LRX- and FER-dependent extracellular sensing coordinates vacuolar size for cytosol homeostasis

Cellular elongation requires the defined coordination of intra- and extracellular processes. The vacuole is the biggest plant organelle and its dimension has a role in limiting cell expansion (Löfke et al., 2015; Scheuring et al., 2016). We reveal that the increase in vacuolar occupancy enables cellular elongation with relatively little enlargement of the cytosole. It remains, however, completely unknown how the vacuolar size is coordinated with other growth-relevant processes. Intriguingly, we show that extracellular constraints impact on the intracellular expansion of the vacuole. The underlying cell wall sensing mechanism requires the interaction of the extracellular leucine-rich repeat extensin (LRX) with the receptor-like kinase Feronia (FER). Our data suggests that LRX links the plasma membrane localised FER with the cell wall, allowing this module to jointly sense and convey extracellular signals to the underlying cell. This mechanism coordinates cell wall acidification/loosening with the increase in vacuolar size, contributing cytosol homeostasis during plant cell expansion.

about 3-fold (Figure 1 c and Suppl. Figure 1 a and b). The 3D imaging of a cytosolic fluorescent protein also confirmed that the cytosol shows limited volume expansion during epidermal elongation (Suppl. Figure 1 a and b). Accordingly, the relative increase in vacuolar size has a dramatic impact on cytosol homeostasis during cellular enlargement. We conclude that the size of the vacuole is important for rapid plant cell expansion, requiring relatively little de-novo production of cytosolic components.
Cell wall acidification is central in activating a cascade of events, ultimately leading to cell wall loosening and subsequent cellular elongation (Fendrych et al., 2016;Barbez et al., 2017). Accordingly, cell wall acidification/loosening (Barbez et al., 2017) correlates with the increase in vacuolar size (Figure 1a-c), but the coordinative mechanism is unknown. We, therefore, investigated the possibility that apoplast acidification/cell wall loosening is sensed and provides a feedback for vacuolar morphogenesis. We either directly acidified the rhizosphere (by lowering the pH of the media) or genetically (SAUR19 induction) as well as pharmacologically (fusicoccin treatment) induced the activity of the plasma membrane H + -ATPase (Spartz et al., 2014;Spartz et al., 2016, Marre, 1979. All these conditions have recently been shown to acidify the pH of epidermal cell walls, consequently inducing cellular elongation (Barbez et al., 2017). We visualized the vacuolar morphology in root epidermal cells in the late meristematic zone, allowing assessment of the impact of cell wall acidification before the onset of elongation. Using the tonoplast stain MDY-64 or the tonoplast marker line pUBQ10::VAMP711-YFP (Scheuring et al., 2015;Löfke et al., 2015), we revealed that cell wall acidification caused a dramatic alteration in vacuolar morphology (Figure 1 d,f,h). Cell wall acidification did not only change the vacuolar appearance, but also induced an increased vacuolar occupancy of the cell (Figure 1 e, g, i). Our data accordingly suggests that cell wall acidification/loosening impacts on vacuolar morphogenesis and size.
The inhibition of pectin methyl esterases (PMEs) leads to reduced cellular elongation, presumably due to stiffening of the cell walls (Wolf et al., 2012). Therefore, we used epigallocatechin gallate (EGCG), which is a natural inhibitor for PMEs (Lewis et al., 2008), to inducibly interfere with the properties of the cell wall. The application of EGCG induced smaller vacuolar structures and reduced the overall vacuolar occupancy of the cell (Figure 2 a and b). Notably, roots that penetrate a relatively stiff medium similarly show smaller vacuoles, when compared to surface-grown roots (Figure 2 c and d). These findings suggest that extracellular constraints restrict intracellular enlargements of vacuoles.
We assume that cell wall sensing integrates vacuolar size. To test this hypothesis, we focused on the receptor-like kinase (RLK) FERONIA (FER), which is required for mechanical cell wall sensing (Shih et al., 2014). The fer-2 and fer-4 loss-of-function mutants Moreover, fer mutant vacuoles were neither affected by EGCG treatments nor by extracellular constraints of the substrate (Figure 2 g and h). Accordingly, we conclude that an extracellular, FER-dependent signal restricts intracellular expansion of the vacuole.
FER is a transmembrane protein, with an extracellular domain for signal perception and a cytoplasmic domain that phosphorylates target molecules (Escobar-Restrepo et al., 2007). Notably, pFER::FERKR-GFP bares a mutation in the kinase domain and was not able to complement the vacuolar phenotype of fer mutants (Suppl. Figure 2 c and d). This data supports a role for the FER kinase activity in restricting intracellular expansion of the vacuole.
FER is a versatile growth integrator in plants with important contributions to a wide range of responses (Li et al., 2016). It remains, however, still unknown how FER contributes to the mechanical sensing of the extracellular space (Shih et al., 2014). Next, we turned our attention to extracellular proteins with a possible role in cell wall sensing and regulation of vacuolar size. Interestingly, leucine-rich repeat extensins (LRXs) are extracellular proteins (Baumberger et al., 2003) and showed co-expression with FER and other FER-related RLKs (Suppl. Figure 3 a and b). LRXs display an N-terminal leucine-rich repeat (LRR) and a Cterminal extensin (EXT) domain (Draeger et al., 2015). While the LRR domain is presumably involved in protein-protein interactions, the EXT domain allows the LRX proteins to bind cell wall components (Ringli, 2010). This interesting domain structure also envisioned the hypothetical role of LRX in cell wall sensing (Humphrey et al., 2007). Intriguingly, lrx3 lrx4 lrx5 triple mutants displayed larger vacuolar lumina ( Notably, truncated LRX proteins that lack the EXT cell wall binding domain partially associate with the plasma membrane (Ndinyanka et al., 2017), suggesting that the N-terminus of LRX4 interacts with some membrane component. Accordingly, we expressed the HAtagged N-terminal part of LRX4 (LRR4-HA) together with a GFP-tagged FER in tobacco.
Co-immunoprecipitation of FER and LRR4 indicated an association of these two proteins in a complex (Figure 4 b).
Most remarkably, we noticed that the expression of a citrine-tagged LRR4 under the Therefore, we analysed the contribution of the BAK1 co-receptor to the regulation of vacuolar form. Importantly, none of the tested bak1 (strong and weak) mutant alleles affected the vacuolar morphology (Figure 4 e). Accordingly, we conclude that the FER/LRXdependent cell wall sensing mechanism is distinct from the previously reported BAK1dependent scaffold mechanism.
The increase in vacuolar volume allows for rapid cell elongation with relatively little de-novo production of cytosolic content. Our data suggests that a cell wall sensing mechanism impacts on the intracellular expansion of the vacuole. This feedback ensures the spatial and temporal coordination of cell wall acidification/loosening with the increase in vacuolar size, which effects cytosol homeostasis during rapid cell expansion.
FER has been previously proposed to be required for cell wall sensing (Shih et al., 2014), but the underlying mechanism remains unknown. Our work proposes LRX as a physical link between the plasma membrane localised FER on one side (via the LRR domain) and the cell wall (via the EXT domain) on the other. Both LRX and FER are essential for constraining the vacuole, suggesting that the interaction of LRX and FER is required for its signalling to the underlying cell. The FER-/LRX-dependent mechanism is independent of the co-receptor BAK1. Therefore, the LRX-dependent cell wall sensing mechanism differs molecularly from the FER scaffold function during immune responses (Stegmann et al., 2017). Accordingly, FER appears to form distinct complexes, which is consistent with the very diverse external signals that can be perceived by the same core component FER. The dominant negative impact of LRR4 suggests the presence of additional components in the FER/LRX complex, assuming that the truncated LRX outcompetes/sequesters some crucial interactor(s). Given the molecular complexity of the FER signalling, it is furthermore likely that the state of LRX/FER interaction also disrupts and/or enables other FER involved interactions (e.g. BAK1).
We propose that an LRX-and FER-dependent module integrates the cell wall status with intracellular expansion of the vacuole. It remains to be seen precisely how LRX/FER signalling at the cell surface leads to the modulation of vacuoles. FER-dependent phosphorylation events impact on RAC/ROP GTPases (Rho-related molecular switches in plants), which are known regulators of actin dynamics (Duan et al., 2010). Notably, the actin cytoskeleton surrounds the vacuole and contributes to the regulation of vacuolar size (Scheuring et al., 2016). Accordingly, it is very tempting to speculate that an actin-dependent process could link the LRX/FER-dependent, extracellular sensing mechanism with the intracellular control of vacuolar expansion.

