Studies On The Molecular Mechanism Of WRKY44-mediated Anthocyanin Biosynthesis And Malate Accumulation In Pear

Due to being a big continental country with diverse climates,China has the most plentiful Pyrus germplasm resources than other countries.In China,most pears are consumed as fresh fruit,and few quantities are used for canning and juice processing.Recently,fruit quality traits have acquired more attention in the Chinese pear industry,where quality traits clearly impact consumers’ overall acceptance of pear fruit and affect its price and market competitiveness.Indeed,consumers want products that have a good appearance and taste and are nutritious.Among these traits,fruit color is the most important quality trait and directly affects consumer appeal.The fruit color results from the accumulation of various anthocyanins,a class of flavonoids,the main water-soluble secondary metabolites.They are also considered natural antioxidants and have a variety of biological functions.Another important trait of fruit quality is acidity,which contributes to fruit taste.Acidity is caused due to the presence of organic acids,which are one of the main metabolites in fruit.Thus,peel color and acidity are important quality traits for pears.Presently,the transcription mechanism of WRKY transcription factors(TFs)for regulating the accumulation of anthocyanin is very limited,while regulating the accumulation of malate remains unclear.1)Identification and phylogenetic analysis of pear PpWRKY44 TFWRKY transcription Factor(TF),one of the main TFs in plants,is an important regulator in many developmental and physiological processes.Pear(Pyrus L.)is an economically important fleshy fruit in temperate regions;however,identifying WRKY TFs involved in anthocyanin biosynthesis and malate accumulation is very limited.This study identified a Group I WRKY TF(PpWRKY44)that may regulate anthocyanin biosynthesis and malate accumulation in pear fruit.We found that PpWRKY44 expression was upregulated in pear calli after two days of light treatment based on RNA-seq data.Phylogenetic analysis revealed that PpWRKY44 is homologous to At WRKY44 and Ph PH3;the last two were reported to regulate proanthocyanidin synthesis in the seed coat and petal cell acidity in petunia,respectively.A fluorescence examination based on GFP detection revealed that PpWRKY44-GFP is localized entirely to the nuclei,demonstrating that PpWRKY44 is a nuclear protein.These results suggest that PpWRKY44 may regulate anthocyanin biosynthesis and malate accumulation in pear fruit.2)PpWRKY44 positively regulates anthocyanin accumulation in pearAnthocyanins are a valuable source of antioxidants in the human diet and contribute to fruit coloration.In red-skinned pears,anthocyanin biosynthesis can be induced by light,in which the MYB–b HLH–WDR complex plays a critically important role in transcriptional regulation.However,the knowledge of WRKY-mediated transcriptional regulation of lightinduced anthocyanin biosynthesis is scarce in red pears.This work functionally characterized a light-inducing WRKY TF,PpWRKY44,in pear.Functional analysis based on overexpressed pear calli showed that PpWRKY44 promoted anthocyanin accumulation.Also,transiently overexpressed PpWRKY44 in pear leaves and fruit peels significantly enhanced the accumulation of anthocyanin,whereas silencing PpWRKY44 in pear fruit peels impaired induction of the accumulation of anthocyanin by light.By chromatin immunoprecipitation and electrophoretic mobility shift assay coupled to a quantitative polymerase chain reaction,we found that PpWRKY44 bound in-vivo and in-vitro to the PpMYB10 promoter,revealing it as a direct downstream target gene.Moreover,PpWRKY44 was activated by PpBBX18,a light signal transduction pathway component.Our results explained the mechanism mediating the impacts of PpWRKY44 on the transcriptional regulation of anthocyanin accumulation,with potential implications for fine-tuning the fruit peel coloration triggered by light in red pears.3)PpWRKY44 positively regulates malate accumulation in pearMalate impacts fruit acidity and plays a vital role in stress tolerance.It has been known that malate accumulation is induced by salinity in various plants as a metabolite in coping with this stress.However,the exact molecular mechanism responsible for salinityinduced malate accumulation and the roles of WRKY TFs in this process remains unclear.Here,we determined that salinity treatment induces malate accumulation in pear calli and plantlets compared to the control.Genetic and biochemical analyses established the key roles of PpWRKY44 and ABRE-BINDING FACTOR3(PpABF3)transcription factors in promoting malate accumulation in response to salinity.We found that PpWRKY44 is involved in salinity-induced malate accumulation by directly binding to W-box on the promoter of malate-associated gene PpALMT9(aluminum-activated malate transporter 9)to activate its expression.A series of in-vivo and in-vitro assays revealed that PpABF3 targeted the G-box cis-element in the promoter of PpWRKY44.Finally,PpABF3 enhanced salinityinduced malate accumulation by regulating PpWRKY44 expression.Taken together,these findings suggest that PpWRKY44 and PpABF3 play positive roles in salinity-induced malate accumulation in pears.This research provides new insights into the molecular mechanism by which salinity affects malate accumulation and fruit quality.In conclusion,the presented study identified and functionally characterized a WRKY TF that regulates anthocyanin biosynthesis and malate accumulation in pear.PpWRKY44 positively regulates light-induced anthocyanin accumulation in red pear fruit by directly activating the PpMYB10 promoter.Additionally,PpWRKY44 is highly expressed downstream of PpBBX18 in response to light.PpWRKY44 also positively regulates salinity-induced malate accumulation in pear by activating the promoter of the malate transporter gene PpALMT9.Moreover,a series of in-vivo and in-vitro assays revealed that PpABF3 targeted the G-box cis-element in the promoter of PpWRKY44.This study explores the function of TFs,specifically PpWRKY44,in regulating fruit quality traits.This study provides new insights into the co-regulation of anthocyanin and malate accumulation in pear.Also,it gives and also provides a potential biotechnological strategy for generating new pear varieties with improved fruit coloring and the fruit taste and flavor of pear.