Inorganic phosphate (Pi) is essential for plant growth and is acquired and distributed by the plasma membrane PHOSPHATE TRANSPORTER 1 proteins (PHT1s). Enhancing the abundance of PHT1s at the cell surface thus ensures plant productivity and sustainable agriculture. CORNICHON HOMOLOG proteins (CNIHs) are conserved eukaryotic cargo receptors that mediate the selective export of membrane proteins from the endoplasmic reticulum (ER). In this study, we identified the Arabidopsis thaliana CNIH5 (AtCNIH5) as a Pi starvation-inducible gene, preferentially expressed in vascular tissues and outer root cell layers above the meristem. AtCNIH5 co-localizes to the AtSAR1A/AtSEC16A/AtSEC24A-labeled ER exit sites and interacts with AtPHT1;1. Loss of AtCNIH5 confers reduced shoot Pi levels under Pi sufficiency due to the reduced translocation of Pi from roots to shoots, as well as decreased Pi uptake under Pi deficiency. The cnih5 mutant exhibits decreased abundance of AtPHT1s but increased abundance of PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (AtPHF1), which enables the exit of PHT1s from the ER. The cnih5 mutant also shows a lower plasma membrane targeting efficiency of split-GFP-tagged AtPHT1;1 in the root hair and the epidermis within the root transition/elongation zone. Consistently, dysfunctional AtCNIH5 suppresses the growth of the phf1 mutant and alleviates Pi toxicity in the Pi overaccumulator mutant pho2. However, in vivo protein-protein interaction and degradation assays indicate that AtCNIH5 is not a direct target of AtPHO2. Our findings show that AtCNIH5 is a low Pi-responsive ER cargo receptor that interplays with AtPHF1 to promote the plasma membrane targeting of AtPHT1s in a cell type-dependent manner.

3',5'-cAMP is a ubiquitous signaling molecule across kingdoms. Although cAMP was identified early in plants, its significance remained underappreciated. Recent discoveries revealed that nuclear auxin receptors (TIR1/AFBs) possess intrinsic adenylyl cyclase (AC) activity. Crucially, the cAMP produced can participate in transcriptional regulation semi-independently of canonical receptor function, transforming our understanding of the regulatory roles of cAMP in plants. This review systematically synthesizes: (i) the molecular basis of cAMP homeostasis; (ii) identification and functions of downstream effectors; and (iii) the role of cAMP in plant development, stress responses, and hormone interactions. We further highlight critical knowledge gaps, including the evolutionary rationale for moonlighting ACs, precise signal transduction mechanisms, and functions of non-canonical cyclic nucleotides. This review provides a conceptual framework for advancing plant cAMP research.

The auxin indole-3-acetic acid (IAA) is essential for plant growth and stress adaptation. Its biosynthesis via the indole-3-acetaldoxime (IAOx) pathway has recently undergone a paradigm shift. Recent genetic and metabolomic studies have fundamentally revised the indole-3-acetaldoxime (IAOx) pathway from a linear route (IAOx→IAN→IAM→IAA) to a dynamic network. This review synthesizes this paradigm shift by integrating evidence from key Arabidopsis studies. Crucially, mutants disrupting multiple downstream enzyme families fail to block IAA overproduction in the IAOx-accumulating superroot 2 (sur2) background. Functioning as a central branching point between auxin and defense metabolism, the tryptophan-derived metabolite IAOx, along with indole-3-acetonitrile (IAN) and indole-3-acetamide (IAM), elicits auxin responses via independent, tissue-specific pathways, with no metabolic requirement for IAM as a universal intermediate. Furthermore, IAN and IAM levels do not increase with massive IAOx accumulation, indicating a bypass route from IAOx to IAA. We conclude that IAOx acts as a central metabolic hub, partitioning flux competitively between growth and defense. Resolving the unknown IAOx-converting enzyme, the signaling roles of IAN/IAM, and the logic of metabolic channeling is vital to understanding how plants integrate hormonal and stress responses.

