SUBCELLULAR STRUCT OF PHAEOCYSTIS AND ITS RELATIONSHIP TO ABSORBTION PROPERTIES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。海洋颜色遥感的出现为海洋学家提供了在全球尺度上对浮游植物色素的时空变化进行天气观测的能力。通过使用生物光学算法，通过了解叶绿素特异性吸收系数、量子产率和入射辐照度，可以从色素生物量估算出总初级产量。最近的几项研究表明，最终影响浮游植物光学特性的细胞特性随着温度、光照和营养限制而发生可预测的变化。实际的亚细胞吸收是在叶绿体类囊体中完成的，光途径的长度是由细胞内部细胞器的分布决定的。在之前的透射电镜研究中，结果表明类囊体的堆叠与光适应有关，当生长在14和259¿mol量子m-2S-1时，类囊体的堆叠变化接近2倍。浮游植物的吸收特性先前已经使用基于单细胞直径和细胞物质吸收的Mie理论建模。该理论假定浮游植物细胞是球形的，色素沉着均匀地分布在整个细胞中。因为吸收是在叶绿体的细胞水平上起作用的，我们选择重建藻类的顶叶叶绿体，南极Phaeocystis。目前的叶绿体形态模型是用透射电子显微镜从薄片中得到的。叶绿体被描述为一个简单的细胞器，它包含与叶绿体膜平行的类囊体膜。目前，我们对叶绿体超微结构的认识仅限于x和y两个方向。本文首次利用全球海洋重要生物南极Phaeocystis - prymnesiophyte的培养物，阐明了叶绿体的三维结构。利用厚截面（1/4 - 3/4 m），我们计算了在两种光照条件下生长的细胞的层析重建，低光照条件（14 &#61549；mol量子m-2 s_1）和高光照条件（259 &#61549；mol量子m-2 s-1），以便了解类囊体膜在极端光照条件下的适应性和连续性。我们也希望了解类芘、含淀粉细胞器和类囊体膜之间的空间关系。在这两种条件下，我们收集了大约28个倾斜序列，并对每种条件下生长的藻类的叶绿体进行了完全重建或分析，这些数据集中大约有5-6个是最好的。我们的研究结果表明，叶绿体是一个复杂的细胞器，它包含连续的横向类囊体膜，这些膜围绕类核蛋白流动。在弱光条件下，叶绿体材料密度较低、堆积周期较高的两种处理下，类囊体膜的表面积和堆积周期发生了显著变化。单个褐囊藻细胞的三维重建是高度复杂的。不同于先前描述的在二维空间中使用薄片的叶绿体的软糖状结构，在类囊体的叶绿体排列与类核蛋白的关系中出现了一个新的视角。以前发表的显微照片强调了类囊体与叶绿体膜的平行性质。相比之下，我们的层析重建揭示了一个高度进化的细胞器，它在x-y维度上显示出复杂的模式，在z维度上是连续的。类囊体通常与叶绿体膜平行，但在三维或z方向上观察，类囊体呈扭曲状。这种排列与线粒体的排列相反，线粒体的嵴通常垂直于线粒体的长轴。尽管如此，类囊体经常破裂或合并到其他类囊体中并形成复杂的结构。我们观察到类囊体的双功能和三功能（在x-y方向），它们在z方向上是连续的。类囊体从一个类囊体分支发出，分支分成几个分支，并在整个z方向上持续存在。这种结构似乎是独立于叶绿体内其他类囊体形成的结构。我们目前还不能假设这些叶绿体膜接点的性质或功能可能起什么作用。这些观察结果与在线粒体中观察到的相似，在线粒体中，嵴通过嵴连接连接到内膜(Perkins等，（1997）J Struct Biol. 119:260-72；Mannella et .,（1994）中国生物医学工程学报，27:278-283)。除了类囊体的新形态特征外，我们还观察到Phaeocystis的一些细胞特征，这些特征可能有助于其生态成功。我们看到在弱光和强光处理下生长的细胞在整体形态上有显著差异。在它们的整体形态上，与在低光照下生长的细胞相比，在高光照下有更多的细胞成分（高尔基体、类囊体和囊泡）的堆积。一般来说，我们发现在弱光条件下，单个堆叠中的类囊体在每个叶绿体中堆积得更密集，并且与每个细胞中增加的色素相称（Moisan和Mitchell (1999) Limnol）。海洋学报，44(4):247-258)和更小的孔直径。这些细胞特征的结合导致了色素包装的增加，正如在低辐照下与高辐照下相比，色素包装参数Q*a （Moisan和Mitchell， 1999）的值更大所观察到的那样。相比之下，在强光处理下，我们看到的类囊体堆积较少，而类pyrenoid体（在叶绿体中央发现的含淀粉的细胞器）似乎更分散。这对细胞的吸收特性有深远的影响，因为类pyrenox是一种高度散射的细胞器，可能导致路径长度放大。

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.