Chloroplast-encoded chlB is required for light-independent protochlorophyllide reductase activity in Chlamydomonas reinhardtii.

A chloroplast-encoded gene, designated chlB, has been isolated from Chlamydomonas reinhardtii, its nucleotide sequence determined, and its role in the light-independent reduction of protochlorophyllide to chlorophyllide demonstrated by gene disruption experiments. The C. reinhardtii chlB gene is similar to open reading frame 563 (orf563) of C. moewusii, and its encoded protein is a homolog of the Rhodobacter capsulatus bchB gene product that encodes one of the polypeptide components of bacterial light-independent protochlorophyllide reduction. To determine whether the chlB gene product has a similar role in light-independent protochlorophyllide reduction in this alga, a series of plasmids were constructed in which the aadA gene conferring spectinomycin resistance was inserted at three different sites within the chlB gene. The mutated chlB genes were introduced into the Chlamydomonas chloroplast genome using particle gun-mediated transformation, and homoplasmic transformants containing the disrupted chlB genes were selected on the basis of conversion to antibiotic resistance. Individual transformed strains containing chlB disruptions were grown in the dark or light, and 17 of the 18 strains examined were found to have a "yellow-in-the-dark" phenotype and to accumulate the chlorophyll biosynthetic precursor protochlorophyllide. RNA gel blot analysis of chlB gene expression in wild-type cells indicated that the gene was transcribed at low levels in both dark- and light-grown cells. The results of these studies support the involvement of the chlB gene product in light-independent protochlorophyllide reduction, and they demonstrate that, similar to its eubacterial predecessors, this green alga requires at least three components (i.e., chlN, chlL, and chlB) for light-independent protochlorophyllide reduction.     

bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants.

We present the nucleotide and deduced amino acid sequences of four contiguous bacteriochlorophyll synthesis genes from Rhodobacter capsulatus. Three of these genes code for enzymes which catalyze reactions common to the chlorophyll synthesis pathway and therefore are likely to be found in plants and cyanobacteria as well. The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. The other three genes are required for portions of the pathway that are shared with chlorophyll synthesis, and they were expected to be common to both pathways. bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL), a reaction that has been observed in all major groups of photosynthetic organisms except angiosperms, where only the light-dependent reaction has been clearly established. The purple bacterial and plant enzymes show 35% identity between the amino acids coded by bchN and chlN (gidA) and 49% identity between the amino acids coded by bchL and chlL (frxC). Furthermore, bchB is 33% identical to ORF513 from the Marchantia polymorpha chloroplast. We present arguments in favor of the probable role of ORF513 (chlB) in protochlorophyllide reduction in the dark. The further similarities of all three subunits of protochlorophyllide reductase and the three subunits of chlorin reductase in bacteriochlorophyll synthesis suggest that the two reductase systems are derived from a common ancestor.     

Analysis of the Amborella trichopoda chloroplast genome sequence suggests that amborella is not a basal angiosperm

Phylogenetic analyses based on comparison of a limited number of genes recently suggested that Amborella trichopoda is the most ancient angiosperm. Here we present the complete sequence of the chloroplast genome of this plant. It does not display any of the genes characteristic of chloroplast DNA of the gymnosperm Pinus thunbergii (chlB, chlL, chlN, psaM, and ycf12). The majority of phylogenetic analyses of protein-coding genes of this chloroplast DNA suggests that Amborella is not the basal angiosperm and not even the most basal among dicots.     

Light-independent chlorophyll biosynthesis: involvement of the chloroplast gene chlL (frxC).

The Chlamydomonas reinhardtii chloroplast gene chlL (frxC) is shown to be involved in the light-independent conversion of protochlorophyllide to chlorophyllide. The polypeptide encoded by chlL contains a striking 53% amino acid sequence identity with the bacteriochlorophyll (bch) biosynthesis bchL gene product in the photosynthetic bacterium Rhodobacter capsulatus. In a previous analysis, we demonstrated that bchL was involved in light-independent protochlorophyllide reduction, thereby implicating chlL in light-independent protochlorophyllide reduction in photosynthetic eukaryotes. To perform a functional/mutational analysis of chlL, we utilized particle gun-mediated transformation to disrupt the structural sequence of chlL at its endogenous locus in the chloroplast genome of Chlamydomonas. Transformants for which the multicopy chloroplast genome was homoplasmic for the disrupted chlL allele exhibit a "yellow-in-the-dark" phenotype that we demonstrated to be a result of the dark accumulation of protochlorophyllide. The presence of a chlL homolog in distantly related bacteria and nonflowering land plants, which are thought to be capable of synthesizing chlorophyll in the dark, was also demonstrated by cross-hybridization analysis. In contrast, we observed no cross-hybridization of a probe of chlL to DNA samples from representative angiosperms that require light for chlorophyll synthesis, in support of our conclusion that chlL is involved in light-independent chlorophyll biosynthesis. The role of chlL in protochlorophyllide reduction as well as recent evidence that both light-independent and light-dependent protochlorophyllide reductases may be of bacterial origin are discussed.     

