The MdMYB44-MdTPR1 repressive complex inhibits MdCCD4 and MdCYP97A3 expression through histone deacetylation to regulate carotenoid biosynthesis in apple.

Carotenoids are photosynthetic pigments and antioxidants that contribute to different plant colors. However, the involvement of TOPLESS (TPL/TPR)-mediated histone deacetylation in the modulation of carotenoid biosynthesis through ethylene-responsive element-binding factor-associated amphiphilic repression (EAR)-containing transcription factors (TFs) in apple (Malus domestica Borkh.) is poorly understood. MdMYB44 is a transcriptional repressor that contains an EAR repression motif. In the present study, we used functional analyses and molecular assays to elucidate the molecular mechanisms through which MdMYB44-MdTPR1-mediated histone deacetylation influences carotenoid biosynthesis in apples. We identified two carotenoid biosynthetic genes, MdCCD4 and MdCYP97A3, that were confirmed to be involved in MdMYB44-mediated carotenoid biosynthesis. MdMYB44 enhanced ß-branch carotenoid biosynthesis by repressing MdCCD4 expression, whereas MdMYB44 suppressed lutein level by repressing MdCYP97A3 expression. Moreover, MdMYB44 partially influences carotenoid biosynthesis by interacting with the co-repressor TPR1 through the EAR motif to inhibit MdCCD4 and MdCYP97A3 expression via histone deacetylation. Our findings indicate that the MdTPR1-MdMYB44 repressive cascade regulates carotenoid biosynthesis, providing profound insights into the molecular basis of histone deacetylation-mediated carotenoid biosynthesis in plants. These results also provide evidence that the EAR-harboring TF/TPL repressive complex plays a universal role in histone deacetylation-mediated inhibition of gene expression in various plants.

Abscisic acid activates transcription factor module MdABI5-MdMYBS1 during carotenoid-derived apple fruit coloration.

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, ß-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 ß-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.

The 14-3-3 protein GRF8 modulates salt stress tolerance in apple via the WRKY18-SOS pathway.

Salinity is a severe abiotic stress that limits plant survival, growth, and development. 14-3-3 proteins are phosphopeptide-binding proteins that are involved in numerous signaling pathways, such as metabolism, development, and stress responses. However, their roles in salt tolerance are unclear in woody plants. Here, we characterized an apple (Malus domestica) 14-3-3 gene, GENERAL REGULATORY FACTOR 8 (MdGRF8), the product of which promotes salinity tolerance. MdGRF8 overexpression improved salt tolerance in apple plants, whereas MdGRF8-RNA interference (RNAi) weakened it. Yeast 2-hybrid, bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays revealed that MdGRF8 interacts with the transcription factor MdWRKY18. As with MdGRF8, overexpressing MdWRKY18 enhanced salt tolerance in apple plants, whereas silencing MdWRKY18 had the opposite effect. We also determined that MdWRKY18 binds to the promoters of the salt-related genes SALT OVERLY SENSITIVE 2 (MdSOS2) and MdSOS3. Moreover, we showed that the 14-3-3 protein MdGRF8 binds to the phosphorylated form of MdWRKY18, enhancing its stability and transcriptional activation activity. Our findings reveal a regulatory mechanism by the MdGRF8-MdWRKY18 module for promoting the salinity stress response in apple.

DNA methylation variation is crucial to restore adventitious rooting ability during in vitro shoot culture-induced rejuvenation in apple rootstock.

In vitro shoot culture has been widely used for restoring adventitious rooting ability in rooting recalcitrant woody perennial species for the past few decades, but its molecular mechanism is largely uncovered. DNA methylation is an essential epigenetic mark that participates in many biological processes. Recent reports suggested a role of DNA methylation in vitro culture in plants. In this study, we characterized the single-base resolution DNA methylome and transcriptome of adult and in vitro shoot culture-induced rejuvenation cuttings of apple rootstock M9T337. We found a global decrease in DNA methylation during rejuvenation, which may be correlated with increased expression of DNA demethylase genes and decreased expression of DNA methyltransferase genes. We additionally documented DNA hypomethylation in 'T337'_R in gene protomer associated with higher transcript levels of several adventitious rooting-related genes. The application of a DNA methylation inhibitor (5-azacytidine) enhanced the adventitious rooting ability and the expression level of adventitious rooting-related genes, such as, MdANT, MdMPK3, MdABCB21, MdCDC48, MdKIN8B, pri-MdMIR156a5 and pri-MdMIR156a12. Together, the DNA hypomethylation is critical for the rejuvenation-dependent adventitious rooting ability in apple rootstock. In addition, increased DNA methylation was also found in thousands of genes in 'T337'_R. We additionally documented that DNA hypermethylation is required for inhibition of adventitious rooting-repressed genes, such as MdGAD5a, encoding glutamate decarboxylase, which can catalyze glutamate decarboxylated to form γ-aminobutyric acid (GABA). Our results revealed that in vitro shoot culture-dependent DNA methylation variation plays important roles in adventitious rooting in apple rootstock.

An apple (Malus domestica) AP2/ERF transcription factor modulates carotenoid accumulation.

