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Fusarium spp., a necrotrophic soil‐borne pathogen, causes root rot disease on many crops. CERK1, as a typical pattern recognition receptor, has been widely studied. However, the function of CERK1 during plant–Fusarium interaction has not been well described. We determined that MdCERK1 is a susceptibility gene in the apple‐Fusarium solani (Fs) interaction, and jasmonic acid (JA) plays a crucial role in this process. MdCERK1 directly targets and phosphorylates the lipoxygenase MdLOX2.1, an enzyme initiating the JA biosynthesis, at positions Ser326 and Thr327. These phosphorylations inhibit its translocation from the cytosol to the chloroplasts, leading to a compromised JA biosynthesis. Fs upregulates MdCERK1 expression during infection. In turn, when the JA level is low, the apple MdWRKY71, a transcriptional repressor of MdCERK1, is markedly upregulated and phosphorylated at Thr99 and Thr102 residues by the MAP kinase MdMMK2. The phosphorylation of MdWRKY71 enhances its transcription inhibition on MdCERK1. Taken together, MdCERK1 plays a novel role in limiting JA biosynthesis. There seems to be an arms race between apple and Fs, in which Fs activates MdCERK1 expression to reduce the JA level, while apple senses the low JA level and activates the MdMMK2‐MdWRKY71 module to elevate JA level by inhibiting MdCERK1 expression. Summary Statement: Chitin Elicitor Receptor Kinase 1, CERK1 plays a well‐known role in plants to initiate the primary defence upon pathogens infection. In addition to this, this study discovered that apple CERK1 interacts with and phosphorylates the lipoxygenase MdLOX2.1 to block the JA biosynthesis. Moreover, the apple MMK2‐WRKY71 module negatively regulates MdCERK1 expression to maintain the JA level. [ABSTRACT FROM AUTHOR]

Summary: In plants under drought stress, sugar content in roots increases, which is important for drought resistance. However, the molecular mechanisms for controlling the sugar content in roots during response to drought remain elusive. Here, we found that the MdDOF3‐MdHT1.2 module‐mediated glucose influx into the root is essential for drought resistance in apple (Malus × domestica). Drought induced glucose uptake from the rhizosphere and up‐regulated the transcription of hexose transporter MdHT1.2. Compared with the wild‐type plants, overexpression of MdHT1.2 promoted glucose uptake from the rhizosphere, thereby facilitating sugar accumulation in root and enhancing drought resistance, whereas silenced plants showed the opposite phenotype. Furthermore, ATAC‐seq, RNA‐seq and biochemical analysis demonstrated that MdDOF3 directly bound to the promoter of MdHT1.2 and was strongly up‐regulated under drought. Overexpression of MdDOF3 in roots improved MdHT1.2‐mediated glucose transport capacity and enhanced plant resistance to drought, but MdDOF3‐RNAihr apple plants showed the opposite phenotype. Moreover, overexpression of MdDOF3 in roots did not attenuate drought sensitivity in MdHT1.2‐RNAi plants, which was correlated with a lower glucose uptake capacity and glucose content in root. Collectively, our findings deciphered the molecular mechanism through which glucose uptake from the rhizosphere is mediated by MdDOF3‐MdHT1.2, which acts to modulate sugar content in root and promote drought resistance. [ABSTRACT FROM AUTHOR]

SUMMARY: Soluble sugar content is a key component in controlling fruit flavor, and its accumulation in fruit is largely determined by sugar metabolism and transportation. When the diurnal temperature range is greater, the fleshy fruits accumulated more soluble sugars and become more sweeter. However, the molecular mechanism underlying this response remains largely unknown. In this study, we verified that low‐temperature treatment promoted soluble sugar accumulation in apple fruit and found that this was due to the upregulation of the Tonoplast Sugar Transporter genes MdTST1/2. A combined strategy using assay for transposase‐accessible chromatin (ATAC) sequencing and gene expression and cis‐acting elements analyses, we identified two C‐repeat Binding Factors, MdCBF1 and MdCBF2, that were induced by low temperature and that might be upstream transcription factors of MdTST1/2. Further studies established that MdCBF1/2 could bind to the promoters of MdTST1/2 and activate their expression. Overexpression of MdCBF1 or MdCBF2 in apple calli and fruit significantly upregulated MdTST1/2 expression and increased the concentrations of glucose, fructose, and sucrose. Suppression of MdTST1 and/or MdTST2 in an MdCBF1/2‐overexpression background abolished the positive effect of MdCBF1/2 on sugar accumulation. In addition, simultaneous silencing of MdCBF1/2 downregulated MdTST1/2 expression and apple fruits failed to accumulate more sugars under low‐temperature conditions, indicating that MdCBF1/2‐mediated sugar accumulation was dependent on MdTST1/2 expression. Hence, we concluded that the MdCBF1/2‐MdTST1/2 module is crucial for sugar accumulation in apples in response to low temperatures. Our findings provide mechanistic components coordinating the relationship between low temperature and sugar accumulation as well as new avenues to improve fruit quality. Significance Statement: This study showed that low temperature induced the expression levels of the transcription factors genes MdCBF1/2 in apple, which could bind to the promoters of the tonoplast sugar transporter genes MdTST1/2 and activate their expression, leading to Glc, Fru, and Suc accumulation at high concentrations in the vacuoles. [ABSTRACT FROM AUTHOR]

Summary: Biosynthesis of flavonoid aglycones and glycosides is well established. However, key genes involved in their catabolism are poorly understood, even though the products of hydrolysis and oxidation play important roles in plant resistance to biotic stress.Here, we report on catabolism of dihydrochalcones (DHCs), the most abundant flavonoids in domesticated apple and wild Malus. Two key genes, BGLU13.1 and PPO05, were identified by activity‐directed protein purification. BGLU13.1‐A hydrolyzed phlorizin, (the most abundant DHC in domesticated apple) to produce phloretin which was then oxidized by PPO05. The process differed in some wild Malus, where trilobatin (a positional isomer of phlorizin) was mainly oxidized by PPO05.The effects of DHC catabolism on apple resistance to biotic stresses was investigated using transgenic plants. Either directly or indirectly, phlorizin hydrolysis affected resistance to the phytophagous pest two‐spotted spider mite, while oxidation of trilobatin was involved in resistance to the biotrophic fungus Podosphaera leucotricha. DHC catabolism did not affect apple resistance to necrotrophic pathogens Valsa mali and Erwinia amylovara.These results suggest that different DHC catabolism pathways play different roles in apple resistance to biotic stresses. The role of DHC catabolism on apple resistance appeared closely related to the mode of invasion/damage used by pathogen/pest. [ABSTRACT FROM AUTHOR]

Glomerella leaf spot (GLS), a fungal disease caused by Colletotrichum fructicola, severely affects apple (Malus domestica) quality and yield. In this study, we found that the transcription factor MdWRKY71 was significantly induced by C. fructicola infection in the GLS‐susceptible apple cultivar Royal Gala. The overexpression of MdWRKY71 in apple leaves resulted in increased susceptibility to C. fructicola, whereas RNA interference of MdWRKY71 in leaves showed the opposite phenotypes. These findings suggest that MdWRKY71 functions as a susceptibility factor for the apple—C. fructicola interaction. Furthermore, MdWRKY71 directly bound to the promoter of the salicylic acid (SA) degradation gene Downy Mildew Resistant 6 (DMR6)‐Like Oxygenase 1 (DLO1) and promoted its expression, resulting in a reduced SA level. The sensitivity of 35S:MdWRKY71 leaves to C. fructicola can be effectively alleviated by knocking down MdDLO1 expression, confirming the critical role of MdWRKY71‐mediated SA degradation via regulating MdDLO1 expression in GLS susceptibility. In summary, we identified a GLS susceptibility factor, MdWRKY71, that targets the apple SA degradation pathway to promote fungal infection. [ABSTRACT FROM AUTHOR]

