Search Results (271)

Peach (Prunus persica) production and the northward expansion of its cultivation boundaries are severely constrained by recurrent extreme climatic events. Based on time-series transcriptomic analysis of two independent parallel treatments (cold stress and ABA application) and qRT-PCR validation, multiple candidate transcription factor genes co-responsive to both cold stress and ABA were identified (PpERF48, PpERF017, PpMYB6, PpWRKY46, PpWRKY40, and PpbHLH35). Among these, PpMYB6 was further characterized through bioinformatic analysis, and transgenic peach callus and Arabidopsis thaliana lines overexpressing PpMYB6 were generated, revealing its dual role in modulating plant growth and conferring low-temperature stress tolerance. Yeast one-hybrid and dual-luciferase reporter assays confirmed that PpMYB6 directly binds to and activates the PpCBF2 promoter. Yeast two-hybrid library screening identified DWARF8 as a candidate interacting protein, pointing to a working hypothesis by which PpMYB6 may negatively regulate vegetative growth via the gibberellin pathway. Together, this study characterizes a novel molecular module associated with cold tolerance and growth balance in peach, providing a promising candidate gene for molecular breeding of cold-resistant cultivars and rootstocks. Full article

Proline dehydrogenase (ProDH) is a rate-limiting enzyme in the proline metabolism cycle that plays an important role in plant stress response and disease resistance. However, the function and mechanism of ProDH1 in the wheat–Puccinia striiformis f. sp. tritici (Pst) interaction remain poorly understood. In this study, a wheat proline dehydrogenase gene TaProDH1 was induced by stripe rust. qRT-PCR analyses showed that the transcript levels of TaProDH1 were highly increased during early infection. The transient expression of TaProDH1 in Nicotiana benthamiana leaves revealed that TaProDH1 increases the content of proline in leaves and induces the accumulation of reactive oxygen species. Silencing TaProDH1 via the barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) system led to compromised wheat resistance to the Pst avirulent pathotype CYR23, significantly increased proline content in the plant, and increased mycelial growth and sporulation of the pathogen. In addition, it was confirmed by the yeast one-hybrid (Y₁H) and dual-luciferase reporter systems that the transcription factor TaMYB30 can activate its transcriptional activity by binding to the TaProDH1 promoter region. In summary, the TaProDH1 gene is a positive regulator of stripe rust resistance in wheat. It is activated by the upstream transcription factor TaMYB30 and accelerates the process of proline metabolism, which is conducive to enhancing the resistance of wheat to stripe rust. Full article

Yellow leaf phenotypes represent an important trait in pepper and enrich genetic germplasm resources. Previous studies have primarily examined leaf yellowing caused by defects in chlorophyll biosynthesis, whereas the functions of chlorophyll degradation-related genes remain less well characterized. In this study, a yellow leaf phenotype (NY) was identified. Transmission electron microscopy and RNA-seq showed that differentially expressed genes were concentrated in photosynthesis- and chlorophyll-related pathways. CaCLH1 was then demonstrated to promote chlorophyll degradation. Yeast one-hybrid analysis identified the CabHLH18-like gene as a direct interactor with the CaCLH1 promoter and as a positive regulator of CaCLH1 transcription. The CabHLH18-like gene was highly expressed in yellow leaf pepper. The CabHLH18-like gene was subsequently isolated and characterized, revealing nuclear localization and transcription factor activity. Functional analysis showed that silencing the CabHLH18-like gene increased chlorophyll content, whereas its overexpression reduced chlorophyll content. These findings indicate that CaCLH1 is a direct downstream target of the CabHLH18-like gene and contributes to CabHLH18-like-gene-mediated regulation of leaf yellowing in pepper. This study refines mechanistic understanding of leaf yellowing and provides a foundation for future research and breeding applications in pepper. Full article

Narcissus tazetta var. chinensis is a monocot with unique floral morphology and heat-induced flowering. In Arabidopsis, the MADS-box gene AGL6 promotes flowering by positively regulating FT expression, but whether this regulatory mechanism is conserved in monocots remains unknown. Here, we cloned the AGL6 homolog NtAGL6 from N. tazetta var. chinensis, analyzed its sequence, phylogeny, expression pattern, and promoter cis-elements, and performed ectopic expression in wild-type and ap1 mutant Arabidopsis. Yeast one-hybrid and transient expression assays were used to test its interaction with the NtFT1 promoter. NtAGL6 was specifically expressed in floral organs and during flower bud differentiation, and its promoter contained heat shock and gibberellin response elements. Ectopic expression of NtAGL6 promoted flowering in Arabidopsis and upregulated FT, SOC1, LFY, and AP1, and partially rescued petal and sepal defects in the ap1 mutant. Although NtAGL6 activated NtFT1 promoter activity in transient assays, yeast one-hybrid assays detected no direct binding. Together, these results indicate that NtAGL6 promotes flowering and affects A-class floral organ identity, but likely regulates FT through an indirect pathway in this monocot. This study provides new insights into the functional divergence of AGL6 in flowering regulation in monocots. Full article

