CLE peptides

Cytokinins are important regulators of cell proliferation and differentiation in plant development. Here, a role for this phytohormone group in soybean nodulation is shown through the exogenous application of cytokinins (6-benzylaminopurine, N 6-(2isopentenyl)-adenine and trans-zeatin) via either root drenching or a petiole feeding technique. Overall, nodule numbers were reduced by treatment with high cytokinin concentrations, but increased with lower concentrations. This was especially evident when feeding the solutions directly into the vasculature via petiole feeding. These findings highlight the importance of cytokinin in nodule development. To further investigate the role of cytokinin in controlling nodule numbers, the IPT gene family involved in cytokinin biosynthesis was characterized in soybean. Bioinformatic analyses identified 17 IPT genes in the soybean genome and homeologous duplicate gene partners were subsequently identified including GmIPT5 and GmIPT6, the orthologs of LjIPT3. Expression of GmIPT5 was upregulated in the shoot in response to nodulation, but this was independent of a functional copy of the autoregulation of nodulation (AON) receptor, GmNARK, which suggests it is unlikely to have a role in the negative feedback system called AON. Legumes also control nodule numbers in the presence of soil nitrogen through nitrate-dependent regulation of nodulation, a locally acting pathway in soybean. Upon nitrate treatment to the root, the tandem duplicates GmIPT3 and GmIPT15 were upregulated in expression indicating a role for these genes in the plant's response to soil nitrogen, potentially including the nitrate-dependent regulation of legume nodulation pathway. Additional roles for cytokinin and their IPT biosynthetic genes in nodulation and the control of nodule numbers are discussed.

Legumes control the nitrogen-fixing root nodule symbiosis in response to external and internal stimuli, such as nitrate, and via systemic autoregulation of nodulation (AON). Overexpression of the CLV3/ESR-related (CLE) pre-propeptideencoding genes GmNIC1 (nitrate-induced and acting locally) and GmRIC1 (Bradyrhizobium-induced and acting systemically) suppresses soybean nodulation dependent on the activity of the nodulation autoregulation receptor kinase (GmNARK). This nodule inhibition response was used to assess the relative importance of key structural components within and around the CLE domain sequences of these genes. Using a site-directed mutagenesis approach, mutants were produced at each amino acid within the CLE domain (RLAPEGPDPHHN) of GmRIC1. This approach identified the Arg1, Ala3, Pro4, Gly6, Pro7, Asp8, His11, and Asn12 residues as critical to GmRIC1 nodulation suppression activity (NSA). In contrast, none of the mutations in conserved residues outside of the CLE domain showed compromised NSA. Chimeric genes derived from combinations of GmRIC1 and GmNIC1 domains were used to determine the role of each pre-propeptide domain in NSA differences that exist between the two peptides. It was found that the transit peptide and CLE peptide regions of GmRIC1 significantly enhanced activity of GmNIC1. In contrast, the comparable GmNIC1 domains reduced the NSA of GmRIC1. Identification of these critical residues and domains provides a better understanding of how these hormone-like peptides function in plant development and regulation.

Peptide signals mediate a variety of cell-to-cell communication crucial for plant growth and development. During Arabidopsis thaliana vascular development, a CLE (CLAVATA3/EMBRYO SURROUNDING REGION-related) family peptide hormone, TDIF (tracheary element differentiation inhibitory factor), regulates procambial cell fate by its inhibitory activity on xylem differentiation. To address if this activity is conserved among vascular plants, we performed comparative analyses of TDIF signaling in non-flowering vascular plants (gymnosperms, ferns and lycophytes). We identified orthologs of TDIF/CLE as well as its receptor TDR/PXY (TDIF RECEPTOR/PHLOEM INTERCALATED WITH XYLEM) in Ginkgo biloba, Adiantum aethiopicum, and Selaginella kraussiana by RACE-PCR. The predicted TDIF peptide sequences in seed plants and ferns were identical to that of A. thaliana TDIF. We examined the effects of exogenous CLE peptide-motif sequences of TDIF in these species. We found that liquid culturing of dissected ...

Plant roots are important for a wide range of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface with the soil environment. Several small signalling peptides and receptor kinases have been shown to affect primary root growth, but very little is known about their role in lateral root development. In this context, the CLE family, a group of small signalling peptides that has been shown to affect a wide range of developmental processes, were the focus of this study. Here, the expression pattern during lateral root initiation for several CLE family members is explored and to what extent CLE1, CLE4, CLE7, CLE26, and CLE27, which show specific expression patterns in the root, are involved in regulating root architecture in Arabidopsis thaliana is assessed. Using chemically synthesized peptide variants, it was found that CLE26 plays an important role in regulating A. thaliana root architecture and interacts with auxin signalling. In addition, through alanine scanning and in silico structural modelling, key residues in the CLE26 peptide sequence that affect its activity are pinpointed. Finally, some interesting similarities and differences regarding the role of CLE26 in regulating monocot root architecture are presented.

Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-likekinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.

LONELY GUY (LOG) genes encode cytokinin riboside 5′-monophosphate phosphoribohydrolases and are directly involved in the activation of cytokinins. To assess whether LOG proteins affect the influence of cytokinin on nodulation, we studied two LOG genes of Medicago truncatula. Expression analysis showed that MtLOG1 and MtLOG2 were upregulated during nodulation in a CRE1-dependent manner. Expression was mainly localized in the dividing cells of the nodule primordium. In addition, RNA interference revealed that MtLOG1 is involved in nodule development and that the gene plays a negative role in lateral root development. Ectopic expression of MtLOG1 resulted in a change in cytokinin homeostasis, triggered cytokinininducible genes and produced roots with enlarged vascular tissues and shortened primary roots. In addition, those 35S:LOG1 roots also displayed fewer nodules than the wild-type. This inhibition in nodule formation was local, independent of the SUPER NUMERIC NODULES gene, but coincided with an upregulation of the MtCLE13 gene, encoding a CLAV-ATA3/EMBRYO SURROUNDING REGION peptide. In conclusion, we demonstrate that in M. truncatula LOG proteins might be implicated in nodule primordium development and lateral root formation.