CEP hormones at the nexus of nutrient acquisition and allocation, root development, and plant-microbe interactions.

A growing understanding is emerging of the roles of peptide hormones in local and long-distance signalling that coordinates plant growth and development as well as responses to the environment. C-TERMINALLY ENCODED PEPTIDE (CEP) signalling triggered by its interaction with CEP RECEPTOR 1 (CEPR1) is known to play roles in systemic nitrogen (N) demand signalling, legume nodulation, and root system architecture. Recent research provides further insight into how CEP signalling operates, which involves diverse downstream targets and interactions with other hormone pathways. Additionally, there is emerging evidence of CEP signalling playing roles in N allocation, root responses to carbon levels, the uptake of other soil nutrients such as phosphorus and sulfur, root responses to arbuscular mycorrhizal fungi, plant immunity, and reproductive development. These findings suggest that CEP signalling more broadly coordinates growth across the whole plant in response to diverse environmental cues. Moreover, CEP signalling and function appear to be conserved in angiosperms. We review recent advances in CEP biology with a focus on soil nutrient uptake, root system architecture and organogenesis, and roles in plant-microbe interactions. Furthermore, we address knowledge gaps and future directions in this research field.

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormone signaling intersect to promote shallow lateral root angles.

Root system architecture (RSA) influences the acquisition of heterogeneously dispersed soil nutrients. Cytokinin and C-TERMINALLY ENCODED PEPTIDE (CEP) hormones affect RSA, in part by controlling the angle of lateral root (LR) growth. Both hormone pathways converge on CEP DOWNSTREAM 1 (CEPD1) and CEPD2 to control primary root growth; however, a role for CEPDs in controlling the growth angle of LRs is unknown. Using phenotyping combined with genetic and grafting approaches, we show that CEP hormone-mediated shallower LR growth requires cytokinin biosynthesis and perception in roots via ARABIDOPSIS HISTIDINE KINASE 2 (AHK2) and AHK3. Consistently, cytokinin biosynthesis and ahk2,3 mutants phenocopied the steeper root phenotype of cep receptor 1 (cepr1) mutants on agar plates, and CEPR1 was required for trans-Zeatin (tZ)-type cytokinin-mediated shallower LR growth. In addition, the cepd1,2 mutant was less sensitive to CEP and tZ, and showed basally steeper LRs on agar plates. Cytokinin and CEP pathway mutants were grown in rhizoboxes to define the role of these pathways in controlling RSA. Only cytokinin receptor mutants and cepd1,2 partially phenocopied the steeper-rooted phenotype of cepr1 mutants. These results show that CEP and cytokinin signaling intersect to promote shallower LR growth, but additional components contribute to the cepr1 phenotype in soil.

CEP peptide and cytokinin pathways converge on CEPD glutaredoxins to inhibit root growth.

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormones act over short and long distances to control plant responses to environmental cues. CEP and cytokinin pathway mutants share phenotypes, however, it is not known if these pathways intersect. We show that CEP and cytokinin signalling converge on CEP DOWNSTREAM (CEPD) glutaredoxins to inhibit primary root growth. CEP inhibition of root growth was impaired in mutants defective in trans-zeatin (tZ)-type cytokinin biosynthesis, transport, perception, and output. Concordantly, mutants affected in CEP RECEPTOR 1 showed reduced root growth inhibition in response to tZ, and altered levels of tZ-type cytokinins. Grafting and organ-specific hormone treatments showed that tZ-mediated root growth inhibition involved CEPD activity in roots. By contrast, root growth inhibition by CEP depended on shoot CEPD function. The results demonstrate that CEP and cytokinin pathways intersect, and utilise signalling circuits in separate organs involving common glutaredoxin genes to coordinate root growth.

The Peptide Hormone Receptor CEPR1 Functions in the Reproductive Tissue to Control Seed Size and Yield.

The interaction of C-TERMINALLY ENCODED PEPTIDES (CEPs) with CEP RECEPTOR1 (CEPR1) controls root growth and development, as well as nitrate uptake, but has no known role in determining yield. We used physiological, microscopic, molecular, and grafting approaches to demonstrate a reproductive tissue-specific role for CEPR1 in controlling yield and seed size. Independent Arabidopsis (Arabidopsis thaliana) cepr1 null mutants showed disproportionately large reductions in yield and seed size relative to their decreased vegetative growth. These yield defects correlated with compromised reproductive development predominantly in female tissues, as well as chlorosis, and the accumulation of anthocyanins in cepr1 reproductive tissues. The thinning of competing reproductive organs to improve source-to-sink ratios in cepr1, along with reciprocal bolt-grafting experiments, demonstrated that CEPR1 acts locally in the reproductive bolt to control yield and seed size. CEPR1 is expressed throughout the vasculature of reproductive organs, including in the chalazal seed coat, but not in other seed tissues. This expression pattern implies that CEPR1 controls yield and seed size from the maternal tissue. The complementation of cepr1 mutants with transgenic CEPR1 rescued the yield and other phenotypes. Transcriptional analyses of cepr1 bolts showed alterations in the expression levels of several genes of the CEP-CEPR1 and nitrogen homeostasis pathways. This transcriptional profile was consistent with cepr1 bolts being nitrogen deficient and with a reproductive tissue-specific function for CEP-CEPR1 signaling. The results reveal a local role for CEPR1 in the maternal reproductive tissue in determining seed size and yield, likely via the control of nitrogen delivery to the reproductive sinks.

