Metabolomics and transcriptomics analysis revealed the response mechanism of alfalfa to combined cold and saline-alkali stress.

Cold and saline-alkali stress are frequently encountered by plants, and they often occur simultaneously in saline-alkali soils at mid to high latitudes, constraining forage crop distribution and production. However, the mechanisms by which forage crops respond to the combination of cold and saline-alkali stress remain unknown. Alfalfa (Medicago sativa L.) is one of the most essential forage grasses in the world. In this study, we analyzed the complex response mechanisms of two alfalfa species (Zhaodong [ZD] and Blue Moon [BM]) to combined cold and saline-alkali stress using multi-omics. The results revealed that ZD had a greater ability to tolerate combined stress than BM. The tricarboxylic acid cycles of the two varieties responded positively to the combined stress, with ZD accumulating more sugars, amino acids, and jasmonic acid. The gene expression and flavonoid content of the flavonoid biosynthesis pathway were significantly different between the two varieties. Weighted gene co-expression network analysis and co-expression network analysis based on RNA-Seq data suggested that the MsMYB12 gene may respond to combined stress by regulating the flavonoid biosynthesis pathway. MsMYB12 can directly bind to the promoter of MsFLS13 and promote its expression. Moreover, MsFLS13 overexpression can enhance flavonol accumulation and antioxidant capacity, which can improve combined stress tolerance. These findings provide new insights into improving alfalfa resistance to combined cold and saline-alkali stress, showing that flavonoids are essential for plant resistance to combined stresses, and provide theoretical guidance for future breeding programs.

Antagonistic activity and mechanism of Bacillus subtilis CG-6 suppression of root rot and growth promotion in Alfalfa.

Root rot is a common disease, that severely affects the yield and quality of alfalfa. Biocontrol is widely used to control plant diseases caused by pathogenic fungi, however, biocontrol strains for alfalfa root rot are very limited. In this study, a Bacillus subtilis CG-6 strain with a significant biocontrol effect on alfalfa root rot was isolated. CG-6 secretes antibacterial enzymes and siderophore, phosphate solubilization and indoleacetic acid (IAA). The inhibition rate of strain CG-6 against Fusarium oxysporum was 87.33%, and it showed broad-spectrum antifungal activity. Inoculation with CG-6 significantly reduced the incidence of alfalfa root rot, the control effect of greenhouse cultivation reached 58.12%, and CG-6 treatment significantly increased alfalfa plant height, root length, fresh weight, and dry weight. The treatment with CG-6 significantly increased the levels of antioxidant enzymes (catalase, peroxidase, superoxide dismutase, and lipoxygenase) in alfalfa leaves by 15.52%-34.03%. Defensive enzymes (chitinase and ß-1,3-glucanase) increased by 24.37% and 28.08%, respectively. The expression levels of regulatory enzyme genes (MsCAT, MsPOD, MsCu, Zn-SOD1, MsCu, Zn-SOD2, MsCu, Zn-SOD3, and MsLOX2) and systemic resistance genes (MsPR1, MsPDF1.2, and MsVSP2) increased by 0.50-2.85 fold, which were higher than those in the pathogen treatment group. Therefore, CG-6 could be used as a potential strain to develop biopesticides against alfalfa root rot.

Comparative Analysis of Chloroplast Pan-Genomes and Transcriptomics Reveals Cold Adaptation in Medicago sativa.

