Auxin responsive factor MdARF17 promotes ethylene synthesis in apple fruits by activating MdERF003 expression.

KEY MESSAGE: Auxin (AUX) promotion of apple fruit ripening is ethylene-dependent, and AUX-MdARF17-MdERF003 plays a role in AUX-promoted ethylene synthesis in apple. Phytohormones play important roles in plant growth and fleshy fruit ripening, and the phytohormone auxin (AUX) can either promote or inhibit the ripening of fleshy fruits. Although AUX can influence ethylene (ETH) synthesis in apple (Malus domestica) fruits by affecting ETH system II, this mechanism remains to be explored. Here, we identified an ETH response factor (ERF) family transcription factor, MdERF003, whose expression could be activated by naphthalene acetic acid. The transient silencing of MdERF003 inhibited ETH synthesis in fruits, and MdERF003 could bind to the MdACS1 promoter. To explore the upstream target genes of MdERF003, we screened the MdARF family members by yeast one-hybrid assays of the MdERF003 promoter, and found that the transcription factor MdARF17, which showed AUX-promoted expression, could bind to the MdERF003 promoter and promote its expression. Finally, we silenced MdERF003 in apple fruits overexpressing MdARF17 and found that MdERF003 plays a role in MdARF17-promoted ETH synthesis in apple. Thus, AUX-MdARF17-MdERF003 promotes ETH synthesis in apple fruits.

Sorbitol metabolism plays a key role in the differential accumulation of sugar in two plum cultivars.

Sugar is vital for plant growth and determines fruit quality via its content and composition. This study explores the differential sugar accumulation in two plum varieties, 'Fengtangli (FTL)' and 'Siyueli (SYL)'. The result showed that 'FTL' fruit displayed higher soluble solids and sugar content at various development stages. Metabolomic analysis indicated increased sorbitol in 'FTL', linked to elevated sorbitol-6-phosphate-dehydrogenase (S6PDH) activity. Transcriptome analysis identified a key gene for sorbitol synthesis, PsS6PDH4, which was significantly higher expressed in 'FTL' than in 'SYL'. The function of the PsS6PDH4 gene was verified in strawberry, apple, and plum fruits using transient overexpression and virus-induced gene silencing techniques. The results showed that overexpression of the PsS6PDH4 gene in strawberry, apple, and plum fruits promoted the accumulation of soluble solids content and sorbitol, while inhibition of the gene reduced soluble solids content and sorbitol content. Meanwhile, analysis of the relationship between PsS6PDH4 gene expression, sorbitol, and soluble solids content in four different plum varieties revealed a significant correlation between PsS6PDH4 gene expression and soluble solids content as well as sorbitol content. This research discovered PsS6PDH4 as a crucial regulator of sugar metabolism in plum, with potential applications in improving fruit sweetness and nutritional value in various fruit species. Understanding these molecular pathways can lead to innovative approaches for enhancing fruit quality, benefiting sustainable agriculture and consumer preferences in the global fruit industry.

Spatiotemporal patterns of leaf nutrients of wild apples in a wild fruit forest plot in the Ili Valley, China.

