Characterization of WHIRLY1 functions in cereal crops

Chloroplasts are major sensors of environmental change, particularly stress perception. Plants growing in fluctuating environments require coordinated regulation of photosynthesis, growth and defence. The current lack of understanding of how chloroplast and nuclear gene expression are co-regulated under abiotic stress conditions is a major impediment to crop improvement. This project, which concerns the functional characterization of WHY1, is based on recent work in the applicant's laboratory that demonstrated this protein as an important player in chloroplast and nuclear biology, with roles in signalling between these organelles. WHY1 is a member of the WHIRLY family of single-stranded DNA-binding proteins that is found in both plastids and the nucleus, fits well into BBSRC's strategic priorities in the area of "sustainably enhancing agricultural production", particularly in the topic of "greater resilience of crops to abiotic stresses". WHY1 acts as a transcription activator for pathogen-related gene expression in the nucleus and as a repressor for the kinesin-like protein 1 that modulates telomere length. In chloroplasts, WHY1 is required for plastid genome stability and plastid gene transcription. However, much remains uncertain concerning how WHY1 participates in the regulation of leaf development and stress tolerance. Moreover, WHY1 appears to be essential for plastid ribosome biogenesis in maize but not in other species. This project combines the expertise of the Foyer lab on WHIRLY proteins with that of Biogemma in cereal transformation and the creation of new traits suitable for plant breeders. These studies will provide new knowledge concerning WHY1 functions in cereals and shed new light on chloroplast to nucleus retrograde signalling mechanisms under optimal and stress conditions. The objectives of this proposal are:
1) To produce and characterise transgenic RNAi wheat plants lacking WHY1. (Milestone 1: generation of transgenic wheat lines with altered WHY1 expression).
2) To generate and characterise transgenic maize plants over-expressing WHY1 in either the mesophyll or bundle sheath chloroplasts. (Milestone 2: generation of transgenic maize lines with altered WHY1 expression)
3) To determine the effects of modified WHY1 expression on plant responses to low nitrogen availability, drought and high light stresses to link these changes to chloroplast to nucleus signalling (Milestone 3: Generation of transcript and metabolite profile data).
The transgenic lines and wild types will be used together with why1 and why2 Mu transposon-induced maize mutants to explore the roles of the WHY1 protein in the chloroplasts and nuclei of mesophyll and bundle sheath cells during leaf development in absence or presence of stress (high light, drought, low nitrogen). The mechanisms of redox regulation of the WHY1 protein in chloroplasts will be characterised. In addition to physiology, biochemistry and cell biology approaches to study photosynthesis and leaf development, the student will use RNAseq and metabolite profiling on specific lines under selected conditions. qPCR techniques and screens with lincomycyin and other inhibitors will be used to characterise chloroplast to nucleus signalling pathways. The effects of WHY1 deficiency on root phenotypes will also be compared in wild type and transgenic lines grown under optimal and stress conditions. Field behaviour and yield impacts will be assessed in selected lines