Analysis of controlling genes for tiller growth of Psathyrostachys juncea based on transcriptome sequencing technology.

BACKGROUND: Tillering is a complicated process in plant and is a significant trait that affects biomass and seed yield of bunch grass Psathyrostachys juncea, a typical perennial forage species. To clarify the regulatory mechanisms of tillering in P. juncea and to explore related candidate genes could be helpful to improve the seed and forage yield of perennial gramineous forages. We selected the tiller node tissues of P. juncea for transcriptome sequencing to determine the differentially expressed genes (DEG) between dense and sparse tillering genotypes. The metabolic pathway was studied, candidate genes were screened, and reference genes stability were evaluated. RESULTS: The results showed that approximately 5466 DEGs were identified between the two genotypes with dense and sparse tillers of P. juncea, which significantly differed in tiller number. Tillering regulation pathways analysis suggested that DEGs closely related to the biosynthesis of three plant hormones, namely auxin (IAA), cytokinin (CTK), and strigolactones (SLs), while "biosynthesis of lignin" and "nitrogen metabolism" have remarkable differences between the dense and sparse tillering genotypes. Meanwhile, the reference gene Actin1, having the best stability, was screened from twelve genes with highest expression level and was used in verification of ten tillering related candidate genes. CONCLUSIONS: The tillering mechanism of perennial grass P. juncea was expounded by transcriptome analysis of tiller node tissues. We demonstrated that dense-tillering genotypes may be distinguished by their low expression patterns of genes involved in SL, IAA, and high expression patterns of genes involved in CTK biosynthesis at the tillering stage, and nitrogen metabolism and lignin biosynthesis can also affect the number of tillers. Furthermore, the expression level of ten tillering related candidate genes were verified using Actin1 as reference gene. These candidate genes provide valuable breeding resources for marker assisted selection and yield traits improvement of P. juncea.

Zinc toxicity response in Ceratoides arborescens and identification of CaMTP, a novel zinc transporter.

Zinc (Zn) is an essential micronutrient for several physiological and biochemical processes. Changes in soil Zn levels can negatively affect plant physiology. Although the mechanism of Zn nutrition has been studied extensively in crops and model plants, there has been little research on steppe plants, particularly live in alkaline soils of arid and semiarid regions. Ceratoides arborescens is used in arid and semiarid regions as forage and ecological restoration germplasm, which is studied can enrich the mechanism of Zn nutrition. The plants were exposed to three different Zn treatments, Zn-deficient (-Zn 0 mM L-1), Zn-normal (Control, 0.015 mM L-1), and Zn-excess (+Zn, 0.15 mM L-1), for 3 weeks. Individual biomass, ion concentrations, photosynthetic system, and antioxidant characteristics were measured. High Zn supply significantly decreased plant biomass and induced chlorosis and growth defects and increased Zn concentration but decreased Fe and Ca concentrations, unlike in controls (p < 0.05). High Zn supply also reduced plant chlorophyll content, which consequently decreased the photosynthesis rate. Increased concentrations of malondialdehyde and soluble sugar and activities of peroxidase and superoxide dismutase could resist the high-level Zn stress. In contrast, low Zn supply did not affect plant growth performance. We also identified a novel protein through RNA transcriptome analysis, named CaMTP, that complemented the sensitivity of a yeast mutant to excessive Zn, which was found to be localized to the endoplasmic reticulum through transient gene expression in Nicotiana benthamiana. The gene CaMTP identified to be highly sensitive to Zn stress is a potential candidate for overcoming mineral stress in dicot crop plants.

Targeted Metabolomics Provide Chemotaxonomic Insights of Medicago ruthenica, with Coupled Transcriptomics Elucidating the Mechanism Underlying Floral Coloration.

