The transcription factor MdBPC2 alters apple growth and promotes dwarfing by regulating auxin biosynthesis.

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.

The MdHSC70-MdWRKY75 module mediates basal apple thermotolerance by regulating the expression of heat shock factor genes.

Heat stress severely restricts the growth and fruit development of apple (Malus domestica). Little is known about the involvement of WRKY proteins in the heat tolerance mechanism in apple. In this study, we found that the apple transcription factor MdWRKY75 responds to heat and positively regulates basal thermotolerance. Apple plants that overexpressed MdWRKY75 were more tolerant to heat stress, while silencing MdWRKY75 caused the opposite phenotype. RNA-seq and reverse transcription quantitative PCR showed that heat shock transcription factor genes (MdHsfs) could be the potential targets of MdWRKY75. Electrophoretic mobility shift, yeast one-hybrid, ß-glucuronidase, and dual-luciferase assays showed that MdWRKY75 can bind to the promoters of MdHsf4, MdHsfB2a, and MdHsfA1d and activate their expression. Apple plants that overexpressed MdHsf4, MdHsfB2a, and MdHsfA1d exhibited heat tolerance and rescued the heat sensitive phenotype of MdWRKY75-Ri3. In addition, apple heat shock cognate 70 (MdHSC70) interacts with MdWRKY75, as shown by yeast two-hybrid, split luciferase, bimolecular fluorescence complementation, and pull-down assays. MdHSC70 acts as a negative regulator of the heat stress response. Apple plants that overexpressed MdHSC70 were sensitive to heat, while virus-induced gene silencing of MdHSC70 enhanced heat tolerance. Additional research showed that MdHSC70 exhibits heat sensitivity by interacting with MdWRKY75 and inhibiting MdHsfs expression. In summary, we proposed a mechanism for the response of apple to heat that is mediated by the 'MdHSC70/MdWRKY75-MdHsfs' molecular module, which enhances our understanding of apple thermotolerance regulated by WRKY transcription factors.

Elongation factor MdEF-Tu coordinates with heat shock protein MdHsp70 to enhance apple thermotolerance.

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.

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

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

Origin and early divergence of tandem duplicated sorbitol transporter genes in Rosaceae: insights from evolutionary analysis of the SOT gene family in angiosperms.

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.

Melatonin confers tolerance to nitrogen deficiency through regulating MdHY5 in apple plants.

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.

The MdCBF1/2-MdTST1/2 module regulates sugar accumulation in response to low temperature in apple.

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.

MdbHLH160 is stabilized via reduced MdBT2-mediated degradation to promote MdSOD1 and MdDREB2A-like expression for apple drought tolerance.

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.

Valsa mali effector Vm_04797 interacts with adaptor protein MdAP-2ß to manipulate host autophagy.

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-2ß 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-2ß is involved in autophagy by interacting with Malus domestica autophagy protein 16 MdATG16 and promoting its accumulation. By degrading MdAP-2ß, 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.

Transcriptional factor MdESE3 controls fruit acidity by activating genes regulating malic acid content in apple.

Acidity is a key factor controlling fruit flavor and quality. In a previous study, combined transcriptome and methylation analyses identified a P3A-type ATPase from apple (Malus domestica), MdMa11, which regulates vacuolar pH when expressed in Nicotiana benthamiana leaves. In this study, the role of MdMa11 in controlling fruit acidity was verified in apple calli, fruits, and plantlets. In addition, we isolated an AP2 domain-containing transcription factor, designated MdESE3, based on yeast one-hybrid (Y1H) screening using the MdMa11 promoter as bait. A subcellular localization assay indicated that MdESE3 localized to the nucleus. Analyses of transgenic apple calli, fruits, and plantlets, as well as tomatoes, demonstrated that MdESE3 enhances fruit acidity and organic acid accumulation. Meanwhile, chromatin immunoprecipitation quantitative PCR (ChIP-qPCR), luciferase (LUC) transactivation assays, and GUS reporter assays indicated that MdESE3 could bind to the ethylene-responsive element (ERE; 5'-TTTAAAAT-3') upstream of the MdMa11 transcription start site, thereby activating its expression. Furthermore, MdtDT, MdDTC2, and MdMDH12 expression increased in apple fruits and plantlets overexpressing MdESE3 and decreased in apple fruits and plantlets where MdESE3 was silenced. The ERE was found in MdtDT and MdMDH12 promoters, but not in the MdDTC2 promoter. The Y1H, LUC transactivation assays, and GUS reporter assays indicated that MdESE3 could bind to the MdtDT and MdMDH12 promoters and activate their expression. Our findings provide valuable functional validation of MdESE3 and its role in the transcriptional regulation of MdMa11, MdtDT, and MdMDH12 and malic acid accumulation in apple.

Age-dependent changes in leaf size in apple are governed by a cytokinin-integrated module.

