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Multiphoton Microscopy
Multiphoton Microscopy
Characterization of Ozone-Sensitive Arabidopsis thaliana Mutants with Confocal Laser Scanning Microscopy
Carina Barth*, Jonas Korlach**, Watt W. Webb**, Patricia Conklin*,
*Boyce Thompson Institute for Plant Research, Tower Road, **School of Applied Physics, Clark Hall, Ithaca, New York 14853
Aerobic organisms such as plants and animals require oxygen for life. But oxygen can also be harmful when reactive oxygen species (ROS) are formed. ROS are deleterious because they can oxidize biomolecules, e.g. lipids, pigments and proteins. In plants, ROS are generated by photosynthesis and respiration. The production of ROS is enhanced under severe stress conditions, e.g. strong light, heat, drought or air pollutants (oxidative stress). Plants possess a complex defense system in order to remove these potentially damaging oxygen species.
To provide a better understanding of protective mechanisms that plants utilize to detoxify harmful oxygen species, Arabidopsis thaliana mutants sensitive to ozone (a generator of ROS in planta) are studied. Mutants were generated by ethyl methanesulfonate (Barczak et al. 1995) and were isolated using a screen for ozone-sensitivity (Conklin et al. 1996). The sensitive to ozone mutant, soz2, was picked for further analyses. Soz2 exhibits a pale phenotype under normal growth conditions (approximately 100 µmol m-2 s-1 photosynthetic active radiation). This paleness is inter-veinal, i.e. leaf areas are a pale green in comparison to wild-type. When treated with ozone (250 ppb/8h), soz2 leaves become even more pale and have a bleached appearance, indicating that soz2 suffered from oxidative stress. It has been suggested that the pale phenotype in soz2 is due to a defect in chlorophyll biosynthesis or a defect in protective pigments and that soz2 is therefore more susceptible to photooxidative stress than wild-type. However, photosynthetic parameters determined under photooxidative stress conditions (e.g. high light in combination with low temperature) were almost identical in wild-type and soz2. Moreover, soz2 and wild-type were comparable in their chloroplast pigment composition, except for a significant lower content of total chlorophyll in soz2.
To elucidate the soz2 defect, confocal laser scanning microscopy was employed. Confocal microscopy three-dimensional image reconstructions revealed that soz2 leaves compared to wild-type leaves were characterized by a lower cell density per leaf area unit, whereas the number of chloroplasts per cell was not altered. These data suggest that chlorophyll biosynthesis is not directly affected in soz2, as previously presumed. The obvious reduction in chlorophyll and thus the pale phenotype of soz2 is most likely due to a reduction in cells per leaf area unit. Based on these data it is speculated that the gene product encoded by soz2 is involved in cell or cell wall development. In order to answer this question, multiphoton microscopy will be used to investigate the cell wall. Several dyes, such as sirofluor, DAPI, calcofluor white, will be tested to identify alterations in the cell wall of soz2. These experiments will aid to understand the physiological and biochemical defect of soz2, and will thus help to identify the soz2 mutation.
Figures:
References:
Barczak A.J., Zhao J., Pruitt K.D. and Last R.L. (1995), "5-Fluoroindole resistance identifies tryptophan synthase beta subunit mutants in Arabidopsis thaliana," Genetics 140, 303-313.
Conklin P. L., Williams E. H. and Last R. L. (1996), "Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant," Proc. Natl. Acad. Sci. USA 93, 9970-9974.
