Ethylene promotes ripening in fruits as well as the biosynthesis of anthocyanins in plants. However, the question of which ethylene response factors (ERFs) interact with the genes along the anthocyanin biosynthesis pathway is yet to be answered. Herein, we conduct an integrated analysis of transcriptomes and metabolome on fruits of two mulberry genotypes (‘Zijin’, ZJ, and ‘Dashi’, DS, with high and low anthocyanin abundance, respectively) at different post-flowering stages. In total, 1035 upregulated genes were identified in ZJ and DS, including MYBA in the MBW complex and anthocyanin related genes such as F3H. A KEGG analysis suggested that flavonoid biosynthesis and plant hormone signaling transduction pathways were significantly enriched in the upregulated gene list. In particular, among 103 ERF genes, the expression of ERF5 showed the most positive correlation with the anthocyanin change pattern across both genotypes and in the post-flowering stages, with a Pearson correlation coefficient (PCC) of 0.93. Electrophoresis mobility shift assay (EMSA) and luciferase assay suggested that ERF5 binds to the promoter regions of MYBA and F3H and transcriptionally activates their gene expression. We elucidated a potential mechanism by which ethylene enhances anthocyanin accumulation in mulberry fruits and highlighted the importance of the ERF5 gene in controlling the anthocyanin content in mulberry species. This knowledge could be used for engineering purposes in future mulberry breeding programs.

Mulberry leaves are excellent for health care, confirmed as a ‘drug homologous food’ by the Ministry of Health, China. The bitter taste of mulberry leaves is one of the main problems that hinders the development of the mulberry food industry. The bitter, unique taste of mulberry leaves is difficult to eliminate by post-processing. In this study, the bitter metabolites in mulberry leaves were identified as flavonoids, phenolic acids, alkaloids, coumarins and L-amino acids by a combined analysis of the metabolome and transcriptome of mulberry leaves. The analysis of the differential metabolites showed that the bitter metabolites were diverse and the sugar metabolites were down-regulated, indicating that the bitter taste of mulberry leaves was a comprehensive reflection of various bitter-related metabolites. Multi-omics analysis showed that the main metabolic pathway related to bitter taste in mulberry leaves was galactose metabolism, indicating that soluble sugar was one of the main factors of bitter taste difference in mulberry leaves. Bitter metabolites play a great role in the medicinal and functional food of mulberry leaves, but the saccharides in mulberry leaves have a great influence on the bitter taste of mulberry. Therefore, we propose to retain bitter metabolites with drug activity in mulberry leaves and increase the content of sugars to improve the bitter taste of mulberry leaves as strategies for mulberry leaf food processing and mulberry breeding for vegetable use.

Mulberry is a valuable woody plant with significant economic importance. It can be propagated through two main methods: cutting and grafting. Waterlogging can have a major impact on mulberry growth and can significantly reduce production. In this study, we examined gene expression patterns and photosynthetic responses in three waterlogged mulberry cultivars propagated through cutting and grafting. Compared to the control group, waterlogging treatments reduced levels of chlorophyll, soluble protein, soluble sugars, proline, and malondialdehyde (MDA). Additionally, the treatments significantly decreased the activities of ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT) in all three cultivars, except for superoxide dismutase (SOD). Waterlogging treatments also affected the rate of photosynthesis (Pn), stomatal conductance (Gs), and transpiration rate (Tr) in all three cultivars. However, no significant difference in physiological response was observed between the cutting and grafting groups. Gene expression patterns in the mulberry changed dramatically after waterlogging stress and varied between the two propagation methods. A total of 10,394 genes showed significant changes in expression levels, with the number of differentially expressed genes (DEGs) varying between comparison groups. GO and KEGG analysis revealed important DEGs, including photosynthesis-related genes that were significantly downregulated after waterlogging treatment. Notably, these genes were upregulated at day 10 in the cutting group compared to the grafting group. In particular, genes involved in carbon fixation were significantly upregulated in the cutting group. Finally, cutting propagation methods displayed better recovery capacity from waterlogging stress than grafting. This study provides valuable information for improving mulberry genetics in breeding programs.

Photosynthesis is one of the most important factors in mulberry growth and production. To study the photosynthetic regulatory network of mulberry we sequenced the transcriptomes of two high-yielding (E1 and E2) and one low-yielding (H32) mulberry genotypes at two-time points (10:00 and 12:00). Re-annotation of the mulberry genome based on the transcriptome sequencing data identified 22,664 high-quality protein-coding genes with a BUSCO-assessed completeness of 93.4%. A total of 6587 differentially expressed genes (DEGs) were obtained in the transcriptome analysis. Functional annotation and enrichment revealed 142 out of 6587 genes involved in the photosynthetic pathway and chloroplast development. Moreover, 3 out of 142 genes were further examined using the VIGS technique; the leaves of MaCLA1- and MaTHIC-silenced plants were markedly yellowed or even white, and the leaves of MaPKP2-silenced plants showed a wrinkled appearance. The expression levels of the ensiled plants were reduced, and the levels of chlorophyll b and total chlorophyll were lower than those of the control plants. Co-expression analysis showed that MaCLA1 was co-expressed with CHUP1 and YSL3; MaTHIC was co-expressed with MaHSP70, MaFLN1, and MaEMB2794; MaPKP2 was mainly co-expressed with GH9B7, GH3.1, and EDA9. Protein interaction network prediction revealed that MaCLA1 was associated with RPE, TRA2, GPS1, and DXR proteins; MaTHIC was associated with TH1, PUR5, BIO2, and THI1; MaPKP2 was associated with ENOC, LOS2, and PGI1. This study offers a useful resource for further investigation of the molecular mechanisms involved in mulberry photosynthesis and preliminary insight into the regulatory network of photosynthesis.

Identifying the early predictive biomarkers or compounds represents a pivotal task for guiding a targeted agricultural practice. Despite the various available tools, it remains challenging to define the ideal compound combination and thereby elaborate an effective predictive model fitting that. Hence, we employed a stepwise feature selection approach followed by a maximum relevance and minimum redundancy (MRMR) on the untargeted metabolism in four mulberry genotypes at different fruit developmental stages (FDSs). Thus, we revealed that 7 out of 226 differentially abundant metabolites (DAMs) explained up to 80% variance of anthocyanin based on linear regression model and stepwise feature selection approach accompanied by an MRMR across the genotypes over the FDSs. Among them, the phosphoenolpyruvate, d-mannose and shikimate show the top 3 attribution indexes to the accumulation of anthocyanin in the fruits of these genotypes across the four FDSs. The obtained results were further validated by assessing the regulatory genes expression levels and the targeted metabolism approach. Taken together, our findings provide valuable evidences on the fact that the anthocyanin biosynthesis is somehow involved in the coordination between the carbon metabolism and secondary metabolic pathway. Our report highlights as well the importance of using the feature selection approach for the predictive biomarker identification issued from the untargeted metabolomics data.