Fig. 2. Integrated Chl a/b biosynthetic pathway depicting the two fully esterified biosynthetic routes. To facilitate understanding of the text,
the biosynthetic routes are designated by the numbers 1, 16, and 17. Biosynthetic routes 16 and 17 branch from biosynthetic routes 1 and 12 of Fig. 1. 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.
The fully esterified Chl a pathway is populated by tetrapyrroles with a methyl propionate residue at position 6 of the macrocycle and a propionic acid residue at position 7 which is
esterified with a long chain fatty alcohol (Rebeiz et al. 1983). This pathway deals with the least understood phase of the intermediary metabolism of Chl
a . In what follows a brief account of what is known about this pathway is presented.
A. Mg-Protoporphyrin IX diester (Mpde)
Mg-proto diesters (Mpdes) (see formulas below) are the first metabolic intermediates of the fully esterified Chl a biosynthetic routes (Fig. 2, routes 16, 17). The fully esterified Chl a pathway is populated by tetrapyrroles with a methyl propionate residue at position 6 of the macrocycle and a propionic acid residue at position 7 which is esterified with one of several different long chain fatty alcohols (LCFAs) (Rebeiz et al, 1983, 2003). The fully esterified biosynthetic routes of the Chl a biosynthetic pathway deal with the least understood phases of the intermediary metabolism of Chl a. In our opinion, the unjustified neglect of this facet of Chl a biosynthesis is motivated by several factors, among which (a) occurrence of metabolic intermediates in very small amounts, (b) slow reaction rates, (c) analytical difficulties, and (d) misconceptions that date back to the late 1930s, which assumed that Pchlide a ester was the major precursore of all chl a in nature.
A fully esterified Mg-Proto diester (Mpde) pool was first detected in etiolated cucumber cotyledons incubated overnight with ALA and 2,2'-dipyridyl (Dpy) in darkness (McCarthy et al, 1981). The novel pool exhibited the chromatographic properties of a fully esterified metalloporphyrin and the spectrofluorometric properties of Mg-protoporphyrin IX. Chemical derivatization coupled to spectrofluorometric and chromatographic analysis identified it as a Mg-Proto diester (Mpde). Mpde was also detected in dark-grown Euglena gracilis and in etiolated cucumber cotyledons incubated in darkness with ALA, in the absence of added Dpy. Upon detection of Mpde, it was suggested to be a metabolic precursor of the fully esterified, heterogeneous, Pchlide a ester pool (McCarthy et al, 1981).
High-pressure liquid chromatographic analysis indicated that the Mpde pool was heterogeneous and consisted of three fully esterified Mg-Protos. Gas-chromatographic /mass spectroscopic analysis of the saponified alcohol fraction of the heterogeneous Mpde pool revealed that the latter consisted of three major long-chain alcohols, none of which was identifiable with known isoprenoids such as, farnesol or phytol (McCarthy et al, 1981).
In addition to the heterogeneity of the long chain alcohols esterifying the propionic acid residue at position 7 of the macrocycle, the Mpde pool exhibited a well pronounced DV-MV chemical heterogeneity (Belanger and Rebeiz, 1982). For example in etiolated cucumber cotyledons incubated in darkness with ALA and Dpy, as well as in dark-grown Euglena gracilis, the Mpde pool consisted of DV and MV Mpde components. In general the proportion of DV Mpde was higher than that of MV Mpde, except in Euglena.
The biosynthetic origin of DV Mpde is not presently clear, and is tentatively assigned to DV Mpe esterification (Fig. 2, route 17). After its detection in cucumber cotyledons and in Euglena cultures (Belanger and Rebeiz, 1982), DV Mpde , has been proposed as a precursor of fully esterified DV Pchlide a, i. e. DV Pchlide a ester (DV Pchlide a E) Fig. 2, route 17. A precursor-product relationship between DV Mpde and DV Pchlide a E remains to be established and is complicated by the presence of Mpde esterases that convert exogenous Mpde to Mpe (Rebeiz, unpublished).
The biosynthetic origin of MV Mpde is not presently clear, and is tentatively assigned to MV Mpe esterification (Fig. 2, route 16). After its detection in cucumber cotyledons and in Euglena cultures where it is the main constituent of the Mpde pool (Belanger and Rebeiz, 1982), MV Mpde has been proposed as a precursor of MV Pchlide a E (Fig. 2, route 16). A precursor-product relationship between MV Mpde and DV Pchlide a E remains to be established and is complicated by the presence of Mpde esterases that convert exogenous Mpde to Mpe (Rebeiz, unpublished).
