High CO₂/hypoxia Responsive DkNAC And DkZF Proteins Synergistically Control Persimmon Fruit Deastringency

Over 700 species of persimmon are planted globally whereas persimmons are onsidered to be originated in the mountainous areas of southern China.China not only has huge germplasm resources of persimmon but alone accounting for 4.1 million tonnes?72%?of world production.Two major types of persimmon are astringent and non-astringent.Astringent persimmons produce abundant amount of tannins in the flesh even the fruits are at maturity stages in which soluble proanthocyanidin?PAs?produce astringency that badly affects the persimmon industry as well consumer acceptance.Apart from other astringency removing technologies?i.e ethylene,ethanol,warm water,high temperature,alternate freezing and thawing?,artificial high-CO₂ atmosphere treatment?AHCA,1%O₂ and 95%CO₂?is the most effective which caused the burst of acetaldehyde and polymerization of SCTs due to the activation on ADH and PDC coding genes,DkADH and DkPDC,which were mainly regulated by DkERFs,DkMYBs and DkWRKYs.However,the other TFs for persimmon deastringency were rarely studied.In the present research,six DkNACs?DkNAC13-18?were isolated from persimmon ruit.Phylogenetic and sequence analyses predicted that DkNAC13/15/18 might be hypoxia-responsive and possibly involved in regulating the persimmon deastringency,according to the functions of their putative homologs.Gene expression analysis indicated that DkNACs 13-18were all regulated by AHCA treatment,with DkNAC13-17 being up-regulated and DkNAC18down regulated.Dual-luciferase assay further indicated that DkNAC13 significantly activated the promoter of DkERF9?an activator of DkPDC2?,meanwhile,DkNAC16 acted as an activator of deastringency related structural gene?DkADH1?.Thus,six high-CO₂/hypoxia-modulated DkNAC genes,with at least two novels DkNAC genes?DkNAC13/16?were characterized as putative activators for deastringency,trans-activating on promoters of DkERF9 and DkADH1 respectively.Meanwhile,five genes encoded C2H2-type zinc finger proteins were isolated and esigned as DkZF1-5.Phylogenetic and sequence analyses suggested that five DkZFs could be clustered into two different subgroups.qPCR analysis indicated that transcript abundances of DkZF1/4 were significantly up-regulated during AHCA treatment at 1 day,DkZF2/5 at 2day while DkZF3 at both 1 and 2 day.Dual-luciferase assay indicated that DkZF1 and DkZF2act as the activators on deastringency related structural genes?DkPDC2 and DkADH1?and transcription factors?DkERF9/10?.Moreover,combinative effects between various transcription factors were investigated which indicated that DkZF1 and DkZF2 synergistically showed the significantly stronger activations on DkPDC2 promoter.Further,yeast two hybrid?Y2H?and bimolecular fluorescence complementation?BiFC?analyses both confirmed that DkZF2 had protein-protein interactions with DkZF1.Thus,these findings firstly pointed out the regulatory mechanisms of zinc finger proteins for persimmon fruit deastringency under AHCA treatment.The possible transcriptional regulatory linkages involved in persimmon fruit eastringency pathways during hypoxia conditions have been advanced significantly but a synergistic response of C2H2-type zinc finger proteins with NAC proteins during these conditions and astringency exclusion still lacks experimental evidences.Dual-luciferase assay specified DkZF2 as major activators of previously characterized DkERF9,DkPDC2,DkADH1 and DkERF10 promoters while on the other hand DkNAC13 can regulate DkERF9promoter.So,through BiFC and Y2H assays we found that DkZF2 had protein-protein interactions with DkNAC13.Keeping in view,we did dual luciferase assay of both proteins on DkERF9 promoter and we found that DkZF2 and DkNAC13 made protein complex and showed additive transcriptional regulation on DkERF9 promoter.In conclusion,this research indicated that Dk ZF2 not only with Dk ZF1 but also with Dk NAC13 made protein complexes and showed additive transcriptional regulations on Dk PDC2 and Dk ERF9 promoters respectively and was strongly linked to hypoxia response and astringency exclusion in persimmon fruits.