Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii

Abstract

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.

Figure 1.

Diagram of linearized plasmids used for insertional mutagenesis. Relevant restriction enzyme sites are shown. Arrows indicate the approximate positions of specific primers used for TAIL-PCR.

Figure 2.

Outline of primary and secondary screening procedures for the isolation of mutants with photosynthesis-related phenotypes. VLL, 3 μmol photons m⁻² s⁻¹; LL, 80 μmol photons m⁻² s⁻¹; HL, 500 μmol photons m⁻² s⁻¹.

Figure 3.

DNA gel-blot analysis of insertional mutants. Arrows indicate bands corresponding to endogenous RBCS2 sequences, and asterisks indicate mutants containing multiple ble insertions. Size standards are shown to the left. A, Mutants generated using pSP124S. Genomic DNA was digested with NcoI, and the probe was a XbaI/BamHI fragment from pSP124S. B, Mutants generated using pMS188. Genomic DNA was digested with NheI, and the probe was a NheI/KpnI fragment from pMS188.

Figure 4.

Agarose gel analysis of tertiary TAIL-PCR products from 39 insertional lines. Size standards are shown to the left.