Mechanisms underlying variation in barley hull adhesion

This research explores a central but little studied problem in plant biology - how do plant surfaces influence plant architecture? We know that the protective cuticle covering the plant body, beyond preventing water loss and pathogen invasion, also ensures that closely growing plant organs remain separate and plays important roles in organ absicssion. However, less is known about the biology underlying plant interfaces that fuse, such as in tubular flowers, or organs that stick together as in the barley grain. In the latter, a species-specific pathway leads to secretion of a special cementing layer onto the outer pericarp cuticle. Loss of this layer, caused by mutations in a single master regulatory factor, NUDUM (NUD), occurred once during barley cultivation, leading to a complete loss of hull adherence or 'naked' grain, ideal for human consumption. However, most barley grown in the UK is used for animal feed and malt where 'covered' grain, retaining the adherent hull, is preferred as hulls protect the germ and aid in filtration after malting. Thus, the changing relationship of the hull to the grain is a critical quality that largely determines barleys downstream uses. Despite this, we understand practically nothing about the steps between NUD expression and the extrusion of the cementing material on the pericarp or the chemistry explaining the adherent properties of the layer.

Genetic variation in newer elite barley malting cultivars is linked to an increasing incidence of a highly undesirable, intermediate phenotype whereby grain partially sheds its hull during harvest or processing, a phenomenon called 'skinning'. However, identifying the causal variation underlying skinning has proved very difficult, due to its environmental sensitivity and lack of robust screening methods, so to date breeders do not have genetic markers to help control this trait. In addition to addressing these important agronomic concerns, we are interested in identifying the genes and genetic mechanisms underlying skinning since these alleles may represent defective steps along unresolved NUD-driven pathway(s). Moreover, by characterising the molecular and chemical changes occuring in skinning, we may reveal the critical features of the cementing layer and/or other grain characters which influence hull adhesion that are lost in skinning mutants.

To circumvent issues of studying the cultivated germplasm for quantitative skinning variation, we have assembled a panel of mutants with stable skinning phenotypes. We screened a wax-deficient collection of mutants in a single near-isogenic background and identified a small subset that show defective hull adhesion. This foundation work provides a robust and genetically powerful platform to dissect the molecular, chemical and genetic mechanisms that explain variation in hull adhesion.

Our panel suggests that specific components of the surface lipid regulatory pathway may be defective in grain that skins. In this proposal, we seek to define the chemical and ultrastructural surfaces changes associated with hull adhesion and how they are altered by skinning loci. We will also reveal related changes in gene expression, both globally and on a tissue-specific level, that promote hull adherence and assess their importance to the skinning phenotype. Furthermore, we will identify individual genes that control skinning and evaluate diversity at these loci in cultivated germplasm, a critical milestone which will resolve which allelic variants track with incidence of skinning and allow us to develop markers for use in breeding. Taken together, our work will reveal the chemical and genetic components that cause partial loss of adherent hulls in barley, closing a vast knowledge gap about a critical grain quality trait, and delivering routes to improved germplasm selection

This project aims to discover the genetic and molecular processes that contribute to the distinctive adherent hull of barley grain. This research not only addresses fundamental questions about adhesion between plant surfaces, but is also relevant to a critical, unfavourable feature of many elite barley malting cultivars which have a genetic defect known as 'skinning' (partial hull shed) which can result in otherwise premium grain being downgraded to feed. Mutations in a single master factor called NUD result in failure to secrete the so-called cementing layer that normally sticks the hull tightly to the developing seed. One possibility is that NUD coordinates expression of genes responsible for the cementing layer and that skinning is the manifestation of defective alleles of these genes. Due to its intermediate, quantitative nature and acute environmental sensitivity, resolving the genetic basis of natural variation in skinning is difficult. We have identified a series of barley mutants in a near-isogenic background that exhibit partial skinning phenotypes. Although the mutated genes are unknown, our skinning mutants are classified as wax-deficient, consistent with our hypothesis that these mutated genes are defective variants of the genes involved in making the cementing layer. They are thus powerful tools for unravelling the complex and commercially important processes that underpin the skinning phenotype. We propose to use these mutants to investigate the structural, chemical and developmental properties that contribute to hull adhesion. We will: define the critical developmental stages, morphologies and cuticular wax chemistries associated with hull adherence; explore the temporal and spatial transcriptional programme associated with adhesion and adhesion defects; identify the mutated genes in the partial skinning near-isogenic lines, explore their contribution to skinning in modern barley cultivars, and develop diagnostics for use in breeding.

This proposal will reveal the mechanisms that control hull adhesion in barley, a feature strongly relevant to the barley breeding, farming and processing industries, especially those on the premium end of the market (malting). A critical issue with many recently released cultivars is that the grain "skins" during harvest. Skinning occurs when the hull fails to adhere tightly to the grain, partially sheds and causes variability in malting and poor downstream processing performance. It can lead to grain lots being rejected at intake to the malthouse. Despite its importance, the mechansisms underlying variation in hull adherence are understudied with the causal genes remaining unknown. This partly reflects subjective phenotyping methods combined with a large environmental effects generating sizable phenotypic plasticity. Our proposed research programme will quantitatively, chemically and structurally characterise both normal and defective hull adhesion and identify its genetic causes, addressing a major problem for industry while providing fundamental insight into the mechanisms of organ adhesion in plants.

As illustrated clearly in the letters of support, our research is relevant to:
i) Breeders. Breeders currently have no way to select for skinning resistant varieties in their breeding program. Defining the contributions made by different alleles of genes that control skinning would enable development of diagnostic molecular markers that can be routinely deployed to control this trait during breeding.
ii) Growers. Losing the premium payment for malting barley (grain for animal feed is worth less) is a serious financial disincentive to growers which can make growing barley uneconomical. However, the level of skinning is hard to predict because of the environmental influence. We will produce practical outputs that allow the development of varieties that do not skin.
iii) Maltsters. Maltsters reject grain lots that suffer excessive skinning and need to find alternative grain sources, causing a financial knock-on all along the supply chain. In addition to breeding tools, our plans to develop a quantitative screening tool - using a barley pearler - could remove subjectivity for maltsters.
iv) Brewers and Distillers. Years when skinning is widespread and prevalent can lead to lack of local supply of high quality malting barley and subsequent increased costs.
v) Public. Pathways to Impact Projects will contribute to better understanding of plant biology and engage with the wider research community. This research has the potential to inform the public about the genetic control of agronomic traits and, in PtI Project 2, its importance for enlightened breeding strategies.
vi) RCo-I Campoli will benefit by continuing her career in cereal genetics and gaining skills in cutting edge biochemical, genetic and bioinformatics analyses. A newly started PhD student and honours students will also benefit from training over the course of the project. Dr. McKim (PI) will ensure that all staff and students develop transferrable skills, such as scientific writing and presenting.
vii) UK economy. Processed barley products contribute more than any other crop to the UK food and drink sector, with the premium 30% of the national barley crop ultimately contributing close to 20 billion pounds annually to the UK economy through beer and whisky. Non-skinning barley varieties will safeguard this important national industry.

We will ensure that developers, growers and users of premium quality barley benefit from the proposed research through the extended reach of the IBH. IBH has cultivated strong links with the international breeding community and has enhanced links to the farming, food and drink sectors. The PI's are well known within the academic sector and have a strong reputation and identity within the global community. Together, we have the relevant expertise, track-record and motivation to ensure the project deliverables are achieved.