The plant endoplasmic reticulum (ER) is a dynamic organelle composed of multiple distinct structural domains, such as cisternae, which are maintained by ER morphogens including the Arabidopsis thaliana Lunapark proteins (LNPs). Cisternae are typically described as sac-like structures connected by tubules. Here we challenge this assumption and propose that cisternae have a more complex structure that modifies ER functionality. This study used state-of-the-art high-resolution confocal and variable-angle epifluorescence microscopy, along with transmission electron microscopy and tomography, on high-pressure frozen Arabidopsis thaliana samples. We found that AtLNP1-stabilised ER forms cisternae composed of dense tubular matrices, whereas AtLNP2 forms cisternae with a uniform, sac-like structure. Furthermore, overexpression of AtLNP proteins alters Golgi morphology, affecting ER-to-Golgi transport and secretion. Our findings reveal that the balance between AtLNP1 and AtLNP2 is critical for ER cisternae organisation and ER functionality in protein production and secretion. This work provides new insights into ER structural plasticity and its functional implications in plant cells.

The plant endoplasmic reticulum (ER) forms a highly dynamic tubular network whose architecture depends on ER-shaping proteins and its interaction with the cytoskeleton. While actin is well known to drive ER movement in plants, how the ER associates with microtubules and how this affects ER network architecture remain poorly understood. Here, we identify Arabidopsis thaliana reticulon 17 (RTN17) as an atypical reticulon that links the ER to the microtubule cytoskeleton. RTN17 features extended, intrinsically disordered N- and C-terminal domains enriched in low-complexity regions, consistent with a scaffolding or hub function. Topology analysis using redox-sensitive roGFP2 constructs shows that both termini face the cytosol, yet RTN17 lacks the amphipathic helix typical of ER-shaping reticulons and does not induce membrane constriction. Instead, RTN17 localises to punctate foci on curved ER membranes, recruits the ER fusogen ROOT HAIR DEFECTIVE3 (RHD3), and co-expression alters ER architecture and dynamics. RTN17 puncta preferentially co-localise with microtubules, and its over-expression promotes ER alignment with the microtubule network.We propose that RTN17 acts as a multifunctional scaffold linking curved ER domains with the microtubule cytoskeleton and localising RHD3 to these sites to regulate ER fusion events. By integrating curvature sensing, cytoskeletal attachment and fusion regulation, RTN17 represents a new class of plant reticulons with scaffolding rather than shaping functions. This work highlights an unrecognised mechanism coordinating ER organisation with the cytoskeleton, providing insights into how plants achieve spatial control of endomembrane architecture and potentially adapt membrane dynamics to developmental or stress cues.

The phytohormone auxin, predominantly as indole-3-acetic acid (IAA), functions as a master regulator that orchestrates plant growth, development, and adaptation to environmental cues [...]

During morphogenesis of multicellular organs, cells in distinct positions need to meet specific functional requirements to form the appropriate tissue type. For example, epidermal tissues need to form at the organ surface. In plants, the epidermis is widely considered essential for morphogenesis due to its role in both restricting and promoting growth. In the root, epidermal cells are partially covered by a protective root cap and partially positioned at the organ surface. Here, we propose that epidermal cells at the organ surface have unique requirements for growth control due to high mechanical tension, while covered epidermal cells are mechanically shielded by the root cap. We present in silico and in vivo evidence that plants use surface mechanical cues to activate a cell-type-specific growth-control program involving the small guanosine triphosphatase (GTPase) RAB-A5c, thereby allowing roots to maintain directional growth at the organ surface. Positional mechanical cues may thus be used to control expression of a sub-population of epidermal genes, linking gene regulation to surface-specific functional requirements.

Plant cytokinesis requires coordination between the actin cytoskeleton, microtubules, and membranes to guide division plane formation and cell plate expansion; how these regulatory factors are coordinated remains unknown. The actin cytoskeleton assembly is controlled by several actin nucleation factors, such as the SCAR/WAVE complex, which regulates actin nucleation and branching through the activation of the ARP2/3 complex. The activity of these actin regulatory proteins is likely influenced by interactions with specific membranes; however, the molecular basis and the biological relevance of SCAR-membrane interactions are also unclear. In this study, we demonstrate that the ER-PM tethering protein VAP27-1 directly interacts with SCAR2 at the ER membrane and that they colocalize to guide cell plate orientation during cell division. In the root meristem, both VAP27-1 and SCAR2 exhibit polarized localization at the cell plates, where the interaction between ER and PM is abundant. VAP27-1 recruits SCAR2 to the cell division plane, where there is a high concentration of actin filaments. In the vap27- 1346 mutant, the densities of cortical ER, SCAR2, and consequently actin filaments are significantly reduced at the cell division plane, affecting cell plate orientation, cell division, and root development. A similar phenomenon is also observed in the scar1234 mutant, suggesting that VAP27 and SCAR proteins regulate cell division through a similar pathway. In conclusion, our data reveal a plant- specific function of VAP27- regulated ER-PM interaction and advance our understanding of plant ER-PM contact site and its role in cell division.

