Sequencing the transcriptome of Kalanchoe fedtschenkoi: a model for Crassulacean acid metabolism embryogenic plantlet formation and the Saxifragales

Plants have evolved three major forms of photosynthetic metabolism known as C3, C4 and Crassulacean acid metabolism (CAM). CAM is found in ~7 % of plant species, whilst C4 occurs in ~3 %. The remaining majority perform C3 photosynthesis. CAM and C4 improve the efficiency of plant water use; that is, the amount of water lost for each molecule of carbon dioxide converted into sugars in photosynthesis. CAM plants possess water-use efficiencies that can be 10-20 times greater than C3 plants. CAM is found in species that inhabit deserts, semi-arid or Mediterranean regions, and seasonally dry environments such as those on the branches of rain forest trees. Whilst whole genome sequencing projects have been completed for several plant species that perform C3 photosynthesis (Arabidopsis thaliana, rice and poplar) and a project is underway to sequence the genome of the C4 crop maize, there is very limited sequence information available for CAM species. It is therefore important that we sequence a CAM species so that we can complete the genomic picture of plant photosynthetic diversity. The Intergovernmental Panel on Climate Change (IPCC) report published this year predicts increasing desertification in already drought-prone regions of the world. CAM species can play an important role in mitigating the effects of climate change in arid and semi-arid zones, and there is now a pressing need to understand the molecular-genetic basis for CAM in order to capitalise on the utility of CAM plants fully. Importantly, our DNA sequencing proposal will unlock the genetic warehouse of novel genes that have evolved to enable plants to survive in desert and semi-arid environments. We will sequence the genes that are active in the leaf of a CAM plant. We will use Kalanchoe fedtschenkoi as our model system for this study. In 1961, an innovative young scientist called Malcolm Wilkins published a report in Nature showing a daily rhythm of carbon dioxide fixation in leaves of K. fedtschenkoi that persisted in constant conditions. This rhythm revealed that carbon dioxide fixation by the CAM pathway was under the control of an internal timekeeper known as a circadian clock. Circadian clocks keep time even in the absence of external environmental input and optimise the efficiency of photosynthesis and increase plant productivity. K. fedtschenkoi has continued to be a species in which significant scientific breakthroughs are made including advances in our understanding of the regulation of metabolic enzymes and the study of plant embryogenesis. K. fedtschenkoi is one of the best CAM species from which to sequence large amounts of genes for a number of reasons: (i) Unlike other CAM species, genes can be introduced into the K. fedtschenkoi genome using straightforward plant transformation procedures. This facet allows us to manipulate the activity of genes of interest and study their function in whole plants. (ii) There is a wealth of established whole plant physiology data for K. fedtschenkoi, which defines the physiological and biochemical details of the circadian rhythm of carbon dioxide fixation. (iii) The study of the biochemistry and molecular biology of CAM is routine in K. fedtschenkoi. There are established protocols for protein purification, enzyme assays and the isolation of pure DNA and RNA. In addition, K. fedtschenkoi is a member of a group of plants (known as the Saxifragales) that are of great interest to research groups working to understand the evolution of plant diversity. As already mentioned, genome sequence databases already exist for several plant species, but detailed gene sequence data is lacking for the Saxifragales. For this reason, our large-scale sequencing of genes from K. fedtschenkoi will provide a greatly needed resource of genetic information for this important group of plants that includes blackcurrants as well as many important garden species that are of great value to the horticultural trade.

The aim of this proposal is to perform deep sequencing of expressed sequence tags (ESTs) from Kalanchoe fedtschenkoi using massively-parallel pyrosequencing. K. fedtschenkoi is an excellent model system for the study of Crassulacean acid metabolism (CAM), plantlet formation via somatic embryogenesis and the genomics of the Saxifragales. K. fedtschenkoi has facilitated several breakthroughs including: (1) a circadian rhythm of nocturnal CO2 fixation; (2) the allosteric regulation of phosphoenolpyruvate carboxylase (PEPc) due to nocturnal phosphorylation by a specific protein kinase, namely PEPc kinase (PPCK); (3) the first PPCK gene, and (4) the circadian control of PPCK transcript abundance. K. fedtschenkoi is an excellent choice for deep EST sequencing because it has a relatively small genome size (~790 Mb) and can be transformed efficiently using Agrobacterium. A major objective of this proposal is to understand the genetic basis of CAM by identifying the genes that are uniquely expressed in CAM leaves. Due to the incredible depth of coverage that can be achieved with massively-parallel pyrosequencing, we will determine the regulatory genes involved in the establishment and circadian co-ordination of the CAM pathway. We will then characterise the regulation of these genes in detail to establish which ones are most fundamental to the establishment of CAM. Next, we will up- or down-regulate the genes in transgenic K. fedtschenkoi and perform detailed phenotypic characterisation of the transgenic lines. Altogether, the research will provide an unrivalled and greatly needed overview of the genetic architecture of CAM, embryogenic plantlet formation, and the Saxifragales that will facilitate major breakthroughs in our understanding of these fundamentally important aspects of plant biology.