Light-dependent Chlorophyll Biosynthesis Research Articles

Plastome Reduction in the Only Parasitic Gymnosperm Parasitaxus Is Due to Losses of Photosynthesis but Not Housekeeping Genes and Apparently Involves the Secondary Gain of a Large Inverted Repeat.

Plastid genomes (plastomes) of parasitic plants undergo dramatic reductions as the need for photosynthesis relaxes. Here, we report the plastome of the only known heterotrophic gymnosperm Parasitaxus usta (Podocarpaceae). With 68 unique genes, of which 33 encode proteins, 31 tRNAs, and four rRNAs in a plastome of 85.3-kb length, Parasitaxus has both the smallest and the functionally least capable plastid genome of gymnosperms. Although the heterotroph retains chlorophyll, all genes for photosynthesis are physically or functionally lost, making photosynthetic energy gain impossible. The pseudogenization of the three plastome-encoded light-independent chlorophyll biosynthesis genes chlB, chlL, and chlN implies that Parasitaxus relies on either only the light-dependent chlorophyll biosynthesis pathway or another regulation system. Nesting within a group of gymnosperms known for the absence of the large inverted repeat regions (IRs), another unusual feature of the Parasitaxus plastome is the existence of a 9,256-bp long IR. Its short length and a gene composition that completely differs from those of IR-containing gymnosperms together suggest a regain of this critical, plastome structure-stabilizing feature. In sum, our findings highlight the particular path of lifestyle-associated reductive plastome evolution, where structural features might provide additional cues of a continued selection for plastome maintenance.

A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly.

Oxygenic photosynthesis relies on accessory factors to promote the assembly and maintenance of the photosynthetic apparatus in the thylakoid membranes. The highly conserved membrane-bound rubredoxin-like protein RubA has previously been implicated in the accumulation of both PSI and PSII, but its mode of action remains unclear. Here, we show that RubA in the cyanobacterium Synechocystis sp PCC 6803 is required for photoautotrophic growth in fluctuating light and acts early in PSII biogenesis by promoting the formation of the heterodimeric D1/D2 reaction center complex, the site of primary photochemistry. We find that RubA, like the accessory factor Ycf48, is a component of the initial D1 assembly module as well as larger PSII assembly intermediates and that the redox-responsive rubredoxin-like domain is located on the cytoplasmic surface of PSII complexes. Fusion of RubA to Ycf48 still permits normal PSII assembly, suggesting a spatiotemporal proximity of both proteins during their action. RubA is also important for the accumulation of PSI, but this is an indirect effect stemming from the downregulation of light-dependent chlorophyll biosynthesis induced by PSII deficiency. Overall, our data support the involvement of RubA in the redox control of PSII biogenesis.

Identification and characterization of candidates involved in production of OMEGAs in microalgae: a gene mining and phylogenomic approach

Optimizing the production of the high-value renewables such as OMEGAs through pathway engineering requires an in-depth understanding of the structure–function relationship of genes involved in the OMEGA biosynthetic pathways. In this preliminary study, our rationale is to identify and characterize the ∼221 putative genes involved in production of OMEGAs using bioinformatic analysis from the Streptophyte (plants), Chlorophyte (green algae), Rhodophyta (red algae), and Bacillariophyta (diatoms) lineages based on their phylogenomic profiling, conserved motif/domain organization and physico-chemical properties. The MEME suite predicted 12 distinct protein domains, which are conserved among these putative genes. The phylogenomic analysis of the putative candidate genes [such as FAD2 (delta-12 desaturase); ECR (enoyl-CoA reductase); FAD2 (delta-12 desaturase); ACOT (acyl CoA thioesterase); ECH (enoyl-CoA hydratase); and ACAT (acetyl-CoA acyltransferase)] with similar domains and motif patterns were remarkably well conserved. Furthermore, the subcellular network prediction of OMEGA biosynthetic pathway genes revealed a unique interaction between the light-dependent chlorophyll biosynthesis and glycerol-3-phosphate dehydrogenase, which predicts a major cross-talk between the key essential pathways. Such bioinformatic analysis will provide insights in finding the key regulatory genes to optimize the productivity of OMEGAs in microalgal cell factories.

Phosphorylation of GENOMES UNCOUPLED 4 Alters Stimulation of Mg Chelatase Activity in Angiosperms.

