N6-methyladenosine (m⁶A) methylation functions as a vital post-transcriptional and epigenetic modification in higher plants regulated by α-ketoglutarate-dependent dioxygenases (ALKBH). However, the role of ALKBH genes in oriental melon (Cucumis melo L.) fruit ripening has not been explored. Therefore, we treated oriental melon with an exogenous m⁶A demethylase inhibitor (mechlorfenamic acid) then analyzed endogenous ethylene production and ripening-related indicators to explore the effects of m⁶A methylation on ripening. Bioinformatics and real-time quantitative PCR analyses were used to determine the impact of ALKBH genes on key ethylene synthesis gene expression. Treatment effectively inhibited endogenous ethylene production, firmness changes, and soluble solid contents, thereby extending fruit ripening. Eight ALKBH gene family members belonging to five major groups were identified in the melon genome. All members were expressed in ripening fruits, with different expression patterns during ripening. CmALKBH6, CmALKBH7, and CmALKBH8 expression was inhibited by an ethylene inhibitor (1-methylcyclopropene). The transient overexpression (OE) of CmALKBH8 in oriental melon led to the increased expression of the ethylene synthesis genes CmACS1, CmACS2, and CmACO1. In summary, the ethylene-regulated gene CmALKBH8 may participate in oriental melon fruit ripening regulation by modulating the methylation levels of ethylene synthesis-related genes. These findings help us better understand how m⁶A methylation regulates melon ripening.

Dehydrins (DHNs; late embryogenesis-abundant D11 family) are a class of hydrophilic proteins involved in plant abiotic stress response. However, there is less information regarding DHN gene function in cucurbit crops. Herein, 34 DHN gene family members were identified and characterized in Cucumis sativus, Cucumis melo, Citrullus lanatus, Benincasa hispida, Lagenaria siceraria, and Cucurbita maxima. The DHN genes in the six cucurbit crops exhibited greater collinearity within subfamilies than between different subfamilies. Responses to stress (including low-temperature, salt, cadmium, and aluminum stress) varied among the DHN members, with a significant alteration in the expression of the acidic SnKn-type DHN gene CmDHN3 in response to aluminum stress. Subcellular localization analysis confirmed that CmDHN3 is expressed in the nucleus and cytoplasm. Virus-induced gene silencing (VIGS) revealed a remarkable decrease in CmDHN3 expression, which markedly increased malondialdehyde content, relative conductivity, and proline content in the roots and leaves of plants under aluminum stress. Transcriptome analysis showed that the decreased CmDHN3 expression reduced the expression of water channel protein-encoding genes. Interactions between CmDHN3 and CmAQP1 (MELO3C007188) and between CmDHN3 and CmAQP2 (MELO3C020774) were confirmed using yeast two-hybrid assays. These results clarify the pathway by which dehydrin genes are involved in the transcriptional-level response of melon to aluminum stress and provide a theoretical basis to comprehensively analyze the functions of this gene family in cucurbit crops.

Aluminum (Al) toxicity is a key constraint on plant growth in acidic soils. To counteract Al stress, plants secrete organic acids such as malate from their roots to chelate Al³⁺, a process facilitated by Al-activated malate transporters (ALMTs). In this study, we identified 15 ALMT genes in the pumpkin (Cucurbita moschata) genome, which were phylogenetically classified into four subclades. Expression analysis revealed that several ALMTs, including CmaALMT5, CmaALMT6, and CmaALMT12, were upregulated in response to increasing Al concentrations. Exogenous application of polyamines (spermine, spermidine, and putrescine) alleviated Al-induced root growth inhibition, correlating with enhanced malate secretion. Notably, each polyamine differentially regulated specific ALMT genes: spermidine elevated the expression of CmaALMT1, CmaALMT6, CmaALMT13, and CmaALMT15; spermine induced CmaALMT1, CmaALMT2, CmaALMT3, CmaALMT11, and CmaALMT14; while putrescine significantly upregulated CmaALMT1, CmaALMT3, and CmaALMT4. These results suggest that polyamines may enhance Al tolerance in pumpkin through gene-specific transcriptional regulation of the ALMT family and promotion of root malate secretion, though further evidence is required.

Aluminum (Al) stress is an important factor that inhibits crop growth in acidic soils and poses a threat to pumpkin (Cucurbita moschata) production. In this study, we investigated the effect of endophyte (endophyte) strain J01 of blueberry (Vaccinium uliginosum) on the growth, development, and transcriptional regulatory mechanisms of pumpkin under aluminum stress. The results showed that the blueberry endophyte strain J01 significantly increased the root length of pumpkin under aluminum stress, promoted the growth of lateral roots, and increased root vigor; strain J01 reduced the content of MDA and the relative conductivity in the root system; strain J01 enhanced the activities of superoxide dismutase and catalase in the root system but inhibited ascorbate peroxidase activity. Transcriptome analysis further revealed that strain J01 significantly regulated the expression of key genes associated with aluminum tolerance, including the upregulation of transporter protein genes (aluminum-activated malate transporter and aquaporin), affecting the gene expression levels of genes encoding antioxidant enzymes (ascorbate peroxidase and glutathione S-transferase) and cell wall modification genes (xyloglucan endotransglucosylase/hydrolase and pectin methylesterase). This study provides a theoretical basis and practical guidance for using microbial resources to improve aluminum tolerance in cucurbit crops.

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.