Characterising CEP peptide hormone and CEPR1 receptor activity in the control of Arabidopsis thaliana root growth and seed yield

Abstract

Plants use numerous metabolite and peptide hormone pathways to respond to environmental changes and to ensure their survival and reproductive success. One important class of hormone are the small signalling peptides encoded by the CEP (C-TERMINALLY ENCODED PEPTIDE) gene family. CEPs have established roles in inhibiting root growth, and in promoting root nitrate uptake and legume nodulation. Specific roles for CEP signalling in aboveground growth and development, however, are not currently known. This thesis investigates the activity of CEPs and their cognate receptor CEPR1 (CEP RECEPTOR 1) in the control of Arabidopsis thaliana root growth and yield production, and determines if some CEP-CEPR1 activity overlaps with known metabolite hormone pathways. CEPs inhibit root growth via CEPR1, however the downstream signalling pathways are not known. To address this, we determined the global transcriptional response of roots to CEP treatment using RNA-sequencing of wild type and cepr1 mutants. Transcripts that were differentially regulated in a CEPR1-dependent manner included those for CEP DOWNSTREAM 1 (CEPD1), CEP ligand genes, transcription factors, root growth inhibitors, metabolite hormone synthesis and response genes, as well as genes involved in nutrient metabolism and source-to-sink nutrient transport. Comparisons to public datasets showed that CEP-responding genes are largely vasculature-expressed and overlap with transcriptional responses to metabolite hormones including auxin and cytokinin. Given the similar roles of cytokinins and CEPs in root growth inhibition and in mediating plant responses to nitrogen (N) levels, we next investigated whether these pathways overlapped in the context of primary root growth inhibition. Several mutants defective in trans-zeatin (tZ)-type cytokinin biosynthesis, and its root-to-shoot translocation, perception, or response, had impaired CEP sensitivity. Conversely, cepr1 mutants displayed reduced tZ sensitivity. Moreover, the cepr1 mutants had higher tZ-type cytokinin levels in roots, consistent with the feedback upregulation of tZ synthesis. Grafting showed that maximal root growth inhibition by CEP required cytokinin receptor activity in the shoot. Finally, we showed that full root growth inhibition by CEP or tZ required the rootward, phloem-mobile glutaredoxins CEPD1 and CEPD2. These results suggest that the systemic CEP and tZ pathways converge on CEPD activity to inhibit root growth. Whilst roles for CEP-CEPR1 in root growth are well established, roles for this pathway in aboveground growth and development are ill-defined. We investigated the basis for the diminished seed yield in cepr1 knockout mutants. Yield reduction in cepr1 was due to compounding factors that result in fewer, smaller mature seeds. Source-sink manipulation and bolt grafting experiments showed that local CEPR1 activity in the reproductive bolt controls seed size and yield. Consistently, CEPR1 expresses throughout the reproductive tissue vasculature, including in important maternal tissues required for seed filling. qRT-PCR showed that cepr1 bolts have reduced expression of key genes required for source-to-sink N remobilisation and delivery to seeds. These analyses support a broader role for CEPR1 throughout the plant in the control of N homeostasis beyond its known roles in root nitrate acquisition. In summary, this thesis uncovers i) the global transcriptional response to CEP-CEPR1 signalling in roots, ii) an intersection between CEP and cytokinin pathways in root growth inhibition, and iii) a specific role for CEPR1 in reproductive tissues in controlling seed yield. A model is proposed whereby CEP-CEPR1 signalling coordinates nutrient pathways with growth throughout the plant and at different developmental stages. Given their effects on nutrient mobilisation pathways, the manipulation of CEP-CEPR1 signalling outputs may provide new avenues for improving nutrient utilisation efficiency and seed yield.