title: Manipulation of carbonic anhydrase activity in the chloroplasts of Arabidopsis thaliana for engineering a CO₂-concentrating mechanism in land plants
Creators: name: Vardakis, Alexandros
Publisher: name: The University of Edinburgh
Year: 2020
Subjects: subject: CCM
subject: crop improvement
subject: Arabidopsis thaliana
subject: photorespiration
subject: Rubisco
subject: Chlamydomonas reinhardtii
subject: carbonic anhydrase

description: Rubisco, the primary photosynthetic carboxylase, uses either CO₂ or O₂ as substrates. Rubisco-catalysed CO₂ assimilation, or carboxylation, results in photosynthetic CO₂ fixation. On the other hand, Rubisco-catalysed oxygenation, along with the ensuing metabolic process of photorespiration, results in reduced photosynthetic CO₂ fixation. Cyanobacteria and most green algae overcome the oxygenation-imposed reduction in photosynthetic efficiency through biophysical CO₂-concentrating mechanisms (CCMs). CCMs actively raise the local [CO₂] around Rubisco, allowing for carboxylation closer to maximum capacity and suppression of the relative frequency of oxygenation. To demonstrate the crop improvement potential of engineering a biophysical CCM in C3 plants, we aim to reconstitute a CCM inspired by the green alga Chlamydomonas reinhardtii into the model C3 plant Arabidopsis thaliana. The present work is concerned with the role of carbonic anhydrases (CAs) in engineering an in planta CCM. Towards this end, we studied in planta expression of CAH3, a Chlamydomonas thylakoid lumen CA that plays a key role in CCM function. Transient expression of a CAH3::eGFP fusion in Nicotiana benthamiana resulted in a predominantly non-chloroplastic eGFP signal, while immunoblotting showed most of the CAH3::eGFP protein did not undergo chloroplast import-associated proteolytic cleavage. Replacement of the CAH3 transit peptide (TP) with a TP from Arabidopsis resulted in a clear chloroplastic eGFP signal, as well as increased chloroplast import-associated proteolytic cleavage into three distinct bands, underlining the importance of TP recognition in targeting heterologous proteins to the chloroplasts of land plants. We also attempted to validate potential interactions of CAH3 with other Chlamydomonas proteins using Y2H. However, the abundance of false positives indicates that care should be taken when studying CAH3 interactions using the classical Y2H assay. The presence of native chloroplastic CAs, namely αCA1, αCA4, βCA1, βCA5 and a chloroplastic βCA2 isoform, has the potential to disrupt the efficiency of an in planta biophysical CCM. Therefore, the present work also aimed to reduce and, if possible, eliminate chloroplastic CA activity from Arabidopsis, and to assess the impact of such interventions, by studying multiple CA mutant lines generated through T-DNA insertional mutagenesis. Growth of the triple CA mutant line αca1 αca4 βca1 was found to be similar to the WT under either 200 or 1150 μmol photons m⁻² s⁻¹, indicating no observable phenotypic defect. A quadruple CA mutant was generated by crossing line αca1 αca4 βca1 with line βca2. Seedling establishment capacity was assessed for lines βca1, αca1 αca4 βca1, and αca1 αca4 βca1 βca2. As per prior literature for line βca1, seedling establishment for multiple CA mutant lines was significantly impaired compared to the WT; however, the number of phenotypic classes in the progeny was greater than previously reported. Importantly, manifestation of the seedling establishment phenotype requires constant illumination, assuaging concerns about the viability of multiple CA lines under field conditions. Finally, elimination of βCA5-associated CA activity is not possible using conventional insertional mutagenesis, as βca5 plants are sterile and slow-growing. Stable expression lines expressing an enzymatically inactive βCA5 variant were generated to examine whether βca5 sterility can be complemented. However, due to low transgene expression, it was not possible to determine whether CA activity is tied to the βca5 phenotype. Despite the challenges presented by βCA5 removal, the generation and growth characterisation of multiple chloroplastic CA lines is a major step towards controlling CA activity in the Arabidopsis chloroplast.