3D electron tomographic and biochemical analysis of ER, Golgi and trans Golgi network membrane systems in stimulated Venus flytrap (Dionaea muscipula) glandular cells

Abstract

BackgroundThe insect-trapping leaves of Dionaea muscipula provide a model for studying the secretory pathway of an inducible plant secretory system. The leaf glands were induced with bovine serum albumin to secrete proteases that were characterized via zymogram activity gels over a 6-day period. The accompanying morphological changes of the endoplasmic reticulum (ER) and Golgi were analyzed using 3D electron tomography of glands preserved by high-pressure freezing/freeze substitution methods.ResultsSecretion of multiple cysteine and aspartic proteases occurred biphasically. The majority of the Golgi was organized in clusters consisting of 3–6 stacks surrounded by a cage-like system of ER cisternae. In these clusters, all Golgi stacks were oriented with their cis-most C1 cisterna facing an ER export site. The C1 Golgi cisternae varied in size and shape consistent with the hypothesis that they form de novo. Following induction, the number of ER-bound polysomes doubled, but no increase in COPII vesicles was observed. Golgi changes included a reduction in the number of cisternae per stack and a doubling of cisternal volume without increased surface area. Polysaccharide molecules that form the sticky slime cause swelling of the trans and trans Golgi network (TGN) cisternae. Peeling of the trans-most cisternae gives rise to free TGN cisternae. One day after gland stimulation, the free TGNs were frequently associated with loose groups of oriented actin-like filaments which were not seen in any other samples.ConclusionsThese findings suggest that the secretory apparatus of resting gland cells is “overbuilt” to enable the cells to rapidly up-regulate lytic enzyme production and secretion in response to prey trapping.Electronic supplementary materialThe online version of this article (10.1186/s40709-018-0086-2) contains supplementary material, which is available to authorized users.

Functional organisation of the endomembrane network in the digestive gland of the Venus flytrap: revisiting an old story with a new microscopy toolbox

“…In accordance with the typical organisation of plant secretory glands (Luttge, 1971), three cell types may be observed: basal cells, which are inserted in the epidermis; endodermoid cells; and secretory cells involved in the synthesis and release of digestive fluid (see also fig. 1B in Gergely et al, 2018).…”

“…Do these vesicles derive from Golgi stacks or are they derived from other structures that could not be well-established in these 2D micrographs? One hypothesis is that they may correspond to trans-Golgi network (TGN) elements described in Gergely et al (2018). Tubular profiles that associate with those vesicular structures profiles were also observed (Figs.…”

“…Data processing (stack formation, image alignment, and trimming and scaling of sections to a common mean and SD) was performed using the IMOD software package (Kremer et al, 1996). The Amira software (Thermo) brush tool was used to select the regions of interest around the Golgi and ER, and the VolRen function was applied and thresholded to show maximum intensity 3D views of Golgi bodies and ER (Borrett & Hughes, 2016;Gergely et al, 2018).…”

“…(F) 2D + T = 2D study comparing three stages of the digestive cycle over time (T). Broken lines: see complementary protocol for TEM-tomography in Gergely et al (2018).…”

“…However, the preference for cryofixation methods over chemical fixation has been emphasised in the last 30 years in order to obtain a better preservation of plant membranes and fewer ultrastructural artefacts in plant cells (Kiss et al, 1990;Samuels et al, 1995;Seguí-Simarro et al, 2004) including Dionaea muscipula (Gergely et al, 2018). In addition, the development of methods to reconstruct a volume from 2D images (Hughes et al, 2014;Borrett & Hughes, 2016;Kittelmann et al, 2016) has been essential to revisiting or improving working hypotheses on cell functional organisation and may therefore be a good tool to explore cell architecture of carnivorous plants.…”