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Abstract: We used 10 simple sequence repeat primers in combination with touchdown PCR to evaluate the genetic relationships among 15 biotypes of Malus prunifolia. Out of 170 detected fragments, 156 (90.74%) were polymorphic. Coefficients of genetic similarity ranged from 0.53 to 0.88, with an average value of 0.66 between pairs of biotypes, which also indicated the high degree of polymorphism within this species. Cluster analysis sorted all biotypes into three groups, which was then confirmed by principal coordinate analysis. A dendrogram showed that some of the clustered biotypes were largely congruent for geographical distribution. However, the DNA patterns for some biotype groups did not demonstrate relative agreement in their origin. Five biotypes arising from the Xinjiang Autonomous Region were dispersed within the dendrogram, reflecting their complex genetic backgrounds. [Copyright &y& Elsevier]

Abstract: Effects of light irradiation on the photosynthetic electron transport chain between P680 and P700 in apple tree leaves was probed with chlorophyll a fluorescence transient and 820 nm transmission measurements during dehydration under different light intensities. The results showed that light accelerated the leaf water-loss rate during dehydration. Leaf dehydration lowered the maximum quantum yield of PSII and the far-red light induced maximal transmission change at 820 nm, but increased the relative variable fluorescence intensity at J-step, especially under increasing irradiation conditions. During leaf dehydration, irradiation lowered the relative variable fluorescence intensity at I-step. At the beginning of leaf dehydration, moderate light accelerated the leaf water-loss rate and then lowered the maximal light-trapping efficiency of P680. Upon further dehydration under moderate light or dehydration under high light, light accelerated the water-loss rate and also directly decreased the maximal light-trapping efficiency of P680. The more significant decrease in the exchange capacity of plastoquinones at the QB site was mainly attributed to the faster water-loss rate under moderate light than in the dark. Under high light, irradiation also directly lowered the capacity. The reoxidation of PQH2 in the dehydrated leaves was enhanced by the light irradiation. The rapidly decreased contents of P700 + plastocyanin were mainly attributed to the faster water-loss rate under light conditions in contrast with that in the dark. The different effects of light irradiations on the photosynthetic electron transport chain might be involved in the acclimation of apple tree leaves to dehydration. [Copyright &y& Elsevier]

In China, the native Lanzhou lily ( Lilium davidii var. unicolor) is used as an edible lily and is an important source of ascorbic acid. However, an efficient transformation system has not been developed for this important crop. Here, we describe the first successful genetic transformation mediated by particle bombardment in this lily variety. We used pCAMBIA-GLDH, which contains the l -galactono-1, 4-lactone dehydrogenase gene from apple, a hygromycin phosphotransferase gene as a selectable marker, and an intron-containing β-glucuronidase gene as a reporter. In all, 35 transgenic lines were selected via GUS assays and PCR; from these, eight were confirmed by Southern analyses. Transgenic plants were transferred to soil and grown under greenhouse conditions. The levels of reduced ascorbate and total ascorbate (reduced ascorbate + oxidized ascorbate) in transgenic plants were found to be two to seven times those in non-transgenic controls. [ABSTRACT FROM AUTHOR]

