Progress 08/15/02 to 08/14/05
Outputs
The genomes of plants contain a high percentage of duplicated genes. This condition permits genes to evolve new functions, without losing old ones, and so it is a major force in plant adaptation. We discovered duplicate genes in maize for the enzyme coproporphyrinogen oxidase (CPX), which is required for the synthesis of heme and chlorophyll. We studied these duplicated enzymes gentically and biochemically to learn whether they had obtained divergent functions during the evolution if maize. Our studies show that the two genes encode almost identical, enzymatically active enzymes, but they play distinctive roles in plant metabolism, because the products of the genes are directed to different locations in the cell. The cpx1 and cpx2 genes of maize are a novel example of duplicated genes which have diverged by deletion and creation of protein targeting information. Chlorophyll and heme have central roles in energy metabolism. In plants, this biosynthetic step has been
reported to be compartmentalized exclusively in the plastids. In sequencing the maize cpx1 and cpx2 genes, we found that only cpx1 contains the targeting information to direct its product (CPX1) into the plastid. These sequences are absent from the cpx2 gene, but minor modifications of upstream sequences had created a new potential targeting signal, directing the CPX2 product to some other subcellular location. We undertook a broad range of experiments to examine the roles and evolution of CPX1 and CPX2 enzymes. We measured gene expression during the growth of maize and found that both genes are ubiquitously expressed, but expression of cpx1 is higher than that of cpx2 in leaf tissue. We also documented that both genes encode active enzymes by showing that either maize gene could replace a mutant coproporphyrinogen enzyme in yeast. To understand where and how the gene products are targeted in the cell, we fused the cpx1 and cpx2 genes to the Green Fluorescent Protein, and introduced
these gene fusions into maize cells. The fusion with cpx1 localized to the plastids, as expected. The cpx2-GFP fusion did not target plastids and appeared to localize to mitochondria. Using reverse genetics, we obtained a mutant that eliminated the expression of cpx1. The phenotype of this mutant provides the most compelling case for functional divergence between the two genes. Seedlings homozygous for a null mutation in the cpx1 gene completely lack chlorophyll and develop necrotic lesions in the light. However the mutant seedlings and callus cultures will grow in tissue culture in the dark, implying that they retain a capacity to produce heme. To understand how cpx2 evolved, we sequenced cpx genes from a variety of grasses, including distant and very close relatives of maize. We found that the cpx2 deletion arose recently (less than 14 MYA) and mitochondrial targeting information sequence is derived from the untranslated part of the gene by a series of very short
insertions/deletions of diurect rrepeated sequences, a genome feature that can be created or eliminated by replication slippage, misalignment in recombination, or the introduction and excision of transposable elements.
Impacts
The porphyrins are a family of molecules that play critical roles in creating and using energy in plants. These molecules are essential in plant growth. However, various porphyrins can be highly toxic to plants, so the synthesis of these compounds is under strict regulation. We have found that corn and its close relatives have a novel form of one of the porphyrin enzymes that is located in a different part of the cell. We are trying to learn how this unique aspect of porphyrin regulation contributes to the productivity of maize and to its ability to grow in different environments. This information could be important in breeding maize for optimal growth in various regions, and it may be a useful trait to transfer to other crop species as well.
Publications
- Pascale Williams, Kristine Hardeman, John Fowler, Carol Rivin 2005 Divergence of duplicated genes in maize: Evolution of contrasting targeting information for enzymes in the porphyrin pathway. Plant Journal, in press
- Pascale Williams, Kristine Hardeman, John Fowler, Carol Rivin. 2003. Re-targeting of a duplicated gene product and the evolution of novel gene function in maize. Abstract. Plant Genetics 2003. Am. Soc. Plant Biol.
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Progress 01/01/04 to 12/31/04
Outputs
Coproporphyrinogen oxidase (CPX or CPO) is an enzyme in the biosynthetic pathway leading to the production of heme and chlorophyll. These two molecules are of critical importance in plants: Chlorophyll is the key component for the use of sunlight as an energy source in photosynthesis and heme is a required co-factor for a large number of enzymes, and is of particular importance in the mitochondria. Previous studies have CPX exclusively in the plastid compartment of the plant cell. We have found that in maize there is a second CPX gene (CPX2) which encodes an enzyme without any plastid-targeting capacity. In transgenic experiments, we showed that this second CPX enzyme appears to localize in the mitochondria. For our on-going analysis of this unusual gene, we have examined close relatives of maize and found that all the wild Zea and Tripsacum species also carry a second CPX gene that is missing plastid-targeting information, indicating that this novel CPX form has been
retained for at least 14 million years of maize evolution. However, we cannot find evidence for a CPX-2 gene in other closely related taxa. We have expressed CPX1 and CPX2 in vitro, and found that each encodes an active enzyme. Antibodies raised to these purified proteins are being used to look at the cell localization and tissue specific expression of the enzymes. Two strategies are being used to discover the physiological importance of CPX2. The first strategy is to obtain a maize line carrying a mutation in this gene. We are looking for this using a reverse-genetic strategy. The second strategy is to transform Nicotiana, a plant lacking CPX2, with the maize gene, and seeing how this changes the ability of the plant to divide its resources between heme and chlorophyll production under conditions of plant stress.
