The pyrenoid as both functional component and regulator of the CCM

Physiological and molecular characterisation of higher plant RBCS hybrid mutants showed that strains that were unable to form a pyrenoid also had very low photosynthetic affinity for Ci and a high CO2-requiring phenotype (Chapter 4). While the pyrenoid itself is likely to play an important role in concentrating CO2 around Rubisco, proteomic studies indicated that pyrenoid loss was also associated with reduced accumulation of other CCM components (Chapter 5).

In the high CO2-requiring cia6 mutant, the normally conspicuous single pyrenoid is fragmented into small, irregular-shaped pyrenoids, containing only 35% of the cell’s Rubisco and this mutation is associated with low affinity for Ci (Ma et al., 2011). The mutation occurs in a gene encoding a SET domain methyltransferase, suggesting that abnormal pyrenoid formation may be the result of incorrect post- translational modifications; however, no methyltransferase activity was shown in vitro. Under high CO2, the affinity of the cia6 mutant for Ci was around two times lower than the respective wild-type and, similarly, our study found that pyrenoid- less spinach RBCS hybrid cells had a three times lower affinity for Ci than wild- type cells. Furthermore, this reduced affinity for Ci was even more pronounced in low CO2-adapted cia6 and spinach RBCS hybrid cells (Ma et al., 2011; Chapter 4). These data suggest that the presence of a single, spheroidal pyrenoid may increase photosynthetic affinity for Ci at high as well as low CO2, although loss of the pyrenoid only appears to affect growth at low CO2.

Another similarity between cia6 and true pyrenoid-less mutants is the reduced accumulation of certain CCM mRNA and protein (CAH4, CCP1 and LCIB) in response to low CO2 (Ma et al., 2011). This is consistent with qRT-PCR and LC- MS/MS data obtained for the low CO2-adapted pyrenoid-less spinach RBCS hybrid mutant identifying weaker induction of Ci transporter gene transcripts and CCM- related proteins (Chapters 4 and 5). Together, these results imply that some form of regulatory feedback exists between pyrenoid formation and CCM induction.

Cross talk between Rubisco packaging – perhaps also in proximity to a carbonic anhydrase – and CCM expression also appears to occur in cyanobacteria, despite the lack of molecular homology between carboxysomes and pyrenoids. For example, carboxysome-less ΔccmM mutants of two different genera (Synechocystis and Synechococcus) fail to accumulate CCM-related gene transcripts to wild-type levels in response to low CO2 (Woodger et al., 2005; Hackenberg et al., 2012). In Synechococcus, the CCM is induced primarily in response to a decreased internal Ci pool so the higher internal Ci accumulation in the ΔccmM mutant is likely to be responsible for the partial suppression of Ci transporter genes (Woodger et al., 2005). On the other hand, in Synechocystis, the binding of photosynthetic and photorespiratory metabolites to CCM-related transcription factors (Nishimura et al., 2008; Daley et al., 2012) provides a different means of sensing and responding to changes in carbon metabolism, including changes in Rubisco packaging.

Further work is required to clarify whether similar mechanisms are occurring in Chlamydomonas pyrenoid mutants. The cia6 mutant, for example, has a reduced ability to accumulate Ci suggesting that a high internal Ci pool is not responsible for the observed suppression of low CO2-inducible genes (Ma et al., 2011). Whether altered Rubisco packaging interferes with other sensing or signalling pathways or whether loss of carbon fixation inhibits CCM gene expression via downregulation of the electron transport chain remains to be determined. Future studies should include: measurements of internal Ci accumulation in pyrenoid-less higher plant RBCS hybrid mutants; determination of metabolite pools in the cia6 and pyrenoid-less higher plant RBCS hybrid mutants; and the detailed time courses investigating the interplay of carbon fixation, light harvesting and CCM induction suggested in Chapter 5. The development or identification of mutants with alterable levels of Rubisco accumulation/packaging would also enable more detailed investigation of possible cross talk between the pyrenoid and CCM. Further discussion of pyrenoid biogenesis and the correct localisation of Rubisco to the pyrenoid will follow in section 6.3.1.

6.1.4.1 Implications for the engineering of a pyrenoid-based CCM in higher plant chloroplasts

Several collaborations are underway to re-engineer photosynthesis by introducing carbon concentrating mechanisms from higher plants and algae into terrestrial C3

plants. For example, efforts are being made to transform rice into a C4 crop (von Caemmerer et al., 2012, C4 rice project, http://c4rice.irri.org/) as well as to engineer carboxysomes and cyanobacterial Ci transporters into higher plants (Price et al., 2012, RIPE project). Work in our group, in collaboration with the John Innes Centre (Norwich), the University of Edinburgh and the Carnegie Institution for Science (Stanford), is focussed on engineering elements of the Chlamydomonas CCM, including the pyrenoid, into higher plants (Meyer and Griffiths, 2013, CAPP project, http://cambridgecapp.wordpress.com/). Modelling of both pyrenoid- and carboxysome-based CCMs indicates that aggregation of Rubisco in these structures is an important element of the native CCM as well as intracellular Ci accumulation in higher plants (Badger et al., 1998; Price et al., 2011; Price et al., 2012; McGrath and Long, 2014). Work on the pyrenoid-negative RBCS mutants has provided further experimental evidence of the essential role of the pyrenoid in CCM activity (Chapters 4 and 5; Meyer et al., 2012).

Following on from this, site-directed mutagenesis experiments are underway to identify the specific Rubisco amino acid residues required for pyrenoid formation. Previous work in our group identified two surface-exposed RBCS α-helices as necessary for pyrenoid formation (Meyer et al., 2012). Substitution of the amino acids that differ between pyrenoid-positive algae and pyrenoid-negative higher plants should determine the minimal sequence requirements for Rubisco aggregation in Chlamydomonas. Given the potential for alterations to RBCS primary structure to alter Rubisco holoenzyme kinetics (Spreitzer, 2003), as well as the relatively poor kinetics observed in mutants with only the RBCS α-helices substituted (Meyer et al., 2012), this highly targeted approach is worth pursuing in parallel with the experiments using native Chlamydomonas RBCS sequences described below.

In the meantime, experiments with Arabidopsis are also being undertaken to test the feasibility of hybrid Rubisco holoenzyme and pyrenoid assembly (Dr Alistair McCormick, University of Edinburgh). Stable as well as transient protoplast expression of Chlamydomonas RBCS1 and RBCS2 genes in an Arabidopsis

RBCS knockdown mutant, expressing only 15% of wild-type Rubisco levels (Izumi

et al., 2012) will first test whether the Chlamydomonas RBCS is able to assemble with the Arabidopsis rbcL into a functional holoenzyme. Secondly, these

experiments will test whether aggregation of Rubisco occurs via Chlamydomonas RBCSs, without the presence of other Chlamydomonas-specific factors, and whether any Rubisco-Rubisco interactions are sufficient to improve the photosynthetic characteristics of these transgenic plants. Encouragingly, the transient expression of β-carboxysomes in Nicotiana has demonstrated the feasibility of assembling novel highly organised protein structures in higher plant chloroplasts (Lin et al., 2014). Finally, using synthetic approaches rather than biological mimicry to re-engineer C3 photosynthesis might also be successful, for example, novel molecular scaffolds may be introduced to tether Rubisco to carbonic anhydrases (MAGIC project, http://magic.psrg.org.uk/).

6.2 Integration of signals via multiple transcription/regulatory