Implications of leaf anatomy and stomatal responses in the Clusia genus for the evolution of Crassulacean acid metabolism

Abstract

The tropical genus Clusia which contains species with C3, Crassulacean acid metabolism and C3/CAM properties is an interesting model to dissect anatomical, physiological and molecular traits that underpin the evolution of CAM photosynthesis. About 7 % of higher plants perform CAM, a specialised photosynthetic pathway, characterized by CO2 uptake during the night mediated via the enzyme phosphoenolpyruvate carboxylase (PEPC) and keeping stomata shut for much of the day. CAM confers high water use efficiency (WUE) and has long been considered an adaptation to drought stress and high irradiance. In some species like Clusia CAM facilitates remarkable photosynthetic plasticity in dealing with changing environments. Thus, CAM plants are important and challenging model organisms for investigating plant responses to global climate change and for examining the anatomical and physiological traits that underpin enhanced water use efficiency. In this thesis strong relationships were found between the magnitude of CAM photosynthesis in eight species of Clusia (C. hilariana, C. alata, C. rosea, C. lanceolata, C. aripoensis, C. grandiflora, C. tocuchensis and C. multiflora) and six leaf anatomical traits (stomatal size, stomatal density, % intercellular air space, length of mesophyll exposed to air space, cell size and specific leaf area) . These relationships point to leaf anatomical features as important in the evolution of CAM, and also have implications for the behaviour of stomata and their response to light. It was found that CAM species of Clusia have lower densities of larger stomata compared with C3 species of Clusia. The CAM species of Clusia still maintain a high WUE and it was hypothesised that this was a consequence of robust circadian control of stomatal conductance which was maintained under different light regimes. To examine the response of CAM stomata to contrasting light regimes and to test if circadian oscillations in stomatal conductance are disrupted under different wavelengths of light, gas exchange measurements were recorded during 48 hours under constant light regimes (either white light, blue light, red light or darkness) for C. rosea a constitutive CAM plant, and C. multiflora, a constitutive C3 plant. It was found that the species responded differently to variation in light regimes and the response of stomata to blue light in the CAM Clusia had not been lost, as proposed by previous workers. The larger stomata of C. rosea responded faster to changes in light intensity during the photoperiod compared with those of C. multiflora, but this did not happen during the night. It was hypothesised that the kinetic responses of the CAM stomata might be important for optimising carbon gain and reducing water loss under changing environmental conditions at the start and end of the day.. Furthermore, circadian control of stomatal conductance was found to be mediated by both photoreceptors and metabolism, including photosynthesis and carbohydrate metabolism in CAM and C3 Clusia plants. A molecular approach was taken to probe the mechanisms underpinning the contrasting responses to light. The differential transcript abundance, of photoreceptors involved in stomatal opening (phototropin 1 and phototropin 2) and circadian regulation (cryptochrome 2 and phytochrome A) was examined for C. rosea and C. multiflora under different constant light regimes using semi-quantitative reverse transcription- PCR and Real Time PCR. Diel expression patterns of phototropins were found to differ between the C3 and CAM species in terms of transcript abundance, the level of control exerted by circadian clock over the transcripts and the response of transcripts to different light regimes. It was concluded that stomatal responses to light in Clusia species must be mediated by a coordinated labour of different photoreceptors to exert control over water loss and CO2 assimilation. Further work is required to assess the expression and regulation of photoreceptors at the stomatal guard cell level. Having more knowledge regarding the function of stomata in CAM plants and their implications for WUE should help inform efforts for improving the water use of crop species in the light of environmental challenges such as desertification and global warming.