Abstract: To elucidate the roles of cell wall-modifying enzymes in apple fruit, we investigated the activity and gene expression of β-galactosidase (β-Gal), α-l-arabinofuranosidase (α-l-Af), polygalacturonase (PG), and pectin methylesterase (PME). Their regulation by ethylene and cold storage (0°C) was also assessed. ‘Golden Delicious’ and ‘Fuji’ fruit showed differences in their rates of respiration and decline of firmness, as well as demonstrating unique regulated effects. Activities of β-Gal and α-l-Af were higher in ‘Golden Delicious’ than in ‘Fuji’ fruit, although both had similar patterns of change. They were dramatically inhibited by 1-methylcyclopropene (1-MCP) and 0°C, and enhanced by exposure to ethephon, with stronger response in ‘Golden Delicious’ fruit. Gene expression of cell wall enzymes also was significantly affected by 0°C, 1-MCP, and ethephon. The difference in α- l-Af expression among treatments in ‘Golden Delicious’ was more significant than in ‘Fuji’ fruit, especially early in storage. In contrast, expression of β-Gal was inhibited by 1-MCP at early stages in ‘Golden Delicious’ fruit and over the entire storage period in ‘Fuji’ fruit, and was significantly enhanced by ethephon treatment in the former but only slightly in the latter. At 0°C, β-Gal mRNA accumulation was inhibited in both cultivars. PG and PME activities increased during softening, and differed at different stages for each cultivar, and were obviously regulated by ethylene and 0°C. PME expression was higher in ‘Golden Delicious’ fruit, with far greater differences between cultivars than that detected for PG. Both PME and PG mRNA were more intensively influenced by ethylene and cold storage in ‘Golden Delicious’ fruit. The results confirm that cell wall enzymes play an important role in fruit softening. Of these, β-Gal and α-l-Af may be more closely related to the storability of apples than PG and PME, especially when fruit ripening and softening begin. [Copyright &y& Elsevier]

Abstract: Ascorbic acid (AsA), as a unique antioxidant and enzyme cofactor, has multiple roles in plants. However, there is very limited information on the mechanism of AsA accumulation and controlling in leaves. In this study, we determined AsA accumulation levels, analyzed expression patterns of the genes involved in synthesizing via l-galactose pathway and recycling as well as enzyme activities in apple (Malus domestica Borkh) leaves with different age. AsA content was found to increase with leaf development, reaching the highest level in 20-day-old leaves. This level was maintained in mature leaves until the dropping in senescent leaves. Comparing with young and senescent leaves, mature leaves had higher capability for AsA synthesis with high expression levels and activity of l-galactose dehydrogenase and l-galactono-1,4-lactone dehydrogenase. The mRNA expression of genes involved in AsA synthesis also showed highest abundance in 20-day-old leaves, though GDP-mannose-3′,5′-epimerase and l-galactose-1-phosphate phosphatase expression reached the highest levels before 20 days old. These results suggest that AsA accumulation in apple leaves mainly occurs during the transition phase from young to mature leaves with high rates of synthesis and recycling, and that l-galactose-1-phosphate phosphatase could play an important role in regulating AsA biosynthesis via the l-galactose pathway. [Copyright &y& Elsevier]

Block copolymers with terpyridyl-metal ion pendant were prepared by ring-opening metathesis polymerization, and self-assembled into nanostructure in the selective solvent. The dielectric properties of polymers could be well regulated by adjusting the content of metal ions. The dielectric constant of Ru(II)-contained block copolymer reached high value of 32.5, the dielectric loss was lowered to 0.01 in the frequency range of 100 Hz to 1 MHz, and the energy storage density was up to 5.2 J·cm−3 at the electric field strength of 325 mV·m−1 accompanied with the charge-discharge efficiency of 83.3%, which was attributed to the electronic polarization and interface polarization from the metal ions and nanostructure. Therefore, the introduction of metal ion into the side chain was an effective way to enhance the dielectric and energy storage properties of polymers. [Display omitted] • Block copolymers with terpyridyl-metal ion complex were synthesized by ROMP. • Ru(II)-contained block copolymer displayed high dielectric constant of 32.5 and low dielectric loss of 0.01. • Ru(II)-contained block copolymer exhibited high energy storage density of 5.2 J cm−3 with the efficiency of 83.3%. [ABSTRACT FROM AUTHOR]