Phenotype analysis
Vacuolar morphology index and occupancy were quantified in 6-d-old seedlings. Confocal images were analyzed using ImageJ (vacuolar morphology index) or processed using Imaris (vacuolar occupancy of cells). To calculate the vacuolar morphology index, the longest and widest distance of the biggest luminal structure was measured and multiplied (Löfke et al., 2015). The atrichoblast cells were quantified before the onset of elongation (late meristematic). To depict this region, the first cell being twice as long as wide was considered as the onset of elongation. Starting from this cell, the next cell towards the meristem was excluded (as it usually shows either partial elongation and/or already substantial vacuolar expansion), and vacuoles of the subsequent 4 cells were quantified as described previously (Scheuring et al., 2016). For the analysis of occupancy, 1 cell in this region was used.
Vacuolar shape/size were quantified in at least 8 roots (unless stated otherwise in the figure legend). MDY-64 and BCECF staining was performed as described previously (Scheuring et al., 2015). Plant rosette phenotype evaluation was performed 3 weeks after germination.

3-D reconstruction of vacuoles
Imaris 8.4.0 was used for the reconstruction of cell and vacuole volumes. Based on the PI channel, every 3rd slice of the z-stack was utilised to define the cell borders using the isoline, magic wand or manual (distance) drawing functions in the manual surface creation tool. After creating the surface corresponding to the entire cell, a masked channel (based on BCECF) was generated by setting the voxels outside the surface to 0. Subsequently, a second surface (based on the masked BCECF channel) was generated automatically with the smooth option checked. The obtained surface was visually compared to the underlying BCECF channel and, if necessary, the surface was fitted to the underlying signal by adjusting the absolute intensity threshold slider. Finally, volumes of both surfaces were extracted from the statistics window.

Confocal Microscopy
For image acquisition a Leica TCS SP5 (DM6000 CS) confocal laser-scanning microscope, equipped with a Leica HCX PL APO CS 63 × 1.20 water-immersion objective, was used.