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.

Insights into plant salt stress signaling and tolerance.

Soil salinization is an essential environmental stressor, threatening agricultural yield and ecological security worldwide. Saline soils accumulate excessive soluble salts which are detrimental to most plants by limiting plant growth and productivity. It is of great necessity for plants to efficiently deal with the adverse effects caused by salt stress for survival and successful reproduction. Multiple determinants of salt tolerance have been identified in plants, and the cellular and physiological mechanisms of plant salt response and adaption have been intensely characterized. Plants respond to salt stress signals and rapidly initiate signaling pathways to re-establish cellular homeostasis with adjusted growth and cellular metabolism. This review summarizes the advances in salt stress perception, signaling, and response in plants. A better understanding of plant salt resistance will contribute to improving crop performance under saline conditions using multiple engineering approaches. The rhizosphere microbiome-mediated plant salt tolerance as well as chemical priming for enhanced plant salt resistance are also discussed in this review.

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.

MYB30 Regulates Submergence Tolerance by Repressing Ethylene Biosynthesis via ACS7 in Arabidopsis.

Floods impose detrimental effects on natural and agro-ecosystems, leading to a significant loss of worldwide crop production. Global climate change has even worsened this situation. Flooding is a continuous process including two stages of submergence and re-oxygenation, and both are harmful to plant growth and development, resulting in a serious decline in crop yield. Therefore, the understanding of plant flooding tolerance and developing flooding-resistant crops are of great significance. Here, we report that the Arabidopsis thaliana (Arabidopsis) R2R3-MYB transcription factor MYB30 participates in plant submergence response through 1-aminocyclopropane-1-carboxylic acid synthase 7 (ACS7) by repressing ethylene (ET) biosynthesis. The MYB30 loss-of-function mutant exhibits reduced submergence tolerance with a higher level of ET production, whereas the MYB30-overexpressing plant displays enhanced submergence tolerance and repressed ET production. The coding gene of ACS7 might be a direct target of MYB30 during the submergence response. MYB30 binds to the promoter of ACS7 and represses its transcription. The ACS7 loss-of-function mutant with defect in ET biosynthesis displays enhanced submergence tolerance, whereas plants overexpressing ACS7 exhibit a submergence-sensitive phenotype. Genetic analysis shows that ACS7 functions downstream of MYB30 in both ET biosynthesis and submergence response. Taken together, our work revealed a novel transcriptional regulation that modulates submergence response in plants.

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.

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.

AIR12 confers cold tolerance through regulation of the CBF cold response pathway and ascorbate homeostasis.

Auxin induced in root culture (AIR12) is a single gene in Arabidopsis and codes for a mono-heme cytochrome b, but it is unknown whether plant AIR12 is involved in abiotic stress responses. MfAIR12 was identified from Medicago falcata that is legume germplasm with great cold tolerance. Transcript levels of MfAIR12 and its homolog MtAIR12 from Medicago truncatula was induced under low temperature. Overexpression of MfAIR12 led to the accumulation of H2 O2 in apoplast and enhanced cold tolerance, which was blocked by H2 O2 scavengers, indicating that the increased cold tolerance was dependent upon the accumulated H2 O2 . In addition, declined cold tolerance was observed in Arabidopsis mutant air12, which could be restored by expressing MfAIR12. Compared to the wild type, higher levels of ascorbic acid and ascorbate redox state, as well as transcripts of the C repeat/dehydration responsive element-binding factor (CBF) transcription factors and their downstream cold-responsive genes, were observed in MfAIR12 transgenic lines, but lower levels of those in air12 mutant. It is suggested AIR12 confers cold tolerance as a result of the altered H2 O2 in the apoplast that is signaling in the regulation of CBF cold response pathway and ascorbate homeostasis.

The cytomegalovirus UL146 gene product vCXCL1 promotes the resistance of hepatic cells to CD8+ T cells through up-regulation of PD-L1.

The HCMV (human cytomegalovirus) encodes numerous proteins which function to evade the immune response, which allows the virus to replicate. Exploring the mechanisms of HCMV immune escape helps to find the strategy to inhibit HCMV replicate. CD8+ T cells play a critical role in the immune response to viral pathogens. However, the mechanisms of HCMV to evade the attack by CD8+ T cells remain largely unknown. Viral CXCL1 (vCXCL1) is the production of HCMV UL146 gene. Here, we found that vCXCL1 promoted the resistance of hepatic cells to CD8+ T cells. vCXCL1 increased the levels of PD-L1 protein expression and mRNA expression. VCXCL1 enhanced the binding of STAT3 transcription factor to the promoter of PD-L1 and increased the activity of PD-L1 promoter. Furthermore, down-regulation of PD-L1 reduced the effects of vCXCL1 on the resistance of hepatic cells to CD8+ T cells. Taken together, vCXCL1 promotes the resistance of hepatic cells to CD8+ T cells through up-regulation of PD-L1. This finding might provide a new mechanism of HCMV immune escape.