Summary: RAD5B belongs to the Rad5/16‐like group of the SNF2 family, which often functions in chromatin remodelling. However, whether RAD5B is involved in chromatin remodelling, histone modification, and drought stress tolerance is largely unclear. We identified a drought‐inducible chromatin remodeler, MdRAD5B, which positively regulates apple drought tolerance. Transposase‐accessible chromatin with high‐throughput sequencing (ATAC‐seq) analysis showed that MdRAD5B affects the expression of 466 drought‐responsive genes through its chromatin remodelling function in response to drought stress. In addition, MdRAD5B interacts with and degrades MdLHP1, a crucial regulator of histone H3 trimethylation at K27 (H3K27me3), through the ubiquitin‐independent 20S proteasome. Chromatin immunoprecipitation‐sequencing (ChIP‐seq) analysis revealed that MdRAD5B modulates the H3K27me3 deposition of 615 genes in response to drought stress. Genetic interaction analysis showed that MdRAD5B mediates the H3K27me3 deposition of drought‐responsive genes through MdLHP1, which causes their expression changes under drought stress. Our results unravelled a dual function of MdRAD5B in gene expression modulation in apple in response to drought, that is, via the regulation of chromatin remodelling and H3K27me3. [ABSTRACT FROM AUTHOR]

High temperatures negatively impact the yield and quality of fruit crops. Exogenous melatonin (MT) application has been shown to enhance heat tolerance, but the response of endogenous MT to heat stress, particularly in perennial fruit trees, remains unclear. The present study investigated the effects of high temperatures on transgenic apple plants overexpressing the MT biosynthesis gene N‐acetylserotonin methyltransferase 9 (MdASMT9). Endogenous MT protected transgenic plants from heat stress by increasing antioxidant enzyme activity and scavenging reactive oxygen species (ROS), and protecting the chloroplasts from damage. Application of MT and overexpression of MdASMT9 also reduced abscisic acid accumulation through promoting MdWRKY33‐mediated transcriptional inhibition of MdNCED1 and MdNCED3, thus inducing stomatal opening for better heat dissipation. Furthermore, MT‐enhanced autophagic activity through promoting MdWRKY33‐mediated transcriptional enhancement of MdATG18a under heat stress. These findings provide new insights into the regulation of endogenous MT and its role in improving basal thermotolerance in perennial fruit trees. Summary Statement: MdASMT9‐mediated melatonin (MT) biosynthesis in apple plants enhanced autophagic activity and protected plants from heat stress. Furthermore, MT‐promoted MdWRKY33‐mediated transcriptional inhibition of MdNCED1 and MdNCED3, thus promoting stomatal opening for better heat dissipation. [ABSTRACT FROM AUTHOR]

SUMMARY: High‐temperature stress results in protein misfolding/unfolding and subsequently promotes the accumulation of cytotoxic protein aggregates that can compromise cell survival. Heat shock proteins (HSPs) function as molecular chaperones that coordinate the refolding and degradation of aggregated proteins to mitigate the detrimental effects of high temperatures. However, the relationship between HSPs and protein aggregates in apples under high temperatures remains unclear. Here, we show that an apple (Malus domestica) chloroplast‐localized, heat‐sensitive elongation factor Tu (MdEF‐Tu), positively regulates apple thermotolerance when it is overexpressed. Transgenic apple plants exhibited higher photosynthetic capacity and better integrity of chloroplasts during heat stress. Under high temperatures, MdEF‐Tu formed insoluble aggregates accompanied by ubiquitination modifications. Furthermore, we identified a chaperone heat shock protein (MdHsp70), as an interacting protein of MdEF‐Tu. Moreover, we observed obviously elevated MdHsp70 levels in 35S: MdEF‐Tu apple plants that prevented the accumulation of ubiquitinated MdEF‐Tu aggregates, which positively contributes to the thermotolerance of the transgenic plants. Overall, our results provide new insights into the molecular chaperone function of MdHsp70, which mediates the homeostasis of thermosensitive proteins under high temperatures. Significance Statement: Our results provide new insights into the connections between molecular chaperones and denatured protein aggregates, which is required to mediate the homeostasis of thermosensitive proteins under high temperature. [ABSTRACT FROM AUTHOR]

SUMMARY: Nitrogen (N) is an essential nutrient for crop growth and development, significantly influencing both yield and quality. Melatonin (MT), a known enhancer of abiotic stress tolerance, has been extensively studied. However, its relationship with nutrient stress, particularly N deficiency, and the underlying regulatory mechanisms of MT on N absorption remain unclear. In this study, exogenous MT treatment was found to improve the tolerance of apple plants to N deficiency. Apple plants overexpressing the MT biosynthetic gene N‐acetylserotonin methyltransferase 9 (MdASMT9) were used to further investigate the effects of endogenous MT on low‐N stress. Overexpression of MdASMT9 improved the light harvesting and heat transfer capability of apple plants, thereby mitigating the detrimental effects of N deficiency on the photosynthetic system. Proteomic and physiological data analyses indicated that MdASMT9 overexpression enhanced the trichloroacetic acid cycle and positively modulated amino acid metabolism to counteract N‐deficiency stress. Additionally, both exogenous and endogenous MT promoted the transcription of MdHY5, which in turn bound to the MdNRT2.1 and MdNRT2.4 promoters and activated their expression. Notably, MT‐mediated promotion of MdNRT2.1 and MdNRT2.4 expression through regulating MdHY5, ultimately enhancing N absorption. Taken together, these findings shed light on the association between MdASMT9‐mediated MT biosynthesis and N absorption in apple plants under N‐deficiency conditions. Significance Statement: In this study, both exogenous and endogenous melatonin improved the tolerance of apple plants to N deficiency. Melatonin promoted the expression of MdNRT2.1 and MdNRT2.4 through regulating MdHY5, thereby improving nitrate absorption. [ABSTRACT FROM AUTHOR]

SUMMARY: Sorbitol is a critical photosynthate and storage substance in the Rosaceae family. Sorbitol transporters (SOTs) play a vital role in facilitating sorbitol allocation from source to sink organs and sugar accumulation in sink organs. While prior research has addressed gene duplications within the SOT gene family in Rosaceae, the precise origin and evolutionary dynamics of these duplications remain unclear, largely due to the complicated interplay of whole genome duplications and tandem duplications. Here, we investigated the synteny relationships among all identified Polyol/Monosaccharide Transporter (PLT) genes in 61 angiosperm genomes and SOT genes in representative genomes within the Rosaceae family. By integrating phylogenetic analyses, we elucidated the lineage‐specific expansion and syntenic conservation of PLTs and SOTs across diverse plant lineages. We found that Rosaceae SOTs, as PLT family members, originated from a pair of tandemly duplicated PLT genes within Class III‐A. Furthermore, our investigation highlights the role of lineage‐specific and synergistic duplications in Amygdaloideae in contributing to the expansion of SOTs in Rosaceae plants. Collectively, our findings provide insights into the genomic origins, duplication events, and subsequent divergence of SOT gene family members. Such insights lay a crucial foundation for comprehensive functional characterizations in future studies. Significance Statement: We found that Rosaceae SOTs, as PLT family members, originated from a pair of tandemly duplicated PLT genes within Class III‐A. Furthermore, our investigation highlights the role of lineage‐specific and synergistic duplications in Amygdaloideae in contributing to the expansion of SOTs in Rosaceae plants. [ABSTRACT FROM AUTHOR]

