This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The advent of Ocean Color remote sensing has provided oceanographers with the ability to observe synoptically the temporal and spatial variability of phytoplankton pigments on a global scale. Through the use of bio-optical algorithms, gross primary production estimates can be estimated from pigment biomass by knowing the chlorophyll-specific absorption coefficient, quantum yield and incident irradiance. Several recent studies have shown that cellular characteristics, which ultimately affect the optical properties of phytoplankton, vary predictably with temperature, light, and nutrient limitation. Actual subcellular absorption is accomplished in the chloroplast thylakoids and the length of the pathway of light is determined by the distribution of internal cellular organelles. In a previous study using transmission electron microscopy, results have shown that thylakoid stacking is related to photoadaptation and showed a near 2 fold change in thylakoid stacking when grown at 14 and 259 mol quanta m-2S-1. The absorption properties of phytoplankton have been previously modeled using Mie theory that is based on the single cell diameter and the absorption of cellular material. The theory assumes that phytoplankton cells are spherical and pigmentation is homogeneously distributed throughout the cell. Because absorption acts at the cellular level within a chloroplast, we have chosen to reconstruct the parietal chloroplasts of the alga, Phaeocystis antarctica. Current models of chloroplast morphology have been derived from thin sections using transmission electron microscopy. The chloroplast is depicted as a simple organelle, which contains thylakoid membranes, which run parallel to the chloroplast membrane. Presently, our understanding of the chloroplast ultrastructure is limited to two dimensions in the x and y directions. We use cultures of the colonial prymnesiophyte, Phaeocystis antarctica, which is an important organism in the global ocean to elucidate the three dimensional structure of the chloroplast for the first time. Using thick sections (1/4 - 3/4 m), we calculate tomographic reconstructions of cells grown under two light conditions, low light (14 mol quanta m-2 s_1) and high irradiance (259 mol quanta m-2 s-1), in order to gain an understanding of the adaptation and continuity of thylakoid membranes in response to extremes in light conditions. We also wish to understand the spatial relationship between the pyrenoid, the starch containing organelle, and thylakoid membranes. Between these two conditions, we collected about 28 tilt series and have fully reconstructed or analyzed the chloroplasts in algae grown about 5-6 of the best of these data sets for each of the two conditions. Our results show that the chloroplast is a complex organelle, which contains continuous transverse thylakoid membranes that flow around the pyrenoid. We observed significant variations in the surface areas and stacking periodicities of thylakoid membranes for the two treatments with higher stacking periodicities and lower density of chloroplast material at low light. The three dimensional reconstructions of the single Phaeocystis cells were highly complex. Unlike the jellybean like structures of previous depictions of chloroplasts derived from perspectives in 2 dimensional space using thin sections, a new perspective in the chloroplast arrangement of thylakoids in relationship to the pyrenoid emerged. Previously published micrographs have emphasized the parallel nature of the thylakoids to the chloroplast membrane. In contrast, our tomographic reconstructions reveal a highly evolved organelle which shows complex patterns in the x-y dimension that are continuous in the z-dimension. Thylakoids generally ran parallel to the chloroplast membrane but observations in the third dimension or z-direction revealed a twisted pattern of the thylakoid. This arrangement is opposite to that found in mitochondria, where the cristae are often perpendicular to the long axis of the mitochondria. Nonetheless, the thylakoids were often seen to break or merge into other thylakoids and form complex structures. We observed bi- and tri-furcations in the thylakoids (in the x-y direction), which were continuous in the z-direction. The thylakoids emanate from one thylakoid branch which bifurcates into several branches and is persistent throughout the z-direction. This structure appears to be an independently formed structure from the other thylakoids within the chloroplast. We are unable to hypothesize at this time what the nature or function of these junctures to the chloroplast membranes might serve. These observations parallel those observed in mitochondria where cristae connect to the inner membranes via cristae junctions (Perkins et al., (1997) J Struct Biol. 119:260-72; Mannella et al., (1994) Microsc Res Tech, 27: 278-283). In addition to new morphological features of thylakoids, we observed several cellular characteristics of Phaeocystis, which may potentially contribute to its ecological success. We saw significant differences in the overall morphology between the cells grown at the low light and high light treatment. In their overall morphology, there is greater packing of cellular components (Golgi, thylakoids, and vesicles) at the high irradiances as compared to those grown at low light levels. In general, we found that the thylakoid stacking within a single stack is much denser per chloroplast under low light conditions and is commensurate with increased pigment per cell (Moisan and Mitchell (1999) Limnol. Oceanogr. 44: 247-258) and smaller cell diameters. The combination of these cellular characteristics led to increased pigment packaging as observed by greater values in the pigment packaging parameter, Q*a (Moisan and Mitchell, 1999) at low irradiances compared to higher irradiances. In contrast, we saw fewer thylakoid stacks under the high light treatment and the pyrenoid body, the starch containing organelle found centrally within the chloroplast, appeared to be much more diffuse. This has profound implications for the absorption properties of the cells because the pyrenoid is a highly scattering organelle that may lead to path length amplification.
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