To elucidate the structural characteristics, phylogeny and biological function of anthocyanin synthase (ANS) and its role in anthocyanin synthesis, members of the strawberry ANS gene family were obtained by whole genome retrieval, and their bioinformatic analysis and expression analysis at different developmental stages of fruit were performed. The results showed that the strawberry ANS family consisted of 141 members distributed on 7 chromosomes and could be divided into 4 subfamilies. Secondary structure prediction showed that the members of this family were mainly composed of random curls and α-helices, and were mainly located in chloroplasts, cytoplasm, nuclei and cytoskeletons. The promoter region of the FvANS gene family contains light-responsive elements, abiotic stress responsive elements and hormone responsive elements, etc. Intraspecific collinearity analysis revealed 10 pairs of FvANS genes, and interspecific collinearity analysis revealed more relationships between strawberries and apples, grapes and Arabidopsis, but fewer between strawberries and rice. Chip data analysis showed that FvANS15, FvANS41, FvANS47, FvANS48, FvANS49, FvANS67, FvANS114 and FvANS132 were higher in seed coat tissues and endosperm. FvANS16, FvANS85, FvANS90 and FvANS102 were higher in internal and fleshy tissues. Quantitative real-time PCR (qRT-PCR) showed that the ANS gene was expressed throughout the fruit coloring process. The expression levels of most genes were highest in the 50% coloring stage (S3), such as FvANS16, FvANS19, FvANS31, FvANS43, FvANS73, FvANS78 and FvANS91. The expression levels of FvANS52 were the highest in the green fruit stage (S1), and FvANS39 and FvANS109 were the highest in the 20% coloring stage (S2). These results indicate that different members of the FvANS gene family play a role in different pigmentation stages, with most genes playing a role in the expression level of the rapid accumulation of fruit coloring. This study lays a foundation for further study on the function of ANS gene family.

To explore the impact of shade treatment on grape berries, ‘Marselan’ grape berries were bagged under different light transmission rates (100% (CK), 75% (A), 50% (B), 25% (C), 0% (D)). It was observed that this treatment delayed the ripening of the grape berries. The individual weight of the grape berries, as well as the content of fructose, glucose, soluble sugars, and organic acids in the berries, was measured at 90, 100, and 125 days after flowering (DAF90, DAF100, DAF125). The results revealed that shading treatment reduced the sugar content in grape berries; the levels of fructose and glucose were higher in the CK treatment compared to the other treatments, and they increased with the duration of the shading treatment. Conversely, the sucrose content exhibited the opposite trend. Additionally, as the weight of the grape berries increased, the content of soluble solids and soluble sugars in the berries also increased, while the titratable acidity decreased. Furthermore, 16 differentially expressed genes (DEGs) were identified in the photosynthesis-antenna protein pathway from the transcriptome sequencing data. Correlation analysis revealed that the expression levels of genes VIT_08s0007g02190 (Lhcb4) and VIT_15s0024g00040 (Lhca3) were positively correlated with sugar content in the berries at DAF100, but negatively correlated at DAF125. qRT-PCR results confirmed the correlation analysis. This indicates that shading grape clusters inhibits the expression of genes in the photosynthesis-antenna protein pathway in the grape berries, leading to a decrease in sugar content. This finding contributes to a deeper understanding of the impact mechanisms of grape cluster shading on berry quality, providing important scientific grounds for improving grape berry quality.

Nitrogen nutrition participates in many physiological processes and understanding the physiological and molecular mechanisms of apple responses to nitrogen is very significant for improving apple quality. This study excavated crucial genes that regulates sugar metabolism in response to nitrogen in apples through physiology and transcriptome analysis, so as to lay a theoretical foundation for improving fruit quality. In this paper, the content of sugar and organic acid in apple fruit at different developmental periods under different nitrogen levels (0, 150, 300, and 600 kg·hm⁻²) were determined. Then, the transcriptomic analysis was performed in 120 days after bloom (DAB) and 150 DAB. The results showed that the fructose and glucose content were the highest at 120 DAB under 600 kg·hm⁻² nitrogen level. Meanwhile, different nitrogen treatments decreased malate content in 30 and 60 DAB. RNA-seq analysis revealed a total of 4537 UniGenes were identified as differentially expressed genes (DEGs) under nitrogen treatments. Among these DEGs, 2362 (52.06%) were up-regulated and 2175 (47.94%) were down-regulated. The gene co-expression clusters revealed that most DEGs were significantly annotated in the photosynthesis, glycolysis/gluconeogenesis, pyruvate metabolism, carbon metabolism, carbon fixation in photosynthetic organisms and plant hormone signal transduction pathways. The key transcription factor genes (ERF, NAC, WRKY, and C2H2 genes) were differentially expressed in apple fruit. Sugar and acid metabolism-related genes (e.g., HXK1, SPS4, SS2, PPC16-2, and MDH2 genes) exhibited significantly up-regulated expression at 120 DAB, whereas they were down-regulated at 150 DAB. Furthermore, the MdSPS4 gene overexpression positively promoted sucrose accumulation in apple callus and fruit. In conclusion, the combinational analysis of transcriptome and the functional validation of the MdSPS4 gene provides new insights into apple responses to different nitrogen levels.

