Connecting vacuolar and plasma membrane transport networks

The coordinated control of ion transport across the two major membranes of differentiated plant cells, the and the vacuolar membranes, is fundamental in cell physiology. The stomata responses to the fluctuating environmental conditions are an illustrative example. Indeed, they rely on the coordination of ion fluxes between the different cell compartments. The cytosolic environment, that is an interface between intracellular compartments, and the activity of the ion transporters localized in the different membranes influence one each other. Here we analyse the molecular mechanisms connecting and modulating the transport processes at both the plasma and the vacuolar membranes of guard cells.

This article is protected by copyright. All rights reserved The electrochemical potential difference (Δµ i ) for an ion i between two sides of a membrane defines the direction and the limits of ion transport reactions across membranes. Δµ i combines the chemical and the electrical potentials of ion i and is defined by: In the following equations we outline the driving force of the major types of transport reactions in cells: passive transport (ion channels), primary active transport (pumps) and secondary active transport (antiporters and symporters). For pumps that catalyse the transport of n ion i against Δµ i coupling it to ATP hydrolysis, the driving force of the transport reaction ( ) is: ∆ For an antiporter that catalyse the exchange of n ions i and m ions j, the driving force ( ) ∆ of the transport reaction is defined as:

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This article is protected by copyright. All rights reserved For a symporters mediating the co-transport of n ions i and m ions j, the driving force of the transport reaction ( ) is defined as: ∆ In all equations is the gas constant, is the absolute temperature, is the charge of the ion or , is the Faraday's constant, is the membrane potential difference between the cytosol (cyt) and outside (out), is the cytosolic (cyt) concentration of the ion or , [ , ] [ , ] is the concentration of the ion or outside (out), and and are the stoichiometric coefficients of ions and , respectively. The outside (out) corresponds to the apoplast, to the vacuolar lumen or to the lumen of other compartments.

I. Introduction
The vacuole occupies the major part of the cellular volume, up to 90% in differentiated plant cells (Krüger & Schumacher, 2018). It is an acidic compartment presenting dynamic morphology, composition and volume. The characteristics of the vacuole change during plant cell development and cellular responses to the environment (Martinoia et al., 2012), making the plasticity of the vacuole an essential property of this organelle. Given its size, in differentiated cells the vacuole is a key player in the building of the turgor pressure and is part of the intracellular signalling network (Martinoia et al., 2012;Peiter, 2011;Roelfsema & Hedrich, 2005 2016;Cosio et al., 2004;Ma et al., 2005;Thomine et al., 2003). In petunia flowers, specific proton pumps in the VM of petal cells generate extremely acidic vacuolar pHs influencing the colour of flowers (Faraco et al., 2014;Verweij et al., 2008). The VM is also fundamental for the activity of the specialized motor cells generating seismonastic leaf movements of Mimosa pudica pulvini (Fleurat-Lessard et al., 1997a,b;Hagihara & Toyota, 2020). Finally, the VM transporters

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This article is protected by copyright. All rights reserved participate in the control of stomata aperture in guard cells (Martinoia, 2018). In these cells, the morphological changes of the vacuole during stomata movements are linked to the transport activity of the VM (Andrés et al., 2014;Tanaka et al., 2007) (Fig. 1b).
The examples listed above demonstrate the importance of the specialised transport capacities of the vacuole in plant cell physiology. However, for proper cellular responses the VM needs to work in concert with the other cell membranes delimiting intracellular compartments. Thus, the processes occurring at the VM and other cellular membranes are interconnected and coordinated.
Given its size, the VM is a central element of the intracellular transport network, and the vacuolar transport processes must be considered integrated in a network of fluxes mutually influencing each other.

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This article is protected by copyright. All rights reserved Overall, these estimations provide the order of magnitude of the ion fluxes occurring in a guard cell.
Therefore, to avoid aberrant ionic concentrations in the cytosol and to generate coherent responses, the transport systems residing in both the VM and the PM of guard cells are coregulated (Fig. 1).

Cytosolic ion concentration, a straightforward way to coordinate fluxes between membranes
The subcellular organisation of guard cells makes the cytosol a thin layer between two large membranes, the PM and VM. Since in open stomata the vacuole occupies nearly 90% of the whole cellular volume (Fig. 1b) the majority of the solutes crossing the PM will also cross the VM during opening. Therefore, the cytosolic compartment, which accounts for only a fraction of the cell,

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This article is protected by copyright. All rights reserved conditions in the cytosol participate in the coordination of ion fluxes between cellular membranes (Fig. 1b).

