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Summary: Malic acid accumulation in the vacuole largely determines acidity and perception of sweetness of apple. It has long been observed that reduction in malate level is associated with increase in ethylene production during the ripening process of climacteric fruits, but the molecular mechanism linking ethylene to malate reduction is unclear.Here, we show that ethylene‐modulated WRKY transcription factor 31 (WRKY31)–Ethylene Response Factor 72 (ERF72)–ALUMINUM ACTIVATED MALATE TRANSPORTER 9 (Ma1) network regulates malate accumulation in apple fruit.ERF72 binds to the promoter of ALMT9, a key tonoplast transporter for malate accumulation of apple, transcriptionally repressing ALMT9 expression in response to ethylene. WRKY31 interacts with ERF72, suppressing its transcriptional inhibition activity on ALMT9. In addition, WRKY31 directly binds to the promoters of ERF72 and ALMT9, transcriptionally repressing and activating ERF72 and ALMT9, respectively. The expression of WRKY31 decreases in response to ethylene, lowering the transcription of ALMT9 directly and via its interactions with ERF72.These findings reveal that the regulatory complex WRKY31 forms with ERF72 responds to ethylene, linking the ethylene signal to ALMT9 expression in reducing malate transport into the vacuole during fruit ripening. See also the Commentary on this article by Martinoia & Neuhaus, 239: 821–823. [ABSTRACT FROM AUTHOR]

Sugars are involved in plant growth, fruit quality, and signaling perception. Therefore, understanding the mechanisms involved in soluble sugar accumulation is essential to understand fruit development. Here, we report that MdPFPβ, a pyrophosphate‐dependent phosphofructokinase gene, regulates soluble sugar accumulation by enhancing the photosynthetic performance and sugar‐metabolizing enzyme activities in apple (Malus domestica Borkh.). Biochemical analysis revealed that a basic helix‐loop‐helix (bHLH) transcription factor, MdbHLH3, binds to the MdPFPβ promoter and activates its expression, thus promoting soluble sugar accumulation in apple fruit. In addition, MdPFPβ overexpression in tomato influenced photosynthesis and carbon metabolism in the plant. Furthermore, we determined that MdbHLH3 increases photosynthetic rates and soluble sugar accumulation in apple by activating MdPFPβ expression. Our results thus shed light on the mechanism of soluble sugar accumulation in apple leaves and fruit: MdbHLH3 regulates soluble sugar accumulation by activating MdPFPβ gene expression and coordinating carbohydrate allocation. [ABSTRACT FROM AUTHOR]

Auxin response factors (ARFs) mediate auxin response gene transcription by directly binding with AuxREs. However, whether ARF family genes involved in anthocyanin accumulation in apple is largely unexplored. In this study, thirty-three MdARF family members were identified in the apple (Malus × domestica) genome (HFTH1 Whole Genome v1.0) based on the sequence similarity with ARF family members of Arabidopsis. Phylogenetic analysis revealed that thirty-three MdARF members were clustered into Class I, Class II and Class III. Functional domain analysis showed that MdARF proteins have some typical domains such as B3-like DNA-binding domain and Auxin_resp domain, as well as motif III and motif IV. Chromosome localisation analysis demonstrated that apple ARF members were widely distributed in the genome. Among these MdARF genes, MdARF2 was significantly correlated with anthocyanin variation according to extracting expression profiles from published RNA-seq data. Further functional identification in apple calli suggested that MdARF2 functions as a transcriptional repressor of anthocyanin biosynthesis. These findings provide evidence that MdARF family members play a role in the regulation of anthocyanin accumulation in apple. [ABSTRACT FROM AUTHOR]

