Baiquan Ma

Fruits of horticultural crops are indispensable sources of nutrients and bioactive compounds, and their flavor quality, a complex phenotypic trait orchestrated by soluble sugars, organic acids, volatile aromas, is a pivotal determinant of their market competitiveness and consumer preference. Enhancing flavor quality has therefore become a primary priority of horticultural crop breeding. WRKY transcription factors (TFs) constitute one of the largest TF families in plants, and are emerging as central regulators of specialized metabolism underlying fruit flavor formation. In this review, we first identify two core evolutionary features of WRKY TFs in horticultural crops: cross-species conserved sucrose-acid regulatory clades and species-specific flavor regulatory clades. We then systematically summarize the regulatory roles of WRKY TFs in the accumulation of sugars, organic acids, volatile aromas, and bitter/pungent substances, and elaborate on the molecular mechanism by which they integrate environmental and hormone signals to coordinate flavor metabolism. We further propose a multi-dimensional regulatory model of WRKY-mediated flavor formation, and discuss future research directions as well as potential applications in molecular breeding for flavor enhancement. This work provides a comprehensive framework for understanding the role of WRKY in flavor regulation and offers guidance for the cultivation of high-quality horticultural crops.

In most fleshy fruits, including apple, cherry, plum, and grape, citric acid content declines during the late stages of fruit development, a process that largely determines fruit flavor. In contrast, pear (Pyrus spp.) exhibits a less common developmental pattern characterized by continued citric acid accumulation during late fruit development. Although similar patterns have been reported in a limited number of other fruit species and germplasms, the molecular mechanisms underlying this developmental trajectory remain largely unknown. Here, comparative analyses of organic acid dynamics among cultivated pear varieties revealed that citric acid predominantly accumulates during late fruit development. Transcriptome analysis combined with M-fuzz soft clustering identified the vacuolar dicarboxylate transporter PutDT and the NAC transcription factor PuNAC2 as candidate genes closely associated with this developmental pattern. Electrophysiological analyses demonstrated that PutDT functions as a pH-dependent vacuolar transporter for both citrate and malate, with a higher affinity for citrate under acidic conditions. Functional analyses further demonstrated that PutDT promotes citric acid accumulation in pear fruit, callus, and a heterologous tomato system. Furthermore, PuNAC2 directly activated PutDT transcription by binding to the SNBE motif in its promoter, and its positive effect on citric acid accumulation depended on PutDT activation. Collectively, these findings reveal a PuNAC2–PutDT regulatory module controlling vacuolar citric acid accumulation during pear fruit development and provide molecular insights into the regulation of fruit acidity, as well as potential molecular targets for acidity improvement.

The roots of most land plants are in symbiosis with arbuscular mycorrhizal (AM) fungi. The fungus promotes nutrient uptake from the soil while receiving plant photosynthate as lipids and sugars. Nutrient exchange must be regulated by both partners, but the mechanisms underlying the regulation of lipid supplement from the plant to the AM fungus remain elusive. Here, we conducted a molecular study on the role of increased abscisic acid (ABA) levels during AM fungus infection in the roots of apple ( Malus spp.). AM fungus induced the expression of two ABA synthesis genes, MdNCED3.1 and 3.2 , in apple roots and increased the ABA content, which promoted the growth of the AM fungus. The effect of ABA on symbiosis was confirmed in transgenic apple roots either overexpressing or silencing MdNCED3.1 or MdNCED3.2 . Transcriptome analysis and transgenic manipulation revealed that the transcription factor MdABF2 played a key role in the ABA-mediated formation of symbiosis during AM infection and that MdABF2 could regulate the expression levels of genes related to fatty acid (FA) synthesis (e.g., MdKASIII ) and translocation (such as MdSTR2 ) in apple roots. Activation of these genes boosted the levels of available fatty acids in the roots and increased the AM fungal colonization and arbuscule development in the roots. These results revealed a molecular pathway in which positive regulation of FA synthesis and transport by the ABA signaling pathway increases supplementation to AM fungi and promotes AM symbiosis.

Browning is a pervasive problem in horticultural products, substantially diminishing the appearance, flavor and nutritional value of fruit, including important fruits like apple (Malus × domestica Borkh.). In this study, we compared the physiological characteristics of the browning-resistant line ‘Rb-18’ with the susceptible variety ‘Fuji’ and found that the polyphenol oxidase (PPO) enzyme activity and phenolic content of Rb-18 were significantly lower than those in Fuji. In addition, the PPO enzyme in Fuji showed a stronger affinity for its substrate, catechol, compared to Rb-18. Through transcriptome and RT-qPCR analyses, MdPPO7 expression was identified as contributing to flesh browning after cutting. Subsequent fruit injection and stable genetic transformation of the MdPPO7 gene into apple fruit and calli determined that syringic acid, procyanidin, phloridzin, chlorogenic acid, gallic acid, catechin, and caffeic act as its catalytic substrates in the process involved in browning. Furthermore, luciferase reporter, yeast one-hybrid, β-glucuronidase reporter assays and chip-qPCR analysis demonstrated that a WRKY transcription factor(MdWRKY3) binds to the promoter region of polyphenol oxidase gene (MdPPO7) and positively regulates its expression to promote apple flesh browning. This study provides insights into the molecular regulatory mechanisms of fruit browning in fresh-cut apples and provides a theoretical basis for the generation of high-quality apple germplasm resources.