Simultaneous regulation of VM and PM transport systems by cytosolic ions
Some ions and metabolites emerge as elements co-regulating solute transport across intracellular membranes ( Fig. 2; Box 2) et al., 2020b). In the future a critical step to decipher the role of Ca 2+ will be to identify the molecular actors mediating its fluxes in plant cells.
Nucleotides, like ATP, also modify the activity of ion transporters in both the PM and the VM (Fig. 2). ATP is the source of energy of the H + -ATPase pumps as P-and V-type H + pumps in the PM and the VM, respectively. Additionally, ATP negatively regulates the activity of the VM anion/H + exchanger CLCa (De Angeli et al., 2009) and of the VM anion channel ALMT9 (Fig. 2) (Zhang et al., 2014). In the PM, ATP blocks Rapid-type anion currents (Frachisse et al., 1999) that, in guard cells, they are mediated by ALMT12 (Fig. 2

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This article is protected by copyright. All rights reserved of the role of nucleotides like ATP, ADP or AMP is their potential to coordinate ion fluxes with the energetic status of the cell.
Recently, malate emerge as a regulator of ion transport systems in both the VM and the PM.
Cytosolic malate concentration depends on the starch degradation/synthesis cycle, on the metabolic consumption, on the vacuolar stocks and on its transport from the apoplast (Santelia & Lawson, 2016). In the last years it was found that the vacuolar anion channels ALMT4 (Eisenach et al., 2017) and ALMT9 (De Angeli et al., 2013) are directly activated by cytosolic malate (Fig.   2). In the PM SLAC1 (Wang & Blatt, 2011;Wang et al., 2018) and ALMT12 (Meyer et al., 2010) channels are also regulated by malate (Fig. 2). These data open to the possibility that cytosolic malate plays a role in the coordination of ion fluxes between both membranes during the opening and closure of stomata.

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This article is protected by copyright. All rights reserved ions and water to close stomata (Fig. 1b). In addition, OST1 inhibits inward K + currents mediated by KAT1 through protein interaction (Fig 2) (Acharya et al., 2013). Besides OST1/SnRK2.6, other kinase proteins were found to regulate the activity of PM and VM ion transport systems. Recently, the vacuolar K + channel TPK1 was shown to be the target of the kinase KIN7 (Isner et al., 2018).
This kinase phosphorylates TPK1 and seems to participate in ABA and CO 2 signalling (Fig. 2) ( Gobert et al., 2007;Isner et al., 2018). Interestingly, KIN7 is localised in both the PM and the VM (Isner et al., 2018). Although its role in the PM is unknown, the dual localisation suggests that it could regulate transport systems in both membranes. Finally, MAPK were also found to target ion channels (Fig. 2)

V. Conclusions and perspectives
The vacuole presents a high functional plasticity and is involved in a multitude of cellular processes. The transporters fluxing molecules across the VM determine the specialized functions of the vacuole in plant cells. In the last decade, the knowledge on the molecular identity of the vacuolar transport systems has considerably expanded. Currently, a consistent number of proteins forming transports systems in the VM is known, highlighting the role of the vacuolar fluxes in plants (Eisenach & De Angeli, 2017;Martinoia et al., 2012;Zhang et al., 2017). Based on this knowledge, a major step will be to decipher how vacuolar transport is integrated within the cell.
Only some flux studies on Commelina communis L. using 86 Rb + as a tracer investigated simultaneously ion fluxes across the PM and the VM (summarized in MacRobbie, 2000).
Otherwise, the transport processes occurring in the VM and in other membranes, such as the PM,

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This article is protected by copyright. All rights reserved influences ion fluxes across both the VM and the PM. Notably, during stomata opening and closure the vacuole undergoes morphological changes and modifications of its relative volume.

Fig. 2
Identified mechanisms co-regulating ion transport systems in the vacuolar membrane (VM) and plasma membrane (PM). Cytosolic Ca 2+ raise induces the activation of different CBL/CIPK kinase complexes that activates the PM anion channels SLAC1, SLAH3, the potassium (K + ) channels AKT1 and GORK, and the K + /H + symporter HAK5. In the VM, CBL/CIPK target the K + exchangers NHX1 and NHX2 and the K + channel TPK1. Cytosolic Ca 2+ can also directly interact and activate vacuolar channels like TPC1 and ALMT6. The ABA signalling induces phosphorylation by OST1 of the PM channels SLAC1, KAT1 and ALMT12, the PIP2;1 aquaporin, and of the VM anion/proton exchangers CLCa. ABA signalling also acts on the vacuolar exchanger CLCc by an unknown pathway, and on the K + channel TPK1 through KIN7 kinase. MAPK kinases activate SLAC1 in the PM and inhibit ALMT4 in the VM. Several cytosolic molecules induce the activation/inhibition of ion transporters like ALMT9, CLCa, and H + pumps. Malate activates the anion channels ALMT4 and ALMT9 in the VM, and ALMT12 and SLAC1 in the PM. ATP is the substrate for the pumping activity of the H + ATPases and is a negative regulator of anion channels in the PM and VM. Figure 1 Tansley Insight 33874 This article is protected by copyright. All rights reserved Accepted Article