Nuclear-encoded Plastid Proteins Research Articles

Apicoplast genome of the coccidian Eimeria tenella

Unicellular apicomplexans possess an algal-originated plastid referred to as an apicoplast. Although apicomplexan parasites are comprised of highly diverse protists, the complete apicoplast genome sequences have only been determined from the hematozoan Plasmodium falciparum and cyst-forming coccidian Toxoplasma gondii. Here, we report the third complete sequence of apicoplast genome from the intestinal coccidian Eimeria tenella that may serve as a new drug target against coccidiosis in the livestock. The AT-rich E. tenella plastid genome is a 35-kb circular element. Its gene organization resembles more closely that of T. gondii than P. falciparum. Although the E. tenella plastid genome contains an almost identical set of genes to that found in P. falciparum and T. gondii, its encoded genes share low or moderate homologies with their counterparts in the other two apicomplexans. With the addition of this coccidian plastid genome sequence, we attempted to reexamine the apicoplast genome evolution and performed phylogenetic reconstructions using maximum likelihood and Bayesian inference (BI) methods based on a concatenated dataset of plastid-encoded rpoB, rpoC1 and rpoC2 proteins. All resulting rpo protein trees placed apicoplast as a sister to Euglena within the green lineage. On the other hand, many recent studies based on the organization of plastid genes and some nuclear-encoded plastid proteins have supported a common red algal ancestry of apicomplexan and dinoflagellate plastids. If the apicoplast indeed originated from a red ancestor, the green relationship of apicomplexan genes would probably imply that the ancestral host that gave rise to the (red) apicoplast might have already contained some primary green plastid genes.

TIGRINA d, required for regulating the biosynthesis of tetrapyrroles in barley, is an ortholog of the FLU gene of Arabidopsis thaliana

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control of steps prior to δ-aminolevulinic acid (ALA) formation. One of the first mutants with a defect in this control had been identified in barley. The tigrina ( tig) d mutant accumulates 10–15-fold higher amounts of protochlorophyllide than wild type, when grown in the dark. The identity of the TIGRINA d protein and its mode of action are not known yet. Initially this protein had been proposed to act as a repressor of genes that encode enzymes involved in early steps of ALA formation, but subsequent attempts to confirm this experimentally failed. Here we demonstrate that the TIGRINA d gene of barley is an ortholog of the FLU gene of Arabidopsis thaliana. The FLU protein is a nuclear-encoded plastid protein that plays a key role in negative feedback control of chlorophyll biosynthesis in higher plants. Sequencing of the FLU gene of barley revealed a frame shift mutation in the FLU gene of the tig d mutant that results in the loss of two tetratricopeptide repeats that in the FLU protein of Arabidopsis are essential for its biological activity. This mutation cosegregates strictly with the tigrina phenotype within the F1 population of a heterozygous tig d mutant, thus providing additional support for the flu gene being responsible for the tigrina phenotype of barley.

Second- and third-hand chloroplasts in dinoflagellates: phylogeny of oxygen-evolving enhancer 1 (PsbO) protein reveals replacement of a nuclear-encoded plastid gene by that of a haptophyte tertiary endosymbiont.

Several dinoflagellate species have plastids that more closely resemble those of an unrelated algal group, the haptophytes, suggesting these plastids have been obtained by tertiary endosymbiosis. Because both groups are photosynthetic, all of the genes for nuclear-encoded plastid proteins might be supplied by the dinoflagellate host or some of them might have been replaced by haptophyte genes. Sequences of the conserved nuclear psbO gene were obtained from the haptophyte Isochrysis galbana, the peridinin-containing dinoflagellate Heterocapsa triquetra, and the 19'hexanoyloxy-fucoxanthin-containing dinoflagellate Karenia brevis. Phylogenetic analysis of the oxygen-evolving-enhancer (PsbO) proteins confirmed that in K. brevis the original peridinin-type plastid was replaced by that of a haptophyte, an alga which had previously acquired a red algal chloroplast by secondary endosymbiosis. It showed clearly that during this tertiary symbiogenesis the original psbO gene in the dinoflagellate nucleus was replaced by a psbO gene from the haptophyte nucleus. The phylogenetic analysis also confirmed that the origin of the peridinin-type dinoflagellate plastid was indeed a red alga.

FLU: a negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana.

