Truncated Photosystem Chlorophyll Antenna Size in the Green Microalga Chlamydomonas reinhardtii upon Deletion of the TLA3-CpSRP43 Gene

The truncated light-harvesting antenna size3 (tla3) DNA insertional transformant of Chlamydomonas reinhardtii is a chlorophyll-deficient mutant with a lighter green phenotype, a lower chlorophyll (Chl) per cell content, and higher Chl a/b ratio than corresponding wild-type strains. Functional analyses revealed a higher intensity for the saturation of photosynthesis and greater light-saturated photosynthetic activity in the tla3 mutant than in the wild type and a Chl antenna size of the photosystems that was only about 40% of that in the wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blot analyses showed that the tla3 strain was deficient in the Chl a/b light-harvesting complex. Molecular and genetic analyses revealed a single plasmid insertion in chromosome 4 of the tla3 nuclear genome, causing deletion of predicted gene g5047 and plasmid insertion within the fourth intron of downstream-predicted gene g5046. Complementation studies defined that gene g5047 alone was necessary and sufficient to rescue the tla3 mutation. Gene g5047 encodes a C. reinhardtii homolog of the chloroplast-localized SRP43 signal recognition particle, whose occurrence and function in green microalgae has not hitherto been investigated. Biochemical analysis showed that the nucleus-encoded and chloroplast-localized CrCpSRP43 protein specifically operates in the assembly of the peripheral components of the Chl a/b light-harvesting antenna. This work demonstrates that cpsrp43 deletion in green microalgae can be employed to generate tla mutants with a substantially diminished Chl antenna size. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.There is current interest and ongoing efforts to renewably generate fuel and chemical products for human consumption through the process of microalgal photosynthesis. Such bioproducts include H₂ (Hankamer et al., 2007; Melis, 2007), biofuel and chemical molecules (Hu et al., 2008; Greenwell et al., 2010; Mata et al., 2010; Melis, 2012), antigens (Dauvillée et al., 2010; Michelet et al., 2011), and high-value biopharmaceuticals (Mayfield et al., 2007). For this effort, sunlight energy conversion in photosynthesis must take place with the utmost efficiency, as this would help to make renewable fuel and chemical processes economically feasible. In plants and algae, the solar energy conversion efficiency of photosynthesis is thus a most critical factor for the economic viability of renewable fuel and chemical production (Melis, 2009, 2012).Green microalgae and other photosynthetic systems tend to develop large arrays of light-harvesting complexes, especially when cultivated under high-density mass culture conditions. This physiological response of the cells reflects an effort to absorb as much sunlight as possible as they compete in a light-limited environment (Kirk, 1994). However, in mass culture with cells possessing large chlorophyll (Chl) antennae, cells at the surface of the reactor would absorb incident sunlight (intensity of 2,500 μmol photons m^?2 s^?1) with rates that far exceed the capacity of the photosynthetic apparatus to utilize them (light saturation of photosynthesis occurs at less than 500 μmol photons m^?2 s^?1). The excess absorbed sunlight energy is dissipated via a process of nonphotochemical quenching to prevent photodamage and photoinhibition phenomena at the thylakoid membrane level (for review, see Müller et al., 2001).It has been shown that high-density cultures of microalgae with a truncated Chl antenna size are photosynthetically more productive under bright sunlight due to the elimination of overabsorption and wasteful dissipation of excess energy (Nakajima and Ueda, 1997, 1999; Melis et al., 1999; Polle et al., 2002, 2003; Melis, 2009). Identification of genes that confer a permanently truncated light-harvesting antenna size phenotype in plants and algae is thus of interest, as they could be applied in efforts to improve solar-to-product conversion efficiencies (Mitra and Melis, 2008; Melis, 2009; Ort et al., 2011). To this end, and to better understand the genetic mechanism that defines the size of the light-harvesting antenna in green microalgae, and also in an effort to generate truncated light-harvesting antenna size (tla) mutants, we generated and screened a library of Chlamydomonas reinhardtii DNA insertional mutagenesis strains. This work presents a molecular, genetic, and physiological analysis of one of these mutants, termed tla3, which exhibited a stably truncated light-harvesting Chl antenna size. The corresponding TLA3 gene was cloned and found to encode a homolog of the chloroplast signal recognition particle protein CpSRP43. Detailed functional analysis revealed that the phenotype of the tla3-ΔCpSRP43 mutant in C. reinhardtii entailed substantial reductions of the light-harvesting Chl antenna size. Accordingly, the cpsrp43 mutant phenotype and the CrCpSRP43 gene can be employed in C. reinhardtii, and possibly other green microalgae and plants, as a tool by which to truncate the Chl antenna size without affecting the function of the photosystems or thylakoid membrane electron transport properties of the chloroplast.