The characteristic red walnut germplasm ‘RW-1′ (Juglans regia L.) exhibits a stable red seed coat phenotype due to the abundant accumulation of anthocyanins and proanthocyanidins (PAs). The regulatory mechanisms underlying PA biosynthesis in red walnut remain poorly studied, which hinders the improvement of walnut color quality. Leucoanthocyanidin reductase (LAR) is a key enzyme in the PA metabolic pathway, while its function in red walnut remains unclear. Here, the leucoanthocyanidin reductase gene JrLAR1, whose expression pattern is consistent with the accumulation trend of PAs, was cloned from the seed coats of red walnut ‘RW-1′, and its function in PA biosynthesis was verified via heterologous overexpression in Arabidopsis thaliana, a well-recognized cross-species LAR functional validation system free of endogenous LAR interference due to absent native homologs. The results showed that the full-length coding sequence (CDS) of JrLAR1 gene is 1104 bp, encoding a 367-amino-acid protein belonging to the NADB_Rossmann superfamily, and the protein shares an extremely high sequence similarity with grape VvLAR2. Heterologous overexpression of JrLAR1 significantly increased total PA content in the leaves and seeds of A. thaliana. Integrated transcriptomic and metabolomic analyses further revealed that JrLAR1 overexpression markedly upregulated the core genes involved in PA metabolism and the transcription factor GL3 in A. thaliana, specifically induced (+)-catechin synthesis, and ultimately promoted the significant accumulation of procyanidin B3. In addition, a set of antioxidant enzyme-encoding genes were substantially upregulated in JrLAR1-overexpressing A. thaliana lines. Yeast one-hybrid and dual-luciferase reporter assays demonstrated that JrEGL1b, a homolog of A. thaliana GL3, can bind to the promoter region of JrLAR1 gene and significantly enhance its transcriptional activity. Transient overexpression of JrLAR1 or JrEGL1b in red walnut leaves significantly promoted PA accumulation, and JrEGL1b overexpression notably upregulated JrLAR1 expression. In conclusion, JrLAR1 plays a crucial role in PA biosynthesis in red walnut and is positively regulated by the transcription factor JrEGL1b. These findings improve the molecular regulatory network of pigment metabolism in red walnut and provide valuable molecular targets for the quality improvement and directional breeding of walnuts. Full article

Chrysanthemum morifolium is an important ornamental crop whose flowering is strictly regulated by photoperiod. However, the molecular mechanisms linking photoperiod signals to floral meristem identity and organ development in Asteraceae remain unclear. In this study, we identified and characterized CmAFL1, a FUL-like MADS-box gene specific to the Asteraceae family, in the short-day cultivar ‘Jinba’. CmAFL1 expression is upregulated under short-day conditions, primarily expressed in the shoot apical meristem during flower bud differentiation and predominantly in the receptacle during organ development. RNA interference-mediated silencing of CmAFL1 resulted in delayed flowering, arrested inflorescence development, malformed tubular florets, plant dwarfism, and excessive lateral branching, indicating its pleiotropic roles in flowering time, plant architecture, and floral organ patterning. At the molecular level, yeast one-hybrid assays revealed that the photoperiod pathway transcription factor CmCOL4 directly binds to the promoter region of CmAFL1. Furthermore, yeast one-hybrid and dual-luciferase assays confirmed that CmAFL1 directly interacts with and activates the promoter of the E-class floral organ identity gene CmSEP3. Together, our findings identify a novel transcriptional regulatory module CmCOL4-CmAFL1-CmSEP3 that potentially links photoperiod signaling to floral meristem identity and organ specification in Chrysanthemums. While further in planta validation is warranted, these findings provide novel insights into photoperiodic flowering regulation in Asteraceae and may serve as a theoretical foundation for molecular breeding strategies targeting flowering time manipulation in ornamental Chrysanthemums. Full article

