@phdthesis{DiezCocero2017,
  author    = {Diez Cocero, Mercedes},
  title     = {Analysis of Rubisco - carbonic anhydrase fusions in tobacco as an approach to reduce photorespiration},
  school      = {Universit{\"a}t Potsdam},
  pages     = {116},
  year      = {2017},
  abstract  = {Rubisco catalyses the first step of CO2 assimilation into plant biomass. Despite its crucial role, it is notorious for its low catalytic rate and its tendency to fix O2 instead of CO2, giving rise to a toxic product that needs to be recycled in a process known as photorespiration. Since almost all our food supply relies on Rubisco, even small improvements in its specificity for CO2 could lead to an improvement of photosynthesis and ultimately, crop yield. In this work, we attempted to improve photosynthesis by decreasing photorespiration with an artificial CCM based on a fusion between Rubisco and a carbonic anhydrase (CA). A preliminary set of plants contained fusions between one of two CAs, bCA1 and CAH3, and the N- or C-terminus of RbcL connected by a small flexible linker of 5 amino acids. Subsequently, further fusion proteins were created between RbcL C-terminus and bCA1/CAH3 with linkers of 14, 23, 32, and 41 amino acids. The transplastomic tobacco plants carrying fusions with bCA1 were able to grow autotrophically even with the shortest linkers, albeit at a low rate, and accumulated very low levels of the fusion protein. On the other hand, plants carrying fusions with CAH3 were autotrophic only with the longer linkers. The longest linker permitted nearly wild-type like growth of the plants carrying fusions with CAH3 and increased the levels of fusion protein, but also of smaller degradation products. The fusion of catalytically inactive CAs to RbcL did not cause a different phenotype from the fusions with catalytically active CAs, suggesting that the selected CAs were not active in the fusion with RbcL or their activity did not have an effect on CO2 assimilation. However, fusions to RbcL did not abolish RbcL catalytic activity, as shown by the autotrophic growth, gas exchange and in vitro activity measurements. Furthermore, Rubisco carboxylation rate and specificity for CO2 was not altered in some of the fusion proteins, suggesting that despite the defect in RbcL folding or assembly caused by the fusions, the addition of 60-150 amino acids to RbcL does not affect its catalytic properties. On the contrary, most growth defects of the plants carrying RbcL-CA fusions are related to their reduced Rubisco content, likely caused by impaired RbcL folding or assembly. Finally, we found that fusions with RbcL C-terminus were better tolerated than with the N-terminus, and increasing the length of the linker relieved the growth impairment imposed by the fusion to RbcL. Together, the results of this work constitute considerable relevant findings for future Rubisco engineering.},
  language  = {en}
}
@article{SchwarteTiedemann2011,
  author    = {Schwarte, Sandra and Tiedemann, Ralph},
  title     = {A Gene Duplication/Loss Event in the Ribulose-1,5-Bisphosphate-Carboxylase/Oxygenase (Rubisco) Small Subunit Gene Family among Accessions of Arabidopsis thaliana},
  series = {Molecular biology and evolution},
  volume    = {28},
  journal   = {Molecular biology and evolution},
  number    = {6},
  publisher = {Oxford Univ. Press},
  address   = {Oxford},
  issn      = {0737-4038},
  doi       = {10.1093/molbev/msr008},
  pages     = {1861 -- 1876},
  year      = {2011},
  abstract  = {Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated.},
  language  = {en}
}