Chlorophyll b Biosynthetic Pathway


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  • XII. The Chl b Biosynthetic Pathway: Chl b Biosynthetic Routes

  • A. Biosynthesis of MV Chl b

  • 1. Conversion of MV Mg-Proto to MV Chl b via MV Pchlide a, MV Chlide a and MV Chl a (Route 1)
  • 2. Conversion of MV Mg-Proto to MV Chl b via MV Chlide a and MV Chlide b (Route 2)
  • 3. Conversion of MV Mg-Proto to MV Chl b via MV Pchlide b and MV Chlide b (Route 3)
  • 4. Conversion of DV Pchlide a to MV Chl b via MV Pchlide b and MV Chlide b (Route 4)
  • 5. Conversion of DV Pchlide a to MV Chl b via MV Pchlide a, MV Chlide a and MV Chlide b (Route 5)
  • 6. Conversion of DV Pchlide a to MV Chl b via MV Pchlide a, MV Chlide a and MV Chl a (Route 6).
  • 7. Conversion of DV Pchlide a to MV Chl b via DV Chlide a, MV Chlide a and MV Chl a (Route 7)
  • 8. Conversion of DV Pchlide a to MV Chl b via DV Chlide a, DV Chl a and MV Chl a (Route 8)

  • B. Biosynthesis of DV Chl b

  • 1. Conversion of DV Pchlide a to DV Chl b via DV Chlide a and DV Chl a (Route 9)
  • 2. Conversion of DV Pchlide a to DV Chl b via DV Chlide a and DV Chlide b (Route 10)

  • C. References

    XII. The Chl b Biosynthetic Pathway

    ChlbPWe2.GIF - 21.07 K

    Fig. 4: Synopsis of all Possible Reactions that May Result in the Formation of MV Chl b.

    The reactions between ALA and DV Proto are shown in more detail in Fig. 1. The various Chl a carboxylic biosynthetic routes are discussed in sections I to VIII. DV= divinyl; MV = monovinyl; Mg-Proto = Mg-protoporphyrin IX; Mpe = Mg-Proto monomethyl ester.


    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.

    A. Biosynthesis of MV Chl b

    1. Conversion of MV Mg-Proto to MV Chl b via MV Pchlide a, MV Chlide a and MV Chl a (Route 1)

    Chlb2Route1.GIF - 4.15 K

    MV Pchlide a
    MV Chl a
    MV Chl b

    Conversion of MV Mg-Proto to MV Chl b via MV Pchlide a, MV Chlide a, and MV Chl a may take place in DMV-LDV-LDMV plant species, such as corn, wheat, oat and barley (see Classification of Green Plants into Various Greening Groups) which possess 4-vinyl Mg-proto reductase activity (see Metabolism of MV Mg-Proto) and (Kim and Rebeiz, 1996; Abd El Mageed et al, 1997). The conversion of MV Mg-Proto to MV Pchlide a has been discussed under Metabolism of MV Mg-Proto and metabolism of MV Mpe. The photoconversion of MV Pchlide a to MV Chlide a has been discussed under Photoconversion of the MV Pchlide a Chromophore to MV Chlide a. The conversion of MV Chlide a to MV Chl a, has been discussed under Conversion of MV Mpe to MV Chl a via MV Pchlide a and MV Chlide a.

    The conversion of MV Chl a to MV Chl b in DMV-LDV-LDMV plant species such as etiolated corn has been reported by Shlyk and coworkers (Shlyk et al 1970, 1971a, 1971b, 1971c). Essentially it was reported that exogenous MV Chl a was convertible to MV Chl b by tissue homogenates prepared from etiolated corn seedlings before and after treatment with white light for 20 seconds. Shlyk and coworkers, championed the idea that under natural conditions, MV Chl b was formed from Young (i. e.newly formed MV Chl a molecules).

    In corn seedlings that were greened for 5 and 7 hours, studies of precursor- product relationships in- vivo between MV Chl a and MV Chl b did not indicate any possible precursor product relationships between these two tetrapyrroles (Ioannides, 1993). This in turn casts doubt about the formation of the bulk of Chl b via biosynthetic route 1 in greening DMV-LDV-LDMV plant species such as barley and corn. More elaborate in vitro studies are required to validate this hypothesis.

