Plastid development in germinating wheat (Triticum aestivum) is enhanced by gibberellic acid and delayed by gabaculine

Etioplast development and protochlorophyllide (Pchlide) accumulation was studied in wheat seedlings ( Triticum aestivum L. cv. Walde, Weibull) grown in darkness on gibberellic acid (GA₃), gabaculine (3-amino-2,3-dihydrobenzoic acid), or on a combination of the two. The results were compared with the features of seedlings grown on water only. GA₃ enhanced shoot growth and promoted etioplast development. A correlation was observed between the appearance of prolamellar bodies (PLBs) and of phototransformable Pchlide. Gabaculine, a known tetrapyrrole biosynthesis inhibitor, delayed growth, slowed down the rate of PLB formation and caused structural alterations of the etioplasts up to 48 h of germination. Gabaculine also delayed the formation of phototransformable Pchlide as well as overall Pchlide biosynthesis, as determined by low-temperature fluorescence emission in vivo. The spectral blue-shift of newly formed chlorophyllide (Chlide) was delayed in irradiated dark-grown gabaculine-grown seedlings, indicating an inhibited dissociation of Chlide and NADPH-Pchlide oxidoreductase (Pchlide reductase: EC 1.3.1.33). Thus there is a close correlation between accumulation of Pchlide and etioplast development, also under conditions when development is enhanced or delayed.     

Effect of BA upon the Amount of Pchlide and NPR protein and the Activity of NPR in Cucumber Cotyledons

When cucumber seedlings (Cucumis sativus L. cv. Aonagajibai) were treated with aqueous solution of BA (10–5mol/1 and 10–8mol/l) by spraying application, the amount of NPR protein was increased and the NPR activity was promoted. The contents of protochlorophyl, lide (Pchlide) and NADPH as the substrates of the enzyme reaction, were also increased remarkably. 2. The amount of Pchlide and NPR protein decreased rapidly when treated with red light. BA plays the role in the protection against the damage of enzyme activity by light and then decreasing the destruction of NPR protein. BA increased the content of Pchlide and NADPH, so the NPR activity was maintained at a higher level although in light condition. 3. Because that BA increased the content of NPR protein and the reaction substrate such as NADPH and Pchlide, the NPR activity was promoted. So, the rate of chlorophyll biosynthesis was rapid, and the content of chlorophyll increased. The leaf turns to dark green.     

Identification of a gene essential for the first committed step in the biosynthesis of bacteriochlorophyll c

Bacteriochlorophylls (BChls) c, d, and e are the major chlorophylls in chlorosomes, which are the largest and one of the most efficient antennae produced by chlorophototrophic organisms. In the biosynthesis of these three BChls, a C-13(2)-methylcarboxyl group found in all other chlorophylls (Chls) must be removed. This reaction is postulated to be the first committed step in the synthesis of these BChls. Analyses of gene neighborhoods of (B)Chl biosynthesis genes and distribution patterns in organisms producing chlorosomes helped to identify a gene (bciC) that appeared to be a good candidate to produce the enzyme involved in this biochemical reaction. To confirm that this was the case, a deletion mutant of an open reading frame orthologous to bciC, CT1077, was constructed in Chlorobaculum tepidum, a genetically tractible green sulfur bacterium. The CT1077 deletion mutant was unable to synthesize BChl c but still synthesized BChl a and Chl a. The deletion mutant accumulated large amounts of various (bacterio)pheophorbides, all of which still had C-13(2)-methylcarboxyl groups. A C. tepidum strain in which CT1077 was replaced by an orthologous gene, Cabther_B0081 [corrected] from "Candidatus Chloracidobacterium thermophilum" was constructed. Although the product of Cabther_B0081 [corrected] was only 28% identical to the product of CT1077, this strain synthesized BChl c, BChl a, and Chl a in amounts similar to wild-type C. tepidum cells. To indicate their roles in the first committed step of BChl c, d, and e biosynthesis, open reading frames CT1077 and Cabther_B0081 [corrected] have been redesignated bciC. The potential mechanism by which BciC removes the C-13(2)-methylcarboxyl moiety of chlorophyllide a is discussed.     