Medicago ruthenica, a wild legume forage widely distributed in the Eurasian steppe, demonstrates high genetic and phenotypic variation. M. ruthenica with a purely yellow flower (YFM), differing from the general phenotype of M. ruthenica with a purple flower (PFM), was recently discovered. The similar characteristics of YFM with Medicago falcata have led to conflicting opinions on its taxonomy using traditional morphological methods. The lack of chemotaxonomy information about M. ruthenica species and the unclear flower coloration mechanisms have hampered their study. Here, we investigated M. ruthenica using targeted metabolomics based on the chemotaxonomy method and elaborated the floral coloration mechanisms using transcriptomics. The identified flavonoids were the same types, but there were different contents in YFM and PFM, especially the contents of cyanidin-3-O-glucoside (C3G), an anthocyanin that causes the purple-reddish color of flowers. The over-accumulation of C3G in PFM was 1,770 times more than YFM. Nineteen anthocyanin-related genes were downregulated in YFM compared with their expression in PFM. Thus, YFM could be defined as a variety of M. ruthenica rather than a different species. The loss of purple flower coloration in YFM was attributed to the downregulation of these genes, resulting in reduced C3G accumulation. The taxonomic characteristics and molecular and physiological characteristics of this species will contribute to further research on other species with similar external morphologies.

Phytotoxic effect and molecular mechanism induced by graphene towards alfalfa (Medicago sativa L.) by integrating transcriptomic and metabolomics analysis.

Although the widespread use of nanoparticles has been reported in various fields, the toxic mechanisms of molecular regulation involved in the alfalfa treated by nanomaterials is still in the preliminary research stage. In this study, Bara 310 SC (Bara, tolerant genotype) and Gold Empress (Gold, susceptible genotype) were used to investigate how the leaves of alfalfa interpret the physiological responses to graphene stress based on metabolome and transcriptome characterizations. Herein, graphene at different concentrations (0, 1% and 2%, w/w) were selected as the analytes. Physiological results showed antioxidant defence system and photosynthesis was significantly disturbed under high environmental concentration of graphene. With Ultra high performance liquid chromatography electrospray tandem mass spectrometry (UPLC-ESI-MS/MS), 406 metabolites were detected and 62/13 and 110/58 metabolites significantly changed in the leaves of Gold/Bara under the 1% and 2%-graphene treatments (w/w), respectively. The most important metabolites which were accumulated under graphene stress includes amino acids, flavonoids, organic acids and sugars. Transcriptomic analysis reveals 1125 of core graphene-responsive genes in alfalfa that was robustly differently expressed in both genotypes. And differential expression genes (DEGs) potentially related to photosynthetic enzymes, antioxidant enzymes, amino acids metabolism, and sucrose and starch metabolic which finding was supported by the metabolome study. Gold was more disturbed by graphene stress at both transcriptional and metabolic levels, since more stress-responsive genes/metabolites were identified in Gold. A comprehensive analysis of transcriptomic and metabolomic data highlights the important role of amino acid metabolism and nicotinate and nicotinamide metabolism pathways for graphene tolerance in alfalfa. Our study provide necessary information for better understanding the phytotoxicity molecular mechanism underlying nanomaterials tolerance of plant.

Utilization of Transcriptome, Small RNA, and Degradome Sequencing to Provide Insights Into Drought Stress and Rewatering Treatment in Medicago ruthenica.

Drought is a major limiting factor in foraging grass yield and quality. Medicago ruthenica (M. ruthenica) is a high-quality forage legume with drought resistance, cold tolerance, and strong adaptability. In this study, we integrated transcriptome, small RNA, and degradome sequencing in identifying drought response genes, microRNAs (miRNAs), and key miRNA-target pairs in M. ruthenica under drought and rewatering treatment conditions. A total of 3,905 genes and 50 miRNAs (45 conserved and 5 novel miRNAs) were significantly differentially expressed in three test conditions (CK: control, DS: plants under drought stress, and RW: plants rewatering after drought stress). The degradome sequencing (AllenScore < 4) analysis revealed that 104 miRNAs (11 novel and 93 conserved miRNAs) were identified with 263 target transcripts, forming 296 miRNA-target pairs in three libraries. There were 38 differentially expressed targets from 16 miRNAs in DS vs. CK, 31 from 11 miRNAs in DS vs. RW, and 6 from 3 miRNAs in RW vs. CK; 21, 18, and 3 miRNA-target gene pairs showed reverse expression patterns in DS vs. CK, DS vs. RW, and RW vs. CK comparison groups, respectively. These findings provide valuable information for further functional characterization of genes and miRNAs in response to abiotic stress, in general, and drought stress in M. ruthenica, and potentially contribute to drought resistance breeding of forage in the future.

