A Once-Hidden Endoplasmic Reticulum Matrix Reveals the Totally Tubular Function of LUNAPARKs in Plants

The endoplasmic reticulum (ER) is an organelle that is vital for many cellular processes, such as protein and lipid synthesis, calcium signaling, detoxification, and movement of other organelles. It forms an intricate meshwork in the cell cortex that iscomprisedofhighlydynamic interconnected tubules and cisternae (“sheets”) that are continually rearranging. In plants, ROOT HAIR DEFECTIVE3 (RHD3), like its homologs in animals and yeast (Saccharomyces cerevisiae), is an ER membrane-localized GTPase and is one player involved in the tight regulation of ER shape that is required for its proper function and normal organismal development (Chen et al., 2011). Overexpression of these GTPases causes excessive fusion of ER tubules. In animals and yeast, the Lunapark (Lnp) protein seems to counteract their action by stabilizing ER tubules. However, the role of plant LUNAPARK (LNP) homologs was debated— until now. Tubules and cisternae have long been viewed as diametrical but continuous and intermorphing structures of the ER, whose proportions change depending on developmental stage and in response to environmental stimuli. Compared to the wild type, lnpmutants display cortical ER that seems to have fewer sheets and larger intertubule spaces (lacunae), whereas overexpression of Arabidopsis (Arabidopsis thaliana) LNP1 or LNP2 in tobacco (Nicotiana tabacum) causes an increase in ER sheets. It was therefore hypothesized that LNPs raise the ratio of sheets to tubules (Kriechbaumer et al., 2018), a role opposite to that of the animal and yeast homologs. Additionally, because of the presence of large clumps of ER membrane in lnp mutants, it was hypothesized that LNPs play a role in the proper distribution of ER throughout the cell (Ueda et al., 2018). The controversy surrounding the function of LNPs in plants stemmed from the previously unrecognized and difficult-todecipher morphology of the ER in lnp mutants.Drawing from thedemonstration with super-resolution microscopy in animal cells thatmanyER sheets are actually clusters of dense tubules that forman “ER matrix” (Nixon-Abell et al., 2016), Sun et al. (2020) have now overcome this barrier by employing Airyscan superresolution and transmission electron microscopy techniques. They established here that lnp mutants exhibit a densely packed ER network, both in the cytosolic ER clumps and in the areas once considered to be closed ER sheets (see figure). The authors also clarified the relationship between LNPs and RHD3. Although the phenotypes were not identical, the lnp1-1 lnp2-1 double mutant, like rhd3 mutants, had shorter root hairs and a smaller overall size compared to the wild type. In addition, interactions between RHD3 and LNP1 or LNP2 proteins were robustly shown using variousmethods. More importantly, the authors reconcile previous subcellular localization results and unveil a previously cryptic finding that, as in animals, LNPs do indeed localize to three-way ER junctions—they just need to be in relatively equal proportions to RHD3 to do so. When RHD3 levels were high, either endogenously in the lnp1-1 lnp2-1mutant (as demonstrated via immunoblot analysis) or by overexpression, the ER formed a dense tubular network in the form of “sheets.” In lnp1-1 and lnp2-1 plants complemented with LNP1pro:LNP1-YFP and LNP2pro:LNP2-YFP, respectively, LNP1 or LNP2 was concentrated in LNPs Stabilize and Localize to Three-Way ER Junctions.