Background: Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew. Results: Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles. Conclusions: The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.

Summary: Proanthocyanidins (PAs) have antioxidant properties and are beneficial to human health. The fruit of apple (Malus × domestica Borkh.), especially the peel, is rich in various flavonoids, such as PAs, and thus is an important source of dietary antioxidants. Previous research on the regulation of PAs in apple has mainly focussed on the transcription level, whereas studies conducted at the post‐transcriptional level are relatively rare. In this study, we investigated the function of mdm‐miR858, a miRNA with multiple functions in plant development, in the peel of apple fruit. We showed that mdm‐miR858 negatively regulated PA accumulation by targeting MdMYB9/11/12 in the peel. During fruit development, mdm‐miR858 expression was negatively correlated with MdMYB9/11/12 expression and PA accumulation. A 5′‐RACE experiment, GUS staining assays and transient luminescent assays indicated that mdm‐miR858 cleaved and inhibited the expression of MdMYB9/11/12. Overexpression of mdm‐miR858 in apple calli, tobacco and Arabidopsis reduced the accumulation of PAs induced by overexpression of MdMYB9/11/12. Furthermore, we found that MdBBX22 bound to the mdm‐miR858 promoter and induced its expression. Overexpression of MdBBX22 induced the expression of mdm‐miR858 to inhibit the accumulation of PAs in apple calli overexpressing MdMYB9/11/12. Under light stress, MdBBX22 induced mdm‐miR858 expression to inhibit PA accumulation and thereby indirectly enhanced anthocyanin synthesis in the peel. The present results revealed that the MdBBX22–miR858–MdMYB9/11/12 module regulates PA accumulation in apple. The findings provide a reference for further studies of the regulatory mechanism of PA accumulation and the relationship between PAs and anthocyanins.

‘Orin’ is a popular apple cultivar, which has a yellow-green appearance, pleasant taste, and unique aroma. However, few studies on the fruit quality characteristics of ‘Orin’ apples have been reported before. In this study, changes of the physiological characteristics were measured at different ripening stages, and the soluble sugars and organic acids were determined by high-performance liquid chromatography (HPLC). Volatile compounds were identified using the headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). During the fruit ripening, the ‘Orin’ apple fruit weight, size, and total soluble solid were gradually increased by contrast with the titratable acidity, and the firmness decreased. The content of four soluble sugars reached the maximum at the 180 days after full bloom (DAFB) stage. Malic acid was measured as the most abundant organic acid in ‘Orin’ apples. Ethyl butyrate, hexyl propanoate, hexyl acetate and butyl acetate belonging to esters with high odor activity values (OAVs) could be responsible for the typical aroma of ‘Orin’ apples. The aim of this work was to provide information on the flavor characteristics of ‘Orin’ apples and promote this apple cultivar for marketing and processing in the future.

Fruit color in apple (Malus domestica Borkh.) is ascribed mainly to the accumulation of anthocyanin pigments, and is an important trait for determining fruit market acceptance. Bagging is a commonly used treatment to enhance the red pigmentation in apple skin. The MdMYB1 transcription factor gene plays an important role in the biosynthesis of anthocyanin in apple after bag removal, but little is known about how MdMYB1 transcription is regulated. In this study, we investigated pigmentation in the non-red skinned cultivars ‘Granny Smith’ and ‘Golden Delicious’ after bag removal. The fruit skins of the two cultivars showed red/pink pigmentation after bag treatment. Transcript levels of MdMYB1, the master regulator of anthocyanin biosynthesis in apple, increased, and showed a correlation with anthocyanin content in both cultivars after bag removal. The MdMYB1 genomic sequences were compared in the two cultivars, which showed that the green-fruited cultivar ‘Granny Smith’ harbors the MdMYB1–1 and MdMYB1–2 alleles, while the yellow-fruited cultivar ‘Golden Delicious’ harbors only MdMYB1–2. A comparison of methylation levels in the 2 kb region upstream of the MdMYB1 ATG between the bag-treated fruits after removal from the bags and the unbagged fruits showed a correlation between hypomethylation and the red-skin phenotype in ‘Granny Smith’. Moreover, ‘Granny Smith’ fruits responded to treatment with 5-aza-2′-deoxycytidine, an inducer of DNA demethylation. An investigation of the MdMYB1 promoter in ‘Granny Smith’ showed reduced methylation in the regions − 2026 to − 1870 bp, − 1898 to − 1633 bp, and − 541 to − 435 bp after bag removal and 5-aza-2′-deoxycytidine treatments. Differences in anthocyanin levels between ‘Granny Smith’ and ‘Golden Delicious’ can be explained by differential accumulation of MdMYB1-specific mRNA. Different levels of MdMYB1 transcripts in the two cultivars are associated with methylation levels in the promoter region. Hypomethylation of the MdMYB1 promoter is correlated with the formation of red pigmentation in ‘Granny Smith’ fruit skins. As a result, red pigmentation in Granny Smith’ was more intense than in ‘Golden Delicious’ fruits after bag removal.

Bagging is commonly used to enhance red pigmentation and thereby improve fruit quality of apples (Malus domestica). The green-skinned apple cultivar ‘Granny Smith’ develops red pigmentation after bagging removal, but the signal transduction pathways mediating light-induced anthocyanin accumulation in apple peel are yet to be defined. The aim of this study was to identify the mechanisms underpinning red pigmentation in ‘Granny Smith’ after bag removal based on transcriptome sequencing. The anthocyanin content in apple peel increased considerably after bag removal, while only trace amounts of anthocyanins were present in the peel of unbagged and bagged fruits. RNA sequencing identified 18,152 differentially expressed genes (DEGs) among unbagged, bagged, and bag-removed fruits at 0, 4, and 10 days after bag removal. The DEGs were implicated in light signal perception and transduction, plant hormone signal transduction, and antioxidant systems. Weighted gene co-expression network analysis of DEGs generated a module of 23 genes highly correlated with anthocyanin content. The deletion of − 2026 to − 1870 bp and − 1062 to − 964 bp regions of the MdMYB1 (LOC103444202) promoter induced a significant decrease in glucuronidase activity and anthocyanin accumulation in apple peel. Bagging treatment can induce red pigmentation in ‘Granny Smith’ via altering the expression patterns of genes involved in crucial signal transduction and biochemical metabolic pathways. The − 2026 to − 1870 bp and − 1062 to − 964 bp regions of the MdMYB1 promoter are essential for MdMYB1-mediated regulation of anthocyanin accumulation in the ‘Granny Smith’ apple cultivar. The findings presented here provide insight into the mechanisms of coloration in the peel of ‘Granny Smith’ and other non-red apple cultivars.

The red color of apples (Malus domestica) is an attractive trait for consumers. The green skinned “Granny Smith” cultivar develops red pigmentation after bagging treatment. DNA methylation plays an important role in various developmental processes in plants. To explore the possible functions of DNA methylation in the pigmentation of bagged “Granny Smith” apples, we first analyzed the anthocyanin content of fruit skin following treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC). The results revealed an increase in anthocyanin content in bagged fruits following 5-aza-dC treatment, while no anthocyanins were detected in unbagged fruits. In addition, 8482 differentially expressed genes between 5-aza-dC-treated and control groups were identified in bagged fruits by RNA sequencing, including genes encoding transcription factors, enzymes related to anthocyanin accumulation, and methylases. Changes in the expression of these genes may be responsible for 5-aza-dC-induced red pigmentation in bagged fruits of “Granny Smith”. The findings provide novel evidence for the involvement of DNA methylation in the red pigmentation of non-red-skinned apples.