Response and mechanism on the growth dynamics of mulberry seedlings to calcium under drought stress

In arid and semi-arid regions, calcium, a heterogeneous element, along with water, affects plant growth. Mulberry (Morus alba L.) is a significant ecological and economic forest species in semi-arid regions. To investigate the effects of Ca addition on the physiological indicators of mulberry seedlings under drought stress, this experiment was set up with water treatments (normal water supply: 70±5% of the maximum water holding capacity; drought stress: 40±5% of the maximum water holding capacity) and Ca addition (Ca0:0 mg·kg-1 Ca2+; Ca400:400 mg·kg-1 Ca2+) two factors. Sampling was carried out five times (S1, S2, S3, S4, and S5) in the two-year growing season of mulberry seedlings for physiological indicators, and S2 for transcriptome and metabolome analyses were performed on the leaves of mulberry seedlings treated with Ca0 and Ca400 under normal water supply conditions and Ca400 under drought stress. Comparative transcriptomic analysis revealed that the differentially expressed genes (DEGs) between Ca400-DC and Ca0-DC were significantly enriched in flavonoid biosynthesis and carotenoid metabolism pathways. The upregulation of 20 key photosynthetic genes in Ca400 inhibited drought. The downregulation of 20 PSI and PSII light-harvesting protein-related genes and 16 downregulation of genes related to subunits on PSI and PSII severely inhibited the photosynthetic ability of mulberry seedlings. Using weighted gene co-expression network analysis (WGCNA), seven candidate genes were identified from eight modules that were highly correlated with phenylpropanoid synthesis in mulberry leaves. These genes may play key regulatory roles in mulberry and could serve as candidate genetic resources for preventing drought. Additionally, three important metabolic pathways were observed in the metabolomics analysis: the phenylpropanoid biosynthesis pathway, photosynthesis pathway, and antenna protein pathway. This study deepens our understanding of the adaptive responses of mulberry seedlings to drought-Ca heterogeneity and provides theoretical guidance for mulberry afforestation and forest management in semi-arid areas.