SUMMARY: Dihydrochalcones (DHCs) including phlorizin (phloretin 2′‐O‐glucoside) and its positional isomer trilobatin (phloretin 4′‐O‐glucoside) are the most abundant phenylpropanoids in apple (Malus spp.). Transcriptional regulation of DHC production is poorly understood despite their importance in insect‐ and pathogen‐plant interactions in human physiology research and in pharmaceuticals. In this study, segregation in hybrid populations and bulked segregant analysis showed that the synthesis of phlorizin and trilobatin in Malus leaves are both single‐gene‐controlled traits. Promoter sequences of PGT1 and PGT2, two glycosyltransferase genes involved in DHC glycoside synthesis, were shown to discriminate Malus with different DHC glycoside patterns. Differential PGT1 and PGT2 promoter activities determined DHC glycoside accumulation patterns between genotypes. Two transcription factors containing MYB‐like DNA‐binding domains were then shown to control DHC glycoside patterns in different tissues, with PRR2L mainly expressed in leaf, fruit, flower, stem, and seed while MYB8L mainly expressed in stem and root. Further hybridizations between specific genotypes demonstrated an absolute requirement for DHC glycoside production in Malus during seed development which explains why no Malus spp. with a null DHC chemotype have been reported. [ABSTRACT FROM AUTHOR]

Valsa mali causes Valsa canker, one of the most destructive diseases on apple trees, leading to severe losses for the apple industry in China. Considering the development of fungicide resistance and the harmful effects of chemical residues, it is urgent to identify alternatives to control this disease. Trans‐cinnamic acid (t‐CA), a compound with good antibacterial, antitumour and anti‐inflammatory properties, is widely used in food, medicine and other industries. However, the antifungal activity of t‐CA against V. mali and its regulatory role in apple defence against biotic stresses are unclear. Based on a metabolomic assay, we found that Malus yunnaensis (Valsa canker‐resistant) twigs infected with V. mali dramatically accumulated t‐CA. Exogenous application of t‐CA effectively inhibited V. mali growth on potato dextrose agar. The EC50 value of t‐CA inhibiting mycelial growth was 200 μg/mL. Malus prunifolia (Valsa canker‐susceptible) leaves and twigs pretreated with t‐CA had significantly enhanced V. mali resistance. The t‐CA application increased salicylic acid but reduced jasmonic acid levels in leaves and twigs. Moreover, the contents of phenolic acids and flavonoids increased in t‐CA‐treated samples. In addition, t‐CA increased the activities of phenylalanine ammonia‐lyase, β‐1,3‐glucanase and chitinase. These results indicate that t‐CA plays a significant role in inhibiting V. mali growth and priming apple defence. [ABSTRACT FROM AUTHOR]

SUMMARY: Salt stress adversely affects the yield and quality of crops and limits their geographical distribution. Studying the functions and regulatory mechanisms of key genes in the salt stress response is important for breeding crops with enhanced stress resistance. Autophagy plays an important role in modulating the tolerance of plants to various types of abiotic stressors. However, the mechanisms underlying salt‐induced autophagy are largely unknown. Cation/Ca2+ exchanger proteins enhance apple salt tolerance by inhibiting Na+ accumulation but the mechanism underlying the response to salt stress remains unclear. Here, we show that the autophagy‐related gene MdATG18a modulated apple salt tolerance. Under salt stress, the autophagic activity, proline content, and antioxidant enzyme activities were higher and Na+ accumulation was lower in MdATG18a‐overexpressing transgenic plants than in control plants. The use of an autophagy inhibitor during the salt treatment demonstrated that the regulatory function of MdATG18a depended on autophagy. The yeast‐one‐hybrid assay revealed that the homeodomain‐leucine zipper (HD‐Zip) transcription factor MdHB7‐like directly bound to the MdATG18a promoter. Transcriptional regulation and genetic analyses showed that MdHB7‐like enhanced salt‐induced autophagic activity by promoting MdATG18a expression. The analysis of Na+ efflux rate in transgenic yeast indicated that MdCCX1 expression significantly promoted Na+ efflux. Promoter binding, transcriptional regulation, and genetic analyses showed that MdHB7‐like promoted Na+ efflux and apple salt tolerance by directly promoting MdCCX1 expression, which was independent of the autophagy pathway. Overall, our findings provide insight into the mechanism underlying MdHB7‐like‐mediated salt tolerance in apple through the MdHB7‐like‐MdATG18a and MdHB7‐like‐MdCCX1 modules. These results will aid future studies on the mechanisms underlying stress‐induced autophagy and the regulation of stress tolerance in plants. [ABSTRACT FROM AUTHOR]

Summary: Understanding the molecular regulation of plant cold response is the basis for cold resistance germplasm improvement. Here, we revealed that the apple histone deacetylase MdHDA6 can perform histone deacetylation on cold‐negative regulator genes and repress their expression, leading to the positive regulation of cold tolerance in apples. Moreover, MdHDA6 directly interacts with the transcription factor MdTCP15. Phenotypic analysis of MdTCP15 transgenic apple lines and wild types reveals that MdTCP15 negatively regulates cold tolerance in apples. Furthermore, we found that MdHDA6 can facilitate histone deacetylation of MdTCP15 and repress the expression of MdTCP15, which positively contributes to cold tolerance in apples. Additionally, the transcription factor MdTCP15 can directly bind to the promoter of the cold‐negative regulator gene MdABI1 and activate its expression, and it can also directly bind to the promoter of the cold‐positive regulator gene MdCOR47 and repress its expression. However, the co‐expression of MdHDA6 and MdTCP15 can inhibit MdTCP15‐induced activation of MdABI1 and repression of MdCOR47, suggesting that MdHDA6 suppresses the transcriptional regulation of MdTCP15 on its downstream genes. Our results demonstrate that histone deacetylase MdHDA6 plays an antagonistic role in the regulation of MdTCP15‐induced transcriptional activation or repression to positively regulate cold tolerance in apples, revealing a new regulatory mechanism of plant cold response. [ABSTRACT FROM AUTHOR]

Glomerella leaf spot (GLS), caused by the fungal pathogen Colletotrichum fructicola, significantly threatens apple production. Some resistances to plant disease are mediated by the accumulation of nucleotide‐binding site and leucine‐rich repeat (NBS‐LRR) proteins that are encoded by a major class of plant disease resistance genes (R genes). However, the R genes that confer resistance to GLS in apple remain largely unclear. Malus hupehensis YT521‐B homology domain‐containing protein 2 (MhYTP2) was identified as an N6‐methyladenosine RNA methylation (m6A) modified RNA reader in our previous study. However, whether MhYTP2 binds to mRNAs without m6A RNA modifications remains unknown. In this study, we discovered that MhYTP2 exerts both m6A‐dependent and ‐independent functions by analysing previously obtained RNA immunoprecipitation sequencing results. The overexpression of MhYTP2 significantly reduced the resistance of apple to GLS and down‐regulated the transcript levels of some R genes whose transcripts do not contain m6A modifications. Further analysis indicated that MhYTP2 binds to and reduces the stability of MdRGA2L mRNA. MdRGA2L positively regulates resistance to GLS by activating salicylic acid signalling. Our findings revealed that MhYTP2 plays an essential role in the regulation of resistance to GLS and identified a promising R gene, MdRGA2L, for use in developing apple cultivars with GLS resistance. [ABSTRACT FROM AUTHOR]

Summary: Low temperature is the main environmental factor affecting the yield, quality and geographical distribution of crops, which significantly restricts development of the fruit industry. The NAC (NAM, ATAF1/2 and CUC2) transcription factor (TF) family is involved in regulating plant cold tolerance, but the mechanisms underlying these regulatory processes remain unclear. Here, the NAC TF MdNAC104 played a positive role in modulating apple cold tolerance. Under cold stress, MdNAC104‐overexpressing transgenic plants exhibited less ion leakage and lower ROS (reactive oxygen species) accumulation, but higher contents of osmoregulatory substances and activities of antioxidant enzymes. Transcriptional regulation analysis showed that MdNAC104 directly bound to the MdCBF1 and MdCBF3 promoters to promote expression. In addition, based on combined transcriptomic and metabolomic analyses, as well as promoter binding and transcriptional regulation analyses, we found that MdNAC104 stimulated the accumulation of anthocyanin under cold conditions by upregulating the expression of anthocyanin synthesis‐related genes, including MdCHS‐b, MdCHI‐a, MdF3H‐a and MdANS‐b, and increased the activities of the antioxidant enzymes by promoting the expression of the antioxidant enzyme‐encoding genes MdFSD2 and MdPRXR1.1. In conclusion, this study revealed the MdNAC104 regulatory mechanism of cold tolerance in apple via CBF‐dependent and CBF‐independent pathways. [ABSTRACT FROM AUTHOR]

