Gregory A. Armstrong

Identification of NADPH:Protochlorophyllide Oxidoreductases A and B: A Branched Pathway for Light-Dependent Chlorophyll Biosynthesis in Arabidopsis thaliana

Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development.

Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.

The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide–F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide–F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide–F655 to nonphotoactive Pchl[ide]–F632, and the amount of Pchlide–F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide–F655.

Greening in the dark : light-independent chlorophyll biosynthesis from anoxygenic photosynthetic bacteria to gymnosperms

Abstract The enzymatic reduction of protochlorophyllide (Pchlide) represents a key regulatory step in Mg-tetrapyrrole pigment biosynthesis among organisms from eubacteria to higher plants. Pchlide is a late precursor of both chlorophylls (Chls) and bacteriochlorophylls (Bchls), the crucial pigments required for charge separation during oxygenic and anoxygenic photosynthesis, respectively. Two biochemically and genetically distinct strategies to reduce Pchlide have arisen during evolution and coexist in many photosynthetic organisms. One strategy relies on NADPH:Pchlide oxidoreductase (POR), a nuclear-encoded, plastid-localized enzyme. POR requires light and NADPH as cofactors for the enzymatic reduction of Pchlide to chlorophyllide during light-dependent Chl biosynthesis. This reaction is lacking in anoxygenic photosynthetic bacteria, but occurs in cyanobacteria, algae and plants, including their most highly evolved representatives, the angiosperms. The main focus of this review will be the genetics and regulation of a light-independent strategy for Pchlide reduction catalyzed by the darkactive Pchlide oxidoreductase (DPOR). The presence of DPOR allows light-independent Bchl biosynthesis in anoxygenic photosynthetic bacteria, and light-independent Chl biosynthesis in cyanobacteria, algae and many plants, angiosperms excepted. DPOR, which is structurally distinct from POR, is specified by three plastid-encoded genes in eukaryotes that green in the dark. The corresponding gene products, ChlB, ChlL and ChlN, are evolutionarily related to the subunits of the eubacterial nitrogenase enzyme complex.

Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 3∶1 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, δ-aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.

Does a light-harvesting protochlorophyllide a/b-binding protein complex exist?

Recent in vitro studies have led to speculation that a novel light-harvesting protochlorophyllide a/b-binding protein complex (LHPP) might exist in dark-grown angiosperms. Structurally, it has been suggested that LHPP consists of a 5:1 ratio of dark-stable ternary complexes of the light-dependent NADPH: protochlorophyllide oxidoreductases A and B containing nonphotoactive protochlorophyllide b and photoactive protochlorophyllide a, respectively. Functionally, LHPP has been hypothesized to play major roles in establishing the photosynthetic apparatus, in protecting against photo-oxidative damage during greening, and in determining etioplast inner membrane architecture. However, the LHPP model is not compatible with other studies of the pigments and the pigment-protein complexes of dark-grown angiosperms. Protochlorophyllide b, which is postulated to be the major light-harvesting pigment of LHPP, has, for example, never been detected in etiolated seedlings. This raises the question: does LHPP exist?

Genetic Analysis and Regulation of Carotenoid Biosynthesis

Carotenoids comprise a large class of natural pigments that are ubiquitous in photosynthetic organisms and are essential for photooxidative protection. A large collection of anoxygenic photosynthetic bacterial carotenoid biosynthesis mutants with novel color phenotypes have been isolated over the past 40 years. Genes involved in carotenoid biosynthesis have recently been mapped, cloned and sequenced. Seven clustered genes (crtA, crtB, crtC, crtD, crtE, crtF, crtI) are required for carotenoid biosynthesis in Rhodobacter (Rb.) capsulatus and Rb. sphaeroides. The crt gene products participate in reactions that convert FPP to spheroidene/spheroidenone. The proposed requirement for the products of Rb. capsulatus ORF160 and ORF 469 in carotenoid biosynthesis conflicts with experiments using their respective Rb. sphaeroides homologs. Nucleotide sequences have been determined for all of the Rb. capsulatus crt genes and for crtD, crtI, crtB and a portion of crtC in Rb. sphaeroides. The Rb. capsulatus crtEF and crtIB genes appear to form multigene operons, the former within the global context of a superoperon in the photosynthesis gene cluster.

Pigment-Protein Complexes, Plastid Development and Photooxidative Protection

Etiolated seedlings of angiosperms, the most highly evolved and diverse group of higher plants, synthesize chlorophyll (Chl) only upon exposure to light (1). In contrast, most oxygenic photosynthetic organisms also green when grown initially in the dark (2). The biochemical basis of light-dependent Chl biosynthesis is the strictly light-and NADPHdependent enzymatic reduction of protochlorophyllide (Pchlide), a late Chl biosynthetic precursor. Pchlide is converted to chlorophyllide (Chlide) by two structurally related but differentially regulated enzymes, NADPH:Pchlide oxidoreductases (POR; EC 1.3.1.33) A and B, in the angiosperms Arabidopsis thaliana (3) and barley (4). Although PORA and PORB are both nuclear-encoded, translated in the cytosol, and ultimately imported into plastids, the PORA and PORB genes display dramatic differences in their regulation by light and developmental state, and the two enzymes have different plastid import requirements (5).

Stepping out of the Dark: How Higher Plants Cope with the Risk of Photooxidative Damage

Most oxygenic photosynthetic organisms have two options to complete the synthesis of chlorophyll. In a light-independent reaction the immediate precursor of chlorophyllide, protochlorophyllide (Pchlide), can be reduced to chlorophyllide through a Pchlidereducing enzyme that consists of three subunits Ch L, Ch B and Ch N that in plants are encoded by chloroplast DNA (1). In addition to this light-independent enzyme they also contain a nuclear DNA-encoded light-dependent NADPH-Pchlide oxidoreductase (POR) (2, 3). In angiosperms, however, only one of these two routes is active. Through the loss of the light-independent enzyme angiosperms are no longer able to use this reaction during the night to avoid the accumulation of Pchlide, a photosensitizing pigment, that upon illumination may lead to severe photooxidative damage (Figure 1). In order to compensate for the lack of the light-independent Pchlide-reducing enzyme angiosperms seem to have evolved at least two protection mechanisms that minimize the risk of photooxidative damage during the transition from the dark to the light. First, in contrast to other plants, some angiosperms — and also some of the gymnosperms — possess a second POR enzyme (2–6) (Figure 1). It is tempting to speculate that angiosperms use this additional POR enzyme as part of a protection strategy against reactive oxygen species, that in particular during seedling development may lead to the rapid bleaching and death of etiolated seedlings after they have been exposed to light. Second, a feed-back inhibitory loop has been described in higher plants through which further synthesis of δ-aminolevulinic acid, a precursor common to all porphyrins, is stopped once a critical level of Pchlide has been reached in the dark (Figure 2). The existence of this regulatory circuit has been deduced from experiments in which exogenous δ-aminolevulinic acid had been applied to dark-grown plants. Large amounts of Pchlide accumulated under these conditions that led to the greening of etiolated seedlings in the dark (7).