Background: Low temperature is one of the main environmental factors that limits crop growth, development, and production.Medicago falcatais an important leguminous herb that is widely distributed worldwide.M. falcatais related to alfalfa but is more tolerant to low temperature than alfalfa. Understanding the low temperature tolerance mechanism ofM. falcatais important for the genetic improvement of alfalfa.Results: In this study, we explored the transcriptomic changes in the roots of low-temperature-treatedM. falcataplants by combining SMRT sequencing and NGS technologies. A total of 115,153 nonredundant sequences were obtained, and 8849 AS events, 73,149 SSRs, and 4189 lncRNAs were predicted. A total of 111,587 genes from SMRT sequencing were annotated, and 11,369 DEGs involved in plant hormone signal transduction, protein processing in endoplasmic reticulum, carbon metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, and endocytosis pathways were identified. We characterized 1538 TF genes into 45 TF gene families, and the most abundant TF family was the WRKY family, followed by the ERF, MYB, bHLH and NAC families. A total of 134 genes, including 101 whose expression was upregulated and 33 whose expression was downregulated, were differentially coexpressed at all five temperature points. PB40804, PB75011, PB110405 and PB108808 were found to play crucial roles in the tolerance ofM. falcatato low temperature. WGCNA revealed that the MEbrown module was significantly correlated with low-temperature stress inM. falcata. Electrolyte leakage was correlated with most genetic modules and verified that electrolyte leakage can be used as a direct stress marker in physiological assays to indicate cell membrane damage from low-temperature stress. The consistency between the qRT-PCR results and RNA-seq analyses confirmed the validity of the RNA-seq data and the analysis of the regulatory mechanism of low-temperature stress on the basis of the transcriptome.Conclusions: The full-length transcripts generated in this study provide a full characterization of the transcriptome ofM. falcataand may be useful for mining new low-temperature stress-related genes specific toM. falcata. These new findings could facilitate the understanding of the low-temperature-tolerance mechanism ofM. falcata.

On the Qinghai-Tibetan Plateau, the high-altitudinal gradients can negatively affect plant distribution and limit plant growth and reproduction. Leymus secalinus (Georgi) Tzvel. is an important forage crop on the Qinghai-Tibetan Plateau and has an excellent ability to fix sand and improve soil. To evaluate the effect of altitude on the physiological characteristics of L. secalinus on the Qinghai-Tibetan Plateau, we measured the lipid peroxidation; chlorophyll a (Chl a), chlorophyll b (Chl b), total carotenoid (Car), soluble protein, proline and soluble sugar contents; and the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in leaves from eight different altitudes in Minhe County and Huangzhong County. The leaves were collected at the initial bloom stage, and the average vertical distance between two adjacent collection sites was approximately 100 meters. The reduction in Chl a and Chl b contents and the increase in Car contents can allow plants to weaken their light absorption and avoid photodamage to the chloroplast. The decrease in malondialdehyde (MDA) content associated with lower lipid peroxidation, and the changes of CAT, SOD and POD activities reflect a higher reactive oxygen species (ROS) scavenging capacity in high-altitude plants. The increase in proline and soluble sugar contents with elevation suggests that proline and soluble sugar may play a key role in the osmotic adjustment of plants in alpine regions. The results suggested that altitudinal gradients negatively affect L. secalinus on the Qinghai-Tibetan Plateau and that the adaptation mechanism and survival strategies of L. secalinus were attributed to the combined effects of multiple protective strategies.

Voltage-dependent anion channels (VDACs) are highly conserved proteins that are involved in the translocation of tRNA and play a key role in modulating plant senescence and multiple pathways. However, the functions of VDACs in plants are still poorly understood. Here, a novel VDAC gene was isolated and identified from alfalfa (Medicago sativa L.). MsVDAC localized to the mitochondria, and its expression was highest in alfalfa roots and was induced in response to cold, drought and salt treatment. Overexpression of MsVDAC in tobacco significantly increased MDA, GSH, soluble sugars, soluble protein and proline contents under cold and drought stress. However, the activities of SOD and POD decreased in transgenic tobacco under cold stress, while the O₂^- content increased. Stress-responsive genes including LTP1, ERD10B and Hxk3 were upregulated in the transgenic plants under cold and drought stress. However, GAPC, CBL1, BI-1, Cu/ZnSOD and MnSOD were upregulated only in the transgenic tobacco plants under cold stress, and GAPC, CBL1, and BI-1 were downregulated under drought stress. These results suggest that MsVDAC provides cold tolerance by regulating ROS scavenging, osmotic homeostasis and stress-responsive gene expression in plants, but the improved drought tolerance via MsVDAC may be mainly due to osmotic homeostasis and stress-responsive genes.

In recent years, drought stress caused by global warming has become a major constraint on agriculture. The thiamine thiazole synthase (THI1) is responsible for controlling thiamine production in plants displaying a response to various abiotic stresses. Nonetheless, most of the THI1 activities in plants remain largely unknown. In this study, we extracted MsTHI1 from alfalfa and demonstrated its beneficial impact on improving the resistance of plants to stress conditions. The highest levels of MsTHI1 expression were identified in alfalfa leaves, triggered by exposure to cold, drought, salt, or alkaline conditions. The upregulation of MsTHI1 in drought-stressed transgenic plants resulted in enhanced accumulation of vitamin B1 (VB1), chlorophyll a (Chl a), chlorophyll b (Chl b), soluble protein, higher soil and plant analyzer development (SPAD) value, and the activity of peroxidase (POD), maintained Fv/Fm, and decreased lipid peroxidation. Moreover, overexpression of MsTHI1 upregulated the transcription of THI4, TPK1, RbcX2, Cu/Zn-SOD, CPK13, and CPK32 and downregulated the transcription of TH1 and CPK17 in transgenic alfalfa under drought stress. These results suggested that MsTHI1 enhances drought tolerance by strengthening photosynthesis, regulating the antioxidant defense system, maintaining osmotic homeostasis, and mediating plant signal transduction.