| Pollination constant and non-astringent(PCNA)type fruits can naturally lose their astringency on the tree without any special treatment and convenient for the fresh consumption,storage,and transportation,whereas almost of the non-PCNA type fruit are inedible without any artificial treatment.The PCNA trait is controlled by different locus between Chinese(CPCNA)and Japanese PCNA(JPCNA)persimmon,which is dominant and recessive against non-PCNA persimmon,respectively.However,the key genes and regulatory network involved in natural deastringency in CPCNA persimmon are still unclear.To gain insight into the molecular events of natural de-astringency in CPCNA persimmon,the RNA-seq and iTRAQ-based quantitative proteomic analysis were used to measure changes in genes and proteins expression at two key stages of natural astringency removal(i.e.10 and 20 weeks after bloom)and water-treated(i.e.40 °C·12h)de-astringency fruits.After data analysis a group of genes that related to persimmon astringency removal were obtained.Based on this data,we performed a target analysis of differential expressed MYB transcription factors and one of most important MYB transcription factor gene DkMYB14 that involved in natural deastringency in CPCNA persimmon was isolated.Finally,molecular mechanisms was characterized for DkMYB14 underlying natural deastringency.The main results are as fellow:1.Transcriptome assembly and identification of differentially expressed genes a nd proteins.The clean data were de novo assembled into a non-redundant set of 135,999 unigenes with a total length of 166,861,433 nt,average length of 1227 nt,and an N50 of 2,071 bp.Among them,3,818 unigenes were differentially expressed during natura l deastringency,and 15,597 unigenes were differentially expressed after water-treated deastringency.There are 4,954 proteins were identified by iTRAQ technology,and 3,041 proteins(61.37%)covered with two different peptides(unique peptide),and 523 differential expressed proteins were identified.2.Functional analysis of differentially expressed genes and proteins.During natural deastringency the significantly enriched GO terms found for the set of up-regulated genes were mainly related to sugar metabolism,fruit color,signal transduction pathway,and ethylene-activated signaling pathway.The GO enriched terms of up-regulated by water-treated genes were related to primary glucosamine biosynthesis and glycolytic process.There are 228 and 633 genes were both up-regulated in processes of natural and water-treated deastringency.The 228 shared up-regulated genes were significantly enriched in the GO terms that were abundant are related to primary sugar metabolism and stress response.The enriched GO terms of 633 shared down-regulated genes were abundant and related to the fruit development,pigmentation,and flavonoid metabolism.The acetaldehyde metabolism was activated both in process of natural and water-treated de-astringency but more remarkable in water treatment.We also found the expression of genes and proteins involved in PA biosynthetic pathway was highly reduced both in processes of natural and water-treated de-astringency.There were 95 and 65 genes and their related proteins were both up-and down-reguated in process of natural deastrigency,meanwhile,54 and 51 genes and their related proteins were shared up-and down-reguated in process of water-treated de-astringency,respectively.The 65 shared down-regulated genes/proteins were mainly related to PA biosynthesis,while 95 shared up-regulated genes/proteins were mainly enriched in sugar metabolism.In de-astringency with warm water treatment,the GO term of 51 shared down-regulated genes/protein were related to primary chorismate biosynthesis process and shikimate biosynthesis were quite abundant.In addition,the significantly enriched GO terms of 54 shared up-regulated genes/proteins were related to the response to heat and stimulus.We found 43 and 136 were especially up-regulated in natural and water-treated deastringency,respectively.And 10 transcription factors(i.e.4 ERF,3 NAC,2 WRKY,and 1 Zinc finger transcription factor)were shared up-regulated in two de-astringency processes(Fig.8A).For down-regulated transcription factors,20 and 122 were especially up-regulated in natural and water-treated deastringency,respectively.And 16 TFs(i.e.2 bHLH,1 bZIP,3 ERF,2 WRKY,and 8 Zinc finger transcription factor)were shared down-regulated in two de-astringency processes.In addition,the transcription factors such as ERF,MYB,NAC,and WRKY are abundantly expressed during natural deastringency.3.Functional characterization of DkMYB14 in CPCNA persimmon.Based on the previous generated RNA-seq data,we found 7 MYB transcription factors that differentially expressed in CPCNA persimmon during natural astringency removal.Due to the important role of MYB transcription factor involved in secondary metabolism especially in flavonoid/PA metabolism,these seven MYB-like unigenes were selected as candidate genes for further study.After RACE experiments,seven MYB CDS were isolated and named them as DkMYB14-20,respectively.Alignment shows that six of them are belong to R2R3-MYB transcription factors,except DkMYB16,which only have a single R3 domain.In C-terminal region two conserved protein motifs C1 and C2 were identified,which is diagnostic for subgroup 4 MYB proteins and contains the EAR-like repressor domain LxLxI.In phylogenetic analysis,DkMYB14 was grouped together with the previously reported phenylpropanoid biosynthesis repressors in subgroup 4 MYB proteins.The DkMYB14,whose expression level was obviously the highest in the seven DkMYBs isolated from CPCNA fruit flesh,showed gradually up-regulated in CPCNA fruit and maintain high expression between 15 to 20 WAB,and lower expressed in whole stage of fruit development in non-PCNA and J-PCNA fruit.These results suggested that DkMYB14 is a candidate regulator involved in PA biosynthesis in CPCNA persimmon fruit.DkMYB14-GFP was specifically localized in the nucleus,indicating that DkMYB14 functions as a transcription factor.Transient overexpression of DkMYB14 in persimmon leaves showed dramatically reduced soluble PA levels and significant increased insoluble PA levels,and the soluble PA levels was significant increased after suppression of DkMYB14.To further examine the roles of DkMYB14 in vivo for their potential role in regulating PA metabolism,DkMYB14 was introduced into the wild type Arabidopsis and similar results was obtained,that soluble PAs was significant reduced with increase of insoluble PAs.After overexpression of DkMYB14,a decrease in transcript accumulation was observed for most of the PA biosynthetic gene and the ADH1 and PDC2 were highly expressed both in the transgenic plants and persimmon leaves.Yeast one-hybrid(Y1H)and transient promoter activity assays demonstrated that DkMYB14 can directly reducing the activity of PA biosynthesis genes promoters of DkANR and DkF3’5’H,meanwhile activating DkPDC2 and DkADH1 expression and promotes insolubilization of soluble PAs in C-PCNA persimmon.Finally,results from the transient overexpression analysis of DkMYB14 in persimmon fruit discs conformed the function of DkMYB14 in persimmon leaves transient overexpression and Arabidopsis transformation but much obvious.All of these results indicated that DkMYB14 is the key gene for regulates natural deastringency in CPCNA persimmon.In summary,through genome-wide screening we obtained many transcription factor genes that were differentially expressed during persimmon fruits deastringency,among which DkMYB14 a first transcription factor gene that reported regulating the natural deastringency in CPCNA persimmon.This finding will contribute to the molecular breeding and genetic improvement of PCNA persimmon in future. |
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