Approximately 80 days after full bloom, well-exposed fruit on the south part of the canopy of mature Liberty/M9 apple (Malus domestica Borkh.) trees were randomly assigned to one of the following two treatments. Some fruits were turned about 180° to expose the original shaded side to full sun, whereas the rest served as untreated controls. On days 0, 1, 2, 4, 7 and 10 after treatment, fruit peel samples were taken from the original shaded side of the treated fruit and both the sun-exposed side and the shaded side of the control fruit at midday, to determine photosynthetic pigments, enzymatic and non-enzymatic antioxidants. Maximum photosystem II (PSII) efficiency, measured as Fv/Fm (maximum fluorescence) of chlorophyll fluorescence at pre-dawn, was higher in the shaded side than the sun-exposed side of the control fruit. Fv/Fm of the original shaded side decreased sharply after 1 day exposure to full sun, and then gradually recovered to a similar value of the sun-exposed side of the control fruit by day 10. The shaded side of the control fruit had much lower xanthophyll cycle pool size, conversion and antioxidant enzymes and also soluble antioxidants of the ascorbate–glutathione cycle than the sun-exposed side. Xanthophyll cycle pool size of the original shaded side increased in response to full sun exposure, reaching a similar value of the sun-exposed side by day 10. Almost all the xanthophyll cycle pool was converted to zeaxanthin and antheraxanthin starting from the first day of exposure to full sun. In contrast, chlorophyll content decreased whereas lutein or neoxanthin content remained unchanged. In response to full sun exposure, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase and total pool size and reduction state of both ascorbate and glutathione, all increased to the corresponding values found in the sun-exposed side of the control fruit over a 10-day period. It is concluded that both xanthophyll cycle and the ascorbate–glutathione cycle in the original shaded side are up-regulated in response to full sun exposure to minimize photo-oxidative damage and contributes to its acclimation to full sun. [Copyright &y& Elsevier]

The objective of this study was to determine how xanthophyll cycle-dependent thermal dissipation and the antioxidant system in the peel of apple fruit respond to the natural light exposure within the tree canopy. Fruit from exterior and interior canopies of both mature ‘Gala’ and ‘Smoothee’ apple trees were sampled at noon and/or predawn to measure chlorophyll fluorescence, xanthophyll cycle pool size and composition, and enzymatic and non-enzymatic antioxidants. Compared with the shaded side, the sun-exposed peel of the fruit had more excess absorbed photon flux density (PFD) as a result of a lower photosystem II operating efficiency and a higher incident PFD at noon. The efficiency of excitation transfer was lower in the sun-exposed peel than in the shaded peel, indicative of higher thermal dissipation. The sun-exposed peel had a larger xanthophyll cycle pool size and a higher conversion state. It also had higher activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, and a larger size and a higher reduction state of the ascorbate pool and the glutathione pool. However, catalase activity was lower in the sun-exposed peel than in the shaded peel. Superoxide dismutase did not show significant trend with regard to fruit peel type or position in the canopy. We conclude that both the xanthophyll cycle and the ascorbate–glutathione pathway in the apple fruit peel are acclimated to the prevailing light exposure within the tree canopy to meet the respective needs for dissipating excess absorbed PFD and detoxifying reactive oxygen species. [Copyright &y& Elsevier]

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 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: 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]

Apple Valsa canker, caused by the ascomycete fungus Valsa mali, employs virulence effectors to disturb host immunity and poses a substantial threat to the apple industry. However, our understanding of how V. mali effectors regulate host defense responses remains limited. Here, we identified the V. mali effector Vm_04797, which was upregulated during the early infection stage. Vm_04797, a secreted protein, suppressed Inverted formin 1 (INF1)-triggered cell death in Nicotiana benthamiana and performed virulence functions inside plant cells. Vm_04797 deletion mutants showed substantially reduced virulence toward apple. The adaptor protein MdAP-2β positively regulated apple Valsa canker resistance and was targeted and degraded by Vm_04797 via the ubiquitination pathway. The in vitro analysis suggested that Vm_04797 possesses E3 ubiquitin ligase activity. Further analysis revealed that MdAP-2β is involved in autophagy by interacting with Malus domestica autophagy protein 16 MdATG16 and promoting its accumulation. By degrading MdAP-2β, Vm_04797 inhibited autophagic flux, thereby disrupting the defense response mediated by autophagy. Our findings provide insights into the molecular mechanisms employed by the effectors of E3 ubiquitin ligase activity in ascomycete fungi to regulate host immunity. Effectors with E3 ubiquitin ligase activity in ascomycete fungi regulate host immunity. [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]

