Apple (Malus domestica Borkh.) red-skin coloration, primarily determined by anthocyanin accumulation, is a critical trait affecting consumer preference and market value. However, the molecular mechanisms underlying distinct red-skin patterns (striped vs. blushed) remain unclear. Here, we performed an integrated multi-omics analysis comparing the red-striped ‘Fuji’ apple, which exhibits alternating light-red stripes (LRS) and dark-red stripes (DRS), with its red-blushed mutant (FBM). Physiological and microstructural analyses showed that FBM and DRS had higher anthocyanin content, thicker cuticles, and denser epidermal cells than LRS. Metabolomic profiling identified key differential metabolites positively associated with fruit blush intensity, which mainly consisted of six anthocyanins, fourteen flavonols, and twelve flavones within flavonoids, alongside fifteen phenolic acids, four amino acids and derivatives, one alkaloid, three lignans and coumarins, as well as two tannins. Transcriptomic analysis uncovered MdMAPKKK17, a mitogen-activated protein kinase kinase kinase gene, whose expression was negatively associated with anthocyanin accumulation across red-skin patterns. Functional validation based on transient and stable overexpression demonstrated that MdMAPKKK17 negatively regulates anthocyanin biosynthesis by interacting with MdMYB10 and suppressing its transcriptional activity. Our findings clarify the metabolic and transcriptional networks governing apple red-skin patterning, identify MdMAPKKK17 as a key repressor of anthocyanin deposition and pattern formation, and provide potential targets for molecular breeding to tailor apple skin coloration to meet market demands and enhance nutritional value.

The 'Harlikar' apple, known for its larger fruit and rich flavor, faces challenges in post-harvest storage. Our study compared the storage characteristics of 'Harlikar' apples at three different harvest stages and found that stage II showed superior storage characteristics. At stage II, the application of 1-methylcyclopropene (1-MCP) proved particularly beneficial, resulting in the slowest decline in apple firmness. By the end of cold storage, apples treated with 1-MCP retained a firmness of 56 N, which was superior when compared to the other two stages. Moreover, 1-MCP treatment was effective in maintaining the soluble solid content of the apples. 1-MCP treatment at stage II significantly delayed cell wall degradation by reducing protopectin and cellulose content. Additionally, the treatment also decreased softening-related enzyme activities, and related gene expression. 1-MCP also influenced key aroma compounds, 2-hexenal and hexyl acetate. These findings suggest that stage II with 1-MCP is optimal for storage.

Astaxanthin, a ketocarotenoid with superior antioxidant properties compared with other carotenoids, is increasingly demanded in feed, food, and pharmaceutical industries. This study aimed to engineer apple (Malus domestica Borkh.) callus and tobacco (Nicotiana benthamiana Domin) leaf for sustainable production of natural (3S,3'S)-astaxanthin. A plant expression vector, pRI101-Flag-BHY-BKT-PSY1-CrtI (BBPC), was constructed to deliver astaxanthin biosynthetic genes PSY1, CrtI, BHY and BKT genes into 'Orin' apple callus and N. benthamiana leaf. Transgenic apple calli synthesized 40.4 mu g/g dry weight (DW) astaxanthin, representing 53.5 % of total carotenoids. Transiently transformed N. benthamiana leaves produced 732.8 mu g/g DW astaxanthin,accounting for about 18.2 % of total carotenoids. Furthermore, supplementing tomato juice (20 mL/L) and carrot juice (30 mg/L) increased astaxanthin levels in calli by 1.7-fold. Moreover, astaxanthin-rich tissues exhibited 2-4 fold higher antioxidant activity (P < 0.05) than controls. Acute toxicity assays in mice revealed no adverse effects. These findings demonstrate the potential of engineered apple callus and N. benthamiana leaf as scalable sources of natural (3S,3'S)-astaxanthin for industrial applications.

