Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells

Abstract

We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC-MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1-GL3/TT8-PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.

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“…The transcriptional levels of the PAL1 and several other anthocyanin pathway genes were increased 1.2-6.7 folds by the overexpression of the PAP1 gene (Borevitz et al 2000;Lillo et al 2008;Tohge et al 2005). In addition, this result supported our recent report that most of the pathway genes involved in the anthocyanin pathway in red cells over expressing PAP1 were dramatically increased leading to alterations of multiple metabolic networks (Shi and Xie 2011). Therefore, the alterations of metabolic profiles of the three molecules resulted from the ectopic expression of PAP1 transgene.…”

“…Those red cells showed dramatically different transcriptional profiles from wild-type cells (Shi and Xie 2011) and these global gene expression alterations were not observed in Tohge's experiments either. Particularly, in red cells, expression levels of a large number of genes involved in glycolysis, amino metabolisms, photosynthesis and other metabolic pathways were significantly decreased (Shi and Xie 2011). In addition, the discrepancy might result from the speciesspecific responses to the over-expressed PAP1 protein.…”

“…Our recent report showed that genome-wide transcriptional programs in red pap1-D Arabidopsis cells cultured in vitro were dramatically different from cultured wild-type cells (Shi and Xie 2011). On the one hand, massive numbers of genes involved in multiple metabolic networks were highly expressed in red cells.…”

“…On the other hand, the expression levels of large number of genes involved in multiple metabolic networks were also reduced in red cells. We hypothesized that the dramatic alterations of genome-wide gene expression must have led to massive alterations of metabolic profiles beyond anthocyanin biosynthesis in red cells and specifically programmed red cells established by the overexpression of PAP1 must have led to transcriptional and metabolic reprogramming (Shi and Xie 2011).…”

“…PAP1 (MYB75), TT8 (transparent testa 8) (bHLH) / GL3 (glabra 3) (bHLH) and TTG1 (transparent testa glabra 1) (WD40) have been demonstrated to form a regulatory complex of MYB-bHLH-WD40 (MBW) controlling anthocyanin biosynthesis in Arabidopsis (Gonzalez et al 2009;Ramsay and Glover 2005). We recently determined that the PAP1-TT8/GL3-TTG1 complex independently regulated anthocyanin biosynthesis in pap1-D cells (Shi and Xie 2011). In addition, there are other MBW regulatory complexes controlling anthocyanin biosynthesis in Arabidopsis (Gonzalez et al 2009).…”