Plants undergo various age-dependent changes in leaf morphology during juvenile to adult vegetative stage. However, the precise molecular mechanisms governing these changes in apple (Malus domestica) remain unknown. Here, we showed that CYTOKININ OXIDASE/DEHYDROGENASE5 (MdCKX5), an age-dependent gene, encodes a functional CKX enzyme and serves as the common downstream target of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor MdSPL14 and WRKY transcription factor MdWRKY24 to control the degradation of cytokinin (CK). As the target of mdm-microRNA156a, MdSPL14 interacts with MdWRKY24 to coordinately repress the transcription of MdCKX5 by forming the age-mediated mdm-miR156a-MdSPL14-MdWRKY24 module, which regulates age-dependent changes in CK during the juvenile-to-adult phase transition. We further demonstrated that MdARR6, a type-A ARABIDOPSIS RESPONSE REGULATOR (ARR), is a negative feedback regulator in the CK signaling pathway. Silencing of MdARR6 in apple resulted in large leaves with smaller epidermal cells and a greater number of epidermal cells. Biochemical analysis showed that the mdm-miR156a-MdSPL14-MdWRKY24 module acts as a transcriptional repressor to directly regulate MdARR6 expression, thus controlling the age-dependent changes in leaf size by reducing CK responses. These findings established a link between the age pathway and CK signaling and revealed the molecular mechanism underlying age-dependent changes during the juvenile-to-adult phase transition; our results also provide targets for the genetic improvement of the vegetative phase transition in apple.

Transcriptional Landscape and Dynamics Involved in Sugar and Acid Accumulation during Apple Fruit Development.

In fleshy fruit, sugars and acids are central components of fruit flavor and quality. To date, the mechanisms underlying transcriptional regulation of sugar and acid during fruit development remain largely unknown. Here, we combined ATAC-seq with RNA-seq to investigate the genome-wide chromatin accessibility and to identify putative transcription factors related to sugar and acid accumulation during apple (Malus domestica) fruit development. By integrating the differentially accessible regions (DARs) and differentially expressed genes (DEGs), we generated a global dataset of promoter-accessibility- and expression-increased genes (PEIGs). Using this strategy, we constructed a transcriptional regulatory network enabling screening for key transcription factors and target genes involved in sugar and acid accumulation. Among these transcription factors, five fruit-specific Dof (DNA binding with one finger) genes were selected to confirm their regulatory effects, and our results showed that they could affect sugar or acid concentration by regulating the expression of sugar or acid metabolism-related genes in apple fruits. Our transcriptional regulatory network provides a suitable platform to identify candidate genes that control sugar and acid accumulation. Meanwhile, our dataset will aid in analyzing other characteristics of apple fruit that have not been illuminated previously. Overall, these findings support a better understanding of the regulatory dynamics during apple fruit development and lay a foundation for quality improvement of apple.

An insertion in the promoter of a malate dehydrogenase gene regulates malic acid content in apple fruit.

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

Transcription factor PbNAC71 regulates xylem and vessel development to control plant height.

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.

Methylation of a MITE insertion in the MdRFNR1-1 promoter is positively associated with its allelic expression in apple in response to drought stress.

Miniature inverted-repeat transposable elements (MITEs) are widely distributed in the plant genome and can be methylated. However, whether DNA methylation of MITEs is associated with induced allelic expression and drought tolerance is unclear. Here, we identified the drought-inducible MdRFNR1 (root-type ferredoxin-NADP+ oxidoreductase) gene in apple (Malus domestica). MdRFNR1 plays a positive role in drought tolerance by regulating the redox system, including increasing NADP+ accumulation and catalase and peroxidase activities and decreasing NADPH levels. Sequence analysis identified a MITE insertion (MITE-MdRF1) in the promoter of MdRFNR1-1 but not the MdRFNR1-2 allele. MdRFNR1-1 but not MdRFNR1-2 expression was significantly induced by drought stress, which was positively associated with the MITE-MdRF1 insertion and its DNA methylation. The methylated MITE-MdRF1 is recognized by the transcriptional anti-silencing factors MdSUVH1 and MdSUVH3, which recruit the DNAJ domain-containing proteins MdDNAJ1, MdDNAJ2, and MdDNAJ5, thereby activating MdRFNR1-1 expression under drought stress. Finally, we showed that MdSUVH1 and MdDNAJ1 are positive regulators of drought tolerance. These findings illustrate the molecular roles of methylated MITE-MdRF1 (which is recognized by the MdSUVH-MdDNAJ complex) in induced MdRFNR1-1 expression as well as the drought response of apple and shed light on the molecular mechanisms of natural variation in perennial trees.

MdHB7-like positively modulates apple salt tolerance by promoting autophagic activity and Na+ efflux.

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.

Overexpression of the FERONIA receptor kinase MdMRLK2 enhances apple cold tolerance.

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.

Mdm-miR160-MdARF17-MdWRKY33 module mediates freezing tolerance in apple.

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 H2 O2 levels and decreased peroxidase (POD) and catalase (CAT) activities in Mdm-miR160e OE plants and MdARF17 RNAi plants and (ii) decreased H2 O2 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.

Transcriptional repression of MdMa1 by MdMYB21 in Ma locus decreases malic acid content in apple fruit.

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.

Dihydrochalcone glycoside biosynthesis in Malus is regulated by two MYB-like transcription factors and is required for seed development.

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.