Malus sieversii, commonly known as wild apples, represents a Tertiary relict plant species and serves as the progenitor of globally cultivated apple varieties. Unfortunately, wild apple populations are facing significant degradation in localized areas due to a myriad of factors. To gain a comprehensive understanding of the nutrient status and spatiotemporal variations of M. sieversii, green leaves were collected in May and July, and the fallen leaves were collected in October. The concentrations of leaf nitrogen (N), phosphorus (P), and potassium (K) were measured, and the stoichiometric ratios as well as nutrient resorption efficiencies were calculated. The study also explored the relative contributions of soil, topographic, and biotic factors to the variation in nutrient traits. The results indicate that as the growing period progressed, the concentrations of N and P in the leaves significantly decreased (P < 0.05), and the concentration of K in October was significantly lower than in May and July. Throughout plant growth, leaf N-P and N-K exhibited hyperallometric relationships, while P-K showed an isometric relationship. Resorption efficiency followed the order of N < P < K (P < 0.05), with all three ratios being less than 1; this indicates that the order of nutrient limitation is K > P > N. The resorption efficiencies were mainly regulated by nutrient concentrations in fallen leaves. A robust spatial dependence was observed in leaf nutrient concentrations during all periods (70.1-97.9% for structural variation), highlighting that structural variation, rather than random factors, dominated the spatial variation. Nutrient resorption efficiencies (NRE, PRE, and KRE) displayed moderate structural variation (30.2-66.8%). The spatial patterns of nutrient traits varied across growth periods, indicating they are influenced by multifactorial elements (in which, soil property showed the highest influence). In conclusion, wild apples manifested differentiated spatiotemporal variability and influencing factors across various leaf nutrient traits. These results provide crucial insights into the spatiotemporal patterns and influencing factors of leaf nutrient traits of M. sieversii at the permanent plot scale for the first time. This work is of great significance for the ecosystem restoration and sustainable management of degrading wild fruit forests.

The transcription factor MdERF023 negatively regulates salt tolerance by modulating ABA signaling and Na+/H+ transport in apple.

KEY MESSAGE: MdERF023 is a transcription factor that can reduce salt tolerance by inhibiting ABA signaling and Na+/H+ homeostasis. Salt stress is one of the principal environmental stresses limiting the growth and productivity of apple (Malus × domestica). The APETALA2/ethylene response factor (AP2/ERF) family plays key roles in plant growth and various stress responses; however, the regulatory mechanism involved has not been fully elucidated. In the present study, we identified an AP2/ERF transcription factor (TF), MdERF023, which plays a negative role in apple salt tolerance. Stable overexpression of MdERF023 in apple plants and calli significantly decreased salt tolerance. Biochemical and molecular analyses revealed that MdERF023 directly binds to the promoter of MdMYB44-like, a positive modulator of ABA signaling-mediated salt tolerance, and suppresses its transcription. In addition, MdERF023 downregulated the transcription of MdSOS2 and MdAKT1, thereby reducing the Na+ expulsion, K+ absorption, and salt tolerance of apple plants. Taken together, these results suggest that MdERF023 reduces apple salt tolerance by inhibiting ABA signaling and ion transport, and that it could be used as a potential target for breeding new varieties of salt-tolerant apple plants via genetic engineering.

Identification of Apple Flower Development-Related Gene Families and Analysis of Transcriptional Regulation.

Apple (Malus domestica Borkh.) stands out as a globally significant fruit tree with considerable economic importance. Nonetheless, the orchard production of 'Fuji' apples faces significant challenges, including delayed flowering in young trees and inconsistent annual yields in mature trees, ultimately resulting in suboptimal fruit yield due to insufficient flower bud formation. Flower development represents a pivotal process influencing plant adaptation to environmental conditions and is a crucial determinant of successful plant reproduction. The three gene or transcription factor (TF) families, C2H2, DELLA, and FKF1, have emerged as key regulators in plant flowering regulation; however, understanding their roles during apple flowering remains limited. Consequently, this study identified 24 MdC2H2, 6 MdDELLA, and 6 MdFKF1 genes in the apple genome with high confidence. Through phylogenetic analyses, the genes within each family were categorized into three distinct subgroups, with all facets of protein physicochemical properties and conserved motifs contingent upon subgroup classification. Repetitive events between these three gene families within the apple genome were elucidated via collinearity analysis. qRT-PCR analysis was conducted and revealed significant expression differences among MdC2H2-18, MdDELLA1, and MdFKF1-4 during apple bud development. Furthermore, yeast two-hybrid analysis unveiled an interaction between MdC2H2-18 and MdDELLA1. The genome-wide identification of the C2H2, DELLA, and FKF1 gene families in apples has shed light on the molecular mechanisms underlying apple flower bud development.

Transcription factor MdbZIP44 targets the promoter of MdPPO2 to regulate browning in Malus domestica Borkh.