    2. Conversion of MV Mg-Proto to MV Chl b via MV Chlide a and MV Chlide b (Route 2)

    Chlb2Route2.GIF - 3.95 K

    MV Chlide a
    MV Chlide b
    MV Chl b

    The conversion of MV Mg-Proto to MV Chl b via MV Pchlide a, MV Chlide a, and MV Chlideb in DMV-LDV-LDMV plant species, such as corn, wheat, oat and barley (see Classification of Green Plants into Various Greening Groups) is discussed below. Such plant species possess 4-vinyl Mg-proto reductase activity (see Metabolism of MV Mg-Proto) and (Kim and Rebeiz, 1996; Abd El Mageed et al, 1997). The conversion of MV Mg-Proto to MV Pchlide a has been discussed under Metabolism of MV Mg-Proto and metabolism of MV Mpe. The photoconversion of MV Pchlide a to MV Chlide a has been discussed under Photoconversion of the MV Pchlide a Chromophore to MV Chlide a.

    To our knowledge, the conversion of MV Chlide a to MV Chlide b in DMV-LDV-LDMV plant species has not been investigated in vitro. In corn seedlings that were greened for15 hours, studies of precursor- product relationships in- vivo between MV Chlide a and MV Chlide b did not indicate any possible precursor product relationships between these two tetrapyrroles (Ioannides, 1993). This in turn casts doubt about the formation of Chl b via biosynthetic route 2 in greening DMV-LDV-LDMV plant species such as barley and corn. More elaborate in vitro studies are required to validate this hypothesis.

    3. Conversion of MV Mg-Proto to MV Chl b via MV Pchlide b and MV Chlide b (Route 3)


    Chlb2Route3.GIF - 3.31 K

    MV Pchlide b
    MV Chlide b
    MV Chl b

    The conversion of MV Mg-Proto to MV Chl b via MV Pchlide a, MV Pchlide b, and MV Chlideb in DMV-LDV-LDMV plant species, such as corn, wheat, oat and barley (see Classification of Green Plants into Various Greening Groups) is discussed below. Such plant species possess 4-vinyl Mg-proto reductase activity (see Metabolism of MV Mg-Proto) and (Kim and Rebeiz, 1996; Abd El Mageed et al, 1997).

    This hypothetical biosynthetic route is proposed on the basis of two distinct observations namely: (a) the discovery of Pchlide b in green plants (Shedbalkar et al, 1991), and (b) the observation that zinc protopheophorbide b (i. e. demetalated zinc Pchlide b) was photoreducible by NADPH-Pchlide oxidoreductase from etiolated wheat to zinc pheophorbide b (i. e demetalated zinc Chlide b) (Schoch et al, 1995). The conversion of exogenous MV Chlide b to MV Chl b in etiolated oat has been reported by Benz and Rudiger (1981). Further studies of precursor-product relationships in vitro will be useful in validating the operation of this hypothetical route in DM-LDV-LDMV plant species.

    4. Conversion of DV Pchlide a to MV Chl b via MV Pchlide b and MV Chlide b (Route 4)


    Chlb2Route4.GIF - 3.74 K
    MV Pchlide b
    MV Chlide b
    MV Chl b

    The conversion of DV Pchlide a to MV Chl b via MV Pchlide a, MV Pchlide b, and MV Chlideb in DMV-LDV-LDMV plant species, such as corn, wheat, oat and barley, and DDV-LDV-LDDV plant species such as cucumber(see Classification of Green Plants into Various Greening Groups) is discussed below. DMV-LDV-LDMV plant species possess very active 4-vinyl Pchlide a reductase activity, while in DDV-LDV-LDDV plant species 4-vinyl Pchlide a reductase activity is expressed after prolonged dark incubation (see Tripathy and Rebeiz, 1988; also see Metabolism of DV Mg-Proto) and Abd El Mageed et al, 1997).

    This hypothetical biosynthetic route is proposed by analogy to biosynthetic route 3. The conversion of exogenous MV Chlide b to MV Chl b in DMV-LDV-LDMV species (oat) and in DDV-LDV-LDDV species (cucumber) has been reported by Benz and Rudiger (1981) in oat, and by Shioi and Sass (1983) in cucumber. Further studies of precursor-product relationships in vitro will be useful in validating the operation of this hypothetical route.

