Generation of drought-resistant transgenic cereals using transcription factors isolated from wheat grain

Tese de doutoramento em Ciências da Saúde, ramo de Medicina, na especialidade de Medicina Interna (Cardiologia), apresentada à Faculdade de Medicina da Universidade de Coimbr

Complex regulation by Apetala2 domain-containing transcription factors revealed through analysis of the stress-responsive TdCor410b promoter from durum wheat

Expression of the wheat dehydrin gene Cor410b is induced several fold above its non-stressed levels upon exposure to stresses such as cold, drought and wounding. Deletion analysis of the TdCor410b promoter revealed a single functional C-repeat (CRT) element. Seven transcription factors (TFs) were shown to bind to this CRT element using yeast one-hybrid screens of wheat and barley cDNA libraries, of which only one belonged to the DREB class of TFs. The remaining six encoded ethylene response factors (ERFs) belong to three separate subfamilies. Analysis of binding selectivity of these TFs indicated that all seven could bind to the CRT element (GCCGAC), and that three of the six ERFs could bind both to the CRT element and the ethylene-responsive GCC-box (GCCGCC). The TaERF4 subfamily members specifically bound the CRT element, and did not bind either the GCC-box or DRE element (ACCGAC). Molecular modeling and site-directed mutagenesis identified a single residue Pro42 in the Apetala2 (AP2) domain of TaERF4-like proteins that is conserved in monocotyledonous plants and is responsible for the recognition selectivity of this subfamily. We suggest that both DREB and ERF proteins regulate expression of the Cor410b gene through a single, critical CRT element. Members of the TaERF4 subfamily are specific, positive regulators of Cor410b gene expression.Omid Eini, Nannan Yang, Tatiana Pyvovarenko, Katherine Pillman, Natalia Bazanova, Natalia Tikhomirov, Serik Eliby, Neil Shirley, Shoba Sivasankar, Scott Tingey, Peter Langridge, Maria Hrmova, Sergiy Lopat

Characterization of the wheat gene encoding a grain-specific lipid transfer protein TdPR61, and promoter activity in wheat, barley and rice

The TaPR61 gene from bread wheat encodes a lipid transfer protein (LTP) with a hydrophobic signal peptide, predicted to direct the TaPR61 protein to the apoplast. Modelling of TaPR61 revealed the presence of an internal cavity which can accommodate at least two lipid molecules. The full-length gene, including the promoter sequence of a TaPR61 orthologue, was cloned from a BAC library of Triticum durum. Quantitative RT-PCR analysis revealed the presence of TaPR61 and TdPR61 mainly in grain. A transcriptional TdPR61 promoter-GUS fusion was stably transformed into wheat, barley, and rice. The strongest GUS expression in all three plants was found in the endosperm transfer cells, the embryo surrounding region (ESR), and in the embryo. The promoter is strong and has similar but not identical spatial patterns of activity in wheat, barley, and rice. These results suggest that the TdPR61 promoter will be a useful tool for improving grain quality by manipulating the quality and quantity of nutrient/lipid uptake to the endosperm and embryo. Mapping of regions important for the promoter function using transient expression assays in developing embryos resulted in the identification of two segments important for promoter activation in embryos. The putative cis-elements from the distal segment were used as bait in a yeast 1-hybrid (Y1H) screen of a cDNA library prepared from the liquid part of the wheat multinucleate syncytium. A transcription factor isolated in the screen is similar to BES1/BLZ1 from Arabidopsis, which is known to be a key transcriptional regulator of the brassinosteroid signalling pathway.Nataliya Kovalchuk, Jessica Smith, Natalia Bazanova, Tatiana Pyvovarenko, Rohan Singh, Neil Shirley, Ainur Ismagul, Alexander Johnson, Andrew S. Milligan, Maria Hrmova, Peter Langridge and Sergiy Lopat

Enhancing abiotic stress tolerance in plants by modulating properties of stress responsive transcription factors

Drought, heat and other abiotic stresses negatively impact growth, development, yield and seed quality of plants. The perception of stress and later adaptation to it occurs via signal transduction pathways that regulate expression of stress-responsive genes. Products of these genes include proteins that are directly involved in plant protection and those that fulfill regulatory function. The latter group includes transcription factors (TFs) and other transcription-related proteins that are investigated using the tools of forward and reverse genetics. Genomics analyses also revealed the importance of other proteins such as protein kinases and phosphatases, enzymes involved in metabolism of phospholipids, signalling molecules, etc. Once the stress response pathways are described, the role of key players in these pathways can be optimised through allele mining, selection and genetic engineering. These approaches offer alternatives to classical breeding and marker-assisted selection. During plant responses to drought, a set of basic leucine zipper (bZIP), homeodomain-leucine zipper (HD-Zip) and WRKY TFs are transcriptionally or post-translationally activated via abscisic acid (ABA)-dependent signal transduction pathways. Despite a surge of data on the significance of plant bZIP, HD-Zip and WRKY TFs in the regulation of drought responses, the three-dimensional (3D) structures of these classes of TFs have been poorly defined. This structural information can be used for rational design of variant TFs that can help in understanding their oligomerisation and post-translational modification patterns, as well as their abilities to recognise target DNA sequences. In turn, this knowledge would permit the commercial application of genetically engineered TFs in agricultural biotechnology, by expression of TF variants and using the wild-type or modified promoter regions of stress-responsive genes. To this end, the aim of this review is to discuss strategies for improving tolerance of cereals to drought and other environmental stresses using molecular variants of the abiotic stress responsive TFs.Maria Hrmova and Sergiy Lopat