Polymorphism of CLE gene sequences in potato

CLE (CLV3/ESR) is one of the most important groups of peptide phytohormones: its members regulate the development of various plant organs and tissues, as well as interaction with some parasites and symbionts and response to environmental factors. In this regard, the identification and study of the CLE genes encoding the peptides of this group in cultivated plants are of great practical interest. Relatively little is known about the functions of CLE peptides in potato, since the CLE genes of the potato Solanum phureja Juz. et Buk. were characterized only in 2021. At the same time, potato includes plenty of tuberous species of the genus Solanum L., both wild and cultivated, and the diversity of its forms may depend on differences in the sequences of CLE genes. In this work, we performed a search for and analysis of the CLE gene sequences in three wild potato species (S. bukasovii Juz., S. verrucosum Schltdl., S. commersonii Dunal) and four cultivated species (S. chaucha Juz. et Buk., S. curtilobum Juz. et Buk., S. juzepczukii Juz. et Buk., S. ajanhuiri Juz. et Buk.). In total, we identified 332 CLE genes in the analyzed potato species: from 40 to 43 genes of this family for each potato species. All potato species taken for analysis had homologues of previously identified S. phureja CLE genes; at the same time, the CLE42 gene, which is absent from the S. phureja genome, is present in all other analyzed potato species. Polymorphism of CLE proteins of S. commersonii is significantly higher than that of other analyzed potato species, due to the fact that S. commersonii grows in places outside the growing areas of other potato species and this potato is probably not one of the ancestors of cultivated potato. We also found examples of polymorphism of domains of CLE proteins that carried different functions. Further study of potato CLE proteins will reveal their role in development, including regulation of productivity in this important agricultural crop.

Transcriptomic analysis of sym28 and sym29 supernodulating mutants of pea (Pisum sativum L.) under complex inoculation with beneficial microorganisms

The garden pea (Pisum sativum L.), like most members of Fabaceae family, is capable of forming symbioses with beneficial soil microorganisms such as nodule bacteria (rhizobia), arbuscular mycorrhizal (AM) fungi and plant growth promoting bacteria (PGPB). The autoregulation of nodulation (AON) system is known to play an important role in controlling both the number of nodules and the level of root colonization by AM via root-to-shoot signaling mediated by CLAVATA/ESR-related (CLE) peptides and their receptors. In the pea, mutations in genes Sym28 (CLV2-like) and Sym29 (CLV1-like), which encode receptors for CLE peptides, lead to the supernodulation phenotype, i.e., excessive nodule formation. The aim of the present study was to analyze the response of pea cv. ‘Frisson’ (wild type) and mutants P64 (sym28) and P88 (sym29) to complex inoculation with rhizobia, AM fungi and PGPB, with regard to biomass accumulation, yield and transcriptomic alterations. The plants were grown in quartz sand for 2 and 4 weeks after inoculation with either rhizobia (Rh) or complex inoculation with Rh + AM, Rh + PGPB, and Rh+AM+PGPB, and the biomass and yield were assessed. Transcriptome sequencing of whole shoots and roots was performed using a modified RNAseq protocol named MACE (Massive Analysis of cDNA Ends). In the experimental conditions, P88 (sym29) plants demonstrated the best biomass accumulation and yield, as compared to the wild type and P64 (sym28) plants, whereas P64 (sym28) had the lowest rate of biomass and seed yield. The transcriptome analysis showed that both supernodulating mutants more actively responded to biotic and abiotic factors than the wild-type plants and demonstrated increased expression of genes characteristic to late stages of nodule development. The roots of P64 (sym28) plants responded to AM+Rh treatment with upregulation of genes encoding plastid proteins, which can be connected with the activation of carotenoid biosynthesis (namely, the non-mevalonate pathway that takes place in root plastids). The more active response to symbionts in P88 (sym29) plants, as compared to cv. ‘Frisson’, was associated with counterregulation of transcripts involved in chloroplast functioning and development in leaves, which accompanies successful plant development in symbiotic conditions. Finally, the effect of retardation of plant aging upon mycorrhization on a transcriptomic level was recorded for cv. ‘Frisson’ but not for P64 (sym28) and P88 (sym29) mutants, which points towards its possible connection with the AON system. The results of this work link the plant’s autoregulation with the responsiveness to inoculation with beneficial soil microorganisms.

Peptide Signaling Pathways Regulate Plant Vascular Development

Plant small peptides, including CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) and Epidermal Patterning Factor-Like (EPFL) peptides, play pivotal roles in coordinating developmental processes through cell-cell communication. Recent studies have revealed that the phloem-derived CLE peptides, CLE41/44 and CLE42, promote (pro-)cambial cell proliferation and inhibit xylem cell differentiation. The endodermis-derived EPFL peptides, EPFL4 and EPFL6, modulate vascular development in the stem. Further, several other peptide ligands CLE9, CLE10, and CLE45 play crucial roles in regulating vascular development in the root. The peptide signaling pathways interact with each other and crosstalk with plant hormone signals. In this mini-review, we summtarize the recent advances on peptides function in vascular development and discuss future perspectives for the research of the CLE and EPFL peptides.

