The increased accumulation of anthocyanin in leafy vegetables improves their commercial and healthy values. Yet, there are limited studies on mechanisms underlying the anthocyanin biosynthesis in leafy vegetables . In this study, transcriptomic analysis of the green-leafed and purple-leafed varieties of Pak Choi was conducted using RNA-Seq technology. The current results not only identified Pak Choi’s vital structural genes involved in anthocyanin biosynthesis, but also revealed the potential regulatory mechanism involved in anthocyanin biosynthesis, transport and accumulation in the purple leafed variety.
One objective of this study is to identify the structural genes involved in anthocyanin biosynthetic pathway. The current result confirmed the presence of all known structural genes in this pathway in Pak Choi, which result was in agreement with previous proposal that anthocyanin biosynthesis pathway and structural genes are relatively conserved across higher plants . Yet, at the transcriptional level, great variations were found among these structural genes between ‘P’ and ‘G’. In the variety ‘P’, most genes in flavonoid pathway had significantly higher expression levels. For examples, our results showed that the expression of Bra017210 and Bra039777, encoding for PAL1 and PAL2 respectively, were both higher in ‘P’ than in ‘G’, suggesting that the phenylpropanoid pathway which is upstream of anthocyanin biosynthesis was more active in the purple leafed variety [25, 26]. 4CL2 was another enzyme catalyzing the formation of hydroxycinnamic acid derivatives , and our study also revealed that the homologous gene of 4CL2 was up-regulated in ‘P’.
In the anthocyanin biosynthetic pathway, structural genes can be classified into two types: Early Biosynthetic Genes (EBGs) and Late Biosynthetic Genes (LBGs) . The EBGs, including CHS, CHI, F3H, F3’H, and F3’5’H, catalyze the production of flavonols and other flavonoid compounds, while the LBGs, including DFR, ANS/LDOX, and the UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGTs), are specifically for anthocyanin biosynthesis [5, 29]. Our results showed that both early biosynthetic genes (Bra037747 and Bra029212) and late biosynthetic genes (Bra027457, Bra013652, Bra019350, Bra003021, Bra035004, and Bra038445) concurrent with the high anthocyanin content in ‘P’. O-methyltransferase (OMT) plays important roles in catalyzing the methylation of anthocyanins . And in ‘P’, the expression level of Caffeic acid 3-O-methyltransferase 1 and Caffeoyl-CoA O-methyltransferase 1 were both higher than those in ‘G’, while Quercetin 3-O-methyltransferase 1 and Caffeic acid 3-O-methyltransferase OS were lower in ‘P’ than in ‘G’. Together, these results confirmed that most structural genes were up-regulated for biosynthesis of anthocyanin which was in accordance with previous studies . On the other side, exceptions did exist for the expression of the anthocyanin structural genes. For example, Bra029212 was expressed at a lower level in ‘P’ than those in ‘G’. We reasoned that the possible reasons for such exceptions were that these genes either did not directly participate in the flavonoid biosynthesis pathway, or their expressions were suppressed due to the high-expression level and compensation effect of their redundant allelic genes. Nevertheless, these structural genes in anthocyanin biosynthesis were associated with the level of anthocyanin contents in ‘P’ and ‘G’.
On the other hand, transcriptional regulation of the structural genes in anthocyanin biosynthesis pathway was an important regulatory strategy for anthocyanin formation and accumulation in Arabidopsis [28, 32]. These regulatory genes can be mainly classified into two groups: positive and negative regulatory genes. Unlike bHLH and WD proteins that may have broader and overlapping regulatory targets, MYB proteins are the key components providing specificity for the subsets of regulated genes . It has been reported that three transcription factors (MYB11, MYB12, and MYB111) activate early biosynthetic genes of the anthocyanin biosynthetic pathway in Arabidopsis . While in Pak Choi, there’s only one ortholog of AtMYB111, but no ortholog of AtMYB11 and AtMYB12 was found either due to the absence of these genes in the Pak Choi genome or because of their extremely low expression level in both of these two Pak Choi varieties. The bHLH gene family regulates anthocyanin biosynthesis through formation of MBW ternary complexes . In Pak Choi, only one bHLH transcriptional factor (TRANSPARENT TESTA8, TT8) was found that potentially involved in the regulation of anthocyanin biosynthetic genes. In Arabidopsis, TT8 not only regulates anthocyanidin production towards the synthesis of proanthocyanidins in seeds, but is also involved in the regulation of anthocyanin biosynthesis in vegetative tissues and cell cultures . Our transcriptional result was also consistent with the functional role of TT8 in Pak Choi. There are several transcription factors including two R3-type single MYB proteins (MYBL2 and CPC) and three members of the LBD (LATERAL ORGAN BOUNDARIES DOMAIN) family that act as negative regulators of activities of WBM complexes decreasing anthocyanin biosynthesis in A. thaliana [5, 36]. Yet, the corresponding orthologs in Pak Choi (Bra016164 and Bra039283) showed higher expression levels in ‘P’ than in ‘G’, probably either due to the different but contradictory function of these two genes in Pak Choi or due to the feedback effect of the high anthocyanin content in ‘P’ that in turn inhibited their expressions.
The flavonoid transporters also involved in the vacuolar transport of anthocyanins and proanthocyanidin precursors and might contribute to the accumulation of anthocyanin in plant as well [37–39]. In Arabidopsis, three genes, TT12, TT19 and AHA10, have been found to be related to the transport of anthocyanins. However, our results showed that Bra009033 was down regulated while genes encoding ATPase 10 and ATPase 1 were up-regulated in purple-leafed variety. Glutathione S-transferases (GSTs) act as non-enzymatic carrier proteins enabling intracellular shuttling of endogenous compounds such as anthocyanin in plants. Previous gene knockout experiments revealed that GSTs were involved in anthocyanin transport  and the deposition of anthocyanin pigment into the vacuole . The differential expressions of the GST encoding genes were found in this study, indicating a possible link between GST and the transportation of anthocyanin in Pak Choi as well.