Geneviève Frick

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.

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).