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Anthocyanins are plant pigments responsible for the colors of many flowers, fruits and storage organs and have roles in abiotic and biotic stress resistance. Anthocyanins and polyphenols are bioactive compounds in plants including potato ( L.) which is the most important non-cereal crop in the world, cultivated for its tubers rich in starch and nutrients. The genetic regulation of the flavonoid biosynthetic pathway is relatively well known leading to the formation of anthocyanins. However, our knowledge of post-transcriptional regulation of anthocyanin biosynthesis is limited. There is increasing evidence that micro RNAs (miRNAs) and other small RNAs can regulate the expression level of key factors in anthocyanin production. In this study we have found strong associations between the high levels of miR828, TAS4 D4(-) and purple/red color of tuber skin and flesh. This was confirmed not only in different cultivars but in pigmented and non-pigmented sectors of the same tuber. Phytochemical analyses verified the levels of anthocyanins and polyphenols in different tissues. We showed that miR828 is able to direct cleavage of the RNA originating from ( ) and initiate the production of phased small interfering RNAs (siRNAs) whose production depends on RNA-dependent RNA polymerase 6 (RDR6). MYB transcription factors were predicted as potential targets of miR828 and TAS4 D4(-) and their expression was characterized. and genes showed decreased expression levels in purple skin and flesh in contrast with high levels of small RNAs in the same tissues. Moreover, we confirmed that and are direct targets of the small RNAs. Overall, this study sheds light on the small RNA directed anthocyanin regulation in potato, which is an important member of the family.

Anthocyanins are responsible for the red pigmentation in the peel of apple ( Malus × domestica Borkh.) fruit. Relatively few studies have investigated anthocyanins at the posttranscriptional level. MicroRNAs play an important role in plant growth and development by regulating gene expression at the posttranscriptional level. In this study, mdm-miR828 showed a relatively low expression level during the rapid fruit coloration period. However, the mdm-miR828 expression level increased in the late fruit coloration stage. Overexpression of mdm-miR828 inhibited anthocyanin synthesis in apple and Arabidopsis . Dual-luciferase and yeast one-hybrid assays showed that MdMYB1 is capable of binding to the promoter of mdm-MIR828b to promote its expression. The results indicate that mdm-miR828 is involved in a feedback regulatory mechanism associated with anthocyanin accumulation in apple. In addition, mdm-miR828 is involved in the inhibition of anthocyanin accumulation in response to high temperature.

Comprehensive research in various plants shows that the metabolic pathway of anthocyanin biosynthesis is affected by environmental factors and regulated by microRNAs through post-transcriptional regulation. In seedlings of Brassica rapa Tsuda, the accumulation of anthocyanin is induced by light. However, the roles of BrmiR828 in the light-induced synthesis of anthocyanin in Brassica rapa remain to be explored. Here, a primary transcript of BrmiR828 was identified to be located on the chromosomes of the A03 sub-genome. Five candidate MYB family genes were predicted as targets of BrmiR828 in the database of Brassica rapa (BRAD, V1.1) by using psRNATarget. The transcript abundance of mature BrmiR828 was reduced in seedlings of Brassica rapa Tsuda under blue light irradiation comparing with dark treatment. However, Real-time PCR showed the transcript level of the five candidate targets, Bra004162, Bra022602, Bra001917, Bra029113, and Bra039763 was up-regulated when the seedlings exposed to blue or UV-A light. Trans-acting siRNA gene 4 (BrTAS4) was also identified to have a higher transcript level under blue and UV-A light irradiation than that in dark treatment. RNA ligase mediated 5′amplification of cDNA ends (RLM-5′ RACE) showed that BrmiR828 can splice the mRNA of Bra039763, Bra022602, and BrTAS4 on binding sites. Phylogenetic analysis of candidate BrMYBs targets along with MYBs from Arabidopsis thaliana showed that Bra039763, Bra004162, Bra001917, Bra029113, and Bra022602 are classified to the same group with AtMYB75, AtMYB114, AtMYB90, AtMYB113, and AtMYB82 which are involved in the anthocyanin biosynthetic pathway. As a result, light-induced down-regulation of BrmiR828 can target BrTAS4, BrPAP1 (Bra039763), MYB82 (Bra022602) to negatively regulate their transcript levels leading to the accumulation of MYB transcription factors that positively regulate anthocyanin biosynthesis in light-exposed seedlings of Brassica rapa.