Jasmonic acid (JA) and gibberellin (GA) coordinate many aspects of plant growth and development, including anthocyanin biosynthesis. However, the crossover points of JA and GA signals and the pathways through which they interact to regulate anthocyanin biosynthesis are poorly understood. Here, we investigated the molecular mechanism by which the zinc finger protein (ZFP) transcription factor Malus domestica ZFP7 (MdZFP7) regulates anthocyanin biosynthesis by integrating JA and GA signals at the transcriptional and post-translational levels. MdZFP7 is a positive regulator of anthocyanin biosynthesis, which fulfills its role by directly activating the expression of MdMYB1 and enhancing the transcriptional activation of MdWRKY6 on the target genes MdDFR and MdUF3GT. MdZFP7 integrates JA and GA signals by interacting with the JA repressor apple JASMONATE ZIM-DOMAIN2 (MdJAZ2) and the GA repressor apple REPRESSOR-of-ga1-3-like 3a (MdRGL3a). MdJAZ2 weakens the transcriptional activation of MdMYB1 by MdZFP7 and disrupts the MdZFP7-MdWRKY6 interaction, thereby reducing the anthocyanin biosynthesis promoted by MdZFP7. MdRGL3a contributes to the stimulation of anthocyanin biosynthesis by MdZFP7 by sequestering MdJAZ2 from the MdJAZ2-MdZFP7 complex. The E3 ubiquitin ligase apple BOI-related E3 ubiquitin-protein ligase 3 (MdBRG3), which is antagonistically regulated by JA and GA, targets the ubiquitination degradation of MdZFP7. The MdBRG3-MdZFP7 module moves the crosstalk of JA and GA signals from the realm of transcriptional regulation and into the protein post-translational modification. In conclusion, this study not only elucidates the node-role of MdZFP7 in the integration of JA and GA signals, but also describes the transcriptional and post-translational regulatory network of anthocyanin biosynthesis with MdZFP7 as the hub.

Apple is classified as a climacteric fruit, characterized by a rapid surge in ethylene levels at the onset of ripening. However, there is significant variation in the degree and timing of ripening and softening among different apple cultivars. Understanding the molecular mechanisms underlying fruit softening and the associated changes in cell wall integrity is crucial for improving fruit quality and extending shelf life. In this study, we conducted a comparative analysis of fruit firmness and ethylene production among seven apple cultivars with different softening patterns during storage. We also examined the expression patterns of genes and enzyme activities involved in cell wall metabolism. Our results showed that fruit firmness was negatively correlated with ethylene production rate, respiration rate, soluble pectin content, cellulase activity, polygalacturonase activity, and β-glucosidase activity, while it was positively correlated with protopectin content and cellulose content. Furthermore, we identified a BBX transcription factor gene, MdBBX25, that was negatively correlated with fruit softening during storage. Overexpression of MdBBX25 resulted in delayed fruit softening by suppressing ethylene biosynthesis and cell wall-related genes, whereas silencing of MdBBX25 accelerated fruit softening by promoting ethylene biosynthesis and cell wall-related genes in apple. Our findings provide insights into the role of MdBBX25 as a negative regulator of fruit softening through the modulation of ethylene biosynthesis and cell wall degradation during storage.

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 β-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.

The lignification process exerts an adverse impact on the peach fruit quality. The identification of a texture-related gene module was achieved through weighted gene co-expression network analysis (WGCNA) based on transcriptomic analysis of two peach fruit varieties, ‘Baili’ and ‘Hongli’. Notably, the WRKY transcription factor PpWRKY65 played a crucial role in regulating lignin biosynthesis in peach fruit. Analyzing the correlation among the expression level of PpWRKY65, the expression level of genes related to lignin synthesis, and the lignin content of peach fruit both under ambient temperature and chilling injury condition, it was found that PpWRKY65 negatively regulated the accumulation of peach fruit lignin by inhibiting the expression of genes related to lignin synthesis. Moreover, the lignin content was significantly lower in transient PpWRKY65-overexpressing fruit, compared to the control. Concurrently, the expressions of lignin biosynthesis genes (Pp4CL7, PpCAD1, PpCOMT, PpF5H) were decreased in fruit overexpressing PpWRKY65.

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 (TF), 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, beta-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 beta-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 TF 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. Abscisic acid activates a TF module that regulates apple color by activating the major carotenoid biosynthesis genes.

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 weakened it. Yeast two-hybrid, bimolecular fluorescence complementation, pull-down, and co-immunoprecipitation 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.

以蓬莱地区神富1号苹果为试材,在不同时期增施矿源腐殖酸钾和硝酸铵钙,探索其对苹果果实着色、品质和产量的影响.结果表明,增施矿源腐殖酸钾提高了苹果果实可溶性固形物、还原糖、Vc和总黄酮含量,促进了果皮着色和果皮花青苷的积累,提高了果树产量,能有效防控果实苦痘病.

SUMMARY 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.

比较了烟富8号苹果带袋采收和不套袋的果实品质和货架期品质变化.结果表明:烟富8号苹果带袋采收果实单果重和果形指数均高于不套袋果实,但差异均不显著;烟富8号带袋采收果实硬度、可溶性固形物含量和还原糖含量、果皮a*值、类胡萝卜素含量和花青苷含量、果皮果肉总黄酮含量等均显著低于不套袋果实;烟富8号带袋采收果实果皮L*、b*值和果实可滴定酸含量均显著高于不套袋果实;烟富8号带袋采收果实维生素C含量和类胡萝卜素含量与不套袋果实无显著差异.在货架期,烟富8号带袋采收果实硬度和还原糖含量、果皮类胡萝卜素含量和花青苷含量、果皮果肉总黄酮含量显著低于不套袋果实,烟富8号带袋采收果肉类胡萝卜素含量显著高于不套袋果实.

