Genetic control of light-independent chlorophyll biosynthesis in C. reinhardtii

Elena Chekunova and Nataliya Savelieva

Department of Genetics of St.-Petersburg State University, St.-Petersburg, 198504, Russia

The green alga C. reinhardtii (Chlamy), in contrast to angiosperms, is capable of synthesizing chlorophyll in the dark. Well known light control step in CBS is a reduction of protochlorophyllide (PD) to chlorphyllide (CD). In Chlamy the light-independent PD to CD conversion involves at least 3 chloroplast and 7 nuclear genes. Mutation in these genes results in "yellow" phenotype of mutant cells (loss chlorophylls and accumulation of PD in the dark). Other light controlled step in CBS vas uncovered by allelic mutations: lts3 and brc-1 in nuclear gene Lts3. The Lts3-mutant cells are green in the light, but accumulate protoporphyrin IX (PP), Mg-PP and PD and formed orange colonies under dark-growing conditions. The LTS3 gene product must affect yet unknown step in dark CBS prior to PD reduction. Green in the dark spontaneous revertant brc-8 was selected from culture of double auxotrophic mutant brc-1,arg7 after transferring it from photoautotrophic to heterotrophic conditions. Genetic analysis revealed that dark CBS was recovered in brc-8 strain by single recessive mutation in the nuclear gene-suppressor Sup3. The brc-8 strain was transformed by plasmid DNA pCB412 containing Arg7 gene, and orange in the dark clone T8-3 was selected from 5153 green transformants. The insertion mutant T8-3 has the same dark phenotype as brc-1 mutant and lost the ability for photoautotrophic growth. Genetic analysis revealed that T8-3 - is the original brc-1 mutant, appeared in the result of disruption of Sup3 mutant gene. The genomic DNA fragment flanking insertion vector in T8-3 strain was determined and gene Sup3 was identified. Possible mechanisms of the genetic regulation of light-independent CBS are discussed as well as processes, important for adaptation of Chlamy cells for light and dark growth conditions.

e-mail address of presenting author: elena_chekunova@mail.ru