Molecular Mechanism Of Ca²⁺-mediated MdCDPK7 Inhibiting The Ethylene Biosynthesis In Apple Fruit

Apple（Malus domestica）is one of the fruits that people often eat.In order to ensure the all seasonal supply,most of the apple fruits are stored and then sold on the market.The plant hormone ethylene plays a critical role in regulating climacteric fruit ripening,including apple.The amount of ethylene produced is inversely proportional to the length of fruit storage time.The higher the ethylene release,the shorter the storage time of fruit.Calcium ions(Ca²⁺)participate in the regulation of various metabolic processes in plant life activities in response to external and internal signals and play a critical role in fruit growth and development.It has been reported that exogenous Ca Cl₂ can decrease ethylene production in apple fruit during storage,delay fruit senescence,and extend the shelf life of the fruit.However,the molecular mechanism of Ca²⁺inhibiting ethylene biosynthesis is unclear.In our study,2%Ca Cl₂was used to treat the?Golden Delicious?apple fruit.The results showed that Ca Cl₂ significantly reduced ethylene production and the expression of ethylene synthesis related genes.The RNA-Seq data revealed that a differentially expressed calmodulin Md CDPK7 was identified.Yeast two hybrid,Co-Immunoprecipitation,and phosphorylation in vitro analysis further confirmed that Ca²⁺affected the phosphorylation of transcription factor Md MADS5 and the key enzyme Md ACO1 by Md CDPK7,promoting their degradation,and reducing the production of ethylene in apple fruit.The main results are as follows:1.Ca Cl₂-treated inhibited ethylene production and promoted the expression of calcium-binding protein Md CDPK7 in apple fruits.Apple fruit（Malus domestica cv.Golden Delicious）was harvested at commercial harvest day（140 days after full bloom,DAFB）and treated with 2%Ca Cl₂.Ca²⁺significantly inhibited the production of ethylene and suppressed the expression of Md ACS1 and Md ACO1,which were the key genes in the ethylene synthesis pathway.The RNA-Seq data revealed that a differentially expressed calmodulin Md CDPK7was identified.The results of RT-q PCR showed that Ca²⁺promoted the expression of Md CDPK7 gene.2.Md CDPK7 interacted with proteins that regulated ethylene synthesis.To clarify the role of Md CDPK7 in regulating ethylene biosynthesis,we used Md CDPK7 as bait to screen proteins interacting with it in a yeast c DNA library of apple fruit.In 79 positive clones,we identified a transcription factor Md MADS5 that may regulate fruit ripening and Md ACO1that was a key enzyme in the ethylene synthesis pathway.3.Ca²⁺promoted Md CDPK7 interacting Md MADS5.To further verify the relationship between Md CDPK7 and Md MADS5,Yeast two-hybrid,Pull down and Co-immunoprecipitation experiments showed Md CDPK7 interacting with Md MADS5in vitro and in vivo.4.Md MADS5 accelerated ripening of apple fruit.A sequencing analysis revealed the presence of MADS-box binding elements（CAr G motifs）in the Md ACS1 and Md ACO1promoters to clarify the role of transcription factor Md MADS5 in ethylene biosynthesis.Yeast one-hybrid,EMSA and Ch IP-PCR verify Md MADS5 can bind to Md ACS1 promoter.Meanwhileβ-glucuronidase（GUS）transactivation assay indicating that Md MADS5 promoted the transcriptional activity of the Md ACS1 promoter.However the result of yeast two-hybrid and Ch IP-q PCR indicated Md MADS5 could not bind to Md ACO1 promoter.To investigate the role of Md MADS5 in regulating ethylene biosynthesis during apple fruit storage,a Pro35S::Md MADS5（Md MADS5-OE）recombinant plasmid was used to overexpress Md MADS5 in apple fruit by Agrobacterium infiltration,and fruit infiltrated with the p RI101plasmid used as a control.Overexpression of Md MADS5 promoted the expression of ethylene synthesis Md ACS1 and ethylene production in apple fruit to accelerate fruit ripening.5.CDPK phosphorylated Md MADS5 and Md ACO1.CDPK phosphorylates the substrate protein by interacting with other proteins.To evaluate this,an in vitro kinase assay with recombinant Md CDPK7-His and Md MADS5-His or Md ACO1-His was performed.The phosphorylation of Md MADS5 and Md ACO1 by Md CDPK7 was markedly increased in the presence of Ca²⁺,indicating that Ca²⁺can promote the phosphorylation of Md ACO1 and Md MADS5 by Md CDPK7.Protein extracts from wild-type and Md CDPK7-overexpression transgenic apple callus were incubated with purified Md MADS5-His or Md ACO1 fusion protein.These results suggest that Md CDPK7 phosphorylated Md MADS5 or Md ACO1,undergos 26S proteasome-mediated protein degradation.6.Md CDPK7 plays a key role in Ca²⁺inhibition of fruit ripening.To investigate whether Md CDPK7 functions in the Ca²⁺-mediated suppression of ethylene biosynthesis during apple fruit storage,Md CDPK7 was silenced in apple fruit.Correspondingly,the production of ethylene and expression of Md ACS1 increased significantly in the Md CDPK7-AS fruit treated with Ca Cl₂ during storage.In conclusion,we constructed the molecular mechanism of Ca²⁺regulation of ethylene biosynthesis in apple fruit.Ca²⁺/Md CDPK7-mediated phosphorylation of Md MADS5,which resulted in the ubiquitination and degradation of Md MADS5,thereby weakening Md ACS1transcription and inhibiting ethylene biosynthesis during storage.In addition,Md CDPK7phosphorylates Md ACO1,a key enzyme in ethylene biosynthesis,to degrade it and inhibit ethylene biosynthesis.