Tetrapyrroles such as chlorophylls and bacteriochlorophylls play a fundamental role in the energy absorption and transduction activities of photosynthetic organisms. Because of these molecules, however, photosynthetic organisms are also prone to photooxidative damage. They had to evolve highly efficient strategies to control tetrapyrrole biosynthesis and to prevent the accumulation of free intermediates that potentially are extremely destructive when illuminated. In higher plants, the metabolic flow of tetrapyrrole biosynthesis is regulated at the step of delta-aminolevulinic acid synthesis. This regulation previously has been attributed to feedback control of Glu tRNA reductase, the first enzyme committed to tetrapyrrole biosynthesis, by heme. With the recent discovery of chlorophyll intermediates acting as signals that control both nuclear gene activities and tetrapyrrole biosynthesis, it seems likely that heme is not the only regulator of this pathway. A genetic approach was used to identify additional factors involved in the control of tetrapyrrole biosynthesis. In Arabidopsis thaliana, we have found a negative regulator of tetrapyrrole biosynthesis, FLU, which operates independently of heme and seems to selectively affect only the Mg(2+) branch of tetrapyrrole biosynthesis. The identity of this protein was established by map-based cloning and sequencing the FLU gene. FLU is a nuclear-encoded plastid protein that, after import and processing, becomes tightly associated with plastid membranes. It is unrelated to any of the enzymes known to be involved in tetrapyrrole biosynthesis. Its predicted features suggest that FLU mediates its regulatory effect through interaction with enzymes involved in chlorophyll synthesis.

Do plastid envelope membranes play a role in the expression of the plastid genome?

A unique biochemical machinery is present within the two envelope membranes surrounding plastids (Joyard et al., Plant Physiol. 118 (1998) 715-723) that reflects the stage of development of the plastid and the specific metabolic requirements of the various tissues. Envelope membranes are the site for the synthesis and metabolism of specific lipids. They are also the site of transport of metabolites, proteins and information between plastids and surrounding cellular compartments. For instance, a complex machinery for the import of nuclear-encoded plastid proteins is rapidly being elucidated. The functional studies of plastid envelope membranes result in the characterization of an increasing number of envelope proteins with unexpected functions. For instance, recent experiments have demonstrated that envelope membranes bind specifically to plastid genetic systems, the nucleoids surrounded by plastid ribosomes. At early stages of plastid differentiation, the inner envelope membrane contains a unique protein (named PEND protein) that binds specifically to plastid DNA. This tight connection suggests that the PEND protein is at least involved in partitioning the plastid DNA to daughter plastids during division. The PEND protein can also provide a physical support for replication and transcription. In addition, factors involved in the control of plastid protein synthesis can become associated to envelope membranes. This was shown for a protein homologous to the E. coli ribosome recycling factor and for the stabilizing factors of some specific chloroplast mRNAs encoding thylakoid membrane proteins. In fact, the envelope membranes together with the plastid DNA are the two essential constituents of plastids that confer identity to plastids and their interactions are becoming uncovered through molecular as well as cytological studies. In this review, we will focus on these recent observations (which are consistent with the endosymbiotic origin of plastids) and we discuss possible roles for the plastid envelope in the expression of plastid genome.

Protein Transport into “Complex” Diatom Plastids Utilizes Two Different Targeting Signals

The plastids found in diatoms and other chromophytic algae are completely enclosed by four membranes in contrast to chloroplasts of higher plants, which are surrounded by only two membranes. The bipartite targeting sequence of diatom nuclear-encoded plastid proteins contains an endoplasmic reticulum signal sequence and, based on sequence comparison, a transit peptide-like domain similar to that which targets proteins into the plastids of higher plants. By performing heterologous import experiments using the precursor of the gamma subunit of the chloroplast ATPase from the diatom Odontella sinensis we were able to show that protein import into diatom plastids is at least a two-step event. We demonstrate that the first step involves co-translational transport through endoplasmic reticulum membranes and that there is an additional targeting step which is similar to the import of precursor proteins into chloroplasts of higher plants and green algae indicating that the transit peptide-like domain of the diatom precursor is functionally equivalent to the respective targeting signal of higher plants. Our results suggest that the transit peptide depending targeting mechanism in plastids has apparently remained relatively unchanged over the course of evolution, with only the peptidase cleavage site significantly modified.

Reduction of coproporphyrinogen oxidase level by antisense RNA synthesis leads to deregulated gene expression of plastid proteins and affects the oxidative defense system.

