A calmodulin-like protein PvCML9 negatively regulates salt tolerance.

Calmodulin-like proteins (CMLs) are unique Ca2+ sensors and play crucial roles in response to abiotic stress in plants. A salt-repressed PvCML9 from halophyte seashore paspalum (Paspalum vaginatum O. Swartz) was identified. PvCML9 was localized in the cytoplasm and nucleus and highly expressed in roots and stems. Overexpression of PvCML9 led to reduced salt tolerance in rice and seashore paspalum, whereas downregulating expression of PvCML9 showed increased salt tolerance in seashore paspalum as compared with the wild type (WT), indicating that PvCML9 regulated salt tolerance negatively. Na+ and K+ homeostasis was altered by PvCML9 expression. Lower level of Na+/K+ ratio in roots and shoots was maintained in PvCML9-RNAi lines compared with WT under salt stress, but higher level in overexpression lines. Moreover, higher levels of SOD and CAT activities and proline accumulation were observed in PvCML9-RNAi lines compared with WT under salt stress, but lower levels in overexpression lines, which altered ROS homeostasis. Based on the above data, mutation of its homolog gene OsCML9 in rice by CRISPR/Cas9 was performed. The mutant had enhanced salt tolerance without affecting rice growth and development, suggesting that OsCML9 gene is an ideal target gene to generate salt tolerant cultivars by genome editing in the future.

Transcriptome Profiling Reveals Molecular Responses to Salt Stress in Common Vetch (Vicia sativa L.).

Common vetch (Vicia sativa L.) is an important annual diploid leguminous forage. In the present study, transcriptomic profiling in common vetch in response to salt stress was conducted using a salt-tolerant line (460) and a salt-sensitive line (429). The common responses in common vetch and the specific responses associated with salt tolerance in 460 were analyzed. Several KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, including plant hormone and MAPK (mitogen-activated protein kinase) signaling, galactose metabolism, and phenylpropanoid phenylpropane biosynthesis, were enriched in both lines, though some differentially expressed genes (DEGs) showed distinct expression patterns. The roots in 460 showed higher levels of lignin than in 429. α-linolenic acid metabolism, carotenoid biosynthesis, the photosynthesis-antenna pathway, and starch and sucrose metabolism pathways were specifically enriched in salt-tolerant line 460, with higher levels of accumulated soluble sugars in the leaves. In addition, higher transcript levels of genes involved in ion homeostasis and reactive oxygen species (ROS) scavenging were observed in 460 than in 429 in response to salt stress. The transcriptomic analysis in common vetch in response to salt stress provides useful clues for further investigations on salt tolerance mechanism in the future.

Overexpression of PvWAK3 from seashore paspalum increases salt tolerance in transgenic Arabidopsis via maintenance of ion and ROS homeostasis.

Seashore paspalum (Paspalum vaginatum O. Swartz) is an important warm-season turfgrass species with extreme salt tolerance, but investigations on its salt tolerance mechanism are limited. A salt induced PvWAK3 from halophyte seashore paspalum was identified in this study. Overexpression of PvWAK3 in Arabidopsis led to increased salt tolerance. Transgenic plants had higher levels of seed germination rate, root length, number of lateral roots, shoot weight, survival rate, Fv/Fm, ETR, and NPQ compared with the wild type (WT) under salt stress. Na+ content was increased and K+ content was decreased after salinity treatment, with lower levels of Na+ and Na+/K+ ratio but higher level of K+ in transgenic plants than in WT under salt stress. The improved maintenance of Na+ and K+ homeostasis was associated with the higher transcript levels of K + -Uptake Permease 4 (KUP4), Potassium Transport 2/3 (AKT2), Salt Overly Sensitive 1 (SOS1) and High-Affinity K + Transporter 5 (HAK5) in transgenic plants compared with WT. Superoxide dismutase (SOD), catalase (CAT) and ascorbate-peroxidase (APX) activities, proline concentration, and P5CS1 transcript were increased after salinity treatment, with higher levels in transgenic lines compared with WT, which led to reduced accumulation of O2·- and H2O2 under salt stress. It is suggested that PvWAK3 regulates salt tolerance positively, which is associated with promoted Na+ and K+ homeostasis, activated antioxidant enzymes, and proline biosynthesis under salt stress.

Transcriptome Analysis of Native Kentucky Bluegrass (Poa pratensis L.) in Response to Osmotic Stress.

Kentucky bluegrass (Poa pratensis L.) is an important cool season turfgrass species with a high cold tolerance, but it is sensitive to drought. It is valuable for the applications of Kentucky bluegrass to improve its drought tolerance. However, little is known about the underlying drought mechanism. In the present study, transcriptomic profiling in the roots and leaves of the Kentucky bluegrass cultivar 'Qinghai', in response to osmotic stress in the form of treatment with 2 h and 50 h of 25% (v/v) PEG-6000, was analyzed. The results showed that a large number of genes were significantly up-regulated or down-regulated under osmotic stress. The majority of genes were up-regulated in leaves but down-regulated in roots after 2 h and 50 h of osmotic stress, among them were 350 up-regulated DEGs and 20 down-regulated DEGs shared in both leaves and roots. GO and KEGG analysis showed that carbohydrate metabolism, polyamine and amino acid metabolism and the plant hormone signaling pathway were enriched in the leaves and roots of 'Qinghai' after osmotic stress. The genes involving in carbohydrate metabolism were up-regulated, and sucrose, trehalose and raffinose levels were consistently increased. The genes involved in polyamine and amino acid metabolism were up-regulated in leaves in response to osmotic stress and several amino acids, such as Glu, Met and Val levels were increased, while the genes involved in photosynthesis, carbon fixation and citrate cycle in leaves were down-regulated. In addition, the genes involved in plant hormone biosynthesis and signal transduction were altered in leaves after osmotic stress. This study provided promising candidate genes for studying drought mechanisms in 'Qinghai' and improving the drought tolerance of Kentucky bluegrass and drought-sensitive crops.

Zinc finger transcription factor MtZPT2-2 negatively regulates salt tolerance in Medicago truncatula.

Zinc finger proteins (ZFPs) are transcription factors involved in multiple cellular functions. We identified a C2H2 type ZFP (MtZPT2-2) in Medicago truncatula and demonstrated that it localizes to the nucleus and inhibits the transcription of 2 genes encoding high-affinity potassium transporters (MtHKT1;1 and MtHKT1;2). MtZPT2-2 transcripts were detected in stem, leaf, flower, seeds and roots, with the highest level in the xylem and phloem of roots and stems. MtZPT2-2 transcription in leaves was reduced after salt stress. Compared with the wild-type (WT), transgenic lines overexpressing MtZPT2-2 had decreased salt tolerance, while MtZPT2-2-knockout mutants showed increased salt tolerance. MtHKT1;1 and MtHKT1;2 transcripts and Na+ accumulation in shoots and roots, as well as in the xylem of all genotypes of plants, were increased after salt treatment, with higher levels of MtHKT1;1 and MtHKT1;2 transcripts and Na+ accumulation in MtZPT2-2-knockout mutants and lower levels in MtZPT2-2-overexpressing lines compared with the WT. K+ levels showed no significant difference among plant genotypes under salt stress. Moreover, MtZPT2-2 was demonstrated to bind with the promoter of MtHKT1;1 and MtHKT1;2 to inhibit their expression. Antioxidant enzyme activities and the gene transcript levels were accordingly upregulated in response to salt, with higher levels in MtZPT2-2-knockout mutants and lower levels in MtZPT2-2-overexpressing lines compared with WT. The results suggest that MtZPT2-2 regulates salt tolerance negatively through downregulating MtHKT1;1 and MtHKT1;2 expression directly to reduce Na+ unloading from the xylem and regulates antioxidant defense indirectly.

Jasmonate biosynthesis enzyme allene oxide cyclase 2 mediates cold tolerance and pathogen resistance.

Allene oxide cyclase (AOC) is a key enzyme in the biosynthesis of jasmonic acid (JA), which is involved in plant growth and development as well as adaptation to environmental stresses. We identified the cold- and pathogen-responsive AOC2 gene from Medicago sativa subsp. falcata (MfAOC2) and its homolog MtAOC2 from Medicago truncatula. Heterologous expression of MfAOC2 in M. truncatula enhanced cold tolerance and resistance to the fungal pathogen Rhizoctonia solani, with greater accumulation of JA and higher transcript levels of JA downstream genes than in wild-type plants. In contrast, mutation of MtAOC2 reduced cold tolerance and pathogen resistance, with less accumulation of JA and lower transcript levels of JA downstream genes in the aoc2 mutant than in wild-type plants. The aoc2 phenotype and low levels of cold-responsive C-repeat-binding factor (CBF) transcripts could be rescued by expressing MfAOC2 in aoc2 plants or exogenous application of methyl jasmonate. Compared with wild-type plants, higher levels of CBF transcripts were observed in lines expressing MfAOC2 but lower levels of CBF transcripts were observed in the aoc2 mutant under cold conditions; superoxide dismutase, catalase, and ascorbate-peroxidase activities as well as proline concentrations were higher in MfAOC2-expressing lines but lower in the aoc2 mutant. These results suggest that expression of MfAOC2 or MtAOC2 promotes biosynthesis of JA, which positively regulates expression of CBF genes and antioxidant defense under cold conditions and expression of JA downstream genes after pathogen infection, leading to greater cold tolerance and pathogen resistance.

Genome-Wide Analysis of the Wall-Associated Kinase (WAK) Genes in Medicago truncatula and Functional Characterization of MtWAK24 in Response to Pathogen Infection.

The wall-associated kinases (WAKs) can perceive and transmit extracellular signals as one kind of unique receptor-like kinases (RLKs) involved in the regulation of cell expansion, pathogen resistance and abiotic stress tolerance. To understand their potential roles and screen some key candidates in Medicago truncatula (M. truncatula), genome-wide identification and characterization of MtWAKs were conducted in this study. A total of 54 MtWAK genes were identified and classified into four groups based on their protein domains. They were distributed on all chromosomes, while most of them were clustered on chromosome 1 and 3. The synteny analysis showed that 11 orthologous pairs were identified between M. truncatula and Arabidopsis thaliana (A. thaliana) and 31 pairs between M. truncatula and Glycine max (G. max). The phylogenetic analysis showed that WAK-RLKs were classified into five clades, and they exhibited a species-specific expansion. Most MtWAK-RLKs had similar exon-intron organization and motif distribution. Multiple cis-acting elements responsive to phytohormones, stresses, growth and development were observed in the promoter regions of MtWAK-RLKs. In addition, the expression patterns of MtWAK-RLKs varied with different plant tissues, developmental stages and biotic and abiotic stresses. Interestingly, plasm membrane localized MtWAK24 significantly inhibited Phytophthora infection in tobacco. The study provides valuable information for characterizing the molecular functions of MtWAKs in regulation of plant growth, development and stress tolerance in legume plants.

Overexpression of CdtCIPK21 from triploid bermudagrass reduces salt and drought tolerance but increases chilling tolerance in transgenic rice.

Calcineurin B-like-interacting protein kinase (CIPK) is a serine/threonine kinase, which transmits the Ca2+ signal sensed by CBL proteins. A CdtCIPK21 showing highly identical to OsCIPK21 in rice was isolated from triploid bermudagrass (Cynodon dactylon × Cynodon transvaalensis). CdtCIPK21 transcript could be detected in roots, rhizomes, stems, stolons, and leaves, with highest level in roots. It was induced by salinity, dehydration and chilling, but reduced by ABA treatment. Transgenic rice plants overexpressing CdtCIPK21 had decreased salt and drought tolerance as well as ABA sensitivity but increased chilling tolerance. Lower SOD and CAT activities was observed in transgenic lines under salinity and drought stress conditions, but higher levels under chilling stress. Similarly, lower levels of proline concentration and P5CS1 and P5CS2 transcripts were maintained in transgenic lines under salinity and drought stresses, and higher levels were maintained under chilling. In addition, transgenic lines had lower transcript levels of ABA-independent genes (OsDREB1A, OsDREB1B, and OsDREB2A) and ABA responsive genes (OsLEA3, OsLIP9, and OsRAB16A) under salinity and drought but higher levels under chilling compared with WT. The results suggest that CdtCIPK21 regulates salt and drought tolerance negatively and chilling tolerance positively, which are associated with the altered ABA sensitivity, antioxidants, proline accumulation and expression of ABA-dependent and ABA-independent stress responsive genes.

Transcriptomic analysis revealed the candidate metabolic pathways and genes associated with cold tolerance in a mutant without anthocyanin accumulation in common vetch (Vicia sativa L.).

Common vetch (Vicia sativa L.) is a leguminous crop used to feed livestock with vegetative organs or fertilize soils by returning to the field. Survival of fall-seeded plants is often affected by freezing damage during overwintering. This study aims to investigate the transcriptomic profiling in response to cold in a mutant with reduced accumulation of anthocyanins under normal growth and low-temperature conditions for understanding the underlying mechanisms. The mutant had increased cold a tolerance with higher survival rate and biomass during overwintering compared to the wild type, which led to increased forage production. Transcriptomic analysis in combination with qRT-PCR and physiological measurements revealed that reduced anthocyanins accumulation in the mutant resulted from reduced expression of serial genes involving in anthocyanin biosynthesis, which led to the altered metabolism, with an increased accumulation of free amino acids and polyamines. The higher levels of free amino acids and proline in the mutant under low temperature were associated with improved cold tolerance. The altered expression of some genes involved in ABA and GA signaling was also associated with increased cold tolerance in the mutant.

Screening of Alfalfa Varieties Resistant to Phytophthora cactorum and Related Resistance Mechanism.

Alfalfa is one of the most important legume forages in the world. Root rot caused by soil-borne pathogens severely restricts the production of alfalfa. The knowledge of the interaction between alfalfa and root rot-pathogens is still lacking in China. Phytophthora cactorum was isolated from symptomatic seedlings of an alfalfa field in Nanjing with high levels of damping-off. We observed the different infection stages of P. cactorum on alfalfa, and found that the purified P. cactorum strain was aggressive in causing alfalfa seed and root rot. The infecting hyphae penetrated the epidermal cells and wrapped around the alfalfa roots within 48 h. By evaluating the resistance of 37 alfalfa cultivars from different countries to P. cactorum, we found Weston is a resistant variety, while Longdong is a susceptible variety. We further compared the activities of various enzymes in the plant antioxidant enzyme system between Weston and Longdong during P. cactorum infection, as well as gene expression associated with plant hormone biosynthesis and response pathways. The results showed that the disease-resistant variety Weston has stronger antioxidant enzyme activity and high levels of SA-responsive PR genes, when compared to the susceptible variety Longdong. These findings highlighted the process of interaction between P. cactorum and alfalfa, as well as the mechanism of alfalfa resistance to P. cactorum, which provides an important foundation for breeding resistant alfalfa varieties, as well as managing Phytophthora-caused alfalfa root rot.

Evaluation of salt tolerance in common vetch (Vicia sativa L.) germplasms and the physiological responses to salt stress.