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]

Polymers with strong polar side groups of combined push-pull azobenzene and triphenylamine were synthesized by ring-opening metathesis polymerization, and exhibited excellent dielectric and energy storage properties mainly due to highly dipolar polarization. The dielectric constant of polymer containing one azobenzene group and one triphenylamine group was 13.5, which was enhanced to 15.7 when one azobenzene group and two triphenylamine groups were incorporated into polymer in virtue of its higher dipole density. Besides, the dielectric loss of polymers was lowered to 0.004–0.025, and the stored-energy density of polymer can reach up to 5.09 J cm−3 at electric field strength of 270 MV m−1 with good charge-discharge efficiency of 86.4%. Therefore, polymers are promising for dielectric and electrical energy storage applications. Image 1 • Polymer with azobenzene and triphenylamine side groups was synthesized by ROMP. • Polymer displayed high dielectric constant of 15.7 and low dielectric loss of 0.01. • Polymer exhibited energy storage density of 5.09 J cm−3 with efficiency of 86.4%. [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]

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]

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]

Auxin plays important roles throughout plant growth and development. However, the mechanisms of auxin regulation of plant structure are poorly understood. In this study, we identified a transcription factor (TF) of the BARLEY B RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family in apple (Malus × domestica), MdBPC2. It was highly expressed in dwarfing rootstocks, and it negatively regulated auxin biosynthesis. Overexpression of MdBPC2 in apple decreased plant height, altered leaf morphology, and inhibited root system development. These phenotypes were due to reduced auxin levels and were restored reversed after exogenous indole acetic acid (IAA) treatment. Silencing of MdBPC2 alone had no obvious phenotypic effect, while silencing both Class I and Class II BPCs in apple significantly increased auxin content in plants. Biochemical analysis demonstrated that MdBPC2 directly bound to the GAGA-rich element in the promoters of the auxin synthesis genes MdYUC2a and MdYUC6b, inhibiting their transcription and reducing auxin accumulation in MdBPC2 overexpression lines. Further studies established that MdBPC2 interacted with the polycomb group (PcG) protein LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) to inhibit MdYUC2a and MdYUC6b expression via methylation of histone 3 lysine 27 (H3K27me3). Silencing MdLHP1 reversed the negative effect of MdBPC2 on auxin accumulation. Our results reveal a dwarfing mechanism in perennial woody plants involving control of auxin biosynthesis by a BPC transcription factor, suggesting its use for genetic improvement of apple rootstock. MdYUC2a and MdYUC6b are targets of H3K27me3 modification mediated by MdBPC2-MdLHP1, which regulates auxin biosynthesis during apple growth. [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]

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]

Drought stress is a key environmental factor limiting the productivity, quality, and geographic distribution of crops worldwide. Abscisic acid (ABA) plays an important role in plant drought stress responses, but the molecular mechanisms remain unclear. Here, we report an ABA-responsive bHLH transcription factor, MdbHLH160, which promotes drought tolerance in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica). Under drought conditions, MdbHLH160 is directly bound to the MdSOD1 (superoxide dismutase 1) promoter and activated its transcription, thereby triggering reactive oxygen species (ROS) scavenging and enhancing apple drought tolerance. MdbHLH160 also promoted MdSOD1 enzyme activity and accumulation in the nucleus through direct protein interactions, thus inhibiting excessive nuclear ROS levels. Moreover, MdbHLH160 directly upregulated the expression of MdDREB2A-like, a DREB (dehydration-responsive element binding factor) family gene that promotes apple drought tolerance. Protein degradation and ubiquitination assays showed that drought and ABA treatment stabilized MdbHLH160. The BTB protein MdBT2 was identified as an MdbHLH160-interacting protein that promoted MdbHLH160 ubiquitination and degradation, and ABA treatment substantially inhibited this process. Overall, our findings provide insights into the molecular mechanisms of ABA-modulated drought tolerance at both the transcriptional and post-translational levels via the ABA–MdBT2–MdbHLH160–MdSOD1/MdDREB2A-like cascade. The ABA-responsive transcription factor MdbHLH160 enhances apple drought tolerance by promoting MdSOD1 and MdDREB2A-like expression and undergoes MdBT2-mediated ubiquitination and degradation. [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]

