Manganese (Mn) is a toxic metal element that adversely affects plant growth. Hydrogen sulfide (H₂S) is considered an important signaling molecule with significant potential in alleviating various abiotic stresses. However, there is limited information available on the role of H₂S in alleviating manganese stress in plants. In this study, the effects of exogenous H₂S and its scavenger, homocysteine thiolactone (HT), on the physiological and biochemical parameters of Malus hupehensis var. pingyiensis seedlings were evaluated. Our results show that H₂S treatment significantly alleviates growth inhibition and oxidative damage induced by manganese stress in Malus hupehensis seedlings, primarily by enhancing antioxidant enzyme activity and up-regulating the ascorbate-glutathione (ASA-GSH) cycle. H₂S treatment increased photosynthetic pigment content and helped maintain osmotic balance in leaves, thereby enhancing key gas exchange parameters and mitigating manganese-induced suppression of photosynthesis. H₂S treatment enhanced the absorption of Ca, Mg, Fe and Zn under manganese stress, significantly reduced manganese accumulation in Malus hupehensis seedlings, and modulated the transcriptional expression of MTPs, facilitating the transfer of manganese to the leaves. Thus, H₂S reduces oxidative damage and promotes growth under Mn stress, highlighting its important role in plant stress tolerance.

Glycogen synthase kinase 3 (GSK3/Shaggy-like) is a highly conserved serine/threonine kinase that orchestrates growth, hormone signaling, and abiotic stress responses in both animals and plants, yet its role in watermelon remains unexplored. In this study, we conducted a whole-genome identification, identifying a total of eight members of the GSK3 gene family (ClGSK3) distributed across seven chromosomes. Phylogenetic and synteny analyses resolved the eight ClGSK3s into four subfamilies that display one-to-one or one-to-many orthology with Arabidopsis and rice GSK3 genes, indicating conserved genomic micro-collinearity across dicots and monocots. Predictions of cis-acting elements and transcriptome data analysis indicate that ClGSK3s may be involved in hormone- and stress-responsive conditions. Protein–protein interaction networks predicted 53 candidate partners for five ClGSK3 proteins; yeast two-hybrid assays subsequently confirmed that ClSK21 associates with three of them—orthologs of the core brassinosteroid (BR)-signaling components BKI1 and BZR1. qRT-PCR revealed that ClSK21, ClSK31, and ClSK41 are rapidly and significantly reprogrammed by BR treatment. Collectively, our data suggest that ClGSK3s modulate fruit development and stress tolerance by integrating hormone-related pathways, especially BR signaling. Future studies are encouraged to integrate genetics and multi-omics approaches to systematically validate the roles of ClGSK3s in hormone signaling and abiotic stress responses.

The regulatory mechanism of brassinolide (BR) signaling in cucurbitaceae crops remains incompletely understood. Previous research demonstrated that the rice genes GW5 and GW5L modulate seed morphology via the BR pathway. However, the conservation of their orthologs in watermelon and their evolutionary trajectory are yet to be elucidated. In this study utilizing the watermelon 97103v2 genome, we identified 15 GW5-LIKE genes. Through structure, phylogenetic tree construction, collinearity, promoter and spatiotemporal expression analysis, we determined that ClGL1 to ClGL3 are the most closely related to GW5 and GW5L. Subsequently, two crucial materials were acquired: the inbred line Jing L6M harboring the homozygous mutant Clgl1, and the near-isogenic line Changhong, a Jing L6M backcross containing the wild-type allele ClGL1. Apart from the disparity in fruit morphology, a clear difference in seed shape was observed between the two. Furthermore, exogenous BR treatment demonstrated that ClGL1 positively regulated the BR signal, aligning with the positive impact of GW5 and GW5L. In conclusion, ClGL1 modulates the morphology of watermelon fruit and seed by enhancing BR signaling, which provides a key gene and theoretical basis for BR signaling evolution and molecular design breeding in Cucurbitaceae.

Glycogen synthase kinase 3 (GSK3/Shaggy-like) belongs to evolutionarily conserved serine/threonine protein kinases that regulate plant morphological development, multi-hormone crosstalk and adaptation to abiotic stresses. However, systematic genome-wide characterization of CmGSK3 is still absent in melon (Cucumis melo L.). This study identified six CmGSK3 members on a whole-genome level, unevenly distributed among four chromosomes. Combined phylogenetic and synteny profiling separated these six genes into four conserved subclades; orthologous links were discovered between melon, Arabidopsis, and rice, revealing evolutionary conservation between monocot and dicot crops. Prediction of promoter cis-regulatory motifs combined with transcriptome datasets suggested that CmGSK3 genes participate in hormone transduction and environmental stress adaptation. Quantitative real-time PCR further verified that exogenous brassinosteroid (BR) application dramatically induced transcriptional accumulation of CmSK21 and CmSK22. Heterologous overexpression of these two genes in wild-type Arabidopsis significantly lowered plant sensitivity to BR, confirming they may function as negative modulators of the BR signaling cascade. Collectively, CmGSK3 members coordinate multiple metabolic routes, dominated by BR-related signal transduction, to manipulate melon growth and stress adaptability. This study establishes the first systematic research on the melon GSK3 family and supplies elite candidate genes for molecular breeding targeting fruit quality and stress resistance improvement in melon.