Chlorophyll a Biosynthetic Pathway

VII. The Chl a Carboxylic Biosynthetic Routes: Conversion of Chlide a to Chl a

A. Chlorophyll a Biosynthetic Heterogeneity

1. Chlorophyll a formation by esterification of chlorophyllide a with geranylgeraniol in etiolated tissues

2. Preferential chlorophyll a formation by esterification of chlorophyllide a with Phytol in green tissues

b. Biosynthesis of MV Chlorophyll a via Route 5 in Etiolated DDV-LDV-LDDV Plants after Exposure to Light, and in Greening DDV-LDV-LDDV Plants during the Initial Dark Phases of the Photoperiod

c. Biosynthesis of MV Chlorophyll a via Routes 2 and 3 in Etiolated DDV-LDV-LDDV Plants after Exposure to Light, and in Greening DDV-LDV-LDDV Plants during the Initial Dark Phases of the Photoperiod

d. Biosynthesis of MV Chlorophyll a via Route 8 in Greening DDV-LDV-LDDV Plants during the Light Phases of the Photoperiod

e. Biosynthesis of MV Chlorophyll a via Route 10 in Greening DMV-LDV-LDMV Plants during the Light Phases of the Photoperiod

f. Biosynthesis of MV Chlorophyll a via Route 12 in Etiolated DMV-LDV-LDMV Plants after Exposure to Light, and in Greening DMV-LDV-LDMV Plants during the Initial Dark Phases of the Photoperiod

g. Biosynthesis of MV Chlorophyll a via Route 13 in Etiolated DMV-LDV-LDMV Plants after Exposure to Light, and in Greening DMV-LDV-LDMV Plants during the Initial Dark Phases of the Photoperiod

4. Biosynthetetic Heterogeneity of DV Chlorophyll a

b. Biosynthesis of DV Chlorophyll a via Route 1 in the Nec 7 Corn Mutant and in Picoplankton of the Euphotic Zone of the World Tropical and Temperate Oceans, and the Mediterranean Sea

Fig. 5: Reactions of the DV and MV Carboxylic Chl a Biosynthetic Routes Between Mg-Proto and Chl a

Most of the Chl a in higher and lower plants is formed by esterification of Chlide a. A minor Chl a fraction esterified with long chain fatty acids (LCFA) other than phytol is also formed from MV Pchlide a E in Section VIII. In this section emphasis will be placed on the biosynthetic heterogeneity of Chl a esterified with phytol.

A. Chlorophyll a Biosynthetic Heterogeneity

The biosynthetic heterogeneity of the Chl a of green plants is extremely complex. In addition to the DV and MV chemical heterogeneity of the Chl a chromophore, and the chemical and spatial biosynthetic heterogeneity of its immediate precursor, Chlide a (see section VI), another layer of biosynthetic heterogeneity is imposed by the esterification process. Indeed, although in green plants, most of the Chl a is esterified with phytol (C20H39OH), conversion of Chlide a to Chlide a-phytol appears to follow different routes in etiolated and green tissues (vide infra).

1. Chlorophyll a formation by esterification of chlorophyllide a with geranylgeraniol in etiolated tissues

In etiolated tissues subjected to a light treatment, formation of Chl a by esterification of Chlide a involves a complex set of reactions. Initially, it was observed that treatment of etiolated bean leaves with 1 min of light followed by dark incubation resulted in the transient appearance of putative Chlide a-granylgeraniol (GG) which was followed by the formation of Chl a-phytol (Ogawa, 1975). Subsequently etiolated wheat seedlings treated with herbicides then exposed to light followed by darkness, resulted in the accumulation of Chl a-GG and Chl a-dihydroxyGG (DHGG) (Rudiger et al 1976). This was followed by the demonstration of Chlide a esterification with GG in a cell-free system from maize shoots (Rudiger et al, 1977).

Further work dealing with the identification of various esterified Chlide a in etiolated tissues subjected to a brief light treatment followed by dark incubation, led to the proposal that during phytylation, Chlide a is first esterified with GG to yield Chl a-GG, which is reduced stepwise to Chl a-DHGG, to -Chl a-tetrahydroGG (THGG) and finally to Chl a-hexahydroGG, i. e. Chl a-phytol (Schoch, 1978).