Apple (Malus domestica Borkh.) is among the most widely planted and economically valuable horticultural crops globally. Over time, the apple fruit's cut surface undergoes browning, and the degree of browning varies among different apple varieties. Browning not only affects the appearance of fruits but also adversely affects their taste and flavor. In the present study, we observed browning in different apple varieties over time and analyzed the expression of genes in the polyphenol oxidase gene family. The results indicated a strong correlation between the browning degree of the fruit and the relative expression of the polyphenol oxidase gene MdPPO2. With the MdPPO2 promoter as bait, the basic leucine zipper (bZIP) transcription factor MdbZIP44 was identified using the yeast single-hybrid screening method. Further investigation revealed that the overexpression of MdbZIP44 in 'Orin' callus could enhance the expression of MdPPO2 and promote browning of the callus. However, knocking out MdbZIP44 resulted in a callus with no apparent browning phenotype. In addition, our results confirmed the interaction between MdbZIP44 and MdbZIP11. In conclusion, the results indicated that MdbZIP44 can induce apple fruit browning by activating the MdPPO2 promoter. The results provide a theoretical basis for further clarifying the browning mechanism of apple fruit.

MbWRKY53, a M. baccata WRKY Transcription Factor, Contributes to Cold and Drought Stress Tolerance in Transgenic Arabidopsis thaliana.

Apple is an important horticultural crop, but various adverse environmental factors can threaten the quality and yield of its fruits. The ability of apples to resist stress mainly depends on the rootstock. Malus baccata (L.) Borkh. is a commonly used rootstock in Northeast China. In this study, it was used as the experimental material, and the target gene MbWRKY53 was screened through transcriptome analysis and Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) after cold and drought treatment. Bioinformatics analysis revealed that this transcription factor (TF) belonged to the WRKY TF family, and its encoded protein was localized in the nucleus. RT-qPCR showed that the gene was more easily expressed in roots and young leaves and is more responsive to cold and drought stimuli. Functional validation in Arabidopsis thaliana confirmed that MbWRKY53 can enhance plant tolerance to cold and drought stress. Furthermore, by analyzing the expression levels of genes related to cold and drought stress in transgenic Arabidopsis lines, it was inferred that this gene can regulate the expression of stress-related genes through multiple pathways such as the CBF pathway, SOS pathway, Pro synthesis pathway, and ABA-dependent pathways, enhancing the adaptability of transgenic Arabidopsis to cold and drought environments.

Effect of Storage Conditions on the Volatilome, Biochemical Composition and Quality of Golden Delicious and Red Delicious Apple (Malus domestica) Varieties.

The effects of normal (NA) and controlled atmosphere (CA) storage and postharvest treatment with 1-methylcyclopropene (1-MCP) before CA storage for 5 months on the volatilome, biochemical composition and quality of 'Golden Delicious' (GD) and 'Red Delicious' (RD) apples were studied. Apples stored under NA and CA maintained and 1-MCP treatment increased firmness in both cultivars. NA storage resulted in a decrease of glucose, sucrose and fructose levels in both cultivars. When compared to CA storage, 1-MCP treatment caused a more significant decrease in sucrose levels and an increase in glucose levels. Additionally, 1-MCP-treated apples exhibited a significant decrease in malic acid content for both cultivars. All storage conditions led to significant changes in the abundance and composition of the volatilome in both cultivars. GD and RD apples responded differently to 1-MCP treatment compared to CA storage; higher abundance of hexanoate esters and (E,E)-α-farnesene was observed in RD apples treated with 1-MCP. While 1-MCP was effective in reducing (E,E)-α-farnesene abundance in GD apples, its impact on RD apples was more limited. However, for both cultivars, all storage conditions resulted in lower levels of 2-methylbutyl acetate, butyl acetate and hexyl acetate. The effectiveness of 1-MCP is cultivar dependent, with GD showing better results than RD.

Magnesium alleviates growth inhibition under low potassium by enhancing photosynthesis and carbon-nitrogen metabolism in apple plants.

