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

The early steps in chloroplast development were characterized in wild-type pea (Pisum sativum L, cv, Alaska) and the lip1 mutant with light-independen t photomorphogenesis. When grown in darkness, the mild type had a long slen der epicotyl and undeveloped leaves while the lip1 mutant had seedlings wit h short epicotyls and well-developed leaves. The leaves of the wild-type Al aska pea had two fluorescence emission maxima of protochlorophyllide (Pchli de) at 630 and 655 nm. Leaves from the lip1 mutant had a fluorescence emiss ion maximum at 632 nm with only a small shoulder at 655 nm, Flash irradiati on of wild-type leaves gave chlorophyllide (Chlide) with a fluorescence pea k 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 l ip1 and wild-type leaves, Sodium dodecyl sulfate-polyacrylamide gel electro phoresis (SDS-PAGE) and immunoblots indicated the presence of FOR (EC 1.3.1 .33) in leaves of both wild-type and lip1 dark-grown seedlings. Just one ba nd 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 prolam ellar bodies (PLBs) were common in dark-grown wildtype etioplasts, whereas lip1 etioplast inner membranes consisted mostly of prothylakoids (PTs), Whe n present in lip1, the PLBs were rudimentary, but often a number of plastog lobuli were assembled in the etioplasts. The high content of FOR was unexpe cted considering the observed lack of PLBs and Pchlide fluorescing at 655 n m, Immunogold labeling showed a distribution of FOR among the PTs, It can b e concluded that the formation and accumulation of FOR in pea is, in itself , insufficient to induce PLB formation or the formation of long-wavelength Pchlide forms. It appears that there also must he other factor(s) of import ance, which are significant for the formation of the PLBs.