REML/BLUP methodology for selection intraspecific hybrids of Paspalum notatum Flügge by multivariate analysis.

The Paspalum genus has potential for further genetic improvement because of its adaptability to different ecosystems and production of high yields for grazing livestock. We estimate the genetic parameters of 195 intraspecific P. notatum hybrids using Restricted Maximum Likelihood (REML), followed by selection based on Best Linear Unbiased Prediction (BLUP) through multivariate analysis. The intraspecific hybrids studied showed considerable genetic variability in the evaluated forage traits, displaying their potential for progression in subsequent stages of the genetic improvement program. Notably, plant height emerged as an important trait for indirect selection to enhance forage production. The use of the REML/BLUP procedure proves to be a robust tool for data analysis, particularly for perennial species. Furthermore, multivariate analysis based on BLUPs should be used in the selection process within breeding programs. Based on the BLUP values, hybrids D3, D16, C17, C2 and B17 were identified as superior for forage production, and they hold promise for future breeding programs for future breeding initiatives aimed at direct selection to improve yield.

Lipid metabolism and antioxidant system contribute to salinity tolerance in halophytic grass seashore paspalum in a tissue-specific manner.

Soil salinization is a growing issue that limits agriculture globally. Understanding the mechanism underlying salt tolerance in halophytic grasses can provide new insights into engineering plant salinity tolerance in glycophytic plants. Seashore paspalum (Paspalum vaginatum Sw.) is a halophytic turfgrass and genomic model system for salt tolerance research in cereals and other grasses. However, the salt tolerance mechanism of this grass largely unknown. To explore the correlation between Na+ accumulation and salt tolerance in different tissues, we utilized two P. vaginatum accessions that exhibit contrasting tolerance to salinity. To accomplish this, we employed various analytical techniques including ICP-MS-based ion analysis, lipidomic profiling analysis, enzyme assays, and integrated transcriptomic and metabolomic analysis. Under high salinity, salt-tolerant P. vaginatum plants exhibited better growth and Na+ uptake compared to salt-sensitive plants. Salt-tolerant plants accumulated heightened Na+ accumulation in their roots, leading to increased production of root-sourced H2O2, which in turn activated the antioxidant systems. In salt-tolerant plants, metabolome profiling revealed tissue-specific metabolic changes, with increased amino acids, phenolic acids, and polyols in roots, and increased amino acids, flavonoids, and alkaloids in leaves. High salinity induced lipidome adaptation in roots, enhancing lipid metabolism in salt-tolerant plants. Moreover, through integrated analysis, the importance of amino acid metabolism in conferring salt tolerance was highlighted. This study significantly enhances our current understanding of salt-tolerant mechanisms in halophyte grass, thereby offering valuable insights for breeding and genetically engineering salt tolerance in glycophytic plants.

ORIGIN OF RECOGNITION COMPLEX 3 controls the development of maternal excess endosperm in the Paspalum simplex agamic complex (Poaceae).

Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex, the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4n×2n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route-based on ORC3 manipulation-to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses.

De novo transcriptome analysis identifies key genes involved in dehydration stress response in kodo millet (Paspalum scrobiculatum L.).

Kodo millet (Paspalum scrobiculatum L.) is a small millet species known for its excellent nutritional and climate-resilient traits. To understand the genes and pathways underlying dehydration stress tolerance of kodo millet, the transcriptome of cultivar 'CO3' subjected to dehydration stress (0 h, 3 h, and 6 h) was sequenced. The study generated 239.1 million clean reads that identified 9201, 9814, and 2346 differentially expressed genes (DEGs) in 0 h vs. 3 h, 0 h vs. 6 h, and 3 h vs. 6 h libraries, respectively. The DEGs were found to be associated with vital molecular pathways, including hormone metabolism and signaling, antioxidant scavenging, photosynthesis, and cellular metabolism, and were validated using qRT-PCR. Also, a higher abundance of uncharacterized genes expressed during stress warrants further studies to characterize this class of genes to understand their role in dehydration stress response. Altogether, the study provides insights into the transcriptomic response of kodo millet during dehydration stress.