Monoterpenes are typical aroma components of muscat grape cultivars, providing pleasant floral and fruity aromas to grapes and wines. However, the molecular mechanism of monoterpene biosynthesis between muscat and non-muscat grape remains unclear. Here, the muscat grape cultivar ‘Jumeigui’ and the non-muscat grape cultivar ‘Kyoho’ were chosen as plant materials for a comprehensive transcriptome and metabolite analysis. The gas chromatography–mass spectrometry (GC–MS) analysis demonstrated that a total of 27 and 23 monoterpene compounds were identified and quantified in the ‘Jumeigui’ and ‘Kyoho’ grape, respectively. ‘Jumeigui’ grape accumulated significantly higher concentrations of monoterpenes than ‘Kyoho’ grape. Furthermore, geraniol, linalool, geranic acid, and β-citronellol might be important odorants contributing to the floral character of the ‘Jumeigui’ grape due to the high levels odor activity values (OAVs). Transcriptome analysis demonstrated that the expression profiles of VvDXS, VvGGPPS.SSU1, VvTPS-b/g showed a positive correlation with monoterpene accumulation in grapes. In addition, the expression patterns of the genes involved in jasmonic acid (JA) synthesis and signal were also positively correlated with monoterpene accumulation. All these results will help guide the functional verification of candidate genes related to monoterpene biosynthesis, as well as identify the master transcriptional and hormonal regulators of this pathway in grapes.

Nudix hydrolases are widely distributed across all classes of organisms and provide the potential capacity to hydrolyze a wide range of organic pyrophosphates. Although Nudix hydrolases are involved in plant detoxification processes in response to abiotic and biotic stresses, the biological functions of Nudix hydrolases remain largely unclear in grapevine. In the present study, a total of 25 putative grapevine Nudix hydrolases (VvNUDXs) were identified by bioinformatics analysis and classified into eight subfamilies based to their preferred substrates. Both tandem and segmental duplications were responsible for the evolution and expansion of the NUDX gene family in grapevine. To investigate the regulatory roles of VvNUDX genes during growth and development, as well as in response to abiotic and biotic stresses in grapevine, the expression patterns were revealed in publicly available microarray data. The spatial and temporal expression patterns of the VvNUDX genes indicated that they might play important roles in multiple developmental processes. Transcriptome and qRT-PCR analyses showed that ten VvNUDX genes were specifically expressed in grapevine berries, suggesting potential roles in grapevine berry development. Expression and phylogenetic analyses demonstrated that VvNUDX1 and VvNUDX3 might be involved in terpenoid biosynthesis in grapevine. Furthermore, most VvNUDX genes active toward the ADP-ribose/NADH showed different patterns in response to various abiotic and biotic stresses, such as salinity and drought, as well as different types of biotic treatments, such as Erysiphe necator, Bois Noir phytoplasma and leaf-roll-associated virus-3 (GLRaV-3). These results indicated that VvNUDX genes were associated with plant detoxification processes in response to abiotic and biotic stresses, and regulate the disease immunity and resistance pathways. The information obtained here may provide good opportunities to explore the physiological functions of VvNUDX genes in berry development and stress response networks in grapevine.

Flavonoids are one of the main pigments in horticultural fruit. The AP2/ERF transcription factors play various roles in higher plants, and thus, MdAP2–34 has been reported to regulate carotenoid accumulation in apple. However, it is unclear whether light interferes with the flavonoid accumulation in apple fruit flesh. Here, we found that apple of ‘Benin Shogun’ fruit flesh had higher levels of flavonoids under light treatment than under dark treatment, with an increase of 48% at 15 days after harvest (DAH) and 39% at 30 DAH. The MdAP2–34 transcript levels exhibited a highly positive correlation with the flavonoid content of ‘Benin Shogun’ fruit under dark and light treatments. Furthermore, the structural genes of flavonol biosynthesis, including MdCHS, MdCHI, MdF3′H, and MdFLS, were positively associated with flavonoid contents of fruit and were upregulated in MdAP2–34-OVX apple calli. The MdAP2–34 stimulated flavonoid accumulation via the flavonoid biosynthetic pathway by directly binding to and activating the MdF3′H promoter activity. Our findings demonstrate the use of multiple MdAP2–34-mediated functions in understanding the complex mechanisms of flavonoid synthesis in apple, which may also be useful in apple breeding.