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 ß-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 ß-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.

Genetic variation in the promoter of an R2R3-MYB transcription factor determines fruit malate content in apple (Malus domestica Borkh.).

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.

The MdXTHB gene is involved in fruit softening in 'Golden Del. Reinders' (Malus pumila).

BACKGROUND: Fruit softening is a major determinant of commercial value and shelf life. A transcriptomic analysis of 'Golden Delicious' and 'Golden Del. Reinders' (a bud mutation of 'Golden Delicious' that readily softens) apple fruit was conducted during storage. RESULTS: A comparative analysis of the obtained expression profiles of fruit between two cultivars identified 1345 upregulated and 3475 downregulated differentially expressed genes (DEGs). The DEGs identified were associated with cellular processes and carbohydrate metabolism and were especially enriched in cell-wall-related genes. Among the cell-wall-related genes, the xyloglucan endotransglucosylase/hydrolases (XTH) gene MdXTHB was significantly upregulated and exhibited high expression levels in 'Golden Del. Reinders' fruit, which had a lower level of firmness relative to 'Golden Delicious'. Overexpression of MdXTHB in both 'Golden Delicious' and 'Fuji', which typically maintain high levels of firmness in storage, exhibited faster rates of softening and an earlier peak of ethylene production than empty-vector-infiltrated fruit did. CONCLUSION: The results of this study indicate that MdXTHB potentially promotes apple fruit softening by degrading the fruit cell wall. This result is also useful to designing further experiments on the molecular regulation of fruit softening in apple. © 2020 Society of Chemical Industry.

Anthocyanin Biosynthesis and Methylation of the MdMYB10 Promoter Are Associated with the Red Blushed-Skin Mutant in the Red Striped-Skin "Changfu 2" Apple.

The color of apple skin, particularly anthocyanin-based coloration, is a key factor determining market acceptance. The mechanisms of anthocyanin accumulation in apples with different skin color patterns (i.e., striped and blushed) were analyzed. In total, 14 anthocyanins and 5 procyanidins were simultaneously assayed in red blushed-skin mutants (CF-B1 and CF-B2) and red striped-skin parents (CF-S1 and CF-S2), and 13 significant differences were revealed. Anthocyanin accumulation was significantly higher in the red blushed-skin apples than it was in the parents. The transcript levels of anthocyanin biosynthesis genes and regulatory factors (MdMYB10, MdbHLH3, and MdWD40) were associated with different skin color patterns during the coloring period at 4, 6, and 8 days after the fruits were debagged. The methylation levels of the MdMYB10 promoter regions -1203 to -779 bp, -1667 to -1180 bp, and -2295 to -1929 bp were associated with different skin color patterns, and there was more methylation in red striped-skin apples. These results improve our understanding of anthocyanin accumulation and its underlying molecular mechanism in apples with different skin color patterns, thereby providing valuable information for apple breeding.

Genome-wide identification and analysis of B-BOX gene family in grapevine reveal its potential functions in berry development.

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.

Genome-wide identification and transcript analysis of TCP transcription factors in grapevine.

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.

Lignin Involvement in Programmed Changes in Peach-Fruit Texture Indicated by Metabolite and Transcriptome Analyses.

Texture is an important component of peach-fruit quality. In the present study, an analysis of metabolite and transcriptome profiles during storage of a nonmelting-flesh cultivar, 'Baili', and a melting-flesh cultivar, 'Hongli', was conducted to explore the molecular mechanisms underlying different fruit textures in peach. Results indicated that higher levels of anthocyanins were present in 'Hongli' peach, whereas lignin monomers and ethylene precursors were higher in 'Baili'. A transcriptome analysis indicated that genes associated with lignin synthesis were more highly expressed in 'Baili' than in 'Hongli', especially Pp4CL2, Pp4CL3, and PpCOMT2. Texture differences between the two varieties may be the result of differential expression of two branches of the phenylpropanoid metabolic pathway. One branch regulates flavonoid metabolism and was highly active in 'Hongli' fruit, whereas the other branch regulates lignin synthesis and was more highly active in 'Baili' fruit.

Soil amendment alters soil physicochemical properties and bacterial community structure of a replanted apple orchard.