An intermediate membrane subfraction in cyanobacteria is involved in an assembly network for Photosystem II biogenesis

Early steps in the biogenesis of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 are thought to occur in a specialized membrane fraction that is characterized by the specific accumulation of the PSII assembly factor PratA and its interaction partner pD1, the precursor of the D1 protein of PSII. Here, we report the molecular characterization of this membrane fraction, called the PratA-defined membrane (PDM), with regard to its lipid and pigment composition and its association with PSII assembly factors, including YCF48, Slr1471, Sll0933, and Pitt. We demonstrate that YCF48 and Slr1471 are present and that the chlorophyll precursor chlorophyllide a accumulates in the PDM. Analysis of PDMs from various mutant lines suggests a central role for PratA in the spatial organization of PSII biogenesis. Moreover, quantitative immunoblot analyses revealed a network of interdependences between several PSII assembly factors and chlorophyll synthesis. In addition, formation of complexes containing both YCF48 and Sll0933 was substantiated by co-immunoprecipitation experiments. The findings are integrated into a refined model for PSII biogenesis in Synechocystis 6803.     

Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)₂ with the catalytic (BchY/BchZ)₂ protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF₄⁻. Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)₂. A second [4Fe-4S] cluster was identified on (BchY/BchZ)₂. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.     

Identification of chlorophyllide a oxygenase in the Prochlorococcus genome by a comparative genomic approach

Chl b is a major photosynthetic pigment of peripheral antenna complexes in chlorophytes and prochlorophytes. Chl b is synthesized by chlorophyllide a oxygenase (CAO), an enzyme that has been identified from higher plants, moss, green algae and two groups of prochlorophytes, Prochlorothrix and Prochloron. Based on these results, we previously proposed the hypothesis that all of the Chl b synthesis genes have a common origin. However, the CAO gene is not found in whole genome sequences of Prochlorococcus although a gene which is distantly related to CAO was reported. If Prochlorococcus employs a different enzyme, a Chl synthesis gene should have evolved several times on the different phylogenetic lineages of Prochlorococcus and other Chl b-containing organisms. To examine these hypotheses, we identified a Prochlorococcus Chl b synthesis gene by using a combination of bioinformatics and molecular genetics techniques. We first identified Prochlorococcus-specific genes by comparing the whole genome sequences of Prochlorococcus marinus MED4, MIT9313 and SS120 with Synechococcus sp. WH8102. Synechococcus is closely related to Prochlorococcus phylogenetically, but it does not contain a Chl b synthesis gene. By examining the sequences of Prochlorococcus-specific genes, we found a candidate for the Chl b synthesis gene and introduced it into Synechocystis sp. PCC6803. The transformant cells accumulated Chl b, indicating that the gene product catalyzes Chl b synthesis. In this study, we discuss the evolution of CAO based upon the molecular phylogenetic studies we performed.     

Temperature dependent reduction of protochlorophyllide in darkness followed by the assembly of active photosystems in pigment mutant C-2A' of Scenedesmus obliquus

In the wild type of Scenedesmus obliquus strain D3 grown heterotrophically, the chlorophyll biosynthesis and thus the reduction of protochlorophyllide to chlorophyllide takes place in darkness. However, in pigment mutant C-2A' of Scenedesmus obliquus only traces of protochlorophyllide are reduced under optimal growth conditions in darkness. By lowering the growth temperature from 33° to 15–25°C, protochlorophyllide can be reduced in darkness. At 20°C this process is about 10 times more active than at 33°C, but reaches only about 13% of the light-dependent chlorophyll biosynthesis. The chlorophylls synthesized at the lower temperatures are inserted into the pigment-protein complexes and photosystem I as well as photosys-tem II capacities are developed. The rate of light-independent protochlorophyllide reduction at lower temperatures is not limited by the enzyme PChlide-oxidoreductase itself, but rather by its substrate, being in turn limited by the amount of 5-amino levulinic acid (ALA) available.     