Potassium (K) and magnesium (Mg) play analogous roles in regulating plant photosynthesis and carbon and nitrogen (C-N) metabolism. Based on this consensus, we hypothesize that appropriate Mg supplementation may alleviate growth inhibition under low K stress. We monitored morphological, physiological, and molecular changes in G935 apple plants under different K (0.1 and 6 mmol L-1) and Mg supply (3 and 6 mmol L-1) conditions. Low K stress caused changes in root and leaf structure, inhibited photosynthesis, and limited the root growth of the apple rootstock. Further study on Mg supplementation showed that it could promote the uptake of K+ and NO3- by upregulating the expression of K+ transporter proteins such as Arabidopsis K+ transporter 1 (MdAKT1), high-affinity K+ transporter 1 (MdHKT1), and potassium transporter 5 (MdPT5) and nitrate transporters such as nitrate transporter 1.1/1.2/2.1/2.4 (MdNRT 1.1/1.2/2.1/2.4). Mg promoted the translocation of 15N from roots to leaves and enhanced photosynthetic N utilization efficiency (PNUE) by increasing the proportion of photosynthetic N and alleviating photosynthetic restrictions. Furthermore, Mg supplementation improved the synthesis of photosynthates by enhancing the activities of sugar-metabolizing enzymes (Rubisco, SS, SPS, S6PDH). Mg also facilitated the transport of sucrose and sorbitol from leaves to roots by upregulating the expression of sucrose transporter 1.1/1.2/4.1/4.2 (MdSUT 1.1/1.2/4.1/4.2) and sorbitol transporter 1.1/1.2 (MdSOT 1.1/1.2). Overall, Mg effectively alleviated growth inhibition in apple rootstock plants under low K stress by facilitating the uptake of N and K uptake, optimizing nitrogen partitioning, enhancing nitrogen use efficiency (NUE) and PNUE, and promoting the photosynthate synthesis and translocation.

Reactive nitrogen species act as the enhancers of glutathione pool in embryonic axes of apple seeds subjected to accelerated ageing.

MAIN CONCLUSION: Reactive nitrogen species mitigate the deteriorative effect of accelerated seed ageing by affecting the glutathione concentration and activities of GR and GPX-like. The treatment of apple (Malus domestica Borkh.) embryos isolated from accelerated aged seeds with nitric oxide-derived compounds increases their vigour and is linked to the alleviation of the negative effect of excessive oxidation processes. Reduced form of glutathione (GSH) is involved in the maintenance of redox potential. Glutathione peroxidase-like (GPX-like) uses GSH and converts it to oxidised form (GSSG), while glutathione reductase (GR) reduces GSSG into GSH. The aim of this work was to investigate the impact of the short-time NOx treatment of embryos isolated from apple seeds subjected to accelerated ageing on glutathione-related parameters. Apple seeds were subjected to accelerated ageing for 7, 14 or 21 days. Isolated embryos were shortly treated with NOx and cultured for 48 h. During ageing, in the axes of apple embryos, GSH and GSSG levels as well as half-cell reduction potential remained stable, while GR and GPX-like activities decreased. However, the positive effect of NOx in the vigour preservation of embryos isolated from prolonged aged seeds is linked to the increased total glutathione pool, and above all, higher GSH content. Moreover, NOx increased the level of transcripts encoding GPX-like and stimulated enzymatic activity. The obtained results indicate that high seed vigour related to the mode of action of NO and its derivatives is closely linked to the maintenance of higher GSH levels.

A non-invasive method for phenotyping scab-tolerant apple plants using volatile organic compounds.