SUMMARY: Malic acid is a major organic acid component of apples and a crucial determinant of fruit organoleptic quality. A candidate gene for malic acid content, designated MdMa1, was previously identified in the Ma locus, which is a major quantitative trait locus (QTL) for apple fruit acidity located on the linkage group 16. Region‐based association mapping to detect candidate genes in the Ma locus identified MdMa1 and an additional MdMYB21 gene putatively associated with malic acid. MdMYB21 was significantly associated with fruit malic acid content, accounting for ~7.48% of the observed phenotypic variation in the apple germplasm collection. Analyses of transgenic apple calli, fruits and tomatoes demonstrated that MdMYB21 negatively regulated malic acid accumulation. The apple fruit acidity‐related MdMa1 and its tomato ortholog, SlALMT9, exhibited lower expression profiles in apple calli, mature fruits and tomatoes in which MdMYB21 was overexpressed, compared with their corresponding wild‐type variety. MdMYB21 directly binds to the MdMa1 promoter and represses its expression. Interestingly, a 2‐bp variation in the MdMYB21 promoter region altered its expression and regulation of its target gene, MdMa1, expression. Our findings not only demonstrate the efficiency of integrating QTL and association mapping in the identification of candidate genes controlling complex traits in apples, but also provide insights into the complex regulatory mechanism of fruit malic acid accumulation. Significance Statement: Fruit acidity is a complex quantitative trait that is regulated by one or more major and/or numerous minor genes. Region‐based association mapping was performed, and identified two candidate genes, MdMa1 and MdMYB21, which were associated with malic acid content in the Ma locus at linkage group (LG)16. Furthermore, MdMYB21 could negatively regulate malic acid accumulation in apple fruits by repressing the MdMa1 expression. [ABSTRACT FROM AUTHOR]

SUMMARY: Cold is one of the main abiotic stresses in temperate fruit crops, affecting the yield and fruit quality of apple in China and European countries. The plant receptor‐like kinase FERONIA is widely reported to be involved in abiotic stresses. However, its function in apple cold resistance remains unknown. Modification of cell wall components and accumulation of soluble sugars and amino acids are important strategies by which plants cope with cold. In this study, expression of the apple FERONIA receptor‐like kinase gene MdMRLK2 was rapidly induced by cold. Apple plants overexpressing MdMRLK2 (35S:MdMRLK2) showed enhanced cold resistance relative to the wild type. Under cold conditions, 35S:MdMRLK2 apple plants had higher amounts of water insoluble pectin, lignin, cellulose, and hemicellulose, which may have resulted from reduced activities of polygalacturonase, pectinate lyase, pectinesterase, and cellulase. More soluble sugars and free amino acids and less photosystem damage were also observed in 35S:MdMRLK2 apple plants. Intriguingly, MdMRLK2 interacted with the transcription factor MdMYBPA1 and promoted its binding to MdANS and MdUFGT promoters, leading to more anthocyanin biosynthesis, particularly under cold conditions. These findings complemented the function of apple FERONIA MdMRLK2 responding to cold resistance. Significance Statement: The function of FERONIA receptor‐like kinase in apple cold resistance is unknown. Here, we complemented the function of apple FERONIA MdMRLK2 responding to cold stress by modulating cell wall components, osmotic adjustment substances, photosystems, and anthocyanin biosynthesis processes. [ABSTRACT FROM AUTHOR]

The fungal disease Glomerella leaf spot (GLS) seriously impacts apple production. As a nonprotein amino acid, γ‐aminobutyric acid (GABA) is widely involved in biotic and abiotic stresses. However, it is not clear whether GABA is involved in a plant's response to GLS, nor is its molecular mechanism understood. Here, we found that exogenous GABA could significantly alleviate GLS, reduce lesion lengths, and increase antioxidant capacity. MdGAD1 was identified as a possible key gene for GABA synthesis in apple. Further analysis indicated that MdGAD1 promoted antioxidant capacity to improve apple GLS resistance in transgenic apple calli and leaves. Yeast one‐hybrid analysis identified the transcription factor MdWRKY33 upstream of MdGAD1. Electrophoretic mobility shift assay, β‐glucuronidase activity, and luciferase activity further supported that MdWRKY33 bound directly to the promoter of MdGAD1. The content of GABA and the transcription level of MdGAD1 in the MdWRKY33 transgenic calli were higher than that of the wild type. When MdWRKY33 transgenic calli and leaves were inoculated with GLS, MdWKRY33 positively regulated resistance to GLS. These results explained the positive regulatory effects of GABA on apple GLS and provided insight into the metabolic regulatory network of GABA. [ABSTRACT FROM AUTHOR]

SUMMARY: Apple (Malus domestica) trees are vulnerable to freezing temperatures. Cold resistance in woody perennial plants can be improved through biotechnological approaches. However, genetic engineering requires a thorough understanding of the molecular mechanisms of the tree's response to cold. In this study, we demonstrated that the Mdm‐miR160–MdARF17–MdWRKY33 module is crucial for apple freezing tolerance. Mdm‐miR160 plays a negative role in apple freezing tolerance, whereas MdARF17, one of the targets of Mdm‐miR160, is a positive regulator of apple freezing tolerance. RNA sequencing analysis revealed that in apple, MdARF17 mediates the cold response by influencing the expression of cold‐responsive genes. EMSA and ChIP‐qPCR assays demonstrated that MdARF17 can bind to the promoter of MdWRKY33 and promotes its expression. Overexpression of MdWRKY33 enhanced the cold tolerance of the apple calli. In addition, we found that the Mdm‐miR160–MdARF17–MdWRKY33 module regulates cold tolerance in apple by regulating reactive oxygen species (ROS) scavenging, as revealed by (i) increased H2O2 levels and decreased peroxidase (POD) and catalase (CAT) activities in Mdm‐miR160e OE plants and MdARF17 RNAi plants and (ii) decreased H2O2 levels and increased POD and CAT activities in MdmARF17 OE plants and MdWRKY33 OE calli. Taken together, our study uncovered the molecular roles of the Mdm‐miR160–MdARF17–MdWRKY33 module in freezing tolerance in apple, thus providing support for breeding of cold‐tolerant apple cultivars. Significance Statement: This study uncovered the molecular roles of the Mdm‐miR160–MdARF17–MdWRKY33 module in freezing tolerance in apple, thus providing support for breeding of cold‐tolerant apple cultivars. [ABSTRACT FROM AUTHOR]

Summary: Drought limits apple yield and fruit quality. However, the molecular mechanism of apple in response to drought is not well known. Here, we report a Cys2/His2 (C2H2)‐type zinc‐finger protein, MdZAT5, that positively regulates apple drought tolerance by regulating drought‐responsive RNAs and microRNAs (miRNAs).DNA affinity purification and sequencing and yeast‐one hybrid analysis identified the binding motifs of MdZAT5, T/ACACT/AC/A/G. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP‐qPCR) and electrophoretic mobility shift assays (EMSAs) showed that MdZAT5 directly binds to the promoters of the drought‐responsive genes including MdRHA2a, MdLEA14, MdTPX1, and MdCAT3, and activates their expression under drought stress.MdZAT5 interacts with and directly targets HYPONASTIC LEAVES1 (MdHYL1). MdZAT5 may facilitate the interaction of MdHYL1 with pri‐miRNAs or MdDCL1 by activating MdHYL1 expression, thereby regulating the biogenesis of drought‐responsive miRNAs. Genetic dissection showed that MdHYL1 is essential for MdZAT5‐mediated drought tolerance and miRNA biogenesis. In addition, ChIP‐qPCR and EMSA revealed that MdZAT5 binds directly to the promoters of some MIR genes including Mdm‐miR171i and Mdm‐miR172c, and modulates their transcription.Taken together, our findings improve our understanding of the molecular mechanisms of drought response in apple and provide a candidate gene for the breeding of drought‐tolerant cultivars. [ABSTRACT FROM AUTHOR]