Soluble sugars are not only an important contributor to fruit quality, but also serve as the osmotic regulators in response to abiotic stresses. Early drought stress promotes sugar accumulation, while specific sugar transporters govern the cellular distribution of the sugars. Here, we show that apple plantlets accumulate soluble sugars in leaf tissues under drought stress. Transcriptional profiling of stressed and control plantlets revealed differential expression of several plasma membrane—or vacuolar membrane-localized sugar transporter genes. Among these, four previously identified vacuolar sugar transporter (VST) genes (MdERDL6–1, MdERDL6–2, MdTST1, and MdTST2) showed higher expression under drought, suggesting their roles in response to drought stress. Promoter cis-elements analyses, yeast one-hybrid, and dual-luciferase tests confirmed that the drought-induced transcription factor MdDREB2A could promote the expression of MdERDL6–1/−2 and MdTST1/2 by binding to their promoter regions. Moreover, overexpressing of each of these four MdVSTs alone in transgenic apple or Arabidopsis plants accumulated more soluble sugars and abscisic acid (ABA), and enhanced drought resistance. Furthermore, apple plants overexpressing MdERDL6–1 also showed reduced water potential, facilitated stomatal closure, and reactive oxygen species scavenging under drought conditions compared to control plants. Overall, our results suggest a potential strategy to enhance drought resistance and sugar accumulation in fruits through manipulating the genes involved in vacuolar sugar transport.

Organic acids are major determinants of fruit flavor and a primary focus of fruit crop breeding. The accumulation of organic acids is determined by their synthesis, degradation, and transport, all of which are manipulated by sophisticated genetic mechanisms. Constant exploration of the genetic basis of organic acid accumulation, especially through linkage analysis, association analysis, and evolutionary analysis, have identified numerous loci in recent decades. In this review, the genetic loci and genes responsible for malate and citrate contents in fruits are discussed from the genetic perspective. Technologies such as gene transformation and genome editing as well as efficient breeding using marker-assisted selection (MAS) and genomic selection (GS) are expected to break the bottleneck of traditional fruit crop breeding and promote fruit quality improvement.

Malic acid is an important flavor determinant in apple (Malus × domestica Borkh.) fruit. One known variation controlling malic acid is the A/G single nucleotide polymorphism in an aluminum-activated malate transporter gene (MdMa1). Nevertheless, there are still differences in malic acid content in apple varieties with the same Ma1 genotype (Ma1/Ma1 homozygous), such as ‘Honeycrisp’ (high malic acid content) and ‘Qinguan’ (low malic acid content), indicating that other loci may influence malic acid and fruit acidity. Here, the F1 (Filial 1) hybrid generation of ‘Honeycrisp’ × ‘Qinguan’ was used to analyze quantitative trait loci for malic acid content. A major locus (Ma7) was identified on chromosome 13. Within this locus, a malate dehydrogenase gene, MDH1 (MdMa7), was the best candidate for further study. Subcellular localization suggested that MdMa7 encodes a cytosolic protein. Overexpression and RNA interference of MdMa7 in apple fruit increased and decreased malic acid content, respectively. An insertion/deletion (indel) in the MdMa7 promoter was found to affect MdMa7 expression and malic acid content in both hybrids and other cultivated varieties. The insertion and deletion genotypes were designated as MA7 and ma7, respectively. The transcription factor MdbHLH74 was found to stimulate MdMa7 expression in the MA7 genotype but not in the ma7 genotype. Transient transformation of fruit showed that MdbHLH74 affected MdMa7 expression and malic acid content in ‘Gala’ (MA7/MA7) but not in ‘Fuji’ (ma7/ma7). Our results indicated that genetic variation in the MdMa7 (MDH1) promoter alters the binding ability of the transcription factor MdbHLH74, which alters MdMa7 (MDH1) transcription and the malic acid content in apple fruit, especially in Ma1/Ma1 homozygous accessions.

Acidity is a key factor controlling fruit flavor and quality. In a previous study, combined transcriptome and methylation analyses identified a P_3A-type ATPase from apple (Malus domestica), MdMa11, which regulates vacuolar pH when expressed in Nicotiana benthamiana leaves. In this study, the role of MdMa11 in controlling fruit acidity was verified in apple calli, fruits, and plantlets. In addition, we isolated an AP2 domain-containing transcription factor, designated MdESE3, based on yeast one-hybrid (Y1H) screening using the MdMa11 promoter as bait. A subcellular localization assay indicated that MdESE3 localized to the nucleus. Analyses of transgenic apple calli, fruits, and plantlets, as well as tomatoes, demonstrated that MdESE3 enhances fruit acidity and organic acid accumulation. Meanwhile, chromatin immunoprecipitation quantitative PCR (ChIP-qPCR), luciferase (LUC) transactivation assays, and GUS reporter assays indicated that MdESE3 could bind to the ethylene-responsive element (ERE; 5ʹ-TTTAAAAT-3ʹ) upstream of the MdMa11 transcription start site, thereby activating its expression. Furthermore, MdtDT, MdDTC2, and MdMDH12 expression increased in apple fruits and plantlets overexpressing MdESE3 and decreased in apple fruits and plantlets where MdESE3 was silenced. The ERE was found in MdtDT and MdMDH12 promoters, but not in the MdDTC2 promoter. The Y1H, LUC transactivation assays, and GUS reporter assays indicated that MdESE3 could bind to the MdtDT and MdMDH12 promoters and activate their expression. Our findings provide valuable functional validation of MdESE3 and its role in the transcriptional regulation of MdMa11, MdtDT, and MdMDH12 and malic acid accumulation in apple.