One of the most devastating diseases of apples is scab, caused by the fungus Venturia inaequalis. Most commercial apple varieties are susceptible to this disease; only a few are resistant. Breeding approaches are being used to develop better apple varieties that are resistant to scab. Volatile organic compounds (VOCs) contribute greatly to a plant's phenotype, and their emission profile largely depends on the genotype. In the non-destructive phenotyping of plants, VOCs can be used as biomarkers. In this study, we assessed non-destructively the scab tolerance potential of resistant (cv. 'Prima') and susceptible (cv. 'Oregon Spur') apple cultivars by comparing their major leaf VOC compositions and relative proportions. A comparison of the leaf VOC profiles of the two cultivars revealed 16 different VOCs, with cis-3-hexenyl acetate (3HA) emerging as a biomarker of cultivar differences. V. inaequalis growth was significantly inhibited in vitro by 3HA treatment. 3HA was significantly effective in reducing scab symptoms on V. inaequalis-inoculated leaves of 'Oregon Spur.' The resistant cultivar 'Prima' also exhibited higher lipoxygenase (LOX) activity and α-linolenic acid (ALA) levels, suggesting that V. inaequalis resistance is linked to LOX activity and 3HA biosynthesis. This study proposes 3HA as a potential biomarker for rapid non-destructive screening of scab-resistant apple germplasm of 'Prima' based on leaf VOCs.

Metabolomics comparison of four varieties apple with different browning characters in response to pretreatment during pulp processing.

Browning commonly appeared in apple processing, which varied in different apple varieties. Present work investigated the metabolomics of four varieties apple of Yataka, Gala, Sansa, and Fuji, which possessed different browning characteristics and related enzymes. Sansa as browning insensitive apple variety, exhibited the least chroma change with the lowest PPO activity and the highest SOD activity among the four apple varieties. Browning inhibition pretreatment increased the activity of SOD and PAL and decreased PPO and POD activity. In addition, metabolomic variances among the four apple varieties (FC), their browning pulp (BR) and browning inhibition pulp (CM) were compared. And the key metabolites were in-depth analyzed to match the relevant KEGG pathways and speculated metabolic networks. There were 487, 644, and 494 significant differential metabolites detected in FC, BR and CM, which were consisted of lipids, benzenoids, phenylpropanoids, organheterocyclic compounds, organic acids, nucleosides, accounting for 23 %, 11 %, 15 %, 16 %, 11 % of the total metabolites. The differential metabolites were matched with 39, 49, and 36 KEGG pathways in FC, BR, and CM, respectively, in which other secondary metabolites biosynthesis metabolism was the most significant in FC, lipid metabolism was the most significant in BR and CM, and energy metabolism was markedly annotated in CM. Notably, Sansa displayed the highest number of differential metabolites in both its BR (484) and CM (342). The BR of Sansa was characterized by flavonoid biosynthesis, while the other three apple varieties were associated with α-linolenic acid metabolism. Furthermore, in browning sensitive apple varieties, the flavonoid and phenylpropanoid biosynthesis pathway was significantly activated by browning inhibition pretreatment. Phenolic compounds, lipids, sugars, organic acids, nucleotides, and adenosine were regulated differently in the four apple varieties, potentially serving as key regulatory sites. Overall, this work provides novel insight for browning prevention in different apple varieties.

The HD-Zip I transcription factor MdHB-7 negatively regulates resistance to Glomerella leaf spot in apple.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola (Cf), has been one of the main fungal diseases afflicting apple-producing areas across the world for many years, and it has led to substantial reductions in apple output and quality. HD-Zip transcription factors have been identified in several species, and they are involved in the immune response of plants to various types of biotic stress. In this study, inoculation of MdHB-7 overexpressing (MdHB-7-OE) and interference (MdHB-7-RNAi) transgenic plants with Cf revealed that MdHB-7, which encodes an HD-Zip transcription factor, adversely affects GLS resistance. The SA content and the expression of SA pathway-related genes were lower in MdHB-7-OE plants than in 'GL-3' plants; the content of ABA and the expression of ABA biosynthesis genes were higher in MdHB-7-OE plants than in 'GL-3' plants. Further analysis indicated that the content of phenolics and chitinase and ß-1, 3 glucanase activities were lower and H2O2 accumulation was higher in MdHB-7-OE plants than in 'GL-3' plants. The opposite patterns were observed in MdHB-7-RNAi apple plants. Overall, our results indicate that MdHB-7 plays a negative role in regulating defense against GLS in apple, which is likely achieved by altering the content of SA, ABA, polyphenols, the activities of defense-related enzymes, and the content of H2O2.

