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 fruits are rich in anthocyanins, which are important secondary metabolites that give mulberries their bright color, favorable taste and high nutritional quality, making them a popular fruit. However, few studies have focused on the molecular mechanism underlying anthocyanin accumulation in mulberries and the gene regulatory networks of anthocyanin biosynthetic pathways remain largely unknown. In this study, we performed RNA sequencing to identify differentially expressed genes (DEGs) associated with anthocyanin accumulation between two mulberry genotypes (‘Zi Jing’, ZJ and ‘Zhen Zhu Bai’, ZZB, with purple and white fruit flesh, respectively) at 5, 18, 27 and 31 days after flower. Using transcriptome analysis, we explored several key DEGs involved in the anthocyanin biosynthetic pathway, including the structural genes: CHS, CHI, F3H, DFR1, DFR2 and ANS, known as MBW complex genes: MYB (M.alba_G0017209), MYB (M.alba_G0017689), bHLH (M.alba_G0012659), bHLH (M.alba_G0009347) and bHLH3 (M.alba_G0016257) and the ethylene response factor: ERF (M.alba_G0016603). Of these, changing trends related to expression pattern and anthocyanin content showed their most positive correlation at the post-flowering stage in both genotypes. Our results indicated that ethylene enhances anthocyanin accumulation in mulberry fruits. Furthermore, qRT-PCR was performed to confirm the above-mentioned genes’ expression (except for MYB (M.alba_G0017689) and bHLH (M.alba_G0009347) was significantly up-regulated under ethylene treatment at 300 mg/L. These findings help uncover the gene regulatory networks of the anthocyanin biosynthetic pathway and will contribute to engineering purposes in future mulberry breeding programs.

Mulberry (Morus L.; Moraceae; Rosales) is an economically important tree with a long history in China and valued for its rich nutrient, flavor content, medicinal value and useful ecological functions. Morus species are widely distributed in Asia, Europe, North and South America and Africa and exhibit obvious differences in morphological characteristics and phenological stages due to being distributed in diverse climate zones. The morphological description of the phenological stages of black mulberry (Morus nigra L.) has been established in Mediterranean climates and this study extended the BBCH scale for application in different Morus species, especially for mulberry trees grown in subtropical monsoon climates. In this study, we used the BBCH (Biologische Bundesanstalt, Bundessortenamt and CHemische Industrie) scale to describe in detail the phenological growth stages for Morus species in Wuhan, China (a subtropical monsoon climate). Based on this general scale, eight principal stages, i.e., bud, leaf and shoot development stage, inflorescence emergence stage, flowering stage, fruit development stage, fruit maturation stage, senescence and beginning of dormancy stage, were described. We provide photographic images of some primary and secondary developmental stages to better define and standardize morphological characteristics and phenological descriptions of these mulberries (Morus spp.). In addition, because the color of mulberry fruit varies from white, purple, black, or pink to red at maturity, with mostly monosexual flowers, we have also presented detailed descriptions of morphological traits of flower sexuality and fruit color among these four species. In addition, two late phenological germplasms (late bud sprouting associated with late blooming and late fruit ripening) were screened out according to the BBCH scale. Overall, this study will contribute to advance the field of mulberry breeding and implementation of agronomic practices and facilitate convenient communication between mulberry cultivators and researchers in different areas.

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.