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BIOENERGETICS AND PHOTOSYNTHESIS

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C₄ Carbonic Anhydrase in Flaveria^1,[C],[OA]

Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia (S.K.T., S.G.T., N.G.F.V.); and School of Biomedical, Biomolecular, and Chemical Sciences, University of Western Australia, Crawley, Western Australia 6009, Australia (M.L.)

C₄ photosynthesis has evolved multiple times from ancestral C₃ species. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO₂ and is involved in both C₃ and C₄ photosynthesis; however, its roles and the intercellular and intracellular locations of the majority of its activity differ between C₃ and C₄ plants. To understand the molecular changes underlying the evolution of the C₄ pathway, three cDNAs encoding distinct β-CAs (CA1, CA2, and CA3) were isolated from the leaves of the C₃ plant Flaveria pringlei. The phylogenetic relationship of the F. pringlei proteins with other embryophyte β-CAs was reconstructed. Gene expression and protein localization patterns showed that CA1 and CA3 demonstrate high expression in leaves and their products localize to the chloroplast, while CA2 expression is low in all organs examined and encodes a cytosolic enzyme. The roles of the F. pringlei enzymes were considered in light of these results, other angiosperm β-CAs, and Arabidopsis (Arabidopsis thaliana) "omics" data. All three F. pringlei CAs have orthologs in the closely related C₄ plant Flaveria bidentis, and comparisons of ortholog sequences, expression patterns, and intracellular locations of their products indicated that CA1 and CA2 have maintained their ancestral role in C₄ plants, whereas modifications to the C₃ CA3 gene led to the evolution of the CA isoform that catalyzes the first step in the C₄ photosynthetic pathway. These changes included the loss of the chloroplast transit peptide and an increase in gene expression, which resulted in the high levels of CA activity seen in the cytosol of C₄ mesophyll cells.

² Present address: ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia.

³ Present address: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Martha Ludwig (mludwig{at}cyllene.uwa.edu.au).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.137513

^* Corresponding author; e-mail mludwig{at}cyllene.uwa.edu.au.

Received February 21, 2009; accepted May 11, 2009; published May 15, 2009.