Auxin plays an important role in plant growth and development; for example, it regulates the elongation and division of plant cells, the formation of plantlet's geotropism and phototropism, and the growth of main lateral roots and hypocotyl. IAA gene is associated with auxin and can response to biotic and abiotic stress in plants. However, the regulatory effect of auxin on anthocyanin accumulation has been rarely reported. In this study, we show that auxin inhibites the accumulation of anthocyanin and decreases the expression of genes related to anthocyanin synthesis in calli, leaves, and seedlings of apple. The expression levels of MdIAA family genes were determined, and we found that MdIAA26 significantly responded to auxin, which also induced MdIAA26 degradation. Functional analysis of MdIAA26 showed that overexpressing MdIAA26 in apple calli and Arabidopsis could promote the accumulation of anthocyanin and up-regulate the genes related to anthocyanin synthesis. Furthermore, the MdIAA26 -overexpressing Arabidopsis could counteract auxin-induced inhibition on anthocyanin accumulation, which indicates that auxin inhibits the accumulation of anthocyanin in apple by degrading MdIAA26 protein. • Exogenous auxin influences anthocyanin accumulation in apple. • MdIAA26 is a positive regulator of anthocyanin accumulation. • Auxin controls anthocyanin accumulation by regulating the stability of MdIAA26 protein. [ABSTRACT FROM AUTHOR]

Apple ring rot is a severe disease that affects the yield and quality of apple fruits worldwide. However, the underlying molecular mechanism that involved in this process still remains largely unexplored. Here, we report that apple POZ/BTB CONTAINING-PROTEIN 1 (MdPOB1), a BTB-BACK domain E3 ligase protein, functions to suppress apple pathogen defense against Botryosphaeria dothidea (B. dothidea). Both in vitro and in vivo assays indicated that MdPOB1 interacted directly with and degraded apple U-box E3 ligase MdPUB29, a well-established positive regulator of plant innate immunity, through the ubiquitin/26S proteasome pathway. A series of transgenic analyses in apple fruits demonstrated that MdPOB1 affected apple pathogen defense against B. dothidea at least partially, if not completely, via regulating MdPUB29. Additionally, it was found that the apple pathogen defense against B. dothidea was correlated with the H2O2 contents and the relative expression of salicylic acid (SA) synthesis- and SA signaling-related genes, which might be regulated via degradation of MdPUB29 by MdPOB1. Overall, our findings provide new insights into the mechanism of the MdPOB1 modulation of apple ring rot resistance, which occur by directly regulating potential downstream target protein MdPUB29 for proteasomal degradation in apple. [ABSTRACT FROM AUTHOR]

Highlights • The research advance on anthocyanins of horticultural products. • We summarize the anthocyanin biosynthetic pathways and transport mechanisms in horticultural plants. • We summarize the environmental factors affecting anthocyanin accumulation in this review. Abstract Anthocyanins, a flavonoid group of polyphenolic compounds, are important human health-promoting pigments responsible for the red and blue colors of horticultural products such as fruits, flowers, and leaves. The anthocyanin biosynthetic and transport pathways are well known. Key regulatory factors involved in these pathways have been characterized in many plant species. Recently, it has been found that regulatory proteins controlling anthocyanin biosynthesis at the transcriptional and post-transcriptional levels are found to be differentially modulated by environmental and biological factors such as light, temperature, sugars, and hormones. In this review, a simplified model for the role of environmental and developmental factors in the modulation of the anthocyanin biosynthesis pathway in horticultural plants is proposed, with emphasis on the coordinated regulation of the key transcriptional MYB-bHLH-WD40 (MBW) complex. [ABSTRACT FROM AUTHOR]

Anthocyanins have essential biological functions, affecting the development of horticultural production. They are synthesized in the cytoplasm through flavonoid metabolic pathways and finally transported into vacuoles for storage. Plant glutathione S-transferases (GSTs) are multifunctional enzymes involved in anthocyanin transportation. In this study, we identified 38 GSTs from the apple (Malus domestica) genome (HFTH1 Whole Genome v1.0) based on the sequence similarity with the GST family proteins of Arabidopsis. These MdGST genes could be grouped into nine chief subclasses: U, F, L, Z, T, GHR, EF1Bγ, TCHQD, and DHAR. The structures, motifs, three-dimensional models, and chromosomal distribution of MdGST genes were further analyzed. Elements which are responsive for some hormones and stress, and others that involve genes related to flavonoid biosynthesis were forecast in the promoter of MdGST. In addition, we identified 32 orthologous gene pairs between apple and Arabidopsis. These genes indicated that numerous apple and Arabidopsis counterparts appeared to be derived from a common ancestor. Amongst the 38 MdGST genes, MdGSTU12 was considerably correlated with anthocyanin variation in terms of extracting expression profiles from reported. Finally, further functional identification in apple transgenic calli and subcellular localization confirmed that MdGSTU12 was of great significance in anthocyanin accumulation in apple. [ABSTRACT FROM AUTHOR]

