Chlorophyll b Biosynthetic Pathway

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  • IX. The Chl b Biosynthetic Pathway: Novel Metabolic Intermediates or End Products

  • A. Monovinyl Protochlorophyllide b (MV Pchlide b)
  • B. Divinyl Protochlorophyllide b (DV Pchlide b)
  • C. Moovinyl Chlorophyllide b (MV Chlide b)
  • D. Divinyl Chlorophyllide b (DV Chlide b)
  • E. Divinyl Chlorophyll b (DV Chl b)
  • D. References

    Fig. 1. Integrated Chl a/b biosynthetic pathway depicting 15 carboxylic routes. To facilitate understanding of the text, various biosynthetic routes are designated by the numbers 1-14. Black boxes with yellow lettering joined by black arrows and numbers refer to biosynthetic routes that occur in etiolated tissues in darkness, or in greening tissues at the beginning of the light phase of the photoperiod. White boxes with green lettering joined by yllowish-brown arrows and numbers refer to biosynthetic routes that occur in green tissues, during the light phases of the photoperiod. Blue frames refer to routes that occurin DDV-LDDV greening groups. Red frames refer to routes that occurin DMV-LDMV greening groups.

    FourteenRoutesCarboxylicPW.jpg - 86601 Bytes


    The demonstration of metabolic pathways is a multistep process. It involves at least three stages: (a) the detection and characterization of metabolic intermediates, (b) the demonstration of precursor-product relationships between putative intermediates, and (c) purification and characterization of enzymes involved in the metabolic interconversions. These criteria will be applied in our evaluation of the experimental evidence that supports the operation of a multibranched Chl b biosynthetic pathway in green(ing) plants.

    During the past two decades, several putative metabolic intermediates of the Chl b biosynthetic pathway have been detected in higher and lower plants. These intermediates are discussed below.

    A. Monovinyl Protochlorophyllide b (MV Pchlide b)

    MV Pchlide b


    The discovery of protochlorophyllide (Pchlide) b in higher plants was reported by Shedbalkar et al in 1991. It was first observed as a fluorescent compound at 77 K in diethyl ether, with Soret excitation and red emission maxima at 463 and 643 nm respectively. These fluorescence properties were identical to those of synthetic Pchlide phytyl ester b. The chemical structure of the latter was confirmed by proton nuclear magnetic resonance, fast atom bombardment mass spectroscopic analysis and chemical derivatization coupled to electronic spectroscopic analysis (Shedbalkar et al, 1991). MV Pchlide b differed from MV Pchlide a by the presence of a formyl instead of a methyl group at position 3 of the macrocycle. The trivial name MV Pchlide b was proposed to differentiate it from MV Pchlide a.

    Determination of the Amount of MV Pchlide b either in the Presence of MV Chl(ide) a and b, or in the presence of MV Pchlide a was achieved by combined spectrofluorometric analysis at room temperature and 77 K (Shedbalkar et al, 1997). In green cucumber seedlings grown under a 14-h light/10-h dark photoperiod, the amount of MV Pchlide b ranged from about 400 to 800 nmoles per 100 mg proteins. MV Pchlide b was not detectable green tissues but not in etiolated tissues or during the early phases of greening of etiolated tissues (Kolossov and Rebeiz, 2004).

    B. Divinyl Protochlorophyllide b (DV Pchlide b)

    DV Pchlide b

    So far it has not been possible to detect DV Pchlide b in plants.

    C. Moovinyl Chlorophyllide b (MV Chlide b)

    MV Chlide b
    Monovinyl chlorophyllide b was first detected in greening (Duggan and Rebeiz, 1981; 1982) and green higher plant tissues (Aronoff, 1981). The pool of MV Chlide b exhibited the spectrofluorometric properties of MV Chl b in diethyl ether at 298 and 77 K, but had the chromatographic mobility and solubility of a monocarboxylic phorbin. The presence of a free carboxylic group and a formyl group was demonstrated by methylation with diazomethane and conversion to a Chlide b oxime upon reaction with hydroxylamine (Duggan and Rebeiz, 1982). The concentration of Chlide b in green tissues was in the same range as that of MV Pchlide a and MV Chlide a. It was estimated that less than 15% of the Chlide b pool could have arisen from chlorophyllase activity in vitro as confirmed by the extent of hydrolysis of 14C-labeled MV Chl b added to green tissues just before pigment extraction (Duggan and Rebeiz, 1982).

    D. Divinyl Chlorophyllide b (DV Chlide b)

    DV Chlide b
    DV Chlide b has so far been detected only in the Nec 2 maize mutant (that used to be known as the ON 8147 mutant) (C. A. Rebeiz, unpublished). This mutation is a lethal mutation, the leaves are pale yellow, and accumulate only DV Chl a and b (Bazzaz, 1981). Nec2 maize leaves accumulate DV Chlide b to the extent of about 1. 00 nmoles per gram of fresh leaves (C. A. Rebeiz, unpublished).