Alfalfa (Medicago sativa) is a perennial forage legume that is widely distributed all over the world; therefore, it has an extremely complex genetic background. Though population structure and phylogenetic studies have been conducted on a large group of alfalfa nuclear genomes, information about the chloroplast genomes is still lacking. Chloroplast genomes are generally considered to be conservative and play an important role in population diversity analysis and species adaptation in plants. Here, 231 complete alfalfa chloroplast genomes were successfully assembled from 359 alfalfa resequencing data, on the basis of which the alfalfa chloroplast pan-genome was constructed. We investigated the genetic variations of the alfalfa chloroplast genome through comparative genomic, genetic diversity, phylogenetic, population genetic structure, and haplotype analysis. Meanwhile, the expression of alfalfa chloroplast genes under cold stress was explored through transcriptome analysis. As a result, chloroplast genomes of 231 alfalfa lack an IR region, and the size of the chloroplast genome ranges from 125,192 bp to 126,105 bp. Using population structure, haplotypes, and construction of a phylogenetic tree, it was found that alfalfa populations could be divided into four groups, and multiple highly variable regions were found in the alfalfa chloroplast genome. Transcriptome analysis showed that tRNA genes were significantly up-regulated in the cold-sensitive varieties, while rps7, rpl32, and ndhB were down-regulated, and the editing efficiency of ycf1, ycf2, and ndhF was decreased in the cold-tolerant varieties, which may be due to the fact that chloroplasts store nutrients through photosynthesis to resist cold. The huge number of genetic variants in this study provide powerful resources for molecular markers.

Deciphering the genetic basis of resistance to soybean cyst nematode combining IBD and association mapping.

KEY MESSAGE: IBD analysis clarified the dynamics of chromosomal recombination during the ZP pedigree breeding process and identified ten genomic regions resistant to SCN race3 combining association mapping. Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most devastating pathogens for soybean production worldwide. The cultivar Zhongpin03-5373 (ZP), derived from SCN-resistant progenitor parents, Peking, PI 437654 and Huipizhi Heidou, is an elite line with high resistance to SCN race3. In the current study, a pedigree variation map was generated for ZP and its ten progenitors using 3,025,264 high-quality SNPs identified from an average of 16.2 × re-sequencing for each genome. Through identity by decent (IBD) tracking, we showed the dynamic change of genome and detected important IBD fragments, which revealed the comprehensively artificial selection of important traits during ZP breeding process. A total of 2,353 IBD fragments related to SCN resistance including SCN-resistant genes rhg1, rhg4 and NSFRAN07 were identified based on the resistant-related genetic paths. Moreover, 23 genomic regions underlying resistance to SCN race3 were identified by genome-wide association study (GWAS) in 481 re-sequenced cultivated soybeans. Ten common loci were found by both IBD tracking and GWAS analysis. Haplotype analysis of 16 potential candidate genes suggested a causative SNP (C/T, - 1065) located in the promoter of Glyma.08G096500 and encoding a predicted TIFY5b-related protein on chr8 was highly correlated with SCN race3 resistance. Our results more thoroughly elucidated the dynamics of genomic fragments during ZP pedigree breeding and the genetic basis of SCN resistance, which will provide useful information for gene cloning and the development of resistant soybean cultivars using a marker-assisted selection approach.

Genome-wide identification and characterization of filamentation temperature-sensitive H (FtsH) genes and expression analysis in response to multiple stresses in Medicago truncatula.

BACKGROUND: Filamentation temperature-sensitive H (FtsH) is an AAA+ ATP-dependent protease that plays a vital role in plant environmental adaption and tolerance. However, little is known about the function of the FtsH gene family in the most important legume model plant, Medicago truncatula. METHODS AND RESULTS: To identify and investigate the potential stress adaptation roles of FtsH gene family in M. truncatula, we conducted a series of genome-wide characterization and expression analyses. Totally, twenty MtFtsH genes were identified, which were unevenly distributed across eight chromosomes and classified into six evolution groups based on their phylogenetic relationships, with each group containing similar structures and motifs. Furthermore, MtFtsH genes exhibited a high degree of collinearity and homology with leguminous plants such as alfalfa and soybean. Multiple cis-elements in the upstream region of MtFtsH genes were also identified that responded to light, abiotic stress, and phytohormones. Public RNA-seq data indicated that MtFtsH genes were induced under both salt and drought stresses, and our transcript expression analysis showed that MtFtsH genes of MtFtsH1, MtFtsH2, MtFtsH4, MtFtsH9, and MtFtsH10 were up-regulated after ABA, H2O2, PEG, and NaCl treatments. These results suggest that MtFtsH genes may play a critical role in drought and high salt stress responses and the adaption processes of plants. CONCLUSIONS: This study provides a systematic analysis of FtsH gene family in M. truncatula, serving as a valuable molecular theoretical basis for future functional investigations. Our findings also extend the pool of potential candidate genes for the genetic improvement of abiotic stress tolerance in legume crops.

Overexpression of CcFALDH from spider plant (Chlorophytum comosum) enhances the formaldehyde removing capacity of transgenic gloxinia (Sinningia speciosa)1.

Formaldehyde can cause leukemia and nasopharyngeal cancer in humans, and is a major indoor air pollutant. In this study, to improve the ability of flowering plants to purify formaldehyde, we cloned the CcFALDH gene encoding formaldehyde dehydrogenase (FALDH) from the spider plant (Chlorophytum comosum), which encodes 379 amino acids with the alcohol dehydrogenase (ADH) structural domain, and used it to transform the flowering plant gloxinia (Sinningia speciosa). The FALDH activity of transgenic gloxinia was 1.8-2.7 times that of wild-type (WT) with a considerable increase in formaldehyde stress tolerance. The activities of the antioxidant enzymes SOD, POD, and CAT of transgenic gloxinia were 1.5-2.0 times those of the WT under formaldehyde stress; H2O2, O2-, and MDA contents were markedly lower than those in WT. Liquid formaldehyde and gaseous formaldehyde were metabolized at 2.1-2.8 and 2.1-2.7 times higher rates in transgenic gloxinia than in WT. Our findings indicate that overexpression of CcFALDH can enhance the capacity of flowering plants to metabolize formaldehyde, which provides a new strategy to tackle the indoor formaldehyde pollution problem.

Isolation and Identification of a Phosphate-Solubilizing Pantoea dispersa with a Saline-Alkali Tolerance and Analysis of Its Growth-Promoting Effects on Silage Maize Under Saline-Alkali Field Conditions.

Phosphate-solubilizing bacteria (PSB) are microorganisms that can dissolve insoluble phosphorus (P) to accessible forms. This study aimed to screen saline-alkali-tolerant PSB and analyze its growth promoting properties, and evaluate its effects on the growth, quality, soil nutrient balance, and enzyme activities of silage maize in the field. We isolated six phosphate-solubilizing strains from rhizosphere soil of silage maize planted in saline-alkali land, and FC-1 with the best P-solubilizing effect was used for further study. The morphological, physiological and biochemical analysis, and 16S rDNA and housekeeping gene atpD sequencing were performed for identification. FC-1 was identified as Pantoea dispersa and had high P solubility. The phosphate solubility of FC-1 using four P sources ranged from 160.79 to 270.22 mg l-1. FC-1 produced indole-3-acetic acid (IAA) and decreased the pH of the growth media by secreting organic acids, including citric acid, malic acid, succinic acid, and acetic acid. The results of a field experiment indicated that FC-1 treatment increased the height, stem diameter, fresh weight, dry weight, starch content, crude protein content, and total P content of silage maize by 9.8, 9.2, 12.6, 11.7, 12.6, 18.3, and 17.4%, respectively. The nitrogen, potassium, phosphorus, and organic matter contents in the rhizosphere soil of silage maize increased by 29.8, 17.1, 17.9, and 25.3%, respectively; urease, catalase, sucrase, and alkaline phosphatase levels also increased by 24.7, 26.7, 24.0, and 19.5%, respectively. FC-1 promoted the growth of silage maize by improving nutrient metabolism and enzyme activities in saline-alkali soil and may be an effective alternative to fertilizers.

Role of FoERG3 in Ergosterol Biosynthesis by Fusarium oxysporum and the Associated Regulation by Bacillus subtilis HSY21.

Ergosterol is an important component of the fungal cell membrane and represents an effective target of chemical pesticides. However, the current understanding of ergosterol biosynthesis in the soybean root rot pathogen Fusarium oxysporum remains limited. In addition, the regular use of fungicides that inhibit ergosterol synthesis will seriously harm the ecological environment and human health. Bacillus subtilis is gradually replacing chemical control as a safe and effective biological agent; to investigate its effect on ergosterol synthesis of F. oxysporum, we verified the biological function of the FoERG3 gene of F. oxysporum by constructing knockout mutants. The results showed that knocking out FoERG3 blocked ergosterol biosynthesis, restricted mycelial growth, and increased the sensitivity to external stressors (NaCl, D-sorbitol, Congo Red, and H2O2). The increased permeability of the cell membrane promoted increased extracellular K+ levels and decreased mitochondrial cytochrome C contents. Treatment with suspension of B. subtilis HSY21 cells resulted in similar damage as observed when treating FoERG3-knockout F. oxysporum cells with ergosterol, which was characterised by deformity and swelling of the mycelium surface; increased membrane permeability; decreased pathogenicity to soybeans; and significantly decreased activities of cellulase, ß-glucosidase, amylase, and pectin-methyl galactosylase. Notably, deleting FoERG3 resulted in a significant lag in the defense-response time of soybeans. Our results suggest that FoERG3 strongly influences the virulence of F. oxysporum and may be used as a potential antimicrobial target by B. subtilis HSY21 to inhibit ergosterol synthesis, which supports the use of B. subtilis as a biological control agent for protecting against F. oxysporum infection.

Vehicle State Joint Estimation Based on Lateral Stiffness.

In this study, a vehicle state joint estimation method based on lateral stiffness was applied to estimate the running states of electric vehicles driven by rear-drive, in-wheel motors. Different from the estimation methods used in other research, the joint estimator designed in this study uses the least-squares (LS) algorithm to estimate the lateral stiffness of the front and rear axles of the vehicle, deploying the high-degree cubature Kalman filter algorithm to estimate the vehicle state. We establish a three-degree-of-freedom nonlinear vehicle model with longitudinal velocity, lateral velocity, and yaw rate, and the lateral stiffness of the front and rear axles as the principal parameters. For the low-speed running state of the vehicle, a linearized magic tire model with high fitting accuracy was used to calculate the lateral force of the entire vehicle. The LS algorithm with a forgetting factor was used to design a lateral stiffness estimator to assess the front-axle and rear-axle lateral stiffness of the entire vehicle. The generalized high-degree cubature Kalman filter (GHCKF) algorithm was used to design the vehicle state estimator and further improve the GHCKF algorithm. A vehicle state estimator, using the square root generalized high-degree cubature Kalman filter (SRGHCKF), was designed. Therefore, the joint estimator, comprising a lateral stiffness estimator and a vehicle state estimator, adopts the LS-GHCKF/SRGHCKF algorithm and enables the estimation of the lateral stiffness, the longitudinal velocity, the lateral velocity, and the yaw rate of the entire vehicle during the driving process. A double lane change and slalom simulation were performed to analyze the feasibility and accuracy of the joint estimation algorithm and verify the results of the LS-GHCKF algorithm and the LS-SRGHCKF algorithm. Further, a low-speed driving experiment was carried out for electric vehicles driven by rear in-wheel motors. The inertial navigation system (INS), the global positioning system (GPS), the real-time kinematic (RTK), and an angle sensor were used to collect real-time vehicle data. The results were compared to verify the feasibility of the joint estimator and the progressiveness of the algorithm. The experimental verification and simulation both show that the vehicle state joint estimator, designed based on the LS-GHCKF/SRGHCKF algorithm, can accurately estimate the real-time state of the vehicle. Additionally, the LS-SRGHCKF algorithm shows better effectiveness and robustness than the LS-GHCKF algorithm.