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: 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]

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]

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]

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]

Background and aims: As the immobility of inorganic phosphorus (P) in soil, the acquisition of P by sessile plants is limited. γ-Aminobutyric acid (GABA) as a signal molecule and a metabolite can regulate plants to cope with various stresses. However, whether GABA could contribute to the adaption to low P stress in apple plants remains unclear. Methods: This study combined different methods to detect the induction of auxin (IAA) synthesis by GABA to improve the tolerance of apple seedlings to low P stress (including growth and development analysis, reactive oxygen species (ROS) clearance effect, root structure analysis, multiple factorial analysis (MFA), gene expression analysis). Results: Exogenous GABA improved the growth of apple seedlings under low P conditions, reduced the ROS accumulation, and promoted the photosynthetic capacity. GABA contributed to the root system architecture and the development of mature area of root tips. In addition, the intervention of exogenous GABA interfered with the homeostasis of endogenous IAA and activated the expression of P starvation induction (PSI) gene, leading to the significantly increase of the P uptake in apple plants. Meanwhile, transgenic roots with overexpressing MdGAD1 enhanced the tolerance of apple seedlings to low P stress, improved the root development through regulating IAA signaling pathway, and significantly improved the P uptake of apple seedlings under low P conditions. Conclusion: All results suggested that GABA could contribute to the adaption of apple seedlings to low P conditions, by decreasing ROS accumulation, maintaining photosynthetic capacity, and increasing IAA level to improve the root development and the P absorption. [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]

Invertases are ubiquitous enzymes that catalyze the unalterable cleavage of sucrose into glucose and fructose, and are crucially involved in plant growth, development and stress response. In this study, a total of 17 putative invertase genes, including 3 cell wall invertases, 3 vacuolar invertases, and 11 neutral invertases were identified in apple genome. Subcellular localization of MdNINV7 and MdNINV11 indicated that both invertases were located in the cytoplasm. Comprehensive analyses of physicochemical properties, chromosomal localization, genomic characterization, and gene evolution of MdINV family were conducted. Gene duplication revealed that whole-genome or segmental duplication and random duplication might have been the major driving force for MdINVs expansion. Selection index values, ω, showed strong evidence of positive selection signatures among the INV clusters. Gene expression analysis indicated that MdNINV1/3/6/7 members are crucially involved in fruit development and sugar accumulation. Similarly, expression profiles of MdCWINV1 , MdVINV1 , and MdNINV1/2/7/11 suggested their potential roles in response to cold stress. Furthermore, overexpression of MdNINV11 in apple calli at least in part promoted the expression of MdCBF1–5 and H 2 O 2 detoxification in response to cold. Overall, our results will be useful for understanding the functions of MdINVs in the regulation of apple fruit development and cold stress response. [ABSTRACT FROM AUTHOR]

Plant epistatic regulation is the DNA methylation, non-coding RNA regulation, and histone modification of gene sequences without altering the genome sequence, thus regulating gene expression patterns and the growth process of plants to produce heritable changes. Epistatic regulation in plants can regulate plant responses to different environmental stresses, regulate fruit growth and development, etc. Genome editing can effectively improve plant genetic efficiency by targeting the design and efficient editing of genome-specific loci with specific nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9). As research progresses, the CRISPR/Cas9 system has been widely used in crop breeding, gene expression, and epistatic modification due to its high editing efficiency and rapid translation of results. In this review, we summarize the recent progress of CRISPR/Cas9 in epigenome editing and look forward to the future development direction of this system in plant epigenetic modification to provide a reference for the application of CRISPR/Cas9 in genome editing. [ABSTRACT FROM AUTHOR]