Hydrogen, carbon and nitrogen isotopic fractionations during chlorophyll biosynthesis in C3 higher plants

We determined hydrogen, carbon and nitrogen isotopic compositions of chlorophylls a and b isolated from leaves of five C3 higher plant species (Benthamidia japonica, Prunus japonica, Acer carpinifolium, Acer argutum and Querus mongloica), and hydrogen and carbon isotopic compositions of phytol and chlorophyllides in the chlorophylls to understand isotopic fractionations associated with chlorophyll biosynthesis in these species. Chlorophylls are depleted in D relative to ambient water by approximately 189 per thousand and enriched in (13)C relative to bulk tissue by approximately 1.6 per thousand. These data can be explained by the contribution of isotopic fractionations during phytol and chlorophyllide biosyntheses. Phytol is more depleted in both D (by approximately 308 per thousand) and (13)C (by approximately 4.3 per thousand), while chlorophyllides are less depleted in D (by approximately 44 per thousand) and enriched in (13)C (by approximately 4.8 per thousand). Such inhomogeneous distribution of isotopes in chlorophylls suggests that (1) the phytol in chlorophylls reflects strong D- and (13)C-depletions due to the isotopic fractionations during the methylerythritol phosphate pathway followed by hydrogenation, and (2) the chlorophyllides reflect D- and (13)C-enrichments in tricarboxylic acid cycle. On the other hand, chlorophylls are slightly ( approximately 1.2 per thousand) depleted in (15)N relative to the bulk tissue, indicating that net isotopic fractionation of nitrogen during chlorophyll biosynthesis is small compared with those of hydrogen and carbon.     

The regular ultrastructure of isolated prolamellar bodies depends on the presence of membrane-bound NADPH-protochlorophyllide oxidoreductase

Light-induced alterations of isolated prolamellar bodies (PLBs) were studied in flash-irradiated suspensions of a PLB-enriched fraction and a mixed membrane fraction isolated from dark-grown seedlings of wheat (Triticum aestivum L. cv. Walde). The mixed membrane fraction consisted of PLB fragments and membrane vesicles originating from the prothylakoids. Ultrastructural and spectral properties, as well as pigment and protein composition of non-irradiated and of flash-irradiated suspensions were studied. The addition of 0.3 mM NADPH prevented spectral shifts towards shorter wavelengths in irradiated as well as in non-irradiated PLB-fractions. as measured by fluorescence emission at – 196°C. In non-irradiated PLB-fractions the amount of phototransformable protochlorophyllide (PChlide) as compared to nonphototransformable PChlide decreased when NADPH was not added. The emission maximum due to chlorophyll(ide) shifted from 696 nm to 680 um in the flashirradiated fractions where no NADPH was added. The amount of chlorophyllous pigments, as well as the amount of NADPH-protochlorophyllide oxidoreductase, decreased during the experimental period of 4 h in the suspensions without added NADPH. especially in the irradiated ones. The ultrastructure of the pelletable material in the different suspensions was analyzed by transmission and scanning electron microscopy. The non-irradiated PLBs appeared as cottonball-like structures in the scanning electron microscope. Without NADPH added more PLBs with an irregular tubular appearance were seen. After irradiation and storage for 1 h in darkness the surface was covered with vesicles. These vesicles were still present after 4 h. In the presence of NADPH no vesicle-formation occurred and the regular network of the PLBs was preserved also after an irradiation which caused transformation of PChlide to chlorophyllide. Thus, the regular structure seems to depend on an ample supply of NADPH. which in turn may be necessary to stabilize the pigment-protein complex in the lipid moiety of the PLB membranes. The formation of vesicles may thus be caused by a loss of this pigment-protein complex in suspensions with a low level of NADPH. The possible significance of an NADPH-dependence in vivo is discussed.