Genome-Wide Analysis of the LATERAL ORGAN BOUNDARIES Domain (LBD) Members in Alfalfa and the Involvement of MsLBD48 in Nitrogen Assimilation.

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a transcription factor family specific to the land plants, have been implicated in multiple biological processes including organ development, pathogen response and the uptake of inorganic nitrogen. The study focused on LBDs in legume forage Alfalfa. The genome-wide analysis revealed that in Alfalfa 178 loci across 31 allelic chromosomes encoded 48 unique LBDs (MsLBDs), and the genome of its diploid progenitor M. sativa spp. Caerulea encoded 46 LBDs. Synteny analysis indicated that the expansion of AlfalfaLBDs was attributed to the whole genome duplication event. The MsLBDs were divided into two major phylogenetic classes, and the LOB domain of the Class I members was highly conserved relative to that of the Class II. The transcriptomic data demonstrated that 87.5% of MsLBDs were expressed in at least one of the six test tissues, and Class II members were preferentially expressed in nodules. Moreover, the expression of Class II LBDs in roots was upregulated by the treatment of inorganic nitrogen such as KNO3 and NH4Cl (0.3 mM). The overexpression of MsLBD48, a Class II member, in Arabidopsis resulted in growth retardance with significantly declined biomass compared with the non-transgenic plants, and the transcription level of the genes involved in nitrogen uptake or assimilation, including NRT1.1, NRT2.1, NIA1 and NIA2 was repressed. Therefore, the LBDs in Alfalfa are highly conserved with their orthologs in embryophytes. Our observations that ectopic expression of MsLBD48 inhibited Arabidopsis growth by repressing nitrogen adaption suggest the negative role of the transcription factor in plant uptake of inorganic nitrogen. The findings imply the potential application of MsLBD48 in Alfalfa yield improvement via gene editing.

Alfalfa MsATG13 Confers Cold Stress Tolerance to Plants by Promoting Autophagy.

Autophagy is a conserved cellular process that functions in the maintenance of physiological and metabolic balance. It has previously been demonstrated to improve plant tolerance to abiotic stress. Numerous autophagy-related genes (ATGs) that regulate abiotic stress have been identified, but there have been few functional studies showing how ATGs confer cold stress tolerance. The cold transcriptome data of the crown buds that experienced overwintering of the alfalfa (Medicago sativa L.) showed that MsATG13 is upregulated in response to cold stress. In the present study, we found that MsATG13 transgenic tobacco enhanced cold tolerance compared to wild-type (WT) plants. Transmission electron microscopy demonstrated that transgenic tobacco overexpressing MsATG13 formed more autophagosomes than WT plants in response to cold stress conditions. The transgenic tobacco increased autophagy levels due to upregulation of other ATGs that were necessary for autophagosome production under cold stress conditions. MsATG13 transgenic tobacco also increased the proline contents and antioxidant enzyme activities, enhancing the antioxidant defense capabilities under cold stress conditions. Furthermore, MsATG13 overexpression decreased levels of superoxide anion radicals and hydrogen peroxide under cold stress conditions. These findings demonstrate the role of MsATG13 in enhancing plant cold tolerance through modulation of autophagy and antioxidant levels.

MsNRAMP2 Enhances Tolerance to Iron Excess Stress in Nicotiana tabacum and MsMYB Binds to Its Promoter.