    5. Conversion of DV Pchlide a to MV Chl b via MV Pchlide a, MV Chlide a and MV Chlide b (Route 5)

    Chlb2Route5.GIF - 3.72 K


    MV Chlide a
    MV Chlide b
    MV Chl b

    The conversion of DV Pchlide a to MV Chl b via MV Pchlide a, MV Chlide a, and MV Chlide b, in DMV-LDV-LDMV plant species, such as corn, wheat, oat and barley, and DDV-LDV-LDDV plant species such as cucumber(see Classification of Green Plants into Various Greening Groups) is discussed below. LDD-LDMV plant species possess very active 4-vinyl Pchlide a reductase activity, while in DDV-LDV-LDDV plant species 4-vinyl Pchlide a reductase activity is expressed after prolonged dark incubation (see Tripathy and Rebeiz, 1988; also see Metabolism of DV Mg-Proto) and Abd El Mageed et al, 1997)

    The conversion of DV Mg-Proto to DV Pchlide a has been discussed under Metabolism of DV Mg-Proto and metabolism of DV Mpe. The conversion of DV Pchlide a to MV Pchlide a has been reported by Tripathy and Rebeiz (1988). The photoconversion of MV Pchlide a to MV Chlide a has been discussed under Photoconversion of the MV Pchlide a Chromophore to MV Chlide a. The lack of conversion of MV Chlide a to MV Chlide b in DMV-LDV-LDMV plant species such as greening corn (Ioannides, 1993) suggests that this route is not functional in such plant species. It may be functional however in DDV-LDV-LDDV plant species such as cucumber. The conversion of exogenous MV Chlide b to MV Chl b in DDV-LDV-LDDV species has been reported by Shioi and Sasa (1983) in cucumber. Further studies of precursor-product relationships in vitro will be useful in validating the operation of this hypothetical route.

    6. Conversion of DV Pchlide a to MV Chl b via MV Pchlide a, MV Chlide a and MV Chl a (Route 6)

    Chlb2Route6.GIF - 3.70 K

    MV Chlide a
    MV Chl a
    MV Chl b

    The possible conversion of DV Pchlide a to MV Chl b via MV Pchlide a, MV Chlide a, and MV Chl a, in DMV-LDV-LDMV plant species such as, corn, wheat, oat, and barley, and in DDV-LDV-LDDV plant species such as cucumber (see Classification of Green Plants into Various Greening Groups) is discussed below. LDD-LDMV plant species possess very active 4-vinyl Pchlide a reductase activity, while in DDV-LDV-LDDV plant species 4-vinyl Pchlide a reductase activity is expressed after prolonged dark-incubation (see Tripathy and Rebeiz, 1988; also see Metabolism of DV Mg-Proto) and Abd El Mageed et al, 1997)

    The conversion of DV Mg-Proto to DV Pchlide a has been discussed under Metabolism of DV Mg-Proto and metabolism of DV Mpe. The conversion of DV Pchlide a to MV Pchlide a has been reported by Tripathy and Rebeiz (1988). The photoconversion of MV Pchlide a to MV Chlide a has been discussed under Photoconversion of the MV Pchlide a Chromophore to MV Chlide a. The lack of conversion of MV Chl a to MV Chl b in DMV-LDV-LDMV plant species such as greening corn (Ioannides, 1993) suggests that this route is not functional in such plant species. It may be functional however in DDV-LDV-LDDV plant species such as cucumber. The conversion of MV Chl a to MV Chl b in DDV-LDV-LDDV species has been reported by Ioannides (1983) in cucumber, in vivo and in vitro. The conversion of MV Chl b to MV Chl a in cucumber etioplasts via putative 7-hydroxymethyl chlorophyll has been reported by Tanaka and coworkers (Ito et al, 1993; 1994; 1996; Ito and Tanaka, 1996; Ohtsuka et al, 1997). This work needs to be independently replicated in other laboratories, and NMR characterization of the 7-hydroxymethyl intermediate need to be performed.

