Systematic analysis of specific and nonspecific auxin effects on endocytosis and trafficking.

The phytohormone auxin and its directional transport through tissues are intensively studied. However, a mechanistic understanding of auxin-mediated feedback on endocytosis and polar distribution of PIN auxin transporters remains limited due to contradictory observations and interpretations. Here, we used state-of-the-art methods to reexamine the auxin effects on PIN endocytic trafficking. We used high auxin concentrations or longer treatments versus lower concentrations and shorter treatments of natural indole-3-acetic acid (IAA) and synthetic naphthalene acetic acid (NAA) auxins to distinguish between specific and nonspecific effects. Longer treatments of both auxins interfere with Brefeldin A-mediated intracellular PIN2 accumulation and also with general aggregation of endomembrane compartments. NAA treatment decreased the internalization of the endocytic tracer dye, FM4-64; however, NAA treatment also affected the number, distribution, and compartment identity of the early endosome/trans-Golgi network, rendering the FM4-64 endocytic assays at high NAA concentrations unreliable. To circumvent these nonspecific effects of NAA and IAA affecting the endomembrane system, we opted for alternative approaches visualizing the endocytic events directly at the plasma membrane (PM). Using total internal reflection fluorescence microscopy, we saw no significant effects of IAA or NAA treatments on the incidence and dynamics of clathrin foci, implying that these treatments do not affect the overall endocytosis rate. However, both NAA and IAA at low concentrations rapidly and specifically promoted endocytosis of photo-converted PIN2 from the PM. These analyses identify a specific effect of NAA and IAA on PIN2 endocytosis, thus, contributing to its polarity maintenance and furthermore illustrate that high auxin levels have nonspecific effects on trafficking and endomembrane compartments.

Auxin analog-induced Ca2+ signaling is independent of inhibition of endosomal aggregation in Arabidopsis roots.

Much of what we know about the role of auxin in plant development derives from exogenous manipulations of auxin distribution and signaling, using inhibitors, auxins, and auxin analogs. In this context, synthetic auxin analogs, such as 1-naphthalene acetic acid (1-NAA), are often favored over the endogenous auxin, indole-3-acetic acid (IAA), in part due to their higher stability. While such auxin analogs have proven instrumental in revealing the various faces of auxin, they display in some cases bioactivities distinct from IAA. Here, we focused on the effect of auxin analogs on the accumulation of PIN proteins in brefeldin A-sensitive endosomal aggregations (BFA bodies), and correlation with the ability to elicit Ca2+ responses. For a set of commonly used auxin analogs, we evaluated if auxin analog-induced Ca2+ signaling inhibits PIN accumulation. Not all auxin analogs elicited a Ca2+ response, and their differential ability to elicit Ca2+ responses correlated partially with their ability to inhibit BFA-body formation. However, in tir1/afb and cngc14, 1-NAA-induced Ca2+ signaling was strongly impaired, yet 1-NAA still could inhibit PIN accumulation in BFA bodies. This demonstrates that TIR1/AFB-CNGC14-dependent Ca2+ signaling does not inhibit BFA body formation in Arabidopsis roots.

Identification of Novel Inhibitors of Auxin-Induced Ca2+ Signaling via a Plant-Based Chemical Screen.

Many signal perception mechanisms are connected to Ca2+-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca2+ signal. However, the cellular targets of auxin-induced Ca2+ are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca2+ entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca2+ and to explore the physiological relevance of Ca2+ in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca2+ entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca2+ signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca2+ channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic stress-induced Ca2+ signals, bepridil specifically inhibited auxin-induced Ca2+ We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca2+ entry and its relevance for auxin responses.

RBOH-mediated ROS production facilitates lateral root emergence in Arabidopsis.

Lateral root (LR) emergence represents a highly coordinated process in which the plant hormone auxin plays a central role. Reactive oxygen species (ROS) have been proposed to function as important signals during auxin-regulated LR formation; however, their mode of action is poorly understood. Here, we report that Arabidopsis roots exposed to ROS show increased LR numbers due to the activation of LR pre-branch sites and LR primordia (LRP). Strikingly, ROS treatment can also restore LR formation in pCASP1:shy2-2 and aux1 lax3 mutant lines in which auxin-mediated cell wall accommodation and remodeling in cells overlying the sites of LR formation is disrupted. Specifically, ROS are deposited in the apoplast of these cells during LR emergence, following a spatiotemporal pattern that overlaps the combined expression domains of extracellular ROS donors of the RESPIRATORY BURST OXIDASE HOMOLOGS (RBOH). We also show that disrupting (or enhancing) expression of RBOH in LRP and/or overlying root tissues decelerates (or accelerates) the development and emergence of LRs. We conclude that RBOH-mediated ROS production facilitates LR outgrowth by promoting cell wall remodeling of overlying parental tissues.

