Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing

Abstract

High-pressure freezing and freeze substitution were used to prepare leaves of rice (Oryza sativa L.) for ultrastructural analysis. Under these preparative conditions, plastid-derived stroma-containing protuberances were preserved and described with the electron microscope for the ®rst time. Similar protuberances were observed previously only in living cells examined with the light microscope. Infoldings of the chloroplast inner envelope were a prominent ultrastructural feature of protuberances. Infoldings were also observed in the main body of the chloroplasts and sometimes appeared contiguous with thylakoid membranes. Protuberances also contained infoldings in the form of bifurcated tubules. Apparent interconnection between protuberances of adjacent plastids was observed only in one instance. A distinct gradient in the staining density of thylakoid lumina appeared to be a function of grana position and orientation relative to the cell wall. Immunocytochemistry was used to determine that the stroma within protuberances contained 1,5-bisphosphate carboxylase/oxygenase enzyme.

Roles for Rice Membrane Dynamics and Plasmodesmata during Biotrophic Invasion by the Blast Fungus

“…Previously published discrepancies in the presence of a plant membrane surrounding blast IH may have been attributable to challenges in preserving membranes with conventional chemical fixatives used for TEM in these studies (Koga and Horino, 1984b;Heath et al, 1992). Even though the HPF/FS TEM used in our study is better for preserving membranes, artifactual breaks in membranes still occur (Bourett et al, 1999). Therefore, TEM images alone could not prove that the EIHM was continuous around IH, especially for extensive IH formed in the later stages of cell colonization.…”

“…This microscopic analysis involved >950 independent infection sites using four different fluorescent probes. Key results were confirmed using TEM with HPF/FS samples, which provided improved preservation of membranes compared with conventional chemical fixation methods (Bourett et al, 1999;Mims et al, 2004). We documented an amazing degree of plasticity in the intracellular biotrophic IH responsible for rice blast disease.…”

Stromules: Recent Insights into a Long Neglected Feature of Plastid Morphology and Function

“…Due to greater ease of generation of stable transgenic lines expressing fluorescent protein, dicot model systems such as Nicotiana and Arabidopsis have most often been used for studies of stromules, but they have also been observed by light or electron microscopy in monocots such as onion (Allium cepa), maize (Zea mays), iris (Iris sp. ), rice (Oryza sativa), and wheat (Triticum aestivum; Bourett et al, 1999;Langeveld et al, 2000;Gray et al, 2001;Gunning, 2005;Sattarzadeh et al, 2010;Hanson and Sattarzadeh, 2011). The presence of stromules is not limited to vascular plants; they have been described in Euglena, Acetabularia, and in a variety of lower plants in the prefluorescent protein literature (for review, see Gray et al, 2001;Kwok and Hanson, 2004a).…”

Plastid tubules of higher plants are tissue-specific and developmentally regulated

“…Though some of the previously reports of extensions may be artifact, a number of the images appear to be high quality and are consistent with our observations at the light microscopy level. Furthermore, chloroplast protuberance in rice leaves were observed when Bourett et al (1999) preserved cells by high-pressure freezing and freeze substitution. Two factors have probably contributed to the paucity of electron micrographs of stromules.…”