    7. Conversion of DV Pchlide a to MV Chl b via DV Chlide a, MV Chlide a and MV Chl a (route 7)

    Chlb2Route7.GIF - 3.66 K

    MV Chlide a
    MV Chl a
    MV Chl b

    The conversion of DV Pchlide a to MV Chl b via DV Chlide a, MV Chlide a, and MV Chl a, cannot take place in greening DMV-LDV-LDMV plant species such as, corn, wheat, oat, and barley which lack the capability of converting DV Chlide a to MV Chlide a via 4-vinyl Chlide a reductase (4VCR) (Abd El Mageed et al, 1997). This route may be operational however in greening DDV-LDV-LDDV plant species such as cucumber (see Classification of Green Plants into Various Greening Groups) which possess 4VCR activity (Abd El Mageed et al, 1997).

    The conversion of DV Mg-Proto to DV Pchlide a has been discussed under Metabolism of DV Mg-Proto and metabolism of DV Mpe. The photoconversion of DV Pchlide a to DV Chlide a has been discussed under Photoconversion of the DV Pchlide a Chromophore to DV Chlide a. The conversion of DV Chlide a to MV Chlide a in greening DDV-LDV-LDDV plant species has been reported by (Abd El Mageed et al, 1997). The conversion of MV Chlide a to MV Chl a, has been discussed under Conversion of MV Mpe to MV Chl a via MV Pchlide a and MV Chlide a. Finally the conversion of MV Chl a to MV Chl b in DDV-LDV-LDDV species has been reported by Ioannides (1983) in cucumber, in vivo and in vitro. The conversion of MV Chl b to MV Chl a in cucumber etioplasts via putative 7-hydroxymethyl chlorophyll has been reported by Tanaka and coworkers (Ito et al, 1993; 1994; 1996; Ito and Tanaka, 1996; Ohtsuka et al, 1997). This work needs to be independently replicated in other laboratories, and NMR characterization of the 7-hydroxymethyl intermediate need to be performed.

    8. Conversion of DV Pchlide a to MV Chl b via DV Chlide a, DV Chl a and MV Chl a (Route 8)

    Chlb2Route8.GIF - 3.63 K

    DV Chl a
    MV Chl a
    MV Chl b
    This MV Chl b biosynthetic route may be operational only in greening DDV-LDV-LDDV plant species such as cucumber (see Classification of Green Plants into Various Greening Groups) which possess 4VCR activity (Abd El Mageed et al, 1997). The conversion of DV Chlide a to DV Chl a has been discussed in section VIII: Conversion of DV Chlide a to DV Chl a. The conversion of DV Chl a to MV Chl a has been reported during post-illumination dark period following the treatment of etiolated cucumber cotyledons with a 2.5 ms actinic light flash (Adra, 1996). The conversion of MV Chl a to MV Chl b in DDV-LDV-LDDV species has been reported by Ioannides (1983) in cucumber, in vivo and in vitro. The conversion of MV Chl b to MV Chl a in cucumber etioplasts via putative 7-hydroxymethyl chlorophyll has been reported by Tanaka and coworkers (Ito et al, 1993; 1994; 1996; Ito and Tanaka, 1996; Ohtsuka et al, 1997). This work needs to be independently replicated in other laboratories, and NMR characterization of the 7-hydroxymethyl intermediate need to be performed

    B. Biosynthesis of DV Chl b

    As was reported in Section X DV Chl b accumulates in the Nec 2 maize mutant (ex-ON 8147) to the extent of about 100 nmoles per gram fresh weight (Rebeiz, Unpublished). It 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). its biosynthesis is discussed below.

    1. Conversion of DV Pchlide a to DV Chl b via DV Chlide a, and DV Chl a (Route 9)

    Chlb2Route9.GIF - 4.07 K

    DV Chlide a
    DV Chl a
    DV Chl b

    Conversion of DV Pchlide a to DV Chl b via DV Chlide a and DV Chl a probably takes place in the Nec 2 corn mutant and in the prochlorophyte picoplankton (see above) which form and accumulate only DV Chl a and DV Chl b. precursor- product relationships in- vivo and in vitro is required however, to validate this hypothesis.