The above hypothesis was confirmed in cell-free systems from various etiolated plant tissues. It was demonstrated that in irradiated etioplast-membrane fractions prepared form oat seedlings, [1-³H]-GG and its monophosphate were incorporated into Chl a only in the presence of exogenous ATP, whereas incorporation of activated [1-³H]-GG pyrophosphate (GG-PP) did not require ATP (Rudiger et al, 1980). In order to distinguish this enzymatic activity from chlorophyllase it was named Chl synthetase. Conversion of Chl-GG in vitro to Chl a-phytol by hydrogenation required the addition of exogenous NADPH. NADH was not a cofactor (Benz et al, 1980). Enzymic hydrogenation of Chl-GG to Chl a-phytol was inhibited by anaerobiosis (Schoch et al, 1980). Substrate specificity investigations indicated that Chl synthetase requires a chlorin derivative that contains Mg as a central metal ion. A hydrogenated ring D was mandatory since Pchlide a with a double bond at position 7-8 of the macrocycle was not a substrate (Benz et al, 1981; Helfrich et al, 1992). Direct esterification of endogenous Chlide a with exogenous phytol in the presence of added ATP, and Mg was also observed in etiolated tissues which led to the proposal that the conversion of Chlide a to Chl a may follow different biosynthetic routes having different substrate and cofactor requirements, depending on the stage of plastid development (Daniell and Rebeiz, 1984) In oat etioplasts, the relative substrate specificities for GG-PP, Phytol-PP and farnesyl-PP amounted to 6, 3, and 1 respectively (Rudiger, 1984).

Chlorophyll synthetase is present mainly in the prothylakoid and prolamellar body of etioplasts (Rudiger, 1984). Prolamellar body disaggregation and Chlide a esterification appear to be closely related phenomena. It appears that Chlide a formed in the prolamellar body can migrate with Pchlide-oxidoreductase to the prothylakoid membranes during light-dependent dissociation of prolamellar bodies (Rudiger, 1984).

2. Preferential Chlorophyll a formation by esterification of chlorophyllide a with Phytol in green tissues

Although illumination of etiolated tissues with white light leads to a slow decrease in Chl synthetase activity (Rudiger, 1993), Chl synthetase activity does not disappear completely, and some activity is still observed in mature chloroplasts (Soll and Schultz, 1981). In spinach chloroplasts, the relative substrate specificity for Chlide a esterification with exogenous GGPP and PhyPP were 1 and 4 respectively a (Sol et al, 1983).

In Arabidopsis thaliana a nuclear encoded gene, G4, was identified which exhibited homology to the product of the Rhodobacter capsulatus bchG locus which is involved in the esterification of bacteriochlorophyllide with GG (Gaubier et al, 1996). The relationship between gene G4 and bchG was confirmed by isolation and sequencing of a corresponding full length cDNA. The gene appears to consist of 14 exons, some of which were very short. Southern and Northern analyses showed that gene G4 is a single copy gene and its transcripts were only detected in green or greening tissues.

3. Biosynthetic heterogeneity of MV chlorophyll a

The formation of MV Chl a via seven different biosynthetic routs is discussed below.

a. Biosynthesis of MV Chlorophyll a by 4-Vinyl Reduction of DV Chlorophyll a via Route 7 in Etiolated DDV-LDV-LDDV Plants after Exposure to Light, and in Greening DDV-LDV-LDDV Plants during the Initial Dark Phases of the Photoperiod

It is presently acknowledged that during the initial phases of greening of etiolated tissues, most of the Chl consists of MV Chl a, located in PSI and PSII (Alberte et al, 1972; Akoyunoglou et al, 1981). As a consequence we propose that the MV Chl a formed via route 7 is destined to PSI and/or PSII Chl-protein complexes.

b. Biosynthesis of MV Chlorophyll a via Route 5 in Etiolated DDV-LDV-LDDV Plants after Exposure to Light, and in Greening DDV-LDV-LDDV Plants during the Initial Dark Phases of the Photoperiod.

Etiolated DDV-LDV-LDDV plants accumulate mostly DV Pchlide a. Upon exposure to light, DV Pchlide a is converted to DV Chlide a by PORA as described in section VID. Thus in etiolated DDV-LDV-LDDV plants subjected to illumination most of the MV Chlide a is formed from DV Chlide a by 4-vinyl reduction. Since in etiolated tissues, MV Chlide a conversion to MV Chl a takes place via esterification with GG (Schoch, 1978), it is our guess that, conversion of the nascent MV Chlide a to MV Chl a via route 5 takes place via Chl a-GG followed by stepwise hydrogenation to Chl a-Phytol as described in section VII1. This route is also most probably functional in DDV-LDV-LDDV plants growing under photoperiodic conditions, at the onset of illumination, during the first few dark cycles of the photoperiod when t-LW-Pchlide a accumulation is substantial (Cohen et al, 1977; Cohen and Rebeiz, 1978). Since during the initial phases of greening of etiolated tissues, most of the Chl consists of MV Chl a, located in PSI and PSII (Alberte et al, 1972; Akoyunoglou et al, 1981), we propose that the MV Chl a formed via route 5 is destined to PSI and/or PSII Chl- protein complexes.

c. Biosynthesis of MV Chlorophyll a via Routes 2 and 3 in Etiolated DDV-LDV-LDDV Plants after Exposure to Light, and in Greening DDV-LDV-LDDV Plants during the Initial Dark Phases of the Photoperiod.

