title: Characterisation of transcription factors with potential roles in the circadian optimisation of Crassulacean acid metabolism in Kalanchoë fedtschenkoi.
Creators: name: Waller, JL
Publisher: name: University of Liverpool Repository
Year: 2016

description: Crassulacean acid metabolism (CAM) is a metabolic adaptation of photosynthesis that is optimised via strict temporal regulation of its biochemistry by the circadian oscillator. CAM plants achieve high water use efficiency and thus thrive in seasonally dry regions unsuitable for C3 food crops such as rice or wheat. Climate change and the associated challenges of global food and energy security, mean that CAM research is currently of urgent and pressing need, as CAM may reveal methods for the generation of more water use efficient crops. The efficiency of CAM is optimised by the circadian clock, through the regulation of PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) expression and nocturnal CO2 fixation, but the signalling pathway between the central clock and CAM has yet to be elucidated. Whole genome sequencing and detailed RNA-seq datasets for C3 and CAM leaves of the model CAM species Kalanchoë fedtschenkoi have enabled the discovery of novel genes that could function to link CAM to the circadian clock. Three CAM-induced and clock-controlled transcription factor (TF) genes were identified from the RNA-seq datasets: MYB-LIKE 439 (KfMYB439), CAM-INDUCED BZIP1 (KfCIB1) and CYCLING DOF FACTOR2 (KfCDF2). Both over-expressor and RNAi knockdown transgenic lines of K. fedtschenkoi were generated for each TF, facilitating the further elucidation of their biological functions. Over 200 transgenic lines were screened for altered expression levels, and changes to the dawn and dusk levels of key CAM metabolites: malate and starch. Four transgenic lines for each TF, two over-expressor and two RNAi lines, were used for detailed phenotypic analysis of CAM-associated traits. Transgenic perturbation of any one of the three TFs caused small but widespread changes to the transcript levels of core clock- and CAM-associated genes. KfMYB439 is a REVEILLE family TF related to the clock gene CIRCADIAN CLOCK ASSOCIATED1 (CCA1). Data revealed that KfMYB439 functioned close to the core circadian oscillator. Mis-regulation of KfMYB439 led to the perturbation of efficient dark CO2 fixation, reduced levels of starch and malate, and reduced productivity during drought. KfCIB1, was found to feed back to influence the core circadian clock as well as regulating CAM. In constant light conditions, KfCIB1 mis-expression led to perturbed timing of KfCCA1 and TIMING OF CAB1 (KfTOC1) . KfCIB1 mis-regulation also impacted on stomatal control. At dusk and dawn, large and rapid changes in stomatal conductance resulted in spikes of CO2. Mis-expression also resulted in small improvements in productivity in water-limited environments. KfCDF2 was found to function not only in the clock control of CAM, but also in the photoperiodic control of flowering time. In terms of CAM and the clock, KfCDF2 mis-expression caused changes to CCA1, TOC1 and PPCK expression, and arrhythmic CO2 fixation in constant conditions. It also impacted on water retention during drought, with both over-expressor and RNAi lines displaying higher succulence than the wild type lines after 90 days of drought. KfCDF2 over-expression in both K. fedtschenkoi and K. laxiflora caused constitutive flowering in long days, whereas wild type plants never flowered. Q-RT-PCR analysis of flowering pathway genes revealed that KfCDF2 over-expression impacted on transcript levels for CONSTANS (CO) and FLOWERING LOCUS T (FT); key proteins in the day-length dependent induction of flowering. Results suggested that all three TFs likely function in the circadian optimisation of CAM, although whether or not the often small effects were direct or indirect will require further work. Future Chromatin Immunoprecipitation and sequencing experiments will reveal the target genes regulated by these TFs, and the identification of other novel CAM-induced genes from the RNA-seq data, will allow the pathway between the circadian clock and CAM to be elucidated in much greater detail.