Fermentation of mulberry juice not only improves its shelf life, but also effectively enhances their flavor and nutritional quality. This study elucidated the phytochemical and flavor characteristics of mulberry juice fermented with Lactiplantibacillus plantarum BXM2, originally isolated from naturally fermented fruit beverage, through widely targeted metabolomics. The fermentation produced the unique flavor of fermented juice and decreased the pH from 4.15 to 3.19. The metabolomic analysis detected 907 non-volatile metabolites, from which 359 significantly different non-volatile metabolites (up 238, down 121) were screened out. Among 731 identified volatile metabolites, 26 flavor substances were the major contributors to the flavor differences between fermented and unfermented mulberry juices. It is hypothesized that lipid metabolism and amino acid catabolism are crucial pathways for the flavor enhancement of mulberry juice fermented with L. plantarum BXM2. Meanwhile, significant increases of the contents of a variety of bioactive substances, such as indole-3-lactic acid, octadeca-9,12,15-trienoic acid, di-/tri-peptides, etc., conferred additional health potential to BXM2-fermented mulberry juice.

Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions, plays various roles in health and medical therapies. However, it is only found in a limited number of plant species in low concentrations, which hinders its development and utilization. Chalcone synthase (CHS) and stilbene synthase (STS) catalyze the same substrates to produce flavonoids and resveratrol, respectively. However, it remains unclear how CHS and STS compete in metabolite synthesis. In this study, two CHS2 mutant cell lines (MT1 and MT2) were generated using CRISPR/Cas9 genome editing. These CHS2 mutant cell lines exhibited abundant mutations in CHS2, leading to the premature termination of protein translation and subsequent CHS2 knockout. Amplicon sequencing confirmed comprehensive CHS2 knockout in MT1, whereas the wild-type sequence remained predominant in the MT2 cell line. Transcriptome and RT-qPCR results showed a significant downregulation of genes involved in flavonoid biosynthesis, including CHS2, CHS3, F3H, F3’H, DFR, FLS, LDOX, among others, resulting in decreased flavonoid accumulation, such as anthocyanins, proanthocyanidins, quercetin, and kaempferol. Conversely, STS genes involved in stilbenoid biosynthesis were upregulated competing with the flavonoid pathway. Consequently, there was a marked increase in stilbenoids, including resveratrol, piceatannol, piceid and pterostilbene, with a 4.1-fold increase in resveratrol and a 5.3-fold increase in piceid (a derivative of resveratrol) observed in CHS2 mutant cell lines. This research demonstrates that CHS2 mutation induces a shift from flavonoid biosynthesis towards stilbenoid biosynthesis, offering new insights into metabolite biosynthesis and regulation, as well as an alternative solution for natural resveratrol production, and a novel breeding approach for eliminating non-target agronomic traits using CRISPR-Cas9.

Vitis vinifera plants are disease-susceptible while Vitis pseudoreticulata plants are disease-resistant; however, the molecular mechanism remains unclear. In this study, the single-stranded DNA- and RNA-binding protein gene Whirly (VvWhy1 and VpWhy1) were cloned from V. vinifera “Cabernet Sauvignon” and V. pseudoreticulata “HD1”. VvWhy1 and VpWhy1 promoter sequences (pVv and pVp) were also isolated; however, the identity of the promoter sequences was far lower than that between the Why1 coding sequences (CDSs). Both Why1 gene sequences had seven exons and six introns, and they had a C-terminal Whirly conserved domain and N-terminal chloroplast transit peptide, which was then verified to be chloroplast localization. Transcriptional expression showed that VpWhy1 was strongly induced by Plasmopara viticola, while VvWhy1 showed a low expression level. Further, the GUS activity indicated pVp had high activity involved in response to Phytophthora capsici infection. In addition, Nicotiana benthamiana transiently expressing pVp::VvWhy1 and pVp::VpWhy1 enhanced the P. capsici resistance. Moreover, Why1, PR1 and PR10 were upregulated in pVp transgenic N. benthamiana leaves. This research presented a novel insight into disease resistance mechanism that pVp promoted the transcription of Why1, which subsequently regulated the expression of PR1 and PR10, further enhancing the resistance to P. capsici.

