Sushuang Liu

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.

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.

Melon (Cucumis melo L.) is a significant cash crop globally and is cherished for its sweet and flavorful fruits, as well as its high nutritional values. However, its yield and quality are limited by various factors, including drought, salinity, and low temperatures. Low temperatures are one of the primary factors influencing the growth and development of melons, diminishing the viability, germination, and growth rate of melon seeds. Concurrently, low temperatures also reduce light absorption efficiency and fruit yields, thereby affecting melon growth and development. Serine hydroxymethyltransferase (SHMT), a conserved phosphopyridoxal-dependent enzyme, plays a crucial role in plant resistance to abiotic stressors. In this study, eight CmSHMT family genes were identified from the melon genome. We predicted their chromosomal locations, physicochemical properties, gene structures, evolutionary relationships, conserved motifs, cis-acting elements of promoters, and tissue-specific expression patterns. The expression levels of CmSHMT family genes in response to low-temperature stress was then analyzd using qRT-PCR. The phylogenetic results indicated that these CmSHMT genes were classified into four subfamilies and were unevenly distributed across five chromosomes, with relatively high conservation among them. Furthermore, our investigation revealed that the promoter regions of the CmSHMT family genes contain many cis-acting elements related to phytohormones, growth, and various stress responses. The relative expression levels of CmSHMT3, CmSHMT4, CmSHMT6, and CmSHMT7 were higher under low-temperature stress compared to the control group. Notably, the promoter region of CmSHMT3 contains cis-acting elements associated with low-temperature response (LTR) and abscisic acid response (ABRE). It is suggested that the mechanism through which CmSHMT3 responds to low-temperature stress treatments may be associated with hormonal regulation. These findings provide a foundation for the further exploration of CmSHMT family genes in melon and their functional roles in response to low-temperature stress, and they provide a theoretical basis for the targeted breeding of superior melon varieties with enhanced tolerance to low temperatures.

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.

The root architecture of a range of host plants is altered in response to Ralstonia solanacearum infection. This work aimed to identify host genes involved in root development during R. solanacearum infection. A deficient mutant of the type III secretion system regulator hrpB was created in R. solanacearum GMI1000. The hrpB mutant was impaired in virulence but showed a similar suppressive effect as wild-type GMI1000 on tomato root development. Based on comparative transcriptome analysis, 209 genes were found that showed the same changed expression pattern in GMI1000 and hrpB mutant infected roots relative to uninoculated roots. Among them, the wall-associated receptor kinase WAKL20 was substantially downregulated in GMI1000 and hrpB mutant infected roots. Knockdown of WAKL20 led to a shorter primary root length and fewer lateral roots in tomato as well as in Nicotiana benthamiana. The WAKL20 is a pivotal target suppressed by R. solanacearum to shape the altered root development during infection.

The pomegranate is one of the most essential crop fruits in the world. 5-Aminolevulinic acid (ALA) regulates the growth and development of 5-year-old pomegranate. However, for plenty of pome crops, conventional information on the physio-chemical characteristics underlying 5-ALA is limited. Therefore, in this study, we applied four different concentrations of 5-ALA treatments (0 mg L⁻¹, 10 mg L⁻¹, 20 mg L⁻¹, 50 mg L⁻¹, and 100 mg L⁻¹), where 0 mg L⁻¹ was considered as a control group, to evaluate the effects of 5-ALA on the physiochemical characteristics of pomegranates. Our results showed that maximal photochemical efficiency (F_V/F_M), photosystem (Φ_PSII), and photochemical quenching (qP) increased at concentrations of 50 mg L⁻¹ and 100 mg L⁻¹ compared with controls. Anthocyanin contents were elevated by 2.27% and 1.33% at the 5-ALA concentrations of 50 mg L⁻¹ and 100 mg L⁻¹. Furthermore, qRT-PCRs of the DEGs, such as punica granatum chalcone synthase (PgCHS), chalcone isomerase (PgCHI), flavanone 3-hydroxylase (PgF3H), dihydroflavonol 4-reductase (PgDFR), anthocyanidin synthase (PgANS), and ultrafine-grained (PgUFG), showed up-regulations, except for chalcone isomerase (PgCHI), after 5-ALA treatments. The fresh weight of the fruit and the weight of the grains were elevated under 50 mg L⁻¹ and 100 mg L⁻¹ concentrations, and both the fresh weight of the fruit and the grain weight were higher than controls. Total sugar (TS) increased by 8.49% and 24.99%, total soluble solids (TSS) increased by 2.02% and 6.07%, soluble proteins increased by 1.16% and 1.15%, and the pH level of juice increased by 0.12% and 0.19%, respectively. In addition, the contents of ascorbic acid, total phenols, and total flavonoids increased by 24.18%, 2.53%, and 1.29%, respectively, compared with controls. Taken together, the antioxidant activities of SOD and POD also increased by 13.33% and 11.95% at concentrations of 100 mg L⁻¹. Our results show that concentrations of 5-ALA treatments at 50 mg L⁻¹ and 100 mg L⁻¹ will be beneficial for crop cultivation.

The glabrous-enhancer-binding protein (GeBP) family is a family of plant-specific transcription factors, whose members share a central DNA-binding domain. Previous studies have already proven that GeBP genes are involved in the control of cell expansion but not cell proliferation in Arabidopsis. However, there has not yet been a versatile analysis of the GeBP genes’ function in soybean (Glycine max L.). Here, we identified and named 9 GmGeBP genes in the soybean genome. These genes were distributed on 7 of the 20 chromosomes and the intron numbers ranged from zero to one. According to the phylogenetic tree, 52 GeBP genes obtained from four plant species were clustered into major four groups. Through the RNA-seq analysis of the nine GmGeBP genes, 8 of 9 GmGeBP genes were be found to expressed differentially across the 14 tissues. Additionally, among nine GmGeBP genes, only GeBP4 were highly expressed in abnormal trichome soybeans, which was predicted to be involved in trichome development. This genome-wide analysis of GmGeBP genes helps to provide an overview of the evolution and functions of two kinds of soybean plants. These results will help to clarify the potential functions and characteristics of GmGeBP genes in the soybean life cycle.

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.

Abiotic stresses such as high temperature, high humidity, and heavy metals are important factors that affect seed development and quality, and restrict yield in soybean. The ATX1-type copper chaperones are an important type of proteins that are used for maintaining intracellular copper ion homeostasis. In our previous study, a copper chaperone protein GmATX1 was identified in developing seeds of soybean under high temperature and humidity (HTH) stresses. In this study, the GmATX1 gene was isolated, and multiple alignment analysis showed that its encoding protein shared high sequence identities with other plant orthologues of copper chaperone proteins containing the HMA domain, and a conserved metal ion-binding site, CXXC. A subcellular localization assay indicated that GmATX1 was localized in the cell membrane and nucleus. An expression analysis indicated that GmATX1 was involved in seed development, and in response to HTH and heavy metal stresses in soybean. GmATX1-silent soybean seedlings were found to be more severely damaged than the control under HTH stress. Moreover, the silencing of GmATX1 reduced antioxidase activity and reactive oxygen species (ROS) scavenging ability in the seedling leaves. The overexpression of GmATX1 in Arabidopsis improved seed vigor and seedling tolerance, and enhanced antioxidase activity and ROS scavenging ability under HTH and heavy metal stresses. Our results indicated that GmATX1 could promote seed vigor and seedling tolerance to HTH and heavy metal stresses in transgenic Arabidopsis, and this promotion could be achieved by enhancing the antioxidase activity and ROS scavenging ability.