Common vetch (Vicia sativa L.) is an important leguminous crop, providing humans with starch from seeds, feeding livestock with vegetative organs, or fertilizing soils by returning to field. It is aimed to evaluate salt tolerance in common vetch collections for breeding programs and to investigate the underlined physiological mechanisms. Relative germination rate and relative seedling growth showed great difference among common vetch collections in response to salt. A lower level of Na+ and higher levels of K+ and K+/Na+ ratio were maintained in both shoots and roots in salt-tolerant collections than in salt-sensitive ones under salt stress. Expression of the genes involved in transportation and redistribution of Na+ and K+ were cooperatively responsible for salt stress. Transcript levels of NHX7, HKT1, AKT2, and HAK17 in leaves and roots were induced after salt stress, with higher transcript levels in salt-tolerant collections compared with the sensitive ones. Proline and P5CS1 transcript levels were increased after salt stress, with higher levels in salt-tolerant collection compared with salt-sensitive ones. Both O2- and H2O2 were accumulated after salt stress, and lower levels were accumulated in salt-tolerant collection compared with salt-sensitive ones. Superoxide dismutase, catalase and ascorbate peroxidase activities were altered in response to salt and higher levels were maintained in salt-tolerant collections compared with salt-sensitive ones. It is suggested that salt tolerance in common vetch is associated with maintenance of K+ and Na+ homeostasis and the associated gene expression and promoted proline accumulation and antioxidant defense system.

Analysis of Elymus nutans seed coat development elucidates the genetic basis of metabolome and transcriptome underlying seed coat permeability characteristics.

The seed coat takes an important function in the life cycle of plants, especially seed growth and development. It promotes the accumulation of nutrients inside the seed and protects the seed embryo from mechanical damage. Seed coat permeability is an important characteristic of seeds, which not only affects seed germination, but also hinders the detection of seed vigor by electrical conductivity (EC) method. This research aimed to elucidate the mechanism of seed coat permeability formation through metabolome and transcriptome analysis of Elymus nutans. We collected the samples at 8, 18, and 28 days post-anthesis (dpa), and conducted a seed inclusion exosmosis experiment and observed the seed coat permeability. Moreover, we analyzed the changes in the metabolome and transcriptome during different development stages. Here, taking 8 dpa as control, 252 upregulated and 157 downregulated differentially expressed metabolites (DEMs) were observed and 886 upregulated unigenes and 1170 downregulated unigenes were identified at 18 dpa, while 4907 upregulated unigenes and 8561 downregulated unigenes were identified at 28 dpa. Meanwhile, we observed the components of ABC transporters, the biosynthesis of unsaturated fatty acids, and phenylalanine metabolism pathways. The key metabolites and genes affecting seed coat permeability were thiamine and salicylic acid. Furthermore, there were 13 and 14 genes with correlation coefficients greater than 0.8 with two key metabolites, respectively, and the -log2Fold Change- of these genes were greater than 1 at different development stages. Meanwhile, pathogenesis-related protein 1 and phenylalanine ammonia-lyase play an important role in regulating the formation of compounds. Our results outline a framework for understanding the development changes during seed growth of E. nutans and provide insights into the traits of seed coat permeability and supply a great significance value to seed production and quality evaluation.

Research Progress and Prospect of Alfalfa Resistance to Pathogens and Pests.

Alfalfa is one of the most important legume forages in the world and contributes greatly to the improvement of ecosystems, nutrition, and food security. Diseases caused by pathogens and pests severely restrict the production of alfalfa. Breeding resistant varieties is the most economical and effective strategy for the control of alfalfa diseases and pests, and the key to breeding resistant varieties is to identify important resistance genes. Plant innate immunity is the theoretical basis for identifying resistant genes and breeding resistant varieties. In recent years, the framework of plant immunity theory has been gradually formed and improved, and considerable progress has been made in the identification of alfalfa resistance genes and the revelation of the related mechanisms. In this review, we summarize the basic theory of plant immunity and identify alfalfa resistance genes to different pathogens and insects and resistance mechanisms. The current situation, problems, and future prospects of alfalfa resistance research are also discussed. Breeding resistant cultivars with effective resistance genes, together with other novel plant protection technologies, will greatly improve alfalfa production.

First report of Alfalfa root rot caused by Fusarium commune in China.