Constitutive expression of a group 3 LEA protein from Medicago falcata (MfLEA3) increases cold and drought tolerance in transgenic tobacco.

KEY MESSAGE: MfLEA3 is involved in protection of catalase activity and confers multiple abiotic stress tolerance. Late embryogenesis abundant (LEA) proteins are involved in plant growth, development and abiotic stress tolerance. A member of group 3 LEA proteins from Medicago sativa subsp. falcata (L.) Arcang, MfLEA3, was investigated in the study. MfLEA3 transcript was induced in response to cold, dehydration, and abscisic acid (ABA), while the cold-induced transcript of MfLEA3 was blocked by pretreatment with inhibitor of ABA synthesis. Constitutive expression of MfLEA3 led to enhanced tolerance to cold, drought, and high-light stress in transgenic tobacco plants. Compared to accumulated reactive oxygen species (ROS) in the wild-type in response to treatments with low temperature, drought, and high light, ROS were not accumulated in transgenic plants. Superoxide dismutase, catalase (CAT), and ascorbate-peroxidase activities were increased in all plants after treatments with the above stresses, while higher CAT activity was maintained in transgenic plants compared with wild-type. However, transcript level of CAT-encoding genes including CAT1, CAT2, and CAT3 showed no significant difference between transgenic plants and wild-type, indicating that the higher CAT activity was not associated with its gene expression. ABA sensitivity and transcripts of several ABA and stress-responsive genes showed no difference between transgenic plant and wild-type, indicating that ABA signaling was not affected by constitutive expression of MfLEA3. The results suggest that MfLEA3 may be involved in the protection of CAT activity and confers multiple abiotic stress tolerance.

An eukaryotic elongation factor 2 from Medicago falcata (MfEF2) confers cold tolerance.

BACKGROUND: An eukaryotic translation elongation factor-2 (eEF-2) plays an important role in protein synthesis, however, investigation on its role in abiotic stress responses is limited. A cold responsive eEF2 named as MfEF2 was isolated from yellow-flowered alfalfa [Medicago sativa subsp. falcata (L.) Arcang, thereafter M. falcata], a forage legume with great cold tolerance, and transgenic tobacco (Nicotiana tabacum L.) plants overexpressing MfEF2 were analyzed in cold tolerance and proteomic profiling was conducted under low temperature in this study. RESULTS: MfEF2 transcript was induced and peaked at 24 h and remained at the high level during cold treatment up to 96 h. Overexpression of MfEF2 in trasngenic tobacco plants resulted in enhanced cold tolerance. Compared to the wild type, transgenic plants showed higher survival rate after freezing treatment, higher levels of net photosynthetic rate (A), maximum photochemical efciency of photosystem (PS) II (Fv/Fm) and nonphotochemical quenching (NPQ) and lower levels of ion leakage and reactive oxygen species (ROS) production after chilling treatment. iTRAQ-based quantitative proteomic analysis identified 336 differentially expressed proteins (DEPs) from leaves of one transgenic line versus the wild type after chilling treatment for 48 h. GO and KEGG enrichment were conducted for analysis of the major biological process, cellular component, molecular function, and pathways of the DEPs involving in. It is interesting that many down-regulated DEPs were grouped into "photosynthesis" and "photosynthesis-antenna", such as subunits of PSI and PSII as well as light harvesting chlorophyll protein complex (LHC), while many up-regulated DEPs were grouped into "spliceosome". CONCLUSIONS: The results suggest that MfEF2 confers cold tolerance through regulating hundreds of proteins synthesis under low temperature conditions. The elevated cold tolerance in MfEF2 transgenic plants was associated with downregulation of the subunits of PSI and PSII as well as LHC, which leads to reduced capacity for capturing sunlight and ROS production for protection of plants, and upregulation of proteins involving in splicesome, which promotes alternative splicing of pre-mRNA under low temperature.

Overexpression of a NF-YC Gene Results in Enhanced Drought and Salt Tolerance in Transgenic Seashore Paspalum.

