In vitro spectrofluorometric determination of chlorophyll de novo synthesis in chlorophyll-deficient phenotypes of Pennisetum glaucum (L.) R.Br

Abstract

Blocks in chlorophyll (Chl) biosynthesis in crude extracts of well-defined chlorophyll-deficient seedlings and green siblings from five Pennisetum glaucum (L.) R.Br. genotypes and the ability of these genotypes to relieve such blocks in crude extract mixtures were studied with low temperature (77 K) fluorescence spectroscopy. Dark-adapted green seedling extracts exhibited F₆₅₇, F₆₈₅, and F₇₃₅ peaks corresponding with protochlorophyllide (Pchlide), Chl from photosystem II and from photosystem I, respectively. Extracts of white seedlings from white stripe mutants and an albino mutant exhibited F₆₅₇ peaks indicative of Pchlide, whereas the extracts of yellow seedlings from yellow stripe mutants exhibited F₆₅₇ and F₆₇₈ peaks representing Pchlide and chlorophyllide (Chlide) accumulation, respectively. Addition of their green sibling's extract resulted in the appearance of F₆₈₅ and F₇₃₅ peaks, suggesting de novo relief of the blocks and photoreduction of Pchlide to Chl(ide). Incubating a mixture of crude extracts of yellow seedlings from two different yellow stripe mutants did not alter their peaks, indicating a lack of reconstitution because of a common limiting factor in the two yellow seedling sources. Likewise, the mixture of crude extract of yellow seedlings with that of white seedlings from white stripe mutants displayed no Chl peaks. Spontaneous greening in the mixture of crude extracts of yellow seedlings with that of albino mutant and its failure in other reconstitution experiments suggests that Chl synthase in albino seedlings enabled the conversion of Chlide to Chl in vitro. The factors governing the in vitro reconstitution and its implications in genetic complementation are discussed.