Regulation of wheat inflorescence architecture by a cation amino acid transporter

An inflorescence is a group of grain-producing flowers that form on a complex arrangement of branches. Domestication and modern breeding have harnessed diversity of branch numbers and arrangements that form on an inflorescence to increase grain production, which has frequently involved modifying the activity of genes that control inflorescence development. This approach has been under-utilised in wheat as very few genes are known to regulate inflorescence architecture in this crop - we aim to identify genes that control wheat inflorescence development to help optimise yield related traits that will contribute to the 60-70% increase in global grain production required by 2050.

The research proposed here will build on our group's breakthrough discovery that a mutation in a gene encoding CATION AMINO ACID TRANSPORTER1 (CAT1) dramatically effects inflorescence development in wheat, and the arrangement of branches, or spikelets, that form on the inflorescence. By deciphering the role of CAT1 during inflorescence development, the project will uncover a previously unknown role for amino acid transporters in plant reproductive biology, and will advance our understanding of molecular processes that regulate yield-related traits in cereals.

We identified CAT1 as a protein that influences inflorescence architecture by performing a genetic screen to identify mutants that form an alternate arrangement of spikelets, known as 'paired spikelets'. One mutant contained mutation in the copy of CAT1 on the D genome (CAT-D1), which promotes paired spikelet development with a dominant inheritance pattern. Plants with one copy of the mutated allele (heterozygous) and two mutated copies (homozygous) form paired spikelets, and homozygous mutants display additional developmental phenotypes including severe leaf curling, additional vascular bundles (transport system within plants) and perturbed lateral root development. There is a copy of CAT1 on the B genome (CAT-B1), but not the A genome.

We will investigate the amino acid transporter activity of CAT-D1 and CAT-B1 to test their functional conservation as proteins that localise to the cell membrane and transport cationic amino acids, and to assess their ability to transport the growth hormone, auxin (Objective 1). The effect of the missense mutation on CAT-D1 function will be investigated by assessing the ability of the mutant protein to transport amino acids and/or auxin, relative to the wild-type protein, using diverse experimental assays (Objective 2). We will investigate genes and molecular processes that act downstream of CAT-D1 to regulate inflorescence development by studying the involvement of auxin response pathways, which is supported by outcomes of our preliminary RNA-seq data (Objective 3). Finally, we will investigate the genetic and environmental regulation of CATD1- dependent control of inflorescence architecture by analysing the interaction between CAT1 with Photoperiod-1 and FLOWERING LOCUS T1, which are major flowering-time genes that regulate spikelet development (Objective 4).

Knowledge and resources (e.g. germplasm, molecular markers) developed during the project will benefit researchers investigating molecular processes that control reproductive and developmental biology of plants, and those studying amino acid transporters in other organisms. The outputs will benefit publicly-funded and commercial breeding programmes by improving our understanding of genes that regulate inflorescence development in wheat, which will also benefit barley, rice and maize breeders. The outcomes will ultimately benefit growers and consumers by contributing to improved food security for the world's growing population, which will enhance society's quality of life and boost the UK economy.

The project will investigate the molecular function of a putative cation amino acid transporter, CATION AMINO ACID TRANSPORTER 1 (CAT1), and its role during inflorescence development in wheat. We will examine the function of CAT1 from the D and B genomes of hexaploid wheat (CAT-D1 and -B1), and determine how a serine to leucine missense mutation (S240L) in CAT-D1 promotes formation of additional 'paired' spikelets.

To investigate protein function and the effect of the S240L mutation, we will investigate of the cellular location of CAT-D1 and CAT-B1, and assess the ability of CAT1 to transport amino acids and/or auxin using yeast complementation and Xenopus-based experiments. We will compare these results to identical assays using the allele that encodes a protein with the S240L mutation. To complement this analysis, we will measure amino acid and auxin levels in inflorescences, roots and leaves from heterozygous and homozygous mutant plants, relative to wild-type.

To investigate the molecular pathways that regulate spikelet development down-stream of CAT-D1, we will analyse genes involved in auxin response pathways that we have identified to be differentially expressed in the cat-d1 mutant plants. We will also generate transgenic plants containing a DR5:VENUS3X reporter to investigate the effect of the mutant cat-d1 allele on auxin responses.

We will investigate the genetic and seasonal regulation of CAT1 expression using variant alleles of major flowering genes, Photoperiod-1 and FT1, and growth of plants under different photoperiods. We will introgress insensitive and knock-out alleles of Ppd-1 and FT1 into the cat-d1 mutant lines and analyse paired spikelet development and inflorescence architecture traits. To analyse seasonal regulation of CAT1, we will measure CAT-D1 and -B1 transcript levels in leaves and inflorescences of plants grown under field conditions at photoperiods defined by 1-hr increments in day-length (e.g. 10 hr light/14 hr dark

The proposed research will generate impacts in multiple ways, which are described below:

INDUSTRY - Breeders will benefit from the discovery of amino acid transporters as proteins that contribute to inflorescence development as it will introduce CAT1 as a gene that can be targeted to optimise yield-related traits. I expect that demonstration of CAT1's role as an amino acid transporter in wheat will support follow-on research that will analyse variant alleles from diverse germplasm, to identify genetic variation that improves amino acid transport; this outcome is significant given that amino acids are an important component of nitrogen uptake in cereals, and is supported by CAT1 associating with a QTL for amino acid content in maize. We will deliver knowledge and resources to breeders in the UK (e.g. KWS-UK, Limagrain, RAGT, Syngenta) and globally (e.g. CIMMYT) through our ongoing interaction with breeding companies and our aligned project with the International Wheat Yield Partnership. Impacts will extend beyond wheat, as our research will be the first to demonstrate the key role of amino acid transporters during inflorescence development in plants - the project outcomes will therefore impact breeders of other major cereals including maize, rice and barley. The PDRA and I will attend maize, rice and barley breeding conferences to transfer the knowledge gained from the project, with the aim of collaborating to uncover CAT1 allelic variation across cereals that may be used to improve the performance of each crop.

Our analysis of the effect of the S240L missense mutation on CAT-D1 function is likely to have impacts for medical biotechnology, as the transmembrane domain that contains S240 shares strong sequence homology with the human homologue (SLC7A1). Mutations in SLC7A1 predispose to fungal infections of the skin, and other amino acid transporters (or solute carriers) have important roles in cancer biology. We will attend conferences in membrane transporter biology to disseminate our results to the broad amino acid transporter community and to form collaborations for future research on CATs.

SOCIETY - Wheat is the UK's most abundantly produced and valuable crop, and is the most widely grown and second most abundant cereal cultivated world-wide; however, new genetic traits need to be developed to increase productivity and sustainability in the face of higher food prices, climate change and natural resource depletion. The project will contribute to improving global food security by identifying genes that regulate key yield-related traits, which will help breeders develop strategies to optimise grain production.

We will disseminate outcomes to the general public, school children and university students to increase awareness of the potential for society to benefit significantly from advances in science, and to inspire the next generation of plant scientists. Dissemination will occur through social media, press releases and our continued participation in outreach events such as Café Science.

EDUCATION - The multi-disciplinary approach to investigate wheat inflorescence development will help strengthen national research capacity by training researchers with unique skills in laboratory- and field-based research, who can move into jobs in industry or government.

We will also contribute to the education and training of junior breeders through our lectures and practical sessions at courses such as the Wheat Genetics Course of the BBSRC-funded Designing Future Wheat ISP. We will also organise a wheat development course to be run alongside the UK's premier small grains cereal meeting (Monogram), with the aim of encouraging more Masters, PhD students and post-doctoral scientists to undertake research in wheat.