The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of protochlorophyll(ide) a [Pchl(ide) a]. Either of two structurally related, but differentially expressed light-dependent NADPH:Pchlide oxidoreductases (PORs), PORA and PORB, can assemble the PLB and form dark-stable ternary complexes containing enzymatically photoactive Pchlide-F655. Here we have examined in detail whether these polypeptides play redundant roles in etioplast differentiation by manipulating the total POR content and the PORA-to-PORB ratio of etiolated Arabidopsis seedlings using antisense and overexpression approaches. POR content correlates closely with PLB formation, the amounts, spectroscopic properties, and photoreduction kinetics of photoactive Pchlide, the ratio of photoactive Pchlide-F655 to non-photoactive Pchl(ide)-F632, and the ratio of divinyl- to monovinyl-Pchl(ide). This last result defines POR as the first endogenous protein factor demonstrated to influence the chemical heterogeneity of Pchl(ide) in angiosperms. It is intriguing that excitation energy transfer between different spectroscopic forms of Pchl(ide) in etiolated cotyledons remains largely independent of POR content. We therefore propose that the PLB contains a minimal structural unit with defined pigment stoichiometries, within which a small amount of non-photoactive Pchl(ide) transfers excitation energy to a large excess of photoactive Pchlide-F655. In addition, our data suggests that POR may bind not only stoichiometric amounts of photoactive Pchlide, but also substoichiometric amounts of non-photoactive Pchl(ide). We conclude that the typical characteristics of etioplasts are closely related to total POR content, but not obviously to the specific presence of PORA or PORB.

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.

The cellular distribution of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase has been determined in ultrathin sections of barley leaves by the method of immunogold labelling. In leaves of etiolated seedlings a large portion of the immunoreactive protein was localized within the prolamellar body. However, approximately one third of the total immunoreactive protein was present outside the plastid in the area of the plasmalemma. During illumination of etiolated seedlings the two polypeptide populations were differentially affected by light. While the concentration of the plastid-localized immunoreactive protein rapidly decreased and was hardly detectable after 16 h of continuous white-light treatment, the concentration of the extraplastidic polypeptide did not decline significantly during this illumination period. A similar distribution pattern of the immunoreactive polypeptide was also found in maize and rye. The chlorophyll-deficient barley mutant xantha-l81 contained the immunoreactive 36000-Mr polypeptide, even though the prolamellar body was not detectable in etioplasts of this mutant. All of the immunoreactive polypeptide was localized outside the plastid in the area of the plasmalemma. Despite the apparent absence of the enzyme protein from the plastid, dark-grown mutant plants contained the same relative concentration of mRNA activity for the NADPH-protochlorophyllide oxidoreductase, which declined rapidly during illumination, as in wild-type plants. The antigenic properties and the apparent molecular weight of the plastid-localized NADPH-protochlorophyllide oxidoreductase and the 36000-Mr immunoreactive polypeptide outside the plastid were so similar as to indicate that the two proteins may be of common origin.