Crassulacean acid metabolism in tropical orchids: integrating phylogenetic, ecophysiological and molecular genetic approaches

Crassulacean Acid Metabolism (CAM) is a water-conserving mode of photosynthesis present in approximately 7% of vascular plant species worldwide. CAM photosynthesis minimizes water loss by limiting CO2 uptake from the atmosphere at night, improving the ability to acquire carbon in water and CO2-limited environments. In neotropical orchids, the CAM pathway can be found in up to 50% of species. To better understand the role of CAM in species radiations and the molecular mechanisms of CAM evolution in orchids, we performed carbon stable isotopic composition of leaf samples from 1,102 species native to Panama and Costa Rica, and character state reconstruction and phylogenetic trait analysis of CAM and epiphytism. When ancestral state reconstruction of CAM is overlain onto a phylogeny of orchids, the distribution of photosynthetic pathways shows that C3 photosynthesis is the ancestral state and that CAM has evolved independently several times within the Orchidaceae. Using phylogenetic trait analysis, we found that divergences in photosynthetic pathway and epiphytism are consistently correlated through evolutionary time and are related to the prevalence of CAM epiphytes in lower elevations and abundant species diversification of high elevation epiphytes. The multiple independent evolutionary origins of CAM in orchids suggest that evolution from C3 to weak and strong CAM might involve relatively few genetic changes. In plants performing CAM, phosphoenolpyruvate carboxylase (PEPC) catalyzes the initial fixation of atmospheric CO2 into C4-dicarboxylic acids forming oxaloacetate and inorganic phosphate as a product. PEPC is a ubiquitous enzyme that belongs to a multigene family with each gene encoding a function- and tissue-specific isoform of the enzyme. CAM-specific PEPC isoforms might have evolved from ancestral nonphotosynthetic C3 isoforms by gene duplication and acquired transcriptional control sequences that mediate increased mRNA expression and leaf-specific or leaf-preferential expression patterns. In order to understand patterns of PEPC family diversification over evolutionary times, PEPC genes families in ten closely-related orchid species from the Subtribe Oncidiinae with a range of photosynthetic pathways from C3-photosynthesis (Oncidium maduroi, Ticoglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) to strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthaginense) were characterized. At least three major changes are hypothesized to have occurred during evolution to adapt the CAM progenitor genes for function in CAM plants: 1) CAM isoform genes in orchids have evolved highly expressed mRNA expression patterns; 2) leaf preferential (or specific) expression patterns; and 3) circadian clock control expression patterns. We found that up to five PEPC isoforms are present in orchids, with one putative CAM-specific PEPC isogene with discrete amino acid changes identified in CAM species based on cDNA clone sampling, and an evident shift in PEPC isoform number from 2-3 isoforms in C3 species, to 3-4 isoforms in weak CAM species, to 4-5 isoforms in strong CAM species. Validation of the isotopic analysis and the molecular genetic analysis of PEPC gene family using 24- hour gas exchange showed that weak CAM species exhibit limited amounts of nocturnal CO2 uptake and fixation when compared to strong CAM species. To understand the molecular mechanisms responsible for the recruitment of CAM-specific genes, 454 sequencing of cDNA prepared from RNA of the strong CAM species Rossioglossum ampliatum was conducted, and resulted in 189 Mb of DNA sequence, 41,115 contigs, and 100,889 singletons. A NimbleGen microarray constructed and used in a C3 species (Oncidium maduroi), a weak CAM species (Oncidium panamense) and a strong CAM species (Rossioglossum ampliatum), showed that C3 and weak CAM species had average hybridization intensities that diverged from the strong CAM species by 2 and 3 percent, respectively. From 13,566 genes that showed a significant 4.6-fold difference in expression levels from the comparisons between CAM, C3 and weak CAM, 4,520 genes showed a greater than 4.6-fold increase in the ratio of CAM/C3 relative transcript abundance, whereas 3,745 genes showed a greater than 4.6- fold decrease in the ratio of CAM/C3 relative transcript abundance. A maximal increase or decrease in relative transcript abundance of more than 1,000- and 500-fold, respectively, was observed. The results of the microarray analysis will serve as a catalogue of gene expression patterns available for future work aimed at understanding CAM specific expression patterns, and can be used to further understand gene regulation by in-depth analysis of the transcriptional control regions responsible for altered gene expression patterns associated with CAM evolution. Several patterns of CAM evolution have been demonstrated in orchids, thus improving our understanding of the functional significance and evolutionary origins of CAM. The results of this project will aid in understanding photosynthetic plasticity in plants