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 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 fruit decay of postharvest frequently occurs, which causes severe waste and quality deterioration. However, the biochemical basis of mulberry fruit decay is not fully understood. The phenotypic and physiological analysis suggest mulberry fruits of the mulberry cultivar, CS1 exhibit better storage resistance compared to those of the mulberry cultivar, D10 during 4 °C storage. Meanwhile, carbohydrates, flavonoids, and lipids are markedly up-regulated in mulberry fruits after 4 °C storage. Amino acids and lipids are specially accumulated in mulberry fruits of the mulberry cultivar, CS1 before or after 4 °C storage. Graphene non-woven fabric reduces mulberry fruit decay while promoting lipid accumulation during 4 °C storage. Furthermore, exogenous fatty acid effectively inhibits mulberry fruit decay during 4 °C storage and the lipid-soluble extract from mulberry fruits has strong antibacterial activity. These findings reveal that lipids are involved in mulberry fruit decay, and provide novel insights into the biochemical underpinnings of mulberry fruit decay of postharvest.

The excessively concentrated ripening period of mulberries causes seasonal surplus. Fruit mulberry (Morus spp.) exhibits the unique trait of “simultaneous flowering and leaf flushing”, rendering budburst timing closely correlated with fruit ripening time. Thus, deciphering the molecular mechanism underlying winter bud dormancy maintenance in fruit mulberry is urgently needed. Herein, an F₁ hybrid population comprising 337 individuals, derived from Morus wittiorum (♀) and ‘322’ (♂), was utilized as research material. Through Bulked Segregant Analysis Sequencing (BSA-Seq), we successfully mapped a dormancy-associated QTL interval designated as LB (Late Burst), spanning 9,990,001–11,990,000 bp on Chromosome 13. Integrating Weighted Gene Co-expression Network Analysis (WGCNA) results, MaSVP was identified as a candidate gene within this interval. Virus-induced gene silencing (VIGS) of MaSVP in winter buds of Morus wittiorum significantly accelerated budburst compared to the control, demonstrating that MaSVP represses winter bud dormancy release and plays a crucial role in regulating dormancy maintenance in fruit mulberry. Dynamic expression profiling of dormancy-related genes revealed that the transcript levels of MaSVP, MaSAPK3, MaCASL2, and MaPYR8 were significantly downregulated (Tukey’s test, p < 0.05) as budburst approached, whereas those of MaFT and MaGA20ox1-D were significantly upregulated (Tukey’s test, p < 0.05). These results indicate that winter bud dormancy maintenance in Morus wittiorum is associated with abscisic acid (ABA) and gibberellin (GA) metabolism. Collectively, this study provides critical insights into the biological basis of winter bud dormancy maintenance in fruit mulberry and offers valuable genetic resources for breeding late-maturing cultivars.