Development of tobacco callus cultures over expressing Arabidopsis PAP1/MYB75 transcription factor and characterization of anthocyanin biosynthesis

Abstract

The Arabidopsis PAP1 gene (At1g56650) encodes the MYB75 transcription factor, which has been demonstrated to essentially regulate the biosynthesis of anthocyanins. Our previous study showed that ectopic expression of the PAP1 gene led to high pigmentation of anthocyanins in all tissues of transgenic tobacco plants. In order to understand the mechanisms of how PAP1 regulates anthocyanin biosynthesis and what can regulate the function of PAP1, we have established PAP1 transgenic tobacco callus cultures. Phenotypically different calli including anthocyanin-producing red and anthocyanin-free white calli lines were differentially induced from the same genotype of PAP1 transgenic plants. RT-PCR analysis showed that the expression of the PAP1 transgene was similar in the two types of calli, indicating that the transgenic red and white calli had differential responses to the regulation of PAP1. The growth of transgenic red calli followed a "sigmoid-like" curve in a 25-day callus culture period, during which the time course obviously impacted the profiles and the average levels of anthocyanins even though the expression of the PAP1 transgene was constitutive. A HPLC-UV-ESI-mass spectrum-based profiling characterized nine anthocyanin molecules (e.g., 595, 579 and 609 m/z) in the transgenic red calli over the course of the culture period. Cyanidin, pelargonidin, and peonidin were the major anthocyanidins identified by HPLC-mass spectrum analysis. We have demonstrated that dark, nitrogen nutrients, and auxin apparently affect the anthocyanin profiles in PAP1 transgenic callus cultures; and suggest that these cell cultures are an appropriate system to study the regulatory function of PAP1 on the anthocyanin biosynthesis at post-transcriptional level in vivo.

Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco

“…4e). Callus cultures induced from PAP1-overexpressing tobacco leaves was also reported to accumulate anthocyanin pigments (Zhou et al 2008). The anthocyanin phenotype indicates that NtAn2 is the key component missing from the MYB-bHLH-WD40 complex in the vegetative tissues of tobacco, which could explain why anthocyanin accumulation is absent or very low in wild-type plants.…”

Comparative Metabolomics of Transgenic Tobacco Plants (Nicotiana tabacum var. Xanthi) Reveals Differential Effects of Engineered Complete and Incomplete Flavonoid Pathways on the Metabolome

“…The results of ectopic expression of the A. thaliana PAP1 gene in tobacco plants might differ from the consequences of its overexpression in its native plant species. The evidence was that we recently observed dramatically differential response of anthocyanin biosynthesis to chemical factors in tissue cultures of PAP1 transgenic Arabidopsis and tobacco (Shi and Xie 2011;Zhou et al 2008). Therefore, the alterations of these non-targeted metabolite profiles most likely resulted from the ectopic expression of PAP1 transgene.…”

“…It was interesting that the overexpression of PAP1 led to massive accumulation of anthocyanins in transgenic tobacco leaf trichomes (Xie et al 2006) but not in leaf trichomes of pap1-D Arabidopsis thaliana (Shi and Xie 2010). In vitro separation of red cells from other cells has demonstrated that the regulatory function of PAP1 expression can be inherited by cell culture developed from mother plants (Zhou et al 2008). Both no anthocyanin-producing white cells and anthocyanin-producing red cells were obtained from transgenic PAP1 tobacco leaves.…”

“…Transcript analysis showed that the level of the PAP1 overexpression in these two types of cells were similar, indicating that the regulatory function of the PAP1 expression was dependent upon cell types. (Zhou et al 2008). The regulatory function of PAP1 is also highly controlled by environmental factors.…”

Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida

“…DFR, which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition (Li et al, 2012). DFR uses NADPH as a cofactor to catalyze the reduction of dihydroflavonols to their respective leucoanthocyanidins, and these are common precursors for anthocyanin and proanthocyanidin biosynthesis (Zhou et al, 2008). The colorless, unstable leucoanthocyanidins are immediate precursors in the synthesis of anthocyanins, which are the main water-soluble pigments found in flowers and fruits.…”