High-quality chromosome-scale de novo assembly of the Paspalum notatum 'Flugge' genome.

BACKGROUND: Paspalum notatum 'Flugge' is a diploid with 20 chromosomes (2n = 20) multi-purpose subtropical herb native to South America and has a high ecological significance. It is currently widely planted in tropical and subtropical regions. Despite the gene pool of P. notatum 'Flugge' being unearthed to a large extent in the past decade, no details about the genomic information of relevant species in Paspalum have been reported. In this study, the complete genome information of P. notatum was established and annotated through sequencing and de novo assembly of its genome. RESULTS: The latest PacBio third-generation HiFi assembly and sequencing revealed that the genome size of P. notatum 'Flugge' is 541 M. The assembly result is the higher index among the genomes of the gramineous family published so far, with a contig N50 = 52Mbp, scaffold N50 = 49Mbp, and BUSCOs = 98.1%, accounting for 98.5% of the estimated genome. Genome annotation revealed 36,511 high-confidence gene models, thus providing an important resource for future molecular breeding and evolutionary research. A comparison of the genome annotation results of P. notatum 'Flugge' with other closely related species revealed that it had a close relationship with Zea mays but not close compared to Brachypodium distachyon, Setaria viridis, Oryza sativa, Puccinellia tenuiflora, Echinochloa crusgalli. An analysis of the expansion and contraction of gene families suggested that P. notatum 'Flugge' contains gene families associated with environmental resistance, increased reproductive ability, and molecular evolution, which explained its excellent agronomic traits. CONCLUSION: This study is the first to report the high-quality chromosome-scale-based genome of P. notatum 'Flugge' assembled using the latest PacBio third-generation HiFi sequencing reads. The study provides an excellent genetic resource bank for gramineous crops and invaluable perspectives regarding the evolution of gramineous plants.

A study of the heterochronic sense/antisense RNA representation in florets of sexual and apomictic Paspalum notatum.

BACKGROUND: Apomixis, an asexual mode of plant reproduction, is a genetically heritable trait evolutionarily related to sexuality, which enables the fixation of heterozygous genetic combinations through the development of maternal seeds. Recently, reference floral transcriptomes were generated from sexual and apomictic biotypes of Paspalum notatum, one of the most well-known plant models for the study of apomixis. However, the transcriptome dynamics, the occurrence of apomixis vs. sexual expression heterochronicity across consecutive developmental steps and the orientation of transcription (sense/antisense) remain unexplored. RESULTS: We produced 24 Illumina TruSeq®/ Hiseq 1500 sense/antisense floral transcriptome libraries covering four developmental stages (premeiosis, meiosis, postmeiosis, and anthesis) in biological triplicates, from an obligate apomictic and a full sexual genotype. De novo assemblies with Trinity yielded 103,699 and 100,114 transcripts for the apomictic and sexual samples respectively. A global comparative analysis involving reads from all developmental stages revealed 19,352 differentially expressed sense transcripts, of which 13,205 (68%) and 6147 (32%) were up- and down-regulated in apomictic samples with respect to the sexual ones. Interestingly, 100 differentially expressed antisense transcripts were detected, 55 (55%) of them up- and 45 (45%) down-regulated in apomictic libraries. A stage-by-stage comparative analysis showed a higher number of differentially expressed candidates due to heterochronicity discrimination: the highest number of differential sense transcripts was detected at premeiosis (23,651), followed by meiosis (22,830), postmeiosis (19,100), and anthesis (17,962), while the highest number of differential antisense transcripts were detected at anthesis (495), followed by postmeiosis (164), meiosis (120) and premeiosis (115). Members of the AP2, ARF, MYB and WRKY transcription factor families, as well as the auxin, jasmonate and cytokinin plant hormone families appeared broadly deregulated. Moreover, the chronological expression profile of several well-characterized apomixis controllers was examined in detail. CONCLUSIONS: This work provides a quantitative sense/antisense gene expression catalogue covering several subsequent reproductive developmental stages from premeiosis to anthesis for apomictic and sexual P. notatum, with potential to reveal heterochronic expression between reproductive types and discover sense/antisense mediated regulation. We detected a contrasting transcriptional and hormonal control in apomixis and sexuality as well as specific sense/antisense modulation occurring at the onset of parthenogenesis.