Summary Softening in fruit adversely impacts their edible quality and commercial value, leading to substantial economic losses during fruit ripening, long‐term storage, long‐distance transportation, and marketing. As the apple fruit demonstrates climacteric respiration, its firmness decreases with increasing ethylene release rate during fruit ripening and postharvest storage. However, the molecular mechanisms underlying ethylene‐mediated regulation of fruit softening in apple remain poorly understood. In this study, we identified a WRKY transcription factor (TF) MdWRKY31, which is repressed by ethylene treatment. Using transgenic approaches, we found that overexpression of MdWRKY31 delays softening by negatively regulating xyloglucan endotransglucosylase/hydrolases 2 (MdXTH2) expression. Yeast one‐hybrid (Y1H), electrophoretic mobility shift (EMSA), and dual‐luciferase assays further suggested that MdWRKY31 directly binds to the MdXTH2 promoter via a W‐box element and represses its transcription. Transient overexpression of ethylene‐induced MdNAC7, a NAC TF, in apple fruit promoted softening by decreasing cellulose content and increasing water‐soluble pectin content in fruit. MdNAC7 interacted with MdWRKY31 to form a protein complex, and their interaction decreased the transcriptional repression of MdWRKY31 on MdXTH2. Furthermore, MdNAC7 does not directly regulate MdXTH2 expression, but the protein complex formed with MdWRKY31 hinders MdWRKY31 from binding to the promoter of MdXTH2. Our findings underscore the significance of the regulatory complex NAC7–WRKY31 in ethylene‐responsive signalling, connecting the ethylene signal to XTH2 expression to promote fruit softening. This sheds light on the intricate mechanisms governing apple fruit firmness and opens avenues for enhancing fruit quality and reducing economic losses associated with softening. [ABSTRACT FROM AUTHOR]

Apple, one of the most common fruits consumed worldwide, has a high economic value. Vacuoles are membrane-bound organelles in plant cells that perform many important functions. An increasing number of studies have reported a variety of proteins in a vacuole, with crucial functions in the transport, storage and metabolism of energy and substances. Here, we successfully extracted the vacuoles and vacuolar proteins from the ‘Orin’ apple fruit calli. We collected intact vacuoles with a density of 100/mm2 by mild osmotic lysis and Ficoll density gradient centrifugation. The result of SDS-PAGE showed that lots of vacuolar proteins were isolated and enriched from vacuole samples, and western blotting showed that the purity of the vacuolar protein was high. Furthermore, we showed GO enrichment and KEGG enrichment of isolated vacuolar proteins and selected 41 vacuolar proteins with known functions and 10 vacuolar proteins with unknown functions for functional annotation from the mass spectrometry analysis. This approach for vacuole isolation and vacuolar protein identification will provide a theoretical basis for further study of the function of vacuoles and vacuolar proteins. [ABSTRACT FROM AUTHOR]

Various abiotic stressors, particularly drought stress, affect plant growth and yield. Zinc finger proteins play an important role in plant abiotic stress tolerance. Here, we isolated the apple MdZAT10 gene, a C2H2-type zinc finger protein, which is a homolog of Arabidopsis STZ/ZAT10. MdZAT10 was localized to the nucleus and highly expressed in leaves and fruit. Promoter analysis showed that MdZAT10 contained several response elements and the transcription level of MdZAT10 was induced by abiotic stress and hormone treatments. MdZAT10 was responsive to drought treatment both at the transcriptional and post-translational levels. MdZAT10 -overexpressing apple calli decreased the expression level of MdAPX2 and increased sensitivity to PEG 6000 treatment. Moreover, ectopically expressed MdZAT10 in Arabidopsis reduced the tolerance to drought stress, and exhibited higher water loss, higher malondialdehyde (MDA) content and higher reactive oxygen species (ROS) accumulation under drought stress. In addition, MdZAT10 reduced the sensitivity to abscisic acid in apple. Ectopically expressed MdZAT10 in Arabidopsis promoted seed germination and seedling growth. These results indicate that MdZAT10 plays a negative regulator in the drought resistance, which can provide theoretical basis for further molecular mechanism research. • MdZAT10 reduced the drought tolerance by regulating the expression of MdAPX2 and increased ROS accumulation in apple. [ABSTRACT FROM AUTHOR]