Pchlide a E is one of the least understood pools of the Chl biosynthetic pathway and its history is steeped in controversy. Fischer and Oestreicher (1940) synthesized the phytyl ester of Pchlide a and showed that it differed from MV Chl a in having two fewer hydrogens at position 7 and 8 of the macrocycle. They named this molecule protochlorophyll. Because of the structural similarity between Pchlide a phytyl ester and Chl a the erroneous notion evolved that Pchlide a phytyl ester was the major immediate photoprecursor of Chl a (Smith, 1948; Koski, 1950). When Granick, (1950) isolated and identified Pchlide a from an X-ray Chlorella mutant inhibited in its capability to form Chl, he considered it to be the immediate precursor of Pchlide a ester. The biological function of Pchlide a as the immediate precursor of chlorophyllide (Chlide) a was not fully understood till seven years later (Wolff and Price, 1957).
Because of the structural similarity between Pchlide a and Pchlide a E it was convenient to propose that Pchlide a was the immediate precursor of Pchlide a E (Granick, 1950). However, as early as 1970, precursor-product relationship studies in vivo, between the biosynthesis of 14C-Pchlide a and 14C-Pchlide a E failed to establish a precursor-product relationship between these two tetrapyrroles. Instead, the results indicated that Pchlide a and Pchlide a E were most probably formed in parallel from a common precursor (Rebeiz et al, 1970). These studies were confirmed by in vitro investigations which also failed to establish precursor product relationships between Pchlide a and Pchlide a E (Ellsworth and Nowak, 1973; Mattheis and Rebeiz, 1977). Later on, more rigorous precursor-product relationship studies between Pchlide a and Pchlide a E were carried out (McCarthy et al, 1982). Comparison of the ratio of 14C-ALA and various 14C-tetrapyrrole substrates incorporation into 14 C-Pchlide a and 14C-Pchlide a E in vitro, allowed the determination of which exogenous 14C-tetrapyrrole substrate was the most likely common precursor of Pchlide a and Pchlide a E. On the basis of these studies, it was proposed that Pchlide a was formed via an acidic (monocarboxylic) biosynthetic route while Pchlide a E was formed via a fully esterified route. It was also proposed that the two routes are weakly linked at the level of Proto, Mg-Proto, Mpe and Pchlide a by porphyrin ester synthetases (McCarthy et al, 1982).
As early as 1958 various researchers started questioning the assumed phytol nature of the long chain fatty alcohol that esterified the propionic acid residue at position 7 of the Pchlide a ester macrocycle (Rebeiz and Castelfranco, 1973). For example gas chromatographic analysis of the hydrolyzed fatty alcohol fraction of Pchlide a ester of etiolated cucumber cotyledons failed to detect any phytol (Rebeiz and Castelfranco, 1973; McCarthy et al, 1981). On the other hand, the Pchlide a ester of etiolated barley leaves was shown to contain geranylgeraniol (GG) instead of phytol (Liljenberg, 1974). The inner seed coat of Cucurbitaceae, a rich source of Pchlide a ester was shown to contain a large number of Pchlide a esters esterified with different long chain alcohols. The latter consisted of farnesol and all possible C20 alcohols including GG and phytol (Shioi and Sasa, 1982). Roots of etiolated wheat, accumulated large amounts of MV Pchlide a esters and lesser amounts of MV Pchlide a. The alcohol moieties of the four accumulated Pchlide a esters consisted of GG, dihydrogeranylgraniol (GHGG), tetrahydrogeranylgraniol (THGG) and phytol (Mc Ewen and Lindsten, 1992).
The Occurrence of DV Pchlide a ester in higher plants was first reported in the inner seed coat of Cucurbita pepo (pumpkin) (Jones, 1966), and was confirmed by Houssier and Sauer (1969). The search for the occurrence of DV Pchlide a ester in other higher plant tissues was however unsuccessful till 11 years later (Belanger and Rebeiz, 1980). Using sensitive spectrofluorometric techniques, it was possible to show that etiolated cucumber cotyledons, a DDV-LDV-LDDV plant species incubated in darkness with ALA, accumulated mainly MV Pchlide a E and detectable, yet small amounts of DV Pchlide a E. The two Pchlide a E were separated by chromatography on thin layers of polyethylene and were characterized by their fluorescence emission and excitation spectra at room temperature and 77 °K. However, these studies were not extended with rigor to other plant species such as wheat, corn and barley. In other words, it is not certain at this stage whether small amounts of DV Pchlide a E also occur in DMV-LMV-LDMV such as johnsongrass and DMV-LDV-LDMV plant species such as barley, wheat and corn.
Because of (a) the DV-MV nature of the Mpde and Pchlide a E pools, and (b) because of the structural similarities between Mpde and Pchlide a E, one fully esterified DV biosynthetic route (Fig. 2 route 17) and one fully esterified MV biosynthetic route (Fig. 2, route 16) are considered to contribute to the formation of Pchlide a Es (Fig. 2). It should be emphasized however, that this hypothesis is based on detection of putative intermediates and structural similarities, but has not been confirmed by demonstration of precursor-product relationships between the Mpde and Pchlide a E components of the two putative routes.
