Redox regulation of chlorophyll biosynthesis

Chlorophyll captures and redirects light-energy and is thus essential for photosynthetic organisms. The demand for chlorophyll differs throughout the day and night and in response to changing light conditions. Moreover, the chlorophyll biosynthesis pathway is up to certain points shared between the different tetrapyrroles; chlorophyll, heme, siroheme and phytochromobilin, for which the cell has different requirements at different time points. Combined with the phototoxic properties of tetrapyrroles which, if not properly protected, can lead to formation of reactive oxygen species (ROS), the need for a strict regulation of the chlorophyll biosynthetic pathway is obvious. Here we describe the current knowledge on regulation of chlorophyll biosynthesis in plants by the chloroplast redox state with emphasis on the Mg-chelatase situated at the branch point between the heme and the chlorophyll pathway. We discuss the proposed role of the Mg-chelatase as a key regulator of the tetrapyrrole pathway by its effect on enzymes both up- and downstream in the pathway and we specifically describe how redox state might regulate the Mg-branch. Finally, we propose that a recently identified NADPH-dependent thioredoxin reductase (NTRC) could be involved in redox regulation or protection of chlorophyll biosynthetic enzymes and describe the possible modes of action by this enzyme.     

Green or red: what stops the traffic in the tetrapyrrole pathway?

Regulation of tetrapyrrole biosynthesis is crucial to plant metabolism. The two pivotal control points are formation of the initial precursor, 5-aminolaevulinic acid (ALA), and the metal-ion insertion step: chelation of Fe(2+) into protoporphyrin IX leads to haem and phytochromobilin, whereas insertion of Mg(2+) is the first step to chlorophyll. Recent studies with mutants and transgenic plants have demonstrated that perturbation of the branch point affects ALA formation. Moreover, one of the signals that controls the expression of genes for nuclear-encoded chloroplast proteins has been shown to be Mg-protoporphyrin-IX. Here, we discuss the regulation of branch-point flux and the relative contributions of the haem and chlorophyll branches to the regulation of ALA synthesis and thus to flow through the tetrapyrrole pathway.     

Regulation and evolution of chlorophyll metabolism

Chlorophylls are the most abundant tetrapyrrole molecules essential for photosynthesis in photosynthetic organisms. After many years of intensive research, most of the genes encoding the enzymes for the pathway have been identified, and recently the underlying molecular mechanisms have been elucidated. These studies revealed that the regulation of chlorophyll metabolism includes all levels of control to allow a balanced metabolic flow in response to external and endogenous factors and to ensure adaptation to varying needs of chlorophyll during plant development. Furthermore, identification of biosynthetic genes from various organisms and genetic analysis of functions of identified genes enables us to predict the evolutionary scenario of chlorophyll metabolism. In this review, based on recent findings, we discuss the regulation and evolution of chlorophyll metabolism.     

Post-translational control of tetrapyrrole biosynthesis in plants, algae, and cyanobacteria

The tetrapyrrole biosynthetic pathway provides the vital cofactors and pigments for photoautotrophic growth (chlorophyll), several essential redox reactions in electron transport chains (haem), N- and S-assimilation (sirohaem), and photomorphogenic processes (phytochromobilin). While the biochemistry of the pathway is well understood and almost all genes encoding enzymes of tetrapyrrole biosynthesis have been identified in plants, the post-translational control and organization of the pathway remains to be clarified. Post-translational mechanisms controlling metabolic activities are of particular interest since tetrapyrrole biosynthesis needs adaptation to environmental challenges. This review surveys post-translational mechanisms that have been reported to modulate metabolic activities and organization of the tetrapyrrole biosynthesis pathway.     

Tetrapyrrole synthesis of photosynthetic chromerids is likely homologous to the unusual pathway of apicomplexan parasites

Most photosynthetic eukaryotes synthesize both heme and chlorophyll via a common tetrapyrrole biosynthetic pathway starting from glutamate. This pathway was derived mainly from cyanobacterial predecessor of the plastid and differs from the heme synthesis of the plastid-lacking eukaryotes. Here, we show that the coral-associated alveolate Chromera velia, the closest known photosynthetic relative to Apicomplexa, possesses a tetrapyrrole pathway that is homologous to the unusual pathway of apicomplexan parasites. We also demonstrate that, unlike other eukaryotic phototrophs, Chromera synthesizes chlorophyll from glycine and succinyl-CoA rather than glutamate. Our data shed light on the evolution of the heme biosynthesis in parasitic Apicomplexa and photosynthesis-related biochemical processes in their ancestors.     