Rosa roxburghii Tratt is characterized by its abundant flavonoid content. However, the mechanisms underlying MYB-mediated regulation of flavonoid biosynthesis in R. roxburghii remain largely unknown. In this study, we found that flavonoid accumulation was markedly higher in fruits. By WGCNA of differentially expressed genes (DEGs) with flavonoid accumulation profiles, we identified RrMYB5 as a key regulatory factor in flavonoid biosynthesis of R. roxburghii. RrMYB5 contained characteristic R2R3 domains and a conserved PA1-type motif YEEYLQALL. It was localized exclusively to the nucleus. The qRT-PCR analysis showed that RrMYB5 was constitutively expressed, with peak expression occurring at the rapid fruit expansion stage. The total soluble flavonoid accumulation in R. roxburghii calli was substantially increased by overexpression of RrMYB5. Further LC–MS-based metabolomic analysis revealed significant enrichment of flavonols and proanthocyanidins in RrMYB5-OE calli. Consistently, the transcript levels of RrLAR (Rr404249) and RrANR (Rr300417) were markedly elevated in RrMYB5-overexpressing calli. Moreover, DAP-seq analysis suggested that RrMYB5 might directly bind the promoters of flavonoid structural genes. Subsequent yeast one-hybrid and dual-luciferase assays confirmed that RrFLS (Rr101307) and RrF3H (Rr306546) were direct downstream targets of RrMYB5. These findings indicated that RrMYB5 promoted the expression of flavonol and flavanol biosynthetic genes through different regulatory routes. Thus, our study elucidates the regulatory role of RrMYB5 in flavonoid biosynthesis and provides a valuable molecular target for improving the quality and utilization of R. roxburghii. Full article

Dendrobium officinale, a perennial medicinal herb rich in flavonoids, exhibits multiple pharmacological properties including antioxidant and hypoglycemic effects. However, its medicinal quality is compromised by various abiotic stresses, such as heat, cold, and osmotic stresses. UDP-glycosyltransferases play critical roles in both flavonoid glycosylation and plant stress tolerance. Here, we report the identification of a heat-responsive glycosyltransferase gene, DoUGT71K2, from D. officinale transcriptomes, and a characterization of its stress-induced expression and transcriptional regulatory mechanisms. Quantitative real-time PCR (qRT-PCR) showed that DoUGT71K2 exhibits tissue-specific high expression in flowers, and its transcript abundance is strongly elevated in response to heat, cold, and abscisic acid (ABA) treatment. The direct promoter-binding and trans-activating function of DoMYB102 on DoUGT71K2 was confirmed via dual-luciferase reporter and yeast one-hybrid (Y1H) assays. These results establish a DoMYB102–DoUGT71K2 regulatory module that couples heat stress response with flavonoid biosynthesis, which provides a molecular basis for improving stress tolerance and medicinal quality in D. officinale through molecular breeding. Full article

Aluminum (Al) toxicity severely limits crop growth in acidic soils. Although 24-epibrassinolide (EBR), a bioactive brassinosteroid analog, is known to regulate plant stress responses, its role in Al tolerance in oriental melon remains unclear. Here, we investigated the effects of exogenous EBR application on Al stress tolerance in oriental melon seedlings, and the molecular regulatory mechanisms underlying the EBR-induced alleviation of aluminum stress were explored. Seedlings were pretreated with 0, 0.01, 0.1, and 1 μM EBR for 24 h prior to exposure to 100 μM AlCl₃. Our results showed that 0.01 μM EBR significantly alleviated Al toxicity by improving root vitality, promoting primary root elongation, reducing hematoxylin staining intensity in root tips, and notably increasing malate exudation. The transcriptional analysis revealed that EBR upregulated CmBES1-6, a core transcription factor in BR signaling, along with Al-responsive genes including CmALMT7, CmALMT8, CmALMT12, CmSTOP3, and CmSTOP5. Silencing of CmBES1-6 though virus-induced gene silencing (VIGS) compromised Al tolerance, as reflected by the reduced expression of these Al-responsive genes. Yeast two-hybrid (Y2H) assays confirmed that CmBES1-6 physically interacts with both CmSTOP3 and CmSTOP5. Furthermore, yeast one-hybrid (Y1H) assays demonstrated that CmBES1-6 directly binds to E-box cis-elements (CANNTG) in the promoters of CmSTOP3 and CmSTOP5. Together, our findings suggest that exogenous EBR promotes BR signaling through CmBES1-6, which is associated with the upregulation of CmSTOP3 and CmSTOP5 and enhanced malate exudation from roots, thereby potentially contributing to improved Al tolerance in oriental melon seedlings. This work implicates a CmBES1-6–CmSTOP3/5 transcriptional module in Al tolerance and provides candidate genes for the genetic improvement of Al resistance in melons cultivated in acidic soils. Full article

