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

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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.

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Abbreviations

2, 4-D:

2, 4-Dichlorophenoxyacetic acid

IAA:

Indole-3-acetic acid

MS:

Murashige and Skoog

NAA:

Naphthalene acetic acid

PAP1:

Production of anthocyanin pigment 1

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Acknowledgments

This work is funded by USDA-NRI, USDA 2006-35318-17431. We thank Dr. Richard Dixon at Noble Foundation for kindly providing us with the PAP1 transgenic tobacco plants. We thank Dr. Wendy Boss for critically reading this manuscript and many suggestions.

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Authors and Affiliations

  1. Department of Plant Biology, North Carolina State University, Raleigh, NC, 27695, USA

    Li-Li Zhou, Hai-Nian Zeng, Ming-Zhu Shi & De-Yu Xie

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  1. Li-Li Zhou
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  2. Hai-Nian Zeng
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Correspondence to De-Yu Xie.

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Fig. S1 Suspension cultures of 6R, 6W, and P3 cells (TIFF 10281 kb)

425_2008_809_MOESM2_ESM.tif

Fig. S2a, a growth curve for 6W calli during the 25-day culture period; b, a growth curve for P3 calli during the 25-day culture period; c: absorbance values of HCl-methanol extraction from P3 calli throughout the whole culture period (HCl-methanol was used as a background control) (TIFF 2683 kb)

425_2008_809_MOESM3_ESM.tif

Fig. S3 a, an LC chromatogram of low resolution showing an overview of profile for anthocyanins in the 6R calli grown in the light/dark (16/8) condition; b, a mass spectrum chromatogram shows the anthocyanins 5 (m/z 595); 7 (m/z 579); and 8 (m/z 609) in the 6R calli grown in the light/dark (16/8) condition; c, an LC chromatogram of low resolution shows an overview of a profile for anthocyanins in the 6R calli grown under dark; d, a mass spectrum chromatogram shows the anthocyanins 5 (m/z 595); 7 (m/z 579); and 8 (m/z 609) in the 6R calli grown in the dark (Note: in order to put the three peaks together, the scale for the heights of peaks 7 and 8 in b and d were magnified five-fold) (TIFF 2292 kb)

425_2008_809_MOESM4_ESM.tif

Fig. S4 Five ion chromatograms show the peak sizes of anthocyanins 5 (m/z 595), 7 (m/z 579), and 8 (m/z 609) in response to five combinations of NH4NO3 and KNO3 concentrations (Note: in order to put the three peaks together, the scale for the heights of the peaks 7 and 8 are magnified five-fold (TIFF 1718 kb)

425_2008_809_MOESM5_ESM.tif

Fig. S5 effects of five combinations of NH4NO3 and KNO3 concentrations on the FW of 6R calli (a) and on average levels of anthocyanins extracted from the 6R calli (b) (The error bar in panel a and b means standard deviation of four replicates) (TIFF 6643 kb)

425_2008_809_MOESM6_ESM.tif

Fig. S6 a, two LC chromatograms show the effects of 0.02 mg/L and 1 mg/L of NAA on peak sizes of anthocyanins in the 6R calli; b, two LC chromatograms show the effects of 0.02 mg/L and 1 mg/L of 2, 4-D on peak sizes of anthocyanins in the 6R calli (TIFF 2115 kb)

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Zhou, LL., Zeng, HN., Shi, MZ. et al. Development of tobacco callus cultures over expressing Arabidopsis PAP1/MYB75 transcription factor and characterization of anthocyanin biosynthesis. Planta 229, 37–51 (2008). https://doi.org/10.1007/s00425-008-0809-y

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