In a forward genetic, GUS-based screen for endodermal barrier mutants (Lee et al., 2013) (Alassimone et al., unpublished), we discovered one mutant, sgn3 that displayed a dramatic defect in endodermal barrier formation, as visualized by penetration of the apoplastic tracer propidium iodide (PI) into the stele along the entire length of the root. This resembled a phenotype of T-DNA insertion lines in an endodermis-enriched gene that we had analyzed in parallel (Figure 1A, Figure 1—figure supplement 1A) (Birnbaum et al., 2003; Brady et al., 2007). Complementation analysis and sequencing confirmed that the causal mutation in sgn3-2 was an early stop codon in the open reading frame of At4g20140. SGN3 encodes for a leucine-rich-repeat receptor-like kinase (LRR-RLK) of subfamily XI that had previously been described as GASSHO1 (GSO1) (Figure 1B, Figure 1—figure supplement 2, Figure 1—source data 1) (Tsuwamoto et al., 2008). In the previous publication, this LRR-RLK was investigated because of embryonic expression of a putative ortholog in Brassica napus, but no single mutant phenotype was found. A phenotype was only observed when gso1 (sgn3) was combined with a mutant of its closest homolog, At5g44700 (named GSO2). This gso1 gso2 double mutant displayed a protodermal phenotype, characterized by a defective cuticle and fusion of cotyledons (Tsuwamoto et al., 2008). True leaves did not differ from wild type. In contrast to SGN3/GSO1, GSO2 was shown to be expressed only in shoots, but not in roots, explaining the exclusive root phenotype in the sgn3 single mutant that we report here and that had escaped detection earlier on. In order to investigate the cause of this phenotype, we investigated formation of the Casparian strip and found that it is still formed and correctly positioned, but that it is repeatedly interrupted, forming irregularly-sized ‘holes’ of several micrometer in length (Figure 1C, Figure 1—figure supplement 1B). Suberin lamellae formation, by contrast, appeared at a position and in a manner very similar to wild type (Figure 1D,E, Figure 1—figure supplement 1C,D).

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Detail of SGN3 T-DNA and EMS mutants.

Detail of SGN3 mutations. sgn3-1 EMS mutant was lost during the screen and is not available anymore.

https://doi.org/10.7554/eLife.03115.004

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We asked whether the Casparian strip cell wall defect is caused by a defect in the formation of the CSD, or whether the function of SGN3 lies downstream of the CASPs, as has been demonstrated for the NADPH oxidase SGN4/RBOHF (Lee et al., 2013). In the sgn3 background, CASP1-GFP showed a pattern of interrupted patches that was strikingly similar to that of the Casparian strip itself. This suggests that it is the incorrect localization of the CASPs that causes the Casparian strip defects of the mutant (Figure 1F). Interestingly, in a novel forward genetic screen in which we scored directly for mutants with altered CASP1-GFP localization (Kalmbach et al., unpublished), we found 14 novel alleles of sgn3 (Figure 1B, Figure 1—figure supplement 2, Figure 1—source data 1). This suggests that SGN3 is one of the major non-redundant genes necessary for CASP localization in the Arabidopsis genome. We could show that not only CASP1-GFP, but all four other CASPs (CASP2-5) display identical defects in sgn3 (Figure 1—figure supplement 1E).

An interrupted CSD should abolish the lateral diffusion barrier in the plasma membrane of endodermal cells that we had previously described (Alassimone et al., 2010). Indeed, a generic plasma membrane protein that is excluded from the CSD in wild type (Alassimone et al., 2010) (Figure 1G) shows a pattern of exclusion in the mutant that represents a perfect negative image to the CASP1-GFP islands (Figure 1G). This illustrates that the lateral diffusion barrier between inner and outer plasma membrane domain is absent in sgn3 mutants, but that the remaining CSD islands are still able to exclude other proteins. To our surprise, this defect did not affect the polar localization of PDR6, a transporter that localizes to the outer polar domain in the endodermis (Figure 1H). This indicates that a strict polarity in the endodermis can be maintained in the absence of a diffusion barrier, further supporting the notion that polarity of transmembrane proteins in plants might simply be maintained by generally low lateral diffusion, high rates of endocytosis, or both (Geldner, 2009).

