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Leaves are the most significant parts of forage crops such as alfalfa. Senescence is the terminal stage of leaf development and is controlled by an integrated myriad of endogenous signals and environmental stimuli. WRKY transcription factors (TFs) play essential roles in regulating leaf senescence; however, only a few studies on the analysis and identification of the WRKY TF family in Medicago Sativa have been reported. In this study, we identified 198 WRKY family members from the alfalfa (M. sativa L.) cultivar ’XinjiangDaye’ using phylogenetic analysis and categorized them into three subfamilies, Groups I, II, and III, based on their structural characteristics. Group II members were further divided into five subclasses. In addition, several hormone- and stress-related cis-acting elements were identified in the promoter regions of MsWRKYs. Furthermore, 14 aging-related MsWRKYs genes from a previous transcriptome in our laboratory were selected for RT-qPCR validation of their expression patterns, and subsequently cloned for overexpression examination. Finally, MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141 were confirmed to cause leaf yellowing in Nicotiana benthaminana using a transient expression system. Our findings lay a groundwork for further studies on the mechanism of M. sativa leaf aging and for the creation of new germplasm resources.

Background Persistent infection with high-risk human papillomavirus (HPV) is closely related to cervical cancer. The epidemiologic characteristics of cervical HPV have regional differences. Therefore, it is necessary to develop the most favorable policies according to the actual situation of each region to prevent and reduce the prevalence of cervical cancer. This retrospective cross-sectional study investigated the prevalence, gene subtypes, and temporal trends of HPV in women undergoing physical examination in Wenzhou, to provide a decision-making basis for further prevention and control of HPV. Methods A total of 31 131 cervical exfoliated cell specimens obtained from physical examinations in Wenzhou, a coastal city of China, from 2015 to 2020 were collected. The age distribution was analyzed using the chi-squared test, and the time change trend was analyzed using the Mann–Kendall trend test. On this basis, the distribution characteristics of the HPV subtypes were analyzed. Results The total prevalence rate was 9.55%, and the prevalence rate in different age groups ranged from 7.77% to 14.16%. The prevalence rate in different years was 8.84%-11.83%. The prevalence rate was bimodal; it was highest in the group 25 years old, followed by the group >61 years old. The top five high-risk gene subtypes were HPV52, HPV58, HPV53, HPV16, and HPV39, whereas the low-risk subtypes were HPV61, HPV81, HPV44, HPV43, and HPV55. Of all the positive samples, 76.03% were infected with a high-risk subtype. Conclusion Most female HPV patients in Wenzhou are infected with high-risk gene subtypes. Therefore, physical examination and screening for HPV should be further strengthened, and the corresponding vaccination policy should focus on high-risk gene subtypes.

Alfalfa sprouts are among the most nutritionally rich foods, and light exposure is a critical factor in determining their biomass and quality. However, detailed metabolic and molecular differences between yellow and green alfalfa sprouts remain unclear. In this study, comprehensive metabolomic and transcriptomic analyses were integrated to evaluate the nutrient composition of alfalfa sprouts during germination with or without light exposure. Differentially expressed genes and differentially accumulated metabolites in green and yellow alfalfa sprouts were significantly enriched in secondary metabolic pathways, such as the isoflavonoid biosynthesis pathway. Green alfalfa sprouts contained a wide variety of lipids, flavonoids, phenolic acids, and terpenoids, among which the top three upregulated were calycosin, methyl gallate, and epicatechin 3-gallate, whereas yellow alfalfa sprouts contained relatively more isoquercitrin. These results provide new insights into the nutritional value and composition of alfalfa sprouts under different germination regimes.