During prolonged dark-incubation of DDV-LDV-LDDV plants, small amounts of MV Pchlide a are formed via routes 2 and 3 as discussed in section VAba. Upon exposure to light, MV Pchlide a is photoconverted to MV Chlide a by PORA. Most probably, conversion of the nascent MV Chlide a to MV Chl a via routes 2 and 3 takes place via Chl a-GG followed by stepwise hydrogenation to Chl a-Phytol as described in section VII1. These route are also functional in DDV-LDV-LDDV plants growing under photoperiodic conditions, at the onset of illumination, during the first few dark cycles of the photoperiod when t-LW-Pchlide a accumulation is substantial (Cohen et al, 1977; Cohen and Rebeiz, 1978).

Since during the initial phases of greening of etiolated tissues, most of the Chl consists of MV Chl a, located in PSI and PSII (Alberte et al, 1972 ; Akoyunoglou et al, 1981), we propose that the MV Chl a formed via routes 2 and 3 is destined to PSI and/or PSII Chl-protein complexes.

d. Biosynthesis of MV Chlorophyll a via Route 8 in Greening DDV-LDV-LDDV Plants during the Light Phases of the Photoperiod

In photoperiodically-grown DDV-LDV-LDDV plants, during the light phase of the photoperiod, MV Chlide a is formed from regenerated DV Pchlide a via DV Chlide a, by a reaction catalyzed by PORB as discussed in section VID2. Based on the prevalence of direct phytylation of MV Chlide a in green tissues (Soll et al, 1983), it is our guess that in route 8, most of then conversion of nascent MV Chlide a to MV Chl a proceeds by direct esterification of MV Chlide a with phytol. Since in nature, most of the Chl a is formed in the light, the size of the MV Chl a pool formed via route 8 is most probably very substantial.

Furthermore, since under continuous illumination, most of the synthesized Chl consists of MV Chl a, and b located in antenna Chl-protein complexes, (Alberte et al, 1972; Akoyunoglou et al, 1981), we propose that the MV Chl a formed via route 8 is destined to LHCII and other antenna Chl-protein complexes.

e. Biosynthesis of MV Chlorophyll a via Route 10 in Greening DMV-LDV-LDMV Plants during the Light Phases of the Photoperiod

In photoperiodically-grown DMV-LDV-LDMV plants, during the light phase of the photoperiod, MV Chlide a is formed from regenerated MV Pchlide a, a reaction catalyzed by PORB as discussed in section VIE2 Based on the prevalence of direct phytylation of MV Chlide a in green tissues (Soll et al, 1983), it is our guess that most of the conversion of nascent MV Chlide a to MV Chl a in route 10, proceeds by direct esterification of MV Chlide a with phytol. Since most of the Chl a is formed in the light, the size of the MV Chl a pool formed via route 7 is most probably very substantial.

Since under continuous illumination, most of the synthesized Chl consists of MV Chl a, and b located in antenna Chl-protein complexes, ( Alberte et al, 1972; Akoyunoglou et al, 1981), we propose that the MV Chl a formed via route 10 is destined to LHCII and other antenna Chl-protein complexes.

f. Biosynthesis of MV Chlorophyll a via Route 12 in Etiolated DMV-LDV-LDMV Plants after Exposure to Light, and in Greening DMV-LDV-LDMV Plants during the Initial Dark Phases of the Photoperiod

Etiolated DMV-LDV-LDMV plants accumulate mostly MV Pchlide a. Upon exposure to light, MV Pchlide a is converted to MV Chlide a by PORA as described in section VIE3. Since in etiolated tissues, MV Chlide a conversion to MV Chl a takes place via esterification with GG (Schoch, 1978), it is our guess that, conversion of the nascent MV Chlide a to MV Chl a via route 12 takes place via Chl a-GG followed by stepwise hydrogenation to Chl a-Phytol as described in section VII1. This route is also most probably functional in DMV-LDV-LDMV plants growing under photoperiodic conditions, at the onset of illumination, during the first few dark cycles of the photoperiod when t-LW-Pchlide a accumulation is substantial Cohen et al, 1977; Cohen and Rebeiz, 1978).

