Insights into ACO genes across Rosaceae: evolution, expression, and regulatory networks in fruit development.

BACKGROUND: ACO (1-aminocyclopropane-1-carboxylic acid) serves as a pivotal enzyme within the plant ethylene synthesis pathway, exerting influence over critical facets of plant biology such as flowering, fruit ripening, and seed development. OBJECTIVE: This study aims to identify ACO genes from representative Rosaceae genomes, reconstruct their phylogenetic relationships by integrating synteny information, and investigate their expression patterns and networks during fruit development. METHODS: we utilize a specialized Hidden Markov Model (HMM), crafted on the sequence attributes of ACO gene-encoded proteins, to systematically identify and analyze ACO gene family members across 12 representative species within the Rosaceae botanical family. Through transcriptome analysis, we delineate the expression patterns of ACO genes in six distinct Rosaceae fruits. RESULTS: Our investigation reveals the presence of 62 ACO genes distributed among the surveyed Rosaceae species, characterized by hydrophilic proteins predominantly expressed within the cytoplasm. Phylogenetic analysis categorizes these ACO genes into three discernible classes, namely Class I, Class II, and Class III. Further scrutiny via collinearity assessment indicates a lack of collinearity relationships among these classes, highlighting variations in conserved motifs and promoter types within each class. Transcriptome analysis unveils significant disparities in both expression levels and trends of ACO genes in fruits exhibiting respiratory bursts compared to those that do not. Employing Weighted Gene Co-Expression Network Analysis (WGCNA), we discern that the co-expression correlation of ACO genes within loquat fruit notably differs from that observed in apples. Our findings, derived from Gene Ontology (GO) enrichment results, signify the involvement of ACO genes and their co-expressed counterparts in biological processes linked to terpenoid metabolism and carbohydrate synthesis in loquat. Moreover, our exploration of gene regulatory networks (GRN) highlights the potential pivotal role of the GNAT transcription factor (Ejapchr1G00010380) in governing the overexpression of the ACO gene (Ejapchr10G00001110) within loquat fruits. CONCLUSION: The constructed HMM of ACO proteins offers a precise and systematic method for identifying plant ACO proteins, facilitating phylogenetic reconstruction. ACO genes from representative Rosaceae fruits exhibit diverse expression and regulative patterns, warranting further function characterizations.

Sorbitol mediates age-dependent changes in apple plant growth strategy through gibberellin signaling.

Plants experience various age-dependent changes during juvenile to adult vegetative phase. However, the regulatory mechanisms orchestrating the changes remain largely unknown in apple (Malus domestica). This study showed that tissue-cultured apple plants at juvenile, transition, and adult phase exhibit age-dependent changes in their plant growth, photosynthetic performance, hormone levels, and carbon distribution. Moreover, this study identified an age-dependent gene, sorbitol dehydrogenase (MdSDH1), a key enzyme for sorbitol catabolism, highly expressed in the juvenile phase in apple. Silencing MdSDH1 in apple significantly decreased the plant growth and GA3 levels. However, exogenous GA3 rescued the reduced plant growth phenotype of TRV-MdSDH1. Biochemical analysis revealed that MdSPL1 interacts with MdWRKY24 and synergistically enhance the repression of MdSPL1 and MdWRKY24 on MdSDH1, thereby promoting sorbitol accumulation during vegetative phase change. Exogenous sorbitol application indicated that sorbitol promotes the transcription of MdSPL1 and MdWRKY24. Notably, MdSPL1-MdWRKY24 module functions as key repressor to regulate GA-responsive gene, Gibberellic Acid-Stimulated Arabidopsis (MdGASA1) expression, thereby leading to a shift from the quick to the slow-growth strategy. These results reveal the pivotal role of sorbitol in controlling apple plant growth, thereby improving our understanding of vegetative phase change in apple.

MdVQ17 negatively regulates apple resistance to Glomerella leaf spot by promoting MdWRKY17-mediated salicylic acid degradation and pectin lyase activity.

Glomerella leaf spot (GLS) is a fungal disease caused by Colletotrichum fructicola, which severely restricts the yield and quality of apples. Valine-glutamine (VQ) proteins are transcriptional regulators involved in the regulation of plant growth and stress responses. However, little is known about the role of VQ proteins in the biotic stress response in apple. Here, a VQ gene, MdVQ17, that was highly induced by C. fructicola infection was identified. Overexpression of MdVQ17 in apple increased susceptibility to C. fructicola and significantly reduced the salicylic acid content and ß-1,3-glucanase and chitinase activities. Based on yeast two-hybrid screening, MdWRKY17, which promotes susceptibility to C. fructicola, was identified as an MdVQ17-interacting protein. Co-expression of MdVQ17 can promote the binding and transcriptional activation activity of MdWRKY17 on the promoter of Downy Mildew Resistant 6 (MdDMR6), thereby promoting MdWRKY17-mediated salicylic acid degradation. Based on DNA affinity purification sequencing, the pectin lyase-encoding gene MdPL-like was identified as a direct target of MdWRKY17. MdWRKY17 can directly bind to the promoter of MdPL-like and activate its transcription, and the binding and activation of MdWRKY17 on the MdPL-like promoter were significantly enhanced by MdVQ17 co-expression. Functional identification showed that MdPL-like promoted pectin lyase activity and susceptibility to C. fructicola. In sum, these results demonstrate that the MdVQ17-MdWRKY17 module mediates the response to C. fructicola infection by regulating salicylic acid accumulation and pectin lyase activity. Our findings provide novel insights into the mechanisms by which the VQ-WRKY complex modulates plant pathogen defense responses.