Alfalfa (Medicago sativa) is the most important perennial forage legume worldwide, with high yields and nutrient quality. Diseases are significant threats to alfalfa and cause substantial yield losses (Fang et al. 2021). In March 2022, a disease with symptoms similar to fusarium wilt was observed in an alfalfa (cultivar Aurora) field in Baima teaching and research base (119°18'07″E, 31°61'47″N) in Nanjing, China. Reddish-brown discoloration of the roots, stele, basal stems and the withering symptoms on the aerial portions are the specific symptoms of fusarium wilt. The disease incidence varied from 3 to 6% in around 0.3 hectares of alfalfa fields surveyed. Fresh tissues from symptomatic alfalfa were cut into pieces (3-5 mm) and surface sterilized with 75% ethanol for 30 seconds, followed by 1% sodium hypochlorite (NaClO) for 120 seconds, and three rinses with sterile distilled water. Tissue pieces were placed on selective potato dextrose agar (PDA) containing 50 mg/L rifampicin and ampicillin. Plates were sealed and incubated at 25°C for 48 to 72 hours. Over 70 tissue fragments plated, 20 isolates from different fragments showed similar colonies. These isolates were purified by single spore isolation and grown on PDA and mung bean medium for morphology identification, molecular identification and pathogenicity test. The colony showed white to pink, abundant, densely aerial mycelium, while its backside was light violet. Macroconidia were hyaline, falcate with septa, and ranged in size from 25.5 to 61.5 × 3.8 to 6.2 µm (n=50). Microconidia were non-septate, hyaline, oval, straight to slightly curved, and were 5.6 to 10.7 × 2.4 to 3.2 µm (n=50). Spherical chlamydospores were 10.6 ± 1.3 µm in size. The rDNA internal transcribed spacers (ITS), RNA polymerase II subunit 2 (RPB2) and translation elongation factor 1-α (TEF) genes were amplified and sequenced (White et al. 1990; Carbone et al. 1999; Wang et al. 2019). Blast analysis showed 99.41% to 100% identity to F. commune sequences in the GenBank database (KU341324.1, MN892350.1, MH341219.1). The pathogenicity test was conducted using the dipping method in roots. Fresh F. commune hyphae were cut into 3 x 3mm agar plugs from a 7 cm PDA plate and inoculated in 200 mL mung bean medium on a shaker at 160 rpm, 25°C for 5 days. Spores were filtered through a cheese cloth, adjusted to 1 × 106 spores/mL with distilled water and used immediately. Roots in two-week-old alfalfa seedlings were soaked in spore suspension for 30 mins before being transplanted to sterile vermiculate with four repeats, while those in sterile water were used as non-inoculated control. The plants were placed in the green house at 25°C with 16 h of light and 8 h of darkness. The symptoms were observed two weeks following the inoculation. 30% of the seedlings withered, and reddish-brown discoloration symptoms were visible on the roots and stem of the unwithered plant. F. commune was isolated from these lesions again, and no disease symptoms were observed in the control plant. F. commune has been reported to cause root and stem rot in many plants worldwide, including tobacco (Zhong et al. 2021), maize (Mezzalama et al. 2021; Xi et al. 2019), soybean (Detranaltes et al. 2022) and sugarcane (Wang et al. 2018). Our results show that F. commune was the causative agent of alfalfa root rot. To our knowledge, this is the first report of F. commune causing root rot in alfalfa. The finding provides insights for disease diagnosis of alfalfa root rot disease and management in the region.

A calmodulin-like protein (CML10) interacts with cytosolic enzymes GSTU8 and FBA6 to regulate cold tolerance.

Calmodulin-like proteins (CMLs) are calcium (Ca2+) sensors involved in plant growth and development as well as adaptation to environmental stresses; however, their roles in plant responses to cold are not well understood. To reveal the role of MsCML10 from alfalfa (Medicago sativa) in regulating cold tolerance, we examined transgenic alfalfa and Medicago truncatula overexpressing MsCML10, MsCML10-RNAi alfalfa, and a M. truncatula cml10-1 mutant and identified MsCML10-interacting proteins. MsCML10 and MtCML10 transcripts were induced by cold treatment. Upregulation or downregulation of MsCML10 resulted in increased or decreased cold tolerance, respectively, while cml10-1 showed decreased cold tolerance that was complemented by expressing MsCML10, suggesting that MsCML10 regulates cold tolerance. MsCML10 interacted with glutathione S-transferase (MsGSTU8) and fructose 1,6-biphosphate aldolase (MsFBA6), and the interaction depended on the presence of Ca2+. The altered activities of Glutathione S-transferase and FBA and levels of ROS and sugars were associated with MsCML10 transcript levels. We propose that MsCML10 decodes the cold-induced Ca2+ signal and regulates cold tolerance through activating MsGSTU8 and MsFBA6, leading to improved maintenance of ROS homeostasis and increased accumulation of sugars for osmoregulation, respectively.

