Light at the end of the tunnel: FRAP assays combined with super resolution microscopy confirm the presence of a tubular vacuole network in meristematic plant cells.

Plant vacuoles play key roles in cellular homeostasis, performing catabolic and storage functions, and regulating pH and ion balance. Despite their essential role, there is still no consensus on how vacuoles are established. A model proposing that the endoplasmic reticulum is the main contributor of membrane for growing vacuoles in meristematic cells has been challenged by a study proposing that plant vacuoles are formed de novo by homotypic fusion of multivesicular bodies (MVBs). Here, we use the Arabidopsis thaliana root as a model system to provide a systematic overview of successive vacuole biogenesis stages, starting from the youngest cells proximate to the quiescent center. We combine in vivo high- and super-resolution (STED) microscopy to demonstrate the presence of tubular and connected vacuolar structures in all meristematic cells. Using customized fluorescence recovery after photobleaching (FRAP) assays, we establish different modes of connectivity and demonstrate that thin, tubular vacuoles, as observed in cells near the quiescent center, form an interconnected network. Finally, we argue that a growing body of evidence indicates that vacuolar structures cannot originate from MVBs alone but receive membrane material from different sources simultaneously.

SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists.

Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response.

Pathogen-induced pH changes regulate the growth-defense balance in plants.

Environmental adaptation of organisms relies on fast perception and response to external signals, which lead to developmental changes. Plant cell growth is strongly dependent on cell wall remodeling. However, little is known about cell wall-related sensing of biotic stimuli and the downstream mechanisms that coordinate growth and defense responses. We generated genetically encoded pH sensors to determine absolute pH changes across the plasma membrane in response to biotic stress. A rapid apoplastic acidification by phosphorylation-based proton pump activation in response to the fungus Fusarium oxysporum immediately reduced cellulose synthesis and cell growth and, furthermore, had a direct influence on the pathogenicity of the fungus. In addition, pH seems to influence cellulose structure. All these effects were dependent on the COMPANION OF CELLULOSE SYNTHASE proteins that are thus at the nexus of plant growth and defense. Hence, our discoveries show a remarkable connection between plant biomass production, immunity, and pH control, and advance our ability to investigate the plant growth-defense balance.

ClCd and ClCf act redundantly at the trans-Golgi network/early endosome and prevent acidification of the Golgi stack.

The trans-Golgi network/early endosome (TGN/EE) serves as the central hub in which exocytic and endocytic trafficking pathways converge and specificity of cargo routing needs to be achieved. Acidification is a hallmark of the TGN/EE and is maintained by the vacuolar H+-ATPase (V-ATPase) with support of proton-coupled antiporters. We show here that ClCd and ClCf, two distantly related members of the Arabidopsis Cl- channel (ClC) family, colocalize in the TGN/EE, where they act redundantly, and are essential for male gametophyte development. Combining an inducible knockdown approach and in vivo pH measurements, we show here that reduced ClC activity does not affect pH in the TGN/EE but causes hyperacidification of trans-Golgi cisternae. Taken together, our results show that ClC-mediated anion transport into the TGN/EE is essential and affects spatiotemporal aspects of TGN/EE maturation as well as its functional separation from the Golgi stack.

Dual-Reporting Transcriptionally Linked Genetically Encoded Fluorescent Indicators Resolve the Spatiotemporal Coordination of Cytosolic Abscisic Acid and Second Messenger Dynamics in Arabidopsis.

Deciphering signal transduction processes is crucial for understanding how plants sense and respond to environmental changes. Various chemical compounds function as central messengers within deeply intertwined signaling networks. How such compounds act in concert remains to be elucidated. We have developed dual-reporting transcriptionally linked genetically encoded fluorescent indicators (2-in-1-GEFIs) for multiparametric in vivo analyses of the phytohormone abscisic acid (ABA), Ca2+, protons (H+), chloride (anions), the glutathione redox potential, and H2O2 Simultaneous analyses of two signaling compounds in Arabidopsis (Arabidopsis thaliana) roots revealed that ABA treatment and uptake did not trigger rapid cytosolic Ca2+ or H+ dynamics. Glutamate, ATP, Arabidopsis PLANT ELICITOR PEPTIDE, and glutathione disulfide (GSSG) treatments induced rapid spatiotemporally overlapping cytosolic Ca2+, H+, and anion dynamics, but except for GSSG, only weakly affected the cytosolic redox state. Overall, 2-in-1-GEFIs enable complementary, high-resolution in vivo analyses of signaling compound dynamics and facilitate an advanced understanding of the spatiotemporal coordination of signal transduction processes in Arabidopsis.

Salinity-Induced Cytosolic Alkaline Shifts in Arabidopsis Roots Require the SOS Pathway.