    2. Conversion of DV Pchlide a to DV Chl b via DV Chlide a and DV Chlide b (Route 10)

    Chlb2Route10.GIF - 3.89 K

    DV Chlide a
    DV Chlideb
    DV Chl b
    Conversion of DV Pchlide a to DV Chl b via DV Chlide a, and DV Chlide b may take place in the Nec 2 corn mutant and in the prochlorophyte picoplankton (see above) which form and accumulate only DV Chl a and DV Chl b. precursor- product relationships in- vivo and in vitro is required however, to validate this hypothesis.

    E. References



    1. Rebeiz, C. A., J. M. Mayasich and B. C. Tripathy (1988). Chloroplast Biogenesis 61: Kinetic analysis of precursor-product relationships in complex biosynthetic pathways. J. Theor. Biol. 133: 319-326.
    2. Tripathy, B. C. and C. A. Rebeiz (1988). Chloroplast Biogenesis 60: Conversion of divinyl protochlorophyllide to monovinyl protochlorophyllide in green(ing) barley, a dark monovinyl light divinyl plant species. Plant Physiol. 87: 89-94.
    3. Brereton, R. G., and M. B. Bazzaz (1983). Positive and negative ion fast atom bombardment mass spectroscopic studies on chlorophylls: Structure of 4-vinyl-4-desethyl chlorophyll b. Tetrahedron, 24: 5775-5778.
    4. Wu, S. M. and C. A. Rebeiz. Chloroplast biogenesis. Molecular structure of chlorophyll b (E489 F666). J. Biol. Chem. 260: 3632-3634
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    7. Shlyk A. A., A. B. Rudoi and A. Yu. Veitskii (1970). Immediate appearance and accumulation of chlorophyll b after a short illumination of etiolated maize seedlings. Photosynthetica. 4: 68-77.
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    9. Shlyk A. A., I. V. Prudnikova, G. E. Savchenko and M. S. Grozovskaya. (1971b). Accumulation of chlorophyll b and protochlorophyllide by a leaf tissue homogenate in the dark. Dokl. Akad. Nauk. SSSR. 200: 222-225
    10. Shlyk A. A. (1971c). Biosynthesis of chlorphyll b. Annu Rev. Plant Physiol. 22: 169-184
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    12. Schoch S., M. Helfrich, B. Wiktorsson, C. Sundqvist, W. Rudiger, and M. Ryberg (1995). Photoreduction of xinc protopheophorbide b with NADPH-protochlorophyllide oxidoreductase from etiolated wheat (Triticum aestivum L.). Eur. J. Biochem. 229: 291-298.
    13. Benz J. and W. Rudiger (1981). Chlorophyll biosynthesis: Various chlorophyllides as exogenous substrates for chlorophyll synthetase. Z. Naturforsch. 36c: 51-57.
    14. Shioi Y. and T. Sasa (1983). Esterification of chlorophyllide b in higher plants. Bioch. Biophys. Acta. 756: 127-131.
    15. Ito H., Y. Tanaka, H. Tsuji, and A. Tanaka (1993). Conversion of chlorophyll b to chlorophyll a by isolated cucumber etioplasts. Arch. Biochem. Biophys. 306: 148-151.
    16. Ito, H; S. Takaichi, H. Tsuji, and A. Tanaka (1994). Properties of synthesis of chlorophyll a from chlorophyll b in cucumber etioplasts. J. Biol. Chem. 269: 22034-22038.
    17. Ito, H. T. Ohtsuka, and A. Tanaka (1996). Conversion of chlorophyll b to chlorophyll a via 7-hydroxymethyl chlorophyll. J. Biol. Chem. 271: 475-1479.
    18. Ito H. and A. Tanaka (1996). Determination of the activity of chlorophyll b to chlorophyll a conversion during greening of etiolated cucumber cotyledons by using pyrochlorophyllide b. Plant Physiol. Biochem. 34: 35-40.
    19. Ohtsuka T., H. Ito, and A. Tanaka (1997). Conversion of chlorophyll b to chlorophyll a and the assembly of chlorophyll with apoproteins by isolated chloroplasts. Plant Physiol. 113: 137-147.
    20. Adra, A. (1996). Development of a cell-free system for the study of the terminal stages of the fully esterified Chlorophyll a biosynthetic routes. M.S. Thesis, university of Illinois, Urbana, PP 73.
    21. 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.
    22. 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.
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