Comparative Physiological and Transcriptome Profiles Uncover Salt Tolerance Mechanisms in Alfalfa.

Salinity is a major limiting factor that affects crop production. Understanding of the mechanisms of plant salt tolerance is critical for improving crop yield on saline land. Alfalfa (Medicago sativa L.) is the most important forage crop, while its salt tolerance mechanisms are largely unknown. The physiological and transcriptomic responses in two contrasting salt tolerant cultivars to salinity stress were investigated in the present study. "Magnum Salt" showed higher salt tolerance than "Adrenalin," with higher relative germination rate, survival rate, biomass and K+/Na+ ratio after salt treatment. Activities of antioxidant enzymes SOD, CAT and GR, and proline concentrations were upregulated to higher levels in roots and shoots in Magnum Salt than in Adrenalin after salinity stress, except for no difference in GR activity in shoots, and lower levels of O2 ⋅- and H2O2 were accumulated in leaves. It was interesting to find that salinity caused a decrease in total unsaturated fatty acid in Adrenalin other than Magnum Salt, C18:2 was increased significantly after salinity in Magnum Salt, while it was unaltered in Adrenalin. High quality RNA sequencing (RNA-seq) data was obtained from samples of Magnum Salt and Adrenalin at different time points (0, 2, and 26 h). Generally, "phagosome," "TCA cycle" and "oxidative phosphorylation" pathways were inhibited by salinity stress. Upregulated DEGs in Magnum Salt were specifically enriched in "fatty acid metabolism," "MAPK signaling" and "hormone signal transduction" pathways. The DEGs involved in ionic homeostasis, reactive oxygen species (ROS) scavenging and fatty acid metabolism could partially explain the difference in salt tolerance between two cultivars. It is suggested that salt tolerance in alfalfa is associated with regulation of ionic homeostasis, antioxidative enzymes and fatty acid metabolism at both transcriptional and physiological level.

Rhizobia-Legume Symbiosis Increases Aluminum Resistance in Alfalfa.

Alfalfa is the most important forage legume with symbiotic nitrogen-fixing nodule in roots, but it is sensitive to aluminum (Al), which limits its plantation in acidic soils. One rhizobia clone of Sinorhizobium meliloti with Al tolerance (AT1) was isolated from the nodule in AlCl3-treated alfalfa roots. AT1 showed a higher growth rate than the standard rhizobia strain Sm1021 under Al-stressed conditions. Alfalfa growth was improved by inoculation with AT1 under Al-stressed conditions, with increased length and fresh weight in shoots and roots. High nitrogenase activity and pink effective nodules were obtained in AT1-inoculated plant roots under Al stress, with increased total nitrogen compared with the non-inoculated control. The application of exogenous NH4+-nitrogen increased the Al resistance in alfalfa. It is suggested that rhizobia's increase of the Al resistance in alfalfa is associated with its improved nitrogen status. Inoculation with Al-tolerant rhizobia is worth testing in an acidic field for improved alfalfa productivity.

A novel LRR-RLK (CTLK) confers cold tolerance through regulation on the C-repeat-binding factor pathway, antioxidants, and proline accumulation.