We had shown previously that during endodermal differentiation, CASP1-GFP changes from a protein that displays endocytic cycling and lateral diffusion to one that is highly stable and immobile in the CSD (Roppolo et al., 2011). In this process, CASP1-GFP localization passes through a ‘string of pearls’ stage, in which individual islands or ‘patches’ of CASP1-GFP are aligned in a band before eventually fusing (Figure 2A). Comparison of 3D-time-lapse observations of endodermal differentiation revealed that sgn3 is not able to progress from this ‘string of pearls’ stage, but only undergoes partial fusion of individual islands, leading to CASP1-GFP patches of heterogenous size (Figure 2A). We further found by fluorescence recovery after photobleaching (FRAP) that these CASP1-GFP islands have similar stability and turnover as wild type (Figure 2B). Another feature of the CSD is its very strong adherence to the cell wall, leading to the long-described phenomenon of ‘band plasmolysis’ in endodermal cells (Krömer, 1903; Behrisch, 1926; Alassimone et al., 2010). The inability of the CASP1-GFP ring to retract from the Casparian strip upon plasmolysis (Figure 2—figure supplement 1) allow for easy visualization of this phenomenon. This adhesion leads to greatly flattened, often fenestrated protoplasts, that looked very unlike the usual, rounded protoplasts of epidermal cells (Alassimone et al., 2010). sgn3 showed the very same inability of CASP1-GFP to retract from the strips, demonstrating that plasma membrane-to-cell wall attachment remains intact in the mutant (Figure 2—figure supplement 1). We conclude that sgn3 does not cause a general defect in CASP1-GFP trafficking, polymerization or cell wall attachment, but rather a specific defect in the progression of already stable CASP islands towards a contiguous band.

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We then generated lines of tagged SGN3 variants in order to investigate its expression and subcellular localization. Only a C-terminal mVenus fusion with a 9.4-kb genomic fragment containing intron, 5′UTR, and the upstream neighboring gene, provided full complementation of the mutant phenotype, indicating functionality of the protein fusion and of its regulatory sequences (Figure 2—figure supplement 2A,B). In transgenic lines of this construct, expression of SGN3 could be observed in endodermal cells shortly after the onset of elongation, preceding the onset of CASP1 expression (Figure 2C, Figure 2—figure supplement 3). This is consistent with a function of SGN3 in establishing correct CASP1 localization. Initially, SGN3 could be found on all cell sides, but it then quickly accumulated in the transversal and anticlinal sides of the plasma membrane, yet became excluded from the CSD itself—a hitherto unknown subcellular localization pattern (Figure 2D–F). In 3D-reconstructions this leads to the appearance of a broader ring-like domain that flanks the more restricted, median CSD (Figure 2F). During the critical phase of CASP domain progression from isolated islands towards a fused ring—at which the sgn3 phenotype becomes apparent–SGN3 is seen to surround individual CASP islands on all sides (Figure 2G). Such localization would fit with a role of SGN3 in promoting fusion of growing CASP islands, such that an uninterrupted band can be formed (Figure 2H).

In order to establish the functional relationships between SGN3 and other previously characterized genes, we combined sgn3 with casp1 casp3 double and esb1 single mutants. ESB1 is an extracellular protein that localizes to the site of Casparian strip formation and whose absence causes a phenotype resembling that of sgn3 insofar as both the Casparian strip itself and CASP1-GFP are localized in interrupted bands (Hosmani et al., 2013). In contrast to sgn3, however, esb1 mutants additionally display increased, delocalized deposition of additional autofluorescent cell wall material as well as enhanced and earlier deposition of suberin (Hosmani et al., 2013). We found ESB1-mCherry in sgn3 to be both present and correctly localized, that is, specifically accumulating at the remaining CASP1-GFP islands (Figure 3A). Thus, ESB1 does not require SGN3 for its accumulation and localization and the Casparian strip discontinuities in esb1 and sgn3 might be caused by independent mechanisms. Consistently, we found that sgn3 esb1 double mutant formed less and/or smaller Casparian strip patches than each single mutant, indicating additivity of the two mutations in this common aspect of their phenotypes (Figure 3B,C). Intriguingly however, sgn3 esb1 double mutants neither display an increased or delocalized deposition of autofluorescent cell wall material, nor an enhanced and earlier formation of suberin. This indicates a full epistasis of sgn3 over esb1 for this phenotype (Figure 3B,D). The same epistatic relationship was found between sgn3 and casp1 casp3 double mutants (Figure 3B–D). The enhanced and ectopic formation of autofluorescence and suberin is thought to be due to a feedback and crosstalk between cell wall components, connecting correct Casparian strip formation to the further deposition of lignin and suberin lamellae formation (Hosmani et al., 2013). The epistasis of sgn3 suggests that the perception/signaling that leads to enhanced autofluorescence and suberin formation in esb1 and casp1 casp3 is mediated by—or at least requires—the SGN3 receptor-like kinase.

Figure 3

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Considering the strength of the barrier phenotype of sgn3 and its inability to initiate potentially compensatory suberization, we were surprised to observe a rather mild growth phenotype of sgn3 under certain conditions (Figure 4A, Figure 4—figure supplement 1A). Yet we noticed that sgn3 growth is extremely sensitive to changes in environmental conditions that are non-stressful to wild type (Figure 4A, Figure 4—figure supplement 1A). We ensured that the endodermal barrier phenotype persisted in developed root systems of rosette stage plants both on soil and on hydroponics, thus excluding that the weak phenotype is caused by an eventual repair of the barrier in older plants (Figure 4—figure supplement 1B). A systematic variation of day length, temperature, and light-intensity revealed that growth differences to wild type are more pronounced at higher temperature, as well as under long-day conditions (Figure 4B, Figure 4—figure supplement 1C). In general, sensitivity of sgn3 to growth conditions is such that we observed everything from a near absence of phenotypes to severely dwarfed plants that did not reproduce when grown in different, standard growth chambers that all well supported wild type growth. We wanted to ascertain that the observed growth phenotypes of sgn3 are indeed caused by the Casparian strip defect in the roots and not by some hypothetical, non-redundant function of SGN3 in shoots. We therefore expressed SGN3 under the control of the CASP1 promoter that shows specific expression in differentiating root endodermis, with some expression in the endodermis of the lower hypocotyl and none detectable anywhere else in the plant. These CASP1::SGN3-GFP sgn3 plants could fully rescue the growth phenotype of sgn3 plants (Figure 4C), demonstrating that it is the root endodermal defect of sgn3 that is causative for the overall growth defect of the mutant.

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The numerous, repeated Casparian strip discontinuities in sgn3 must generate an apoplastic bypass for both water and solutes in the mutant root. Especially under conditions of high transpiration, this would be expected to lead to a strong, uncontrolled and possibly detrimental influx of elements into the transpiration stream. We observed that transpiration rate per surface area in sgn3 mutants are very similar to wild type (Figure 4D) revealing that there is no compensatory downregulation of transpiration. Another predicted consequence of interrupted Casparian strips is an inability to build up root pressure, a process necessary for vascular transport of nutrients and water in the absence of transpiration and for transport into non-transpiring organs (Wegner, 2014). Root pressure build-up in the dead xylem vessels is thought to involve an osmotic gradient, generated by living neighbors, that draws water into the vessels. Pressure build-up, however, is thought to crucially depend on the presence of the Casparian strips as an apoplastic diffusion barrier, without which xylem pressure should quickly dissipate into the cortex. We indeed found that root pressure, as estimated by xylem sap exudation from hypocotyls after decapitation, was lower in the sgn3 mutant (Figure 4E). sgn3 also showed a decreased rate of leaf guttation, a process more indirectly dependent on root pressure, but that can be observed less invasively (Figure 4F). As a more direct measure of apoplastic water flow, we inserted excised roots in a pressure chamber and found a significant increase in root hydrostatic hydraulic conductivity (Lp_r-h) in sgn3 mutants compared to wild type (Figure 4G). The defects of Casparian strip formation may allow an apoplastic bypass for water in pressurized sgn3 roots thereby explaining its increased Lp_r-h. Our findings are the first genetic evidence that Casparian strips can be relevant for root pressure buildup and impose a hydraulic resistance; the magnitude of the observed effects nevertheless indicates that an intact Casparian strip is not an absolute requirement and that unmodified walls and/or suberized cells can generate a sufficiently high resistance to radial waterflow so that the positive root pressure can be maintained. sgn3 might be very useful to dissect the mechanisms of root pressure formation and its role in nutrient transport in the future.

In order to identify some potential stress and/or compensatory responses in the sgn3 mutant, we generated a general transcriptional profile of sgn3 mutant rosettes, grown under conditions in which wild type and sgn3 were indistinguishable. Only little differences between sgn3 and wild type were observed. Yet, all of the seven more highly expressed genes in sgn3 were found to be also induced by potassium starvation, suggesting a latent, weak potassium deficiency stress under these conditions (Figure 5A, Figure 5—source data 1). Based on this indication, we tested well-established transcriptional read-outs of potassium deficiency in roots, the potassium influx transport proteins AKT1 and HAK5, and found that both were upregulated in sgn3 (Figure 5B,C).

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Transcriptional differences between wild type and sgn3 shoots.

Genes affected in sgn3-3 transcriptomic analysis in leaves (p < 0.15) and responses of those genes to abiotic stress. Fold change and p-value marked by asterisk were obtained in Genevestigator for K starvation, drought, nitrate starvation, Fe deficiency, S deficiency, P deficiency, and salt (Hruz et al., 2008).

https://doi.org/10.7554/eLife.03115.015

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Figure 5—source data 2

Overview of ionomic experiments.

Ionomic analysis performed on sgn3 leaves (sgn3-3, sgn3-4, and sgn3-19 T-DNA insertion lines) in three different labs (Hokkaido, Lausanne and Aberdeen), two growth systems (hydroponic and soil) and two growth conditions (short days and long days). Values are presented as mean ± SD. t-tests were performed to determine the significant differences to WT (corresponding p-values are indicated).

https://doi.org/10.7554/eLife.03115.016

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In order to directly measure how sgn3 root barrier defects affect elemental homeostasis in leaves, we undertook a number of elemental profiling experiments using inductively coupled plasma–mass spectrometry (ICP-MS) on rosette leaves in different laboratories under different growth conditions. Surprisingly, concentrations of most elements remained essentially unaltered in sgn3. Despite the dramatic apoplastic bypass in sgn3 roots, the mutants managed to maintain wild type levels of sodium (Na), calcium (Ca), or boron (B), for example. Among the measured transition elements (Mo, Co, Mn, Zn, Fe, Cu, Zn) only zinc showed a significant decrease in some measurements (Figure 5D, Figure 5—source data 2). However, matching the transcriptional profiling results, we invariably found lower levels of potassium (K) in the mutant (ranging from 1.4–3.0 fold reduction). Inversely, levels of magnesium (Mg) and cesium (Cs) accumulated to higher levels than in wild type, ranging from 1.5–2.1 and 1.3- to 1.4-fold increase, respectively (Figure 5D, Figure 5—source data 2). These findings nicely corroborated the results of our microarrays, but left us wondering about the absence of potassium deficiency symptoms in sgn3, since the mutant phenotypes under most conditions are simply those of a reduction and delay in growth.

