Abstract:
Coproporphyrinogen III oxidase (CPX) is an enzyme involved in the biosynthesis
of tetrapyrroles, catalyzing the oxidative decarboxylation of coproporphyrinogen III to
protoporphyrinogen IX. In bacteria and yeast this enzyme is cytosolic, in animals it is
mitochondrial, and in each it functions to produce heme. However, in all plants
investigated, the initial steps of tetrapyrrole production, including those catalyzed by
CPX, occur exclusively in the chloroplast stroma. Protoporphyrinogen IX is modified in
the plastid envelope to produce chlorophyll and heme, or channeled to the mitochondria
for heme production.
The maize genome has two CPX-encoding genes: CPX-1 has a putative amino-terminal
chloroplast transit peptide, while CPX-2 has an amino-terminal sequence that is
more consistent with mitochondrial transit peptides. Both genes are transcribed in all
tissues tested but they have distinct expression patterns. The transcription level of cpx-1
is greater than that of cpx-2 in photosynthetic tissues. Neither gene shows light-dependent
expression.
I have attempted to address the possibility that the two CPX-encoding genes have
different roles in maize, and that their products have different cellular locations. One
approach was the identification and characterization of cpx mutants to observe whether
mutants of cpx-1 and cpx-2 have different phenotypes. A transposable element-disrupted
cpx-1 allele co-segregates with homozygous dek seed and yellow-lethal seedling
phenotypes, consistent with a tetrapyrrole deficiency. In collaboration with Pioneer
HiBred Intl., we identified additional cpx mutants. A plant with a cpx-2 mutant allele
appears to develop normally as a homozygote; cpx-1 may complement this deficiency.
Another approach was the immunological identification of CPX in protein preparations
from isolated chloroplasts and mitochondria. CPX was detected in both the chloroplasts
and mitochondria of wild-type plants; however, mitochondrial CPX was not detected in
cpx2-578 homozygotes. A third approach was to determine whether the amino-terminal
sequence of CPX-2 would mobilize a fusion GFP protein into mitochondria. Maize
epidermal cells expressing this construct have mitochondrial-sized foci of GFP
localization with some general cytoplasmic background staining. The data supports the
theory that CPX-1 is chloroplast-localized and participates in tetrapyrrole biosynthesis in
that location. CPX-2 is the first mitochondrial-localized CPX enzyme identified in a plant
and may function in tetrapyrrole production or the decontamination of excess
coproporphyrinogen III.