Leucine-rich repeat-receptor-like kinase (LRR-RLK) is a large subfamily of plant RLKs; however, its role in cold tolerance is still unknown. A novel cold tolerance LRR-RLK gene (MtCTLK1) in Medicago truncatula was identified using the transgenic lines overexpressing MtCTLK1 (MtCTLK1-OE) and mtctlk1 lines with Tnt1 retrotransposon insertion. Compared with the wild-type, MtCTLK1-OE lines had increased cold tolerance and mtctlk1 showed decreased cold tolerance. The impaired cold tolerance in mtctlk1 could be complemented by the transgenic expression of MtCTLK1 or its homolog MfCTLK1 from Medicago falcata. Antioxidant enzyme activities and proline accumulation as well as transcript levels of the associated genes were increased in response to cold, with higher levels in MtCTLK1-OE or lower levels in mtctlk1 lines as compared with wild type. C-Repeat-Binding Factors (CBFs) and CBF-dependent cold-responsive genes were also induced in response to cold, and higher transcript levels of CBFs and CBF-dependent cold-responsive genes were observed in MtCTLK1-OE lines whereas lower levels in mtctlk1 mutants. The results validate the role of MtCTLK1 or MfCTLK1 in the regulation of cold tolerance through the CBF pathway, antioxidant defense system and proline accumulation. It also provides a valuable gene for the molecular breeding program to improve cold tolerance in crops.

Genome-Wide Identification and Expression Analysis of the Aux/IAA and Auxin Response Factor Gene Family in Medicago truncatula.

Aux/IAA and auxin response transcription factor (ARF) genes are key regulators of auxin responses in plants. A total of 25 MtIAA and 40 MtARF genes were identified based on the latest updated Medicago truncatula reference genome sequence. They were clustered into 10 and 8 major groups, respectively. The homologs among M. truncatula, soybean, and Arabidopsis thaliana shared close relationships based on phylogenetic analysis. Gene structure analysis revealed that MtIAA and MtARF genes contained one to four concern motifs and they are localized to eight chromosomes, except chromosome 6 without MtARFs. In addition, some MtIAA and MtARF genes were expressed in all tissues, while others were specifically expressed in specific tissues. Analysis of cis-acting elements in promoter region and expression profiles revealed the potential response of MtIAA and MtARF genes to hormones and abiotic stresses. The prediction protein-protein interaction network showed that some ARF proteins could interact with multiple Aux/IAA proteins, and the reverse is also true. The investigation provides valuable, basic information for further studies on the biological functions of MtIAA and MtARF genes in the regulation of auxin-related pathways in M. truncatula.

A novel Medicago truncatula calmodulin-like protein (MtCML42) regulates cold tolerance and flowering time.

Calmodulin-like proteins (CMLs) are one of the Ca2+ sensors in plants, but the functions of most CMLs remain unknown. The regulation of cold tolerance and flowering time by MtCML42 in Medicago truncatula and the underlying mechanisms were investigated using MtCML42-overexpressing plants and cml42 Medicago mutants with a Tnt1 retrotransposon insertion. Compared with the wild type (WT), MtCML42-overexpressing lines had increased cold tolerance, whereas cml42 mutants showed decreased cold tolerance. The impaired cold tolerance in cml42 could b complemented by MtCML42 expression. The transcript levels of MtCBF1, MtCBF4, MtCOR413, MtCAS15, MtLTI6A, MtGolS1 and MtGolS2 and the concentrations of raffinose and sucrose were increased in response to cold treatment, whereas higher levels were observed in MtCML42-overexpressing lines and lower levels were observed in cml42 mutants. In addition, early flowering with upregulated MtFTa1 and downregulated MtABI5 transcripts was observed in MtCML42-overexpressing lines, whereas delayed flowering with downregulated MtFTa1 and upregulated MtABI5 was observed in cml42. MtABI5 expression could complement the flowering phenotype in the Arabidopsis mutant abi5. Our results suggest that MtCML42 positively regulates MtCBF1 and MtCBF4 expression, which in turn upregulates the expression of some COR genes, MtGolS1 and MtGolS2, which leads to raffinose accumulation and increased cold tolerance. MtCML42 regulates flowering time through sequentially downregulating MtABI5 and upregulating MtFTa1 expression.