    DV Chlide b may also be present in the prochlorophyte picoplankton of the subtropical waters of the North Atlantic as well as in the picoplankton of the euphotic zone of the world tropical and temperate oceans, and the Mediterranean sea, where DV Chl a and b are the predominant Chl species (Veldhuis and Kraay, 1990; Chisholm et al, 1990; Chisholm et al, 1992; Goerike and Repeta, 1992).

    DV Chlide b exhibits the same electronic spectroscopic properties as DV Chl b (see below) but differs from the latter by its solubility in more polar organic solvents and its chromatographic mobility.

    E. DV Chl b

    DV Chl b
    The possible occurrence of DV Chl b in green plants was predicted after the discovery of DV Chl a (Rebeiz, et al, 1980). It was next detected in the Nec 2 maize mutant (ex-ON 8147) by electronic spectroscopy (Bazzaz, 1981). Its chemical structure was ascertained by fast atom mass spectroscopic (Brereton and Bazzaz, 1983), and 1H NMR analysis (Wu and Rebeiz, 1985). It accumulates to the extent of about 100 nmoles per gram fresh weight of Nec 2 leaves (Rebeiz, Unpublished).

    DV Chl b also accumulates in the prochlorophyte picoplankton of the subtropical waters of the North Atlantic as well as in the picoplankton of the euphotic zone of the world tropical and temperate oceans, and the Mediterranean sea, where DV Chl a and b are the predominant Chl species (Veldhuis and Kraay, 1990; Chisholm et al, 1990; Chisholm et al, 1992; Goerike and Repeta, 1992)

    D. References



    1. Shedbalkar V.P., I. M. Ioannides, and C. A. Rebeiz (1991). Chloroplast Biogenesis. Detection of monovinyl protochlorophyll(ide) b in plants. J. Biol. Chem. 266 : 17151-17157
    2. Ioannides, M. I., V. P. Shedbalkar, and C. A. Rebeiz (1997). Quantitative determination of 2-monovinyl protochlorphyll(ide) b by spectrofluorometry. Anal. Biochem. 249: 241-244.
    3. Klossov, Vladimir. L., Rebeiz, Constantin A. (2004) Chloroplast Biogenesis 88. Protochlorophyllide b occurs in green but not in etiolated plants. J. Biol. Chem. 278: 49675-49678.
    4. Duggan, J. X. and C. A. Rebeiz. Detection of a naturally occurring chlorophyllide b pool in higher plants. Plant Physiol. 67 (suppl):267
    5. Duggan, J. X. and C. A. Rebeiz. Chloroplast Biogenesis 38. Quantitative detection of a chlorophyllide b pool in higher plants. Biochim. Biophys. Acta. 714: 248-260.
    6. Aronoff, S. (1981). Chlorophyllide b. Biochem. Biophys. Res. Commun. 102: 108-112.
    7. Rebeiz, C. A., F. C. Belanger, G. Freyssinet and D. G. Saab (1980). Chloroplast Biogenesis. XXIX. The occurrence of several novel chlorophyll a and b chromophores in plants. Biochim. Biophys. Acta. 590: 234-247
    8. Bazzaz, M. B., (1981) New chlorophyll chromophores isolated from a chlorophyll deficient mutant of maize. Photobiochem. Photobiosphys. 2: 199-207.
    9. Wu, S. M. and Rebeiz, C. A. Chloroplast biogenesis. Molecular structure of chlorophyll b (E489 F666). J. Biol. Chem. 260:3632-3634.
    10. Brereton, R. G., and Bazzaz, M. B. (1983) Positive and negative fast atom bombardment mass spectroscoppic studies on chlorophylls: Structure of 4-vinyl-4-desethyl chlorophyll b. Tetrahedron Lett. 24: 5775-5778.
    11. Veldhuis, M. J. W., and G. W. Kraay, (1990) Vertical distribution of pigment composition of a picoplanktonic prochlorophyte in the subtropical north Atlantic: A combined study of pigments and flow cytometry. Mar. Ecol. Progr Ser. 68: 121-127.
    12. Chisholm, S. W., R. J. Olson, E. R. Zettler, R. Goericke, W. J. B., and N. A. Welschmeyer, (1990) A novel free-living prochlorophyte abundant in the oceanic euphotic zone. Nature. 334: 340-343.
    13. Chisholm, S. W., S. Frankel, R. Goerike, R. Olson, R. Palenic, B. Urbach, J. Waterbury, and E. Zettler, (1992) Prochlorococcus marinus nov.gen. sp.: an oxyphototropic marine prokaryote containing divinyl chlorophyll a and b. Arch. Mikrobiol. 157: 297-300.
    14. Goerike, R., and D. Repeta, (1992) The pigments of Prochlorococcus marinus. The presence of divinyl-chlorophyll a and b in a marine prochlorophyte. Limnol. Oceanogr. 37: 425-433.

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