Mutants of Chlamydomonas reinhardtii resistant to very high light

A collection of very high-light-resistant, VHL(R), mutants that grow in 5% CO₂ under 1500 to 2000 μmol photons m^-2 s^-1 has been isolated from both the CC-125 wild-type strain of Chlamydomonas reinhardtii and from the chloroplast psbA mutant A251L* with an Ala to Leu substitution in the D1 protein. Neither of the parental strains survives long-term exposure to very high light intensities (VHL; above 1500 μmol photons m^-2 s^-1). This paper characterizes the genetic, physiological and biochemical properties of two nuclear mutants isolated from wild-type (S4 and S9), and two derived from A251L* (L5 and L30). All four mutants grow photoautotrophically in 5% CO₂ at rates similar to their respective parental strains under high light (HL; 600 μmol photons m^-2 s^-1) and maintain high growth rates under VHL. Total chlorophyll per biomass, or per cell, declines in both mutant and parental strains grown in HL compared with LL, and decreases further in mutants grown under VHL. All four VHL(R) mutants have lower light and CO₂-saturated rates of photosynthetic O₂ evolution per total chlorophyll than wild-type or A251L* under low light (LL; 70 μmol photons m^-2 s^-1) or HL. The VHL(R) mutants S4 and S9 show greater nonphotochemical fluorescence quenching than wild-type when grown and measured under HL or VHL. In contrast, A251L*, L5 and L30 exhibit low nonphotochemical quenching under all conditions. Although xanthophyll pigments, including lutein, are elevated on a chlorophyll basis in both wild-type and S9 cells grown in HL compared to LL, they are unchanged on a biomass basis. The de-epoxidation state is very low even in HL. Preliminary analyses suggest that other VHL(R) mutants behave similarly. Evidently, enhanced photoprotection via the xanthophyll cycle is not involved in survival of several different VHL(R) mutants under saturating light and CO₂. Photosystem II properties and electron transfer rates in all VHL(R) mutants grown in 5% CO₂ under LL do not differ from those of their respective parental strains. These experiments also demonstrate that slow photosystem II electron transfer in strain A251L* due to altered D1 structure, which is present in L5 and L30 isolated from this strain, does not preclude selection of VHL(R) mutants. The ability of these mutants to survive under VHL in 5% CO₂ evidently involves processes other than photoprotection via the xanthophyll cycle and resistance to photodamage via alterations in D1 function.