硅藻是地球上最重要的浮游植物之一。它们包含至少一万个物种,占全球初级生产的20%。由于一系列内共生事件和水平基因转移,硅藻已经形成了一个“次级质体”,由四个含有一个大的相分离室的膜包围,称为pyrenoid。然而,这种独特的叶绿体形态的生理意义尚不清楚。基本生理参数的表征,例如各种亚细胞区室中的局部pH值,应有助于更好地了解次级质体独特结构的生理作用。估计局部pH的一种有前途的方法是pH敏感的绿色荧光蛋白的原位表达。这里,我们首先开发了一种分子工具,用于通过在细胞质中异源表达pHFloriorin2来绘制硅藻毛指藻的原位局部pH,潮周体区室(PPC;两组内外叶绿体包膜之间的空间),叶绿体基质,和拟核蛋白矩阵。我们的数据表明,PPC和类蛋白基质比相邻区域更酸性,细胞质和叶绿体基质。最后,从重组pHluorin2蛋白的比率荧光估计每个区室的绝对pH值,pH值分别约为7.9、6.8、8.0和7.5,对于细胞质,PPC,基质,和Tranornutum细胞的类蛋白,指示pH梯度的发生以及其边界处相关的电化学电势。
Diatoms are one of the most important phytoplankton on Earth. They comprise at least ten thousand species and contribute to up to 20% of the global primary production. Because of serial endosymbiotic events and horizontal gene transfers, diatoms have developed a \"secondary plastid\" bounded by four membranes containing a large phase-separated compartment, termed the pyrenoid. However, the physiological significance of this unique chloroplast morphology is poorly understood. Characterization of fundamental physiological parameters such as local pH in various subcellular compartments should facilitate a greater understanding of the physiological roles of the unique structure of the secondary plastid. A promising method to estimate local pH is the in situ expression of the pH-sensitive green fluorescent protein. Here, we first developed the molecular tool for the mapping of in situ local pH in the diatom Phaeodactylum tricornutum by heterologously expressing
pHluorin2 in the cytosol, periplastidal compartment (PPC; the space in between two sets of outer and inner chloroplast envelopes), chloroplast stroma, and the pyrenoid matrix. Our data suggested that PPC and the pyrenoid matrix are more acidic than the adjacent areas, the cytosol and the chloroplast stroma. Finally, absolute pH values at each compartment were estimated from the ratiometric fluorescence of a recombinant
pHluorin2 protein, giving pH values of approximately 7.9, 6.8, 8.0, and 7.5 respectively, for the cytosol, PPC, stroma, and pyrenoid of the P. tricornutum cells, indicating the occurrence of pH gradients and the associated electrochemical potentials at their boundary.