We then tested growth of sgn3 plants on a nutrient-poor, gravel-like substrate, which we watered with a nutrient solution with or without potassium. Wild type plants coped well with conditions of low potassium nutrition, with most plants displaying no or only very weak chlorosis on older rosette leaves, indicative of potassium deficiency (Figure 5E,F). Consistently, wild type also maintained higher potassium concentration than sgn3 under these conditions (Figure 5—figure supplement 1). The sgn3 mutant in contrast, developed severe chlorosis of its rosette leaf margins, characteristic of strong potassium deficiency (Marschner and Marschner, 2012). sgn3 therefore has an impaired capacity to accumulate sufficient potassium under conditions of low supply of this element.

The deposition of cell wall material with subcellular precision is crucial for the correct function of many plant cell types (Roppolo and Geldner, 2012). How this localized deposition is achieved is only rarely understood in any mechanistic detail, with the notable exception of wall deposition in metaxylem cells (Oda and Fukuda, 2012). Localized wall deposition should implicate the formation of microdomains in the plasma membrane that localize cell wall biosynthetic activity. Walls, in turn, signal back to the cell, and this process informs about mechanical stresses, wall polymer breakdown or for regulated expansion growth (Cheung and Wu, 2011; Lindner et al., 2012). The precisely localized, ring-like Casparian strip can be used as a model for studying localized wall deposition (Lee et al., 2013). The SGN3 LRR-RLK appears to be involved in both the formation of the CSD and the signaling of defective Casparian strips. Lack of SGN3 causes an inability of CASP1-GFP patches to fuse into a contiguous band, without affecting the positioning of the patches along a median ring, nor the stability or functionality of the already established patches. SGN3 localization to the transversal and anticlinal, but not the periclinal membrane domains further increases the complexity of subdomains present at the endodermal plasma membrane. It embeds the forming CASP domain into a larger subdomain within which the narrower CSD is formed. Direct interaction of CASPs and SGN3 could take place early during CASP accumulation at the membrane and occur at the rims of forming CASP patches, possibly promoting patch growth and fusion. This can now be tested in future experiments. A second, currently unrelated role for SGN3 is the induction of increased lignin and suberin formation in other Casparian strip mutants, such as esb1 or casp1 casp3. We speculate that SGN3 acts as a kinase that reports CSD integrity, signaling an upregulation of lignin, and suberin production. A surveillance of Casparian strip integrity could be of great importance for unimpeded root function and would parallel the surveillance of tight junctions that has been reported for animal epithelia (Balda and Matter, 2009).

We could demonstrate that exclusive expression of SGN3 in the differentiating root endodermis is sufficient to rescue the overall growth defect of sgn3. This indicates that the endodermal function that we describe here is the only specific, non-redundant activity of this receptor in the plant. The earlier described function of SGN3/GSO1 in the formation of an embryonic cuticle by contrast is fully redundant with that of its homolog GSO2. The dramatic cotyledonary phenotype of the double mutant is bound to have secondary consequences on seedling growth, making it very difficult to dissect primary from secondary effects. A recent investigation of root meristem defects of the double mutants, for example, could entirely be explained as secondary consequences of the primary defect in embryonic cuticle formation (Racolta et al., 2014). It is nevertheless important to speculate how the two functions of endodermal Casparian strip formation and protodermal cuticle formation could be explained by a common molecular activity. By genetic analysis, the GSO1 GSO2 pair has been assigned to a signaling pathway that depends on an endosperm-expressed subtilase (ABNORMAL LEAF-SHAPE1, ALE1) (Xing et al., 2013). In a currently speculative scenario, subtilases might be involved in processing of peptide hormones that could act as ligands for SGN3/GSO1, promoting formation of a functional cuticle. Yet, the endodermal, lignified Casparian strip and the protodermal, cutin-based embryonic cuticle are two chemically different cell wall modifications that also show distinct modes and sites of deposition within the primary cell wall. These differences make it difficult to perceive a common denominator for SGN3 action in these two cell types. Moreover, our data suggest that a primary function of SGN3/GSO1 is the formation of a continuous band of CASP membrane proteins. No CASP homologs have been implicated in cuticle formation up-to-now, nor are there any indications for the presence of a highly scaffolded membrane protein domain in the epidermis/protodermis that would resemble the CSD. Another SGN3/GSO1 function that we could establish is the promotion of suberin formation in response to a defective Casparian strip. Enhanced suberin formation in esb1 appears to be entirely dependent on SGN3 activity, although the normal developmental progression of suberin formation is not affected in the mutant. Suberin and cutin are chemically closely related polymers whose monomers are formed by sets of homologous enzymes. A common role of SGN3 might be to mediate a cross-talk between cell wall components, possibly regulating suberin production in endodermis and cutin production in epidermis/protodermis. Identification of the SGN3 ligands and downstream kinase substrates will be crucial for an understanding of the common mechanism that might underlie these apparently divergent functions.

