Engineering more water-use efficient crops: functional genomics of weak and strong Crassulacean acid metabolism in diverse species of the genus Kalanc

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

Crassulacean acid metabolism (CAM) is an adaptation of photosynthesis found in a diverse range of plant species that inhabit arid and semi-arid environments. CAM plants can achieve water use efficiencies up to ten-times greater than C3 species. Our over-arching goal is to develop a systems level view of CAM by developing a detailed understanding of the genes, proteins and metabolites involved in this valuable adaptation. Comprehensive knowledge of the CAM 'parts-list' will permit forward engineering of CAM into C3 crops. In particular, we are collaborating with US scientists on a plant synthetic biology project that aims to introduce CAM into poplar trees (http://cambiodesign.org).
We have performed whole genome and transcriptome sequencing (RNA-seq) for our model CAM systems, Kalanchoë fedtschenkoi and K. laxiflora, including detailed analysis of patterns of gene transcript abundance in CAM and C3 tissues of both species. This gene discovery work has identified candidate genes for engineering CAM into C3 crops. In order to refine the list of candidate CAM genes, recent work in the Hartwell lab has focused on silencing each candidate CAM gene in transgenic lines of Kalanchoë in order to refine the minimal parts-list for efficient CAM (e.g. Dever et al., 2015 Plant Physiology; Boxall et al., 2017 The Plant Cell).
In addition, we have recently used detailed gas exchange analysis to identify both weak and strong CAM species belonging to the genus Kalanchoë. The weak CAM species, K. gracilipes, is relatively thin leaved and uses C3 photosynthesis when well-watered, but weak CAM when drought stressed. It occupies a basal/ ancestral position in the Kalanchoë molecular phylogeny. By contrast, strong CAM species of Kalanchoë, such as K. hildebrandtii, are highly succulent and rely on CAM under all environmental conditions. These strong CAM species are also the most derived in evolutionary terms. These discoveries set the stage for comparative genomic analysis of CAM evolution in Kalanchoë, which will allow us to identify the genetic and epigenetic changes that are associated with the transition from weak and inducible CAM through to strong CAM in this diverse genus.
This project will focus on comparative analysis of our existing K. fedtschenkoi and K. laxiflora genomes and RNA-seq data with the newly decoded genomes and transcriptomes of K. gracilipes and K. hildebrandtii, which will be sequenced as part of our ongoing research in the coming months funded by our US Dept. of Energy "CAM biodesign" project. The sequences will be available prior to the start of this proposed PhD project. In the long term, this work will make a substantial contribution to the development of more drought tolerant, water use efficient bioenergy crops and new biofuel feedstock crops suitable for desert cultivation.

Publications

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Description James Hartwell's lab used detailed gas exchange analysis to identify both weak and strong CAM species belonging to the genus Kalanchoe. The weak CAM species, Kalanchoe gracilipes, is relatively thin-leaved and uses C3 photosynthesis when well-watered, but weak CAM when drought-stressed. My project is focussed on comparative analysis of existing K. fedtschenkoi and K. laxiflora genomes with the newly decoded genome and transcriptome of K. gracilipes.
This is the first time that Kalanchoe gracilipes has been used in a scientific capacity. So far, I have managed to develop a regenerative protocol and a stable transformation protocol for Kalanchoe gracilipes. This has enabled me to stably transform Kalanchoe gracilipes with a key CAM gene from a strong CAM species. This construct has also been transformed into Kalanchoe laxiflora and Arabidopsis thaliana. This will allow us to investigate if the presence of this gene (and its promoter sequence) has any phenotypic effects. I have already performed a droughted vs well watered 24h timecourse (taking samples every 2h) for K. gracilipes. This will allow us to do similar work with the transgenic lines and compare with a baseline.
In addition to this, I have now also got bisulphite sequence data for K. fedtschenkoi, K. laxiflora and K. gracilipes at dawn and dusk timepoints. This will allow us to investigate whether there is a change in methylation in specific genes and also over the whole genome between species and over time.
Exploitation Route My work could act as a foundation for investigating Kalanchoe gracilipes. As it has not been used in a scientific context before, a regenerative and transformation protocol will allow further functional and comparative genomics work to be performed. This may allow a more in-depth study of the weak CAM in comparison to more obligate CAM species. My work with PPC and PPCK may also provide more evidence that these genes are vital for the CAM process and should be included for the forward engineering of CAM into C3 crops.
Sectors Agriculture, Food and Drink,Energy