emilie.knight@jic.ac.uk

Over the last few decades, bread wheat (Triticum aestivum) has been selected for high yield. This has come at a cost because of the trade-off between yield and protein content. We are interested in improving grain protein content without hindering yield. To this purpose, we study the processes involved in wheat senescence (when nutrients get remobilised from the vegetative tissues to the maturing spike); the timing of which directly impacts yield and protein content.

Here we investigate the potential role in senescence of three transcription factors (TF), which stood out in a gene regulatory model of senescence: NAC3 and two RWP-RK genes; using TILLING mutants, gene editing and a protoplast-based method to identify the downstream target genes of these TF.

NAC3 was found to interact with NAM-B1, a known senescence regulator, through a yeast two-hybrid experiment. Double mutants of NAC3 obtained from the crossbreeding of two tetraploid TILLING lines with a deleterious mutation for each copy of the gene showed a delay in senescence. To confirm the role of NAC3 in regulating senescence, we have generated gene edited wheat plants cv. Cadenza, using sgRNAs targeting all three copies of NAC3. We have obtained several T0 plants with edits in all 3 copies of NAC3. Senescence phenotyping of homozygous knock-out T1 plants will be carried out in the next few months.

RWP-RK genes are regulators of nitrogen metabolism and are potential new candidate genes in the senescence process. Indeed, one of these genes has predicted shared targets with NAM-A1, a TF known to have a key role in senescence. We are generating double and triple knock-out mutants in tetraploid (cv. Kronos) and hexaploid (cv. Cadenza) wheat plants, using TILLING lines. Senescence phenotyping of these mutants is underway and shows a trend towards an involvement in the senescence process.

Finally, we are adapting the TARGET method from Arabidopsis to accurately identify targets of TF of interest within wheat cells. Protoplasts are transformed with a construct carrying a TF known to be involved in senescence.

Transformed cells are selected and treated with chemicals causing the TF to move to the nucleus or not, thereby allowing for RNA sequencing to determine true primary targets of the TF and eliminating secondary targets and background noise.

These experiments will identify new genes to manipulate senescence and provide a step towards improving yield and grain protein content simultaneously.