title: Engineering more water-use efficient crops: Establishment of Kalanchoë gracilipes as a tractable model species for the molecular genetic study of weak, inducible Crassulacean acid metabolism
Creators: name: Foster, Bree
Publisher: name: University of Liverpool Repository
Year: 2028

description: Crassulacean acid metabolism (CAM) is an evolutionary adaptation of photosynthesis that allows plants to carry out primary atmospheric CO2 fixation at night, store the carbon in the form of malic acid, and break down the malate during the day to re-fix the CO2. Thus, during the day period of CAM, CO2 is concentrated around RuBisCO, increasing its efficiency to fix CO2 into sugars. CAM has the additional benefit of reducing water loss via transpiration as the stomata open at night and close during the hotter and drier part of the day. This allows CAM species to grow and thrive in arid or water-limited environments. This adaptive trait has driven efforts to genetically engineer CAM into C3 crop and biofuel species in order to increase their resistance to drought in hotter and drier climates that are anticipated to result from anthropogenic climate change. Efforts to characterise CAM at the molecular genetic level through comparative transcriptional analysis between C3 and CAM states, and complementary mutagenesis studies in CAM plants, are critical to providing a detailed understanding of the CAM pathway and thus, to providing the genetic blueprint for engineering CAM into C3 species. The Kalanchoë genus represents a potential model system to understand and reconstruct the origins of the CAM pathway since it shows a clear evolutionary spectrum from facultative C3-CAM intermediates through to strong, obligate CAM. Strong, obligate CAM is studied using the model CAM species, Kalanchoë fedtschenkoi and Kalanchoë laxiflora. However, despite the range in photosynthetic physiotypes in Kalanchoë, there is a lack of model species to study weak, inducible or facultative CAM. One example of a potential future model inducible CAM Kalanchoë species is Kalanchoë gracilipes. Prior to the start of this PhD project, an annotated genome and an in-depth RNA-seq dataset including information on drought-response and tissue development were produced for K. gracilipes. This has allowed for genotypic and transcriptomic comparisons of a C3-CAM species (K. gracilipes) with an obligate CAM species (K. fedtschenkoi) for the first time in Kalanchoë. During this PhD, K. gracilipes has been confirmed as a weak, inducible CAM species with detailed temporal characterisation of physiological, biochemical and molecular properties in response to drought. A tissue-culture based regenerative protocol has been developed and progress has been made towards a stable transformation system in K. gracilipes. Additionally, the existence of annotated genomes for K. fedtschenkoi, K. laxiflora and K. gracilipes allowed for genome-wide comparisons of methylation levels and an investigation into how methylation might be associated with CAM. This PhD has established that K. gracilipes is a valuable model for studying weak, inducible CAM in the Kalanchoë genus and will provide an invaluable genetic resource for functional genomic studies in CAM.