A full-length cDNA sequence encoding coproporphyrinogen oxidase was inserted in inverse orientation behind a CaMV promoter and transferred to tobacco (Nicotiana tabacum) by standard transformation techniques. Transformants showed reduced coproporphyrinogen oxidase activity and accumulation of photosensitive coproporphyrin(ogen), indicating antisense RNA expression. An inverse correlation was observed between the level of coproporphyrinogen oxidase and transformant phenotype. The latter is characterized by a broad range of growth retardation and necrosis, indicating oxidative leaf damage. Coproporphyrinogen is an apparent chromophore and its excitation finally leads to the production of reactive oxygen. Evidence is presented that indicates a direct correlation between the accumulation of non-metabolized coproporphyrinogen and oxidative damage to cellular structural components. Enzymatic and non-enzymatic antioxidants were investigated. Whereas superoxide dismutase activity increased in transgenic plants, catalase and ascorbate peroxidase activity remained constant. Tocopherol, rather than carotene or zeaxanthin, seemed to be involved in detoxification, indicating the putative localization and allocation of coproporphyrinogen. Expression of coproporphyrinogen oxidase antisense RNA did not significantly influence the level of other enzymes in the chlorophyll metabolic pathway, but deregulated gene expression of nuclear encoded plastid proteins. Accumulation of coproporphyrinogen and/or the resulting effects, such as oxidative stress, impairs a plastid/nuclear signal which may adapt gene expression to the plastid state.

Enzymatic product formation impairs both the chloroplast receptor-binding function as well as translocation competence of the NADPH: protochlorophyllide oxidoreductase, a nuclear-encoded plastid precursor protein.

The key enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), is a nuclear-encoded plastid protein. Its posttranslational transport into plastids of barley depends on the intraplastidic availability of one of its substrates, protochlorophyllide (PChlide). The precursor of POR (pPOR), synthesized from a corresponding full-length barley cDNA clone by coupling in vitro transcription and translation, is enzymatically active and converts PChlide to chlorophyllide (Chlide) in a light- and NADPH-dependent manner. Chlorophyllide formed catalytically remains tightly but noncovalently bound to the precursor protein and stabilizes a transport-incompetent conformation of pPOR. As shown by in vitro processing experiments, the chloroplast transit peptide in the Chlide-pPOR complex appears to be masked and thus is unable to physically interact with the outer plastid envelope membrane. In contrast, the chloroplast transit peptide in the naked pPOR (without its substrates and its product attached to it) and in the pPOR-substrate complexes, such as pPOR-PChlide or pPOR-PChlide-NADPH, seems to react independently of the mature region of the polypeptide, and thus is able to bind to the plastid envelope. When envelope-bound pPOR-PChlide-NADPH complexes were exposed to light during a short preincubation, the enzymatically produced Chlide slowed down the actual translocation step, giving rise to the sequential appearance of two partially processed translocation intermediates. However, ongoing translocation induced by feeding the chloroplasts delta-aminolevulinic acid, a precursor of PChlide, was able to override these two early blocks in translocation, suggesting that the plastid import machinery has a substantial capacity to denature a tightly folded, envelope-bound precursor protein. Together, our results show that pPOR with Chlide attached to it is impaired both in the ATP-dependent step of binding to a receptor protein component of the outer chloroplast envelope membrane, as well as in the PChlide-dependent step of precursor translocation.

Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.

Regulation of Plastid Gene Expression during Photooxidative Stress

We have used the carotenoid biosynthesis inhibitor norflurazon to study the relationship between chloroplast and nuclear gene expression and the mechanisms by which plastid mRNA accumulation is regulated in response to photooxidative stress. By treating 4-week-old hydroponic spinach plants (Spinacea oleracea), we were able to determine the response at two distinct stages of chloroplast development. For all parameters studied, differences were found between the norflurazon-treated young and mature leaves. Young leaves lost essentially all pigment content in the presence of norflurazon, whereas mature leaves retained more than 60% of their chlorophyll and carotenoids. The accumulation of plastid mRNA was determined for several genes, and we found a decrease in mRNA levels for all genes except psbA in herbicide-treated young leaves. For genes such as atpB, psbB, and psaA, there was a corresponding change in the relative level of transcription, but for psbA and rbcL, transcription and mRNA accumulation were uncoupled. In norflurazon-treated mature leaves, all plastid mRNAs except psaA accumulated to normal levels, and transcription levels were either normal or higher than corresponding controls. This led to the conclusion that plastid mRNA accumulation is regulated both transcriptionally and posttranscriptionally in response to photooxidative stress. Although direct photooxidative damage is confined to the plastid and peroxisome, there is a feedback of information controlling the transcription of nuclear-encoded plastid proteins. Considerable evidence has accumulated implicating a "plastid factor" in this control. Therefore, the expression of several nuclear-encoded plastid proteins and the corresponding mRNAs were determined. Although the levels of both the small subunit of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b-binding protein and corresponding mRNAs were reduced, a 28-kilodalton chloroplast RNA-binding protein and corresponding mRNA were at normal levels in norflurazon-treated plants. Changes in mRNA and protein levels were not the result of a general loss due to photooxidation but rather the result of selective stabilization of certain components. The response of both genomes to photooxidative stress is discussed in terms of the postulated plastid factor.