Calcium is an organizer of cell polarity in plants.

Cell polarity is a fundamental property of pro- and eukaryotic cells. It is necessary for coordination of cell division, cell morphogenesis and signaling processes. How polarity is generated and maintained is a complex issue governed by interconnected feed-back regulations between small GTPase signaling and membrane tension-based signaling that controls membrane trafficking, and cytoskeleton organization and dynamics. Here, we will review the potential role for calcium as a crucial signal that connects and coordinates the respective processes during polarization processes in plants. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Salicylic acid interferes with clathrin-mediated endocytic protein trafficking.

Removal of cargos from the cell surface via endocytosis is an efficient mechanism to regulate activities of plasma membrane (PM)-resident proteins, such as receptors or transporters. Salicylic acid (SA) is an important plant hormone that is traditionally associated with pathogen defense. Here, we describe an unanticipated effect of SA on subcellular endocytic cycling of proteins. Both exogenous treatments and endogenously enhanced SA levels repressed endocytosis of different PM proteins. The SA effect on endocytosis did not involve transcription or known components of the SA signaling pathway for transcriptional regulation. SA likely targets an endocytic mechanism that involves the coat protein clathrin, because SA interfered with the clathrin incidence at the PM and clathrin-deficient mutants were less sensitive to the impact of SA on the auxin distribution and root bending during the gravitropic response. By contrast, SA did not affect the ligand-induced endocytosis of the flagellin sensing2 (FLS2) receptor during pathogen responses. Our data suggest that the established SA impact on transcription in plant immunity and the nontranscriptional effect of SA on clathrin-mediated endocytosis are independent mechanisms by which SA regulates distinct aspects of plant physiology.

Constitutive Active CPK30 Interferes With Root Growth and Endomembrane Trafficking in Arabidopsis thaliana.

Calcium-dependent protein kinases (CPK) are key components of a wide array of signaling pathways, translating stress and nutrient signaling into the modulation of cellular processes such as ion transport and transcription. However, not much is known about CPKs in endomembrane trafficking. Here, we screened for CPKs that impact on root growth and gravitropism, by overexpressing constitutively active forms of CPKs under the control of an inducible promoter in Arabidopsis thaliana. We found that inducible overexpression of an constitutive active CPK30 (CA-CPK30) resulted in a loss of root gravitropism and ectopic auxin accumulation in the root tip. Immunolocalization revealed that CA-CPK30 roots have reduced PIN protein levels, PIN1 polarity defects and impaired Brefeldin A (BFA)-sensitive trafficking. Moreover, FM4-64 uptake was reduced, indicative of a defect in endocytosis. The effects on BFA-sensitive trafficking were not specific to PINs, as BFA could not induce aggregation of ARF1- and CHC-labeled endosomes in CA-CPK30. Interestingly, the interference with BFA-body formation, could be reverted by increasing the extracellular pH, indicating a pH-dependence of this CA-CPK30 effect. Altogether, our data reveal an important role for CPK30 in root growth regulation and endomembrane trafficking in Arabidopsis thaliana.

Corrigendum: Constitutive active CPK30 interferes with root growth and endomembrane trafficking in Arabidopsis thaliana.

[This corrects the article DOI: 10.3389/fpls.2022.862398.].

Calcium Ion Dynamics in Roots: Imaging and Analysis.

Calcium sensors are indispensable tools to study the role of Ca2+ and visualize Ca2+ dynamics during biological processes. Over the past years, the field of Ca2+ imaging has strongly expanded by the development of a wide palette of sensors and optimization of sample handling. Here, we provide guidelines for imaging of the Ca2+ sensor R-GECO1 in Arabidopsis thaliana roots which can be interpolated to other intensiometric Ca2+ sensors. Furthermore, we demonstrate a procedure for image analysis of the acquired time-lapse recordings.

The ins and outs of Ca2+ in plant endomembrane trafficking.

Trafficking of proteins and lipids within the plant endomembrane system is essential to support cellular functions and is subject to rigorous regulation. Despite this seemingly strict regulation, endomembrane trafficking needs to be dynamically adjusted to ever-changing internal and environmental stimuli, while maintaining cellular integrity. Although often overlooked, the versatile second messenger Ca2+ is intimately connected to several endomembrane-associated processes. Here, we discuss the impact of electrostatic interactions between Ca2+ and anionic phospholipids on endomembrane trafficking, and illustrate the direct role of Ca2+ sensing proteins in regulating endomembrane trafficking and membrane integrity preservation. Moreover, we discuss how Ca2+ can control protein sorting within the plant endomembrane system. We thus highlight Ca2+ signaling as a versatile mechanism by which numerous signals are integrated into plant endomembrane trafficking dynamics.