Melatonin enhances plant tolerance to various environmental stressors. Although exogenous application of melatonin has been investigated, the role of endogenous melatonin metabolism in the response of apples to drought stress and nutrient utilization remains unclear. Here, we investigated the effects of ectopically expressing the human melatonin synthase gene HIOMT on transgenic apple plants under drought stress conditions. The tolerance of transgenic apple lines that ectopically expressed HIOMT improved significantly under drought conditions. After 10 days of natural drought stress treatment, the transgenic apple plants showed higher relative water content, chlorophyll levels and Fv/Fm , and lower relative electrolyte leakage and hydrogen peroxide accumulation, than wild-type plants. The activities of peroxidase, superoxide dismutase and catalase, as well as genes in the ascorbate–glutathione cycle, increased more in transgenic apple plants than in the wild-type. The ectopic expression of HIOMT also markedly alleviated the inhibitory effects of long-term drought stress on plant growth, photosynthetic rate and chlorophyll concentrations in apple plants. The uptake and utilization of 15N increased markedly in the transgenic lines under long-term moderate drought stress. Drought stress sharply reduced the activity of enzymes involved in nitrogen metabolism, but ectopic expression of HIOMT largely reversed that response. The expression levels of genes of nitrogen metabolism and uptake were more upregulated in transgenic apple plants than the wild-type. Overall, our study demonstrates that ectopic expression of HIOMT enhanced the tolerance of apple plants to drought stress, and transgenic apple plants showed improved growth due to higher nutrient utilization efficiency under drought conditions. [ABSTRACT FROM AUTHOR]

Drought is a major abiotic stress limiting the growth and production of apple trees worldwide. The receptor-like kinase FERONIA is involved in plant growth, development and stress responses; however, the function of FERONIA in apple under drought stress remains unclear. Here, the FERONIA receptor kinase gene MdMRLK2 from apple (Malus domestica) was shown to encode a plasma membrane-localized transmembrane protein and was significantly induced by abscisic acid and drought treatments. 35S::MdMRLK2 apple plants showed less photosystem damage and higher photosynthetic rates compared with wild-type (WT) plants, after withholding water for 7 days. 35S::MdMRLK2 apple plants also had enhanced energy levels, activated caspase activity and more free amino acids, than the WT, under drought conditions. By performing yeast two-hybrid screening, glyceraldehyde-3-phosphate dehydrogenase and MdCYS4, a member of cystatin, were identified as MdMRLK2 interaction partners. Moreover, under drought conditions, the 35S::MdMRLK2 apple plants were characterized by higher abscisic acid (ABA) content. Overall, these findings demonstrated that MdMRLK2 regulates apple drought tolerance, probably via regulating levels of energetic matters, free amino acids and ABA. [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]

As a precursor of aromatic compounds, fatty acids play important roles in apple fruit quality; however, the genetic and molecular basis underlying fatty acid synthesis and metabolism is largely unknown. In this study, we conducted a genome-wide association study (GWAS) of seven fatty acids using genomic data of 149 Malus accessions and identified 232 significant signals (–log10 P >5) associated with 99 genes from GWAS of four fatty acids across 2 years. Among these, a significant GWAS signal associated with linoleic acid was identified in the transcriptional regulator SUPERMAN-like (SUP) MD13G1209600 at chromosome 13 of M. × domestica. Transient overexpression of MdSUP increased the contents of linoleic and linolenic acids and of three aromatic components in the fruit. Our study provides genetic and molecular information for improving the flavor and nutritional value of apple. [ABSTRACT FROM AUTHOR]