Highlights • MdDEP1 is a Yang's cycle gene and involved in ethylene biosynthesis. • The apple Yang cycle's gene MdDEP1 is involved in the stress responses to salinity and drought. • MdDEP1 triggers early flowering in Arabidopsis. Abstract DEHYDRATASE-ENOLASE-PHOSPHATASE−COMPLEX1 (DEP1), a trifunctional enzyme with dehydratase, enolase, and phosphatase activities, initially characterizes with its enzymatic function in the recycling of the ethylene precursor methionine (Met). Here, we identified an apple MdDEP1 gene, which encodes a protein highly homologous to Arabidopsis Yang's cycle gene AtDEP1. GUS staining of 35S::pMdDEP1-GUS transgenic Arabidopsis showed that MdDEP1 is widely expressed in Arabidopsis tissues especially in vascular systems. Additionally, ectopic expression of MdDEP1 alters salt and drought tolerance, and triggers early flowering in Arabidopsis on the medium. The increase of salt tolerance in MdDEP1 -expressing plants was realized through upregulation of the typical salt overly sensitive (SOS) pathway-related genes, including AtSOS1 , AtSOS2 , and AtSOS3 ; and that MdDEP1 controlled flowering time mainly by influencing the expression of flowering-associated genes. Collectively, these findings provide new functions of the DEP1 protein and its underlying mechanisms, in which MdDEP1 confers salt and drought-tolerance, and early flowering phenotypes. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is an important hormone involved in plant growth and adaptation to environmental stress. Abscisic acid-responsive elements (ABRE)-binding transcription factors (ABFs) are important transcription factors involved in the ABA signaling pathway that participate in various biological processes in plants. The functions of ABF members are largely unknown in Malus domestica. In this study, sixteen Malus domestica ABF (MdABF) family members were identified in the apple (Malus × domestica) genome. The phylogenetic tree, chromosomal locations, promoter region cis-acting elements, gene structures, and conserved motifs of these MdABF members were analyzed. Subsequently, gene expression level analysis revealed that MdABFs had differential gene expression patterns in response to multiple abiotic factors, and most MdABF genes were highly expressed in leaves. According to the gene expression pattern analysis, we found that MdABF1 may play an important role in various stress responses. Subsequently, the MdABF1 gene was cloned and transgenic Arabidopsis thaliana material was obtained for further research. Subcellular localization revealed that the transcription factor MdABF1 was localized to the nucleus. Moreover, MdABF1- overexpression in Arabidopsis reduced its resistance to drought, however, there was no observed difference under salt exposure treatment. Meanwhile, expression of MdABF1 promoted early flowering and leaf senescence under drought conditions. These results indicate that MdABF1 may be a pivotal regulator of plant growth under drought conditions. Our findings lay the foundation for further exploration of the functions of MdABF in plant growth and development. • Sixteen ABF family members from apple were analyzed for bioinformatics and expression patterns. • We identified MdABF1 and demonstrated that it was locatized in nucleus. • Expression of MdABF1 in Arabidopsis reduced the drought resistance and promoted flowering and leaf senescence under drought. [ABSTRACT FROM AUTHOR]