Genome-wide analysis of Claviceps paspali: insights into the secretome of the main species causing ergot disease in Paspalum spp.

BACKGROUND: The phytopatogen Claviceps paspali is the causal agent of Ergot disease in Paspalum spp., which includes highly productive forage grasses such as P. dilatatum. This disease impacts dairy and beef production by affecting seed quality and producing mycotoxins that can affect performance in feeding animals. The molecular basis of pathogenicity of C. paspali remains unknown, which makes it more difficult to find solutions for this problem. Secreted proteins are related to fungi virulence and can manipulate plant immunity acting on different subcellular localizations. Therefore, identifying and characterizing secreted proteins in phytopathogenic fungi will provide a better understanding of how they overcome host defense and cause disease. The aim of this work is to analyze the whole genome sequences of three C. paspali isolates to obtain a comparative genome characterization based on possible secreted proteins and pathogenicity factors present in their genome. In planta RNA-seq analysis at an early stage of the interaction of C. paspali with P. dilatatum stigmas was also conducted in order to determine possible secreted proteins expressed in the infection process. RESULTS: C. paspali isolates had compact genomes and secretome which accounted for 4.6-4.9% of the predicted proteomes. More than 50% of the predicted secretome had no homology to known proteins. RNA-Seq revealed that three protein-coding genes predicted as secreted have mayor expression changes during 1 dpi vs 4 dpi. Also, three of the first 10 highly expressed genes in both time points were predicted as effector-like. CAZyme-like proteins were found in the predicted secretome and the most abundant family could be associated to pectine degradation. Based on this, pectine could be a main component affected by the cell wall degrading enzymes of C. paspali. CONCLUSIONS: Based on predictions from DNA sequence and RNA-seq, unique probable secreted proteins and probable pathogenicity factors were identified in C. paspali isolates. This information opens new avenues in the study of the biology of this fungus and how it modulates the interaction with its host. Knowledge of the diversity of the secretome and putative pathogenicity genes should facilitate future research in disease management of Claviceps spp.

Hybridization, polyploidy and clonality influence geographic patterns of diversity and salt tolerance in the model halophyte seashore paspalum (Paspalum vaginatum).

In plant species, variation in levels of clonality, ploidy and interspecific hybridization can interact to influence geographic patterns of genetic diversity. These factors commonly vary in plants that specialize on saline habitats (halophytes) and may play a role in how they adapt to salinity variation across their range. One such halophyte is the turfgrass and emerging genomic model system seashore paspalum (Paspalum vaginatum Swartz). To investigate how clonal propagation, ploidy variation, and interspecific hybridization vary across ecotypes and local salinity levels in wild P. vaginatum, we employed genotyping-by-sequencing, cpDNA sequencing and flow cytometry in 218 accessions representing > 170 wild collections from throughout the coastal southern United States plus USDA germplasm. We found that the two morphologically distinct ecotypes of P. vaginatum differ in their adaptive strategies. The fine-textured ecotype is diploid and appears to reproduce in the wild both sexually and by clonal propagation; in contrast, the coarse-textured ecotype consists largely of clonally-propagating triploid and diploid genotypes. The coarse-textured ecotype appears to be derived from hybridization between fine-textured P. vaginatum and an unidentified Paspalum species. These clonally propagating hybrid genotypes are more broadly distributed than clonal fine-textured genotypes and may represent a transition to a more generalist adaptive strategy. Additionally, the triploid genotypes vary in whether they carry one or two copies of the P. vaginatum subgenome, indicating multiple evolutionary origins. This variation in subgenome composition shows associations with local ocean salinity levels across the sampled populations and may play a role in local adaptation.

The Halophyte Seashore Paspalum Uses Adaxial Leaf Papillae for Sodium Sequestration.