CEP receptor signalling controls root system architecture in Arabidopsis and Medicago.

Root system architecture (RSA) influences the effectiveness of resources acquisition from soils but the genetic networks that control RSA remain largely unclear. We used rhizoboxes, X-ray computed tomography, grafting, auxin transport measurements and hormone quantification to demonstrate that Arabidopsis and Medicago CEP (C-TERMINALLY ENCODED PEPTIDE)-CEP RECEPTOR signalling controls RSA, the gravitropic set-point angle (GSA) of lateral roots (LRs), auxin levels and auxin transport. We showed that soil-grown Arabidopsis and Medicago CEP receptor mutants have a narrower RSA, which results from a steeper LR GSA. Grafting showed that CEPR1 in the shoot controls GSA. CEP receptor mutants exhibited an increase in rootward auxin transport and elevated shoot auxin levels. Consistently, the application of auxin to wild-type shoots induced a steeper GSA and auxin transport inhibitors counteracted the CEP receptor mutant's steep GSA phenotype. Concordantly, CEP peptides increased GSA and inhibited rootward auxin transport in wild-type but not in CEP receptor mutants. The results indicated that CEP-CEP receptor-dependent signalling outputs in Arabidopsis and Medicago control overall RSA, LR GSA, shoot auxin levels and rootward auxin transport. We propose that manipulating CEP signalling strength or CEP receptor downstream targets may provide means to alter RSA.

CEP3 levels affect starvation-related growth responses of the primary root.

CEPs (C-TERMINALLY ENCODED PEPTIDEs) inhibit Arabidopsis primary root growth by unknown mechanisms. We investigated how CEP3 levels control primary root growth. CEP3 peptide application decreased cell division, S-phase cell number, root meristematic cell number, and meristem zone (MZ) size in a dose- and CEP RECEPTOR1-dependent manner. Grafting showed that CEP3-dependent growth inhibition requires root and shoot CEPR1. CEP3 induced mitotic quiescence in MZ cells significantly faster than that induced by nutrient limitation alone. CEP3 also inhibited the restoration of S-phase to mitotically quiescence cells by nutrient resupply without quantitatively reducing TARGET OF RAPAMYCIN (TOR) kinase activity. In contrast, cep3-1 had an increased meristem size and S-phase cell number under nitrogen (N)-limited conditions, but not under N-sufficient conditions. Furthermore, cep3-1 meristematic cells remained in S-phase longer than wild-type cells during a sustained carbon (C) and N limitation. RNA sequencing showed that CEP3 peptide down-regulated genes involved in S-phase entry, cell wall and ribosome biogenesis, DNA replication, and meristem expansion, and up-regulated genes involved in catabolic processes and proteins and peptides that negatively control meristem expansion and root growth. Many of these genes were reciprocally regulated in cep3-1. The results suggest that raising CEP3 induces starvation-related responses that curtail primary root growth under severe nutrient limitation.

CEP-CEPR1 signalling inhibits the sucrose-dependent enhancement of lateral root growth.

Lateral root (LR) proliferation is a major determinant of soil nutrient uptake. How resource allocation controls the extent of LR growth remains unresolved. We used genetic, physiological, transcriptomic, and grafting approaches to define a role for C-TERMINALLY ENCODED PEPTIDE RECEPTOR 1 (CEPR1) in controlling sucrose-dependent LR growth. CEPR1 inhibited LR growth in response to applied sucrose, other metabolizable sugars, and elevated light intensity. Pathways through CEPR1 restricted LR growth by reducing LR meristem size and the length of mature LR cells. RNA-sequencing of wild-type (WT) and cepr1-1 roots with or without sucrose treatment revealed an intersection of CEP-CEPR1 signalling with the sucrose transcriptional response. Sucrose up-regulated several CEP genes, supporting a specific role for CEP-CEPR1 in the response to sucrose. Moreover, genes with basally perturbed expression in cepr1-1 overlap with WT sucrose-responsive genes significantly. We found that exogenous CEP inhibited LR growth via CEPR1 by reducing LR meristem size and mature cell length. This result is consistent with CEP-CEPR1 acting to curtail the extent of sucrose-dependent LR growth. Reciprocal grafting indicates that LR growth inhibition requires CEPR1 in both the roots and shoots. Our results reveal a new role for CEP-CEPR1 signalling in controlling LR growth in response to sucrose.

CEP peptide hormones: key players in orchestrating nitrogen-demand signalling, root nodulation, and lateral root development.

Secreted peptide hormones play pivotal roles in plant growth and development. So far, CEPs (C-TERMINALLY ENCODED PEPTIDEs) have been shown to act through CEP receptors (CEPRs) to control nitrogen (N)-demand signalling, nodulation, and lateral root development. Secreted CEP peptides can enter the xylem stream to act as long-distance signals, but evidence also exists for CEPs acting in local circuits. Recently, CEP peptide species varying in sequence, length, and post-translational modifications have been identified. A more comprehensive understanding of CEP biology requires insight into the in planta function of CEP genes, CEP peptide biogenesis, the components of CEP signalling cascades and, finally, how CEP peptide length, amino-acid composition, and post-translational modifications affect biological activity. In this review, we highlight recent studies that have advanced our understanding in these key areas and discuss some future directions.