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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. [ABSTRACT FROM AUTHOR]

In order to analyze in depth the release behavior of added flavor components in tobacco matrix using the solid-gas partition coefficient, a PRV-HS-GC-MS method was developed to determine the partition coefficients of monomeric flavors in cut tobacco matrix based on the changing comparison method combined with HS-GC-MS analysis. The partition coefficients (Kcut/air) of six ester flavors in cut tobacco matrix (solid phase) and gas phase were determined by the PRV-HS-GC-MS method. The results showed that: 1) The relative standard deviation (RSD) of Kcut/air of the six ester flavors at 90-110 °C ranged from 0.92% to 9.14%. The developed method was convenient and accurate with good repeatability. 2) The Kcut/air values of the six monomer ester flavors in cut tobacco were negatively correlated with temperature. The prediction equations established on the basis of Kcut/air values in low-temperature regions could predict Kcut/air values that were difficult to determine in high-temperature regions. The relative errors between the actually determined and predicted Kcut/air values at 90-110 °C were between 1.04% and 5.32%, indicating good prediction performance. 3) The Kcut/air values could be used as a parameter for the release performance of monomer flavors in cut tobacco matrix to quantitatively characterize the differences in monomer flavor release behavior, and it provided a new strategy to study the release behavior of tobacco monomer flavors. [ABSTRACT FROM AUTHOR]

As an economically important legume, Vicia sativa is commonly used as fodder for livestock and in health‐promoting foods for human consumption; however, cold stress is one of the most important limitations to its utilization at high altitudes. The shoot tissues of three cold‐resistant (Lan1, Lan2, and Lan3) and three cold‐sensitive (368, 521, and 538) accessions were collected after cold (4°C) treatment for 0, 6, and 48 h to assess their molecular regulatory mechanisms during cold stress via integrative analyses of their transcriptomes and metabolomes. Our Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the biosynthesis of phenylpropanoids was the most enriched pathway in the cold stress response of V. sativa. A total of 18 differential metabolites were obtained in the biosynthesis of the phenylpropanoid pathway, and 722 differentially expressed genes (DEGs) showed a significant positive correlation with naringenin and p‐coumaric acid following a weighted gene co‐expression network analysis, while 84 DEGs showed significant negative associations. Moreover, four DEGs were overexpressed in yeast, and it was indicated that their biological function is to confer cold tolerance. These results provide a valuable resource for understanding the underlying molecular mechanism of V. sativa's response to cold stress and furthering the development of breeding research. [ABSTRACT FROM AUTHOR]

Aquaporins (AQP) are distributed ubiquitously in plants, and they play important roles in multiple aspects of plant growth and development, as well as in plant resistance to various environmental stresses. In this study, 43 MsAQP genes were identified in the forage crop Medicago sativa. All the MsAQP proteins were clustered into four subfamilies based on sequence similarity and phylogenetic relationship, including 17 TIPs, 14 NIPs, 9 PIPs and 3 SIPs. Analyses of gene structure and conserved domains indicated that the majority of the deduced MsAQP proteins contained the signature transmembrane domains and the NPA motifs. Analyses on cis-acting elements in the promoter region of MsAQP genes revealed the presence of multiple and diverse stress-responsive and hormone-responsive cis-acting elements. In addition, by analyzing the available and comprehensive gene expression data of M. truncatula, we screened ten representative MtAQP genes that were responsive to NaCl or drought stress. By analyzing the sequence similarity and phylogenetic relationship, we finally identified the corresponding ten salt- or drought-responsive AQP genes in M. sativa, including three MsTIPs, three MsPIPs and four MsNIPs. The qPCRs showed that the relative expression levels of these ten selected MsAQP genes responded differently to NaCl or drought treatment in M. sativa. Gene expression patterns showed that most MsAQP genes were preferentially expressed in roots or in leaves, which may reflect their tissue-specific functions associated with development. Our results lay an important foundation for the future characterization of the functions of MsAQP genes, and provide candidate genes for stress resistance improvement through genetic breeding in M. sativa. [ABSTRACT FROM AUTHOR]