Identification and characterization of regulatory pathways involved in early flowering in the new leaves of alfalfa (Medicago sativa L.) by transcriptome analysis.

BACKGROUND: Alfalfa (Medicago sativa L.) is a perennial legume extensively planted throughout the world as a high nutritive value livestock forage. Flowering time is an important agronomic trait that contributes to the production of alfalfa hay and seeds. However, the underlying molecular mechanisms of flowering time regulation in alfalfa are not well understood. RESULTS: In this study, an early-flowering alfalfa genotype 80 and a late-flowering alfalfa genotype 195 were characterized for the flowering phenotype. Our analysis revealed that the lower jasmonate (JA) content in new leaves and the downregulation of JA biosynthetic genes (i.e. lipoxygenase, the 12-oxophytodienoate reductase-like protein, and salicylic acid carboxyl methyltransferase) may play essential roles in the early-flowering phenotype of genotype 80. Further research indicated that genes encode pathogenesis-related proteins [e.g. leucine rich repeat (LRR) family proteins, receptor-like proteins, and toll-interleukin-like receptor (TIR)-nucleotide-binding site (NBS)-LRR class proteins] and members of the signaling receptor kinase family [LRR proteins, kinases domain of unknown function 26 (DUF26) and wheat leucine-rich repeat receptor-like kinase10 (LRK10)-like kinases] are related to early flowering in alfalfa. Additionally, those involved in secondary metabolism (2-oxoglutarate/Fe (II)-dependent dioxygenases and UDP-glycosyltransferase) and the proteasome degradation pathway [really interesting new gene (RING)/U-box superfamily proteins and F-box family proteins] are also related to early flowering in alfalfa. CONCLUSIONS: Integrated phenotypical, physiological, and transcriptomic analyses demonstrate that hormone biosynthesis and signaling pathways, pathogenesis-related genes, signaling receptor kinase family genes, secondary metabolism genes, and proteasome degradation pathway genes are responsible for the early flowering phenotype in alfalfa. This will provide new insights into future studies of flowering time in alfalfa and inform genetic improvement strategies for optimizing this important trait.

Sequence characteristics of Medicago truncatula cyclophilin family members and function analysis of MsCYP20-3B involved in axillary shoot development.

Cyclophilins (CYPs) belonging to the immunophilin family are present in all organisms and widely distributed in various cells associated with the activity of peptidyl-prolyl cis/trans isomerase. Plant CYPs are members of a multi-gene family and are involved in a series of biological processes. However, little is known about their structure, evolution, developmental expression and functional analysis in Medicago truncatula. In this study, a total of 33 CYP genes were identified and found to be unevenly distributed on eight chromosomes. Among them, 21 are single-domain and 12 are multi-domain proteins, and most were predicted to be localized in the cytosol, nucleus or chloroplast. Phylogenetic and gene structure analysis revealed seven segmental gene pairs, indicating that segmental duplication probably made a large contribution to the expansion of MtCYP gene family. Furthermore, gene expression analysis revealed that about 10 MtCYP genes (were) highly expressed involved in vegetative and reproduction tissues in M. truncatula, and MsCYP20-3B was mainly upregulated in stems, leaves and flower buds in alfalfa (Medicago sativa). Overexpression of MsCYP20-3B was shown to regulate axillary shoot development associated with higher jasmonic acid and abscisic acid contents in M. truncatula. Our study suggests the importance of the CYP genes family in development, reproduction and stress responses, and provides a reference for future studies and application of CYP genes for alfalfa genetic improvement.

Identification and Characterization of Regulatory Pathways Controlling Dormancy Under Lower Temperature in Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa L.), a kind of high-quality perennial legume forage, is widely distributed in the northern regions of China. In recent years, low temperatures have frequently occurred and limited alfalfa productivity and survival in early spring and late fall. However, the underlying molecular mechanisms of alfalfa response to cold tolerance are not well-documented. In this study, dormancy and non-dormancy alfalfa standard varieties were characterized under low-temperature stress. Our analysis revealed that plant height of the dormancy genotype was strongly inhibited by low temperature; flavonoids content, and higher expression of flavonoids biosynthesis genes (chalcone synthase, leucoanthocyanidin dioxygenase, and flavonoid 3'-monooxygenase) may play essential roles in response to low-temperature stress in dormancy genotype alfalfa. Further analyses revealed that receptor-like kinase family genes (such as cysteine-rich RLK10, lectin protein kinase, and S-locus glycoprotein like kinase), RNA and protein synthesis genes (RNA polymerases, ribosomal protein, and protein phosphatase 2C family protein), and proteasome degradation pathway genes (such as F-box family protein, RING/U-box superfamily protein, and zinc finger family protein) also highly upregulated and contributed to cold tolerance phenotype in dormancy genotype alfalfa. This will provide new insights into future studies for cold tolerance in alfalfa and offer new target genes for further functional characterization and genetic improvement of alfalfa.

Cyclophilin AtROC1S58F confers Arabidopsis cold tolerance by modulating jasmonic acid signaling and antioxidant metabolism.

Cyclophilins (CYPs), a class of proteins with a conserved peptidyl-prolyl cis-trans isomerase domain, are widely involved in the regulation of plant growth and development, as well as in the response to abiotic stresses including cold. In our previous study, we identified an Arabidopsis gain-of-function mutant ROC1S58F with enhanced cold-tolerance and enhanced expression of jasmonic acid (JA) and oxidative stress responsive genes. Here, we show the underlying molecular mechanisms for the improved cold tolerance observed in the ROC1S58F mutant. Compared to the WT, the ROC1S58F mutant showed an increased survival rates and a reduced level of electrolyte leakage and endogenous JA content under the freezing treatment. Correspondingly, the JA biosynthesis genes (AtAOC1 and AtOPR3) and signaling genes (AtJAZ5, AtJAZ10 and AtMYB15) are down-regulated in the ROC1S58F mutant compared with the WT. Moreover, both the transcripts and activities of the ROS-scavenging enzymes (SOD/POD/MDHAR) increased in cold-stressed ROC1S58F mutant, which might mitigate the ROS-induced oxidative stress and contribute to the mutant freezing tolerance. Taken together, our findings indicate that AtROC1S58F confers Arabidopsis freezing tolerance by modulating JA signaling and antioxidant metabolism jointly. This research thus provides a molecular mechanism for AtROC1S58F-conferred freezing resistance in Arabidopsis and offers guidance for crop breeding towards an improved cold tolerance.