Since during the initial phases of greening of etiolated tissues, most of the Chl consists of MV Chl a, located in PSI and PSII (Alberte et al, 1972; Akoyunoglou et al, 1981), we propose that the MV Chl a formed via route 12 is destined to PSI and/or PSII Chl-protein complexes.

g. Biosynthesis of MV Chlorophyll a via Route 13 in Etiolated DMV-LDV-LDMV Plants after Exposure to Light, and in Greening DMV-LDV-LDMV Plants during the Initial Dark Phases of the Photoperiod

Etiolated DMV-LDV-LDMV plants accumulate mostly MV Pchlide a. In addition in some plant species such as corn, they accumulate smaller yet significant amounts of DV Pchlide a, Upon exposure to light, the small amounts of DV Pchlide a are converted to DV Chlide a by PORA as described in section VID3. Thus In etiolated DMV-LDV-LDMV plants subjected to illumination small amounts of MV Chlide a are formed from DV Chlide a by 4-vinyl reduction as discussed for DDV-LDV-LDDV plants in section VIF1. Since in etiolated tissues, MV Chlide a conversion to MV Chl a takes place via esterification with GG Schoch, 1978, it is our guess that, conversion of the nascent MV Chlide a to MV Chl a via route 13 takes place via Chl a-GG followed by stepwise hydrogenation to Chl a-Phytol as described in section VII1. This route is also most probably functional in DDV-LDV-LDDV plants growing under photoperiodic conditions, at the onset of illumination, during the first few dark cycles of the photoperiod when t-LW-Pchlide a accumulation is substantial Cohen et al, 1977 ; Cohen and Rebeiz, 1978.

Since during the initial phases of greening of etiolated tissues, most of the Chl consists of MV Chl a, located in PSI and PSII (Alberte et al, 1972; Akoyunoglou et al, 1981), we propose that the MV Chl a formed via route 13 is destined to PSI and/or PSII Chl-protein complexes.

4. Biosynthetetic Heterogeneity of DV Chlorophyll a

In normal higher plants DV Chl a (XXXXI) is a transient intermediate during MV Chl a formation. Under certain circumstances however, DV Chl a is the main Chl a that accumulates and participates in photosynthesis.

a. Transient Formation of DV Chlorophyll a via Route 1 in Etiolated DDV-LDV-LDDV Plants after Exposure to Light, and in Greening DDV-LDV-LDDV Plants during the Initial Dark Phases of the Photoperiod

In etiolated DDV-LDV-LDDV tissues, it has been repeatedly observed that when the mixed MV-DV Pchlide a is photoconverted into a mixed MV-DV Chlide a pool by a 2.5 ms light pulse, some of the nascent DV Chlide a is rapidly converted to DV Chl a during the first 30 s of dark incubation (Rebeiz et al, 1983) . More recently we have observed that within 10 s the nascent DV Chlide a is rapidly esterified to DV Chl a. In the ensuing 30 s a decrease in DV Chl a is accompanied by a stochiometric rise in MV Chl a (Adra and Rebeiz, 1998). The nature of the lonfg chain fatty acid (LCFA) of the nascent DV Chl a at position 7 of the macrocycle is unknown. This route is also most probably functional in DDV-LDV-LDDV plants growing under photoperiodic conditions, at the onset of illumination, during the first few dark cycles of the photoperiod when t-LW-Pchlide a accumulation is substantial Cohen et al, 1977; Cohen and Rebeiz.

b. Biosynthesis of DV Chlorophyll a via Route 1 in the Nec 7 Corn Mutant and in Picoplankton of the Euphotic Zone of the World Tropical and Temperate Oceans, and the Mediterranean Sea

The major fate of DV Chlide a resides in its conversion to MV Chlide a and MV Chl a (vide supra). However under certain circumstances, DV Chlide a is massively converted to DV Chl a by esterification. For example in the Nec 7 corn mutant (Bazzaz, 1981), the major fate of DV Chlide a is its conversion to DV Chl a (Rebeiz, et al, 1983, 2003). So is the case 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 (Chisholm et al, 1988, 1992; Goerike and Repeta, 1992; Veldhuis and Kraay, 1990). The nature of the LCFA at position 7 of the macrocycle and the details of esterification are unknown.

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