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Analysis of coproporphyrinogen III oxidase in maize : the genes, their expression, and the localization of their products

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Graduation date: 2000Coproporphyrinogen III oxidase (CPX) is an enzyme involved in the biosynthesis\ud of tetrapyrroles, catalyzing the oxidative decarboxylation of coproporphyrinogen III to\ud protoporphyrinogen IX. In bacteria and yeast this enzyme is cytosolic, in animals it is\ud mitochondrial, and in each it functions to produce heme. However, in all plants\ud investigated, the initial steps of tetrapyrrole production, including those catalyzed by\ud CPX, occur exclusively in the chloroplast stroma. Protoporphyrinogen IX is modified in\ud the plastid envelope to produce chlorophyll and heme, or channeled to the mitochondria\ud for heme production.\ud The maize genome has two CPX-encoding genes: CPX-1 has a putative amino-terminal\ud chloroplast transit peptide, while CPX-2 has an amino-terminal sequence that is\ud more consistent with mitochondrial transit peptides. Both genes are transcribed in all\ud tissues tested but they have distinct expression patterns. The transcription level of cpx-1\ud is greater than that of cpx-2 in photosynthetic tissues. Neither gene shows light-dependent\ud expression.\ud I have attempted to address the possibility that the two CPX-encoding genes have\ud different roles in maize, and that their products have different cellular locations. One\ud approach was the identification and characterization of cpx mutants to observe whether\ud mutants of cpx-1 and cpx-2 have different phenotypes. A transposable element-disrupted\ud cpx-1 allele co-segregates with homozygous dek seed and yellow-lethal seedling\ud phenotypes, consistent with a tetrapyrrole deficiency. In collaboration with Pioneer\ud HiBred Intl., we identified additional cpx mutants. A plant with a cpx-2 mutant allele\ud appears to develop normally as a homozygote; cpx-1 may complement this deficiency.\ud Another approach was the immunological identification of CPX in protein preparations\ud from isolated chloroplasts and mitochondria. CPX was detected in both the chloroplasts\ud and mitochondria of wild-type plants; however, mitochondrial CPX was not detected in\ud cpx2-578 homozygotes. A third approach was to determine whether the amino-terminal\ud sequence of CPX-2 would mobilize a fusion GFP protein into mitochondria. Maize\ud epidermal cells expressing this construct have mitochondrial-sized foci of GFP\ud localization with some general cytoplasmic background staining. The data supports the\ud theory that CPX-1 is chloroplast-localized and participates in tetrapyrrole biosynthesis in\ud that location. CPX-2 is the first mitochondrial-localized CPX enzyme identified in a plant\ud and may function in tetrapyrrole production or the decontamination of excess\ud coproporphyrinogen III

Year: 1999
OAI identifier: oai:ir.library.oregonstate.edu:1957/32976
Provided by: ScholarsArchive@OSU
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