Salinity is a growing issue worldwide, with nearly 30% of arable land predicted to be lost due to soil salinity in the next 30 years. Many grass crops that are vital to sustain the world's caloric intake are salt sensitive. Studying mechanisms of salt tolerance in halophytic grasses, plants that thrive in salt conditions, may be an effective approach to ultimately improve salt-sensitive grass crops. Seashore paspalum (Paspalum vaginatum) is a halophytic Panicoid grass able to grow in salt concentrations near that of seawater. Despite its widespread cultivation as a sustainable turfgrass, the mechanism underlying its ability to retain high Na+ concentrations in photosynthetic tissue while maintaining growth remains unknown. We examined the leaf structure and ion content in P. vaginatum 'HI10', which shows increased growth under saline conditions, and Paspalum distichum 'Spence', which shows reduced growth under salt, to better understand the superior salt tolerance of cv HI10. A striking difference between cv HI10 and cv Spence was the high steady-state level of K+ in cv HI10. Imaging further showed that the adaxial surface of both cv HI10 and cv Spence contained dense costal ridges of papillae. However, these unicellular extensions of the epidermis were significantly larger in cv HI10 than in cv Spence. The cv HI10 papillae were shown to act as Na+ sinks when plants were grown under saline conditions. We provide evidence that leaf papillae function as specialized structures for Na+ sequestration in P. vaginatum, illustrating a possible path for biotechnological improvement of salt-sensitive Panicoid crops with analogous leaf structures.

Carbohydrate saving or biomass maintenance: which is the main determinant of the plant's long-term submergence tolerance?

It is hypothesized that plant submergence tolerance could be assessed from the decline of plant biomass due to submergence, as biomass integrates all eco-physiological processes leading to fitness. An alternative hypothesis stated that the consumption rate of carbohydrate is essential in differing tolerance to submergence. In the present study, the responses of biomass, biomass allocation, and carbohydrate content to simulated long-term winter submergence were assessed in four tolerant and four sensitive perennials. The four tolerant perennials occur in a newly established riparian ecosystem created by The Three Gorges Dam, China. They had 100% survival after 120 days' simulated submergence, and had full photosynthesis recovery after 30 days' re-aeration, and the photosynthetic rate was positively related to the growth during the recovery period. Tolerant perennials were characterized by higher carbohydrate levels, compared with the four sensitive perennials (0% survival) at the end of submergence. Additionally, by using a method which simulates posterior estimates, and bootstraps the confidence interval for the difference between strata means, it was found that the biomass response to post-hypoxia, rather than that to submergence, could be a reliable indicator to assess submergence tolerance. Interestingly, the differences of changes in carbohydrate content between tolerant and sensitive perennials during submergence were significant, which were distinct from the biomass response, supporting the hypothesis that tolerant perennials could sacrifice non-vital components of biomass to prioritize the saving of carbohydrates for later recovery. Our study provides some insight into the underlying mechanism(s) of perennials' tolerance to submergence in ecosystems such as temperate wetland and reservoir riparian.

Relationship between the Phenylpropanoid Pathway and Dwarfism of Paspalum seashore Based on RNA-Seq and iTRAQ.

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.

Comparative transcriptome profiling provides insights into plant salt tolerance in seashore paspalum (Paspalum vaginatum).