Impacts
The porphyrins are a family of molecules that play critical roles in creating and using energy in plants. These molecules are essential in plant growth. However, various porphyrins can be highly toxic to plants, so the synthesis of these compounds is under strict regulation. We have found that corn and its close relatives have a novel form of one of the porphyrin enzymes that is located in a different part of the cell. We are trying to learn how this unique aspect of porphyrin regulation contributes to the productivity of maize and to its ability to grow in different environments. This information could be important in breeding maize for optimal growth in various regions, and it may be a useful trait to transfer to other crop species as well.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
This research concerns the roles of duplicate maize genes encoding the enzyme coproporphyrinogen oxidase (coprogen oxidase), an enzyme in the synthesis of chlorophyll and heme. In plants studied so far, this enzyme activity has been found exclusively in the plastids. However, our studies of the maize coprogen oxidase gene sequences predict that one protein product localizes to the plastids (cpx1), while the other is targeted to the mitochondria (cpx2). This prediction is supported by the localization of CPX-GFP fusion proteins expressed as trans-genes. Mutants were isolated in each gene, and these have different phenotypes, indicating that the genes play different biological roles. The hypotheses we are currently testing address the overall question of whether duplication of the coprogen oxidase genes during maize evolution has led to creation of a novel metabolic function in the porphyrin biosynthetic pathway. Our specific research goals and progress are: 1)
Determine whether mitochondrially localized CPX2 protein has coprogen oxidase enzymatic activity. We have cloned both the cpx2 and cpx2 genes into yeast expression vectors and used these genes to complement a yeast strain that lacks coprogen oxidase activity. So, both genes encode an active enzyme. We are using western analysis to look for the presence of CPX1 in purified chloroplast proteins and the presence of CPX2 in protein extracts from mitochondria. We are using null mutants of CPX1 as controls to confirm that bands seen in mitochondrial extracts are indeed cpx2 gene products. Our preliminary data shows that extracts enriched for mitochondria are enriched for a protein detected by anti-CPX serum that is slightly larger than the cross-reacting protein found in purified chloroplasts. This size difference is predicted by gene sequences, again supporting the hypothesis. We still need to confirm the identity of this protein by using cpx2 null mutants as a negative control. We also
want to demonstrate CPX2 enzyme activity in purified mitochondria. 2) Evaluate the phenotype of single and double mutants of cpx1 and cpx2 to determine whether CPX2 participates in heme biosynthesis or acts to protect against phototoxic intermediates in the mitochondrion. We created several different double mutant segregating lines in the summer maize nursery. We will cross these to confirm that the phenotypes segregate with the different cpx alleles. 3) Test for the presence of mitochondrial coprogen oxidase in species other than maize, to learn if this is a trait that has been retained / selected in closely related, and more distantly related grasses. Using PCR , we have analyzed genomic DNA from various cereal species, and from the Zea and Tripsacum genera that are closely related to maize. Only cpx1-type genes were found in all monocots, but Zea and Tripsacum species carry cpx2 form as well, indicating that both genes present in the tetraploid founder-species of this group of
plants. We have sequenced several of these genes and confirmed that the cpx1-types have chloroplast-targeting signals while the cpx2-types are consistent with mitochondrial targeting.
Impacts
The porphyrins are a family of molecules that play critical roles in creating and using energy in plants. These molecules are essential in plant growth. However, various porphyrins can be highly toxic to plants, so the synthesis of these compounds is under strict regulation. We have found that corn and its close relatives have a novel form of one of the porphyrin enzymes that is located in a different part of the cell. We are trying to learn how this unique aspect of porphyrin regulation contributes to the productivity of maize and to its ability to grow in different environments. This information could be important in breeding maize for optimal growth in various regions, and it may be a useful trait to transfer to other crop species as well.
Publications
- Pascale Williams, Kristine Hardeman, John Fowler, Carol Rivin. 2003. Re-targeting of a duplicated gene product and the evolution of novel gene function in maize. Abstract. Plant Genetics 2003. Am. Soc. Plant Biol.
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Progress 08/15/02 to 11/14/02
Outputs
This is a new project that is focused on duplicate genes (CPX1 and CPX2) encoding the enzyme coproporphyrinogen III oxidase (coprogen) in maize. Coprogen is required for the synthesis of chlorophyll and heme, and it is has previously been found exclusively in the plastid. However, experiments in our lab suggested that the CPX2 product is targeted to the mitochondrion instead. Our overall goal is to understand the role of CPX2, since coprogen has no obvious substrates in the mitochondrion. We are currently working towards these four specific aims: 1. Completion and confirmation the sequence of the CPX2 gene and cDNA. From the genomic clone, we knew only the first 5 exons of CPX2. We isolated and sequenced a complete cDNA clone. Further RT-PCR and genomic PCR were used to confirm the sequences. 2. Demonstrating the existance of a CPX2 protein in the mitochondria of maize. Using antisera to barley coprogen, we performed western blot analysis on proteins from purified
plastids and partially purified mitochondria. The antiserum detected a single band of approximately 39 kDa in purified plastid proteins and in stromal subfractions. The partially purified mitochondrial proteins show an additional band at approximately 35 kDa. These are the predicted sizes of CPX1 (plastid) and CPX2 (mitochondria). 3. Functional testing of the CPX2 protein. CPX2 appears to encode a bonafide coprogen enzyme. To test that enzymatically, we are in the process of complementing a S. cerevisiae coprogen mutant with the maize CPX1 and CPX2 enzymes. We have constructed yeast plasmids carrying the maize CPX cDNA sequences driven by a yeast promoter. If these are functional enzymes, the yeast coprogen mutant should be able to grow in the absence of added heme. 4. Isolating mutants of CPX2 and studying their phenotypes. To determine what role CPX2 may play, we are analyzing putative transposon insertion mutants both molecularly and by phenotype. Several of these mutant families
display segregation for growth defects, including extreme stunting and slow growth. We are testing whether these characters co-segregate with the mutant CPX2 allele.
Impacts
We hypothesize that the unique mitochondrially-targeted CPO enzyme of maize plays a protective role, by scavenging and detoxifying tetrapyrrole intermediates in high light environments. This maize gene may then be useful in maintaining crop productivity of other species as well.
Publications
- No publications reported this period
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