Coconut (Cocos nucifera L.) is an important tropical horticultural crop valued for its edible endosperm with high oil content. However, the molecular mechanisms governing carbon partitioning and the regulation of glycolytic genes during endosperm development remain largely unclear. Fructose-1,6-bisphosphate aldolase (FBA), a key enzyme in glycolysis, plays a central role in carbohydrate metabolism, yet its transcriptional regulatory mechanisms in coconut have not been elucidated. In this study, the CoFBA promoter (proFBA) was isolated and used for yeast one-hybrid screening, leading to the identification of a GATA transcription factor, CoGATA2. Subcellular localization analysis confirmed that CoGATA2 is localized in the nucleus. Yeast one-hybrid assays, electrophoretic mobility shift assays (EMSA), and transient expression in coconut protoplasts demonstrated that CoGATA2 directly binds to a proFBA fragment containing the predicted GATA motif and represses CoFBA expression. Moreover, virus-induced gene silencing (VIGS) of CoGATA2 in coconut callus resulted in significant upregulation of CoFBA expression and increased fructose-1,6-bisphosphate (FBP) levels. Collectively, these findings demonstrate that CoGATA2 functions as a transcriptional repressor of CoFBA and that this regulation correlates with altered FBP levels in coconut callus. However, the precise mechanism by which FBP accumulation occurs and its relationship to glycolytic flux require further investigation. This study provides new insights into the transcriptional regulation of a key glycolytic gene in coconut and offers a foundation for future efforts to manipulate carbon partitioning in this important horticultural crop. Full article

Alkaline soil limits soybean production. This study elucidates the molecular mechanism by which the Gshdz4-GmU2AFb-GmCML27 module regulates soybean alkaline tolerance. Transcriptome analysis, yeast one-hybrid and dual-luciferase assays confirm that the HD-Zip transcription factor Gshdz4 binds to the CAATAA motif in the GmU2AFb promoter and activates its transcription. Subcellular localization verifies the nuclear distribution of GmU2AFb. Overexpression of GmU2AFb improves alkaline tolerance by increasing antioxidant enzyme activities and proline levels, reducing MDA accumulation, facilitating root development, and upregulating alkaline-responsive genes, including GmSOD1, while gene knockout impairs stress resistance. Combined Y2H, BiFC and LCI assays validate the nuclear protein interaction between GmU2AFb and the calcium-binding protein GmCML27, and overexpression of GmCML27 also enhances antioxidant capacity and root growth in soybean. Co-overexpression of the two genes generates obvious synergistic effects; compared with single-gene overexpression lines, co-transgenic plants possess higher antioxidant levels and elevated transcription of downstream alkaline-tolerant genes, accompanied by alleviated growth inhibition under alkaline stress. In summary, Gshdz4 transcriptionally activates GmU2AFb, and the interaction between GmU2AFb and GmCML27 connects RNA splicing with calcium signaling pathways to synergistically trigger downstream defense responses and promote root development, thereby enhancing soybean alkaline tolerance at multiple layers. This work provides candidate genes for molecular breeding of alkali-resistant soybean. Full article

Ulva prolifera is the main causative species of marine green tides and exhibits extreme tolerance to intertidal abiotic stress. However, the underlying molecular mechanisms remain largely unclear. In this study, we cloned and characterized an MYB-related transcription factor, UpMYB-PHL, from U. prolifera. Expression analysis showed that UpMYB-PHL is rapidly and significantly induced by high-salt stress. Furthermore, heterologous overexpression of UpMYB-PHL in the model microalga Chlamydomonas reinhardtii significantly improved its salt tolerance and biomass. By yeast one-hybrid and dual-luciferase assays, we demonstrated that UpMYB-PHL directly binds to and activates the promoter of the phosphate transporter gene UpPHT1, which is a typical target gene of phosphate response (PHR) transcription factor and participates in salt stress responses of plants. Interestingly, yeast two-hybrid assays revealed that UpMYB-PHL physically interacts with UpGAPDH, a core enzyme in energy metabolism. Taken together, our findings reveal a novel regulatory network in which UpMYB-PHL coordinates phosphorus transporter and energy metabolism in response to salt stress in U. prolifera. This study provides a vital molecular explanation for the rapid adaptation and massive growth of U. prolifera under severe intertidal salt stress. Full article