Regardless of the fact that SGN3 has other roles in the plant, the sgn3 single mutant defect reported here remains exquisitely specific to the endodermis. This is supported by the fact that it is only in the endodermis where SGN3 expression is non-overlapping with its close homolog, GSO2 (Tsuwamoto et al., 2008). Moreover, exclusive expression of SGN3 in the endodermis is able to rescue the overall growth defects observed in aerial parts (this study). In addition to its specificity, the sgn3 phenotype turns out to be strong and robust, that is, it displays penetration of tracer along the whole length of a seedling root and the phenotype is stable in the different growth conditions that we tested. Finally, sgn3 does not show any of the enhanced suberin formation that has been observed in other mutants and which confounds interpretation of phenotypes. We therefore propose that the sgn3 mutant is uniquely suited to obtain insights into the specific role of Casparian strips in plants. This mutant might be very useful for investigating not only root nutrient and water uptake, but also for addressing the role of the endodermal barrier in pathogen resistance, hormonal transport, stress, or general growth responses.

Our first analysis of the sgn3 mutant has revealed an unexpected robustness of Arabidopsis growth towards a complete lack of its main root diffusion barrier. While still able to grow and complete its life cycle, the mutant nevertheless displays greatly exacerbated growth reductions to non-stressful changes in environmental conditions. This indicates the presence of homeostatic backup systems in the plant that are incompletely compensating for the absence of Casparian strips. Homeostasis of many elements is kept in the sgn3 mutant, and it is intriguing that it is specifically potassium that shows a reproducibly strong decrease in concentration. Potassium (K) is the only macronutrient (N,P,K,S) that is not integrated into polymers and that remains highly mobile within the plants. It is being used as a counter ion in many transport process and is recycled back to the root along with the phloem sap. At the same time, concentrations of potassium required for plant growth are high and potassium often needs to be concentrated 10–100 fold over the concentration found in the soil (Marschner and Marschner, 2012; Wang and Wu, 2013). It therefore makes a lot of sense that potassium is an element that cannot be kept at sufficiently high levels in Casparian strip defective plants. We propose that potassium located in the stelar apoplast is continuously lost to the outer cell layers and the soil in the absence of functional Casparian strips. Upregulation of potassium influx carriers in cortex/epidermis, as we have observed in sgn3 roots, would certainly be able to recuperate large parts of the potassium being lost. However, a chronic condition of very low external potassium would lead to a breakdown in this transporter-mediated ‘potassium recycling’ and eventually cause the deficiency phenotypes that we observe. From this scenario, it follows that there should be a higher rate of potassium flux out-of and into the root stele in a sgn3 mutant and that potassium homeostasis in the mutant is extremely dependent on increased activity of potassium transporters. We are currently working on substantiating this ‘potassium recycling’ model by specific interference with and visualization of potassium transporters in the mutant. Further analysis of sgn3 will be instructive for a better understanding of root potassium transport in general and this analysis should also be extended to other macronutrients in the future.

