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.

Oat is a food and forage crop species widely cultivated worldwide, and it is also an important forage grass in plateau regions of China, where there is a high level of ultraviolet radiation and sunlight. Screening suitable reference genes for oat under UV-B and high-light stresses is a prerequisite for ensuring the accuracy of real-time quantitative PCR (qRT–PCR) data used in plant adaptation research. In this study, eight candidate reference genes (sulfite oxidase, SUOX; victorin binding protein, VBP; actin-encoding, Actin1; protein PSK SIMULATOR 1-like, PSKS1; TATA-binding protein 2-like, TBP2; ubiquitin-conjugating enzyme E2, UBC2; elongation factor 1-alpha, EF1-α; glyceraldehyde-3-phosphate dehydrogenase 1, GAPDH1;) were selected based on previous studies and our oat transcriptome data. The expression stability of these reference genes in oat roots, stems, and leaves under UV-B and high-light stresses was first calculated using three frequently used statistical software (geNorm, NormFinder, and BestKeeper), and then the comprehensive stability of these genes was evaluated using RefFinder. The results showed that the most stably expressed reference genes in the roots, stems, and leaves of oat under UV-B stress were EF1-α, TBP2, and PSKS1, respectively; the most stably expressed reference genes in the roots, stems, and leaves under high-light stress were PSKS1, UBC2, and PSKS1, respectively. PSKS1 was the most stably expressed reference gene in all the samples. The reliability of the selected reference genes was further validated by analysis of the expression of the phenylalanine ammonia-lyase (PAL) gene. This study highlights reference genes for accurate quantitative analysis of gene expression in different tissues of oat under UV-B and high-light stresses.

Thioredoxin (TRX) is a small molecule protein that participates in the redox process and plays a decisive role in various functions of plants. However, the role of TRX in Medicago sativa (alfalfa), a widely cultivated perennial herb of legume, is still poorly understood. Here, we isolated MsTRX from alfalfa and determined the characteristics in improving salt tolerance by assaying the phenotype and physiological changes and the expression of stress-response genes in transgenic tobacco. The expression of MsTRX was similar in alfalfa roots, leaves, and inflorescences, and was downregulated in response to cold, drought, and salt treatment. The overexpression of MsTRX in tobacco promoted the accumulation of soluble sugar (SS) and proline; enhanced the activity of peroxidase (POD); and induced the upregulation of beta-amylase 1 (BAM1), lipid-transfer protein 1 (LTP1), candidate signal molecules/sensor relay proteins (CBSX3), superoxide dismutase [Cu-Zn] (Cu/Zn-SOD), superoxide dismutase [Mn] (Mn-SOD), protein gamma response 1 (GR1), dehydrin DHN1-like (ERD10B), and serine/threonine-protein kinase (SnRK2), as well as the downregulation of phyB activation-tagged suppressor1 (BAS1) and serine/threonine-protein kinase that phosphorylates LHCII protein 7 (STN7) under salt stress. These results indicated that MsTRX improves salt tolerance via maintaining osmotic homeostasis, scavenging reactive oxygen species (ROS), and regulating the transcription of stress-response genes in plants. In our study, we provided a new understanding of how MsTRX improves salt stress in plants and how MsTRX can be included in future breeding programs to improve salt tolerance in alfalfa.

Drought is a critical limiting factor for crop yield, posing a substantial threat to global food security and negatively influencing plant growth and development. As a crop used as both a grain and forage, the yield of oat (Avena sativa) is significantly affected by drought. Glutamine synthetase (GS) is a crucial enzyme in plant nitrogen metabolism and plays an essential role in nitrogen utilization, growth regulation, and yield formation. We used bioinformatics analysis to identify the GS gene family in oats, and employed molecular biology, genetics and plant physiology methods to investigate the drought-resistant function of AsGS2-2C . In this study, 11 oat AsGS genes were identified, and the gene family and expression patterns were analyzed. Our findings revealed that a majority of AsGS genes were upregulated under drought and salt stress, whereas they were downregulated in response to cold stress and abscisic acid treatment. Cloning and functional analysis of the AsGS2-2C gene revealed that transgenic tobacco overexpressing AsGS2-2C presented increased tolerance to drought stress in phenotype. Physiological analyses revealed an increase in antioxidant enzyme activities and a reduction in membrane damage in AsGS2-2C-overexpressing plants. Under drought conditions, the expression of stress-responsive genes ( Cu/Zn-SOD, MnSOD, CBL1, GR1, GAPC, Gln1-5 , and BI-1 ) was significantly elevated in AsGS2-2C transgenic tobacco. Interestingly, ACR11, GLU, ERD10B, Hxk3 and Ltp1 exhibited initial upregulation followed by subsequent downregulation. These findings provide valuable insights into the molecular mechanisms underlying drought tolerance mediated by AsGS2-2C in oat, offering potential targets for crop improvement against drought.

A high salt environment seriously affects the physiological metabolism and yield of plants. Hordeum brevisubulatum (Trin.) Link has high biomass and important ecological, feeding and economic values, but its growth conditions have serious saline-alkali effects. The NHX gene family plays a vital role in regulating intracellular Na⁺/K⁺ balance, pH homeostasis, and vesicle and protein transport in plants. In this study, the HbNHX2 gene was cloned from Hordeum brevisubulatum and functionally characterized through phenotypic, physiological, and molecular analyses in transgenic tobacco. Expression profiling revealed that HbNHX2 was most abundant in spikes and least abundant in root tips, and the expression level was significantly induced under salt stress. Overexpression of HbNHX2 led to decreased malondialdehyde (MDA) and superoxide anion (O²⁻) levels, while it enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Additionally, the levels of glutathione (GSH), soluble proteins, proline, and chlorophyll were also increased. Several stress-responsive genes, including CBL1, ERF2, BI-1, Cu/Zn-SOD, Mn-SOD, POD, GR1, KC1, TPK1, TIP, P5CS, BAS1, STN7 and LTP1, were significantly upregulated, while SERK3B was downregulated. These findings suggest that HbNHX2 enhances plant salt tolerance by maintaining osmotic balance, scavenging reactive oxygen species (ROS), and regulating stress-responsive gene expression. This study provides new insights into the molecular mechanism of salt tolerance in Hordeum brevisubulatum and lays a foundation for breeding salt-tolerant forage crops.

Oat, an important cereal and forage crop, is significantly affected by drought stress during production. However, the molecular mechanisms underlying oat’s response to drought stress remain largely unknown. In this study, K-means clustering classified 28 oat varieties into drought-tolerant (Muda, Mengshi No. 1) and drought-sensitive (Heike, Haywire) groups, with grey relational analysis further verifying MS as the most drought-tolerant and HK as the most drought-sensitive variety. Under drought stress, drought-tolerant and drought-sensitive varieties showed notable differences in leaf chlorophyll content, osmoregulation substances, and the activities of antioxidant enzymes. Transcriptomic analysis showed that 1915 differentially expressed genes (DEGs) were shared among all comparisons between treatment groups and the control group. KEGG pathway analysis revealed enrichment in pathways such as plant–pathogen interactions, plant hormone signal transduction, and starch and sucrose metabolism. In the signal transduction of plant hormones, eight PP2C genes associated with ABA signaling were increased, indicating that oats might respond to drought by enhancing metabolic activities via the ABA signaling pathway. WGCNA identified gene modules significantly associated with physiological traits. Notably, Mantel tests revealed that six core genes exhibited a positive correlation with CAT activity in the drought-tolerant variety, while showing an opposite trend in the sensitive variety. This study provides insights into the mechanisms of drought tolerance in oats and aids in the molecular breeding of drought-tolerant varieties.