Nematicidal activity and action mode of a methyl-accepting chemotaxis protein from Pseudomonas syringae against Caenorhabditis elegans.

The conventional phytopathogen Pseudomonas syringae reportedly possesses several virulence determinants against Caenorhabditis elegans; however, their action mechanisms remain elusive. This study reports the nematicidal activity and action receptor of a methyl-accepting chemotaxis protein (MCP03) of a wild-type P. syringae MB03 against C. elegans. Purified MCP03 exhibited nematicidal toxicity against C. elegans at a half-lethal concentration of 124.4 µg mL-1, alongside detrimental effects on the growth and brood size of C. elegans. Additionally, MCP03-treated worms exhibited severe pathological destruction of the intestine and depressed wrinkles of the cuticle. Yeast two-hybrid assays identified a subunit of COP9 signalosome, namely CSN-5, which functioned as an MCP03 action receptor. In vitro pull-down verified the binding interaction between MCP03 and CSN-5. RNA interference assays confirmed that MCP03 antagonizes CSN-5, thereby adversely affecting the brood size and cuticle integrity of C. elegans. Following MCP03 infection, the expression of genes related to reproduction, growth, and cuticle formation, such as kgb-1, unc-98, and col-117, was considerably downregulated, indicating pathological changes in MCP03-treated nematodes. Therefore, we proposed that MCP03 antagonizes CSN-5, causing lethality as well as detrimental effects on the fertility, growth, and morphogenesis of C. elegans, which can provide new insights into the signaling pathways and mechanisms underlying the nematicidal action of MCP03 toward C. elegans.

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.

Naringenin Ultrafine Powder Was Prepared by a New Anti-Solvent Recrystallization Method.

Raw naringenin directly isolated from plants is significantly limited by its poor dissolution rate and low bioavailability for clinical and in vivo studies. This study reported a method for the preparation of naringenin ultrafine powder (NUP) using a novel anti-solvent recrystallization process; preliminary experiments were conducted using six single-factor experiments. The response surface Box-Behnken (BBD) design was used to optimize the level of factors. The optimal preparation conditions of the DMP were obtained as follows: the feed rate was 40.82 mL/min, the solution concentration was 20.63 mg/mL, and the surfactant ratio was 0.62%. The minimum average particle size was 305.58 ± 0.37 nm in the derived optimum conditions. A scanning electron microscope was used to compare and analyze the appearance and morphology of the powder before and after preparation. The characterization results of FTIR, TG and XRD showed that no chemical change occurred in the powder before and after preparation. Through the simulated gastrointestinal juice digestion experiment, it was confirmed that the absorption rate of NUP was 2.96 times and 4.05 times higher than raw naringenin, respectively. Therefore, the results showed that the reduction in the particle size through the use of low-speed recrystallization could improve the absorption rate and provided a feasible approach for the further applications.

Proteomic analysis reveals responsive mechanisms for saline-alkali stress in alfalfa.

Saline-alkali stress is a major abiotic stress that limits plant growth, yield, and geographical distribution. Alfalfa is a perennial legume with the largest planting area in the world because of its high protein content, good palatability, and long utilization life. However, saline-alkali stress seriously affects alfalfa yield and quality. To better understand the saline-alkali stress response mechanisms of alfalfa, an isobaric tags proteomics method was used to compare and analyse alfalfa under saline-alkali stress for 0, 1, and 7 days, and 126 (1 vs. 0 days) and 1869 (7 vs. 0 days) differentially abundant proteins (DAPs) were found. Through integrative analysis with differentially expressed genes (DEGs), we found correlated DEGs-DAPs of RNA and protein with similar expression trends at the mRNA and protein levels; these were mainly involved in ABA and Ca2+ signal pathways, regulation of photosynthesis, ROS scavenging, secondary metabolism, and transcription factors (TFs) related to saline-alkali stress. Some genes not exhibiting such trends may have been regulated post-transcriptionally. Furthermore, through transgenic experiments, MsFTL was found to significantly improve the saline-alkali tolerance of plants. Overall, our findings provide important clues for understanding the molecular mechanisms underlying the response of alfalfa to saline-alkali stress.

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 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.

Overexpression of an alfalfa glutathione S-transferase gene improved the saline-alkali tolerance of transgenic tobacco.

Abiotic stresses restrict the productivity and quality of agricultural crops. Glutathione S-transferase (GST) utilizes glutathione to scavenge reactive oxygen species (ROS) that result from abiotic stresses. This study aimed to determine the expression pattern of the MsGSTU8 gene and its effects on saline-alkali tolerance. MsGSTU8, from alfalfa (Medicago sativa 'Zhaodong'), was transformed into transgenic tobacco (Nicotiana tabacum) and overexpressed to determine its effects on saline-alkali tolerance. The gene products in alfalfa localized to the cytoplasm and the transcript levels were higher in the leaves than the roots and stems. Expression was strongly induced by cold, drought, salt and saline-alkali stresses as well as abscisic acid (ABA) treatments. The transgenic tobacco lines had significantly higher transcription levels of the abiotic stress-related genes and higher GST activity than the wild types. Transgenic tobacco lines with saline-alkali treatments maintained their chlorophyll content, showed improved antioxidant enzyme activity and soluble sugar levels, reduced ion leakage, O2 .-, H2O2 accumulation and malondialdehyde content. Our results indicate that overexpression of MsGSTU8 could improve resistance to saline-alkali stresses by decreasing the accumulation of ROS and increasing the levels of antioxidant enzymes. Furthermore, they suggest that MsGSTU8 could be utilized for transgenic crop plant breeding.

Overexpression of the alfalfa WRKY11 gene enhances salt tolerance in soybean.

The WRKY transcription factors play an important role in the regulation of transcriptional reprogramming associated with plant abiotic stress responses. In this study, the WRKY transcription factor MsWRKY11, containing the plant-specific WRKY zinc finger DNA-binding motif, was isolated from alfalfa. The MsWRKY11 gene was detected in all plant tissues (root, stem, leaf, flower, and fruit), with high expression in root and leaf tissues. MsWRKY11 was upregulated in response to a variety of abiotic stresses, including salinity, alkalinity, cold, abscisic acid, and drought. Overexpression of MsWRKY11 in soybean enhanced the salt tolerance at the seedling stage. Transgenic soybean had a better salt-tolerant phenotype, and the hypocotyls were significantly longer than those of wild-type seeds after salt treatment. Furthermore, MsWRKY11 overexpression increased the contents of chlorophyll, proline, soluble sugar, superoxide dismutase, and catalase, but reduced the relative electrical conductivity and the contents of malonaldehyde, H2O2, and O2-. Plant height, pods per plant, seeds per plant, and 100-seed weight of transgenic MsWRKY11 soybean were higher than those of wild-type soybean, especially OX2. Results of the salt experiment showed that MsWRKY11 is involved in salt stress responses, and its overexpression improves salt tolerance in soybean.

De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress.

Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen species and malondialdehyde and a decreased content of chlorophyll, indicating that anti-oxidation and detoxification play an important role in response to saline-alkaline stress. Overall, the transcriptome analysis provided novel insights into the saline-alkaline stress tolerance response mechanisms in alfalfa.