Color is an important trait for horticultural crops. Carotenoids are one of the main pigments for coloration and have important implications for photosynthesis in plants and benefits for human health. Here, we identified an APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factor named MdAP2-34 in apple (Malus domestica Borkh.). MdAP2-34 expression exhibited a close correlation with carotenoid content in ‘Benin Shogun’ and ‘Yanfu 3’ fruit flesh. MdAP2-34 promotes carotenoid accumulation in MdAP2-34-OVX transgenic apple calli and fruits by participating in the carotenoid biosynthesis pathway. The major carotenoid contents of phytoene and β-carotene were much higher in overexpressing MdAP2-34 transgenic calli and fruit skin, yet the predominant compound of lutein showed no obvious difference, indicating that MdAP2-34 regulates phytoene and β-carotene accumulation but not lutein. MdPSY2-1 (phytoene synthase 2) is a major gene in the carotenoid biosynthesis pathway in apple fruit, and the MdPSY2-1 gene is directly bound and transcriptionally activated by MdAP2-34. In addition, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. Our findings will advance and extend our understanding of the complex molecular mechanisms of carotenoid biosynthesis in apple, and this research is valuable for accelerating the apple breeding process.

Deciphering the mechanism of malate accumulation in apple (Malus domestica Borkh.) fruits can help to improve their flavor quality and enhance their benefits for human health. Here, we analyzed malate content as a quantitative trait that is determined mainly by genetic effects. In a previous study, we identified an R2R3−MYB transcription factor named MdMYB44 that was a candidate gene in qtl08.1 (quantitative trait locus mapped to chromosome 8) of fruit malate content. In the present study, we established that MdMYB44 negatively regulates fruit malate accumulation by repressing the promoter activity of the malate-associated genes Ma1 (Al-Activated Malate Transporter 9), Ma10 (P-type ATPase 10), MdVHA-A3 (V-type ATPase A3), and MdVHA-D2 (V-type ATPase D2). Two single-nucleotide polymorphisms (SNPs) in the MdMYB44 promoter, SNP A/G and SNP T/−, were experimentally shown to associate with fruit malate content. The TATA-box in the MdMYB44 promoter in the presence of SNP A enhances the basal activity of the MdMYB44 promoter. The binding of a basic-helix–loop–helix transcription factor MdbHLH49 to the MdMYB44 promoter was enhanced by the presence of SNP T, leading to increased MdMYB44 transcript levels and reduced malate accumulation. Furthermore, MdbHLH49 interacts with MdMYB44 and enhances MdMYB44 activity. The two SNPs could be used in combination to select for sour or non-sour apples, providing a valuable tool for the selection of fruit acidity by the apple breeding industry. This research is important for understanding the complex molecular mechanisms of fruit malate accumulation and accelerating the development of germplasm innovation in apple species and cultivars.

Background: The plant-specific TCP transcription factors play different functions in multiple processes of plant growth and development. TCP family genes have been identified in several plant species, but no comprehensive analysis of the TCP family in grapevine has been undertaken to date, especially their roles in fruit development. Results: A total of 18 non-redundant grapevine TCP (VvTCP) genes distributing on 11 chromosomes were identified. Phylogenetic and structural analysis showed that VvTCP genes were divided into two main classes - class I and class II. The Class II genes were further classified into two subclasses, the CIN subclass and the CYC/TB1 subclass. Segmental duplication was a predominant duplication event which caused the expansion of VvTCP genes. The cis-acting elements analysis and tissue-specific expression patterns of VvTCP genes demonstrated that these VvTCP genes might play important roles in plant growth and development. Expression patterns of VvTCP genes during fruit development and ripening were analyzed by RNA-Seq and qRT-PCR. Among them, 11 VvTCP genes were down-regulated during different fruit developmental stages, while only one VvTCP genes were up-regulated, suggesting that most VvTCP genes were probably related to early development in grapevine fruit. Futhermore, the expression of most VvTCP genes can be inhibited by drought and waterlogging stresses. Conclusions: Our study establishes the first genome-wide analysis of the grapevine TCP gene family and provides valuable information for understanding the classification and functions of the TCP genes in grapevine.

Background: The B-BOX (BBX) proteins are the class of zinc-finger transcription factors and can regulate plant growth, development, and endure stress response. In plants, the BBX gene family has been identified in Arabidopsis, rice, and tomato. However, no systematic analysis of BBX genes has been undertaken in grapevine. Results: In this study, 24 grapevine BBX (VvBBX) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Phylogenetic analysis divided VvBBX genes into five subgroups. Numerous cis-acting elements related to plant development, hormone and/or stress responses were identified in the promoter of the VvBBX genes. The tissue-specific expressional dynamics of VvBBX genes demonstrated that VvBBXs might play important role in plant growth and development. The transcript analysis from transcriptome data and qRT-PCR inferred that 11 VvBBX genes were down-regulated in different fruit developmental stages, while three VvBBX genes were up-regulated. It is also speculated that VvBBX genes might be involved in multiple hormone signaling (ABA, ethylene, GA3, and CPPU) as transcriptional regulators to modulate berry development and ripening. VvBBX22 seems to be responsive to multiple hormone signaling, including ABA, ethylene GA3, and CPPU. Some VvBBX genes were strongly induced by Cu, salt, waterlogging, and drought stress treatment. Furthermore, the expression of VvBBX22 proposed its involvement in multiple functions, including leaf senescence, abiotic stress responses, fruit development, and hormone response. Conclusions: Our results will provide the reference for functional studies of BBX gene family, and highlight its functions in grapevine berry development and ripening. The results will help us to better understand the complexity of the BBX gene family in abiotic stress tolerance and provide valuable information for future functional characterization of specific genes in grapevine.

Carotenoids are major pigments contributing to fruit coloration. We previously reported that the apple (Malus domestica Borkh.) mutant fruits of ‘Beni Shogun’ and ‘Yanfu 3’ show a marked difference in fruit coloration. However, the regulatory mechanism underlying this phenomenon remains unclear. In this study, we determined that carotenoid is the main factor influencing fruit flesh color. We identified an R1-type MYB transcription factor, MdMYBS1, which was found to be highly associated with carotenoids and abscisic acid (ABA) contents of apple fruits. Overexpression of MdMYBS1 promoted, and silencing of MdMYBS1 repressed, β-branch carotenoids synthesis and ABA accumulation. MdMYBS1 regulates carotenoid biosynthesis by directly activating the major carotenoid biosynthetic genes encoding phytoene synthase (MdPSY2-1) and lycopene β-cyclase (MdLCYb). 9-cis-epoxycarotenoid dioxygenase 1 (MdNCED1) contributes to ABA biosynthesis, and MdMYBS1 enhances endogenous ABA accumulation by activating the MdNCED1 promoter. In addition, the basic leucine zipper domain transcription factor ABSCISIC ACID-INSENSITIVE5 (MdABI5) was identified as an upstream activator of MdMYBS1, which promotes carotenoid and ABA accumulation. Furthermore, ABA promotes carotenoid biosynthesis and enhances MdMYBS1 and MdABI5 promoter activities. Our findings demonstrate that the MdABI5-MdMYBS1 cascade activated by ABA regulates carotenoid-derived fruit coloration and ABA accumulation in apple, providing avenues in breeding and planting for improvement of fruit coloration and quality.