Pollmann et al. 10.1073/pnas.0610934104. |
Fig. 8. Identification of pigments. Pigments were extracted from etiolated wild-type, flu, and Atoep16 plants subjected to HPLC on a C18-reversed phase column as described in Methods, and the peaks eluting at 12.5 and 15 min, respectively, in turn were subjected to absorbance measurements and mass spectrometry. (A) The absorption spectrum of the peak eluting at 12.5 min is indicative of Pchlide b, whereas that eluting at 15 min is indicative of Pchlide a. (B) Confirmation of the identity of Pchlide b eluting at 12.5 min by matrix-assisted laser desorption/ionization mass spectroscopy (a) and theoretical isotopic distribution of C35H30N4O6Mg corresponding to Pchlide b (b). The matrix used was terthiophene (Mr 248.4, not shown). (C) Mass spectrometry data for Pchlide a eluting at 15 min (a) and theoretical isotopic distribution of C35H32N4O5Mg corresponding to Pchlide a (b). Note that the spectra are identical to those published by Schoch et al. (1), Scheumann et al. (2), and Xue et al. (3).
1. Schoch S, Helfrich M, Wiktorsson B, Sundqvist C, Rüdiger W, Ryberg M (1995) Eur J Biochem 229:291-298.
2. Scheumann V, Klement H, Helfrich M, Oster U, Schoch S, Rüdiger W (1999) FEBS Lett 445:445-448.
3. Xu H, Vavilin D, Vermaas W (2001) Proc Natl Acad Sci USA 98:14168-14173.
SI Methods
Construction of transA-GFP fusion proteins
Construction of transA-GFP, consisting of the transit peptide of pPORA (transA) fused to the GFP, was made by PCR-based cloning (1). The following primer pairs were used: primer 1 (5'-AAACTCGAGCATGGCTCTCCAGCTTCTC-3') plus 2 (5'-AAACCATGGTCGGCGACGACGCGGTCG-3') and clone A7 (2). The DNA for transB was amplified with primers 3 (5'-AAACTCGAGCATGGCTCTTCAGGCGGCC-3') and 4 (5'-AAACCATGGCCGGCGACGCCGGGGTTG-3') and clone L2 as template (3). All of the different PCR fragments were subcloned into pGEM-TEasy (Promega); then the inserts were removed with NcoI and XhoI and ligated into NcoI and SalI pretreated 35W -sGFP(S65T) vector (4).
1. Innis MA, Gelfand DH, Sninsky JJ, White TJ (1990) PCR Protocols (Academic, San Diego).
2. Schulz R, Steinmüller K, Klaas M, Forreiter C, Rasmussen S, Hiller C, Apel K (1989) Mol Gen Genet 217:355-361.
3. Holtorf H, Reinbothe S, Reinbothe C, Bereza B, Apel K (1995) Proc Natl Acad Sci USA 92:3254-3258.
4. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY).