Anthocyanin Biosynthesis in pap1-D Plants and PAP1/MYB75-Programmed Cells of Arabidopsis thaliana

Anthocyanins are a group of colorful and bioactive natural pigments with numerous important physiological and ecological functions in plants. In general, anthocyanins attract pollinators and seed dispersers, protect plants from high light irradiation and scavenge free radicals produced in cells in stress conditions. In addition, anthocyanins are highly valuable nutraceuticals with multiple benefits to human health. Over the past many years, anthocyanin biosynthesis has been studied in many plants including model plants and crops. However, the diversity of anthocyanin molecules in the plant kingdom and the mechanisms leading to various structures remain largely unclear. Also, the mechanisms of genetic and environmental regulation of anthocyanin biosynthesis need further investigation.;PAP1 is a master regulator of anthocyanin biosynthesis in the model plant Arabidopsis whose overexpression results in the high accumulation of anthocyanins in pap1-D plants. The pap1-D mutants are an ideal model system for elucidating the molecular diversity of anthocyanins and the effect on transcriptome and metabolome alterations under the overexpression of PAP1. To analyze the number and composition of different anthocyanin molecules that potentially could be produced by Arabidopsis, we investigated anthocyanin profiles of both pap1-D and WT plants in nine growth conditions including three different light intensities and three media with different nitrogen levels. Twenty cyanin molecules including five previously unknown were identified from pap1-D plants in our study. Among them, A11 was likely the first cyanin formed after multiple modification steps. In addition, an increase in light intensity was found to enhance the levels and molecular diversity of cyanins in both pap1-D and WT plants while the response to three different media conditions was different in pap1-D and WT plants.;Several anthocyanin-producing callus cell lines were isolated and established from pap1-D plants through tissue culture. These pap1-D cell lines are the first anthocyanin-producing cell lines established and reported from Arabidopsis and serve as an appropriate model system to study the metabolic specificity and the environmental regulation of anthocyanin biosynthesis. LC-MS analysis identified seven anthocyanin molecules produced in the pap1-D cells, one of which has not been identified from both pap1-D and WT plants. Genome-wide transcriptome and semi-quantitative and quantitative RT-PCR analyses showed that anthocyanin biosynthesis in pap1-D cells is likely regulated by the TTG1-TT8/GL3-PAP1 complex. In addition, gene expression profiles between pap1-D and wild-type cells revealed large alterations in transcriptome.;To provide a more detailed investigation of transcriptome and metabolic changes in pap1-D and wild-type cells, we conducted transcriptome profiling and GC-MS based metabolic analysis from three time points during one entire culture period in both pap1-D and WT calli. Besides anthocyanin biosynthesis, gene expression and metabolites of many other metabolic pathways were found to be altered. For example, photosynthesis genes were significantly down-regulated in pap1-D cells at all three time points. In addition, the expression of anthocyanin modification genes was found to be consistent with the production of specific anthocyanin molecules in pap1-D cells. Also, different pap1-D cell lines with varying anthocyanin levels were established. The effect of nitrogen re-supplementation on anthocyanin biosynthesis was studied using pap1-D cells. Our data suggest that PAP1 and TT8 likely play primary roles in regulating anthocyanin biosynthesis in pap1-D calli. Our results also suggest additional functions of MYBL2 in repressing anthocyanin biosynthesis in high nitrogen conditions and in anthocyanin-producing cells likely through quantitative competitions with PAP1 to determine the production of anthocyanins in cells.