Functional genomics of nocturnal carboxylation in Crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is an evolutionary adaptation of photosynthesis by which plant photosynthetic organs carry out phosphoenolpyruvate carboxylase (PPC)-catalysed CO2 fixation during the night, store nocturnally-fixed carbon in the form of malate, break down the stored malate during the day, and re-fix the CO2 thus internally generated. The internal supply of CO2 as substrate for Rubisco-catalysed fixation during the day has the potential to greatly reduce the demand for CO2 from the atmosphere. This allows CAM species growing in arid or waterlimited niches to mitigate water loss from transpiration by closing their stomatal pores during the hotter and drier day. This adaptive trait has motivated efforts to genetically engineer CAM into economically-important C3 species in order to increase their resistance to drought in the hotter and drier climates that it is anticipated will result from anthropogenic climate change. Efforts to characterise CAM at the molecular genetic level through reverse genetics, guided by genomic and transcriptomic analysis, are ongoing by the Hartwell group, University of Liverpool, using Kalanchoë species as CAM model species. Two membrane-bound metabolite transporter proteins, a vacuolar aluminium-activated malate transporter (ALMT) family protein and a tonoplast dicarboxylate transporter (TDT), have been suggested to play a role in mediating cytosol-to-vacuolar lumen malate transport in CAM, during nocturnal malate accumulation and diurnal malate breakdown, respectively. Prior to the start of this PhD project, Kalanchoë fedtschenkoi lines were generated in which KfALMT or KfTDT was targeted for transcriptional silencing by a transgenic hairpin RNA in conjunction with endogenous RNA interference (RNAi) pathways. Phenotypic characterisation of KfALMT RNAi lines showed that CAM was suppressed at the level of nocturnal CO2 fixation and diel malate turnover in these lines, with downstream effects on diel patterns of carbohydrate metabolism. Vacuolar membranes were extracted from two KfALMT RNAi lines for in vitro functional characterisation of ALMT. This showed that suppression of KfALMT transcript greatly reduced the H+ -malate coupled transport activity of the tonoplasts, providing a protein function correlate to CAM suppression in vivo. CAM suppression via KfALMT RNAi was also found to perturb the entrainment of the core circadian clock, which is thought to regulate the diel CAM cycle, and the clock-controlled, CAM-regulatory phosphoenolpyruvate kinase (PPCK). Multi-level CAM suppression was not found in KfTDT RNAi lines, but preliminary phenotyping suggested that TDT could act as vacuolar citrate transporter. Malate dehydrogenase (MDH) is required to catalyse the conversion of oxaloacetate (OAA), the product of PPC-catalysed nocturnal CO2 fixation, into malate in order to accumulate the latter at night in CAM. However, it is unknown which of the plant cell's multiple MDH isoforms mediates this metabolic flux. Isogene-specific silencing of MDH via RNAi in transgenic Kalanchoë laxiflora was attempted. Preliminary phenotypic characterisation of MDH RNAi lines found some evidence of a small degree of CAM suppression in cytosolic NAD-MDH (KlCMDH) RNAi and plastidic NAD-MDH (KlPdMDH) RNAi lines. It was suggested that the two enzymes could act synergistically in partially independent and parallel pathways to mediate nocturnal accumulation of malate in CAM.