Plants have evolved elaborate mechanisms to sense, respond to and overcome the detrimental effects of high soil salinity. The role of calcium transients in salinity stress signaling is well established, but the physiological significance of concurrent salinity-induced changes in cytosolic pH remains largely undefined. Here, we analyzed the response of Arabidopsis roots expressing the genetically encoded ratiometric pH-sensor pHGFP fused to marker proteins for the recruitment of the sensor to the cytosolic side of the tonoplast (pHGFP-VTI11) and the plasma membrane (pHGFP-LTI6b). Salinity elicited a rapid alkalinization of cytosolic pH (pHcyt) in the meristematic and elongation zone of wild-type roots. The pH-shift near the plasma membrane preceded that at the tonoplast. In pH-maps transversal to the root axis, the epidermis and cortex had cells with a more alkaline pHcyt relative to cells in the stele in control conditions. Conversely, seedlings treated with 100 mM NaCl exhibited an increased pHcyt in cells of the vasculature relative to the external layers of the root, and this response occurred in both reporter lines. These pHcyt changes were substantially reduced in mutant roots lacking a functional SOS3/CBL4 protein, suggesting that the operation of the SOS pathway mediated the dynamics of pHcyt in response to salinity.

An integrative view on vacuolar pH homeostasis in Arabidopsis thaliana: Combining mathematical modeling and experimentation.

The acidification of plant vacuoles is of great importance for various physiological processes, as a multitude of secondary active transporters utilize the proton gradient established across the vacuolar membrane. Vacuolar-type H+ -translocating ATPases and a pyrophosphatase are thought to enable vacuoles to accumulate protons against their electrochemical potential. However, recent studies pointed to the ATPase located at the trans-Golgi network/early endosome (TGN/EE) to contribute to vacuolar acidification in a manner not understood as of now. Here, we combined experimental data and computational modeling to test different hypotheses for vacuolar acidification mechanisms. For this, we analyzed different models with respect to their ability to describe existing experimental data. To better differentiate between alternative acidification mechanisms, new experimental data have been generated. By fitting the models to the experimental data, we were able to prioritize the hypothesis in which vesicular trafficking of Ca2+ /H+ -antiporters from the TGN/EE to the vacuolar membrane and the activity of ATP-dependent Ca2+ -pumps at the tonoplast might explain the residual acidification observed in Arabidopsis mutants defective in vacuolar proton pump activity. The presented modeling approach provides an integrative perspective on vacuolar pH regulation in Arabidopsis and holds potential to guide further experimental work.

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.

NHX-type Na+(K+)/H+ antiporters are required for TGN/EE trafficking and endosomal ion homeostasis in Arabidopsis thaliana.

The regulation of ion and pH homeostasis of endomembrane organelles is critical for functional protein trafficking, sorting and modification in eukaryotic cells. pH homeostasis is maintained through the activity of vacuolar H+-ATPases (V-ATPases) pumping protons (H+) into the endomembrane lumen, and counter-action by cation/proton exchangers, such as the NHX family of Na+(K+)/H+ exchangers. In plants, V-ATPase activity at the trans-Golgi network/early endosome (TGN/EE) is important for secretory and endocytic trafficking; however, the role of the endosomal antiporters NHX5 and NHX6 in endomembrane trafficking is unclear. Here we show through genetic, pharmacological and live-cell imaging approaches that double knockout of NHX5 and NHX6 results in the impairment of endosome motility and protein recycling at the TGN/EE, but not in the secretion of integral membrane proteins. Furthermore, we report that nhx5 nhx6 mutants are partially insensitive to osmotic swelling of TGN/EE induced by the monovalent cation ionophore monensin, and to late endosomal swelling by the phosphatidylinositol 3/4-kinase inhibitor wortmannin, demonstrating that NHX5 and NHX6 function to regulate the luminal cation composition of endosomes.

Ureide Permease 5 (AtUPS5) Connects Cell Compartments Involved in Ureide Metabolism.

Allantoin is a purine oxidative product involved in long distance transport of organic nitrogen in nodulating legumes and was recently shown to play a role in stress tolerance in other plants. The subcellular localization of enzymes that catalyze allantoin synthesis and degradation indicates that allantoin is produced in peroxisomes and degraded in the endoplasmic reticulum (ER). Although it has been determined that allantoin is mostly synthesized in roots and transported to shoots either for organic nitrogen translocation in legumes or for plant protection during stress in Arabidopsis (Arabidopsis thaliana), the mechanism and molecular components of allantoin export from root cells are still unknown. AtUPS5 (Arabidopsis UREIDE PERMEASE 5) is a transmembrane protein that transports allantoin with high affinity when expressed in yeast. The subcellular fate of splicing variants AtUPS5L (long) and AtUPS5S (short) was studied by tagging them with fluorescent proteins in their cytosolic loops. The capability of these fusion proteins to complement the function of the native proteins was demonstrated by nutritional and salt stress experiments. Both variants localized to the ER, but the AtUPS5L variant was also detected in the trans-Golgi network/early endosome and at the plasma membrane. AtUPS5L and AtUPS5S localization indicates that they could have different roles in allantoin distribution between subcellular compartments. Our data suggest that under nonstress conditions UPS5L and UPS5S may function in allantoin degradation for nutrient recycling, whereas under stress, both genes may be involved in vesicular export allowing allantoin translocation from roots to shoots.