Arsenic, a toxic and carcinogenic metalloid, is a pervasive environmental contaminant that threatens human health through contaminated water and food. The efflux of As(III) via ACR3 transporters is an ancient detoxification mechanism conserved across prokaryotes, fungi, and plants, with the notable exception of angiosperms. Despite their evolutionary significance, plant ACR3s remain largely uncharacterized. Here, we demonstrate that MpACR3, the ACR3 orthologue from the liverwort Marchantia polymorpha , functions as a metalloid/proton antiporter, conferring resistance to arsenicals and moderate tolerance to antimony. Additionally, we uncover an arsenic-sensing domain within MpACR3 that regulates its intracellular trafficking. Under normal conditions, MpACR3 sorting to the plasma membrane is delayed, resulting in its retention within Golgi bodies. However, As(III) binding to three cysteine residues in the N-terminal cytosolic domain induces a conformational change that facilitates MpACR3 trafficking to the plasma membrane. Furthermore, mutational analysis of a conserved arginine-based motif reveals that the N-terminal domain not only controls MpACR3 accumulation at the plasma membrane but also modulates its transport activity. Importantly, this arsenic-sensing domain is conserved among plant ACR3 transporters, suggesting a plant-specific adaptation to arsenic toxicity. ### Competing Interest Statement The authors have declared no competing interest.

Plant systems are emerging as sustainable platforms for producing recombinant therapeutic proteins. This review highlights the role of the plant secretory pathway – particularly the ER and Golgi – in protein folding and glycosylation. Engineering ER structure and targeting human glycosylation enzymes to specific Golgi compartments enhances protein quality. Plant-based systems offer scalable, low-cost alternatives with improved safety for biopharmaceutical production.

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation of organized smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterized and this hampers their potential development for plant synthetic biology. Here, we characterize a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the morphology of the final compartment. We show that a cytosol-facing oligomerization domain is an essential component for compartment formation. Using fluorescence recovery after photobleaching, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment, and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions.

The recent significant progress in developmental bio-imaging of live multicellular organisms has been greatly facilitated by the development of light sheet fluorescence microscopy (LSFM). Both commercial and custom LSFM systems offer the best means for long-term rapid data collection over a wide field of view at single-cell resolution. This is thanks to the low light exposure required for imaging and consequent limited photodamage to the biological sample, and the development of custom holders and mounting techniques that allow for specimens to be imaged in near-normal physiological conditions. This method has been successfully applied to plant cell biology and is currently seen as one of the most efficient techniques for 3D time-lapse imaging for quantitative studies. LSFM allows one to capture and quantify dynamic processes across various levels, from plant subcellular compartments to whole cells, tissues, and entire plant organs. Here we present a method to carry out LSFM on Arabidopsis leaves expressing fluorescent markers targeted to the ER. We will focus on a protocol to mount the sample, test the phototoxicity of the LSFM system, set up a LSFM experiment, and monitor the dynamics of the ER during heat shock.

Simultaneous stoichiometric expression of multiple genes plays a major part in modern research and biotechnology. Traditional methods for incorporating multiple transgenes (or “gene stacking”) have drawbacks such as long time frames, uneven gene expression, gene silencing, and segregation derived from the use of multiple promoters. 2A self-cleaving peptides have emerged over the last two decades as a functional gene stacking method and have been used in plants for the co-expression of multiple genes under a single promoter. Here we describe design features of multicistronic polyproteins using 2A peptides for co-expression in plant cells and targeting to the endoplasmic reticulum (ER). We designed up to quad-cistronic vectors that could target proteins in tandem to the ER. We also exemplify the incorporation of self-excising intein domains within 2A polypeptides, to remove residue additions. These features could aid in the design of stoichiometric protein co-expression strategies in plants in combination with targeting to different subcellular compartments.