Multiple deletions of small Cab-like proteins in the cyanobacterium Synechocystis sp. PCC 6803: consequences for pigment biosynthesis and accumulation

Deletion of the genes for four or five small Cab-like proteins (SCPs) in photosystem (PS) I-less and PS I-less/PS II-less strains of Synechocystis sp. PCC 6803 caused a large decrease in the chlorophyll and carotenoid content of the cells without accumulation of early intermediates in the chlorophyll biosynthesis pathway, suggesting limited chlorophyll availability. The PS II/PS I ratio increased upon deletion of multiple SCPs in a wild type background, similar to what is observed in the presence of subsaturating concentrations of gabaculin, an inhibitor of an early step in the tetrapyrrole biosynthesis pathway. Upon deletion of multiple SCPs, neither 77 K fluorescence emission properties of phycobilisomeless thylakoids from the PS I-less/PS II-less strain nor the energy trapping efficiency of PS II were affected, indicating that under steady-state conditions SCPs do not bind much chlorophyll and do not serve as PS II antenna. Under conditions where protochlorophyllide reduction and thus chlorophyll synthesis were inhibited, chlorophyll disappeared quickly in a mutant lacking all five SCPs. This implies a role of SCPs in stabilization of chlorophyll-binding proteins and/or in reuse of chlorophylls. Under these conditions of inhibited reduction of protochlorophyllide, the accumulation kinetics of this intermediate were greatly altered in the absence of the five SCPs. This indicates an alteration of tetrapyrrole biosynthesis kinetics by SCPs. Based on this and other evidence, we propose that SCPs bind carotenoids and transiently bind chlorophyll, aiding in the supply of chlorophyll to nascent or reassembling photosynthetic complexes, and regulate the tetrapyrrole biosynthesis pathway as a function of the demand for chlorophyll.     

Tetrapyrrole‐based drought stress signalling

Tetrapyrroles such as chlorophyll and heme play a vital role in primary plant metabolic processes such as photosynthesis and respiration. Over the past decades, extensive genetic and molecular analyses have provided valuable insights into the complex regulatory network of the tetrapyrrole biosynthesis. However, tetrapyrroles are also implicated in abiotic stress tolerance, although the mechanisms are largely unknown. With recent reports demonstrating that modified tetrapyrrole biosynthesis in plants confers wilting avoidance, a component physiological trait to drought tolerance, it is now timely that this pathway be reviewed in the context of drought stress signalling. In this review, the significance of tetrapyrrole biosynthesis under drought stress is addressed, with particular emphasis on the inter‐relationships with major stress signalling cascades driven by reactive oxygen species (ROS) and organellar retrograde signalling. We propose that unlike the chlorophyll branch, the heme branch of the pathway plays a key role in mediating intracellular drought stress signalling and stimulating ROS detoxification under drought stress. Determining how the tetrapyrrole biosynthetic pathway is involved in stress signalling provides an opportunity to identify gene targets for engineering drought‐tolerant crops.     

Implication of chlorophyll biosynthesis on chloroplast-to-nucleus retrograde signaling

The biogenesis and function of chloroplast are controlled both by anterograde mechanisms involving nuclear-encoded proteins targeted to chloroplast and by retrograde signals from plastid to nucleus contributing to regulation of nuclear gene expression. A number of experimental evidences support the implication of chlorophyll biosynthesis intermediates on the retrograde signaling, albeit an earlier-postulated direct link between accumulation of chlorophyll intermediates and changes in nuclear gene expression has recently been challenged. By characterization of Arabidopsis mutants lacking the chloroplast localized NADPH-thioredoxin reductase (NTRC) we have recently proposed that imbalanced activity of chlorophyll biosynthesis in developing cells modifies the chloroplast signals leading to alterations in nuclear gene expression. These signals appear to initiate from temporal perturbations in the flux through the pathway from protoporphyrin to protochlorophyllide rather than from the accumulation of a single intermediate of the tetrapyr-role pathway.Key words: chloroplast biogenesis, NADPH-thioredoxin reductase, porphyrins, ROS, signaling, tetrapyrrole, thioredoxinOrchestrated regulation of gene expression in the nucleus and plastids is crucial for the proper biogenesis of the organelle during the development and for the acclimation of plants to environmental cues. Multiple potential candidates for initiating plastidial signals have been recognized, including intermediates of the tetrapyrrole biosynthetic pathway, redox state of chloroplast electron transfer components and reactive oxygen species (ROS). These multiple signaling pathways are likely to interact with each others, resulting in a complex signaling network between plastid and nucleus (reviewed in ref. ¹).     

Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco

Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an amino-terminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and, consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.     

FLU, a negative feedback regulator of tetrapyrrole biosynthesis, is physically linked to the final steps of the Mg(++)-branch of this pathway

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control. Two effectors of feedback inhibition have been identified, heme and the FLU protein. Inhibition by heme implicates the Fe-branch via regulation of the initial step of tetrapyrrole synthesis. In the present work a FLU-containing chloroplast membrane complex was identified, that besides FLU comprises the four enzymes catalyzing the final steps of chlorophyll synthesis. The results support the notion that FLU links chlorophyll synthesis and the target of feedback control, glutamyl-tRNA reductase, thereby allowing also the Mg-branch to control the initial step of tetrapyrrole synthesis.     

Chloroplast biogenesis 89: development of analytical tools for probing the biosynthetic topography of photosynthetic membranes by determination of resonance excitation energy transfer distances separating metabolic tetrapyrrole donors from chlorophyll a acceptors

The thorough understanding of photosynthetic membrane assembly requires a deeper knowledge of the coordination and regulation of the chlorophyll (Chl) and thylakoid apoprotein biosynthetic pathways. As a working hypothesis we have recently proposed three different Chl-thylakoid apoprotein biosynthesis models: a single-branched Chl biosynthetic pathway (SBP)-single location model, a SBP-multilocation model, and a multibranched Chl biosynthetic pathway (MBP)-sublocation model. The detection of resonance excitation energy transfer between tetrapyrrole precursors of Chl, and several Chl-protein complexes, has made it possible to test the validity of the proposed Chl-thylakoid apoprotein biosynthesis models by resonance excitation energy transfer determinations. In this work, resonance excitation energy transfer techniques that allow the determination of distances separating tetrapyrrole donors from Chl-protein acceptors in green plants by using readily available electronic spectroscopic instrumentation are developed. It is concluded that the calculated distances are compatible with the MBP-sublocation model and incompatible with the operation of the SBP-single location Chl-protein biosynthesis model.     

Divergent regulation of the HEMA gene family encoding glutamyl-tRNA reductase in Arabidopsis thaliana: expression of HEMA2 is regulated by sugars,but is independent of light and plastid signalling

The synthesis of 5-aminolevulinic acid (ALA) is a key regulatory step for the production of hemes and chlorophyll via the tetrapyrrole synthesis pathway. The first enzyme committed to ALA synthesis is glutamyl-tRNA reductase encoded in Arabidopsis by a small family of nuclear-encoded HEMA genes. To better understand the regulation of the tetrapyrrole synthesis pathway we have made a detailed study of HEMA2 expression with transgenic Arabidopsis thaliana L. Col. plants carrying chimeric HEMA2 promoter:gusA fusion constructs. Our results show that the HEMA2 promoter directs expression predominantly to roots and flowers, but that HEMA2 is also expressed at low levels in photosynthetic tissues. Deletion analysis of the HEMA2 promoter indicates that a ca. 850 bp fragment immediately upstream of the HEMA2 coding region is sufficient to drive regulated gusA expression. In contrast to HEMA1, HEMA2 is not up-regulated by red, far-red, blue, UV or white light. In addition, elimination of a promotive plastid signal by Norflurazon-induced photobleaching of plastids had no effect on HEMA2 expression while being required for normal white-light induction of HEMA1. HEMA2 expression in the cotyledons is inhibited by the presence of sucrose or glucose, but not fructose, and this response is light-independent. HEMA1 expression in cotyledons is also inhibited by sugars, but in a strictly light-dependent manner. The roles of HEMA1 and HEMA2 in meeting cellular tetrapyrrole requirements are discussed.     

Photodynamic herbicides: 1. Concept and phenomenology

A new approach to the design of conceptually and phenomenologically new herbicides is described. It involves the joint utilization of tetrapyrrole precursors, such as δ-aminolaevulinic acid (a biodegradable amino acid) and activators of the chlorophyll biosynthetic pathway, such as 2,2′-dipyridyl, in order to induce treated plants to biosynthesize and accumulate massive amounts of tetrapyrrole intermediates of the chlorophyll biosynthetic pathway in the dark (i.e. at night). During the subsequent light period (daylight) the accumulated tetrapyrroles act as potent photodynamic sensitiziers, which in turn result in the death of susceptible plants in a matter of hours. We have therefore proposed to name herbicides that act via this mechanism as photodynamic herbicides, or more pictorially as laser herbicides. From a limited survey of agricultural plant and weed species it appears that photodynamic herbicides exhibit a very pronounced organ, age and species-dependent selectivity. For example, dicotyledonous weeds such as mustard, red-root pigweed, common purslane and lambsquarter are very susceptible while monocotyledonous plants such as corn, wheat, barley and oats are not. The biochemical basis of this selectivity seems to lie, among other things, in the rates of tetrapyrrole turnover and in a differential enhancement by the applied chemicals of the monovinyl and divinyl tetrapyrrole biosynthetic pathways in the various species. A survey of various groups of chemicals (herbicides and other selected biochemicals) that are likely to exhibit photodynamic herbicidal properties is currently under investigation.     

