Developmental roles of miR156/172-regulated transcription factors in barley

Cereal grain forms the basis of our food supply. During domestication and subsequent breeding, cereal architecture or body plan was often drastically modified to produce plants which produce higher grain yeilds. Growth of flowering plants, including cereals, reflects the progression of growth phases which influence architecture, broadly moving from vegetative juvenile growth to vegetative adult and finally to the reproductive phase. In temperate cereals, vegetative growth involves production of a main shoot giving rise to leaves and additional shoots, while in reproductive phase, shoots form a spike tip on which develop rows of grain. Although produced at the end of the life cycle, grain yield often reflects earlier developmental events. For instance, yield may be affected by the number of shoots which form fertile spikes and/or the amount of photosynthetic energy related to the number of leaves set. To meet the rapid, rising cereal demand, a sophisticated molecular understanding of genes regulating plant architecture is required for breeders to quickly and confidently select for better-performing crops - yet the depth of knowledge about gene function in temperate cereals such as wheat and barley, both dominant global crops, is especially thin, reflecting the traditional recalcitrance of these species to molecular study. However, recent accelerated generation of sophisticated genomic and molecular tools and resources for barley hold great promise to unlock the developmental genetics of temperate cereal architecture by using this crop as a developmental model.

My proposed research will capitalize on exciting work in other plant model systems which highlights the role of a conserved developmental phase network in the regulation of multiple yield-related architectural traits. This network is driven by antagonism between two microRNA (miRNA) gene families, miR156 and miR172, which encode short regulatory RNA molecules: miR156 is abundant early in the life-cycle to promote juvenile characteristics, like leaf production, however, over time miR156 levels fall, inhibited by rising levels of miR172, associated with adult and reproductive traits. These miRNAs function through repression of multiple target transcription factors, proteins that themselves regulate genes to control specific developmental traits. My research ambition is to understand which traits are controlled by individual miR156/172-regulated transcription factors in temperate cereals, in particular, barley, and how these factors control gene expression in order to inform future breeding efforts.

In fact, I have already found that a gene encoding a miR172-regulated transcription factor in barley is a master regulator of internode elongation in the stem and spike, thereby directly influencing grain density and plant height, both important agronomic traits. Height influences lodging (falling over) of top-heavy, grain-laden crops, making the control of plant height desirable. This gene is the first transcription factor implicated in internode growth in barley and I predict that it functions by controlling the expression of a suite of downstream genes, which could also be potential breeding targets. In this project, I will employ gene expression, biochemical and sequencing techniques to definitively identify these target genes. Moreover, I will examine possible interactions with other pathways involved in internode growth, as a first step towards building a regulatory gene network explaining internode growth. In addition, I will determine where and when other miR156/172-regulated transcription factors in barley are expressed. Finally, I will use powerful transgenic approaches to tease apart individual contributions to other traits controlled by these transcription factors. I anticipate that through this research, new genes involved in important traits for farmers will be identified and characterized, acting to inform crop breeding.

Understanding the molecular control of development in the temperate cereals, including wheat and barley, is essential for predictive, directed breeding strategies to meet projected demand in grain yield. Plants form their architecture over a progression of phases, each associated with specific developmental events, driven by an antagonistic network between two miRNA families, miR156 and miR172. Initial high levels of miR156 promote juvenile traits by repressing the SQUAMOSA-BINDING PROTEIN-like (SPL) family; over time miR156 declines as miR172 levels increase to promote adult and reproductive features through downregulation of the APETALA2 (AP2)-like family. Compelling evidence from multiple species suggest these factors regulate agronomic traits, yet we know little about individual functions in temperate cereals.

To tackle this knowledge gap, my proposal takes advantage of accelerated development of genomic resources to use barley as a developmental model. Firstly, I will capitalize on my work showing that a barley AP2-like gene, HvAP2, regulates internode length, a key agronomic trait. I will determine the interactions between HvAP2 and other genetic and environmental factors affecting internode growth. I will use integrated ChIP-seq and comparative microarray gene expression analyses to identify downstream mediators of HvAP2 function. These data will be integrated into the first model describing the genetic, mechanistic control of internode growth in a temperate cereal, which will then be functionally tested. Secondly, I will discern functional specialisation of individual HvAP2-like and HvSPL function and temporal kinetics of miRNA regulation by using bioinformatics, expression analyses and transgenics. Project outcomes will decipher mechanisms underlying genetic control of internode growth, highlight further downstream targets and reveal links between miR156/172-regulated transcription factors and specific traits.

This proposal seeks to uncover the genetic mechanisms regulating the development of barley, a key globel crop and a member of the cereals, grasses which provide the bulk of caliries to the human diet. Cereal production must increase by 33% of today's levels by 2050 to meet projected needs (FAO) and barley will play an important role in meeting this demand, especially as the most stress-tolerant of the grasses. Over the long term, sustainable yield increases must derive from significantly accelerated breeding of higher-yielding cereal architectures to avoid alternatives such as increased land and fertilizer use, directly relating to the BBSRC priority of Crop Science (Food Security). This research will advance these goals by characterizing genes and genetic mechanisms involved in morphological and developmental events underlying agronomically important traits, thus helping breeders accurately target beneficial alleles. These outputs will be relevant to: i) commercial plant scientists and cereal breeders ii) farmers and communities iii) the UK economy iv) general public and v) research staff.

i) This research is anticipated to significantly improve understanding of genetic regulation underlying numerous morphological traits of import for commercial plant scientists and cereal breeders involved in improving crop performance. Elucidation of the miR156/172-regulated transcription factor network is expected to resolve phase specific functions of individual family members, highlighting multiple potential breeding targets and their regulation by miRNA. Moreover, this project will reveal downstream mediators of HvAP2 control of internode elongation, providing further targets for this critical developmental event. Sequence resources currently available and the estimated increase in sequence information available across hundreds of cultivars by the end of this proposal mean that these loci can be targeted quickly.

ii) and iii) Barley contributes more than any other crop to Scotland's agricultural output, and serves as malt for whisky, the UK's most valuable food and drink export, contributing close to four billion pounds annually to the UK economy. By acting to molecularly-inform barley breeding, this project will contribute to sustainable economic success and increasing exports in the UK long beyond the end of this grant, thus profiting the livelihoods of famers and the enhancing the health of their communities and general public. This research is anticipated to benefit farmers and communities within Scotland and UK, but be relevant worldwide, reflecting the global dominance of cereals to world food supply.

iv) Moreover, this research has potential to inform the public about the genetic control of agronomic traits and its importance for enlightened breeding strategies. To that end, I am involved in science outreach efforts at the University of Dundee Botanic Garden, co-coordinating the creation of an outreach hub called the Genetics Garden whose aim is to highlight the link between phenotype, genetics and breeding. I will also pursue opportunities separately at public speaking series to discuss the importance of transgenic work for functional tests of gene action and as a part of breeding strategies for sustainable crop improvement.

v) Finally, this research will benefit the PDRA by offering training in the latest molecular biology techniques to interrogate transcription factor function and exposure to world-class crop research. A current PhD student who started in Oct 2012 as well as several honours students will also benefit from this training over the course of the project. In addition, I will ensure that all staff and students develop transferable skills, such as scientific writing and presentation skills as well as bioinformatic literacy either through training on site or through workshops within the UK.