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple trees in China and other East Asian countries. The plant receptor‐like kinase FERONIA is involved in plant cell growth, development, and immunity. However, little is known about the function of FERONIA in apple defence against V. mali. In this study, we found that MdMRLK2 was highly induced by V. mali in twigs of V. mali‐susceptible Malus mellana but not in those of the resistant species Malus yunnaensis. 35S:MdMRLK2 apple plants showed compromised resistance relative to wild‐type (WT) plants. Further analyses indicated that 35S:MdMRLK2 apple plants had enhanced abscisic acid (ABA) levels and reduced salicylic acid (SA) levels relative to the WT on V. mali infection. MdMRLK2 overexpression also suppressed polyphenol accumulation and inhibited the activities of phenylalanine ammonia‐lyase (PAL), β‐1,3‐glucanase (GLU), and chitinase (CHT) during V. mali infection. Moreover, MdMRLK2 interacted with MdHIR1, a hypersensitive‐induced response protein, and suppressed the MdHIR1‐mediated hypersensitive reaction (HR), probably by impairing MdHIR1 self‐interaction. Collectively, these findings demonstrate that overexpression of MdMRLK2 compromises Valsa canker resistance, probably by (a) altering ABA and SA levels, (b) suppressing polyphenol accumulation, (c) inhibiting PAL, GLU, and CHT activities, and (d) blocking MdHIR1‐mediated HR by disrupting MdHIR1 self‐interaction. [ABSTRACT FROM AUTHOR]

Summary: Although the N6‐methyladenosine (m6A) modification is the most prevalent RNA modification in eukaryotes, the global m6A modification landscape and its molecular regulatory mechanism in response to drought stress remain unclear.Transcriptome‐wide m6A methylome profiling revealed that m6A is mainly enriched in the coding sequence and 3′ untranslated region in response to drought stress in apple, by recognizing the plant‐specific sequence motif UGUAH (H=A, U or C). We identified a catalytically active component of the m6A methyltransferase complex, MdMTA. An in vitro methyl transfer assay, dot blot, LC‐MS/MS and m6A‐sequencing (m6A‐seq) suggested that MdMTA is an m6A writer and essential for m6A mRNA modification.Further studies revealed that MdMTA is required for apple drought tolerance. m6A‐seq and RNA‐seq analyses under drought conditions showed that MdMTA mediates m6A modification and transcripts of mRNAs involved in oxidative stress and lignin deposition. Moreover, m6A modification promotes mRNA stability and the translation efficiency of these genes in response to drought stress. Consistently, MdMTA enhances lignin deposition and scavenging of reactive oxygen species under drought conditions.Our results reveal the global involvement of m6A modification in the drought response of perennial apple trees and illustrate its molecular mechanisms, thereby providing candidate genes for the breeding of stress‐tolerant apple cultivars. [ABSTRACT FROM AUTHOR]

Summary: SUMOylation is involved in various aspects of plant biology, including drought stress. However, the relationship between SUMOylation and drought stress tolerance is complex; whether SUMOylation has a crosstalk with ubiquitination in response to drought stress remains largely unclear. In this study, we found that both increased and decreased SUMOylation led to increased survival of apple (Malus × domestica) under drought stress: both transgenic MdSUMO2A overexpressing (OE) plants and MdSUMO2 RNAi plants exhibited enhanced drought tolerance. We further confirmed that MdDREB2A is one of the MdSUMO2 targets. Both transgenic MdDREB2A OE and MdDREB2AK192R OE plants (which lacked the key site of SUMOylation by MdSUMO2A) were more drought tolerant than wild‐type plants. However, MdDREB2AK192R OE plants had a much higher survival rate than MdDREB2A OE plants. We further showed SUMOylated MdDREB2A was conjugated with ubiquitin by MdRNF4 under drought stress, thereby triggering its protein degradation. In addition, MdRNF4 RNAi plants were more tolerant to drought stress. These results revealed the molecular mechanisms that underlie the relationship of SUMOylation with drought tolerance and provided evidence for the tight control of MdDREB2A accumulation under drought stress mediated by SUMOylation and ubiquitination. [ABSTRACT FROM AUTHOR]

SUMMARY: Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole‐3‐acetic acid (IAA) conjugating enzyme, to be a negative regulator of water‐deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water‐deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water‐deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water‐deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water‐deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water‐deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees. Significance Statement: We characterized MdGH3.6 in the response of apple to water‐deficit stress. We found that MdGH3.6 was a direct target of MdMYB94 and a negative regulator of water‐deficit stress tolerance in apple. The negative regulation of water‐deficit stress tolerance may stem from the combined roles of MdGH3.6 in the negative regulation of ROS detoxification and cuticular wax. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees. [ABSTRACT FROM AUTHOR]

SUMMARY: Fructose (Fru) content is a key determinant of fruit sweetness and quality. An F1 hybrid population of the apple cultivars 'Honeycrisp' × 'Qinguan' was used to investigate the quantitative trait locus (QTL) regions and genes controlling Fru content in fruit. A stable QTL on linkage group (LG) 01 in 'Honeycrisp' was detected on the single nucleotide polymorphism (SNP) genetic linkage maps. In this region, a sorbitol dehydrogenase (SDH) gene, MdSDH2, was detected and showed promoter variations and differential expression patterns between 'Honeycrisp' and 'Qinguan' fruits as well as their hybrids. A SNP variant (A/G) in the MdSDH2 promoter region (SDH2p‐491) affected the binding ability of the transcription factor MdABI3, which can affect the expression of MdSDH2. Promoter sequences with an A nucleotide at SDH2p‐491 had stronger binding affinity for MdABI3 than those with a G. Among 27 domesticated apple cultivars and wild relatives, this SNP (A/G) was associated with Fru content. Our results indicate that MdSDH2 can alter Fru content as the major regulatory gene and that ABA signaling might be involved in Fru content accumulation in apple fruit. Significance Statement: Here, we detected a stable overlapping QTL region of fructose content in apple fruit with a SNP genetic map, and confirmed that A/G SNP variation in the promoter of the sorbitol dehydrogenase gene MdSDH2 in this QTL region affects binding of the transcription factor MdABl3, which regulates MdSDH2 expression and fructose accumulation. Our results demonstrate the major role of the candidate gene MdSDH2 in regulating fructose content of apple fruit. [ABSTRACT FROM AUTHOR]

Summary: N6‐methyladenosine (m6A) reader protein plays an important role in trichome morphology, developmental timing and morphogenesis in Arabidopsis. However, the function of m6A readers in plant‐microbe interaction remains unclear. Here, a Malus YTH‐domain family protein MhYTP2 was initially characterized as an m6A reader. MhYTP2 overexpression increased mRNA m6A modification level and translation efficiency. The m6A in the exon regions appeared to destabilize the mRNAs, whereas m6A in the untranslated regions positively correlated with the associated mRNA abundance. MhYTP2 overexpression enhanced apple powdery mildew resistance, possibly by rapidly degrading the bound mRNAs of MdMLO19 and MdMLO19‐X1 and improving the translation efficiency of the antioxidant genes. To conclude, the results shed light on the apple m6A profile, the effect of MhYTP2 on m6A profile, and the m6A roles in MdMLO19 and MdMLO19‐X1 mRNAs stability and glutamate dehydrogenase 1‐like MdGDH1L mRNA translation efficiency. [ABSTRACT FROM AUTHOR]

