Isolation and Characterization of DkPK Genes Associated with Natural Deastringency in C-PCNA Persimmon

Abstract

Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) is considered to be an important germplasm resource for the breeding of PCNA cultivars, though its molecular mechanisms of astringency removal remain to be elucidated. Previously, we showed that the abundance of pyruvate kinase gene transcripts increased rapidly during astringency removal in C-PCNA persimmon fruit. Here, we report the full-length coding sequences of six novel DkPK genes from C-PCNA persimmon fruit isolated based on a complementary DNA (cDNA) library and transcriptome data. The expression patterns of these six DkPK genes and correlations with the soluble proanthocyanidin (PA) content were analyzed during various fruit development stages in different types of persimmon, with DkPK1 showing an expression pattern during the last stage in C-PCNA persimmon that was positively correlated with a decrease in soluble PAs. Phylogenetic analysis revealed that DkPK1 belongs to cytosolic-1 subgroup, and subcellular localization analysis confirmed that DkPK1 is located in the cytosol. Notably, tissue expression profiling revealed ubiquitous DkPK1 expression in different persimmon organs, with the highest expression in seeds. Furthermore, transient over-expression of DkPK1 in persimmon leaves resulted in a significant decrease in the content of soluble PAs but a significant increase in the transcript levels of pyruvate decarboxylase genes (DkPDC1, -3, -4, -5), which catalyze the conversion of pyruvate to acetaldehyde. Thus, we propose that an acetaldehyde-based coagulation effect reduces the content of soluble PAs. Taken together, our results suggest that DkPK1 might be involved in the natural removal of astringency at the last developmental stage in C-PCNA persimmon. This is the first report to identify several novel full-length DkPK genes as well as their potential roles in the natural loss of astringency in C-PCNA persimmon.

DkPK Genes Promote Natural Deastringency in C-PCNA Persimmon by Up-regulating DkPDC and DkADH Expression

“…Acetaldehyde metabolism-related genes ADH , PDC , and pyruvate kinase ( PK ) are considered key candidates for involvement in the natural removal of astringency during the last developmental stage in C-PCNA persimmon ( Guan et al, 2016 ; Mo et al, 2016 ). PDC , a gene downstream of PK , catalyzes the generation of acetaldehyde from pyruvate.…”

“…ADH catalyzes the reversible reaction between acetaldehyde and ethanol. The DkADH1 , DkPDC1 , and DkPDC2 genes were up-regulated specifically at late stages of C-PCNA fruit development, during which soluble PA contents decrease rapidly as a result of the coagulation of soluble PAs into insoluble PAs ( Guan et al, 2016 ; Mo et al, 2016 ). Transient over-expression of DkADH1 and DkPDC2 could lead to a significant decrease in the content of soluble PAs in persimmon leaves ( Mo et al, 2016 ).…”

miRNA858b Inhibits Proanthocyanidin Accumulation by Repression ofDkMYB19andDkMYB20in Persimmon

“…We found DkMYB19 could interact with a DkPK2 protein, that is a pyruvate kinase gene maybe a potential roles in the natural loss of astringency in C-PCNA persimmon via the up-regulation of DkPDC and DkADH expression during the last developmental stage (Guan et al ., 2016, 2017). The pGBDT7-DkMYB19 and pGADT7-DkPK2 fusion protein were co-transformed to the Y2H furthermore verified the interactions (Fig.…”

“…The complete open reading frame (ORF) of DkMYB19 / DkMYB20 without the termination codon was amplified using the respective primers (Table S1) in order to conducted the fusion constructs 35S-DkMYB19::GFP, 35S-DkMYB20::GFP according to Guan et al . (2016) and the 35S-GFP vector was acted as a positive control.…”

“…DkMYB6 was identificated as a putative transcriptional activator, induced by high CO2, which is involved in persimmon fruit deastringency, by operating on both DkPDC structural genes and DkERF transcription factors (Fang et al ., 2016). Meanwhile, pyruvate kinase genes ( DkPK ) are involved in the natural loss of astringency in Chinese PCNA persimmon via enhancement of the transcript levels of the DkPDC and DkADH genes (Guan et al ., 2016; 2017). Furthermore, Xu et al .…”