Metallothioneins (MTs) are polypeptide-encoded genes involved in plant growth, development, seed formation, and diverse stress response. High temperature and humidity stress (HTH) reduce seed development and maturity of the field-grown soybean, which also leads to seed pre-harvest deterioration. However, the function of MTs in higher plants is still largely unknown. Herein, we isolated and characterized the soybean metallothionein II gene. The full-length fragment is 255 bp and encodes 85 amino acids and contains the HD domain and the N-terminal non-conservative region. The subcellular location of the GmMT-II-GFP fusion protein was clearly located in the nucleus, cytoplasm, and cell membrane. The highest expression of the GmMT-II gene was observed in seeds both of the soybean Xiangdou No. 3 and Ningzhen No. 1 cultivars, as compared to other plant tissues. Similarly, gene expression was higher 45 days after flowering followed by 30, 40, and 35 days. Furthermore, the GmMT-II transcript levels were significantly higher at 96 and 12 h in the cultivars Xiangdou No. 3 and Ningzhen No. 1 under HTH stress, respectively. In addition, it was found that when the Gm1-MMP protein was deleted, the GmMT-II could bind to the propeptide region of the Gm1-MMP, but not to the signal peptide region or the catalytic region. GmMT-II overexpression in transgenic Arabidopsis increased seed germination and germination rate under HTH conditions, conferring enhanced resistance to HTH stress. GmMT-II overexpressing plants suffered less oxidative damage under HTH stress, as reflected by lower MDA and H₂O₂ content and ROS production than WT plants. In addition, the activity of antioxidant enzymes namely SOD, CAT, and POD was significantly higher in all transgenic Arabidopsis lines under HTH stress compared wild-tpye plants. Our results suggested that GmMT-II is related to growth and development and confers enhanced HTH stress tolerance in plants by reduction of oxidative molecules through activation of antioxidant activities. These findings will be helpful for us in further understanding of the biological functions of MT-II in plants.

Chickpeas (Cicer arietinum L.) are an important legume crop known for their rich nutrient content, including proteins, carbohydrates, and minerals. Thus, they are enjoyed by people worldwide. In recent years, the production scale of chickpeas has been growing gradually. The planting area of chickpeas represents roughly 35–36% of the total planting area, and the output of the beans is roughly 47–48%. However, the growth and development process of chickpeas is limited by a number of factors, including high temperature, drought, salt stress, and so forth. In particular, high temperatures can reduce the germination rate, photosynthesis, seed setting rate, and filling rate of chickpeas, restricting seed germination, plant growth, and reproductive growth. These changes lead to a decrease in the yield and quality of the crop. Heat shock proteins (HSPs) are small proteins that play an important role in plant defense against abiotic stress. Therefore, in the present study, HSP20 gene family members were identified based on the whole-genome data of chickpeas, and their chromosomal positions, evolutionary relationships, promoter cis-acting elements, and tissue-specific expression patterns were predicted. Subsequently, qRT-PCR was used to detect and analyze the expression characteristics of HSP20 genes under different temperature stress conditions. Ultimately, we identified twenty-one HSP20 genes distributed on seven chromosomes, and their gene family members were found to be relatively conserved, belonging to ten subfamilies. We also found that CaHSP20 promoter regions have many cis-acting elements related to growth and development, hormones, and stress responses. In addition, under high-temperature stress, the relative expression of CaHSP20-17, CaHSP20-20, CaHSP20-7, CaHSP20-3, and CaHSP20-12 increased hundreds or even thousands of times as the temperature increased from 25 °C to 42 °C. Among them, excluding CaHSP20-5, the other five genes all contain 1-2 ABA cis-regulatory elements. This finding indicates that CaHSP20s are involved in the growth and development of chickpeas under heat stress, and the mechanisms of their responses to high-temperature stress may be related to hormone regulation. The results of the present study lay the foundation for exploring HSP20 gene family resources and the molecular mechanisms of heat resistance in chickpeas. Our results can also provide a theoretical basis for breeding high-temperature-resistant chickpea varieties and provide valuable information for the sustainable development of the global chickpea industry.

Poecilia reticulata is an important species in the global ornamental fish trade, known for its environmental adaptability and bright adult coloration. However, research on sex-dependent background color choice in this species remains limited, constraining the understanding of visual-environment requirements across sexes. In this study, we used a circular six-color maze (red, yellow, blue, green, black, and white) to independently test male and female P. reticulata at first sexual maturity. The results showed that both sexes spent proportionally more time in blue and green zones and less time in red zones, with no sex differences observed. This indicates a convergent pattern of immediate color choice between sexes at this stage. This cross-sex convergent choice pattern may reflect similar short-term visual-environment requirements between sexes. This study provides descriptive baseline data on immediate color choice, which can serve as a reference for subsequent investigations into long-term stable color preferences in this species. The sex-separated testing approach also offers a methodological reference for similar studies in other species.

Pine wilt disease is a devastating forest disease caused by Bursaphelenchus xylophilus infection. The associated bacterium Pseudomonas fluorescens and its flagellin protein have been implicated in this process, but the role of the flagellin-encoding gene fliC in pathogenesis remains unclear. Current gene knockout strategies in P. fluorescens are limited by low transformation efficiency and cumbersome procedures, which constrain functional studies of this gene. To address this methodological gap, we established a fliC knockout method based on the pT18sacB-sacB counterselection system. Short homologous arms were constructed by splice overlap extension PCR, and the knockout mutant ΔfliC was obtained through S17-1 λpir-mediated conjugation and sucrose counterselection. The mutant was verified by junction PCR, internal PCR, and sequencing. Using Pinus massoniana callus as the experimental material, we preliminarily compared the wild-type strain and the ΔfliC mutant in individual pathogenicity and in promoting complex infection by B. xylophilus. The ΔfliC mutant showed delayed symptom onset and lower disease severity than the wild type, and its ability to promote complex infection also appeared reduced. These results provide preliminary evidence that fliC contributes to symptom development in this callus assay. The established fliC knockout technique provides a methodological basis for further investigating the role of associated bacteria in pine wilt disease.

Screening green and high-efficiency bacteriostatic agents is crucial for the sustainable development of fruit and vegetable industry. Long-term application of chemical fungicides has caused increasingly serious problems including pathogen resistance, pesticide residues and environmental pollution. Meanwhile, single botanical preparations are restricted by narrow antibacterial spectrum, unstable active ingredients and single action targets. In view of the above problems, this study prepared compound botanical bacteriostatic agent FLR-1 by compounding extracts of Forsythia suspensa, linseed and rosemary. Its chemical constituents, antibacterial activity and action mechanism were systematically analyzed via LC–MS metabolomics, Oxford cup method and gradient dilution method. The results revealed that agent FLR-1 contained ten categories of metabolites such as sugars and glycosides, steroids, lipids and organic acids. It exhibited prominent inhibitory and bactericidal effects against 10 fungal strains and 2 bacterial strains at concentrations of 2.5–5 μL/mL, presenting broad-spectrum antibacterial activity across different microbial groups. Mechanistic studies confirmed that agent FLR-1 exerted its efficacy independent of extreme acid–base stress. Instead, it destroyed the integrity of pathogen cell membranes, increased membrane permeability and triggered the leakage of intracellular nucleic acids to inhibit and eliminate pathogens. This study clarified the direct antibacterial mechanism of multisource plant metabolites via combined action and multi-target membrane damage. It provides a novel strategy to overcome the limitations of single botanical agents, and offers theoretical references for the development of novel high-efficiency and low-toxicity compound botanical bacteriostatic agents as well as the prevention and control of pre-harvest and post-harvest diseases of fruits and vegetables.