Targeting chloroplast bicarbonate transporters BicA and SbtA to chloroplasts of Nicotiana for enhanced CO2 fixation

Title : Targeting chloroplast bicarbonate transporters BicA and SbtA to chloroplasts of Nicotiana for enhanced CO2 fixation

Abstract:

In C3 plants, the photorespiratory process that occurs because of the oxygenase activity of the primary CO₂ capturing enzyme Ribulose 1,5 bisphosphate Carboxylase/Oxygenase (RubisCO) renders majority of our crop plants poor in terms of productivity. A persistent lower CO₂ concentration due to diffusive resistance at the chloroplast results in reduced RubisCO carboxylation and increased oxygenation, thereby enhancing the expenditure of metabolic energy to recycle carbon and nitrogen via photorespiration. This situation is worsened again by increases in temperature due to a drop in the affinity of RubisCO for CO₂ and an increase in the oxygenase reaction, a situation that is quite important in the context of climate change. Inspirations are hence drawn from lower organisms which possess carbon concentration mechanisms (CCM) to turbo-charge RubisCOs CO₂ supply. It has been proposed that the installation of CCMs into chloroplasts will improve photosynthetic yield and water use efficiency and reduce losses due to heat stress in C3 plants. In this context, one strategy involves transfer of cyanobacterial bicarbonate transporters such as BicA and SbtA to inner envelope membrane of the chloroplast. In our attempt to attain this objective, chimeric genetic elements containing transit peptide (TP) elements from Arabidopsis thaliana and two cyanobacterial bicarbonate genes BicA and SbtA (TP6/12+bicA gene and TP3/4+sbtA gene) were prepared and fused upstream of the gus and gfp reporter genes into a plant expression vector pRI 101-AN and pCAMBIA1302 respectively. All the recombinant constructs were transformed into Agrobacterium tumefaciens GV3101. pRI constructs were further used to transform Nicotiana tabacum plants via co-culture of Agrobacterium cells with plant leaves/callus and the protein expression was analysed by histochemical GUS assay. Upon obtaining a positive GUS assay, we determined the subcellular location of the targeted proteins by confocal microscopy. To this end, all the pCAMBIA constructs were transiently transformed into Nicotiana benthamiana leaves by agroinfiltration. Four days post agroinfiltration, protoplast samples were prepared from transformed leaves and GFP expression was analyzed under confocal microscope. We found chloroplastic location for TP4+SbtA. In the case of TP12+BicA construct, protein expression was majorly detected in either cytosol or in golgi or in endoplasmic reticulum with extremely low fluorescence. For determining the exact location of expressed proteins, the results obtained are being confirmed by western blotting. The constructs depicting correct localization and significant levels of expression would be further used to generate stable transgenic plants and their growth rate and photosynthetic performance would be evaluated

Biography:

Sandhya Mehrotra earned her Ph.D from National Botanical Research Institute, Lucknow, India in 2003. She then joined as a post doctoral fellow at Nara Institute of Technology and Science, Nara, Japan where she studied carbon metabolic pathways in lower organisms. She joined Birla Institute of Technology and Sciences, Pilani, Rajasthan, India in November 2008 and has been serving as a faculty member in the Department of Biological Sciences since then. Her research mainly focuses on carbon fixation, RubisCO and related genes, transcription control and designer promoters and lipase mediated biofuel production. She has more than 30 publications in journals of repute and serves as editor and reviewer of several journals from time to time.