The contribution of Pchlide a E to the greening process is suggested by the pattern of Pchlide a E formation under natural photoperiodic greening conditions. Since Pchlide a E was observed to accumulate noticeably during the first four dark cycles of the photoperiod, it was suggested by Cohen et al that it may well contribute to Chl a biosynthesis and accumulation at the onset of light (i. e. at dawn) during the first few days of photoperiodic greening (Cohen et al, 1977). Although the level of Pchlide a E dropped after the fourth dark cycle, it was always detectable in most green plants during all stages of greening during the light phase of the photoperiod (Rebeiz, unpublished).
a. Photoconversion of MV Pchlide a E to MV Chlide a E
Addition of two trans-hydrogens across the 7-8 position of the MV Pchlide a ester macrocycle would result in the conversion of MV Pchlide a ester to MV Chl a. Several laboratories have reported such a reaction in higher plants (Rebeiz and Castelfranco, 1973; Liljenberg, 1974; Lancer et al, 1976; Belanger and Rebeiz, 1980), and lower plants (Sasa and Sugahara, 1976; Kotzabasis et al, 1989). Since other researchers have not been able to detect the photoconversion of Pchlide a ester, Rudiger and Schoch (1991) suggested that such discrepancies may be due to age of seedlings or the very rapid esterification of Chlide a to Chl a during the light treatment. The latter possibility is unlikely as the photoconversion of Pchlide a ester has been also observed at temperatures of -15 to 2 Celsius (Rebeiz and Castelfranco, 1973, Liljenberg, 1974). In our opinion, failure to observe the photoconversion of Pchlide a E to Chl a stems from two considerations: (a) The photoconversion is only partial and very small amounts of Chl a are formed, (b) detection of such small amounts of Chl a depend a great deal on the sensitivity of the instrumentation in use. . We have recently reexamined the photoconversion of Pchlide a E to Chlide a E in isolated cucumber etioplasts. Reaction products were determined by HPLC coupled to high resolution spectrofluorometric detection. It was possible to show that isolated etioplasts of barley and corn subjected to a 2.5 ms flash of light at room temperature followed by immediate precipitation with ammoniacal acetone at various temperatures resulted in the detection of several Chlide a Es (Fig. 6 B). However, illumination of frozen etioplasts at -18 C did not photoreduce the Pchlide a E pool (Adra, 1996). These results confirmed the partial photoconvertibility of Pchlide a E at room temperatures, but raised the possibility that the enzyme responsible for (photo)reduction of Pchlide a E was much more sensitive to low temperatures than conventional Pchlide a oxidoreductases.
Separations were performed on a PE Pecosphere 3 x 3C, C-18 reversed phase, 4 x 0.5 cm column. Elution was with an isocratic, solvent system that consisted of H2O:acetone:methanol (5:20:75 v/v/v. P = Pchlide a; PE = Pchlide a ester; CE= Chlide a ester; F = Relative fluorescence emission intensity; RT = retention time.
On the basis of spectrophotometric and spectrofluorometric analysis, it is presently assumed that the fully esterified tetrapyrrole pool of etiolated tissues consists exclusively of Pchlide a Es. Recently, it was conjectured that should small amounts of other fully esterified tetrapyrroles be present, their detection would be obscured by the presence of the much larger amounts of Pchlide a Es. To test this hypothesis, HPLC analysis of etiolated tissues extracts followed by on line spectrofluorometric monitoring of all eluting peaks was performed. As expected, high resolution spectrofluorometric analysis of the fully esterified tetrapyrrole pools of etiolated barley and corn detected only MV Pchlide a E. However HPLC analysis revealed that the fully esterified Pchlide a pools of corn and barley consisted of several fully esterified tetrapyrrole components. On-line spectrofluorometric analysis of the fully esterified components indicated that they consisted of several different Pchlide a Es and very small amounts of Chlide a E (Fig. 6A). Formation of the latter implied the involvement of a light-independent Chlide a E biosynthetic step in higher plants during dark germination which is depicted by biosynthetic route 16D (Fig. 2). Recently, the detection of Chlide a E has also been reported by others in etiolated plant tissues (Skribanek et al, 2000).
b. Photoconversion of DV Pchlide a E to DV Chlide a E
In Fig. 2, the photoconversion of DV Pchlide a ester to DV Chl a is assigned to a fully esterified DV Chl a biosynthetic route. This assignment is based on the detection of DV Chl a formation immediately following a 47 ms actinic white light treatment of etiolated cucumber cotyledons, at room temperature (Belanger and Rebeiz, 1980). It was assumed that the small amounts of DV Chl a were as a consequence of the photoconversion of small amounts of DV Pchlide a ester. It has since come to our attention, that at room temperature, in-vivo, conversion of newly formed DV Chlide a to DV Chl a is extremely rapid (see section VIIA4a). As a consequence the possible photoconversion of DV Pchlide a ester to DV Chl a should be re-confirmed with isolated plastids at subzero temperatures.
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