Protochlorophyllide, NADPH‐protochlorophyllide oxidoreductase, and chlorophyll formation in the lip1 mutant of pea

Abstract

The early steps in chloroplast development were characterized in wild‐type pea (Pisum sativum L. cv. Alaska) and the lip1 mutant with light‐independent photomorphogenesis. When grown in darkness, the wild type had a long slender epicotyl and undeveloped leaves while the lip1 mutant had seedlings with short epicotyls and well‐developed leaves. The leaves of the wild‐type Alaska pea had two fluorescence emission maxima of protochlorophyllide (Pchlide) at 630 and 655 nm. Leaves from the lip1 mutant had a fluorescence emission maximum at 632 nm with only a small shoulder at 655 nm. Flash irradiation of wild‐type leaves gave chlorophyllide (Chlide) with a fluorescence peak at 692 nm exhibiting a Shibata shift to 682 nm. The Chlide formed in lip1 leaves was diminutive with a maximum at 677 nm. Continuous irradiation led to the formation of a significant amount of Chlide, the esterification of which was considerably delayed in the lip1 plants. The transcript level of NADPH‐protochlorophyllide oxidoreductase (POR) was the same in dark‐grown lip1 and wild‐type leaves. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblots indicated the presence of POR (EC 1.3.1.33) in leaves of both wild‐type and lip1 dark‐grown seedlings. Just one band at 36 kDa was detectable. The level of the protein both in wild type and the lip1 mutant was stable during 6 h of irradiation. Large regular prolamellar bodies (PLBs) were common in dark‐grown wild‐type etioplasts, whereas lip1 etioplast inner membranes consisted mostly of prothylakoids (PTs). When present in lip1, the PLBs were rudimentary, but often a number of plastoglobuli were assembled in the etioplasts. The high content of POR was unexpected considering the observed lack of PLBs and Pchlide fluorescing at 655 nm. Immunogold labeling showed a distribution of POR among the PTs. It can be concluded that the formation and accumulation of POR in pea is, in itself, insufficient to induce PLB formation or the formation of long‐wavelength Pchlide forms. It appears that there also must be other factor(s) of importance, which are significant for the formation of the PLBs.

Regulation of Etioplast Pigment-Protein Complexes, Inner Membrane Architecture, and Protochlorophyllide a Chemical Heterogeneity by Light-Dependent NADPH:Protochlorophyllide Oxidoreductases A and B

“…4). These results are in accordance with other studies of higher plants, including barley (Granick and Gassman, 1970;Adra and Rebeiz, 1998;Scheumann et al, 1999;Seyyedi et al, 1999) and support the consensus view that etiolated angiosperms contain exclusively Pchl(ide) a, most of which is photoactive (Armstrong et al, 2000).…”

“…That other unidentified factors contribute to the formation of etioplast inner membranes is suggested by the phenotype of the lip1 constitutive photomorphogenic mutant of pea, an angiosperm that seems to contain only a single POR gene (Spano et al, 1992). By analogy to the pleiotropic cop1 mutant of Arabidopsis (Lebedev et al, 1995;Sperling et al, 1998), lip1 displays a light-grown seedling morphology even when germinated in the dark and its plastids lack PLBs and normal amounts of photoactive Pchlide-F655 (Seyyedi et al, 1999). However, unlike cop1, the lip1 mutant retains roughly wild-type amounts of POR polypeptide, suggesting that this mutant is defective in some other component(s) required for etioplast differentiation.…”

“…4C), photoactive Pchlide is the main pigment form in mature etioplasts in situ (Granick and Gassman, 1970;Adra and Rebeiz, 1998;Seyyedi et al, 1999). Taking into account that POR possesses a single NADPH-binding site, each dark-stable ternary complex should reduce only one photoactive Pchlide molecule per light flash.…”

The relationship between different spectral forms of the protochlorophyllide oxidoreductase complex and the structural organisation of prolamellar bodies isolated from Zea mays

“…Mixtures of these two lipids are also known to form bicontinuous cubic phases in model systems (Sen et al 1982;Brentel et al 1985;Lindblom and Rilfors 1989). The existence of PLB mutants that lack organised PLB (Sperling et al 1998;Seyyedi et al 1999), however, strongly suggests that the presence of such lipids is a necessary but not a sufficient condition for PLB formation.…”

When Proteomics Reveals Unsuspected Roles: The Plastoglobule Example

“…Figure 1). Plastoglobules can be found in diverse types of plastids, from proplastids (for review, see Nagata et al, 2002) to gerontoplasts (Kovacs et al, 2008) or etioplasts (Seyyedi et al, 1999). Although the origin of plastoglobules remains unclear, they may be closely linked to thylakoid development and dismantlement.…”