Growth-regulating factors (GRFs) play crucial roles in plant growth and stress response. To date, there have been no reports of the analysis and identification of the GRF transcription factor family in alfalfa. In this study, we identified 27 GRF family members from alfalfa (Medicago sativa L.) “Xinjiang Daye”, and analyzed their physicochemical properties. Based on phylogenetic analysis, these MsGRFs were divided into five subgroups, each with a similar gene structure and conserved motifs. MsGRFs genes are distributed on 23 chromosomes, and all contain QLQ and WRC conserved domains. The results of the collinearity analysis showed that all MsGRFs are involved in gene duplication, including multiple whole-genome duplication or segmental duplication and a set of tandem duplication, indicating that large-scale duplication is important for the expansion of the GRF family in alfalfa. Several hormone-related and stress-related cis-acting elements have been found in the promoter regions of MsGRFs. Some MsGRFs were highly expressed in young leaves and stems, and their expression decreased during development. In addition, the leaf size of different varieties was found to vary, and MsGRF1 to 4, MsGRF18 to 20, and MsGRF22 to 23 were differentially expressed in large and small leaf alfalfa varieties, suggesting that they are critical in the regulation of leaf size. The results of this study can benefit further exploration of the regulatory functions of MsGRFs in growth and development, and can identify candidate genes that control leaf size development.

In plants, the leaf is an essential photosynthetic organ, and is the primary harvest in forage crops such as alfalfa (Medicago sativa). Premature leaf senescence caused by environmental stress can result in significant yield loss and quality reduction. Therefore, the stay-green trait is important for improving the economic value of forage crops. Alkaline stress can severely damage leaf cells and, consequently, cause leaf senescence. To understand the molecular regulatory mechanisms and identify vital senescence-associated genes under alkaline stress, we used high-throughput sequencing to study transcriptional changes in Medicago truncatula, a model plant for forage crops. We identified 2,165 differentially expressed genes, 985 of which were identical to those in the dark-induced leaf senescence group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the 985 genes were mainly enriched in nutrient cycling processes such as cellular amino acid metabolic processes and organic substance catabolic processes, indicating nutrient redistribution. The other 1,180 differentially expressed genes were significantly enriched in the oxidoreductase complex, aerobic respiration, and ion transport. Our analysis showed the two gene sets guiding the coupled physiological and biochemical alterations play different roles under alkaline stress with a coordinated and integrated way. Many transcription factor families were identified from these differentially expressed genes, including MYB, WRKY, bHLH, and NAC which have particular preference involved in stress resistance and regulation of senescence. Our results contribute to the exploration of the molecular regulatory mechanisms of leaf senescence in M. truncatula under alkaline stress and provide new candidate genes for future breeding to improve the biomass and quality of forage crops.

The Banshan archaeological site is one of the most important Early Pleistocene Paleolithic sites in the Nihewan Basin in North China. Based on analyses of pollen, grain size and charcoal of 56 samples from a sedimentary profile, we reconstructed the environment of the Banshan site before and after the interval of hominin activity (1.340–1.290 Ma). The results show that before the appearance of hominin activity (1.340–1.324 Ma), the climate of the region was initially cold and wet and then cold and dry. The regional vegetation was mainly Pinus and Picea forest in the earlier stage, and steppe dominated by arid-tolerant plants such as Artemisia and Chenopodiaceae in the later stage. During the period of hominin activity (1.324–1.318 Ma), the climate was warm and wet, the vegetation was mainly Pinus forest, and the site was a lakeside environment which would have provided resources such as food and water for hominins. During 1.318–1.310 Ma, the climate was warm and wet, the lake continued to expand and the lake level rose, which may have forced the hominins to migrate outside the area. During 1.310–1.290 Ma, the climate changed from warm and humid to cold and arid, accompanied by the change of the regional vegetation from forest to forest-grassland. Hominin activity at the Banshan site occurred during the interval of climate change from cold and dry to warm and wet, and it ended with the rise of the ancient lake level at Nihewan and the deterioration of the climate.