Cytokinins modulate the steady-state levels of light-dependent and light-independent proteins and mRNAs in tobacco cell suspensions

Cytokinin modulation of chloroplast differentiation in light-grown tobacco ( Nicotiana tabacum cv Wisconsin 38) cell suspensions was previously shown to involve the control of specific nuclear-encoded plastid proteins, including light-harvesting complex chlorophyll a/ b binding protein (Cab) and the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (SSU). The steady-state levels of the corresponding mRNAs was enhanced in response to the hormone. Patterns of gene expression in response to cytokinin and light were further compared. Cytokinin-deprived, light-grown tobacco cells were subcultured, either in the dark or under white light, in a kinetin-supplemented medium or in a cytokinin-free medium. Total cell proteins were separated by two-dimensional electrophoresis. Amongst more than 500 distinguishable silver stained polypeptide spots, only about twenty were found to vary as a function of culture time and of light or cytokinin suppl. These variations were quantified by image analysis. Various interactions between light and cytokinin in modulating the steady-state levels of some polypeptides were observed. Independently of light, cytokinin increased or decreased the levels of some polypeptides. These polypeptides were different from pathogenesis-related polypeptides detected in virus infected tobacco leaves. In vitro translation products of poly(A) +RNAs purified from cells harvested during the growth phase were fractionated by two-dimensional electrophoresis: some translatable mRNAs were modulated by kinetic but not by light. Northern blot and slot-blot hybridization experiments showed that kinetin enhanced the steady-levels of Cab and SSU encoding mRNAs in light-grown cells only and had no detectable effect on these mRNAs in the dark.

Light-harvesting proteins of diatoms: their relationship to the chlorophyll a/b binding proteins of higher plants and their mode of transport into plastids.

We have cloned and characterized members of a gene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex, a light-harvesting complex associated with photosystem II of diatoms and brown algae. Three cDNA clones encoding proteins associated with this complex in the diatom Phaeodactylum tricornutum have been isolated. As deduced from the nucleotide sequences, these light-harvesting proteins show homology to the chlorophyll a/b binding polypeptides of higher plants. Specifically, the N-terminal regions of the fucoxanthin, chlorophyll a/c-binding proteins are homologous to the chlorophyll a/b binding proteins in both the third membrane-spanning domain and the stroma-exposed region between membrane-spanning domains 2 and 3. Like the chlorophyll a/b-binding proteins, the mature fucoxanthin, chlorophyll a/c polypeptides have three hydrophobic alpha-helical domains which could span the membrane bilayer. The similarities between the two light-harvesting proteins might reflect the fact that both bind chlorophyll molecules and/or might be important for maintaining certain structural features of the complex. There is little similarity between the N-terminal sequences of the primary translation products of the fucoxanthin, chlorophyll a/c proteins and any transit sequences that have been characterized. Instead, the N-terminal sequences have features resembling those of signal sequences. Thus either transit peptides used in P. tricornutum show little resemblance to those of higher plants and green algae or the nuclear-encoded plastid proteins enter the organelle via a mechanism different from that used in higher plants.

The complexity of gene expression in higher plants

During light-induced chloroplast formation in higher plants the synthesis of several nuclear encoded plastid proteins is under the control of phytochrome. Light acting through the phytochrome system is able both to increase the transcription of certain nuclear genes and to decrease the transcription of others. It has been generally assumed that regulation by phytochrome alone would be sufficient to account for the observed light-dependent changes in nuclear gene expression during chloroplast formation. However, it has recently become evident that the light-dependent control of nuclear gene expression may be far more complex than originally expected. There are at least two other factors that in addition to phytochrome may affect nuclear gene expression: (1) changes in chromatin organization from an inactive to a transcriptionally active state, and (2) a plastid-derived factor that seems to be involved in the transcriptional control of some nuclear genes encoding plastid-specific proteins. Although the light-dependent control of transcription has been studied intensively for nuclear genes, much less is known about the light-dependent control of plastid gene expression. The P700 chlorophyll a protein of photosystem I is a major membrane protein whose massive accumulation is induced by light and whose genes have been located on the plastid DNA. In barley a high concentration of mRNA for the P700 chlorophyll a protein was detected within the total RNA as well as within the polysomal fraction of etioplasts and remained almost constant during greening. Based on these results it can be inferred that the accumulation of the P700 chlorophyll a protein during light-dependent chloroplast development in barley is not coupled to its transcript concentration but is controlled at a translational — or post-translational - level. The possible function of protochlorophyllide as photoreceptor in this light-dependent control of plastid gene expression is discussed.