BACKGROUND: Seashore paspalum (Paspalum vaginatum), a halophytic warm-seasoned perennial grass, is tolerant of many environmental stresses, especially salt stress. To investigate molecular mechanisms underlying salinity tolerance in seashore paspalum, physiological characteristics and global transcription profiles of highly (Supreme) and moderately (Parish) salinity-tolerant cultivars under normal and salt stressed conditions were analyzed. RESULTS: Physiological characterization comparing highly (Supreme) and moderately (Parish) salinity-tolerant cultivars revealed that Supreme's higher salinity tolerance is associated with higher Na+ and Ca2+ accumulation under normal conditions and further increase of Na+ under salt-treated conditions (400 mM NaCl), possibly by vacuolar sequestration. Moreover, K+ retention under salt treatment occurs in both cultivars, suggesting that it may be a conserved mechanism for prevention of Na+ toxicity. We sequenced the transcriptome of the two cultivars under both normal and salt-treated conditions (400 mM NaCl) using RNA-seq. De novo assembly of about 153 million high-quality reads and identification of Open Reading Frames (ORFs) uncovered a total of 82,608 non-redundant unigenes, of which 3250 genes were identified as transcription factors (TFs). Gene Ontology (GO) annotation revealed the presence of genes involved in diverse cellular processes in seashore paspalum's transcriptome. Differential expression analysis identified a total of 828 and 2222 genes that are responsive to high salinity for Supreme and Parish, respectively. "Oxidation-reduction process" and "nucleic acid binding" are significantly enriched GOs among differentially expressed genes in both cultivars under salt treatment. Interestingly, compared to Parish, a number of salt stress induced transcription factors are enriched and show higher abundance in Supreme under normal conditions, possibly due to enhanced Ca2+ signaling transduction out of Na+ accumulation, which may be another contributor to Supreme's higher salinity tolerance. CONCLUSION: Physiological and transcriptome analyses of seashore paspalum reveal major molecular underpinnings contributing to plant response to salt stress in this halophytic warm-seasoned perennial grass. The data obtained provide valuable molecular resources for functional studies and developing strategies to engineer plant salinity tolerance.

Coexpression and Transcriptome analyses identify active Apomixis-related genes in Paspalum notatum leaves.

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.

Up-regulation of lipid metabolism and glycine betaine synthesis are associated with choline-induced salt tolerance in halophytic seashore paspalum.

Choline may affect salt tolerance by regulating lipid and glycine betaine (GB) metabolism. This study was conducted to determine whether alteration of lipid profiles and GB metabolism may contribute to choline regulation and genotypic variations in salt tolerance in a halophytic grass, seashore paspalum (Paspalum vaginatum). Plants of Adalayd and Sea Isle 2000 were subjected to salt stress (200-mM NaCl) with or without foliar application of choline chloride (1 mM). Genotypic variations in salt tolerance and promotive effects of choline application on salt tolerance were associated with both the up-regulation of lipid metabolism and GB synthesis. The genotypic variations in salt tolerance associated with lipid metabolism were reflected by the differential accumulation of phosphatidylcholine and phosphatidylethanolamine between Adalayd and Sea Isle 2000. Choline-induced salt tolerance was associated with of the increase in digalactosyl diacylglycerol (DGDG) content including DGDG (36:4 and 36:6) in both cultivars of seashore paspalum and enhanced synthesis of phosphatidylinositol (34:2, 36:5, and 36:2) and phosphatidic acid (34:2, 34:1, and 36:5), as well as increases in the ratio of digalactosyl diacylglycerol: monogalactosyl diacylglycerol (DGDG:MGDG) in salt-tolerant Sea Isle 2000. Choline regulation of salt tolerance may be due to the alteration in lipid metabolism in this halophytic grass species.

Phytoremediation potential of Cd and Pb-contaminated soils by Paspalum fasciculatum Willd. ex Flüggé.

The phytoremediation capacity of Paspalum fasciculatum Willd. ex Flüggé, was evaluated in soils from a gold mine contaminated with cadmium (Cd) and lead (Pb), using three concentration levels of each metal (15, 30, and 50 mg kg-1). Their ability to assimilate Cd and Pb in its different tissues was evaluated during 90 days of exposure. Plant growth behavior, accumulation of Cd and Pb, and translocation (TF) and bioaccumulation (BAF) factors were also determined. During the first 60 days of exposure, Cd had an inductive effect on the growth of P. fasciculatum; however, after 90 days, this metal had begun to show toxic effects. Plants showed a similar pattern of accumulating Cd and Pb in their tissues with concentrations decreasing in the order roots > leaves > stem. However, the accumulated concentrations of Cd were generally higher than those of Pb with the highest metal uptakes being observed during the first 30 days of exposure. P. fasciculatum was shown to have a phytostabilization effect with regard to Cd, high concentrations of metals in tissues and little translocation, whereas it showed phytoextraction capacity for Pb. In addition, it can increase pH and organic matter in the soil rhizosphere.

Batch and fed-batch fermentation of optically pure D (-) lactic acid from Kodo millet (Paspalum scrobiculatum) bran residue hydrolysate: growth and inhibition kinetic modeling.

