Progress 10/01/05 to 09/30/07
Outputs
There were three goals of the project: 1) to establish a biochemical assay for the cyclase that is dependent on Crd1 and use reverse genetics to establish the role of Crd1 in the cyclase reaction, 2) to distinguish the two isoforms of Chlamydomonas in terms of structure, localization or function, and 3) establish whether xantha-l or viridis-k mutants of barley correspond to the Chlamydomonas CRD1 gene. In the last progress report, we had described the use of antisense technology in Arabidopsis to show that the Arabidopsis homologue of Chlamydomonas Crd1 was indeed required for the synthesis of divinyl protochlorophyllide. The knock-down plants accumulate the substrate of the reaction, Mg-Protoporphyrin IX monomethylester when they are fed delta-aminolevulinic acid. In collaboration with Mats Hansson who visited UCLA to work on the cyclase, we established a biochemical assay for the cyclase that was dependent on both soluble and membrane fractions from barley plastids.
The goal is to use this assay to identify other components of the reaction by biochemical fractionation. We also used co-immunoprecipitation methods to pull-down associated proteins. Thus far, neither approach has yielded a specific reaction partner. A candidate component has now been suggested to us by genomic and reverse genetic approaches in the Arabidopsis system in the laboratory of Poul Erik Jensen. We are generating tools (i.e. global antibodies) that would allow us to assess the role of this candidate in the cyclase reaction. The ability to move between model organisms should allows us to take advantage of existing mutants and materials, and the parallel approach should solidify the conclusions. Regarding the structure of the two isoforms of Chlamydomonas, we initiated a collaboration with Brian Fox on this aspect. He is an expert on di-iron enzymes and is involved with a large structural genomics project. The Arabidopsis, barley and Synechocystis enzymes have been included in
the project as well to increase the probability of success. Cell fractionation methods have been developed to separate the envelope and thylakoid membranes of Chlamydomonas. However, no difference in the localization of Crd1 vs. Cth1 was noted. We are presently using immunogold labeling techniques to validate the biochemical work, at which point the results will be prepared for publication. Goal 3 has been completed. Sequence analysis of xantha-l alleles in barley confirmed that the plants carried mutations in the Crd1 homolog.
Impacts
The cyclase enzyme, which is highly conserved, catalyzes a key step in Chlorophyll biosynthesis and is unique to aerobic photosynthetic organisms. We established previously that this enzyme is also sensitive to iron nutrition and may in fact play a role in signaling iron status to the photosynthetic apparatus. Elucidation of the structure and distinguishing the composition of this enzyme is therefore important for understanding the molecular basis of iron-deficiency chlorosis and the modification of the light harvesting in an iron poor environment.
Publications
- Rzeznicka, K., Walker, C.J., Westergren, T., Kannangara, G.C., von Wettstein, D., Merchant, S., Gough, S.P., Hansson, M. (2005) Xantha-l encodes one of at least three subunits of the aerobic Mg-protoporphyrin IX monomethyl ester cyclase in the chlorophyll biosynthetic pathway. Proc. Natl. Acad. Sci. USA 102: 5886-5891.
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Progress 09/15/02 to 09/14/07
Outputs
OUTPUTS: We used antisense technology to establish that the Arabidopsis homolog of the Chlamydomonas CRD1 gene was indeed required for the synthesis of divinyl protochlorophyllide. The knock-down plants accumulate the substrate of the reaction, Mg-Protoporphyrin IX monomethylester when they are fed delta-aminolevulinic acid. The Arabidopsis gene was designated CHL27. The two Chlamydomonas proteins are therefore called alternatively CHL27A for CRD1 and CHL27B for CTH1. The barley mutant xantha-l and viridis-k mutants were hypothesized to be defective in the cyclase. Although the plants were not missing the protein, sequence analysis in the group of Mats Hansson confirmed that the xantha-l locus corresponded to the CHL27 gene. We collaborated with Prof. Mats Hansson and Prof. Poul Erik Jensen in order to identify other components of the reaction but these approaches were not successful. We generated anti-peptide antibodies against a putative component, called NTRC. The goal was to
prepare a global antibody that could be used in both model organisms as well as crop species, especially barley where mutants of chlorophyll biosynthesis are known. However, the antibodies were not specific and therefore not useful. We therefore focussed our attention on the localization of the CRD1 subunit of the enzyme for which excellent antibodies are available that cross-react with the protein from algae, bacteria and plants. These antibodies have been made available to researchers world-wide. We noted that Arabidopsis CHL27 localizes to both thylakoid and envelope membranes. This suggests that the pathway for chlorophyll biosynthesis is branched at the level of sub-organellar compartmentation. We developed methods for fractionating envelope and thylakoid membranes of Chlamydomonas and found that the protein also shows dual localization in this system as well. Interestingly, the two isoforms appear to have non-identical function based on the phenotype of the crd1 strain that is
missing only one isoform. Furthermore, under various nutritional and environmental stress situations, the two genes are differently expressed. One student has been trained on this project, Mr. Michael Allen, and he will complete his Ph.D. in Biochemistry in June 2008.
