The role of genes encoding wheat HD-Zip I transcription factors in response to drought and frost in transgenic wheat

Abstract

Drought and frost are common abiotic stresses that negatively affect plant development, growth and yield worldwide. To survive, plants have their own defence mechanisms to adapt to harsh environmental conditions. Transcription factors (TFs), as regulators of gene expression, compose an important part of the plant defence system. TFs from homeodomain leucine zipper class I (HD-Zip I) are suggested to participate in regulatory networks of gene expression to abiotic stresses in plants. However, little information is known about the roles of HD-Zip I TFs in response to abiotic stresses in wheat. Hence, the roles of three wheat genes TdHDZI-3, TdHDZipI-4 and TaHDZipI-5, encoding γ-clade TFs of HD-Zip I were characterised in transgenic wheat. In this study, the TaHDZipI-5 gene product showed its transcriptional activating activity and its activation domain located at the C-terminus using an in-yeast trans-activation assay. The TaHDZipI-5 TF was suggested to have homo-dimerization ability and may hetero-dimerize with TaHDZipI-3. The differences between DNA interactions in homo- and hetero-dimers were analysed using 3D models, and the hetero-dimerisation was indicated to be more stable than the homo-dimerisation form. The overexpression of TaHDZipI-5 transgene driven by the constitutive promoter Ubiquitin, conferred drought and cold tolerance to transgenic wheat. However, a series of undesired phenotypic features, such as reduced plant size and biomass, delayed flowering and decreased grain yields, occurred in transgenic plants with constitutive overexpression of the TaHDZipI-5 transgene. Two stress-inducible promoters, OsWRKY71 and TdCor39, which were previously employed to avoid the negative influences of TaDREB3 gene expression on phenotype and yield in transgenic barley, were then used for relieving the negative effects made by the overexpression of the TaHDZipI-5 transgene. The attempt to improve the phenotypes of transgenic wheat was not as successful as intended. A possible reason might be that the selected promoters may not meet strict spatial requirements for the overexpression of the TaHDZipI-5. However, the inducible overexpression of TaHDZipI-5 driven by each of the promoters conferred frost tolerance to transgenic wheat. In the second part of the study, promoters of genes encoding HD-Zip I TFs TdHDZipI-3 (HDZI-3) and TdHDZipI-4 (HDZI-4), were characterised under drought and cold stresses using transgenic wheat. A novel CBF/DREB transcription factor protein TaCBF5L was isolated from roots of drought-stressed wheat, using Y1H method with DRE cis-element as a bait. The TaCBF5L gene was transformed into wheat plants for the characterisation of the HDZI-3 and HDZI-4 promoters. Based on the phenotypic results, the TaCBF5L transgene driven by HDZI-4 significantly improved grain yield, seed number and biomass under severe drought. In addition, both HDZI-3 and HDZI-4 promoters improved cold tolerance of transgenic wheat by increasing the plant survival rates, though no improvement was observed in plant yield phenotypes under well-watered conditions or moderate drought. To better understand the activity of the two promoters in transgenic wheat under different stresses, we tried to find downstream genes of TaCBF5L. Several stress-related genes encoding LEA/DHN/COR were found to be downstream regulated by TaCBF5L under severe drought and cold conditions. It is notable that these potential downstream genes showed different expression under different abiotic stresses. It is probably because TaCBF5L co-operates with other TFs under drought and frost conditions, which could result in divergent expression of downstream genes. In summary, the functions of the three γ-clade HD-Zip I wheat genes were characterised under drought and cold conditions, which could further explain how wheat plants give response to stress. Besides, these genes can be used as potential tools in bio-engineering to improve the plant stress-tolerance and grain yields under hostile environment. However, the interactions of the genes and corresponding TFs comprise a complicated transcriptional network and the mechanisms still need to be further explored in the future.