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PLANT PHYSIOLOGY , Vol 105, Issue 2 545-554, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Cleavage of Chlorophyll-Porphyrin (Requirement for Reduced Ferredoxin and Oxygen)
S. Ginsburg, M. Schellenberg and P. Matile
Department of Plant Biology, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland
The chemical structures of some colorless catabolites that accumulate in
senescent leaves have been established recently (B. Krautler, B. Jaun, W.
Amrein, K. Bortlik, M. Schellenberg, P. Matile [1992] Plant Physiol Biochem
30: 333-346; W. Muhlecker, B. Krautler, S. Ginsburg, P. Matile [1993] Helv
Chim Acta 76: 2976-2980). Such studies suggest that oxygenolytic cleavage
of chlorophyll-porphyrin may occur by the action of a dioxygenase. We have
attempted to demonstrate such an enzyme activity and to explore the
requirements of the cleavage reaction in a reconstituted system of
chloroplast (Chlpl) components prepared from senescent rape (Brassica napus
L.) cotyledons. Intact senescent Chpls (also referred to as gerontoplasts)
contain small amounts of two fluorescent chlorophyll catabolites, Bn-FCC-1
and Bn-FCC-2, probably representing primary cleavage products. Upon the
incubation of Gpls in the presence of glucose-6-phosphate (Glc6P) or ATP,
these catabolites (predominantly FCC-1) were produced in organello. In a
reconstituted system of thylakoids and stroma fraction the FCCs
(predominantly FCC-2) were produced in the presence of ferredoxin (Fd) and
cofactors (NADPH, Glc6P) helping to keep Fd in the reduced state. Reduced
Fd could not be replaced by other electron donors, suggesting that the
putative dioxygenase requires Fd for the operation of its redox cycle.
Production of FCC-2 did not occur in the absence of oxygen and it was
inhibited by chelators of Fe2+. The contributions to the production of FCCs
from both parts of the reconstituted system, thylakoids and stroma, are
heat labile. The enzymic process in the thylakoids yields pheophorbide a,
the presumptive precursor of FCCs. However, native senescent thylakoids
could not be replaced as a "substrate" by free pheophorbide a. The stromal
enzyme appears to have an affinity for senescent thylakoids; thus, "loaded"
thylakoids capable of FCC production in the presence of Fd and cofactors
were obtained upon homogenization of senescent cotyledons in a medium
containing sorbitol and ascorbate. Such thylakoids were inactive if
prepared from mature green cotyledons. As senescence was induced, the
capacity to generate FCCs appeared and peaked when about half of the
chlorophyll had disappeared from the cotyledons. The effectiveness of a
relevant inhibitor showed that cytoplasmic protein synthesis was required
for inducing the catabolic machinery in the loaded thylakoids. Thylakoids
from mature Chlpls were ineffective as substrate of the stromal enzyme
prepared from Gpls. However, senescent thylakoids yielded FCCs if
challenged with stroma from either Chlpls or Gpls. Therefore, the stromal
part of the system is likely to be a constitutive enzyme, and the
pace-setting step of the pathway of chlorophyll breakdown seems to be
located in the thylakoids.
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