Compost amendment reportedly improved apple tree growth in replant soils. However, its effects should be evaluated at different soil depths and locations. This study investigated the impact of soil improvement with compost on soil physicochemical properties and bacterial community structure of a replanted apple orchard in comparison with the original orchard without compost improvement. The V1-V3 region of the bacterial 16S rRNA gene was subjected to high-throughput 454 pyrosequencing, and data were analyzed using the Mothur pipeline. The results showed that the soil improvement benefited tree growth and fruit quality during the study period. The compost amendment markedly increased tree height and stem diameter by a range of 6.1%-21.0% and 4.0%-14.0%, respectively. Fruit yield (9.5%), average weight (9.6%), and soluble solid content (5.6%) were also increased by compost amendment compared to those of the unimproved treatment. The pH, organic matter, and available N, P, and K contents were significantly increased by 5.7%-21.9%, 0.2%-62.9%, 9.3%-29.3%, 36.7%-64.5%, and 17.2%-100.3% in the compost improved soil. The pyrosequencing data showed that the soil improvement changed the bacterial community structure at all soil depths (0-20 cm and 20-40 cm) and locations (in-row and inter-row) considered; e.g., the relative abundance of Proteobacteria (20.2%), Bacteroidetes (2.5%), and Cyanobacteria (1.0%) was increased while that of Chloroflexi (5.5%), Acidobacteria (5.2%), Nitrospirae (4.5%), Gemmatimonadetes (3.8%), and Actinobacteria (1.8%) was decreased. The relative abundance of some dominant genera Burkholderia (2.3%), Pseudomonas (1.0%), and Paenibacillus (0.5%) were enhanced in the compost improved soil. Moreover, other dominant genera such as Nitrospira (6.4%), Gemmatimonas (2.2%), and Phenylobacterium (0.3%) were reduced by the application of compost. Our results indicate that soil improvement benefits the growth of tree and fruit quality, and is likely mediated by increased soil pH, organic matter, and available nutrient contents and beneficial bacterial community composition.

Differential gene expression analysis of 'Chili' (Pyrus bretschneideri) fruit pericarp with two types of bagging treatments.

Preharvest bagging is a simple, grower-friendly and safe physical protection technique commonly applied to many fruits, and the application of different fruit bags can have various effects. To explore the molecular mechanisms underlying the fruit quality effects of different bagging treatments, digital gene expression (DGE) profiling of bagged and unbagged 'Chili' (Pyrus bretschneideri Rehd.) pear pericarp during development was performed. Relative to unbagged fruit, a total of 3022 and 769 differentially expressed genes (DEGs) were detected in the polyethylene (PE)-bagged and non-woven fabric-bagged fruit, respectively. DEGs annotated as photosynthesis-antenna proteins and photosynthesis metabolism pathway were upregulated in non-woven fabric-bagged fruit but downregulated in the PE-bagged fruit. Non-woven fabric bagging inhibited lignin synthesis in 'Chili' pear pericarp by downregulating DEGs involved in phenylpropanoid biosynthesis; consequently, the fruit lenticels in non-woven fabric-bagged fruit were smaller than those in the other treatments. The results indicate that the non-woven fabric bagging method has a positive effect on the appearance of 'Chili' pear fruit but neither of the two bagging treatments is conducive to the accumulation of soluble sugar.

1,5-anhydro-D-fructose and its derivatives: biosynthesis, preparation and potential medical applications.

1,5-Anhydro-D-fructose (AF) was first found in fungi and red algae. It is produced by the degradation of glycogen, starch and maltosaccharides with α-1,4-glucan lyase (EC 4.2.2.13). In vivo, AF is metabolized to 1,5-anhydro-D-glucitol (AG), ascopyrone P (APP), microthecin and other derivatives via the anhydrofructose pathway. The genes coding for the enzymes in this pathway have been cloned, enabling the large-scale production of AF and related products in a cell-free reactor. The possible applications of these products in medicine have been evaluated using both in vitro and in vivo systems. Thus AF is a useful anticariogenic agent as it inhibits the growth of the oral pathogen Streptococcus mutans, impairing the production of plaque-forming polysaccharides and lactic acid. AF also shows anti-inflammatory and anticancer effects. AG is used as a diabetic marker for glycemic control. AG also stimulates insulin secretion in insulinoma cell lines. in vivo, APP has been shown to lengthen the life span of cancer-afflicted mice. It interferes with tumor growth and metastasis by its cidal effects on fast multiplying cells. Microthecin inhibits the growth of the human pathogen Pseudomonas aeruginosa PAO1, particularly under anaerobic conditions. The pharmaceutical usefulness of the other AF metabolites 1,5-anhydro-D-mannitol,1-deoxymannojirimycin, haliclonol, 5-epipentenomycin I, bissetone, palythazine, isopalythazine, and clavulazine remains to be investigated. In this review AF and its metabolites as the bioactive natural products for their pharmaceutical potentials are discussed.

Examination of 1,5-anhydro-D-fructose and the enolone ascopyrone P, metabolites of the anhydrofructose pathway of glycogen and starch degradation, for their possible application in fruits, vegetables, and beverages as antibrowning agents.

The anhydrofructose pathway describes the degradation of glycogen and starch to 1,5-anhydro-D-fructose (1,5AnFru) and its further conversion to the enolone ascopyrone P (APP) via the transit intermediate ascopyrone M. The two products, 1,5AnFru and APP, were examined in this study for their effects in controlling the browning of selected fruits, vegetables, and beverages. The results showed that 1,5AnFru had an antibrowning effect in green tea and was able to slow turbidity development in black currant wine. APP proved to be an antibrowning agent comparable to kojic acid. It showed an antibrowning effect in a range of agricultural products, such as various cultivars of apple, pear, potato, lettuce, and varieties of green tea in an efficacy concentration range from 300 to 500 ppm. Mechanism studies indicated that, like kojic acid, APP showed inhibition toward plant polyphenol oxidase and was able to decolor quinones.