Proteomic analysis of heterosis in the leaves of sorghum-sudangrass hybrids.

Sorghum-sudangrass hybrids are widely used for forage and silage in the animal husbandry industry due to their hardiness. The heterozygous first generation of sorghum-sudangrass hybrids displays performance superior to their homozygous, parental inbred lines. In order to study the molecular details underlying its heterosis, the leaves of sorghum-sudangrass hybrids and their parents were compared using mass spectrometry-based proteomics. Results showed that among the 996 proteins that were identified, 32 proteins showed 'additive accumulation expression patterns', indicating that the protein abundance in sorghum-sudangrass hybrids showed no significant difference from the average of their parents. Additionally, 74 proteins showed 'nonadditive accumulation expression patterns' (the proteins abundance in the hybrids showed significant difference from the average of their parents). Both additive and nonadditive proteins were mainly involved in photosynthesis and carbohydrate metabolism. More upregulated additive and nonadditive proteins were in the hybrids than in their parents, suggesting that additive and nonadditive proteins are essential to the vigor of sorghum-sudangrass hybrids. The nonadditive proteins were enriched in photosynthesis, carbohydrate metabolism, and protein oligomerization, but the additive proteins were not enriched in any pathway, which indicated that the nonadditive proteins could be greater contributors to heterosis than additive proteins. Furthermore, the highly activated photosynthetic pathway in nonadditive proteins implies that photosynthesis in hybrids is heightened to assimilate more organic matter, resulting in an increased yield. Our results provide a proof-of-concept that reveals the molecular components of heterosis in sorghum-sudangrass hybrid leaves and serves as an important step for future genetic manipulation of specific proteins to improve the performance of hybrids.

[Development of EST-SSR marker and genetic diversity analysis in Sorghum bicolor×Sorghum sudanenes].

A total of 57 498 non-redundant ESTs were identified from 210 878 ESTs of Sorghum in NCBI by sequence analysis. In all, 3 338 SSRs were distributed in 3 116 ESTs with an average frequency of one SSR per 11.28 kb, which included 215 SSR motifs. Analysis of SSR motifs revealed that the trinucleotides were major motifs, accounting for 68.33%.The dinucleotides motifs accounted for 17.97%. There were 1 694 sequences from 3 338 EST-SSR sequences could be designed into primers and the proportion was 50.75%. Fourteen primers were selected to amplify EST-SSR loci with 50 collections of Sorghum bicolor × S. sudanenes, 7 collections of S. bicolor and 3 collections of S. sudanenes. Seventy-two allele variations were detected and the frequency was 5.14 gene loci per primer. The polymorphism index of each primer was in the range of 0.54-0.93. The genetic distance ranged from 0.1646 to 0.6398. This showed abundant genetic diversity in the materials. The materials were divided into 5 groups with clustering analysis of EST-SSR data. Each group included the varieties with similar parents or similar regional distribution. Meanwhile, 4 specific molecular markers were found. Primer D1763 was specific in the registered variety GB-4-2 which was the progeny of S. bicolor 314A × S. sudanenes White Skull. The marker was specific in justification of the germ difference. These results showed that the EST-SSR was an effective marker for genetic diversity analysis and specificity studies on S. bicolor × S. sudanenes.