氯化钠处理对原壳小球藻生长及油脂积累的影响

采用Basal培养基,通过光学显微镜、电子显微镜、激光共聚焦显微镜以及尼罗红染色定量等方法研究了不同浓度氯化钠(0、150、300、600 mmol/L)对小球藻属原壳小球藻的生长状态、脂滴分布、总脂含量的影响。结果表明,添加不同浓度的氯化钠对原壳小球藻的生长有明显的影响,随着氯化钠浓度的增加,小球藻的生长速度受到明显的抑制,600 mmol/L氯化钠处理时生长几乎完全被抑制。在显微镜下观察,可见氯化钠浓度的增加会导致小球藻聚集成团,这种现象在150 mmol/L和300 mmol/L氯化钠培养下比较明显;通过电子显微镜下观察,可以发现培养初期,随着氯化钠浓度的增加,小球藻细胞壁增厚,脂滴增多。通过尼罗红染色对脂含量进行定量,处理初期脂滴的合成量在600 mmol/L时最高,但到后期,随着藻生物量的增加,150 mmol/L和300 mmol/L处理下脂合成量逐渐升高,而对照小球藻脂合成量基本不变。稳定期后,从生物量(干重)和脂总量来看,300 mmol/L氯化钠培养处理的小球藻虽然生物量只有对照的73.55%,但是总脂含量却是对照的2.22倍,可见一定浓度的氯化钠处理一定时间可显著提高原壳小球藻的油脂含量。     

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.     

Evolutionary dynamics of light-independent protochlorophyllide oxidoreductase genes in the secondary plastids of cryptophyte algae

Plastid genes encoding light-independent protochlorophyllide oxidoreductase (LIPOR) subunits were isolated from cryptophyte algae, the first example of such genes in plastids of secondary endosymbiotic origin. The presence of functional and nonfunctional copies of LIPOR genes in cryptophytes suggests that light-independent chlorophyll biosynthesis is a nonessential pathway in these organisms.     

Functional evaluation of a nitrogenase-like protochlorophyllide reductase encoded by the chloroplast DNA of Physcomitrella patens in the cyanobacterium Leptolyngbya boryana

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of the two components, L-protein (a ChlL dimer) and NB-protein (a ChlN-ChlB heterotetramer), to catalyze Pchlide reduction in Chl biosynthesis. While nitrogenase is distributed only among certain prokaryotes, the probable structural genes for DPOR are encoded by chloroplast DNA in lower plants. Here we show functional evaluation of DPOR encoded by chloroplast DNA in a moss Physcomitrella patens by the complementation analysis of the cyanobacterium Leptolyngbya boryana and the heterologous reconstitution of the moss L-protein and the cyanobacterial NB-protein. Two shuttle vectors to overexpress chlL and chlN-chlB from P. patens were introduced into the cyanobacterial chlL- and chlB-lacking mutants, respectively. Both transformants restored the ability to perform Chl biosynthesis in the dark, indicating that the chloroplast-encoded DPOR components form an active complex with the cyanobacterial components. The L-protein of P. patens was purified from the cyanobacterial transformant, and DPOR activity was reconstituted in a heterologous combination with the cyanobacterial NB-protein. The specific activity of the L-protein from P. patens was determined to be 118 nmol min(-1) mg (-1), which is even higher than that of the cyanobacterial L-protein (76 nmol min(-1) mg (-1)). Upon exposure to air, the activity of the L-protein from P. patens decayed with a half-life of 30 s, which was eight times faster than that of the cyanobacterial L-protein (240 s). These results suggested that the chloroplast-encoded L-protein functions as efficiently as the cyanobacterial L-protein but is more oxygen labile than the cyanobacterial L-protein.     

Chloroplast-encoded chlB gene from Pinus thunbergii promotes root and early chlorophyll pigment development in Nicotiana tabaccum

Chlorophyll biosynthesis is catalyzed by two multi subunit enzymes; a light-dependent and a light-independent protochlorophyllide oxidoreductase. The light-independent enzyme consists of three subunits (ChlL, ChlN and ChlB) in photosynthetic bacteria and plastids in which the chlB gene encodes the major subunit that catalyzes the reduction of protochlorophyllide to chlorophyllide. We report here stable integration of the chlB gene from Pinus thunbergii into the chloroplast genome of tobacco. Using helium-driven biolistic gun, transplastomic clones were developed in vitro. The stable integration and homoplasmy for transgenes was confirmed by using PCR and Southern blotting techniques. Nodal cuttings of the homoplasmic transgenic and untransformed wild type shoots were cultured on MS medium in the dark. As expected, shoots developed from the cuttings of the wild type plants in the dark showed etiolated growth with no roots whereas shoots from the cuttings of the transgenic plants developed early and more roots. Upon shifting from dark to light in growth room, leaves of the transgenic shoots showed early development of chlorophyll pigments compared to the wild type shoots. Further, photosynthetically indistinguishable transgenic shoots also showed significant difference in root development from untransformed wild type shoots when cuttings were grown in the light. Therefore, it may be concluded that the chlB gene is involved, directly or indirectly, in the root development of tobacco. Further, the gene promotes early development of chlorophyll pigments, upon illumination from dark, in addition to its role in the light-independent chlorophyll formation when expressed together with subunits L&N in other organisms.     