Light regulates anthocyanins synthesis in plants. Upon exposure to visible light, the inhibition of photosynthetic electron transfer significantly lowered the contents of anthocyanins and the expression levels of key genes involved in anthocyanins synthesis in plum fruit peel. Meanwhile, the expression levels of PsmMDH2 (encoding the malate dehydrogenase in mitochondria) and PschMDH (encoding the malate dehydrogenase in chloroplasts) decreased significantly. The contents of anthocyanins and the levels of the key genes involved in anthocyanin synthesis decreased significantly with the treatment of 1‐MCP (an inhibitor of ethylene perception) but were enhanced by the exogenous application of ethylene. The ethylene treatment could also recover the anthocyanin synthesis capacity lowered by the photosynthetic electron transfer inhibition. Silencing PsmMDH2 and PschMDH significantly lowered the contents of anthocyanins in plum fruit. At low temperature, visible light irradiation induced anthocyanin accumulation in Arabidopsis leaves. However, the mmdh, chmdh, and etr1‐1 mutants had significantly lower anthocyanins content and expressions of the key genes involved in anthocyanins synthesis compared to wild type. Overall, the present study demonstrates that both photosynthesis and respiration were involved in the regulation of anthocyanin synthesis in visible light. The visible light regulates anthocyanin synthesis by controlling the malate metabolism via MDHs and the ethylene signaling pathway. [ABSTRACT FROM AUTHOR]

Salinity is a major environmental constraint that substantially limits global agricultural productivity. HD‐Zip I transcription factors are involved in plant responses to salt stress, but little is known about the HD‐Zip I genes in apple (Malus domestica). Here, we characterized the function of an apple HD‐Zip I gene (MdHB7‐like) and report that its expression is induced by salt stress. To further explore its role in salt stress, we created MdHB7‐like overexpressing and RNAi transgenic apple plants. The overexpression of MdHB7‐like improved the photosynthetic performance and reduced ROS and Na+ accumulation under salt stress. Plants that overexpressed MdHB7‐like also showed increased accumulation of proline and soluble sugars, which may have played an important role in their salt stress tolerance. RNAi suppression of MdHB7‐like had the opposite effects. Together, our results demonstrate that MdHB7‐like is an important regulator of salt tolerance in apple. Our results provide new insights for future research on the mechanisms by which MdHB7‐like promotes salt tolerance and provide a potential target for molecular breeding in apple. [ABSTRACT FROM AUTHOR]

Summary: Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high 14C‐fructose and 14C‐glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up‐regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5‐edited mutant of the MdHT2.2‐expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content. [ABSTRACT FROM AUTHOR]

Summary: Acidity is one of the main determinants of fruit organoleptic quality. Here, comparative transcriptome analysis was conducted between two cultivars that showed a significant difference in fruit acidity, but contained homozygous non‐functional alleles at the major gene Ma1 locus controlling apple fruit acidity. A candidate gene for fruit acidity, designated M10, was identified. The M10 gene encodes a P‐type proton pump, P3A‐ATPase, which facilitates malate uptake into the vacuole. The Ma10 gene is significantly associated with fruit malate content, accounting for ~7.5% of the observed phenotypic variation in apple germplasm. Subcellular localization assay showed that the Ma10 is targeted to the tonoplast. Overexpression of the Ma10 gene can complement the defect in proton transport of the mutant YAK2 yeast strain and enhance the accumulation of malic acid in apple callus. Moreover, its ectopic expression in tomato induces a decrease in fruit pH. These results suggest that the Ma10 gene has the capacity for proton pumping and plays an important role in fruit vacuolar acidification in apple. Our study provides useful knowledge towards comprehensive understanding of the complex mechanism regulating apple fruit acidity. [ABSTRACT FROM AUTHOR]

Summary: Apple (Malus × domestica) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold‐hardy cultivars. This lack of progress is due at least partly to lack of understanding of the molecular mechanisms of freezing tolerance in apple. In this study, we evaluated the potential roles for two R2R3 MYB transcription factors (TFs), MYB88 and the paralogous FLP (MYB124), in cold stress in apple and Arabidopsis. We found that MYB88 and MYB124 positively regulate freezing tolerance and cold‐responsive gene expression in both apple and Arabidopsis. Chromatin‐Immunoprecipitation‐qPCR and electrophoretic mobility shift assays showed that MdMYB88/MdMYB124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 (MdCSP3) and CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1) genes. Dual luciferase reporter assay indicated that MdCCA1 but not MdCSP3 activated the expression of MdCBF3 under cold stress. Moreover, MdMYB88 and MdMYB124 promoted anthocyanin accumulation and H2O2 detoxification in response to cold. Taken together, our results suggest that MdMYB88 and MdMYB124 positively regulate cold hardiness and cold‐responsive gene expression under cold stress by C‐REPEAT BINDING FACTOR (CBF)‐dependent and CBF‐independent pathways. [ABSTRACT FROM AUTHOR]

Summary: Cytosine methylation is an essential feature of epigenetic regulation and is involved in various biological processes. Although cytosine methylation has been analysed at the genomic scale for several plant species, there is a general lack of understanding of the dynamics of global and genic DNA methylation in plants growing in environments challenged with biotic and abiotic stresses. In this study, we mapped cytosine methylation at single‐base resolution in the genome of commercial apple (Malus x domestica), and analysed changes in methylation patterns associated with water deficit in representative drought‐sensitive and drought‐tolerant cultivars. We found that the apple genome exhibits ~54%, ~38% and ~8.5% methylation at CG, CHG and CHH sequence contexts, respectively. We additionally documented changes in gene expression associated with water deficit in an attempt to link methylation and gene expression changes. Global methylation and transcription analysis revealed that promoter‐unmethylated genes showed higher expression levels than promoter‐methylated genes. Gene body methylation appears to be positively correlated with gene expression. Water deficit stress was associated with changes in methylation at a multitude of genes, including those encoding transcription factors (TFs) and transposable elements (TEs). These results present a methylome map of the apple genome and reveal widespread DNA methylation alterations in response to water deficit stress. These data will be helpful for understanding potential linkages between DNA methylation and gene expression in plants growing in natural environments and challenged with abiotic and biotic stresses. [ABSTRACT FROM AUTHOR]

Summary: Autophagy is a major and conserved pathway for delivering and recycling unwanted proteins or damaged organelles to be degraded in the vacuoles. AuTophaGy‐related (ATG) protein 18a has been established as one of the essential components for autophagy occurrence in Arabidopsis thaliana. We previously cloned the ATG18a homolog from Malus domestica (MdATG18a) and monitored its responsiveness to various abiotic stresses at the transcriptional level. However, it is still unclear what its function is under abiotic stress in apple. Here, we found that heterologous expression of MdATG18a in tomato plants markedly enhanced their tolerance to drought. Overexpression (OE) of that gene in apple plants improved their drought tolerance as well. Under drought conditions, the photosynthesis rate and antioxidant capacity were significantly elevated in OE lines when compared with the untransformed wild type (WT). Transcript levels of other important apple ATG genes were more strongly up‐regulated in transgenic MdATG18a OE lines than in the WT. The percentage of insoluble protein in proportion to total protein was lower and less oxidized protein accumulated in the OE lines than in the WT under drought stress. This was probably due to more autophagosomes being formed in the former. These results demonstrate that overexpression of MdATG18a in apple plants enhances their tolerance to drought stress, probably because of greater autophagosome production and a higher frequency of autophagy. Those processes help degrade protein aggregation and limit the oxidation damage, thereby suggesting that autophagy plays important roles in the drought response. [ABSTRACT FROM AUTHOR]

Abstract: Nitrogen (N) availability is an essential factor for plant growth. Recycling and remobilization of N have strong impacts on crop yield and quality under N deficiency. Autophagy is a critical nutrient‐recycling process that facilitates remobilization under starvation. We previously showed that an important AuTophaGy (ATG) protein from apple, MdATG18a, has a positive role in drought tolerance. In this study, we explored its biological role in response to low‐N. Overexpression of MdATG18a in both Arabidopsis and apple improved tolerance to N‐depletion and caused a greater accumulation of anthocyanin. The increased anthocyanin concentration in transgenic apple was possibly due to up‐regulating flavonoid biosynthetic and regulatory genes (MdCHI, MdCHS, MdANS, MdPAL, MdUFGT, and MdMYB1) and higher soluble sugars concentration. MdATG18a overexpression enhanced starch degradation with up‐regulating amylase gene (MdAM1) and up‐regulated sugar metabolism related genes (MdSS1, MdHXKs, MdFK1, and MdNINVs). Furthermore, MdATG18a functioned in nitrate uptake and assimilation by up‐regulating nitrate reductase MdNIA2 and 3 high‐affinity nitrate transporters MdNRT2.1/2.4/2.5. MdATG18a overexpression also elevated other important MdATG genes expression and autophagosomes formation under N‐depletion, which play key contributions to above changes. Together, these results demonstrate that overexpression of MdATG18a enhances tolerance to N‐deficiencies and plays positive roles in anthocyanin biosynthesis through greater autophagic activity. [ABSTRACT FROM AUTHOR]