Iron(Fe) is a trace metal element necessary for plant growth, but excess iron is harmful to plants. Natural resistance-associated macrophage proteins (NRAMPs) are important for divalent metal transport in plants. In this study, we isolated the MsNRAMP2 (MN_547960) gene from alfalfa, the perennial legume forage. The expression of MsNRAMP2 is specifically induced by iron excess. Overexpression of MsNRAMP2 conferred transgenic tobacco tolerance to iron excess, while it conferred yeast sensitivity to excess iron. Together with the MsNRAMP2 gene, MsMYB (MN_547959) expression is induced by excess iron. Y1H indicated that the MsMYB protein could bind to the "CTGTTG" cis element of the MsNRAMP2 promoter. The results indicated that MsNRAMP2 has a function in iron transport and its expression might be regulated by MsMYB. The excess iron tolerance ability enhancement of MsNRAMP2 may be involved in iron transport, sequestration, or redistribution.

Soybean genetic resources contributing to sustainable protein production.

KEY MESSAGE: Genetic resources contributes to the sustainable protein production in soybean. Soybean is an important crop for food, oil, and forage and is the main source of edible vegetable oil and vegetable protein. It plays an important role in maintaining balanced dietary nutrients for human health. The soybean protein content is a quantitative trait mainly controlled by gene additive effects and is usually negatively correlated with agronomic traits such as the oil content and yield. The selection of soybean varieties with high protein content and high yield to secure sustainable protein production is one of the difficulties in soybean breeding. The abundant genetic variation of soybean germplasm resources is the basis for overcoming the obstacles in breeding for soybean varieties with high yield and high protein content. Soybean has been cultivated for more than 5000 years and has spread from China to other parts of the world. The rich genetic resources play an important role in promoting the sustainable production of soybean protein worldwide. In this paper, the origin and spread of soybean and the current status of soybean production are reviewed; the genetic characteristics of soybean protein and the distribution of resources are expounded based on phenotypes; the discovery of soybean seed protein-related genes as well as transcriptomic, metabolomic, and proteomic studies in soybean are elaborated; the creation and utilization of high-protein germplasm resources are introduced; and the prospect of high-protein soybean breeding is described.

Saline-alkali stress reduces soil bacterial community diversity and soil enzyme activities.

Saline-alkalisation of the soil environment and microorganism is a global challenge. However, relevant studies on the effects of saline-alkali stress on soil bacterial communities are limited. In this study, we investigated the effects of saline-alkali stress on the carbon source metabolic utilisation of the microbial community, bacterial diversity, and composition in soil using Biolog Ecoplate and 16S rRNA gene amplicon sequencing. Biolog Ecoplate results showed that saline-alkali stress decreased the metabolic activity and functional diversity, and changed the utilisation characteristics of carbon sources in soil microorganisms. Particularly, high level of saline-alkali stress significantly decreased the utilisation of carbohydrates and amino acids carbon sources. The results of 16S rRNA gene amplicon sequencing showed that high level of saline-alkali stress significantly reduced the diversity of soil bacterial communities. In addition, high level of saline-alkali stress significantly decreased the relative abundances of some key bacterial taxa, such as Gemmatimonas, Sphingomonas, and Bradyrhizobium. Furthermore, as saline-alkali content increased, the soil catalase, protease, urease, and sucrase activities also significantly decreased. Collectively, these results provide new insight for studies on the changes in the soil bacterial community and soil enzyme activity under saline-alkali stress.

Alfalfa (Medicago sativa L.) MsCML46 gene encoding calmodulin-like protein confers tolerance to abiotic stress in tobacco.