Drought stress is an important factor limiting apple production. γ-Aminobutyric acid (GABA) exists widely in plants and participates in the response to abiotic stress as a metabolite or signaling molecule. The role of exogenous GABA in apple plants, response to long-term drought stress remains unclear. Our study confirmed that exogenous GABA affects the drought resistance of apple plants under long-term drought stress. We found that 1 mM exogenous GABA improved the resistance of apple seedlings to long-term drought stress. The plants showed better growth, less reactive oxygen radical accumulation, less damage to cell membranes and greater active photosynthetic capacity. Under long-term drought stress, exogenous GABA facilitated GABA shunt, resulting in more accumulation of organic acids, namely citric acid, succinic acid and malic acid, in roots and stems of apple seedlings. In addition, exogenous GABA upregulated the expression of cellulose-related genes and lignin-related genes, and activated secondary cell wall-related transcription factors to synthesize more cellulose and lignin. A multiple factorial analysis confirmed that the GABA shunt and the biosynthesis of cellulose and lignin substantially contributed to the growth of apple seedlings with the application of exogenous GABA under long-term drought stress. Our results suggested that exogenous GABA improved the resistance of apple seedlings to long-term drought stress by enhancing GABA shunt and secondary cell wall biosynthesis. [ABSTRACT FROM AUTHOR]

The photoprotective role of anthocyanin remains controversial. In this study, we explored the effects of anthocyanin on photosynthesis and photoprotection using transgenic 'Galaxy Gala' apple plants overexpressing MdMYB10 under high light stress. The overexpression of MdMYB10 dramatically enhanced leaf anthocyanin accumulation, allowing more visible light to be absorbed, particularly in the green region. However, through post-transcriptional regulation, anthocyanin accumulation lowered leaf photosynthesis in both photochemical reaction and CO2 fixation capacities. Anthocyanin accumulation also led to a decreased de-epoxidation state of the xanthophyll cycle and antioxidant capacities, but this is most likely a response to the light-shielding effect of anthocyanin, as indicated by a higher chlorophyll concentration and lower chlorophyll a/b ratio. Under laboratory conditions when detached leaves lost carbon fixation capacity due to the limitation of CO2 supply, the photoinhibition of detached transgenic red leaves was less severe under strong white, green, or blue light, but it became more severe in response to strong red light compared with that of the wild type. In field conditions when photosynthesis was performed normally in both green and transgenic red leaves, the degree of photoinhibition was comparable between transgenic red leaves and wild type leaves, but it was less severe in transgenic young shoot bark compared with the wild type. Taken together, these data show that anthocyanin protects plants from high light stress by absorbing excessive visible light despite reducing photosynthesis. [ABSTRACT FROM AUTHOR]

Drought resistance in plants is influenced by multiple signaling pathways that involve various transcription factors, many target genes, and multiple types of epigenetic modifications. Studies on epigenetic modifications of drought focus on DNA methylation and histone modifications, with fewer on chromatin remodeling. Changes in chromatin accessibility can play an important role in abiotic stress in plants by affecting RNA polymerase binding and various regulatory factors. However, the changes in chromatin accessibility during drought in apples are not well understood. In this study, the landscape of chromatin accessibility associated with the gene expression of apple (GL3) under drought conditions was analyzed by Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA-seq. Differential analysis between drought treatment and control identified 23,466 peaks of upregulated chromatin accessibility and 2447 peaks of downregulated accessibility. The drought-induced chromatin accessibility changed genes were mainly enriched in metabolism, stimulus, and binding pathways. By combining results from differential analysis of RNA-seq and ATAC-seq, we identified 240 genes with higher chromatin accessibility and increased gene expression under drought conditions that may play important functions in the drought response process. Among them, a total of nine transcription factor genes were identified, including ATHB7, HAT5, and WRKY26. These transcription factor genes are differentially expressed with different chromatin accessibility motif binding loci that may participate in apple response to drought by regulating downstream genes. Our study provides a reference for chromatin accessibility under drought stress in apples and the results will facilitate subsequent studies on chromatin remodelers and transcription factors. [ABSTRACT FROM AUTHOR]

