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)
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.
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.
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.
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.
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.