CSIRO PUBLISHING www.publish.csiro.au/journals/fpb Functional Plant Biology, 2007, 34, 247–267 Physiological, anatomical and biochemical characterisation of photosynthetic types in genus Cleome (Cleomaceae) Elena V. Voznesenskaya A , Nuria K. Koteyeva A , Simon D. X. Chuong B , Alexandra N. Ivanova A , Jo ˜ ao Barroca C , Lyndley A. Craven D and Gerald E. Edwards C,E A Laboratory of Anatomy and Morphology, V. L. Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov Street 2, 197376, St Petersburg, Russia. B Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. C School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA. D Australian National Herbarium, Centre for Plant Biodiversity Research, GPO Box 1600, Canberra, ACT 2601, Australia. E Corresponding author. Email: edwardsg@wsu.edu This paper originates from an International Symposium in Memory of Vincent R. Franceschi, Washington State University, Pullman, Washington, USA, June 2006. Abstract. C 4 photosynthesis has evolved many times in 18 different families of land plants with great variation in leaf anatomy, ranging from various forms of Kranz anatomy to C 4 photosynthesis occurring within a single type of photosynthetic cell. There has been little research on photosynthetic typing in the family Cleomaceae, in which only one C 4 species has been identified, Cleome gynandra L. There is recent interest in selecting and developing a C 4 species from the family Cleomaceae as a model C 4 system, since it is the most closely related to Arabidopsis,aC 3 model system (Brown et al. 2005). From screening more than 230 samples of Cleomaceae species, based on a measure of the carbon isotope composition (δ 13 C) in leaves, we have identified two additional C 4 species, C. angustifolia Forssk. (Africa) and C. oxalidea F.Muell. (Australia). Several other species have δ 13 C values around -17‰ to -19‰, suggesting they are C 4 -like or intermediate species. Eight species of Cleome were selected for physiological, anatomical and biochemical analyses. These included C. gynandra, a NAD–malic enzyme (NAD–ME) type C 4 species, C. paradoxa R.Br., a C 3 –C 4 intermediate species, and 6 others which were characterised as C 3 species. Cleome gynandra has C 4 features based on low CO 2 compensation point (Ŵ), C 4 type δ 13 C values, Kranz-type leaf anatomy and bundle sheath (BS) ultrastructure, presence of C 4 pathway enzymes, and selective immunolocalisation of Rubisco and phosphoenolpyruvate carboxylase. Cleome paradoxa was identified as a C 3 –C 4 intermediate based on its intermediate Ŵ (27.5 μmol mol -1 ), ultrastructural features and selective localisation of glycine decarboxylase of the photorespiratory pathway in mitochondria of BS cells. The other six species are C 3 plants based on Ŵ, δ 13 C values, non-Kranz leaf anatomy, and levels of C 4 pathway enzymes (very low or absent) typical of C 3 plants. The results indicate that this is an interesting family for studying the genetic basis for C 4 photosynthesis and its evolution from C 3 species. Additional keywords: C 3 plants, C 4 plants, C 3 –C 4 intermediate photosynthesis, chloroplast ultrastructure, immunolocalisation, NAD–ME type, photosynthetic enzymes. Introduction The genus Cleome s.l., which includes more than 200 species according to some classifications, is a member of the family Cleomaceae. Cleomaceae, which is pantropical in distribution and conspicuous in tropical, seasonally dry habitats, is sister to families Capparaceae and Brassicaceae based on recent phylogenetic studies (Hall et al. 2002; Sanchez-Acebo 2005). Arabidopsis thaliana L., a C 3 species in the family Brassicaceae, is an established model system for studies in plant biology. Brown et al. (2005) proposed developing a C 4 model system with a C 4 species from the family Cleomaceae, which among the known occurrence of C 4 in several families would be most closely related to Arabidopsis. This would allow comparisons of genomes and molecular analyses between the two systems in efforts to identify the genes required for development of C 4 photosynthesis. However, there has been little research on leaf anatomy and the types of photosynthesis among members of the family Cleomaceae. Sankhla et al. (1975) reported that Gynandropsis pentaphylla (DC.) had C 4 -type carbon isotope composition and Kranz leaf anatomy. In the same year, Gynandropsis speciosa DC. was reported to have a low CO 2 compensation point (Ŵ), characteristic of © CSIRO 2007 10.1071/FP06287 1445-4408/07/040247