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Adaptive changes in crops contribute to the diversity of agronomic traits, which directly or indirectly affect yield. The change of pubescence form from appressed to erect is a notable feature during soybean domestication. However, the biological significance and regulatory mechanism underlying this transformation remain largely unknown. Here, we identified a major-effect locus, PUBESCENCE FORM 1 ( PF1 ), the upstream region of Mao1 , that regulates pubescence form in soybean. The insertion of a Ty3/ Gypsy retrotransposon in PF1 can recruit the transcription factor GAGA-binding protein to a GA-rich region, which up-regulates Mao1 expression, underpinning soybean pubescence evolution. Interestingly, the proportion of improved cultivars with erect pubescence increases gradually with increasing latitude, and erect-pubescence cultivars have a higher yield possibly through a higher photosynthetic rate and photosynthetic stability. These findings open an avenue for molecular breeding through either natural introgression or genome editing toward yield improvement and productivity.

(1) Background: Alfalfa is an important legume forage throughout the world. Although alfalfa is considered moderately tolerant to salinity, its production and nitrogen-fixing activity are greatly limited by salt stress. (2) Methods: We examined the physiological changes and proteomic profiles of alfalfa with active nodules (NA) and without nodules (NN) under NaCl treatment. (3) Results: Our data suggested that NA roots showed upregulation of the pathways of abiotic and biotic stress responses (e.g., heat shock proteins and pathogenesis-related proteins), antioxidant enzyme synthesis, protein synthesis and degradation, cell wall degradation and modification, acid phosphatases, and porin transport when compared with NN plants under salt stress conditions. NA roots also upregulated the processes or proteins of lipid metabolism, heat shock proteins, protein degradation and folding, and cell cytoskeleton, downregulated the DNA and protein synthesis process, and vacuolar H+-ATPase proteins under salt stress. Besides, NA roots displayed a net H+ influx and low level of K+ efflux under salt stress, which may enhance the salt tolerance of NA plants. (4) Conclusions: The rhizobium symbiosis conferred the host plant salt tolerance by regulating a series of physiological processes to enhance stress response, improve antioxidant ability and energy use efficiency, and maintain ion homeostasis.

Cytokinin oxidase/dehydrogenase (CKXs) are involved in various physiological processes, including cytokinins (CKs) catabolism, root system architecture and response to abiotic stresses in plants. Alfalfa (Medicago sativa) is a widely cultivated forage which is frequently threatened by high salinity, and the potential role of CKXs in alleviating the salt stress in alfalfa lacked attention. In this study, we isolated a CKX gene from alfalfa, MsCKX (MK177192), and identified its biological functions by overexpressing it in Arabidopsis. MsCKX shares high sequence identity with CKX from other legume plants, especially Medicago truncatula (98%). MsCKX was clearly tissue-specific, and it was mainly expressed in roots. In addition, the expression of MsCKX increased under salt stress and abscisic acid (ABA) treatment. Overexpression of MsCKX gene increased the activity of CKX, which led to an enlarged root system in transgenic Arabidopsis plants. Overexpression of MsCKX gene enhanced salt tolerance of transgenic plants by maintaining a higher K+/Na+ ratio, enhancing the activities of antioxidant enzymes to scavenge ROS and increasing the expression levels of stress-related genes (P5CS1, DREB2, ion transporters and H+ pumps). Taken together, these results shed light on the roles of MsCKX involved in salt tolerance and may have applications in salt-resistant breeding of alfalfa.

Aquaporins (AQPs) are channel proteins that facilitate water transport across cell membranes and play important roles in many biological processes. However, most AQP functions are still poorly understood in the plant kingdom. Here, MsPIP2;2 was isolated and identified from alfalfa (Medicago sativa). MsPIP2;2 was localized to the plasma membrane, and its expression was induced by salt and abscisic acid (ABA) treatment. Overexpression of MsPIP2;2 in Arabidopsis increased the seed germination rate, seedling root length, survival rate, proline content and antioxidant defence activity and decreased cell membrane damage and reactive oxygen species (ROS) accumulation compared to those in WT under salt stress. The salt tolerance of MsPIP2;2 was affected by Ca2+ and pH in transgenic Arabidopsis plants. MsPIP2;2-overexpressing plants maintained a better K+/Na+ ratio and higher Ca2+ content under salt stress. The higher K+/Na+ maintenance in transgenic plants was mainly achieved by increasing Na+ efflux and K+ retention in roots via regulating the expression of the related ion channel genes. Stress-responsive genes, including P5CS1, RD29A, DREB2 and KIN2, were upregulated in transgenic plants under salt stress. These results suggest that MsPIP2;2 confers salt tolerance by regulating antioxidant defence system-mediated ROS scavenging, K/Na ion homeostasis and stress-responsive gene expression in plants.