Abstract Background Downregulation of genes involved in lignin biosynthesis and related biochemical pathways has been used as a strategy to improve biofuel production. Plant C1 metabolism provides the methyl units used for the methylation reactions carried out by two methyltransferases in the lignin biosynthetic pathway: caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT). Mutations in these genes resulted in lower lignin levels and altered lignin compositions. Reduced lignin levels can also be achieved by mutations in the C1 pathway gene, folylpolyglutamate synthetase1 (FPGS1), in both monocotyledons and dicotyledons, indicating a link between the C1 and lignin biosynthetic pathways. To test if lignin content can be further reduced by combining genetic mutations in C1 metabolism and the lignin biosynthetic pathway, fpgs1ccoaomt1 double mutants were generated and functionally characterized. Results Double fpgs1ccoaomt1 mutants had lower thioacidolysis lignin monomer yield and acetyl bromide lignin content than the ccoaomt1 or fpgs1 mutants and the plants themselves displayed no obvious long-term negative growth phenotypes. Moreover, extracts from the double mutants had dramatically improved enzymatic polysaccharide hydrolysis efficiencies than the single mutants: 15.1% and 20.7% higher than ccoaomt1 and fpgs1, respectively. The reduced lignin and improved sugar release of fpgs1ccoaomt1 was coupled with changes in cell-wall composition, metabolite profiles, and changes in expression of genes involved in cell-wall and lignin biosynthesis. Conclusion Our observations demonstrate that additional reduction in lignin content and improved sugar release can be achieved by simultaneous downregulation of a gene in the C1 (FPGS1) and lignin biosynthetic (CCOAOMT) pathways. These improvements in sugar accessibility were achieved without introducing unwanted long-term plant growth and developmental defects.

Leaves are the most critical portion of forage crops such as alfalfa (Medicago sativa). Leaf senescence caused by environmental stresses significantly impacts the biomass and quality of forages. To understand the molecular mechanisms and identify the key regulator of the salt stress-induced leaf senescence process, we conducted a simple and effective salt stress-induced leaf senescence assay in Medicago truncatula, which was followed by RNA-Seq analysis coupled with physiological and biochemical characterization. By comparing the observed expression data with that derived from dark-induced leaf senescence at different time points, we identified 3,001, 3,787, and 4,419 senescence-associated genes (SAGs) for salt stress-induced leaf senescence on day 2, 4, and 6, respectively. There were 1546 SAGs shared by dark and salt stress treatment across the three time points. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the 1546 SAGs were mainly related to protein and amino acids metabolism, photosynthesis, chlorophyll metabolism, and hormone signaling during leaf senescence. Strikingly, many different transcription factors (TFs) families out of the 1546 SAGs, including NAC, bHLH, MYB, and ERF, were associated with salt stress-induced leaf senescence processes. Using the transient expression system in Nicotiana benthamiana, we verified that three functional NAC TF genes from the 1546 SAGs were related to leaf senescence. These results clarify SAGs under salt stress in M. truncatula and provide new insights and additional genetic resources for further forage crop breeding.

Switchgrass (Panicum virgatum L.) is a lignocellulosic perennial grass with great potential in bioenergy field. Lignocellulosic bioenergy crops are mostly resistant to cell wall deconstruction, and therefore yield suboptimal levels of biofuel. The one-carbon pathway (also known as C1 metabolism) is critical for polymer methylation, including that of lignin and hemicelluloses in cell walls. Folylpolyglutamate synthetase (FPGS) catalyzes a biochemical reaction that leads to the formation of folylpolyglutamate, an important cofactor for many enzymes in the C1 pathway. In this study, the putatively novel switchgrass PvFPGS1 gene was identified and its functional role in cell wall composition and biofuel production was examined by RNAi knockdown analysis. The PvFPGS1-downregulated plants were analyzed in the field over three growing seasons. Transgenic plants with the highest reduction in PvFPGS1 expression grew slower and produced lower end-of-season biomass. Transgenic plants with low-to-moderate reduction in PvFPGS1 transcript levels produced equivalent biomass as controls. There were no significant differences observed for lignin content and syringyl/guaiacyl lignin monomer ratio in the low-to-moderately reduced PvFPGS1 transgenic lines compared with the controls. Similarly, sugar release efficiency was also not significantly different in these transgenic lines compared with the control lines. However, transgenic plants produced up to 18% more ethanol while maintaining congruent growth and biomass as non-transgenic controls. Severity of rust disease among transgenic and control lines were not different during the time course of the field experiments. Altogether, the unchanged lignin content and composition in the low-to-moderate PvFPGS1-downregulated lines may suggest that partial downregulation of PvFPGS1 expression did not impact lignin biosynthesis in switchgrass. In conclusion, the manipulation of PvFPGS1 expression in bioenergy crops may be useful to increase biofuel potential with no growth penalty or increased susceptibility to rust in feedstock.