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

植物已经进化出三种主要的光合代谢形式，即C3、C4和天冬酸代谢（CAM）。CAM在7%的植物中存在，而C4在3%的植物中存在。其余大部分进行C3光合作用。CAM和C4提高了植物水分利用效率；也就是说，在光合作用中，每个二氧化碳分子转化为糖所损失的水量。CAM植物的用水效率比C3植物高10-20倍。CAM存在于生活在沙漠、半干旱或地中海地区以及季节性干燥环境（如雨林树木的树枝上）的物种中。虽然一些进行C3光合作用的植物物种（拟南芥、水稻和杨树）的全基因组测序项目已经完成，而且C4作物玉米的基因组测序项目正在进行中，但CAM物种的序列信息非常有限。因此，我们对CAM物种进行测序是很重要的，这样我们就可以完成植物光合多样性的基因组图谱。政府间气候变化专门委员会（IPCC）今年发表的报告预测，在世界上已经容易发生干旱的地区，荒漠化将会加剧。在干旱和半干旱区，CAM物种在缓解气候变化的影响方面发挥着重要作用，现在迫切需要了解CAM的分子遗传学基础，以便充分利用CAM植物的效用。重要的是，我们的DNA测序方案将打开新基因的基因仓库，这些基因已经进化到使植物能够在沙漠和半干旱环境中生存。我们将对CAM植物叶片中活跃的基因进行测序。我们将使用kalanche fedtschenkoi作为本研究的模型系统。1961年，一位名叫马尔科姆·威尔金斯（Malcolm Wilkins）的富有创新精神的年轻科学家在《自然》（Nature）杂志上发表了一篇报告，报告显示，在恒定的条件下，费氏叶锦鸡的叶片每天都有固定二氧化碳的节奏。这种节律表明，CAM途径对二氧化碳的固定是在生物钟的内部计时器的控制下进行的。即使在没有外部环境输入的情况下，生物钟也能保持时间，并优化光合作用的效率，提高植物的生产力。fedtschenkoi一直是一个取得重大科学突破的物种，包括我们对代谢酶调控和植物胚胎发生研究的进展。fedtschenkoi是最适合对大量基因进行测序的CAM物种之一，原因如下：(i)与其他CAM物种不同，基因可以通过直接的植物转化程序导入K. fedtschenkoi基因组。这方面使我们能够操纵感兴趣的基因的活性，并研究它们在整个植物中的功能。(2) fedtschenkoi的全植物生理数据丰富，确定了二氧化碳固定昼夜节律的生理生化细节。（iii）对白蛉CAM的生物化学和分子生物学研究是常规的。目前已有蛋白质纯化、酶分析和纯DNA和RNA分离的既定方案。此外，K. fedtschenkoi是一组植物（被称为Saxifragales）的成员，对研究植物多样性进化的研究小组非常感兴趣。如前所述，一些植物物种的基因组序列数据库已经存在，但缺乏详细的萨克斯花属植物的基因序列数据。因此，我们对fedtschenkoi的大规模基因测序将为这一重要植物群提供非常需要的遗传信息资源，包括黑加仑以及许多对园艺贸易具有重要价值的重要园林物种。

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.