An investigation of the protein interactions underpinning CEP-CEPR1 signalling and the evolutionary conservation of this pathway in crops

Abstract

To survive a diverse range of environmental conditions, plants have developed numerous intercellular signalling pathways. The small peptide hormones encoded by the CEP (C-TERMINALLY ENCODED PEPTIDE) gene family are one such pathway. CEPs and their receptor CEPR1 (CEP RECEPTOR 1) have characterised roles in regulating root system architecture, root nitrate uptake and nodulation in legumes. Previous studies of ligand-binding receptor kinases (RKs) in plants have revealed a reliance on co-receptors for signal transduction, but it is not known whether CEP-CEPR1 signalling relies on interaction partners. Here I describe genetic evidence demonstrating that SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASEs (SERKs) function redundantly to regulate CEP-CEPR1 control of primary and lateral root growth, as well as downstream transcriptional targets. CEPR1 interacted with several SERKs in a ligand-independent manner when transiently co-expressed in Nicotiana benthamiana. I also found that several previously characterised SERK-interacting residues in other ligand-binding RKs were conserved in CEPR1, suggesting that these may be important for the CEP-CEPR1-SERK interaction. Thus, I established that SERKs, as co-receptors, play essential roles in the CEP-mediated signalling pathway. In addition, I employed an untargeted proteomic approach to identify novel interaction partners of CEPR1. Several CEPR1-associated proteins were identified using immunoprecipitation coupled with mass spectrometry, including the Arabidopsis thaliana SUCROSE-PROTON SYMPORTER 2 (SUC2). CEPR1 and SUC2 have overlapping expression profiles in the sieve elements and companion cells of the phloem. The relationship between CEPR1 and SUC2 was characterised in two heterologous expression systems, N. benthamiana and Xenopus laevis oocytes. Reciprocal co-immunoprecipitations of CEPR1-CITRINE and SUC2-HA3 expressed in N. benthamiana leaves provided an independent validation of a CEPR1-SUC2 interaction. Heterologous expression of CEPR1 and SUC2 in X. laevis oocytes was used to demonstrate that CEPR1 promotes SUC2-mediated sucrose uptake. Together, these results suggest a novel role for CEP-CEPR1 signalling in regulating SUC2-mediated sucrose transport, which may have implications for our understanding of how sucrose allocation is coordinated to balance root and shoot growth. Whilst roles for CEP-CEPR1 in regulating root phenotypes and nitrate uptake are well established in both A. thaliana and M. truncatula, it is not clear the extent to which this role is functionally conserved in other more agriculturally relevant crop species. To address this, I investigated the conservation of CEP-CEPR1 signalling in five agriculturally important crop species, namely Solanum lycopersicum, Glycine max, Oryza sativa, Hordeum vulgare, and Zea mays. To explore the evolutionary conservation of CEP-CEPR1 signalling in agriculturally important crop species, I transformed the Arabidopsis cepr1-1 mutant with CEPR1 orthologs from Soybean, Tomato, Maize, Rice, and Barley, and assessed their capacity to complement the cepr1-1 mutant phenotypes. CEPR1 orthologs from the monocot species Barley, Maize, and Rice were all able to complement the cepr1-1 mutant phenotypes, whereas orthologs from the dicot species S. lycopersicum and G. max were not. Together, these findings provide the first experimental evidence that CEPR1 is functionally conserved in important agricultural species and provides some preliminary insights into which conserved residues are important for CEPR1 function. In summary, this thesis uncovers i) the essential role of SERKs in the CEP-CEPR1 signalling pathway, ii) the direct interaction between CEPR1 and SUC2 that promotes sucrose transport, and iii) the functional conservation of CEPR1 in several crop species. A model is proposed whereby CEP-CEPR1 signalling, via interaction with SERKs and SUC2, coordinates root growth and nutrient uptake in response to nutrient availability. Show more