Light-dependent chlorophyll a biosynthesis upon chlL deletion in wild-type and photosystem I-less strains of the cyanobacterium Synechocystis sp. PCC 6803

Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL ^- mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL ^- strain, greening occurred at the same rate at two different light intensities (5 and 50 E m^-2s^-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding chelator protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.     

The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron

The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by a 951?bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5′ and 3′ splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522?nt), separating the catalytic core from the 3′ splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3′ splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of -aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL ^–/scpE ^– strain, whereas PChlide accumulated in the PS I-less/chlL ^–/scpB ^– strain. In the PS I-less/chlL ^– control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.     

Chloroplast chlB gene is required for light-independent chlorophyll accumulation in Chlamydomonas reinhardtii

Light-independent chlorophyll synthesis occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have identified a new chloroplast gene, chlB, that is required for the light-independent accumulation of chlorophyll in the green alga Chlamydomonas reinhardtii. The chlB gene was cloned, sequenced, and then disrupted by performing particle gun-mediated chloroplast transformation. The resulting homoplasmic mutant was unable to accumulate chlorophyll in the dark and thus exhibited a yellow-in-the-dark phenotype. The chlB gene encodes a polypeptide of 688 amino acid residues, and is distinct from two previously characterized chloroplast genes (chlN and chlL) also required for light-independent chlorophyll accumulation in C. reinhardtii. Three unidentified open reading frames in chloroplast genomes of liverwort, black pine, and Chlamydomonas moewusii were also identified as chlB genes, based on their striking sequence similarities to the C. reinhardtii chlB gene. A chlB-like gene is absent in chloroplast genomes of tobacco and rice, consistent with the lack of light-independent chlorophyll synthesis in these plants. Polypeptides encoded by the chloroplast chlB genes also show significant sequence similarities with the bchB gene product of Rhodobacter capsulatus. Comparisons among the chloroplast chlB and the bacterial bchB gene products revealed five highly conserved sequence areas that are interspersed by four stretches of highly variable and probably insertional sequences.     

ThechlL (frxC) gene: Phylogenetic distribution in vascular plants and DNA sequence fromPolystichum acrostichoides (Pteridophyta) andSynechococcus sp. 7002 (Cyanobacteria)

We examinedchlL (frxC) gene evolution using several approaches. Sequences from the chloroplast genome of the fernPolystichum acrostichoides and from the cyanobacteriumSynechococcus sp. 7002 were determined and found to be highly conserved. A complete physical map of the fern chloroplast genome and partial maps of other vascular plant taxa show thatchlL is located primarily in the small single copy region as inMarchantia polymorpha. A survey of a wide variety of non-angiospermous vascular plant DNAs shows thatchlL is widely distributed but has been lost in the pteridophytePsilotum and (presumably independently) within the Gnetalean gymnosperms.The namefrxC was originally used to denote a gene encoding a product with probable Fe : S cluster binding activity. This activity was postulated due to the amino acid sequence similarity between this product and the Fe : S-binding nitrogenase iron proteinnifH. Fe : S-binding is a property shared by ferredoxins, which are denoted by the prefix frx. However, this gene does not encode a ferredoxin. It is much larger than any known ferredoxin, it binds its Fe : S cluster between two halves of a homodimer (Fujita & al. 1989,Burke & al. 1993 a, c) instead of within a single subunit, and it lacks the pattern of clustered cysteines present in all ferredoxins (Meyer 1988). Therefore, we use the namechlL to recognize the sequence and functional similarities to the bacterial PChlide reductase subunit,bchL. Similar usage has been adopted for this (Suzuki & Bauer 1992) and other (Choquet & al. 1992,Burke & al. 1993b) PChlide reductase subunits.     

Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana

In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron–sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.     

