**5. Manipulation for high levels of anthocyanins**

Anthocyanins are water-soluble flavonoids that not only contribute to color of the fresh potatoes, but high anthocyanin content also enhances the antioxidant benefits on human health [105–108]. The major anthocyanidins in purple potato are cyanidin, petunidin, pelargonidin, peonidin, and malvidin, while red potatoes contain cyanidin, pelargonidin, and peonidin [109–111]. Transcriptome analysis has identified

#### **Figure 5.**

*Anthocyanin biosynthetic pathway. The representative enzymes are CHS, chalcone, synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3*′*H, flavonoid 3*′*-hydroxylase; F3*′*5*′*H, flavonoid 3*′*,5*′*-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase and UFGT, flavonoid 3-O-glucosyltransferase. The figure is adapted and modified from Mattoo et al. [115].*

104 potentially important genes that may play an important role in anthocyanin biosynthesis in potato [112]. Several studies [113, 114] have investigated the impact of altered expression of some of the key genes in the anthocyanin biosynthesis pathway (**Figure 5** and **Table 4**).

The biosynthesis of anthocyanins (**Figure 5**) initiates from 4-coumaroyl-CoA and malonyl CoA catalyzed by the enzyme chalcone synthase (CHS) to synthesize naringenin chalcone which is then converted to naringenin by chalcone isomerase (CHI). Naringenin is converted to dihydrokaempferol, catalyzed by flavanonone 3′-hydroxylase (F3H). Flavonoid 3′-hydroxylase (F3′H) thereafter hydroxylates dihydrokaempferol (DHK) into dihydroquercitin (DHQ ) or to dihydro-myricetin (DHM) which is catalyzed by flavonoid 3′,5′ –hydroxylase (F3′5′H). All three dihydroflavonols DHK, DHM and DHQ are independently converted to colorless leucoanthocyanidins by the enzyme dihydroflavonol 4-reductase (DFR). The next enzymatic reaction involves the enzyme anthocyanidin synthase (ANS), which converts all three


#### **Table 4.**

*Genes used in the modification of anthocyanin content in transgenic potatoes.*

leucoathocyanidins to colored anthocyanidins [120]. Stobiecki et al. [113] demonstrated that overexpression of DFR gene under the control of the CaMV35S promoter showed increased tuber anthocyanin content along with a 4-fold increase in petunidin and pelargonidin derivatives in red skinned potato cv. 'Desiree'. However, overexpression of DFR in the potato cv 'Prince Hairy', which has a white tuber, resulted in change in the flower color from light blue to purple, but no change in tuber color [116]. Likewise, the overexpression of F3′5′H in the cv. 'Desiree' resulted in plants with purple-colored tubers and stems [114].

In addition to genes involved in the biosynthesis of anthocyanins, several transcription factors have been associated with anthocyanin biosynthesis [118, 119, 121, 122]. Stushnoff et al. [121] studied the gene expression pattern associated with the accumulation of purple tuber anthocyanins using microarray. A total of 27 genes were identified those were differentially expressed in purple and white tuber tissues. One of these genes coded for a novel single-domain MYB transcription factor (StMYBA1) that has been shown to influence anthocyanin-pigment production in potato [121, 122]. *StMYBA1* from potato was transformed into tobacco under the control of the CaMV 35S promoter and the resultant transformants showed anthocyanin accumulation in all tissues of transgenic tobacco lines [118]. In other studies, it has been demonstrated that there are two distinct classes of MYB transcription factors which negatively regulate anthocyanin accumulation: R3 MYB and R2R3 MYB repressors [119]. R2R3 MYB transcription factors; StAN1 and StbHLH1 are responsible for the coordinated regulation of the skin and flesh pigmentation, as well as anthocyanin biosynthetic pathway genes in white regions in potato [122–124]. Therefore, manipulation of specific transcription factor genes associated with anthocyanin synthesis through transgenic or CRISPR tools may be useful in enhancing nutritionally important traits of pigmented tuber flesh.
