The Generation Gap - Mechanisms of maternal control on grain

Understanding grain production in cereals is vital to safeguard food security in our changing world. Barley is a widely grown global cereal of vast economic importance to our country, and a powerful experimental system to identify and characterise processes that influence grain yield and quality. We recently revealed the importance of two master regulatory factors in barley which alter the balance between growth and survival of protective and nutritive maternal tissues versus the starchy, calorie-rich filial tissues filling the grain. We hypothesise that shifting this balance away from maternal tissues by limiting their size and promoting their elimination, provides the nutrients and space needed to increase grain size and weight. We speculate that the regulation of this balance may differ between wild and cultivated barleys, potentially explaining improved grain of cultivated barley. We predict that this process works by controlling gene expression and signalling across grain tissues, but the exact mechanisms remain unclear.

Part of the reason for this knowledge gap is that testing our predictions is tricky: grain is a mix of these tissues and regulatory factors act both early and late, complicating and confounding accurate assessments of tissue and time-specific events and their effects on grain. We propose to surmount these challenges by applying two leading-edge technologies in barley. First, we will use single cell RNA sequencing to measure gene expression within individual cells followed by computational clustering to build cell populations and analysis of how these change throughout grain development. Second, we will apply new inducible expression systems to change the activity of regulatory factors at specific times to tease apart the importance of early versus late functions on grain parameters. We will also combine these approaches to assess the influence of regulatory factors activated in specific tissues on the gene expression in neighbouring and distant tissues. Finally, we will explore how these cell populations differ between wild and cultivated barleys and test the functional relevance of wild and cultivated regulatory factors to grain parameters. Taken together, our work will define the developmental trajectories of grain tissues and their responses to regulatory factors controlling maternal versus filial growth and survival, significantly advancing our understanding of cereal grain development.

This proposal investigates the contribution of pre- and post-fertilisation developmental events in maternal tissues to grain development in barley. We build on our recent discovery that two transcription factors in barley, HvAP2 and HvMADS29, control grain size and shape, and regulate the growth, differentiation and/or death of the hull and ovary tissues. Here, we will evaluate a potential mechanistic relationship of maternal regulators to coordinate tissue growth and survival in the grain.
We hypothesise that tissue transcriptomes will reflect this relationship and predict that these are responsive to regulation by HvAP2 and HvMADS29. To test this prediction we will define, for the first time, cell-specific transcriptomes within pre- and post-fertilisation cereal ovaries by single cell RNAseq (scRNAseq) and computational clustering to build cell populations with similar transcriptomic states and analyse how these states change over time and in response to loss of HvAP2 or HvMADS29 function. Furthermore, we will exploit a heat-inducible Cre-lox gene expression system to restore maternal regulators at specific developmental stages in loss of function mutants to assess responses in the grain size and shape and in cell-specific transcriptomes.
We also hypothesise that grain shape differences in wild compared to cultivated barley may reflect altered maternal tissue developmental processes. We will explore this idea by characterising maternal tissue development by histology approaches followed by scRNAseq on wild barley tissues. Comparative analyses with cultivated grain will reveal conserved and diverged developmental transcriptomic trajectories which may contribute to differences in wild compared to cultivated grain. We will select diverse allelic variation in wild maternal tissue regulators and assess their function in cultivated germplasm by accelerating introgression lines.