangles (▼). (b) Ribbon representations of homo‐dimeric TaHDZipI‐3 and TaHDZipI‐5, and hetero‐dimeric TaHDZipI‐3/TaHDZipI‐5 models in complex with the HDZ1 cis‐element; blue descriptions and atomic colour representations are used for HDZ1. The ribbons of TaHDZipI‐3 and TaHDZipI‐5 are coloured in blue and yellow, respectively. DNA‐interacting residues are shown in sticks, and DNA sugar‐phosphate backbones are coloured in cpk‐orange. Water molecules are shown as red spheres. Interactions (less than 3.5 Å; Table S3) between residues and HDZ1 are shown in black dashed lines. Arrows point to differences in folding of α‐helices between TaHDZipI‐3 and TaHDZipI‐5.

Figure 5

Growth characteristics and yield components of control wild‐type (WT) (Triticum aestivum cv. Gladius) and T₃ transgenic wheat transformed with pUbi‐TaHDZipI‐5 under well‐watered (black boxes) and under drought conditions (grey boxes). Flowering time of transgenic plants was compared to the average flowering time of 16 control WT plants, which is represented as day 0. Differences between transgenic lines and WT plants in each of the well‐watered and drought conditions were tested using unpaired Student's t‐tests (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

Comparison of drought and frost tolerance of wild‐type (WT) (Triticum aestivum cv. Gladius) and transgenic wheat transformed with pUbi‐TaHDZipI‐5. (a) Drought tolerance of two independent transgenic lines (T₂ progeny), sizes of which were similar to those of the control WT plants’, is shown as the survival rate of plants recovered after the terminal drought stress, followed by rewatering. (b, c) Survival rates of plants recovered after seedling‐stage frost: (b) T₁ progeny and (c) T₃ progeny of transgenic plants. Error bars represent ± SD of three independent experiments. Values represent means ± SE (n varies for each column and is shown in each case directly on the graphs). Significant differences between transgenic lines and WT plants were tested using an unpaired Student's t‐test (*P < 0.05). (d) The expression levels of the TaHDZipI‐5 transgene in leaves of T₃ plants in unstressed control WT and transgenic plants, sampled prior to frost treatment. Error bars represent ± SD of three technical replicates.

Figure 7

Characteristics of transgenic wheat transformed with pWRKY71‐TaHDZipI‐5. (a) Comparisons of plant growth and yield characteristics of wild‐type (WT) and transgenic T₂ plants under well‐watered conditions (black boxes) and mild drought (grey boxes). (b) Transgene expression levels in control WT and transgenic T₄ plants at 23 °C (control) and 4 °C (cold). (c) Frost tolerances of WT and transgenic wheat transformed with pWRKY71‐TaHDZipI‐5 are shown as the survival rate of plants recovered after the terminal frost treatment. Error bars represent ± SD for three independent experiments. Differences between transgenic lines and WT plants were tested in the unpaired Student's t‐test (*P < 0.05, **P < 0.01, *** for P < 0.001).

Figure 8

Characteristics of transgenic wheat transformed with pCor39‐TaHDZipI‐5. (a) Comparison of plant growth and yield characteristics of wild‐type (WT) and transgenic T₂ plants, under well‐watered conditions (black boxes) and mild drought (grey boxes). (b) Transgene expression levels in WT and transgenic T₄ plants at 23 °C (control) and 4 °C (cold). (c) Frost tolerance of WT and transgenic wheat transformed with pCor39‐TaHDZipI‐5 is shown as the survival rate of plants recovered after the terminal frost treatment. Error bars represent ± SD for three independent experiments. Differences between transgenic lines and WT plants were tested in the unpaired Student's t‐test (*P < 0.05, **P < 0.01, *** for P < 0.001).