Melatonin mediates many physiological processes in plants. We investigated its role in regulating growth, potassium uptake, and root system architecture under three types of stress: salinity or a deficiency of all nutrients in Malus hupehensis Rehd., as well as a K deficiency in Malus rockii Rehd. Each treatment caused a reduction in growth rates and disrupted the absorption of potassium. However, pretreatment with 0.1 mmol/L melatonin significantly alleviated such inhibitions. The addition of melatonin also upregulated genes for antioxidant enzymes involved in the ascorbate-glutathione cycle ( MdcAPX, MdDHAR1, MdDHAR2, MdMDHAR, and MdcGR) and helped decrease the accumulation of H2O2 while improving the expression of K transporters and genes for the CBL1- CIPK23 pathway. These results indicated that melatonin can regulate the ROS signal and activate the CBL1- CIPK23 pathway to regulate the expression of a potassium channel protein gene, thereby promoting the absorption of potassium ions. Our findings demonstrate that inducing melatonin production is an important mechanism for plant defenses that can serve as a platform for possible applications in agricultural or related fields of research. [ABSTRACT FROM AUTHOR]

Melatonin regulates growth in many plants; however, the mechanism remains unclear. In this study, exogenous melatonin feeding resulted in both promotional (≤10 μ m) and inhibitory (≥100 μ m) effects on maize seedling growth. Initial analyses suggested positive correlations between the amount of melatonin and sucrose synthesis and hydrolysis-related gene expression, enzyme activities, and sucrose metabolites. However, assays of photosynthetic rate, hexokinase (HxK) activity, expression of photosynthetic marker genes, and HxK-related genes showed opposite effects under 10 μ m (positive) and 100 μ m (negative) melatonin treatments. Similarly, 10 μ m melatonin accelerated starch catabolism at night, whereas 100 μ m melatonin significantly decreased this process and led to starch accumulation in photosynthetic tissues. Furthermore, expression analysis of genes related to sucrose phloem loading resulted in a slight upregulation of sucrose transporters ( SUT1 and SUT2) when seedlings were induced with 10 μ m melatonin, while treatment with 100 μ m melatonin resulted in significant downregulation of these sucrose transporter genes ( SUT1 and SUT2), as well as tie-dyed2 ( Tdy2) and sucrose export defective 1. Taken together, these results suggest that low doses of melatonin benefit maize seedling growth by promoting sugar metabolism, photosynthesis, and sucrose phloem loading. Conversely, high doses of melatonin inhibit seedling growth by inducing the excessive accumulation of sucrose, hexose and starch, suppressing photosynthesis and sucrose phloem loading. [ABSTRACT FROM AUTHOR]

Melatonin has been reported to promote plant growth and development. Our experiments with Arabidopsis thaliana showed that exogenous applications of this molecule mediated invertase inhibitor (C/VIF)- regulated invertase activity and enhanced sucrose metabolism. Hexoses were accumulated in response to elevated activities by cell wall invertase (CWI) and vacuolar invertase (VI). Analyses of sugar metabolism-related genes revealed differential expression during plant development that was modulated by melatonin. In particular, C/VIF1 and C/VIF2 were strongly down-regulated by exogenous feeding. We also found the elevated CWI activity in melatonintreated Arabidopsis improved the factors (cellulose, xylose, and galactose) for cell wall reinforcement and callose deposition during Pseudomonas syringae pv. tomato DC3000 infection, therefore, partially induced the pathogen resistance. However, CWI did not involve in salicylic acid (SA)-regulated defense pathway. Taken together, this study reveals that melatonin plays an important role in invertase-related carbohydrate metabolism, plant growth, and pathogen defense. [ABSTRACT FROM AUTHOR]

The beneficial effect that melatonin has against mitochondrial dysfunctioning seems to be linked to mitophagy. Roles for melatonin have been demonstrated in promoting health and preventing disease, as well as activating the process of autophagy in general. However, no reports have been made about how the application of melatonin regulates that process when plants are exposed to oxidative stress. We investigated the influence of different concentrations of melatonin (0.0, 0.5, 5.0, 10.0, or 50.0 μ m) on autophagy under methyl viologen ( MV)-induced oxidative stress. Arabidopsis seedlings that were pretreated with 5 or 10 μ m melatonin underwent relatively strong induction of autophagy, as evidenced by the number of monodansylcadaverine ( MDC)-stained autophagosomes in root samples. Pretreatment with 10 μ m melatonin also alleviated MV-induced photo-oxidation damage and significantly reduced the accumulation of oxidized proteins. Those responses might have been due to the strong upregulation of genes that involved in At ATG8- PE conjugation pathway, which enhanced the capacity for autophagy. Histochemical staining revealed that both [ABSTRACT FROM AUTHOR]

Despite the relationship between melatonin and aging, the overall changes and regulation of proteome profiling by long-term melatonin exposure during leaf senescence is not well understood. In this study, leaf senescence in Malus hupehensis plants was delayed when exogenous melatonin was regularly applied to the roots for 2 months compared with natural leaf senescence. Proteins of samples 0 and 50 day for both treatments were extracted and labeled with TMT regents before being examined via NanoLC-MS/MS. The proteomics data showed that 622 and 309 proteins were altered by senescence and melatonin, respectively. Our GO analysis by Blast2GO revealed that most of the altered proteins that are involved in major metabolic processes exhibited hydrolase activity and were mainly located in the plastids. These proteins were classified into several senescence-related functional categories, including degradation of macromolecules, redox and stress responses, transport, photosynthesis, development, and other regulatory proteins. We found that melatonin treatment led to the downregulation of proteins that are normally upregulated during senescence. The melatonin-related delay in senescence might have occurred due to the altering of proteins involved in processes associated with senescence. And as well, there are many unknown regulatory proteins possibly being involved in the melatonin's function. This study is the first to demonstrate changes at the proteome level in response to exogenous melatonin in plants. Our findings provide a set of informative and fundamental data about the role of melatonin in apple leaf senescence. [ABSTRACT FROM AUTHOR]

Melatonin has an important anti-aging role in plant physiology. We tested the effects of long-term melatonin exposure on metabolic status and protein degradation during natural leaf senescence in trees of Malus hupehensis Rehd. The 2-month regular supplement of 100 μ m melatonin to the soil once every 6 days altered the metabolic status and delayed protein degradation. For example, leaves from treated plants had significantly higher photosynthetic activity, chlorophyll concentrations, and levels of three photosynthetic end products (sorbitol, sucrose, and starch) when compared with the control. The significant inhibition of hexose (fructose and glucose) accumulation possibly regulated the signaling of Md HXK1, a gene for which expression was also repressed by melatonin during senescence. The plants also exhibited better preservation of their nitrogen, total soluble protein, and Rubisco protein concentrations than the control. The slower process of protein degradation might be a result of melatonin-linked inhibition on the expression of apple autophagy-related genes ( ATGs). Our results are the first to provide evidence for this delay in senescence based on the metabolic alteration and protein degradation. [ABSTRACT FROM AUTHOR]