Recent overview of the Mg branch of the tetrapyrrole biosynthesis leading to chlorophylls

In plants, chlorophylls (chlorophyll a and chlorophyll b) are the most abundant tetrapyrrole molecules and are essential for photosynthesis. The first committed step of chlorophyll biosynthesis is the insertion of Mg²⁺ into protoporphyrin IX, and thus subsequent steps of the biosynthesis are called the Mg branch. As the Mg branch in higher plants is complex, it was not until the last decade—after many years of intensive research—that most of the genes encoding the enzymes for the pathway were identified. Biochemical and molecular genetic analyses have certainly modified the classic metabolic map of tetrapyrrole biosynthesis, and only recently have the molecular mechanisms of regulatory pathways governing chlorophyll metabolism been elucidated. As a result, novel functions of tetrapyrroles and biosynthetic enzymes have been proposed. In this review, I summarize the recent findings on enzymes involved in the Mg branch, mainly in higher plants.     

Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis

delta-Aminolevulinic acid (ALA), the first committed precursor to the tetrapyrrole components of hemes and chlorophylls, is synthesized by two different routes in the photosynthetic phytoflagellate Euglena gracilis: directly from glutamate, mediated by a 5-carbon pathway, and via condensation of glycine and succinyl-CoA, catalyzed by the enzyme ALA synthase. The physiological roles of the two pathways were determined by administration of specifically 14C-labeled ALA precursors to cultures growing under different physiological conditions. Relative activities of the ALA synthase and 5-carbon pathways were monitored by incorporation of radioactivity from [2-14C] glycine and [1-14C]glutamate into highly purified protoheme, heme a and chlorophyll a derivatives. Wild type cells grown photoautotrophically or photoheterotrophically synthesized chlorophyll and incorporated radioactivity from [1-14C]glutamate into the tetrapyrrole nucleus of the pigment. [2-14C]Glycine was incorporated primarily into the nontetrapyrrole-derived portions of chlorophyll. In the same cultures both [2-14C]glycine and [1-14C]glutamate were efficiently incorporated into protoheme, while only [2-14C] glycine was incorporated into heme a. In dark-grown wild type or light-grown aplastidic cells, no chlorophyll was formed, and both protoheme and heme a were labeled exclusively from [2-14C]glycine. These results indicate: (a) ALA synthase and the 5-carbon pathway operate simultaneously in growing green cells; (b) the 5-carbon pathway provides ALA for chloroplast protoheme and chlorophyll, and is associated with chloroplast development; (c) ALA synthase provides ALA only for nonplastid heme biosynthesis; and (d) the two ALA pathways are separately compartmentalized along with complete sets of enzymes for subsequent tetrapyrrole synthesis from each ALA pool. The protoheme that was synthesized from [1-14C] glutamate had a higher specific radioactivity than chlorophyll synthesized from the same precursor. This result together with calculated specific radioactivities of the products synthesized during the incubation period, suggest that both protoheme and heme a undergo metabolic turnover.     

Tetrapyrrole regulation of nuclear gene expression

Tetrapyrroles are the structural backbone of chlorophyll and heme, and are essential for primary photochemistry, light harvesting, and electron transport. The biochemistry of their synthesis has been studied extensively, and it has been suggested that some of the tetrapyrrole biochemical intermediates can affect nuclear gene expression. In this review, tetrapyrrole biosynthesis, which occurs in the chloroplast, and its regulation will be covered. An analysis of the intracellular location of tetrapyrrole intermediates will also be included. The focus will be on tetrapyrrole intermediates that have been suggested to affect gene expression. These include Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester. Recent evidence also suggests a specific signaling role for the H subunit of Mg-chelatase, an enzyme that catalyzes the insertion of Mg into the tetrapyrrole ring. Since gene expression studies have been done in plants and green algae, our discussion will be limited to these organisms. This revised version was published online in June 2006 with corrections to the Cover Date.