The apple rootstock Malus prunifolia (Willd.) Borkh. is widely used for apple production. Because polyploid plants are often more tolerant to abiotic stress than diploids, we wondered whether polyploidy induction in M. prunifolia might improve its stress tolerance, particularly to high salinity. We used a combination of colchicine and dimethyl sulfoxide (DMSO) to induce chromosome doubling in M. prunifolia and identified the resulting polyploids by stomatal observations and flow cytometry. We found the best way to induce polyploidy in M. prunifolia was to use 2% DMSO and 0.05% colchicine for 2 days for leaves or 0.02% colchicine for stem segments. The results of hydroponic salt treatment showed that polyploid plants were more salt tolerant and had greater photosynthetic efficiency, thicker leaf epidermis and palisade tissues, and shorter but denser root systems than diploids. During salt stress, the polyploid leaves and roots accumulated less Na+, showed upregulated expression of three salt overly sensitive (SOS) pathway genes, and produced fewer reactive oxygen species. The polyploid plants also had considerably higher ABA and jasmonic acid levels than diploid plants under salt stress. Under normal growth conditions, gibberellins (GAs) levels were much lower in polyploid leaves than in diploid leaves; however, after salt treatment, polyploid leaves showed upregulation of essential GAs synthesis genes. In summary, we developed a system for the induction of polyploidy in M. prunifolia and response to salt stress of the resulting polyploids, as reflected in leaf and root morphology, changes in Na+ accumulation, antioxidant capacity and plant hormone levels. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is associated with gastric disorders. In this study, the anti‐H. pylori capacity of natural products from "Winter Red" crabapple flowers were evaluated, including flavonoids, organic acids, terpenoids, and phenolic acids. Among these products, kaempferol showed the highest antibacterial capacity. Structure–activity relationship assays indicated that all hydroxyls contributed to the antibacterial capacity of kaempferol, with the most important being hydroxyls in the A‐ring. Kaempferol had comparable anti‐H. pylori capacities to clarithromycin and amoxicillin. Both kaempferol and the two antibiotics might damage the cell membrane of H. pylori. However, the RNA‐sequence assay demonstrated that their antibacterial mechanisms were different. The ATP‐binding cassette transporters, flagellar assembly, and fatty acid metabolism were the major pathways in H. pylori cells responding to kaempferol treatment. We suggest that crabapple containing abundant kaempferol may benefit humans by inhibiting gastric H. pylori. Practical applications: The anti‐Helicobacter pylori capacity of natural products including flavonoids, organic acids, terpenoids, and phenolic acids from "Winter Red" crabapple flowers were evaluated in the present study. Among these products, kaempferol showed the highest antibacterial capacity. More importantly, kaempferol had comparable anti‐H. pylori capacities to clarithromycin and amoxicillin, but in a manner distinct from the antibiotics. We suggest that crabapple flowers containing abundant kaempferol may be processed into various products for the patients who have gastric disorders caused by H. pylori. Or the extracted kaempferol from crabapples or other plants could be tested for the clinical treatment of gastric H. pylori. [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]

Apples with red fruit flesh are desirable for breeding because they contain more health-beneficial compounds. That color trait is also considered more attractive to consumer. To obtain experimental evidence about the molecular mechanism for this pigmentation, we investigated how the practice of bagging fruit might affect levels of phenolic compounds as well as the expression of anthocyanin biosynthetic and regulatory genes. Two red-fleshed varieties from Xinjiang, P.R. China, were studied – ‘Xiahongrou’ and ‘No.1 Hongxun’. HPLC analysis showed that the contents of anthocyanins in both peel and flesh were decreased by bagging, but were increased again when fruits were re-exposed to sunlight. Except for F3H in ‘No.1 Hongxun’, transcription levels for most anthocyanin biosynthetic genes in the flesh were enhanced after bag removal. MYB10 is a key transcriptional factor in the anthocyanin pathway of these two apple varieties, particularly in the flesh. These results suggest that the development of red coloring when bags are removed could be a result of light-induced coordinated expression by MYB10 and anthocyanin biosynthetic genes in red-fleshed apples. [ABSTRACT FROM AUTHOR]