A low-cost Kodo millet bran residue was utilized as feedstock for the production of D (-) lactic acid (DLA) using Lactobacillus delbrueckii NBRC3202 under anaerobic condition. Data culled from a series of batch fermentation processes with different initial Kodo millet bran residue hydrolysate (KMBRH) and DLA concentrations were used for kinetic model development. Both simulated and experimental data were in good agreement for cell growth, KMBRH utilization, and DLA formation. The values of kinetic constants specific growth rate, (µm = 0.17 h-1); growth (αP = 0.96 g.g-1) and non-growth (ßP = 1.19 g.g-1.h-1) associated constant for DLA production and the maximum specific KMBRH utilization rate, (qG, max = 1.18 g.g-1.h-1) were in good agreement with the literature reports. Kinetic analysis elucidated that L. delbrueckii growth was predominantly influenced by KMBRH limitation and highly sensitive to DLA inhibition. Fed-batch fermentation studies demonstrated the existence of substrate and product inhibition paving the scope for process intensification.

Small RNA-seq reveals novel regulatory components for apomixis in Paspalum notatum.

BACKGROUND: Apomixis is considered an evolutionary deviation of the sexual reproductive pathway leading to the generation of clonal maternal progenies by seeds. Recent evidence from model and non-model species suggested that this trait could be modulated by epigenetic mechanisms involving small RNAs (sRNAs). Here we profiled floral sRNAs originated from apomictic and sexual Paspalum notatum genotypes in order to identify molecular pathways under epigenetic control that might be involved in the transition from sexuality to agamospermy. RESULTS: The mining of genes participating in sRNA-directed pathways from floral Paspalum transcriptomic resources showed these routes are functional during reproductive development, with several members differentially expressed in apomictic and sexual plants. Triplicate floral sRNA libraries derived from apomictic and a sexual genotypes were characterized by using high-throughput sequencing technology. EdgeR was apply to compare the number of sRNA reads between sexual and apomictic libraries that map over all Paspalum floral transcripts. A total of 1525 transcripts showed differential sRNA representation, including genes related to meiosis, plant hormone signaling, biomolecules transport, transcription control and cell cycle. Survey for miRNA precursors on transcriptome and genome references allowed the discovery of 124 entities, including 40 conserved and 8 novel ones. Fifty-six clusters were differentially represented in apomictic and sexual plants. All differentially expressed miRNAs were up-regulated in apomictic libraries but miR2275, which showed different family members with opposed representation. Examination of predicted miRNAs targets detected 374 potential candidates. Considering sRNA, miRNAs and target surveys together, 14 genes previously described as related with auxin metabolism, transport and signaling were detected, including AMINO ACID/AUXIN PERMEASE 15, IAA-AMIDO SYNTHETASE GH3-8, IAA30, miR160, miR167, miR164, miR319, ARF2, ARF8, ARF10, ARF12, AFB2, PROLIFERATING CELL FACTOR 6 and NITRATE TRANSPORTER 1.1. CONCLUSIONS: This work provides a comprehensive survey of the sRNA differential representation in flowers of sexual and apomictic Paspalum notatum plants. An integration of the small RNA profiling data presented here and previous transcriptomic information suggests that sRNA-mediated regulation of auxin pathways is pivotal in promoting apomixis. These results will underlie future functional characterization of the molecular components mediating the switch from sexuality to apomixis.

Heterochronic reproductive developmental processes between diploid and tetraploid cytotypes of Paspalum rufum.

