Modified function of a stem cell regulator in monocots and dicots

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The majority of our food crops come from flowering plants, which are referred to as angiosperms. Angiosperms are further divided into the dicots, which include many woody species, and the monocots, which include important cereal crops such as wheat and barley. One of the major differences between the monocot and dicot clades is the organisation of the stem. In dicots, the stem is characterised by a group of undifferentiated cells that are referred to as the cambium, which is responsible for most radial growth. The cambium is present in a ring around the stem and cells that divide within the cambium ultimately form the woody plant tissue at the centre of the stem (xylem), or a second transport tissue, the phloem, towards the outer part of the stem. Cell division in the cambium is controlled by a signalling mechanism referred to as TDIF-PXY. TDIF is a small ligand protein, and PXY a receptor protein that is localised to the plasma membrane of cambium cells. When TDIF binds to PXY, a signal is passed to the nucleus which promotes cell division. Monocots do not have a cambium. However, TDIF and PXY are still both encoded for within monocot genomes. This raises the question of what the function of TDIF and PXY is in monocots. To address this question, we deleted the PXY gene from barley. We found no changes to xylem or phloem formation, nor to radial growth, but we did observe fewer larger cells in barley mutant stems. Thus we hypothesised that barley PXY acts to promote cell divisions in a dividing cell population referred to as the intercalary meristem. Intercalary meristems predominate on monocots. They are areas of cell division within the stem that contribute to growth by adding length. The purpose of this proposal is to test the hypothesis that TDIF-PXY regulates the intercalary meristem in monocots. We propose to test this hypothesis by determining cell type specific expression domains of TDIF and PXY genes in barley stems. We already know that TDIF and PXY are expressed in stems, but we don't know in exactly which cell type. To perform this analysis we will use two methods. In the first, we will probe for mRNA derived from PXY and TDIF genes to visualise which cells it is present in. In the second, we will generate so-called reporter genes, where the DNA sequences that define where a gene is expressed (in this case, those for TDIF, PXY, and PXY-like genes) are placed upstream of a gene that can produce a coloured dye. We will further test our hypothesis by removing any TDIF-PXY redundancy from barley using genome editing. Redundancy is where several related genes can perform a similar function, such that mutating a single gene has only a minor effect on phenotype. We have already generated a pxy mutant but other closely related genes may contribute to PXY function. Removing those related genes may lead to clear changes to plant morphology which will help determine TDIF-PXY function in monocots. However, imaging changes to morphology in plant stems is challenging in barley. Thus to fully characterise cell- and tissue-specific changes we will use X-ray computer tomography. This method is a non-destructive technique for visualizing interior features within solid objects, and we propose to use a machine specifically developed for biological samples. This will allow us to fully characterise changes to morphology in the plant lines used in this proposal.Finally, we will determine the consequences of loss of TDIF-PXY on robust plant growth in challenging environmental conditions. Our preliminary data has suggested that TDIF-PXY is required to maintain robust growth under water limited conditions, so we will test the effectiveness of carbon assimilation in the plant lines generated on this proposal when limited amounts of water are available. Global heating is predicted to reduce water availability for crops. But understanding the relationships between plant development and physiology may provide indicators of how we can