KEY MESSAGE: MsCML46 enhances tolerance to abiotic stresses through alleviating osmotic stress and oxidative damage by regulating the expression of stress-related genes to optimize osmolytes levels and antioxidant enzyme activity in transgenic tobacco. Abiotic stresses are major environmental factors that constraint crop productivity worldwide. Various stimuli regulate intracellular calcium levels and calcium-mediated signal transduction, and cellular responses. Ca2+ signals are perceived by different Ca2+ receptors. Calmodulin-like protein (CML) is one of the best-characterized Ca2+ sensors which shares sequence similarity with highly conserved calmodulin (CaM) ubiquitously expressed in plants. Currently, the molecular and physiological functions of CMLs are largely unknown. In this study, the MsCML46 was characterized in alfalfa (Medicago sativa cv. Zhaodong) under freezing stress. Results showed that MsCML46 was localized to the cytoplasm of Arabidopsis, and its expression was strongly elevated by cold, drought, salt, saline-alkali, and ABA treatments. Overexpressing MsCML46 in tobacco enhanced tolerance to freezing, drought, and salt stresses as evidenced by improved contents of osmotic regulatory solutes and antioxidant enzyme activity but decreased reactive oxygen species (ROS) accumulation. Furthermore, cold, drought, and salt stresses increased the expression of stress-related genes in transgenic tobacco. MsCML46 binds free Ca2+ to promote signal transduction and maintain higher K+/Na+ ratio. In this way, it protects intracellular homeostasis under sodium ion toxicity. These results suggest that MsCML46 plays a crucial role in resisting abiotic stresses and can be exploited in genetic engineering for crops.

Bacillus subtilis HSY21 can reduce soybean root rot and inhibit the expression of genes related to the pathogenicity of Fusarium oxysporum.

Soybean root rot occurs globally and seriously affects soybean production. To avoid the many disadvantages of chemical fungicides, the addition of Bacillus is gradually becoming an alternative strategy to tackle soybean root rot. However, the molecular mechanism of phytopathogenic fungi in this process by Bacillus inhibition is rarely reported. In this study, we isolated a strain of B. subtilis HSY21 from soybean rhizosphere soil, which had an inhibition rate of 81.30 ± 0.15% (P < 0.05) against Fusarium oxysporum. The control effects of this strain against soybean root rot under greenhouse and field conditions were 63.83% and 57.07% (P < 0.05), respectively. RNA-seq analysis of F. oxysporum after treatment with strain HSY21 revealed 1445 downregulated genes and 1561 upregulated genes. Among them, genes involved in mycelial growth, metabolism regulation, and disease-related enzymes were mostly downregulated. The activities of cellulase, ß-glucosidase, α-amylase, and pectin-methyl- galacturonase as well as levels of oxalic acid and ergosterol in F. oxysporum were significantly decreased after HSY21 treatment. These results demonstrated that B. subtilis HSY21 could effectively control F. oxysporum by inhibiting its growth and the expression of pathogenic genes, thus indicating that this strain may be an ideal candidate for the prevention and control of soybean root rot.

Inhibition of Mitochondrial ATP Synthesis and Regulation of Oxidative Stress Based on {SbW8 O30 } Determined by Single-Cell Proteomics Analysis.

The 10-nuclear heteroatom cluster modified {SbW8 O30 } was successfully synthesized and exhibited inhibitory activity (IC50 =0.29 µM). Based on proteomics analysis, Na4 Ni2 Sb2 W2 -SbW8 inhibited ATP production by affecting the expression of 16 related proteins, hindering metabolic functions in vivo and cell proliferation due to reactive oxygen species (ROS) stress. In particular, the low expression of FAD/FMN-binding redox enzymes (relative expression ratio of the experimental group to the control=0.43843) could be attributed to the redox mechanism of Na4 Ni2 Sb2 W2 -SbW8 , which was consistent with the effect of polyoxometalates (POMs) and FMN-binding proteins on ATP formation. An electrochemical study showed that Na4 Ni2 Sb2 W2 -SbW8 combined with FMN to form Na4 Ni2 Sb2 W2 -SbW8 -2FMN complex through a one-electron process of the W atoms. Na4 Ni2 Sb2 W2 -SbW8 acted as catalase and glutathione peroxidase to protect the cell from ROS stress, and the inhibition rates were 63.3 % at 1.77 µM of NADPH and 86.06 % at 10.62 µM of 2-hydroxyterephthalic acid. Overall, our results showed that POMs can be specific oxidative/antioxidant regulatory agents.

Genome-wide analysis of the TCP transcription factor genes in five legume genomes and their response to salt and drought stresses.