GENOMES UNCOUPLED 4 (GUN4) is a positive regulator of light-dependent chlorophyll biosynthesis. GUN4 activates Mg chelatase (MgCh) that catalyzes the insertion of an Mg2+ ion into protoporphyrin IX. We show that Arabidopsis (Arabidopsis thaliana) GUN4 is phosphorylated at Ser 264 (S264), the penultimate amino acid residue at the C terminus. While GUN4 is preferentially phosphorylated in darkness, phosphorylation is reduced upon accumulation of Mg porphyrins. Expression of a phosphomimicking GUN4(S264D) results in an incomplete complementation of the white gun4-2 null mutant and a chlorotic phenotype comparable to gun4 knockdown mutants. Phosphorylated GUN4 has a reduced stimulatory effect on MgCh in vitro and in vivo but retains its protein stability and tetrapyrrole binding capacity. Analysis of GUN4 found in oxygenic photosynthetic organisms reveals the evolution of a C-terminal extension, which harbors the phosphorylation site of GUN4 expressed in angiosperms. Homologs of GUN4 from Synechocystis and Chlamydomonas lack the conserved phosphorylation site found in a C-terminal extension of angiosperm GUN4. Biochemical studies proved the importance of the C-terminal extension for MgCh stimulation and inactivation of GUN4 by phosphorylation in angiosperms. An additional mechanism regulating MgCh activity is proposed. In conjunction with the dark repression of 5-aminolevulinic acid synthesis, GUN4 phosphorylation minimizes the flow of intermediates into the Mg branch of the tetrapyrrole metabolic pathway for chlorophyll biosynthesis.

Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L. Purification and substrate specificity.

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.

Influence of polyamine inhibitors on light-independent and light-dependent chlorophyll biosynthesis and on the photosynthetic rate

Abstract The mutant C-2A′ of the unicellular green alga Scenedesmus obliquus behaves at 32°C like higher plants in that it accumulates protochlorophyllide in darkness and converts it to chlorophyll when exposed to light. By lowering the growth temperature from 32 to 22°C, protochlorophyllide can be reduced in darkness. Thus this organism allowed us to study the effects of the inhibition of several enzymes of polyamine biosynthesis on light-dependent and light-independent chlorophyll biosynthesis. Reduction of the intracellular level of putrescine by the use of 1,4-diamino-2-butanone inhibitor blocked the light-independent chlorophyll biosynthesis (dramatically increasing the photochlorophyllide levels with parallel reduction of chlorophyll). The influence of the inhibitor in question is even more dramatic in light-dependent chlorophyll biosynthesis, where small concentrations of 1,4-diamino-2-butanone resulted in considerable reduction of chlorophyll during illumination. The above reduction of chlorophyll was accompanied by an increase in photosynthetic activity and respiration rate, which suggests the formation of a photosynthetic apparatus that behaves similarly to one adapted to high light intensities. Decreases in the intracellular levels of spermidine and spermine by the inhibitors cyclohexylamine and 1,3-diaminopropane respectively, although not showing changes in light-independent chlorophyll biosynthesis, do exhibit a regular reduction of light-dependent chlorophyll biosynthesis and a decrease in the photosynthetic activity as well as in the respiration rate. These characteristics are typical for a photosynthetic apparatus adapted to low light intensities with a higher antenna composition per reaction centre. The possible effects of the reduction of putrescine, spermidine and spermine levels on the stabilization-destabilization of photosynthetic subcomplexes are discussed.

The inhibitory effect of 4,5-dioxovalerate on 5-aminolevulinate dehydratase and its implication in the regulation of light-dependent chlorophyll formation in pigment mutant C-2A′ of Scenedesmus obliquus

Besides the known role of 4,5-dioxovalerate as an intermediate in the C 5 -pathway [1], a novel regulatory function in light-dependent chlorophyll biosynthesis is described. Considerable amounts of protochlorophyllide accumulate in dark grown cultures of the yellow mutant C-2A′ of Scenedesmus [2]. This accumulation is almost completely blocked in darkness by the addition of 4,5-dioxovalerate in vivo. Likewise, light-dependent chlorophyll biosynthesis is strongly inhibited by the addition of this compound during greening. The considerable increase of protochlorophyllide formation in darkness upon the addition of 5-aminolevulinate (Kotzabasis, K. and Senger, H. (1989) Z. Naturforsch., in press) is also drastically reduced by external 4,5-dioxovalerate. It is shown by in vitro experiments that concentrations of dioxovalerate, above the physiologically relevant level, inhibit 5-aminolevulinate dehydratase. The K i -value was determined to be 60±5 μM. From these results it is concluded that besides the predominant control of gli t-RNA-ligase by protochlorophyllide [4] a second regulatory mechanism is involved in chlorophyll biosynthesis. Under excessive concentrations of 5-aminolevulinate and 4,5-dioxovalerate further prophobilinogen and subsequently chlorophyll biosynthesis is inhibited.