Karin Schumacher

Vacuolar H + -ATPase Activity Is Required for Endocytic and Secretory Trafficking in Arabidopsis

In eukaryotic cells, compartments of the highly dynamic endomembrane system are acidified to varying degrees by the activity of vacuolar H+-ATPases (V-ATPases). In the Arabidopsis thaliana genome, most V-ATPase subunits are encoded by small gene families, thus offering potential for a multitude of enzyme complexes with different kinetic properties and localizations. We have determined the subcellular localization of the three Arabidopsis isoforms of the membrane-integral V-ATPase subunit VHA-a. Colocalization experiments as well as immunogold labeling showed that VHA-a1 is preferentially found in the trans-Golgi network (TGN), the main sorting compartment of the secretory pathway. Uptake experiments with the endocytic tracer FM4-64 revealed rapid colocalization with VHA-a1, indicating that the TGN may act as an early endosomal compartment. Concanamycin A, a specific V-ATPase inhibitor, blocks the endocytic transport of FM4-64 to the tonoplast, causes the accumulation of FM4-64 together with newly synthesized plasma membrane proteins, and interferes with the formation of brefeldin A compartments. Furthermore, nascent cell plates are rapidly stained by FM4-64, indicating that endocytosed material is redirected into the secretory flow after reaching the TGN. Together, our results suggest the convergence of the early endocytic and secretory trafficking pathways in the TGN.

A defined range of guard cell calcium oscillation parameters encodes stomatal movements

Oscillations in cytosolic calcium concentration ([Ca2+]cyt) are central regulators of signal transduction cascades, although the roles of individual [Ca2+]cyt oscillation parameters in regulating downstream physiological responses remain largely unknown. In plants, guard cells integrate environmental and endogenous signals to regulate the aperture of stomatal pores and [Ca2+]cyt oscillations are a fundamental component of stomatal closure. Here we systematically vary [Ca2+]cyt oscillation parameters in Arabidopsis guard cells using a ‘calcium clamp’ and show that [Ca2+]cyt controls stomatal closure by two mechanisms. Short-term ‘calcium-reactive’ closure occurred rapidly when [Ca2+]cyt was elevated, whereas the degree of long-term steady-state closure was ‘calcium programmed’ by [Ca2+]cyt oscillations within a defined range of frequency, transient number, duration and amplitude. Furthermore, in guard cells of the gca2 mutant, [Ca2+]cyt oscillations induced by abscisic acid and extracellular calcium had increased frequencies and reduced transient duration, and steady-state stomatal closure was abolished. Experimentally imposing [Ca2+]cyt oscillations with parameters that elicited closure in the wild type restored long-term closure in gca2 stomata. These data show that a defined window of guard cell [Ca2+]cyt oscillation parameters programs changes in steady-state stomatal aperture.

Endocytic and Secretory Traffic in Arabidopsis Merge in the Trans-Golgi Network/Early Endosome, an Independent and Highly Dynamic Organelle

This study examines secretory and endocytotic trafficking in Arabidopsis by tracking the movement of a brassinosteroid receptor and a boron exporter through the endomembrane system. Both endocytotic and secretory cargo travel through the trans-Golgi network/early endosome (TGN/EE), and the TGN/EE is shown to be an independent organelle that only transiently associates with the Golgi. Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Plant proton pumps

Chemiosmotic circuits of plant cells are driven by proton (H+) gradients that mediate secondary active transport of compounds across plasma and endosomal membranes. Furthermore, regulation of endosomal acidification is critical for endocytic and secretory pathways. For plants to react to their constantly changing environments and at the same time maintain optimal metabolic conditions, the expression, activity and interplay of the pumps generating these H+ gradients have to be tightly regulated. In this review, we will highlight results on the regulation, localization and physiological roles of these H+‐ pumps, namely the plasma membrane H+‐ATPase, the vacuolar H+‐ATPase and the vacuolar H+‐PPase.

A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies

Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P(UBQ10) ) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K(+) channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P(UBQ10) promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P(UBQ10) promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant advantages for the study of membrane dynamics and cellular development, as well as events associated with environmental stimuli in guard cells and nutrient acquisition in roots.

Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation.

The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H+-ATPase (V-ATPase) and the vacuolar H+-pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn2+/H+-antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na+/H+-antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vha-a2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth.

The Endosomal System of Plants: Charting New and Familiar Territories

Endocytosis is defined as the uptake of molecules from the extracellular milieu through the formation of a vesicle at the plasma membrane (PM). This transport event circumscribes both soluble (fluid phase endocytosis) and membrane-bound cargos. The initial internalization process is followed by a

Multivesicular Bodies Mature from the Trans -Golgi Network/Early Endosome in Arabidopsis

This work examines the origin and fate of multivesicular bodies (MVBs)/late endosomes, tracing their formation back to the trans-Golgi network (TGN)/early endosome and showing that their maturation into MVBs requires V-ATPase activity and ESCRT for the formation of the intraluminal vesicles and annexins for the release of MVBs from the TGN as transport carriers that fuse with the vacuole. The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole. The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference–mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole.

Plant Cytokinesis Requires De Novo Secretory Trafficking but Not Endocytosis

During plant cytokinesis membrane vesicles are efficiently delivered to the cell-division plane, where they fuse with one another to form a laterally expanding cell plate. These membrane vesicles were generally believed to originate from Golgi stacks. Recently, however, it was proposed that endocytosis contributes substantially to cell-plate formation. To determine the relative contributions of secretory and endocytic traffic to cytokinesis, we specifically inhibited either or both trafficking pathways in Arabidopsis. Blocking traffic to the division plane after the two pathways had converged at the trans-Golgi network disrupted cytokinesis and resulted in binucleate cells, whereas impairment of endocytosis alone did not interfere with cytokinesis. By contrast, inhibiting ER-Golgi traffic by eliminating the relevant BFA-resistant ARF-GEF caused retention of newly synthesized proteins, such as the cytokinesis-specific syntaxin KNOLLE in the ER, and prevented the formation of the partitioning membrane. Our results suggest that during plant cytokinesis, unlike animal cytokinesis, protein secretion is absolutely essential, whereas endocytosis is not.

Comparative sequence analysis reveals extensive microcolinearity in the lateral suppressor regions of the tomato, Arabidopsis, and Capsella genomes.

A 57-kb region of tomato chromosome 7 harboring five different genes was compared with the sequence of the Arabidopsis genome to search for microsynteny between the genomes of these two species. For all five genes, homologous sequences could be identified in a 30-kb region located on Arabidopsis chromosome 1. Only two inversion events distinguish the arrangement of the five genes in tomato from that in Arabidopsis. Inversions were not detected when the arrangement of the five Arabidopsis genes was compared with the arrangement in the orthologous region of Capsella, a plant closely related to Arabidopsis. These results provide evidence for microcolinearity between closely and distantly related dicotyledonous species. The degree of microcolinearity found can be exploited to localize orthologous genes in Arabidopsis and tomato in an unambiguous way.