Development and mapping of AFLP markers linked to the sorghum fertility restorer gene <Emphasis Type="Italic">rf4</Emphasis>

The restoration of male fertility in the sorghum IS1112 C (A3) male-sterile cytoplasm is through a two-gene gametophytic system involving complementary action of the restoring alleles Rf3 and Rf4. To develop markers suitable for mapping rf4, AFLP technology was applied to bulks of sterile and fertile individuals from a segregating BC₃F₁ population. Three AFLP markers linked to rf4 were identified and subsequently converted to STS/CAPS markers, two of which are co-dominant. Based on a population of 378 BC₁F₁ individuals, two STS/CAPS markers, LW7 and LW8, mapped to within 5.31 and 3.18 cM, respectively, of rf4, while an STS marker, LW9, was positioned 0.79 cM on the flanking side of rf4. Markers LW8 and LW9 were used to screen sorghum BAC libraries to identify the genomic region encoding rf4. A series of BAC clones shown to represent a genomic region of linkage group E were identified by the rf4-linked markers. A contig of BAC clones flanking the LW9 marker represent seed clones on linkage group E, from which fine mapping of the rf4 locus and chromosome walking can be initiated. Received: 20 June 2001 / Accepted: 3 August 2001     

Variations in biochemical parameters of Heterocapsa sp. and Olisthodiscus luteus grown in 12:12 light:dark cycles II. Changes in pigment composition

Photosynthetic pigment composition was studied in batch cultures of Heterocapsa sp. and Olisthodiscus luteus growing exponentially in a 12:12 light:dark cycle. Both species divided in the dark. The synthesis of pigments was continuous for both species. However for chlorophyll c and peridinin, in Heterocapsa sp., and chlorophyll c and fucoxanthin, in O. luteus, (pigments belonging to light harvesting complexes) the synthesis was significantly higher during the light period. Concentrations per total cell volume (TCV) of chlorophyll a, chlorophyll c, peridinin and diadinoxanthin in Heterocapsa sp., and chlorophyll a, chlorophyll c, fucoxanthin and violaxanthin in O. luteus, showed a maximum at the onset of light and decreased during the light period. The values of the chlorophyll a:chlorophyll c, chlorophyll a:peridinin and chlorophyll a:fucoxanthin ratios are compared with data reported in the literature.     

Enzymes of the glycollate pathway in relation to greening in Euglena gracilis

Summary The kinetics of chlorophyll formation during the greening of dark-grown Euglena gracilis was accompanied by marked increases in activity of the enzymes of glycollate metabolism; glycollate: DCPIP¹ oxidoreductase and phosphoglycollate phosphatase (E.C. 3.1.3.18.). Inhibitors of protein synthesis indicated a de novo synthesis of these enzymes during the development of the photosynthetic system. The inhibitory effects of chloramphenicol and cycloheximide, together with a non-aqueous localisation of glycollate: DCPIP oxidoreductase in photoautotrophically-grown cells, indicated that this enzyme is synthesized and located in the cytoplasm, while phosphoglycollate phosphatase was synthesized in the chloroplast. Glycollate: DCPIP oxidoreductase did not increase above the low level in heterotrophic cells when exogenous glycollate, in the presence or absence of glucose, was supplied in the dark.     

Identification of the chlB Gene and the Gene Product Essential for the Light-Independent Chlorophyll Biosynthesis in the Cyanobacterium Plectonema boryanum

We cloned a 6.0-kb HindIII fragment from the cyanobacteriumPlectonema boryanum using the chloroplast chlB (ORF513) geneof the liverwort (Marchantia polymorpha) as a probe. An openreading frame (ORF508) encoding a polypeptide of 508 amino acidresidues was found within the nucleotide sequence of the 4,437-bpHindIII-EcoRV subfragment. The deduced amino acid sequence ofORF508 shows very high similarity to that encoded by the liverwortchlB gene (72.7%). A mutant, YFB14, in which ORF508 was inactivatedby the insertion of a kanamycin-resistance cartridge, was unableto synthesize chlorophyll, accumulating protochlorophyllidein darkness while synthesizing chlorophyll normally in the light.Thus, the chlB gene is the third gene that is essential forthe light-independent reduction of protochlorophyllide. Theother two genes are chlL and chlN, and the results suggest thatthe light-independent protochlorophyllide reductase consistsof at least three subunits, which are encoded by chlL, chlNand chlB. Using an antiserum prepared against a ChlB-6xHis fusionprotein expressed in Escherichia coli, we detected a proteinwith an apparent molecular weight of 58,000 in the membranefraction of the cyanobacterium. These results indicate thateither the cytoplasmic or thylakoid membranes could be the siteof the light-independent reduction of protochlorophyllide. (Received November 16, 1995; Accepted February 7, 1996)     