The MdNAC72-MdABI5 module acts as an interface integrating jasmonic acid and gibberellin signals and undergoes ubiquitination-dependent degradation regulated by MdSINA2 in apple.

Jasmonic acid (JA) and gibberellin (GA) coordinately regulate plant developmental programs and environmental cue responses. However, the fine regulatory network of the cross-interaction between JA and GA remains largely elusive. In this study, we demonstrate that MdNAC72 together with MdABI5 positively regulates anthocyanin biosynthesis through an exquisite MdNAC72-MdABI5-MdbHLH3 transcriptional cascade in apple. MdNAC72 interacts with MdABI5 to promote the transcriptional activation of MdABI5 on its target gene MdbHLH3 and directly activates the transcription of MdABI5. The MdNAC72-MdABI5 module regulates the integration of JA and GA signals in anthocyanin biosynthesis by combining with JA repressor MdJAZ2 and GA repressor MdRGL2a. MdJAZ2 disrupts the MdNAC72-MdABI5 interaction and attenuates the transcriptional activation of MdABI5 by MdNAC72. MdRGL2a sequesters MdJAZ2 from the MdJAZ2-MdNAC72 protein complex, leading to the release of MdNAC72. The E3 ubiquitin ligase MdSINA2 is responsive to JA and GA signals and promotes ubiquitination-dependent degradation of MdNAC72. The MdNAC72-MdABI5 interface fine-regulates the integration of JA and GA signals at the transcriptional and posttranslational levels by combining MdJAZ2, MdRGL2a, and MdSINA2. In summary, our findings elucidate the fine regulatory network connecting JA and GA signals with MdNAC72-MdABI5 as the core in apple.

Apple SINA11-JAZ2 module is involved in jasmonate signaling response.

The E3 ubiquitin ligase MdSINA11 targets the jasmonate ZIM domain protein MdJAZ2 for ubiquitination and degradation through the 26S proteasome pathway, thereby initiating jasmonate signaling and jasmonic acid-triggered anthocyanin biosynthesis in apple.

Differential effects of pectin-based dietary fibre type and gut microbiota composition on in vitro fermentation outcomes.

Interactions between human gut microbiota and dietary fibres (DF) are influenced by the complexity and diversity of both individual microbiota and sources of DF. Based on 480 in vitro fermentations, a full factorial experiment was performed with six faecal inocula representing two enterotypes and three DF sources with nanometer, micrometer, and millimeter length-scales (apple pectin, apple cell walls and apple particles) at two concentrations. Increasing DF size reduced substrate disappearance and fermentation rates but not biomass growth. Concentrated DF enhanced butyrate production and lactate cross-feeding. Enterotype differentiated final microbial compositions but not biomass or fermentation metabolite profiles. Individual donor microbiota differences did not influence DF type or concentration effects but were manifested in the promotion of different functional microbes within each population with the capacity to degrade the DF substrates. Overall, consistent effects (independent of donor microbiota variation) of DF type and concentration on kinetics of substrate degradation, microbial biomass production, gas kinetics and metabolite profiles were found, which can form the basis for informed design of DF for desired rates/sites and consequences of gut fermentation. These results add further evidence to the concept that, despite variations between individuals, the human gut microbiota represents a community with conserved emergent properties.

Movement of ACC oxidase 3 mRNA from seeds to flesh promotes fruit ripening in apple.