BACKGROUND AND AIMS: Apomixis is an asexual reproductive mode via seeds that generate maternal clonal progenies. Although apomixis in grasses is mainly expressed at the polyploid level, some natural diploid genotypes of Paspalum rufum produce aposporous embryo sacs in relatively high proportions and are even able to complete apomixis under specific conditions. However, despite the potential for apomixis, sexuality prevails in diploids, and apomixis expression is repressed for an as yet undetermind reason. Apomixis is thought to derive from a deregulation of one or a few components of the sexual pathway that could be triggered by polyploidy and/or hybridization. The objectives of this work were to characterize and compare the reproductive development and the timing of apospory initial (AI) emergence between diploid genotypes with potential for apomixis and facultative apomictic tetraploid cytotypes of P. rufum. METHODS: Reproductive characterization was performed by cytoembryological observations of cleared ovaries and anthers during all reproductive development steps and by quantitative evaluation of the ovule growth parameters. KEY RESULTS: Cytoembryological observations showed that in diploids, both female and male reproductive development is equally synchronized, but in tetraploids, megasporogenesis and early megagametogenesis are delayed with respect to microsporogenesis and early microgametogenesis. This delay was also seen when ovary growth was taken as a reference parameter. The analysis of the onset of AIs revealed that they emerge during different developmental periods depending on the ploidy level. In diploids, the AIs appeared along with the tetrad (or triad) of female meiocytes, but in tetraploids they appeared earlier, at the time of the megaspore mother cell. In both cytotypes, AIs can be seen even during megagametogenesis. CONCLUSIONS: Overall observations reveal that female sexual reproductive development is delayed in tetraploids as compared with diploid genotypes, mainly at meiosis. In tetraploids, AIs appear at earlier sexual developmental stages than in diploids, and they accumulate up to the end of megasporogenesis. The longer extension of megasporogenesis in tetraploids could favour AI emergence and also apomixis success.

Responses of the grass Paspalum distichum L. to Hg stress: A proteomic study.

Paspalum distichum L. was tested to evaluate its ability to phytoremediate mercury (Hg) contaminated soil over a 60-d period by analysis of the total Hg concentrations in roots and leaves. Hg concentration in Hg-contamination soil decreased by 70.0 µg g-1 after 60 day of grass cultivation and Hg was readily taken up by the roots (4.51 ±â€¯1.90 µg g-1) rather than the leaves (0.35 ±â€¯0.02 µg g-1). In addition, a comparative proteomic study was performed to unravel the protein expression involved in the Hg stress response in P. distichum L. A total of 49 proteins were classified as differentially proteins in the roots by the 'top three' proteomic analysis, of which 32 were up-regulated and 17 down-regulated in response to Hg stress. These changed proteins were classified by gene ontology analysis into five complex molecular functions involving photosynthesis and energy metabolism (31%), oxidative stress (14%), protein folding (16%), sulfur compound metabolism (10%), metal binding, and ion transport (29%). Moreover, the protein expression patterns were consistent with the metabolism pathway results. Overall, the results contribute to our understanding of the molecular mechanisms of the Hg response in P. distichum and we propose a theoretical basis for the phytoremediation of Hg-contaminated soils.

Investigation of mitochondrial-derived plastome sequences in the Paspalum lineage (Panicoideae; Poaceae).

BACKGROUND: The grass family (Poaceae), ca. 12,075 species, is a focal point of many recent studies that aim to use complete plastomes to reveal and strengthen relationships within the family. The use of Next Generation Sequencing technology has revealed intricate details in many Poaceae plastomes; specifically the trnI - trnL intergenic spacer region. This study investigates this region and the putative mitochondrial inserts within it in complete plastomes of Paspalum and other Poaceae. RESULTS: Nine newly sequenced plastomes, seven of which contain an insert within the trnI - trnL intergenic spacer, were combined into plastome phylogenomic and divergence date analyses with 52 other species. A robust Paspalum topology was recovered, originating at 10.6 Ma, with the insert arising at 8.7 Ma. The alignment of the insert across Paspalum reveals 21 subregions with pairwise homology in 19. In an analysis of emergent self-organizing maps of tetranucleotide frequencies, the Paspalum insert grouped with mitochondrial DNA. CONCLUSIONS: A hypothetical ancestral insert, 17,685 bp in size, was found in the trnI - trnL intergenic spacer for the Paspalum lineage. A different insert, 2808 bp, was found in the same region for Paraneurachne muelleri. Seven different intrastrand deletion events were found within the Paspalum lineage, suggesting selective pressures to remove large portions of noncoding DNA. Finally, a tetranucleotide frequency analysis was used to determine that the origin of the insert in the Paspalum lineage is mitochondrial DNA.