miR828 in Arabidopsis triggers the cleavage of Trans - Acting SiRNA Gene 4 ( TAS4 ) transcripts and production of small interfering RNAs ( ta -siRNAs). One siRNA, TAS4 -siRNA81(−), targets a set of MYB transcription factors including PAP1 , PAP2, and MYB113 which regulate the anthocyanin biosynthesis pathway. Interestingly, miR828 also targets MYB113, suggesting a close relationship between these MYBs, miR828, and TAS4 , but their evolutionary origins are unknown. We found that PAP1 , PAP2 , and TAS4 expression is induced specifically by exogenous treatment with sucrose and glucose in seedlings. The induction is attenuated in abscisic acid (ABA) pathway mutants, especially in abi3 - 1 and abi5 - 1 for PAP1 or PAP2 , while no such effect is observed for TAS4 . PAP1 is under regulation by TAS4 , demonstrated by the accumulation of PAP1 transcripts and anthocyanin in ta -siRNA biogenesis pathway mutants. TAS4 -siR81(−) expression is induced by physiological concentrations of Suc and Glc and in pap1 - D , an activation-tagged line, indicating a feedback regulatory loop exists between PAP1 and TAS4 . Bioinformatic analysis revealed MIR828 homologues in dicots and gymnosperms, but only in one basal monocot, whereas TAS4 is only found in dicots. Consistent with this observation, PAP1 , PAP2 , and MYB113 dicot paralogs show peptide and nucleotide footprints for the TAS4 -siR81(−) binding site, providing evidence for purifying selection in contrast to monocots. Extended sequence similarities between MIR828, MYB s, and TAS4 support an inverted duplication model for the evolution of MIR828 from an ancestral gymnosperm MYB gene and subsequent formation of TAS4 by duplication of the miR828* arm. We obtained evidence by modified 5′-RACE for a MYB mRNA cleavage product guided by miR828 in Pinus resinosa . Taken together, our results suggest that regulation of anthocyanin biosynthesis by TAS4 and miR828 in higher plants is evolutionarily significant and consistent with the evolution of TAS4 since the dicot—monocot divergence.

Plant microRNAs (miRNAs) are RNAs of 20–22 nucleotides in length with sequence complementarities to specific mRNAs that are targeted for either cleavage or translational repression. They play critical regulatory roles in plant growth and development. In this study, the functionality of miR828 in the regulation of anthocyanin biosynthesis has been investigated. RNA blotting analysis has confirmed the presence of a conserved 22 nucleotide miR828 in both dicot and monocot plants. Moreover, it has revealed that miR828 is constitutively expressed in different tissues of Arabidopsis. Subsequently, a 35S:pre-miR828 construct has been created and transformed into Arabidopsis. Expression analysis has shown that levels of pre-miR828 and mature miR828 transcripts are increased, while levels of MYB75, MYB90, and MYB113 transcripts, encoding MYB transcription factors that positively regulate anthocyanin biosynthesis, are repressed in transgenic plants. As a result, anthocyanin levels are reduced, as are transcription levels of genes that are directly involved in anthocyanin biosynthesis, including PAL, CHS, CHI, F3H, F3′H, DFR, and LDOX. In addition, overexpression of miR828 in Arabidopsis inhibits transcription of yet another MYB factor, MYB82, indicating that MYB82 is likely to be involved in the anthocyanin biosynthesis pathway.

MicroRNAs (miRNAs) are small noncoding RNAs which post-transcriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. miRNAs play multiple roles in the growth, development and stress responses in plants. However, little is known of the wounding-responsive miRNAs and their regulation. Here, we investigated the expression patterns of microR828 (miR828) on wounding in sweet potato (Ipomoea batatas cv Tainung 57). The expression of miR828 was only detected in leaves, and was induced by wounding rather than by ethylene, hydrogen peroxide (H2O2), methyl jasmonate or nitric oxide (NO). Moreover, cyclic guanosine monophosphate (cGMP) was necessary for miR828 accumulation in leaves on wounding. Two miR828 target candidates, named IbMYB and IbTLD, were obtained by cDNA cloning, and their mRNA cleavage caused by miR828 was confirmed by cleavage site mapping, agro-infiltration and transgenics studies. The reduction in IbMYB and IbTLD expression coincided with the induction of miR828, demonstrating thatIbMYB and IbTLD might be miR828 targets. Furthermore, transgenic sweet potato overexpressing miR828 precursor affected lignin and H2O2 contents. These results showed that cGMP could regulate wounding-responsive miR828, which repressed the expression of IbMYB and IbTLD. Subsequently, lignin and H2O2 were accumulated to participate in defense mechanisms.