Summary The plant endoplasmic reticulum (ER) is crucial to the maintenance of cellular homeostasis. The ER consists of a dynamic and continuously remodelling network of tubules and cisternae. Several conserved membrane proteins have been implicated in formation and maintenance of the ER network in plants, such as RHD3 and the reticulon proteins. Despite the recent work in mammalian and yeast cells, the detailed molecular mechanisms of ER network organization in plants remain largely unknown. Recently, novel ER network‐shaping proteins called Lunapark (LNP) have been identified in yeast and mammalian cells.Here we identify two Arabidopsis LNP homologues and investigate their subcellular localization via confocal microscopy and potential function in shaping the ER network using protein–protein interaction assays and mutant analysis.We show that AtLNP1 overexpression in tobacco leaf epidermal cells mainly labels cisternae in the ER network, whereas AtLNP2 labels the whole ER. Overexpression of LNP proteins results in an increased abundance of ER cisternae and lnp1 and lnp1lnp2 amiRNA lines display a reduction in cisternae and larger polygonal areas.Thus, we hypothesize that AtLNP1 and AtLNP2 are involved in determining the network morphology of the plant ER, possibly by regulating the formation of ER cisternae.

In non-plant systems, chromatin association with the nuclear periphery affects gene expression, where interactions with nuclear envelope proteins can repress and interactions with nucleoporins can enhance transcription. In plants, both hetero- and euchromatin can localize at the nuclear periphery, but the effect of proximity to the nuclear periphery on gene expression remains largely unknown. This study explores the putative function of Seh1 and Nup50a nucleoporins on gene expression by using the Lac Operator / Lac Repressor (LacI-LacO) system adapted to Arabidopsis thaliana. We used LacO fused to the luciferase reporter gene (LacO:Luc) to investigate whether binding of the LacO:Luc transgene to nucleoporin:LacI protein fusions alters luciferase expression. Two separate nucleoporin-LacI-YFP fusions were introduced into single insert, homozygous LacO:Luc Arabidopsis plants. Homozygous plants carrying LacO:Luc and a single insert of either Seh1-LacI-YFP or Nup50a-LacI-YFP were tested for luciferase activity and compared to plants containing LacO:Luc only. Seh1-LacI-YFP increased, while Nup50a-LacI-YFP decreased luciferase activity. Seh1-LacI-YFP accumulated at the nuclear periphery as expected, while Nup50a-LacI-YFP was nucleoplasmic and was not selected for further study. Protein and RNA levels of luciferase were quantified by western blotting and RT-qPCR, respectively. Increased luciferase activity in LacO:Luc+Seh1-LacI-YFP plants was correlated with increased luciferase protein and RNA levels. This change of luciferase expression was abolished by disruption of LacI-LacO binding by treating with IPTG in young seedlings, rosette leaves and inflorescences. This study suggests that association with the nuclear periphery is involved in the regulation of gene expression in plants.

The actin cytoskeleton is the driver of gross ER remodelling and the movement and positioning of other membrane-bound organelles such as Golgi bodies. Rapid ER membrane remodelling is a feature of most plant cells and is important for normal cellular processes, including targeted secretion, immunity and signalling. Modifications to the actin cytoskeleton through pharmacological agents such as Latrunculin B and phalloidin, or disruption of normal myosin function also affect ER structure and/or dynamics. Here, we investigate the impact of changes in the actin cytoskeleton on structure and dynamics on the ER as well as in return the impact of modified ER structure on the architecture of the actin cytoskeleton. By expressing actin markers that affect actin dynamics, or expressing of ER-shaping proteins that influence ER architecture, we found that the structure of ER-actin networks is closely inter-related; affecting one component is likely to have a direct effect on the other. Therefore, our results indicate that a complicated regulatory machinery and cross-talk between these two structures must exist in plants to co-ordinate the function of ER-actin network during multiple subcellular processes. In addition, when considering organelle structure and dynamics, the choice of actin marker is essential in preventing off-target organelle structure and dynamics modifications.