Overexpression of FERONIA receptor kinase MdMRLK2 regulates lignin accumulation and enhances water use efficiency in apple under long-term water deficit condition.

Water use efficiency (WUE) is crucial for apple tree fitness and survival, especially in response to climatic changes. The receptor-like kinase FERONIA is reportedly an essential regulator of plant stress responses, but its role in regulating WUE under water deficit conditions is unclear. Here, we found that overexpressing the apple FERONIA receptor kinase gene, MdMRLK2, enhanced apple WUE under long-term water deficit conditions. Under drought treatment, 35S::MdMRLK2 apple plants exhibited higher photosynthetic capacity and antioxidant enzyme activities than wild-type (WT) plants. 35S::MdMRLK2 apple plants also showed increased biomass accumulation, root activity, and water potential compared to WT plants. Moreover, MdMRLK2 physically interacts with and phosphorylates cinnamoyl-CoA reductase 1, MdCCR1, an enzyme essential for lignin synthesis, at position Ser260. This interaction likely contributed to increased vessel density, vascular cylinder area, and lignin content in 35S::MdMRLK2 apple plants under drought conditions. Therefore, our findings reveal a novel function of MdMRLK2 in regulating apple WUE under water deficit conditions.

Glycosylation mode of phloretin affects the morphology and stress resistance of apple plant.

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

MdSINA2-MdNAC104 Module Regulates Apple Alkaline Resistance by Affecting γ-Aminobutyric Acid Synthesis and Transport.

Soil alkalization is an adverse factor limiting plant growth and yield. As a signaling molecule and secondary metabolite, γ-aminobutyric acid (GABA) responds rapidly to alkaline stress and enhances the alkaline resistance of plants. However, the molecular mechanisms by which the GABA pathway adapts to alkaline stress remain unclear. In this study, a transcription factor, MdNAC104 is identified, from the transcriptome of the alkaline-stressed roots of apple, which effectively reduces GABA levels and negatively regulates alkaline resistance. Nevertheless, applying exogenous GABA compensates the negative regulatory mechanism of overexpressed MdNAC104 on alkaline resistance. Further research confirms that MdNAC104 repressed the GABA biosynthetic gene MdGAD1/3 and the GABA transporter gene MdALMT13 by binding to their promoters. Here, MdGAD1/3 actively regulates alkaline resistance by increasing GABA synthesis, while MdALMT13 promotes GABA accumulation and efflux in roots, resulting in an improved resistance to alkaline stress. This subsequent assays reveal that MdSINA2 interacts with MdNAC104 and positively regulates root GABA content and alkaline resistance by ubiquitinating and degrading MdNAC104 via the 26S proteasome pathway. Thus, the study reveals the regulation of alkaline resistance and GABA homeostasis via the MdSINA2-MdNAC104-MdGAD1/3/MdALMT13 module in apple. These findings provide novel insight into the molecular mechanisms of alkaline resistance in plants.

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.

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.

Transposable elements in Rosaceae: insights into genome evolution, expression dynamics, and syntenic gene regulation.

Transposable elements (TEs) exert significant influence on plant genomic structure and gene expression. Here, we explored TE-related aspects across 14 Rosaceae genomes, investigating genomic distribution, transposition activity, expression patterns, and nearby differentially expressed genes (DEGs). Analyses unveiled distinct long terminal repeat retrotransposon (LTR-RT) evolutionary patterns, reflecting varied genome size changes among nine species over the past million years. In the past 2.5 million years, Rubus idaeus showed a transposition rate twice as fast as Fragaria vesca, while Pyrus bretschneideri displayed significantly faster transposition compared with Crataegus pinnatifida. Genes adjacent to recent TE insertions were linked to adversity resistance, while those near previous insertions were functionally enriched in morphogenesis, enzyme activity, and metabolic processes. Expression analysis revealed diverse responses of LTR-RTs to internal or external conditions. Furthermore, we identified 3695 pairs of syntenic DEGs proximal to TEs in Malus domestica cv. 'Gala' and M. domestica (GDDH13), suggesting TE insertions may contribute to varietal trait differences in these apple varieties. Our study across representative Rosaceae species underscores the pivotal role of TEs in plant genome evolution within this diverse family. It elucidates how these elements regulate syntenic DEGs on a genome-wide scale, offering insights into Rosaceae-specific genomic evolution.

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

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

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.

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 and differentially expressed genes, we generated a global data set of promoter-accessibility and expression-increased genes. 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, 5 fruit-specific 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 data set 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.

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.

MdWRKY71 promotes the susceptibility of apple to Glomerella leaf spot by controlling salicylic acid degradation.

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.

The MdVQ37-MdWRKY100 complex regulates salicylic acid content and MdRPM1 expression to modulate resistance to Glomerella leaf spot in apples.

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.

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.

CERK1 compromises Fusarium solani resistance by reducing jasmonate level and undergoes a negative feedback regulation via the MMK2-WRKY71 module in apple.

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.

Identification of two key genes involved in flavonoid catabolism and their different roles in apple resistance to biotic stresses.

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.

Glucose uptake from the rhizosphere mediated by MdDOF3-MdHT1.2 regulates drought resistance in apple.

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.

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.