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds

Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.     

Substrate recognition of nitrogenase-like dark operative protochlorophyllide oxidoreductase from Prochlorococcus marinus

Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL(2) and a heterotetrameric protein complex (ChlNB)(2). The ChlL(2) dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB)(2) complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB)(2). For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A-C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.     

Influence of irradiance on chlorophyll synthesis in Picea abies calli cultures

Dark-grown seedlings of Picea abies (L) Karst. are able to accumulate the highest amounts of chlorophyll (Chl) and its precursor protochlorophyllide (Pchlide) in all Pinaceae, but calli derived from 14-d-old green cotyledons of P. abies are completely white during the cultivation in the dark. Pchlide reduction is catalysed in the dark by light-independent protochlorophyllide oxidoreductase (DPOR). This enzyme complex consists of three protein subunits ChlL, ChlN and ChlB, encoded by three plastid genes chlL, chlN and chlB. Using semiquantitative RT-PCR, we observed very low expression of chlLNB genes in dark-grown calli. It seems, that chlLNB expression and thus Chl accumulation could be modulated by light in P. abies calli cultures. This hypothesis is supported by the fact, that we observed low contents of glutamyl-tRNA reductase and Flu-like protein, which probably affected Chl biosynthetic pathway at the step of 5-aminolevulinic acid formation. ChlB subunit was not detected in dark-grown P. abies calli cultures. Our results indicated limited ability to synthesize Chl in callus during cultivation in the dark.     

Reconstitution of light-independent protochlorophyllide reductase from purified bchl and BchN-BchB subunits. In vitro confirmation of nitrogenase-like features of a bacteriochlorophyll biosynthesis enzyme

Protochlorophyllide reductase catalyzes the reductive formation of chlorophyllide from protochlorophyllide during biosynthesis of chlorophylls and bacteriochlorophylls. The light-independent (dark) form of protochlorophyllide reductase plays a key role in the ability of gymnosperms, algae, and photosynthetic bacteria to green (form chlorophyll) in the dark. Genetic and sequence analyses have indicated that dark protochlorophyllide reductase consists of three protein subunits that exhibit significant sequence similarity to the three subunits of nitrogenase, which catalyzes the reductive formation of ammonia from dinitrogen. However, unlike the well characterized features of nitrogenase, there has been no previous biochemical characterization of dark protochlorophyllide reductase. In this study, we report the first reproducible demonstration of dark protochlorophyllide reductase activity from purified protein subunits that were isolated from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. Two of the three subunits (Bchl and BchN) were expressed in R. capsulatus as S tag fusion proteins that facilitated affinity purification. The third subunit (BchB) was co-purified with the BchN protein indicating that BchN and BchB proteins form a tight complex. Dark protochlorophyllide reductase activity was shown to be dependent on the presence of all three subunits, ATP, and the reductant dithionite. The similarity of dark protochlorophyllide reductase to nitrogenase is discussed.     

The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii

The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynthesis in the dark in Chlamydomonas reinhardtii, was precisely replaced by either the Klebsiella pneumoniae nifH (encoding the structural component of nitrogenase Fe protein) or the Escherichia coli uidA reporter gene encoding beta-glucuronidase. Homoplasmic nifH or uidA transformants were identified by Southern blots after selection on minimal medium plates for several generations. All the uidA transformants had the "yellow-in-the-dark" phenotype characteristic of chlL mutants, whereas homoplasmic nifH transformants exhibited a partial "green-in-the-dark" phenotype. NifH protein was detected in the nifH transformants but not in the wild-type strain by Western blotting. Fluorescence emission measurements also showed the existence of chlorophyll in the dark-grown nifH transformants, but not in the dark-grown uidA transformants. The nifH transplastomic form of C. reinhardtii that lacks the chlL gene can still produce chlorophyll in the dark, suggesting that the nifH product can at least partially substitute for the function of the putative "chlorophyll iron protein" encoded by chlL. Thus, introducing nitrogen fixation gene directly into a chloroplast genome is likely to be feasible and providing a possible way of engineering chloroplasts with functional nitrogenase. Notably, to introduce foreign genes without also introducing selective marker genes, a novel two-step chloroplast transformation strategy has been developed.