The plant-specific WUSCHEL-related homeobox（WOX） genes are crucial for plant growth and development.Here,we systematically identified the MdWOX gene family in apple at the genome-wide level,and analyzed the phylogenetic relationships,conserved motifs,gene structure,and syntenic relationships of the MdWOX genes.A total of 18 MdWOX genes were identified and phylogenetic analysis placed them into three clades.The phylogenetic relationships among the WOXs were further supported by the analyses of gene structure and conserved motifs.Chromosomal distribution and synteny analysis revealed that whole-genome and segmental duplications have played key roles in MdWOX gene family expansion.Moreover,the MdWOX genes exhibit tissue-specific expression patterns and MdWOX4a,MdWOX4b,MdWOX5b,MdWOX11/12a,and MdWOX11/12b may play essential roles in adventitious root development.The adventitious rooting ability was enhanced in MdWOX4b transgenic tobacco lines.The results of this study provide useful information for future functional studies on MdWOXs in the development of apple rootstocks.

Fruit softening is a complex process that accelerates postharvest spoilage and shortens the shelf life of fruit. The precise mechanisms underlying fruit softening remain elusive, especially in terms of genetic function. Here, we analyzed in deep the RNA-Seq data of ‘Hongli’ (readily softens after harvest) and ‘Baili’ (slowly softens after harvest) peach fruit. We identified polygalacturonase (PpPG) gene in the ‘plum3’ module as a potential fruit softening biomarker in peach through weighted gene co-expression network analysis (WGCNA). Through transcription factor-target gene regulatory network analysis and molecular approaches, we identified an ethylene response factor (ERF) transcript factor, PpERF/ABR1, as a transcriptional activator that binds directly to the promoter of PpPG. Moreover, transient overexpression of PpERF/ABR1 also enhanced PpPG expression, thereby resulting in fruit softening. These results provide a theoretical basis for the molecular mechanism of ERF in the transcription regulation of cell wall-related genes during fruit softening.

分析了中国矮砧苹果的施肥现状与存在问题,提出基于叶分析为主的矮砧苹果叶营养诊断取样时期和诊断方法,并根据诊断结果提出矮砧苹果水肥一体化技术,在规模化矮砧苹果生产园示范推广,实现了优质与高效生产.

[目的]确定威海地区栽种的金冠、威海金和烟富3号苹果果实的适宜采收期及其采收参考标准.[方法]对3个品种不同发育时期果实横切面进行淀粉染色,构建碘-淀粉染色图谱,测定硬度、可溶性固形物含量、乙烯释放速率和呼吸速率等相关指标,同时利用电子鼻分析发育过程中的发挥性物质,以进一步确立适宜采收期.[结果]金冠、威海金和烟富3号苹果果实纵横经分别在9月30日、10月30日和10月20日达最大值;此时可溶性固形物含量分别为13.94％、14.61％和12.94％;挥发性物质可以被电子鼻很好地区分;呼吸速率均处于较低值;乙烯释放速率处于缓慢上升期;且此时淀粉染色结果显示淀粉的水解部位已向外扩散,其中威海金和烟富3号淀粉染色已扩散至果皮区域.[结论]通过碘-淀粉染色图谱和相关的生理指标及挥发性物质分析,将金冠、威海金和烟富3号苹果果实的适宜采收期分别定为9月30日、10月30日和10月20日,此时期对应的淀粉染色等级分别均为7级,其可以作为采收的参考标准.

以胶东半岛老的果园中土壤为试材,采用逐步稀释法,涂平板于孟加拉红培养基中,根据形态和分子方法鉴定分离木霉菌.分离出的木霉菌与苹果主要病害对峙培养在不同pH的培养基上,观察不同pH条件下,对真菌病害的抑制效果.土壤有机质和pH是目前胶东果园的主要问题,采用正交法设计,研究了不同的土壤条件对木霉菌生长的影响.结果 表明:在不同pH条件下,木霉菌抑制苹果病原真菌的效果不同,木霉菌在不同的pH条件下对腐烂病(Valsa mali)和炭疽病(Colletotrichum gloeosporioides)均可以抑制,但对轮纹病(Ph ysalospora piricola Nose)只有在pH 6.0时没有效果外,其它pH时都对轮纹病有抑制作用.在土壤pH 6.5和土壤有机质含量为75 g·kg-1时,最有利于木霉菌定殖.同时还发现木霉菌对土壤的pH具有调节作用,无论在碱性条件下,还是酸性条件下,均可以使土壤pH趋于中性,另外木霉菌可以增加土壤有机质含量,特别是在高土壤有机质含量的情况下,增加更加显著.总之,分离出的木霉菌对苹果腐烂病、炭疽病和轮纹病抑制效果好.土壤pH和有机质含量对木霉菌的生长有影响,高土壤有机质含量和中性土壤有利于木霉菌定殖.另外,木霉菌能有效地改良土壤有机质含量和土壤pH.