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.

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.

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.

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.

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.

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.

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.

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.

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.

Intraspecific ecological niche divergence and reproductive shifts foster cytotype displacement and provide ecological opportunity to polyploids.

Background and Aims: Niche divergence between polyploids and their lower ploidy progenitors is one of the primary mechanisms fostering polyploid establishment and adaptive divergence. However, within-species chromosomal and reproductive variability have usually been neglected in community ecology and biodiversity analyses even though they have been recognized to play a role in the adaptive diversification of lineages. Methods: We used Paspalum intermedium, a grass species with diverging genetic systems (diploidy vs. autopolyploidy, allogamy vs. autogamy and sexuality vs. apomixis), to recognize the causality of biogeographic patterns, adaptation and ecological flexibility of cytotypes. Chromosome counts and flow cytometry were used to characterize within-species genetic systems diversity. Environmental niche modelling was used to evaluate intraspecific ecological attributes associated with environmental and climatic factors and to assess correlations among ploidy, reproductive modes and ecological conditions ruling species' population dynamics, range expansion, adaptation and evolutionary history. Key Results: Two dominant cytotypes non-randomly distributed along local and regional geographical scales displayed niche differentiation, a directional shift in niche optima and signs of disruptive selection on ploidy-related ecological aptitudes for the exploitation of environmental resources. Ecologically specialized allogamous sexual diploids were found in northern areas associated with higher temperature, humidity and productivity, while generalist autogamous apomictic tetraploids occurred in southern areas, occupying colder and less productive environments. Four localities with a documented shift in ploidy and four mixed populations in a zone of ecological transition revealed an uneven replacement between cytotypes. Conclusions: Polyploidy and contrasting reproductive traits between cytotypes have promoted shifts in niche optima, and increased ecological tolerance and niche divergence. Ecologically specialized diploids maintain cytotype stability in core areas by displacing tetraploids, while broader ecological preferences and a shift from sexuality to apomixis favoured polyploid colonization in peripheral areas where diploids are displaced, and fostered the ecological opportunity for autotetraploids supporting range expansion to open southern habitats.

A reference floral transcriptome of sexual and apomictic Paspalum notatum.

BACKGROUND: Paspalum notatum Flügge is a subtropical grass native to South America, which includes sexual diploid and apomictic polyploid biotypes. In the past decade, a number of apomixis-associated genes were discovered in this species through genetic mapping and differential expression surveys. However, the scarce information on Paspalum sequences available in public databanks limited annotations and functional predictions for these candidates. RESULTS: We used a long-read 454/Roche FLX+ sequencing strategy to produce robust reference transcriptome datasets from florets of sexual and apomictic Paspalum notatum genotypes and delivered a list of transcripts showing differential representation in both reproductive types. Raw data originated from floral samples collected from premeiosis to anthesis was assembled in three libraries: i) sexual (SEX), ii) apomictic (APO) and iii) global (SEX + APO). A group of physically-supported Paspalum mRNA and EST sequences matched with high level of confidence to both sexual and apomictic libraries. A preliminary trial allowed discovery of the whole set of putative alleles/paralogs corresponding to 23 previously identified apomixis-associated candidate genes. Moreover, a list of 3,732 transcripts and several co-expression and protein -protein interaction networks associated with apomixis were identified. CONCLUSIONS: The use of the 454/Roche FLX+ transcriptome database will allow the detailed characterization of floral alleles/paralogs of apomixis candidate genes identified in prior and future work. Moreover, it was used to reveal additional candidate genes differentially represented in apomictic and sexual flowers. Gene ontology (GO) analyses of this set of transcripts indicated that the main molecular pathways altered in the apomictic genotype correspond to specific biological processes, like biotic and abiotic stress responses, growth, development, cell death and senescence. This data collection will be of interest to the plant reproduction research community and, particularly, to Paspalum breeding projects.

Effect of cadmium on physiological parameters of cereal and millet plants-A comparative study.

Metal load is an abiotic stress that becomes stronger by continual industrial production, wastage, and long-range transport of contaminants. It deteriorates the conditions of agricultural soil that leads to lower growth of cereals as well as decreasing nutritional value of harvested grains. Cadmium (Cd) entry by food chain also affects the health of population. The present study is focused on finding out the superior cereal variety under increasing Cd regime. The plants were grown in increasing Cd levels (0-1000 µM) in the medium and were investigated on 15th day of the exposure. Various parameters like antioxidative enzymes and osmoprotectant levels were studied in both roots and shoots. Cd accumulation in plant organs was determined by atomic absorption spectrophotometry (AAS). Analysis of stress tolerance mechanisms through reactive oxygen species (ROS) scavenging and better partitioning of Cd in roots indicated kodo millet to be more stress tolerant than wheat.

Identification and Validation of Reference Genes for Seashore Paspalum Response to Abiotic Stresses.

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.

An apomixis-linked ORC3-like pseudogene is associated with silencing of its functional homolog in apomictic Paspalum simplex.

Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio.

Relative DNA content in diploid, polyploid, and multiploid species of Paspalum (Poaceae) with relation to reproductive mode and taxonomy.

It is generally accepted that polyploids have downsized basic genomes rather than additive values with respect to their related diploids. Changes in genome size have been reported in correlation with several biological characteristics. About 75 % of around 350 species recognized for Paspalum (Poaceae) are polyploid and most polyploids are apomictic. Multiploid species are common with most of them bearing sexual diploid and apomictic tetraploid or other ploidy levels. DNA content in the embryo and the endosperm was measured by flow cytometry in a seed-by-seed analysis of 47 species including 77 different entities. The relative DNA content of the embryo informed the genome size of the accession while the embryo:endosperm ratio of DNA content revealed its reproductive mode. The genome sizes (2C-value) varied from 0.5 to 6.5 pg and for 29 species were measured for the first time. Flow cytometry provided new information on the reproductive mode for 12 species and one botanical variety and supplied new data for 10 species concerning cytotypes reported for the first time. There was no significant difference between the mean basic genome sizes (1Cx-values) of 32 sexual and 45 apomictic entities. Seventeen entities were diploid and 60 were polyploids with different degrees. There were no clear patterns of changes in 1Cx-values due to polyploidy or reproductive systems, and the existing variations are in concordance with subgeneric taxonomical grouping.

Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses.

Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes in sexual and apomictic Paspalum notatum.

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.