PARTICIPANTS: Sabeeha Merchant, principal investigator, designed experiments, analyzed data, supervised personnel and prepared publications Michael Allen, Ph.D. student, designed and executed experiments, analyzed data Tomas Westergren, post-doctoral fellow, executed experiments Stephen Tottey, post-doctoral fellow, executed experiments, analyzed data Mats Hansson, collaborator, designed experiments, analyzed data, supervised personnel and prepared publications Poul Erik Jensen, collaborator, designed experiments, analyzed data, supervised personnel and prepared publications Mr. Allen will obtain a Ph.D. in June 2008 with expertise in plant biochemistry.
TARGET AUDIENCES: The target audience for this work is the scientific community. The synthesis and degradation of chlorophyll is of great interest to a broad range of researchers because of the importance of chlorophyll for energy capture and the potential toxicity of tetrapyrrole products for humans.
PROJECT MODIFICATIONS: None
Impacts
The discovery of one component of the reaction that converts Mg-protoporphyrin IX monomethylester to divinylprotochlorophyllide is a major result of this project. The existence of this enzyme has been known for a long time but its identity was unknown. Furthermore, this enzyme was known to be a target for iron-deficiency. The discovery that CHL27 is a di-iron enzyme allowed us to provide a molecular rationale for the impact of iron-deficiency on chlorophyll biosynthesis. The assignment of the CHL27 gene to the xantha-l locus in barley was a second outcome of the project. The occurrence of multiple homologs of CHL27 in cyanobacteria and linkage to genes encoding other oxygen-dependent enzymes in tetrapyrrole metabolism, such as heme oxygenase and a short chain dehydrogenase suggests that there are aspects of the regulation of chlorophyll biosynthesis in response to the environment that are still not well-understood.
Publications
- Tottey, S., Block, M., Allen, M., Westergren, T., Albrieux, C., Scheller, H.V., Merchant, S., Jensen, P.E. (2003) Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide, Proc. Natl. Acad. Sci. USA, 100:16119-24.
- Rzeznicka, K., Walker, C.J., Westergren, T., Kannangara, G.C., von Wettstein, D., Merchant, S., Gough, S.P., Hansson, M. (2005) Xantha-l encodes one of at least three subunits of the aerobic Mg-protoporphyrin IX monomethyl ester cyclase in the chlorophyll biosynthetic pathway. Proc. Natl. Acad. Sci. USA 102: 5886-5891.
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Progress 10/01/02 to 09/30/03
Outputs
We had proposed that a candidate di-iron enzyme (Crd1) that was identified in Chlamydomonas as one target of copper-deficiency responses corresponded to the aerobic oxidative cyclase in chlorophyll biosynthesis. A key objective was the direct reverse genetic demonstration of this point. This was accomplished through an antisense approach in Arabidopsis in collaboration with Poul Erik Jensen. Specifically, while ALA-fed wild-type plants accumulate protochlorophyllide upon prolonged incubation in the dark, plants accumulating less Crd1 accumulate MgProtoporphyrin IX monomethylester instead. The Arabidopsis gene was named CHL27 to recognize its function in the pathway. Interestingly, CHL27 localizes to both thylakoid and envelope membranes, which suggests that chlorophyll biosynthesis is branched at the level of compartmentalization. We hypothesize that individual chlorophyll proteins may receive their chlorophyll cofactors via different pathways and mechanisms.
Chlamydomonas contains two versions of CHL27 - Crd1 and Cth1, which display reciprocal patterns of expression with respect to copper nutritional status. Crd1 accumulates to greater abundance in -Cu cells and Cth1 in +Cu cells. The fact that crd1 mutants display copper conditional loss of photosystem I and its associated antenna indicates a specialization of function, perhaps at the level of sub-chloroplast location of the enzyme. This hypothesis is presently being tested by comparison of Crd1 vs. Cth1 localization in thylakoid and envelope membranes prepared from wild-type, crd1 and sct1 strains of Chlamydomonas grown in +Cu vs. -Cu medium. In the future, we would use a reverse genetic approach to assess Cth1 function. The barley xantha and viridis mutants were analyzed by immunoblot analyses and determined to accumulate CHL27 at levels comparable to wild-type plants. It is concluded that the mutants may carry point mutations in CHL27 or they may be affected in proteins that interact
with CHL27, but they do not represent a deletion or nonsense mutation in barley CHL27.
Impacts
Through our work we have not only identified one part of an elusive enzyme, namely the aerobic oxidative cyclase, but we have also identified the protein whose activity is sensitive to iron nutritional state. The ongoing work on the role of differently localized forms of this enzyme could reveal multiple mechanisms for assembly of individual chlorophyll proteins.
Publications
- Tottey, S., Block, M., Allen, M., Westergren, T., Albrieux, C., Scheller, H.V., Merchant, S., Jensen, P.E. (2003) Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide, Proc. Natl. Acad. Sci. USA, 100:16119-24.
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