We examined whether exogenously applied melatonin could improve resistance to Marssonina apple blotch (Diplocarpon mali) by apple [Malus prunifolia (Willd.) Borkh. cv. Donghongguo]. This serious disease leads to premature defoliation in the main regions of apple production. When plants were pretreated with melatonin, resistance was increased in the leaves. We investigated the potential roles for melatonin in modulating levels of hydrogen peroxide (H2O2), as well the activities of antioxidant enzymes and pathogenesis-related proteins during these plant-pathogen interactions. Pretreatment enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhance the activities of plant defence-related enzymes, possibly improving disease resistance. Because melatonin is safe and beneficial to animals and humans, exogenous pretreatment might represent a promising cultivation strategy to protect plants against this pathogen infection. [ABSTRACT FROM AUTHOR]

To examine the potential roles of melatonin in drought tolerance, we tested the effects of its long-term exogenous application on 'Hanfu' apple ( Malus domestica Borkh.). When 100 μ m melatonin was added to soils under drought conditions, the resultant oxidative stress was eased and leaf senescence was delayed. This molecule significantly reduced chlorophyll degradation and suppressed the up-regulation of senescence-associated gene 12 ( SAG12) and pheophorbide a oxygenase ( PAO). Such treatment also alleviated the inhibition of photosynthesis brought on by drought stress. We also investigated quenching and the efficiency of Photosystem II ( PSII) photochemistry under dark and light conditions and found that melatonin helped to maintain better function of PSII under drought. The addition of melatonin also controlled the burst of hydrogen peroxide, possibly through direct scavenging and by enhancing the activities of antioxidative enzymes and the capacity of the ascorbate-glutathione cycle. Thus, understanding this effect of melatonin on drought tolerance introduces new possibilities to use this compound for agricultural purposes. [ABSTRACT FROM AUTHOR]

As an indoleamine molecule, melatonin mediates many physiological processes in plants. We investigated its role in regulating growth, ion homeostasis, and the response to oxidative stress in Malus hupehensis Rehd. under high-salinity conditions. Stressed plants had reduced growth and a marked decline in their net photosynthetic rates and chlorophyll contents. However, pretreatment with 0.1 μ m melatonin significantly alleviated this growth inhibition and enabled plants to maintain an improved photosynthetic capacity. The addition of melatonin also lessened the amount of oxidative damage brought on by salinity, perhaps by directly scavenging H2O2 or enhancing the activities of antioxidative enzymes such as ascorbate peroxidase, catalase, and peroxidase. We also investigated whether melatonin might control the expression of ion-channel genes under salinity. Here, MdNHX1 and MdAKT1 were greatly up-regulated in the leaves, which possibly contributed to the maintenance of ion homeostasis and, thus, improved salinity resistance in plants exposed to exogenous melatonin. [ABSTRACT FROM AUTHOR]

The objectives of this study were to test the effects of exogenous melatonin on apple ( Malus domestica Borkh. cv. Golden Delicious) leaves and investigate its possible physiological role in delaying leaf senescence. Detached leaves treated with 10 m m melatonin solutions clearly showed a slowing in their process of dark-induced senescence, as evidenced by both biochemical and molecular parameters. Melatonin delayed the normal reduction in chlorophyll content and maximum potential photosystem II efficiency ( Fv/ Fm). It also suppressed the transcript levels of a key chlorophyll degradation gene, pheide a oxygenase ( PAO), and the senescence-associated gene 12 ( SAG12). This outcome was thought to be because of the enhanced antioxidant capabilities of melatonin. Indeed, H2O2 accumulation was inhibited by exogenous melatonin, which might have resulted from direct reactive oxygen species scavenging by melatonin and a great enhancement of ascorbate peroxidase (APX; ), which acted on both mRNA and protein activity levels. Melatonin treatment led to the maintenance of higher contents of ascorbic acid (AsA) and glutathione (GSH) but less dehydroascorbate (DHA) and oxidized glutathione (GSSG) compared with the control, possibly through its regulation of the AsA-GSH cycle. [ABSTRACT FROM AUTHOR]

Phloretin has different glycosylation modes in plants. Phlorizin (phloretin 2′‐O‐glucoside) is one of the glycosylation products of phloretin, and accumulates abundantly in apple plants. However, it is still unclear whether phlorizin is more beneficial for apple plants compared with other glycosylation products of phloretin. We created transgenic apple plants with different glycosylation modes of phloretin. In transgenic plants, the accumulation of phlorizin was partly replaced by that of trilobatin (phloretin 4′‐O‐glucoside) or phloretin 3′,5′‐di‐C‐glycoside. Compared with wild type, transgenic plants with less phlorizin showed dwarf phenotype, larger stomatal size, higher stomatal density and less tolerance to drought stress. Transcriptome and phytohormones assay indicate that phlorizin might regulate stomatal development and behaviour via controlling auxin and abscisic acid signalling pathways as well as carbonic anhydrase expressions. Transgenic apple plants with less phlorizin also showed less resistance to spider mites. Apple plants may hydrolyse phlorizin to produce phloretin, but cannot hydrolyse trilobatin or phloretin 3′,5′‐di‐C‐glycoside. Compared with its glycosylation products, phloretin is more toxic to spider mites. These results suggest that the glycosylation of phloretin to produce phlorizin is the optimal glycosylation mode in apple plants, and plays an important role in apple resistance to stresses. [ABSTRACT FROM AUTHOR]

SUMMARY 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. [ABSTRACT FROM AUTHOR]

Summary Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is considered one of the most destructive diseases affecting apples. The VQ‐WRKY complex plays a crucial role in the response of plants to biotic stresses. However, our understanding of the defensive role of the VQ‐WRKY complex on woody plants, particularly apples, under biotic stress, remains limited. In this study, we elucidated the molecular mechanisms underlying the defensive role of the apple MdVQ37‐MdWRKY100 module in response to GLS infection. The overexpression of MdWRKY100 enhanced resistance to C. fructicola, whereas MdWRKY100 RNA interference in apple plants reduced resistance to C. fructicola by affecting salicylic acid (SA) content and the expression level of the CC‐NBS‐LRR resistance gene MdRPM1. DAP‐seq, Y1H, EMSA, and RT‐qPCR assays indicated that MdWRKY100 inhibited the expression of MdWRKY17, a positive regulatory factor gene of SA degradation, upregulated the expression of MdPAL1, a key enzyme gene of SA biosynthesis, and promoted MdRPM1 expression by directly binding to their promotors. Transient overexpression and silencing experiments showed that MdPAL1 and MdRPM1 positively regulated GLS resistance in apples. Furthermore, the overexpression of MdVQ37 increased the susceptibility to C. fructicola by reducing the SA content and expression level of MdRPM1. Additionally, MdVQ37 interacted with MdWRKY100, which repressed the transcriptional activity of MdWRKY100. In summary, these results revealed the molecular mechanism through which the apple MdVQ37‐MdWRKY100 module responds to GLS infection by regulating SA content and MdRPM1 expression, providing novel insights into the involvement of the VQ‐WRKY complex in plant pathogen defence responses. [ABSTRACT FROM AUTHOR]

Melatonin mediates many physiological processes in plants. The problem of apple replant disease is unsolved. Our study objectives were to evaluate the regulatory effect of melatonin on plant resistance to this challenge and investigate the preliminary mechanism by which melatonin helps alleviate the effects of this disease. Two‐year‐old trees of "Fuji" apple (Malus domestica), grafted onto rootstock M.26, were grown in "replant" soil for 6 months in the absence or presence of a 200 μmol/L melatonin supplement. The addition of melatonin to the soil significantly increased the rates of plant growth and net photosynthesis and chlorophyll concentrations under replant conditions. This molecule elevated the levels of K in leaves and roots and enhanced the activity of soil enzymes. Such supplementation also changed the composition of the bacterial and fungal communities in the soil. We concluded that the application of melatonin to a replant soil can protect their chloroplasts from oxidative damage and release the apple root from membrane damage, and also lead to increased soil enzyme activity and soil quality while altering the composition of bacterial and fungal communities. These changes can then promote seedling growth, stimulate photosynthesis, and elevate K levels, thereby alleviating the effects of apple replant disease. [ABSTRACT FROM AUTHOR]