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BIOENERGETICS AND PHOTOSYNTHESIS

Rubisco Activase Is Required for Optimal Photosynthesis in the Green Alga Chlamydomonas reinhardtii in a Low-CO₂ Atmosphere¹

Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803

This report describes a Chlamydomonas reinhardtii mutant that lacks Rubisco activase (Rca). Using the Ble^R (bleomycin resistance) gene as a positive selectable marker for nuclear transformation, an insertional mutagenesis screen was performed to select for cells that required a high-CO₂ atmosphere for optimal growth. The DNA flanking the Ble^R insert of one of the high-CO₂-requiring strains was cloned using thermal asymmetric interlaced-polymerase chain reaction and inverse polymerase chain reaction and sequenced. The flanking sequence matched the C. reinhardtii Rca cDNA sequence previously deposited in the National Center for Biotechnology Information database. The loss of a functional Rca in the strain was confirmed by the absence of Rca mRNA and protein. The open reading frame for Rca was cloned and expressed in pSL18, a C. reinhardtii expression vector conferring paromomycin resistance. This construct partially complemented the mutant phenotype, supporting the hypothesis that the loss of Rca was the reason the mutant grew poorly in a low-CO₂ atmosphere. Sequencing of the C. reinhardtii Rca gene revealed that it contains 10 exons ranging in size from 18 to 470 bp. Low-CO₂-grown rca1 cultures had a growth rate and maximum rate of photosynthesis 60% of wild-type cells. Results obtained from experiments on a cia5 rca1 double mutant also suggest that the CO₂-concentrating mechanism partially compensates for the absence of an active Rca in the green alga C. reinhardtii.

² These authors contributed equally to the paper.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.032078.

^* Corresponding author; e-mail btmoro{at}lsu.edu; fax 225-578-2597.

Received August 20, 2003; returned for revision September 2, 2003; accepted September 9, 2003.

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