Proanthocyanidins (PAs) are a class of polyphenolic compounds recognized for their potent antioxidant, anti-cancer, anti-inflammatory, and cardioprotective properties. However, the production of PAs from natural sources is often limited by high costs, resource wastage, and environmental damage. In this study, we investigated the overexpression of VdLAR1, along with phenotypic observation, metabolite determination, light quality treatment, and RT-qPCR analysis, in spine grape cells. The results demonstrated a significant increase in the contents of proanthocyanidins and flavonoids in pVdLAR1-overexpressing transgenic cell lines, while anthocyanin levels showed a decreasing trend. Furthermore, the treatment with white and blue light on the T5 cell line resulted in enhanced accumulation of proanthocyanidins, catechins, and flavonoids, whereas anthocyanins and epicatechins exhibited a declining pattern. Thus, short-wavelength light promoted the accumulation of metabolites, with the proanthocyanidin content in the T5 transformed cell line reaching 2512.0 μg/g (FW) during blue light incubation. RT-qPCR analysis revealed that the key genes involved in the biosynthesis of proanthocyanidin and anthocyanin were upregulated in the transgenic spine grape cell lines, with VdLAR1 expression increasing by several hundredfold, far surpassing the expression levels of LDOX and ANR. The VdLAR1 overexpression markedly improved substrate competitiveness within the metabolic pathway, promoting catechin biosynthesis while inhibiting the production of epicatechins and anthocyanins. This finding provides compelling evidence that LAR1 is a crucial gene for catechin biosynthesis. This research establishes both theoretical and practical foundations for the regulation and development of natural proanthocyanidins, addressing issues related to high costs, safety concerns, resource wastage, and environmental damage associated with their production.

Anthocyanins are significant secondary metabolites that are essential for plant growth and development, possessing properties such as antioxidant, anti-inflammatory, and anti-cancer activities and cardiovascular protection. They offer significant potential for applications in food, medicine, and cosmetics. However, since anthocyanins are mainly obtained through plant extraction and chemical synthesis, they encounter various challenges, including resource depletion, ecological harm, environmental pollution, and the risk of toxic residuals. To address these issues, this study proposes a plant cell factory approach as a novel alternative solution for anthocyanin acquisition. In this study, the VdCHS2 gene was successfully transformed into spine grape cells, obtaining a high-yield anthocyanin cell line designated as OE1. Investigations of the light spectrum demonstrated that white light promoted spine grape cell growth, while short-wavelength blue light significantly boosted anthocyanin production. Targeted metabolomics analysis revealed that the total anthocyanin content in the OE1 cell line reached 11 mg/g, representing a 60% increase compared to the WT. A total of 54 differentially accumulated metabolites were identified, among which 44 were upregulated. Overexpression of the CHS gene enhanced the expression of downstream genes involved in anthocyanin biosynthesis, resulting in the differential expression of CHI, F3Hb, F3′5′H, DFR4, and LDOX. This led to the differential accumulation of anthocyanin monomers, predominantly consisting of 3-O-glucosides and 3-O-galactosides, thereby causing alterations in anthocyanin levels and composition. Furthermore, the OE1 cell line increased the activity of various antioxidant enzymes, improved the clearance of reactive oxygen species, and reduced the levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). The subsequent cultivation of the transformed OE1 cell line, in conjunction with cell suspension culture, established a plant cell factory for anthocyanin production, significantly increasing anthocyanin yield while shortening the culture duration. This study elucidates the molecular mechanisms through which the VdCHS2 gene influenced anthocyanin accumulation and compositional variations. Additionally, it established a model for a small-scale anthocyanin plant cell factory, thereby providing a theoretical and practical foundation for the targeted synthesis of anthocyanin components and the development and utilization of plant natural products.