Q-type C2H2 zinc finger proteins (ZFPs), the largest family of transcription factors, have been extensively studied in plant genomes. However, the genes encoding this transcription factor family have not been explored in grapevine genomes. Therefore, in this study, we conducted a genome-wide identification of ZFP genes in three species of grapevine, namely Vitis vinifera, Vitis riparia, and Vitis amurensis, based on the sequence databases and phylogenetic and their conserved domains. We identified 52, 54, and 55 members of Q-type C2H2 ZFPs in V. vinifera, V. riparia, and V. amurensis, respectively. The physical and chemical properties of VvZFPs, VrZFPs, and VaZFPs were examined. The results showed that these proteins exhibited differences in the physical and chemical properties and that they all were hydrophobic proteins; the instability index showed that the four proteins were stable. The subcellular location of the ZFPs in the grapevine was predicted mainly in the nucleus. The phylogenetic tree analysis of the amino acid sequences of VvZFP, VaZFP, VrZFP, and AtZFP proteins showed that they were closely related and were divided into six subgroups. Chromosome mapping analysis showed that VvZFPs, VrZFPs, and VaZFPs were unevenly distributed on different chromosomes. The clustered gene analysis showed that the motif distribution was similar and the sequence of genes was highly conserved. Exon and intron structure analysis showed that 118 genes of ZFPs were intron deletion types, and the remaining genes had variable numbers of introns, ranging from 2 to 15. Cis-element analysis showed that the promoter of VvZFPs contained multiple cis-elements related to plant hormone response, stress resistance, and growth, among which the stress resistance elements were the predominant elements. Finally, the expression of VvZFP genes was determined using real-time quantitative PCR, which confirmed that the identified genes were involved in response to methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), and low-temperature (4 °C) stress. VvZFP10-GFP and VvZFP46-GFP fusion proteins were localized in the nucleus of tobacco cells, and VvZFP10 is the most responsive gene among all VvZFPs with the highest relative expression level to MeJA, ABA, SA and low-temperature (4 °C) stress. The present study provides a theoretical basis for exploring the mechanism of response to exogenous hormones and low-temperature tolerance in grapes and its molecular breeding in the future.

Skin color is an important trait that is mainly determined by the content and composition of anthocyanins in apples. In this study, a new bud mutant (RM) from ‘Oregon Spur II’ (OS) of Red Delicious apple was obtained to reveal the mechanism underlying red color formation. Results showed that the total anthocyanin content in RM was significantly higher than that in OS with the development of fruit. Through widely-targeted metabolomics, we found that cyanidin-3-O-galactoside was significantly accumulated in the fruit skin of RM. Transcriptome analysis revealed that the structural gene MdF3H and MdMYB66 transcription factor were significantly up-regulated in the mutant. Overexpression of MdMYB66 in apple fruit and apple callus significantly promoted anthocyanin accumulation and significantly increased the expression level of MdMYB66 and structural genes related to anthocyanin synthesis. Y1H and LUC analysis verified that MdMYB66 could specifically bind to the promoter of MdF3H. The results of the double luciferase activity test showed that MdMYB66 activated MdF3H 3.8 times, which led to increased anthocyanin contents. This might explain the phenotype of red color in RM at the early stage. Taken together, these results suggested that MdMYB66 was involved in regulating the anthocyanin metabolic pathways through precise regulation of gene expression. The functional characterization of MdMYB66 provides insight into the biosynthesis and regulation of anthocyanins.

Sucrose phosphate synthase (SPS) is an important link in the process of sugar metabolism. In addition, it is also involved in abiotic stresses in plants. In order to study the SPS gene family and its role in abiotic stress, we identified the MdSPS gene family members by bioinformatics methods such as correlation analysis, the HMM method, and the Clustering method, and analyzed the transient expression of MdSPS genes by quantitative real-time fluorescence analysis (qRT-PCR). The MdSPS gene family consists of a total of 19 members divided into three subfamilies distributed on 14 chromosomes in apples. The MdSPS gene family has 12 collinearity gene pairs, indicating significant duplication. Most members of this family contain a large number of plant hormone response elements, light-inducible elements, and abiotic stress response elements 2kb upstream of the promoter. Codon bias analysis shows that there are 28 high-frequency codons and no codons with strong preference in this family. Gene chip results showed that only MdSPS2, MdSPS3, MdSPS11, and MdSPS17 were up-regulated in roots, and they were all members of subfamily C. The qRT-PCR analysis showed that all members of this family responded significantly to drought stress, salt stress, and low temperature stress. Interestingly, the relative expression of MdSPS12 was significantly down-regulated under salt stress and low temperature stress. In addition, the expression of MdSPS3, MdSPS8, MdSPS11, and MdSPS17 was more than 20 fold higher than that of the control under drought stress, salt stress, and low temperature stress. These four genes could be candidates for molecular breeding in the MdSPS family.