Protochlorophyllide b does not occur in barley etioplasts

Abstract

Barley (Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection 6 1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH:protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7 I -hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80^84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.z 1999 Federation of European Biochemical Societies.

In Situ Conversion of Protochlorophyllideb to Protochlorophyllide a in Barley

“…In the present study, we readdressed the experimental design of Scheumann et al (21). We demonstrate that Chl(ide) b reductase (which may alternatively be named 7-formyl reductase; see below) is indeed able to convert Pchlide b to Pchlide a in situ.…”

“…To test whether this enzyme activity, which we tentatively named 7-formyl reductase to indicate this broad substrate specificity, would also be able to convert Pchlide b to Pchlide a in situ, we followed the experimental design of Scheumann et al (21). Briefly, intact barley etioplasts were isolated on a Percoll gradient, sedimented by centrifugation, lysed, and incubated with ZnPPb, the zinc analog of Pchlide b (32).…”

“…Either a step gradient was used, starting with 34% 25 mM aqueous ammonium acetate, 15% acetone, and 51% methanol (buffer A), increasing to 16% H 2 O, 60% acetone, and 24% methanol within 20 min (buffer B), and finally to 100% acetone another 4 min later, or linear gradients from buffer A to buffer B. Absorbance measurements were made at 455 nm, which corresponds to the Soret band of Pchlide b, to detect and quantify Pchlide a and Pchlide b levels. At this wavelength, the extinction coefficients of Pchlide b and Pchlide a are 5-fold different (21). As internal standards, we used synthetic Pchlides a and b, which were prepared from Chlides a and b with an excess of 2,3-dichloro-5,6-dicyanobenzoquinone as described in Refs.…”

“…n-Octyl-␤-D-glucoside treatment of isolated prolamellar bodies was performed as described in Ref. 21. After solubilization, the assay mixtures were centrifuged, and the resulting supernatant and membrane fractions were analyzed separately as specified herein.…”

A Role for TIC55 as a Hydroxylase of Phyllobilins, the Products of Chlorophyll Breakdown during Plant Senescence

“…Two of them are involved in chlorophyll biosynthesis, i.e., CAO that catalyzes the oxidation of chlorophyll(ide) a to chlorophyll(ide) b (Tanaka et al, 1998), and PTC52 that has been proposed to carry out the same oxidation, but using protochlorophyllide a as substrate. However, an in vivo role of protochlorophyllide b has been questioned (Scheumann et al, 1999;Armstrong et al, 2000). The other two Rieske-type oxygenases are involved in chlorophyll degradation, i.e., PAO, which is responsible for porphyrin ring opening of pheophorbide a (Pruzinská et al, 2005), and TIC55.…”