Seashore paspalum (Paspalum vaginatum O. Swartz) is an important warm-season turfgrass species. In this study we generated transgenic seashore paspalum overexpressing CdtNF-YC1, a nuclear factor Y transcription factor from hybrid bermudagrass (Cynodon dactylon × Cynodon transvaalensis). DNA blot hybridization and qRT-PCR analysis showed that CdtNF-YC1 was integrated into the genomes of transgenic seashore paspalum plants and expressed. Reduced relative water content (RWC) and survival rate and increased ion leakage were observed in both wild type (WT) and transgenic plants after drought stress, while transgenic plants had higher levels of RWC and survival rate and lower ion leakage than the WT. Maximal photochemical efficiency of photosystem II (F v/F m), chlorophyll concentration and survival rate were decreased after salt stress, while higher levels were maintained in transgenic plants than in WT. In addition, an increased Na+ content and decreased or unaltered K+ in leaves and roots were observed after salt treatment, while lower level of Na+ and higher levels of K+ and K+/ Na+ ratio were maintained in transgenic plants than in WT. The results indicated that overexpressing CdtNF-YC1 resulted in enhanced drought and salt tolerance in transgenic plants. Transcript levels of stress responsive genes including PvLEA3, PvP5CS1, PvABI2, and PvDREB1B were induced in response to drought and salt stress, and higher levels were observed in transgenic seashore paspalum than in WT. The results suggest that the enhanced drought and salt tolerance in transgenic seashore paspalum is associated with induction of a series of stress responsive genes as a result of overexpression of CdtNF-YC1.

Alteration of serum high-mobility group protein 1 (HMGB1) levels in children with enterovirus 71-induced hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a common pediatric disease caused by enterovirus infection. It typically presents as a fever along with flat, discolored spots and bumps on the hands, feet, and mouth. Compared with other viruses, enterovirus 71 (EV71)-induced HFMD is more prone to cause severe complications in children, such as brainstem encephalitis, cardiopulmonary disorders, and even death. More in-depth studies are still necessary to understand the characteristics of EV71-induced HFMD, although some related research has been reported so far. High-mobility group box 1 (HMGB1) is an inflammatory cytokine that can upregulate other inflammatory factors through its receptors, such as Toll-like receptors and the receptor for advanced glycation endproducts.We prospectively investigated the alteration of serum HMGB1, interleukin (IL)-6, and tumor necrosis factor (TNF)-α levels before and after treatment in 82 children with HFMD.We found that the serum HMGB1, IL-6, and TNF-α levels were significantly increased in EV71-induced HFMD, and that these changes were more serious in the severe and critical HMFD groups; however, there was no significant difference in the HMGB1 level between the normal control and mild HMFD groups. Moreover, the serum HMGB1 level was positively correlated with the alteration of serum IL-6 and TNF-α concentrations.These results suggest that HMGB1 is involved in the inflammatory pathogenesis of EV71-induced HFMD and that the serum level of HMGB1 could be applied as a clinical indicator for the severity of HFMD, and also a sign for the recovery prognosis of HFMD.

Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth.

Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca(2+) in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth.

[Plasma levels of D-dimer and von Willebrand factor and the therapeutic effect of compound glycyrrhizin in children with cytomegalovirus hepatitis].

OBJECTIVE: To study the significance of plasma D-dimer and von Willebrand factor (vWF) and the therapeutic effect of compound glycyrrhizin in children with cytomegalovirus (CMV) hepatitis. METHODS: Twenty healthy children, 16 asymptomatic cases with CMV infection and 52 cases of CMV hepatitis (21 cholestatic and 31 non-cholestatic) were enrolled. The 52 children with CMV hepatitis were randomly administered with conventional treatment alone or conventional treatment plus compound glycyrrhizin treatment. Plasma D-dimer and vWF levels were measured before and after treatment. RESULTS: Plasma D-dimer and vWF levels in the CMV hepatitis group were markedly higher than those in the healthy control and asymptomatic CMV infection groups (P<0.01). The cholestatic hepatitis group had more increased plasma D-dimer and vWF levels compared with the non-cholestatic hepatitis group (P<0.01). Plasma D-dimer and vWF levels in the CMV hepatitis group were markedly reduced after conventional or compound glycyrrhizin treatment (P<0.01). Compound glycyrrhizin treatment decreased more significantly plasma D-dimer and vWF levels compared with the conventional treatment in children with CMV hepatitis (P<0.01). CONCLUSIONS: The detection of plasma D-dimer and vWF is useful in the early assessment of liver damage in children with CMV hepatitis. Compound glycyrrhizin can decrease obviously plasma D-dimer and vWF levels and might thus provide protective effects against liver damage.