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.     

An IS4-encoded protein is synthesized in minicells

Summary A protein of M_r 47,000 is synthesized in Escherichia coli minicells, when these harbor a multicopy plasmid carrying IS4 in either orientation and between different flanking sequences. The protein corresponds to the sequence predicted from the known DNA sequence of IS4, as shown by partial N-terminal radiolabel protein sequence analysis. Its apparent molecular weight, however, as determined from its electrophoretic mobility in SDS polyacrylamide gels, is smaller than predicted. When compared with other plasmid-encoded proteins, the IS4-encoded protein is synthesized in minicells in small amounts. Its synthesis has not been detected in a DNA-dependent cell-free system.     

Pigment Content of Selected Planktonic Algae in Response to Simulated Natural Light Fluctuations and a Short Photoperiod

The lipophilic photosynthetic pigments in Limnothrix redekei, Planktothrix agardhii (cyanobacteria), Stephanodiscus minutulus, Synedra acus (diatoms), Scenedesmus acuminatus, and Scenedesmus armatus (chlorophycean) all isolated from an eutrophic lake were quantitatively determined by HPLC. The algae were grown semi-continuously under nutrient sufficient conditions at 20°C at a 12/12 h light/dark cycle with constant irradiance or with simulated natural light fluctuations as well as at a 6/18 h light/dark cycle with constant irradiance, all at the same daily light exposure. The zeaxanthin and the myxoxanthophyll contents of cyanobacteria were not influenced by fluctuating light, a short photoperiod or a different sampling time. The chlorophyll b/a ratio, the lutein/chlorophyll a ratio, and the neoxanthin content of chlorophycean as well as the chlorophyll c/a and the fucoxanthin/chlorophyll a ratio of diatoms were only slightly influenced by these factors. Therefore in some cases marker pigment contents and in other cases marker pigment/chlorophyll a ratios may be more useful for quantifying the relative importance of different taxonomic groups in natural phytoplankton. Simulated natural light fluctuations or the length of the photoperiod only slightly influenced the pigment content or the marker pigment/chlorophyll a ratio.     

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene*

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T₂ transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.     

Chloroplast development and the synthesis of chlorophyll a and b and chlorophyll protein complexes I and II in the dark in Tradescantia albiflora (Kunth)

Continued synthesis of chlorophyll a and chlorophyll b occurs in Tradescantia albiflora Kunth on transfer to darkness. This synthesis continues for several days and may result in a doubling of chlorophyll content per leaf. It is accompanied by continued cell division and development of normal chloroplast ultrastructure, including stacked thylakoids.     

A novel insight into the regulation of light-independent chlorophyll biosynthesis in <Emphasis Type="Italic">Larix decidua</Emphasis> and <Emphasis Type="Italic">Picea abies</Emphasis> seedlings

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment–protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.     

Synthesis of chlorophyll <Emphasis Type="Italic">b</Emphasis>: Localization of chlorophyllide <Emphasis Type="Italic">a</Emphasis>oxygenase and discovery of a stable radical in the catalytic subunit

Background  

Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly.     

The chloroplastchIL gene of the green algaChlorella vulgaris C-27 contains a self-splicing group I intron

ThechiL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome ofChlorella vulgaris strain C-27 contains the first example of a splitchiL gene, which is interrupted by a 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5 and 3 splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3 splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3 splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.