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Calmodulin-like proteins (CMLs) are unique Ca 2+ sensors and play crucial roles in response to abiotic stress in plants. A salt-repressed PvCML9 from halophyte seashore paspalum (Paspalum vaginatum O. Swartz) was identified. PvCML9 was localized in the cytoplasm and nucleus and highly expressed in roots and stems. Overexpression of PvCML9 led to reduced salt tolerance in rice and seashore paspalum, whereas downregulating expression of PvCML9 showed increased salt tolerance in seashore paspalum as compared with the wild type (WT), indicating that PvCML9 regulated salt tolerance negatively. Na + and K + homeostasis was altered by PvCML9 expression. Lower level of Na + /K + ratio in roots and shoots was maintained in PvCML9-RNAi lines compared with WT under salt stress, but higher level in overexpression lines. Moreover, higher levels of SOD and CAT activities and proline accumulation were observed in PvCML9-RNAi lines compared with WT under salt stress, but lower levels in overexpression lines, which altered ROS homeostasis. Based on the above data, mutation of its homolog gene OsCML9 in rice by CRISPR/Cas9 was performed. The mutant had enhanced salt tolerance without affecting rice growth and development, suggesting that OsCML9 gene is an ideal target gene to generate salt tolerant cultivars by genome editing in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Seashore paspalum is a major warm-season turfgrass requiring frequent mowing. The use of dwarf cultivars with slow growth is a promising method to decrease mowing frequency. The present study was conducted to provide an in-depth understanding of the molecular mechanism of T51 dwarfing in the phenylpropane pathway and to screen the key genes related to dwarfing. For this purpose, we obtained transcriptomic information based on RNA-Seq and proteomic information based on iTRAQ for the dwarf mutant T51 of seashore paspalum . The combined results of transcriptomic and proteomic analysis were used to identify the differential expression pattern of genes at the translational and transcriptional levels. A total of 8311 DEGs were detected at the transcription level, of which 2540 were upregulated and 5771 were downregulated. Based on the transcripts, 2910 proteins were identified using iTRAQ, of which 392 (155 upregulated and 237 downregulated) were DEPs. The phenylpropane pathway was found to be significantly enriched at both the transcriptional and translational levels. Combined with the decrease in lignin content and the increase in flavonoid content in T51, we found that the dwarf phenotype of T51 is closely related to the abnormal synthesis of lignin and flavonoids in the phenylpropane pathway. CCR and HCT may be the key genes for T51 dwarf. This study provides the basis for further study on the dwarfing mechanism of seashore paspalum . The screening of key genes lays a foundation for further studies on the molecular mechanism of seashore paspalum dwarfing.

In the past decades, the grasses of the Paspalum genus have emerged as a versatile model allowing evolutionary, genetic, molecular, and developmental studies on apomixis as well as successful breeding applications. The rise of such an archetypal system progressed through integrative phases, which were essential to draw conclusions based on solid standards. Here, we review the steps adopted in Paspalum to establish the current body of knowledge on apomixis and provide model breeding programs for other agronomically important apomictic crops. In particular, we discuss the need for previous detailed cytoembryological and cytogenetic germplasm characterization; the establishment of sexual and apomictic materials of identical ploidy level; the development of segregating populations useful for inheritance analysis, positional mapping, and epigenetic control studies; the development of omics data resources; the identification of key molecular pathways via comparative gene expression studies; the accurate molecular characterization of genomic loci governing apomixis; the in-depth functional analysis of selected candidate genes in apomictic and model species; the successful building of a sexual/apomictic combined breeding scheme.

Background: Paspalum notatum exhibits both sexual and apomictic cytotypes and, thus, is considered a good model for studies of apomixis because it facilitates comparative approaches. In this work, transcriptome sequencing was used to compare contrasting P. notatum cytotypes to identify differential expression patterns and candidate genes involved in the regulation of expression of this trait., Results: We built a comprehensive transcriptome using leaf and inflorescence from apomictic tetraploids and sexual diploids/tetraploids and a coexpression network based on pairwise correlations between transcript expression profiles. We identified genes exclusively expressed in each cytotype and genes differentially expressed between pairs of cytotypes. Gene Ontology enrichment analyses were performed to better interpret the data. We de novo assembled 114,306 reference transcripts. In total, 536 candidate genes possibly associated with apomixis were detected through statistical analyses of the differential expression data, and several interacting genes potentially linked to the apomixis-controlling region, genes that have already been reported in the literature, and their neighbors were transcriptionally related in the coexpression network., Conclusions: Apomixis is a highly desirable trait in modern agriculture due to the maintenance of the characteristics of the mother plant in the progeny. The reference transcriptome, candidate genes and their coexpression network identified in this work represent rich resources for future grass breeding programs.

