Manipulating RuBisCO accumulation in the green alga, Chlamydomonas reinhardtii

Abstract

The nuclear factor, Maturation/stability of RbcL (MRL1), regulates the accumulation of the chloroplast rbcL gene transcript in Chlamydomonas reinhardtii by stabilising the mRNA via its 5' UTR. An absence of MRL1 in algal mrl1 mutants leads to a complete absence of RuBisCO large subunit protein and thus a lack of accumulation of the RuBisCO holoenzyme. By complementing mrl1 mutants by random transformation of the nuclear genome with the MRL1 cDNA, different levels of rbcL transcript accumulate. We also observe that RuBisCO Large Subunit accumulation is perturbed. Complemented strains accumulating as little as 15% RuBisCO protein can grow phototrophically while RuBisCO in this range is limiting for phototrophic growth. We also observe that photosynthetic activity, here measured by the quantum yield of PSII, appears to be a determinant for phototrophic growth. In some strains that accumulate less RuBisCO, a strong production of reactive oxygen species is detected. In the absence of RuBisCO, oxygen possibly acts as the PSI terminal electron acceptor. These results show that random transformation of MRL1 into mrl1 mutants can change RuBisCO accumulation allowing a range of phototrophic growth phenotypes. Furthermore, this technique allows for the isolation of strains with low RuBisCO, within the range of acceptable photosynthetic growth and reasonably low ROS production. MRL1 is thus a potential tool for applications to divert electrons away from photosynthetic carbon metabolism towards alternative pathways.

Proton Gradient Regulation 5-Mediated Cyclic Electron Flow under ATP- or Redox-Limited Conditions: A Study of ƊATPase pgr5 and ƊrbcL pgr5 Mutants in the Green Alga Chlamydomonas reinhardtii

“…This finding was especially apparent for the DrbcL pgr5 double mutant, where with respect to the fluorescence phenotype, pgr5 could be considered epistatic to rbcL. The effect of the pgr5 mutation on DrbcL was surprising to us, because we previously attributed the rise in fluorescence in Rubiscoless strains to PSI acceptor side limitations (Johnson, 2011). If this result were the case, the combination of the Rubisco mutant with that of pgr5 would have been expected to exacerbate rather than alleviate the acceptor side limitation, unless an alternative electron sink was present in the mutant.…”

“…5). This result is because the origins of the photosensitivity are different: in DrbcL, photosensitivity has been attributed, in large part, to the production of reactive O 2 species at the acceptor side of PSI Johnson, 2011), whereas photosensitivity in DAtpase is caused by multiple phenomena. The ATPase mutant is probably both acceptor and donor side limited, with effects on PSII stability caused by an overacidification of the thylakoid lumen in both C. reinhardtii and plants (Majeran et al, 2001;Rott et al, 2011).…”

Localized control of oxidized RNA

“…ΔrbcL and ΔRBCS are non-photosynthetic, but fully viable under heterotrophic conditions (Satagopan and Spreitzer, 2004). Because Rubisco mutants are highly light-sensitive (Johnson, 2011), all cultures were grown and tested under heterotrophic conditions [in the dark on Trisacetate phosphate (TAP) medium (Gorman and Levine, 1965), at 24°C, with orbital shaking]. To generate the complemented ΔRBCS strain, the wild-type RBCS2 gene (on plasmid pSS2) (Khrebtukova and Spreitzer, 1996) was introduced into ΔRBCS by glass-bead-mediated transformation as described previously (Purton, 2007).…”

PPR proteins of green algae

“…In contrast, PPR17, 18,19,20,21,22,23,24,25, and 26 are specific of the most ancestral group, the Mamiellophyceae, with an ortholog found in each of the five genomes but not in other algae. These genes may have arisen during the evolution of Mamiellophyceae, or be ancestral in Chlorophyta but lost in the other branches.…”

“…Subsequently, a new series of mrl1 alleles was characterized, and complemented strains with various levels of restoration were used to study phototrophic growth and ROS production as a function of RuBisCO level. 24 The Chlamydomonas MRL1 protein is part of a high MW complex of approximately 800 kDa, whose size was shifted to 550-600 kDa after Rnase I treatment or in a ΔrbcL mutant, indicating that it includes the target mRNA as a Distance (in nucleotide) between the first nucleotide of the target sequence (GAGAAGAAAA) and the A of the petA initiation codon. "None" indicates that the target sequence was not found upstream of petA within 5 kb.…”