Fruit color is a key quality indicator for apples and directly influences their market value. The process of fruit ripening encompasses various physiological and biochemical changes, such as the breakdown of chlorophyll and the buildup of anthocyanins and carotenoids. This study investigated the mechanism of chlorophyll degradation in apple peels using ‘Granny Smith’ varieties. The experiments involving the treatment with methyl jasmonate (MeJA) indicated that a concentration of 10 µM MeJA led to a reduction in chlorophyll degradation, while a higher concentration of 1500 µM MeJA enhanced this degradation, which aligned with the variations observed in the expression of genes associated with chlorophyll degradation. The key chlorophyll degradation gene MdSGR1 was cloned and found to be induced by methyl jasmonate. MdSGR1 encodes a 283-amino-acid protein belonging to the stay-green superfamily. The promoter possesses inducible cis-acting elements that respond to methyl jasmonate, low temperature and light, while the protein is localized to chloroplasts. Overexpression and silencing vectors were constructed. Overexpression of MdSGR1 induced chlorosis in tobacco leaves and ‘Granny Smith’ apple peels, decreased chlorophyll content, and upregulated related gene expression. Conversely, silencing MdSGR1 produced opposite effects. Arabidopsis thaliana plants overexpressing MdSGR1 exhibited low chlorophyll content, reduced photosynthetic rate, upregulated expression of genes associated with chlorophyll degradation. The results of yeast one-hybrid and dual-luciferase reporter assays indicated that the MdMYC2 transcription factor interacts with the promoter region of MdSGR1. In conclusion, MdSGR1 is crucial for the degradation of chlorophyll in apple peel, and it is regulated both by the MdMYC2 transcription factor and different concentrations of MeJA. This study preliminarily elucidated the regulatory mechanism of methyl jasmonate on chlorophyll degradation in fruit peel, and these findings provide an important theoretical basis for controlling degreening and color quality in apple fruit. Full article

Cadmium (Cd) toxicity poses a significant threat to crop production and food safety. Although proline is known to enhance plant tolerance to Cd, the molecular mechanisms regulating Cd detoxification through proline accumulation remain unclear. This study identifies the proline transporter TaPROT2 as a crucial positive regulator of Cd tolerance in wheat. We demonstrate that overexpression of TaPROT2 directly promotes proline accumulation in transgenic wheat while simultaneously activating the antioxidant enzyme system, thereby reducing both Cd accumulation and translocation. Using electrophoretic mobility shift assays (EMSA), yeast one-hybrid (Y1H) assays, and luciferase reporter assays, we confirmed that TaERF3 directly binds to the GCC-box element in the TaPROT2 promoter, thereby activating its transcription. Furthermore, overexpression of TaERF3 enhances the expression of TaPROT2, leading to increased proline accumulation and decreased Cd content. In summary, our study reveals a novel TaERF3-TaPROT2 module that promotes proline accumulation, reduces Cd accumulation, and enhances Cd tolerance, providing a promising target for breeding low-Cd wheat. Full article

Pine wilt disease, caused by Bursaphelenchus xylophilus, poses a serious threat to global pine forest ecosystems and forestry production. Thaumatin-like proteins (TLPs), which belong to the PR-5 family, are known to participate in plant defense, but their roles in pine have not been well characterized. In this study, a comprehensive genome-wide analysis of the TLP gene family was conducted in Pinus taeda. A total of 116 TLP genes were identified and classified into four major clades based on phylogenetic analysis. Gene structure and conserved motif analyses revealed that members within the same clade generally exhibited similar exon–intron organization patterns and conserved motif compositions. Promoter analysis identified numerous cis-regulatory elements associated with stress responses and phytohormone signaling. Transcriptome data from different stages of pine wood nematode infection identified eight TLP genes that exhibited continuous differential expression, and their expression patterns were further confirmed by qRT-PCR. A multilayer regulatory network highlighted MYB and other transcription factors as key upstream regulators, and yeast one-hybrid assays confirmed MYB-mediated regulation. Together, these findings improve our understanding of the TLP gene family in P. taeda and offer valuable candidate genes and regulatory information for future studies on pine resistance to pine wilt disease. Full article