The introgression of apomixis in major seed crops, would guarantee self-seeding of superior heterotic seeds over generations. In the grass species Paspalum simplex, apomixis is controlled by a single locus in which recombination is blocked. In the perspective of isolating the genetic determinants of apomixis, we report data on sequencing, in silico mapping and expression analysis of some of the genes contained in two cloned genomic regions of the apomixis locus of P. simplex. In silico mapping allowed us to identify a conserved synteny group homoeologous to the apomixis locus, located on a telomeric position of chromosomes 12, 8, 3 and 4 of rice, Sorghum bicolor, Setaria italica and Brachypodium distachyum, respectively, and on a more centromeric position of maize chromosome 1. Selected genes of the apomixis locus expressed sense and antisense transcripts in reproductively committed cells of sexual and apomictic ovules. Some of the genes considered here expressed apomixis-specific allelic variants which showed partial non-overlapping expression patterns with alleles shared by sexual and apomictic reproductive phenotypes. Our findings open new routes for the isolation of the genetic determinants of apomixis and, in perspective, for its introgression in crop grasses.

Seashore paspalum ( Paspalum vaginatum ) is among the most salt- and cadmium-tolerant warm-season perennial grass species widely used as turf or forage. The objective of this study was to select stable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) analysis of seashore paspalum in response to four abiotic stresses. The stability of 12 potential reference genes was evaluated by four programs (geNorm, NormFinder, BestKeeper, and RefFinder). U2AF combined with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed stable expression in Cd-treated leaves and cold-treated roots. U2AF and FBOX were the most stable reference genes in Cd-treated roots and cold-treated leaves. In Polyethylene Glycol (PEG)- or salt-treated roots, the reference gene U2AF paired with either ACT or CYP were stable. SAND and CACS exhibited the most stability in salt-treated leaves, and combining UPL , PP2A , and EF1a was most suitable for PEG-treated leaves. The stability of U2AF and instability of UPL and TUB was validated by analyzing the expression levels of four target genes ( MT2a , VP1 , PIP1 , and Cor413 ), and were shown to be capable of detecting subtle changes in expression levels of the target genes in seashore paspalum. This study demonstrated that FBOX , U2AF , and PP2A could be used in future molecular studies that aim to understand the mechanisms of abiotic stress tolerance in seashore paspalum., Competing Interests: The authors declare no conflict of interest.

Background: In flowering plants, apomixis (asexual reproduction via seeds) is widely believed to result from failure of key regulators of the sexual female reproductive pathway. In the past few years, both differential display and RNA-seq comparative approaches involving reproductive organs of sexual plants and their apomictic counterparts have yielded extensive lists of candidate genes. Nevertheless, only a limited number of these genes have been functionally characterized, with few clues consequently available for understanding the molecular control of apomixis. We have previously identified several cDNA fragments with high similarity to genes involved in RNA biology and with differential amplification between sexual and apomictic Paspalum notatum plants. Here, we report the characterization of one of these candidates, namely, N69 encoding a protein of the S-adenosyl-L-methionine-dependent methyltransferases superfamily. The purpose of this work was to extend the N69 cDNA sequence and to characterize its expression at different developmental stages in both sexual and apomictic individuals., Results: Molecular characterization of the N69 cDNA revealed homology with genes encoding proteins similar to yeast and mammalian trimethylguanosine synthase/PRIP-interacting proteins. These proteins play a dual role as ERK2-controlled transcriptional coactivators and mediators of sn(o)RNA and telomerase RNA cap trimethylation, and participate in mammals and yeast development. The N69-extended sequence was consequently renamed PnTgs1-like. Expression of PnTgs1-like during reproductive development was significantly higher in floral organs of sexual genotypes compared with apomicts. This difference was not detected in vegetative tissues. In addition, expression levels in reproductive tissues of several genotypes were negatively correlated with facultative apomixis rates. Moreover, in situ hybridization observations revealed that PnTgs1-like expression is relatively higher in ovules of sexual plants throughout development, from premeiosis to maturity. Tissues where differential expression is detected include nucellar cells, the site of aposporous initials differentiation in apomictic genotypes., Conclusions: Our results indicate that PnTgs1-like (formerly N69) encodes a trimethylguanosine synthase-like protein whose function in mammals and yeast is critical for development, including reproduction. Our findings also suggest a pivotal role for this candidate gene in nucellar cell fate, as its diminished expression is correlated with initiation of the apomictic pathway in plants.

The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene plays a fundamental role in somatic embryogenesis of angiosperms, and is associated with apomixis in Poa pratensis. The objective of this work was to isolate, characterize and analyze the expression patterns of SERK genes in apomictic and sexual genotypes of Paspalum notatum. A conserved 200-bp gene fragment was amplified from genomic DNA with heterologous primers, and used to initiate a chromosomal walking strategy for cloning the complete sequence. This procedure allowed the isolation of two members of the P. notatum SERK family; PnSERK1, which is similar to PpSERK1, and PnSERK2, which is similar to ZmSERK2 and AtSERK1. Phylogenetic analyses indicated that PnSERK1 and PnSERK2 represent paralogous sequences. Southern-blot hybridization indicated the presence of at least three copies of SERK genes in the species. qRT-PCR analyses revealed that PnSERK2 was expressed at significantly higher levels than PnSERK1 in roots, leaves, reproductive tissues and embryogenic calli. Moreover, in situ hybridization experiments revealed that PnSERK2 displayed a spatially and chronologically altered expression pattern in reproductive organs of the apomictic genotype with respect to the sexual one. PnSERK2 is expressed in nucellar cells of the apomictic genotype at meiosis, but only in the megaspore mother cell in the sexual genotype. Therefore, apomixis onset in P. notatum seems to be correlated with the expression of PnSERK2 in nucellar tissue.