@article{liu_shi_xie_2014, title={Regulation of anthocyanin biosynthesis in Arabidopsis thaliana red pap1-D cells metabolically programmed by auxins}, volume={239}, ISSN={["1432-2048"]}, DOI={10.1007/s00425-013-2011-0}, abstractNote={Red pap1-D cells of Arabidopsis thaliana have been cloned from production of anthocyanin pigmentation 1-Dominant (pap1-D) plants. The red cells are metabolically programmed to produce high levels of anthocyanins by a WD40-bHLH-MYB complex that is composed of the TTG1, TT8/GL3 and PAP1 transcription factors. Here, we report that indole 3-acetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) regulate anthocyanin biosynthesis in these red cells. Seven concentrations (0, 0.2, 0.4, 2.2, 9, 18 and 27 μM) were tested for the three auxins. IAA and 2,4-D at 2.2-27 μM reduced anthocyanin levels. NAA at 0-0.2 μM or above 9 μM also decreased anthocyanin levels, but from 0.4 to 9 μM, it increased them. HPLC-ESI-MS analysis identified seven cyanin molecules that were produced in red pap1-D cells, and their levels were affected by auxins. The expression levels of ten genes, including six transcription factors (TTG1, EGL3, MYBL2, TT8, GL3 and PAP1) and four pathway genes (PAL1, CHS, DFR and ANS) involved in anthocyanin biosynthesis were analyzed upon various auxin treatments. The resulting data showed that 2,4-D, NAA and IAA control anthocyanin biosynthesis by regulating the expression of TT8, GL3 and PAP1 as well as genes in the anthocyanin biosynthetic pathway, such as DFR and ANS. In addition, the expression of MYBL2, PAL1 and CHS in red pap1-D and wild-type cells differentially respond to the three auxins. Our data demonstrate that the three auxins regulate anthocyanin biosynthesis in metabolically programmed red cells via altering the expression of transcription factor genes and pathway genes.}, number={4}, journal={PLANTA}, author={Liu, Zhong and Shi, Ming-Zhu and Xie, De-Yu}, year={2014}, month={Apr}, pages={765–781} }
 @article{zhou_shi_xie_2012, title={Regulation of anthocyanin biosynthesis by nitrogen in TTG1-GL3/TT8-PAP1-programmed red cells of Arabidopsis thaliana}, volume={236}, ISSN={["1432-2048"]}, DOI={10.1007/s00425-012-1674-2}, abstractNote={Nitrogen nutrients can regulate anthocyanin biosynthesis in Arabidopsis thaliana. In this investigation, we report the nitrogen regulation of anthocyanin biosynthesis activated by TTG1-GL3/TT8-PAP1 in red pap1-D cells. To understand the mechanisms of nitrogen regulation, we employed red pap1-D cells and wild-type cells (as a control) to examine the effects of different nitrogen treatments on anthocyanin biosynthesis. In general, the higher concentrations of ammonium and high total nitrogen tested (e.g., 58.8 and 29.8 mM total nitrogen consisting of NH(4)NO(3) and KNO(3)) reduced the levels and molecular diversity of anthocyanins; in contrast, the lower concentrations of ammonium and total nitrogen conditions (e.g., 9.4 mM KNO(3) and the depletion of nitrogen) increased the levels and molecular diversity of anthocyanins. An expression analysis of the main regulatory and pathway genes showed that at conditions of higher concentrations of ammonium and total nitrogen, the expression levels of PAP1 and TT8 decreased, but the expression levels of LBD37, 38 and 39, three negative regulators of anthocyanin biosynthesis, increased. In addition, the expression levels of the main pathway genes decreased. In contrast, at conditions of lower concentrations of ammonium and total nitrogen, the expression levels of PAP1, TT8 and the main pathway genes increased, whereas those of LBD37, 38 and 39 decreased. These results show that nitrogen regulation of anthocyanin biosynthesis in red cells undergoes a mechanism by which nitrogen controls the expression of genes encoding both main components of the TTG1-GL3/TT8-PAP1 complex and negative regulators. Based on these observations, we propose that the regulatory mechanism of nitrogen may occur via two pathways to control the expression of genes encoding positive and negative regulators in red pap1-D cells.}, number={3}, journal={PLANTA}, author={Zhou, Li-Li and Shi, Ming-Zhu and Xie, De-Yu}, year={2012}, month={Sep}, pages={825–837} }
 @article{shi_xie_2011, title={Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells}, volume={233}, ISSN={["1432-2048"]}, DOI={10.1007/s00425-010-1335-2}, abstractNote={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.}, number={4}, journal={PLANTA}, author={Shi, Ming-Zhu and Xie, De-Yu}, year={2011}, month={Apr}, pages={787–805} }
 @article{shi_xie_2010, title={Features of anthocyanin biosynthesis in pap1-D and wild-type Arabidopsis thaliana plants grown in different light intensity and culture media conditions}, volume={231}, ISSN={["1432-2048"]}, DOI={10.1007/s00425-010-1142-9}, abstractNote={The number of different anthocyanin molecules potentially produced by Arabidopsis thaliana and which anthocyanin molecule is the first product of anthocyanidin modification remain unknown. To accelerate the understanding of these questions, we investigated anthocyanin biosynthesis in rosette leaves of both pap1-D and wild-type (WT) A. thaliana plants grown in nine growth conditions, which were composed of three light intensities (low light, middle light, and high light) and three media derived from MS medium (medium-1, 2, and 3). These nine growth conditions differentially affected the levels of anthocyanins and pigmentation patterns of rosette leaves, which were closely related to the diversification levels of cyanin structures. The combined growth conditions of high light and either medium-2 or medium-1 induced the most molecular diversity of anthocyanin structures in rosette leaves of pap1-D plants. Twenty cyanin molecules, including five that were previously unknown, were characterized by HPLC-ESI-MS and HPLC-TOF-MS analyses. We detected that the A. thaliana anthocyanin molecule A11 was most likely the first cyanin derived from the multiple modification steps of cyanidin. In addition, in the same growth condition, rosette leaves of pap1-D plants produced much higher levels and more diverse molecular profiling of cyanins than those of WT plants. The transcript levels of PAP1, PAL1, CHS, DFR, and ANS cDNAs were much higher in pap1-D rosette leaves than in WT ones. Furthermore, on the same agar-solidified medium, an enhancement of light intensity increased levels and molecular diversity of cyanins in both pap1-D and WT rosette leaves. In the same light intensity condition, the responses of anthocyanin levels and profiling to medium alternation were different between pap1-D and WT plants.}, number={6}, journal={PLANTA}, author={Shi, Ming-Zhu and Xie, De-Yu}, year={2010}, month={May}, pages={1385–1400} }
 @article{zhou_zeng_shi_xie_2008, title={Development of tobacco callus cultures over expressing Arabidopsis PAP1/MYB75 transcription factor and characterization of anthocyanin biosynthesis}, volume={229}, ISSN={0032-0935 1432-2048}, url={http://dx.doi.org/10.1007/s00425-008-0809-y}, DOI={10.1007/s00425-008-0809-y}, abstractNote={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.}, number={1}, journal={Planta}, publisher={Springer Science and Business Media LLC}, author={Zhou, Li-Li and Zeng, Hai-Nian and Shi, Ming-Zhu and Xie, De-Yu}, year={2008}, month={Sep}, pages={37–51} }