Xenia, the phenomenon in which the pollen genotype directly affects the phenotypic characteristics of maternal tissues (i.e., fruit ripening), has applications in crop production and breeding. However, the underlying molecular mechanism has yet to be elucidated. Here, we investigated whether mobile mRNAs from the pollen affect the ripening and quality-related characteristics of the fruit using cross-pollination between distinct Malus domestica (apple) cultivars. We demonstrated that hundreds of mobile mRNAs originating from the seeds are delivered to the fruit. We found that the movement of one of these mRNAs, ACC oxidase 3 (MdACO3), is coordinated with fruit ripening. Salicylic acid treatment, which can cause plasmodesmal closure, blocks MdACO3 movement, indicating that MdACO3 transcripts may move through the plasmodesmata. To assess the role of mobile MdACO3 transcripts in apple fruit, we created MdACO3-GFP-expressing apple seeds using MdACO3-GFP-overexpressing pollen for pollination and showed that MdACO3 transcripts in the transgenic seeds move to the flesh, where they promote fruit ripening. Furthermore, we demonstrated that MdACO3 can be transported from the seeds to fruit in the fleshy-fruited species tomato and strawberry. These results underscore the potential of mobile mRNAs from seeds to influence fruit characteristics, providing an explanation for the xenia phenomenon. Notably, our findings highlight the feasibility of leveraging diverse pollen genomic resources, without resorting to genome editing, to improve fruit quality.

A Functional InDel in the WRKY10 Promoter Controls the Degree of Flesh Red Pigmentation in Apple.

MYB transcription factors have been linked to anthocyanin synthesis and various color phenotypes in plants. In apple, MYB10 confers a red-flesh phenotype due to a minisatellite insertion in its R6 promoter, but R6:MYB10 genotypes exhibit various degrees of red pigmentation in the flesh, suggesting the involvement of other genetic factors. Here, it is shown that MdWRKY10, a transcription factor identified via DNA pull-down trapping, binds to the promoter of MdMYB10 and activates its transcription. MdWRKY10 specifically interacts with the WDR protein MdTTG1 to join the apple MYB-bHLH-WDR (MBW) complex, which significantly enhances its transcriptional activation activity. A 163-bp InDel detected in the promoter region of the alleles of MdWRKY10 in a hybrid population of identical heterozygous genotypes regarding R6 by structural variation analysis, contains a typical W-box element that MdWRKY10 binds to for transactivation. This leads to increased transcript levels of MdWRKY10 and MdMYB10 and enhanced anthocyanin synthesis in the flesh, largely accounting for the various degrees of flesh red pigmentation in the R6 background. These findings reveal a novel regulatory role of the WRKY-containing protein complex in the formation of red flesh apple phenotypes and provide broader insights into the molecular mechanism governing anthocyanin synthesis in plants.

Four glycosyltransferase genes are responsible for synthesis and accumulation of different flavonol glycosides in apple tissues.

Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species.

Fungal invasion-induced accumulation of salicylic acid promotes anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module in apple fruits.

In the field, necrosis area induced by pathogens is usually surrounded by a red circle in apple fruits. However, the underlying molecular mechanism of this phenomenon remains unclear. In this study, we demonstrated that accumulated salicylic acid (SA) induced by fungal infection promoted anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module in apple (Malus domestica). Inoculating apple fruits with Valsa mali or Botryosphaeria dothidea induced a red circle surrounding the necrosis area, which mimicked the phenotype observed in the field. The red circle accumulated a high level of anthocyanins, which was positively correlated with SA accumulation stimulated by fungal invasion. Further analysis showed that SA promoted anthocyanin biosynthesis in a dose-dependent manner in both apple calli and fruits. We next demonstrated that MdNPR1, a master regulator of SA signaling, positively regulated anthocyanin biosynthesis in both apple and Arabidopsis. Moreover, MdNPR1 functioned as a co-activator to interact with and enhance the transactivation activity of MdTGA2.2, which could directly bind to the promoters of anthocyanin biosynthetic and regulatory genes to promote their transcription. Suppressing expression of either MdNPR1 or MdTGA2.2 inhibited coloration of apple fruits, while overexpressing either of them significantly promoted fruit coloration. Finally, we revealed that silencing either MdNPR1 or MdTGA2.2 in apple fruits repressed SA-induced fruit coloration. Therefore, our data determined that fungal-induced SA promoted anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module, resulting in a red circle surrounding the necrosis area in apple fruits.