• MdCIB1interacts with MdCRY2 and participates in the regulation of flowering. • Overexpression of MdCIB1 enhances tolerance to PEG 6000 treatment. • Ectopic expression of MdCIB1 enhances drought stress tolerance. • MdCIB1 transgenic Arabidopsis alleviates ROS accumulation. Drought restricts the plant growth and productivity worldwide. The basic helix-loop-helix (bHLH) transcription factors play key roles in response to various abiotic stresses. In this study, we identified and characterized a bHLH transcription factor, cryptochrome-interacting bHLH 1 (MdCIB1), from apple (Malus domestica). MdCIB1 had a typical bHLH domain and was localized in the nucleus. Moreover, MdCIB1 also interacted with MdCRY2, and participated in blue light response and flowering time regulation. RT-qPCR analysis showed that PEG 6000 notably induced the transcript level of MdCIB1. MdCIB1 -overexpressing apple calli displayed improved tolerance to PEG 6000 treatment. Ectopic expression of MdCIB1 in Arabidopsis improved drought stress resistance, as reflected by better physiological indexes under drought stress, such as longer roots, higher chlorophyll levels and survival rate, and lower water loss rate of leaves than Col control. Further investigation demonstrated that MdCIB1 played a positive regulator in ABA sensitivity and ABA-mediated stomatal closure. In addition, MdCIB1 transgenic Arabidopsis exhibited higher proline content, lower malondialdehyde (MDA) content, and H 2 O 2 and O 2 − accumulation, as well as increased the activities of antioxidant enzymes under drought stress, which were able to protect plants from osmotic and oxidative damages. Besides, the transcripts of ROS scavenging-related genes and stress-responsive genes were also significantly upregulated in MdCIB1 transgenic lines. In summary, our study demonstrated that MdCIB1 plays a positive role in drought resistance. [ABSTRACT FROM AUTHOR]

• MdBT2 is a negative regulator in response to drought stress. • MdNAC143 is a positive regulator in response to drought stress. • MdBT2 interacts with MdNAC143 and promotes its degradation via the 26S proteasome pathway. • MdBT2 confers sensitivity to drought at least dependent on MdNAC143. Drought stress is a severe source of abiotic stress that can affect apple yield and quality, yet the underlying molecular mechanism of the drought stress response and the role of MdBT2 in the process remain unclear. Here, we find that MdBT2 negatively regulates the drought stress response. Both in vivo and in vitro assays indicated that MdBT2 interacted physically with and ubiquitinated MdNAC143, a member of the NAC TF family that is a positive regulator under drought stress. In addition, MdBT2 promotes the degradation of MdNAC143 via the 26S proteasome system. A series of transgenic assays in apple calli and Arabidopsis verify that MdBT2 confers susceptibility to drought stress at least in part by the regulation of MdNAC143. Overall, our findings provide new insight into the mechanism of MdBT2, which functions antagonistically to MdNAC143 in regulating drought stress by regulating the potential downstream target protein MdNAC143 for proteasomal degradation in apple. [ABSTRACT FROM AUTHOR]

• Ethylene signaling pathway is involved in the plant defense response to pathogens. • ERFs are located downstream of the ethylene signaling pathway, and involved in the ethylene signaling pathway resistance to pathogens. • The apple ethylene response factor MdERF11 plays a significant role in activating phytoimmunity response to Botryosphaeria dothidea. Ethylene response factor (ERF) is a plant-specific transcription factor involved in many biological processes including root formation, hypocotyl elongation, fruit ripening, organ senescence and stress responses, as well as fruit quality formation. However, its underlying mechanism in plant pathogen defense against Botryosphaeria dothidea (B. dothidea) remains poorly understood. Here, we isolate MdERF11, an apple nucleus-localized ERF transcription factor, from apple cultivar ' Royal Gala '. qRT-PCR assays show that the expression of MdERF11 is significantly induced in apple fruits after B. dothidea infection. Overexpression of MdERF11 gene in apple calli significantly increases the resistance to B.dothidea infection, while silencing MdERF11 in apple calli results in reduced resistance. Ectopic expression of MdERF11 in Arabidopsis also exhibits enhanced resistance to B. dothidea infection compared to that of wild type. Infections in apple calli and Arabidopsis leaves by B. dothidea respectively cause an increase in endogenous levels of salicylic acid (SA) followed by induction of SA synthesis-related and signaling-related gene expression. Taken together, these findings illustrate a potential mechanism by which MdERF11 elevates plant pathogen defense against B. dothidea by regulating SA synthesis pathway. [ABSTRACT FROM AUTHOR]