Cargando…
In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis
BACKGROUND: Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg che...
Autores principales: | , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2006
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1578556/ https://www.ncbi.nlm.nih.gov/pubmed/16953878 http://dx.doi.org/10.1186/1746-4811-2-15 |
_version_ | 1782130300542779392 |
---|---|
author | Sawers, Ruairidh JH Farmer, Phyllis R Moffett, Peter Brutnell, Thomas P |
author_facet | Sawers, Ruairidh JH Farmer, Phyllis R Moffett, Peter Brutnell, Thomas P |
author_sort | Sawers, Ruairidh JH |
collection | PubMed |
description | BACKGROUND: Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of ChlI, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through in vitro characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in Nicotiana benthamiana for rapidly assaying mutant variants of the maize CHLI protein in vivo. RESULTS: Transient expression of mutant maize ChlI alleles in N. benthamiana resulted in the formation of chlorotic lesions within 4 d of inoculation. Immunoblot analyses confirmed the accumulation of maize CHLI protein suggesting that the chlorosis observed resulted from an interaction between maize CHLI and endogenous components of the N. benthamiana chlorophyll biosynthetic pathway. On the basis of this assay, PCR-based cloning techniques were used to rapidly recombine polymorphisms present in the alleles studied allowing confirmation of causative lesions. A PCR-based mutagenesis was conducted and clones assayed by transient expression. A number of novel allelic variants of maize ZmChlI were generated and analyzed using this assay, demonstrating the utility of this technique for fine mapping. CONCLUSION: Transient expression provides a convenient, high-throughput, qualitative assay for functional variation in the CHLI protein. Furthermore, we suggest that the approach used here would be applicable to the analysis of other plastid-localized proteins where gain-of-function mutations will result in readily observable mutant phenotypes. |
format | Text |
id | pubmed-1578556 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-15785562006-09-27 In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis Sawers, Ruairidh JH Farmer, Phyllis R Moffett, Peter Brutnell, Thomas P Plant Methods Methodology BACKGROUND: Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of ChlI, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through in vitro characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in Nicotiana benthamiana for rapidly assaying mutant variants of the maize CHLI protein in vivo. RESULTS: Transient expression of mutant maize ChlI alleles in N. benthamiana resulted in the formation of chlorotic lesions within 4 d of inoculation. Immunoblot analyses confirmed the accumulation of maize CHLI protein suggesting that the chlorosis observed resulted from an interaction between maize CHLI and endogenous components of the N. benthamiana chlorophyll biosynthetic pathway. On the basis of this assay, PCR-based cloning techniques were used to rapidly recombine polymorphisms present in the alleles studied allowing confirmation of causative lesions. A PCR-based mutagenesis was conducted and clones assayed by transient expression. A number of novel allelic variants of maize ZmChlI were generated and analyzed using this assay, demonstrating the utility of this technique for fine mapping. CONCLUSION: Transient expression provides a convenient, high-throughput, qualitative assay for functional variation in the CHLI protein. Furthermore, we suggest that the approach used here would be applicable to the analysis of other plastid-localized proteins where gain-of-function mutations will result in readily observable mutant phenotypes. BioMed Central 2006-09-05 /pmc/articles/PMC1578556/ /pubmed/16953878 http://dx.doi.org/10.1186/1746-4811-2-15 Text en Copyright © 2006 Sawers et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Sawers, Ruairidh JH Farmer, Phyllis R Moffett, Peter Brutnell, Thomas P In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis |
title | In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis |
title_full | In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis |
title_fullStr | In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis |
title_full_unstemmed | In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis |
title_short | In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis |
title_sort | in planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1578556/ https://www.ncbi.nlm.nih.gov/pubmed/16953878 http://dx.doi.org/10.1186/1746-4811-2-15 |
work_keys_str_mv | AT sawersruairidhjh inplantatransientexpressionasasystemforgeneticandbiochemicalanalysesofchlorophyllbiosynthesis AT farmerphyllisr inplantatransientexpressionasasystemforgeneticandbiochemicalanalysesofchlorophyllbiosynthesis AT moffettpeter inplantatransientexpressionasasystemforgeneticandbiochemicalanalysesofchlorophyllbiosynthesis AT brutnellthomasp inplantatransientexpressionasasystemforgeneticandbiochemicalanalysesofchlorophyllbiosynthesis |