title: Visualization and characterization of prolamellar bodies with atomic force microscopy.
abstract: Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1-2μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl2 also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.
authors.affiliation: Laboratory of Biological Physics, Institute of Physics PAS, al. Lotników 32/46, 02-668 Warsaw, Poland.
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Grzyb
authors.forename: Katalin
authors.initials: K
authors.lastname: Solymosi
authors.forename: Kazimierz
authors.initials: K
authors.lastname: Strzałka
authors.forename: Beata
authors.initials: B
authors.lastname: Mysliwa-Kurdziel
mesh_terms: D002735:Chlorophyllides / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism* / Q000648:ultrastructure; D003624:Darkness; D008627:Mercuric Chloride / Q000633:toxicity; D018625:Microscopy, Atomic Force / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism; D018515:Plant Leaves / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D010940:Plant Proteins / Q000378:metabolism; D011107:Polylysine / Q000378:metabolism; D011521:Protochlorophyllide / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014908:Triticum / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D002735:Chlorophyllides; D010940:Plant Proteins; D011521:Protochlorophyllide; D011107:Polylysine; D008627:Mercuric Chloride; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase
keywords: AFM; Chlide; F655/F633; Fluorescence spectra; LPOR; PLB; PLBs; PT; Pchlide; Protochlorophyllide photoreduction; TEM; atomic force microscopy; chlorophyllide; light-dependent protochlorophyllide oxidoreductase; prolamellar bodies; prothylakoids; protochlorophyllide; the ratio of the fluorescence intensity at 655nm to the intensity at 633nm; transmission electron microscopy