Phosphorus is one of the essential macronutrients required by plant growth and development, but phosphate resources are finite and diminishing rapidly because of the huge need in global agriculture. In this study, 11 genes were found in the Phosphate Transporter 1 (PHT1) family of Medicago truncatula. Seven genes of the PHT1 family were available by qRT-PCR. Most of them were expressed in roots, and almost all genes were induced by low-phosphate stress in the nodule. The expression of MtPT6 was relatively high in nodules and induced by low-phosphate stress. The fusion expression of MtPT6 promoter-GUS gene in M. truncatula suggested that the expression of MtPT6 was induced in roots and nodules by phosphate starvation. In roots, MtPT6 was mainly expressed in vascular tissue and tips, and it was also expressed in cortex under low-phosphate stress; in nodules, it was mainly expressed in vascular bundles, cortical cells, and fixation zone cells. MtPT6 had a close relationship with other PHT1 family members according to amino acid alignment and phylogenetic analysis. Subcellular localization analysis in tobacco revealed that MtPT6 protein was localized to the plasma membrane. The heterologous expression of MtPT6 in Arabidopsis knockout mutants of pht1.1 and pht1.4 made seedlings more susceptible to arsenate treatment, and the phosphate concentrations in pht1.1 were higher in high phosphate condition by expressing MtPT6. We conclude that MtPT6 is a typical phosphate transporter gene and can promote phosphate acquisition efficiency of plants.

Many beneficial effects of symbiotic rhizobia on leguminous plants have been reported. Here we report a novel effect of rhizobia on forage quality in alfalfa. We found that nodulated alfalfa showed an increase in lignin content and a decrease in digestibility in comparison with non-nodulated plants. Detailed studies revealed that nodulation resulted in an increase in monolignol G unit and S unit. An overall increase in lignin content in nodulated alfalfa was associated with more lignified tissues in the stem and an upregulation of transcript levels of several lignin biosynthesis genes. We hypothesize that an increase in lignin content in nodulated alfalfa is a result of defensive response in plants to rhizobial invasion.

Salt stress negatively affects alfalfa (Medicago sativa L.) production and biological nitrogen fixation. We investigated whether rhizobium symbiosis has an effect on host plant tolerance to salt stress. We determined the survival rate, oxidative damage level, activities of antioxidant enzymes, and contents of osmotic solutes in the leaf and root of 4-month-old alfalfa with active nodules, inactive nodules or without nodules, and under short-term salt stress. Alfalfa with active nodules showed higher survival rate. Higher survival rate was associated with reduced lipid peroxidation, higher activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) as well as higher concentrations of reduced glutathione (GSH) and soluble sugar, especially in roots under salt stress. Variance analysis indicated nodulation affected the activities of SOD, CAT, POD, and APX along with concentrations of GSH, soluble sugar, and soluble protein. Inoculation also resulted in higher basal levels of superoxide anion radical (O2 −·) without salt stress. Rhizobium symbiosis had a positive effect on alfalfa salt tolerance by improving the activity of antioxidant enzymes and osmotic adjustment capacity.

The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized from alfalfa and validated for its function of tolerance toward drought and salt stresses by heterologous expression in Nicotiana tabacum. Zeaxanthin epoxidase (ZEP) plays important roles in plant response to various environment stresses due to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the expression characteristics and the biological functions of ZEP in alfalfa (Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned, characterized and overexpressed in Nicotiana tabacum. The open reading frame of MsZEP contains 1992 bp nucleotides and encodes a 663-amino acid polypeptide. Amino acid sequence alignment indicated that deduced MsZEP protein was highly homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP was grouped into a branch with other legume plants. Real-time quantitative PCR revealed that MsZEP gene expression was clearly tissue-specific, and the expression levels were higher in green tissues (leaves and stems) than in roots. MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment, while the expression levels in roots showed different trends. Besides, the results showed that nodules could up-regulate the MsZEP expression under non-stressful conditions and in the earlier stage of different abiotic stress. Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to drought and salt stress by affecting various physiological pathways, ABA levels and stress-responsive genes expression. Taken together, these results suggested that the MsZEP gene may be involved in alfalfa responses to different abiotic stresses and nodules, and could enhance drought and salt tolerance of transgenic tobacco by heterologous expression.

Glutathione S-transferases are important enzymes in protecting cells by scavenging reactive oxygen species induced by various stresses. In this study, a novel GST gene, MsGST (KM044312), was cloned and characterized from alfalfa. The open reading frame of MsGST contains 660bp nucleotides, encoding 219 amino acid residues. Amino acid sequence alignment indicated that the deduced MsGST protein was highly homologous to other plant tau class GST sequences. According to amino acid phylogenetic analysis, the MsGST gene was clustered into the same branch with other legume plants. Real-time quantitative PCR (qRT-PCR) revealed that the expression levels of MsGST were up-regulated in both shoots and roots under ABA treatment and various stresses, including salt, drought, cold and heat stress. The effect of nodules on MsGST gene expression indicated that the induction of MsGST expression by abiotic stress is independent of rhizobium symbiosis. In conclusion, the MsGST gene may be involved in response to different abiotic stresses in alfalfa.

Dehydration responsive element binding (DREB) transcription factors play an important role in the regulation of stress-related genes. These factors contribute to resistance to different abiotic stresses. In the present study, a novel DREB transcription factor, BdDREB2, isolated from Buchloe dactyloides, was cloned and characterized. The BdDREB2 protein was estimated to have a molecular weight of 28.36kDa, a pI of 5.53 and a typical AP2/ERF domain. The expression of BdDREB2 was involved in responses to drought and salt stresses. Overexpression of BdDREB2 in tobacco showed higher relative water and proline content, and was associated with lower MDA content under drought stress, suggesting that the transgenic tobacco may tolerate drought stress better. Results demonstrate that BdDREB2 may play an important role in the regulation of abiotic stress responses, and mediate many physiological pathways that enhance stress tolerance in plants.