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.