The teosinte branched1, cycloidea, and proliferating cell factor family (TCP) proteins, plant-specific transcription factors, are involved in the regulation of plant development; however, the TCP gene family of legumes has been based primarily on a single crop. Here, a total of 55, 22, 26, 21, and 25 genes containing the VQ motif were identified from the genomes of Glycine max, Cicer arietinum, Phaseolus vulgaris, Medicago truncatula, and Lotus japonicus, respectively. Based on the phylogenetic analysis, we divided these TCP genes into three distinct subfamilies: PCF, CYC/TB1, and CIN. The conserved domain analysis indicated that the TCP gene family members contain the bHLH and R domains. The TCP genes from the same evolutionary branches of legumes shared similar motifs and structures. The promoter analysis revealed that cis-elements were related to stress responses, phytohormone responses, and physical and reproductive growth regulation. In addition, the TCP genes presented different expression patterns in the five legumes. Most of them showed specific expression patterns during development. The results of qRT-PCR indicated that the TCP genes played regulatory roles in both salt and drought stresses. The present study provides novel and detailed information regarding the legume TCP gene family, which aids in functional characterisation of the TCP genes in other plants.

Alfalfa MsCBL4 enhances calcium metabolism but not sodium transport in transgenic tobacco under salt and saline-alkali stress.

KEY MESSAGE: MsCBL4 expression in tobacco enhanced its salt and saline-alkali stress tolerance by regulating calcium accumulation in roots, indicating the important role of calcium metabolism in plant saline-alkali stress tolerance The calcineurin B-like (CBL) family of proteins play important roles in plant abiotic stress tolerance and signal transduction. CBL4 is known to participate in the Salt Overly Sensitive pathway; however, little is currently known regarding the mechanisms underlying the response of CBL4 to saline-alkali stress. In this study, we cloned and characterized the alfalfa MsCBL4 gene. We found that MsCBL4 showed the highest expression in root tissues and was induced by salt and saline-alkali stress, with the latter causing higher induction. Overexpression of MsCBL4 in tobacco enhanced salt and saline-alkali stress tolerance and reduced the Na+/K+ ratio in roots of transgenic lines. Salt (30 and 300 mM NaCl) and saline-alkali (30 mM NaHCO3) stress assays performed for MsCBL4 transgenic tobacco lines revealed a substantial influx of sodium ions in roots under saline-alkali stress and indicated that the expression of MsCBL4 had little influence on sodium ion content reduction. In contrast, in roots subjected to saline-alkali stress, calcium accumulation occurred and was significantly enhanced by the overexpression of MsCBL4. Physiological and biochemical analyses indicated that MsCBL4 plays an important role in saline-alkali stress tolerance via its influence on the regulation of calcium transport and accumulation. These results provide novel insights into the saline-alkali stress tolerance mechanisms of plants.

Genome-wide identification and expression analysis of the AAAP family in Medicago truncatula.

The amino acid/auxin permease (AAAP) gene family plays an important role in the long-distance amino acid transport pathway and takes part in various stages of plant growth and development. However, little is known about the AAAP gene family in Medicago truncatula. Here, we identified 86 putative MtAAAP family members using genome sequence information. Based on phylogenetic analysis, these MtAAAP genes were categorized into eight distinct subfamilies. The MtAAAP genes were mapped on 8 chromosomes and duplication events appeared widely, with 19 and 21 pairs of MtAAAP genes showing segment and tandem duplication events, respectively. Ratio of Ka/Ks indicated that duplicated genes underwent purifying selection. Analysis of RNA-seq data showed that MtAAAP genes exhibited specific expression patterns among different tissues and abiotic stress, indicating that MtAAAP members were involved in plant developmental regulation and stress responses. Expression patterns of 16 MtAAAP genes under abiotic stress were verified by qRT-PCR. The present study provides a foundation for the functional analysis of MtAAAPs in developmental regulation and stress responses.