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Potato (Solanum tuberosum L.): Identification and Expression Analysis

CLE genes encode a group of small secretory peptides, which regulate cell proliferation and differentiation in plants. CLE genes have been studied in many plants; however, little is known about this gene family in potato. In this study, we characterized members of the CLE gene family in potato Solanum tuberosum (StCLE) and comprehensively analyzed their phylogenetic relationships, structure, and expression patterns. Using available transcriptomic data, we found a relative high expression level of StCLE8, StCLE12, and StCLE13 in stolons and tubers. Real-time PCR analysis showed that the StCLE23 gene was upregulated by water deficiency, whereas the expression of StCLE4 and StCLE10 was induced by nitrogen supply. Besides that, using data from transcriptomic studies obtained previously for plants with the induction the StBEL5 gene, a positive regulator of tuber development, we found that StCLE4 was among genes upregulated in response to StBEL5 induction, suggesting that StCLE4 could be a target of StBEL5 transcription factor. However, we did not reveal a direct binding of StBEL5 to the regulatory sequences of StCLE4 using yeast one-hybrid assay. Taken together, our data provide basic information for future functional studies of CLE peptides in potato growth and tuberization and in response to various environmental stimuli.

The CLV3 Homolog in Setaria viridis Selectively Controls Inflorescence Meristem Size

The CLAVATA pathway controls meristem size during inflorescence development in both eudicots and grasses, and is initiated by peptide ligands encoded by CLV3/ESR-related (CLE) genes. While CLV3 controls all shoot meristems in Arabidopsis, evidence from cereal grasses indicates that different meristem types are regulated by different CLE peptides. The rice peptide FON2 primarily controls the size of the floral meristem, whereas the orthologous peptides CLE7 and CLE14 in maize have their most dramatic effects on inflorescence and branch meristems, hinting at diversification among CLE responses in the grasses. Setaria viridis is more closely related to maize than to rice, so can be used to test whether the maize CLE network can be generalized to all members of subfamily Panicoideae. We used CRISPR-Cas9 in S. viridis to knock out the SvFON2 gene, the closest homolog to CLV3 and FON2. Svfon2 mutants developed larger inflorescence meristems, as in maize, but had normal floral meristems, unlike Osfon2, suggesting a panicoid-specific CLE network. Vegetative traits such as plant height, tiller number and leaf number were not significantly different between mutant and wild type plants, but time to heading was shorter in the mutants. In situ hybridization showed strong expression of Svfon2 in the inflorescence and branch meristems, consistent with the mutant phenotype. Using bioinformatic analysis, we predicted the co-expression network of SvFON2 and its signaling components, which included genes known to control inflorescence architecture in maize as well as genes of unknown function. The similarity between SvFON2 function in Setaria and maize suggests that its developmental specialization in inflorescence meristem control may be shared among panicoid grasses.

Nitrate-Induced CLE Peptide Systemically Inhibits Nodulation in Medicago truncatula

Legume plants form nitrogen-fixing nodules in symbiosis with soil bacteria rhizobia. The number of symbiotic nodules is controlled at the whole-plant level with autoregulation of nodulation (AON), which includes a shoot-acting CLV1-like receptor kinase and mobile CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION-related) peptides that are produced in the root in response to rhizobia inoculation. In addition to rhizobia-induced CLE peptides, nitrate-induced CLE genes have been identified in Lotus japonicus and Glycine max, which inhibited nodulation when overexpressed. However, nitrate-induced CLE genes that systemically suppress nodulation in AON-dependent manner have not been identified in Medicago truncatula. Here, we found that MtCLE35 expression is activated by both rhizobia inoculation and nitrate treatment in M. truncatula, similarly to L. japonicus CLE genes. Moreover, we found that MtCLE35 systemically suppresses nodulation in AON-dependent manner, suggesting that MtCLE35 may mediate nitrate-induced inhibition of nodulation in M. truncatula.

Characterisation of Medicago truncatula CLE34 and CLE35 in nodulation control

Legume plants form a symbiosis with N2-fixing soil rhizobia, resulting in new root organs called nodules that enable N2-fixation. Nodulation is a costly process that is tightly regulated by the host through Autoregulation of Nodulation (AON) and nitrate-dependent regulation of nodulation. Both pathways require legume-specific CLAVATA/ESR-related (CLE) peptides. Nitrogen-induced nodulation-suppressing CLE peptides have not previously been characterised in Medicago truncatula, with only rhizobia-induced MtCLE12 and MtCLE13 identified. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control pathways. The nodulation-suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum. Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE-domain. Overexpression of MtCLE12, MtCLE13 and MtCLE35 inhibits nodulation. Together, our findings indicate that MtCLE12 and MtCLE13 have a distinct role in AON, while MtCLE35 regulates nodule numbers in a rhizobia- and nitrate-dependent manner. MtCLE34 likely had a similar role to MtCLE35 but its function was lost due to a nonsense mutation resulting in the loss of the mature peptide.