The manifestation of repetitive developmental programmes during plant growth can be adjusted in response to various environmental cues. During root development, this means being able to precisely control root growth and lateral root development. Small signalling peptides have been found to play roles in many aspects of root development. One member of the CEP (C-TERMINALLY ENCODED PEPTIDE) gene family has been shown to arrest root growth. Here we report that CEP genes are widespread among seed plants but are not present in land plants that lack true branching roots or root vasculature. We have identified 10 additional CEP genes in Arabidopsis. Expression analysis revealed that CEP genes are regulated by environmental cues such as nitrogen limitation, increased salt levels, increased osmotic strength, and increased CO2 levels in both roots and shoots. Analysis of synthetic CEP variants showed that both peptide sequence and modifications of key amino acids affect CEP biological activity. Analysis of several CEP over-expression lines revealed distinct roles for CEP genes in root and shoot development. A cep3 knockout mutant showed increased root and shoot growth under a range of abiotic stress, nutrient, and light conditions. We demonstrate that CEPs are negative regulators of root development, slowing primary root growth and reducing lateral root formation. We propose that CEPs are negative regulators that mediate environmental influences on plant development.

The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin; however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally diverse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.

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.

Plant root systems arise de novo from a single embryonic root. Complex and highly coordinated developmental networks are required to ensure the formation of lateral organs maximizes plant fitness. The Arabidopsis root is well-suited to dissection of regulatory and developmental networks due to its highly ordered, predictable structure. A myriad of regulatory signaling networks control the development of plant roots, from the classical hormones such as auxin and cytokinin to short-range positional signaling molecules that relay information between neighboring cells. Small signaling peptides are a growing class of regulatory molecules involved in many aspects of root development including meristem maintenance, the gravitropic response, lateral root development, and vascular formation. Here, recent findings on the roles of regulatory peptides in these aspects of root development are discussed.

SummaryThe Brassicaceae contains the most diverse collection of agriculturally important crop species of all plant families. Yet, this is one of the few families that do not form functional symbiotic associations with mycorrhizal fungi in the soil for improved nutrient acquisition. The genes involved in this symbiosis were more recently recruited by legumes for symbiotic association with nitrogen-fixing rhizobia bacteria. This study applied second-generation sequencing (SGS) and analysis tools to discover that two such genes, NSP1 (Nodulation Signalling Pathway 1) and NSP2, remain conserved in diverse members of the Brassicaceae despite the absence of these symbioses. We demonstrate the utility of SGS data for the discovery of putative gene homologs and their analysis in complex polyploid crop genomes with little prior sequence information. Furthermore, we show how this data can be applied to enhance downstream reverse genetics analyses. We hypothesize that Brassica NSP genes may function in the root in other plant-microbe interaction pathways that were recruited for mycorrhizal and rhizobial symbioses during evolution.

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