The ER is a highly dynamic network of tubules and membrane sheets. Hence imaging this organelle in its native and mobile state is of great importance. Here we describe methods of labeling the native ER using fluorescent proteins and lipid dyes as well as methods for immunolabeling on plant tissue.

Single-particle tracking (SPT) of biomolecules in the plant endoplasmic reticulum has the potential to inform on the formation of protein-protein complexes, metabolons, and the transport of molecules through both the ER membrane and lumen. Plant cells are particularly challenging for observing and tracking single molecules due to their unique structure, size, and considerable autofluorescence. However, by using variable-angle or highly inclined epifluorescence microscopy (VAEM) and transient expression in tobacco, it is possible to observe single-particle dynamics in the ER. Selecting the appropriate fluorophore, and ensuring the correct fluorophore density in the ER, is essential for successful SPT. By using tuneable fluorophores, which can be photoconverted and photoactivated, it is possible to vary the density of visible fluorophores in the ER dynamically. Here we describe methods to prepare plant samples for VAEM and two methods for determining and analyzing single-particle tracks from VAEM time series.

Microsomes are vesicles derived from the endoplasmic reticulum (ER) when cells are broken down in the lab. These microsomes are a valuable tool to study a variety of ER functions such as protein and lipid synthesis in vitro. Here we describe a protocol to isolate ER-derived microsomes Arabidopsis thaliana seedlings and exemplify the use of these purified microsomes in enzyme assays with the auxin precursors tryptophan (Trp) or indole-3-pyruvic acid (IPyA) to quantify auxin synthetic capacity in microsomal and cytosolic fractions.

Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant-produced human therapeutics. This obstacle can be overcome by creating a plant-based system capable of 'humanising' proteins of interest resulting in a glycosylation profile of synthetic plant-produced proteins as it would occur in mammalian systems. For this, the human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N-acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant-specific ones. Using high-resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial-Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans-Golgi cisternae. Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N-linked glycosylation and adds specific sugar molecules onto the proteins. Here we show the expression of four human glycosylation enzymes, which are required for N-linked glycosylation, in plant cells. In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis-, medial and trans-Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial-Golgi attacks and the other two in the trans-Golgi stacks. Using high-resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.

Metabolons are protein complexes that contain all the enzymes necessary for a metabolic pathway but also scaffolding proteins. Such a structure allows efficient channeling of intermediate metabolites form one active site to the next and is highly advantageous for labile or toxic intermediates. Here we describe two methods currently used to identify metabolons via protein-protein interaction methodology: immunoprecipitations using GFP-Trap®_A beads to find novel interaction partners and potential metabolon components and FRET-FLIM to test for and quantify protein-protein interactions in planta.

The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.

The endoplasmic reticulum (ER) links to multiple organelles through membrane contact sites (MCS), which play critical roles in signal transduction, cell homeostasis and stress response. However, studying the behaviour and functions of MCS in plants is still challenging, partially due to the lack of site-specific markers. Here, we used an optogenetic reporter, LiMETER (Light-inducible Membrane-Tethered cortical ER), to study the structure and dynamics of ER-PM contact sites (EPCS) in plants. Upon blue light activation, LiMETER is recruited to the EPCS rapidly, while this process is reversible when blue light is turned off. Compared with other EPCS reporters, LiMETER specifically and reversibly labels the contact sites, causing little side-effects on the ER structure and plant development. With its help, we re-examined the formation of ER-PM connections induced by cell-intrinsic factors or extracellular stimuli. We found that EPCSs are preferably localised at ER tubules and the edge of ER cisternae, and their number increased significantly under abiotic stress conditions. The abundance of ER and PM interaction is also developmental dependent, suggesting a direct link between ER-PM interaction, ER function and cell homeostasis. Taken together, we showed that LiMETER is an improved marker for functional and microscopical studies of ER-PM interaction, demonstrating the effectiveness of optogenetic tools in future research.