Abstract
Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in persimmon.
Highlights
Persimmon (Diospyros kaki), one of the major fruit crops in Asia, accumulates large amounts of PA in its fruit “tannin cells”,’ which causes a strong astringency sensation when fresh fruits are consumed
We found that miR858b was sharply downregulated by 93.357% compared to the control, while DkMYB19 and DkMYB20 were upregulated significantly, together with the significant upregulation of DkDHD/SDH, DkPAL, DkCHI, DkF3 H, DkF3 5 H, and DkLAR, and the transcript abundances for DkDFR and DkANR were higher in the STTM858b infiltrated leaves without a statistically significant extent
The expression levels in the three DkMYB20 lines revealed that the upregulation of DkMYB20 promoted the expression of the following PA biosynthesis pathway structural genes: DkDHD/SDH, DkCHS, DkCHI, DkF3 H, DkF3 5 H, DkDFR, DkANS, DkANR, and DkLAR (Figure 4C). All of these results indicated that DkMYB19 and DkMYB20 directly acted as regulators of PA pathway genes and controlled PA biosynthesis in persimmon
Summary
Persimmon (Diospyros kaki), one of the major fruit crops in Asia, accumulates large amounts of PA in its fruit “tannin cells”,’ which causes a strong astringency sensation when fresh fruits are consumed. DkMYC1 may be an important bHLH TF that regulates PA biosynthesis (Su et al, 2012; Naval et al, 2016; Nishiyama et al, 2018), while DkbZIP5 could directly regulate DkMYB4 in an ABAdependent manner by recognizing ABA-responsive elements in the promoter region of DkMYB4 (Akagi et al, 2012). It follows that DkMYB2 or DkMYB4 could interact with DkMYC1 and DkWDR1 to form ternary MBW complexes that are involved in regulating PA accumulation in fruit (Naval et al, 2016)
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