What could be the reasons for the lack of effects on many other elements? It can be expected that similar compensatory responses as seen for potassium exist for other elements. If these elements need to be less concentrated over their external concentration—or if they are less mobile within the plant—the compensatory responses might be sufficient for maintaining homeostasis in the mutant. Interestingly, we found an increase in the levels of the divalent cation magnesium, while concentrations of the divalent calcium, for example, were not affected in sgn3 mutants. This could be explained by a lower concentration gradient between the xylem sap and the external environment and a lower mobility of Calcium within the cell wall space (Clarkson, 1984; White and Broadley, 2003). Nevertheless, calcium concentration within the symplast is kept extremely low for cellular signaling purposes. Therefore, calcium delivery to aerial tissues is thought to occur either entirely through the apoplast–using the few apoplastic ‘bypasses’ of the endodermis at the root tip or at lateral root emergence sites (White, 2001)—or to employ a very short symplastic route passing exclusively through endodermal cells (Clarkson, 1984). In both scenarios, it is surprising that the opening of a massive apoplastic bypass in the sgn3 mutant has no effect on calcium homeostasis, while it did affect levels of magnesium. Possibly, magnesium is more mobile in the apoplast than Calcium and undergoes increased apoplastic transport into the transpiration stream in sgn3 (Thibault and Rinaudo, 1985). Identifying conditions in which calcium homeostasis also breaks down in sgn3 would greatly advance our understanding of the calcium uptake mechanisms in plants.

The sgn3 mutant represents a powerful new tool to better understand root uptake and transport mechanism of most other plant nutrients as well. Our initial analysis of the sgn3 mutant challenges the current notions of the role of the Casparian strip as a required barrier for most or all elements. The ability to selectively disrupt this strict endodermal barrier will allow for direct investigations of other mechanisms that maintain and buffer ion homeostasis. Unmodified cell walls, by their properties as ion exchange matrices, in conjunction with vacuolar storage of elements, can play pivotal roles in maintaining homeostasis of many elements. Cytoplasmic magnesium levels, for example, might well remain unaltered in sgn3, the increased magnesium levels being entirely absorbed by the vacuole. Having taken away the ‘first lock’ of the Casparian strip now puts the focus on the existence of ‘second locks’ that can be studied using the sgn3 mutant as a sensitized background.

Arabidopsis thaliana ecotype Columbia was used for all experiments. For details of knockout mutants, see Table 1.

For generation of expression constructs, Gateway Cloning Technology (Invitrogen, Carlsbad, CA) or standard molecular biology procedures were used. pESB1::ESB1-mCherry plasmid (Hosmani et al., 2013) was transformed in sgn3-3 background. pSGN3 (5583 bp before ATG)::SGN3 gDNA-mVenus was cloned by integrating a 9.4-kb genomic fragment including the intron and the 5′UTR into a Basta-resistance pGREENII vector containing mVenus. pCASP1::NLS-GFP-GUS and pCASP4::NLS-GFP-GUS reporters were cloned into a Basta-resistance pGREENII vector and contain 1048 bp and 695 bp promoter fragments, respectively. pCASP1::SGN3 cDNA-GFP was cloned with Gateway using a 1207 promoter fragment followed by SGN3 cDNA without the unique intron and GFP. pCASP1 (1207 bp)::PDR6 gDNA-Venus-4G (4 glycine extension) was cloned using Gateway in a pB7m34 GW,3 expression vector (http://gateway.psb.ugent.be). pCASP1 (1207 bp)::mCherry-SYP122 was constructed with Gateway by cloning the AtSYP122 (At3g52400) cDNA into pH7m42 GW,3. pCASP1::CASP2-GFP-4G, pCASP1::CASP3-GFP-4G, pCASP1::CASP4-mCherry and pCASP1::CASP5-GFP-4G were cloned with Gateway using the pB7m34 GW,3 expression clone. Transgenic plants were generated by introduction of the plant expression constructs into an Agrobacterium tumefaciens strain GV3101 and transformation was done by floral dipping (Clough and Bent, 1998).

For in vitro assays, plants were germinated on 0.5 MS (Murashige and Skoog) agar plates after 2 days in dark at 4°C. Seedlings were grown vertically in growth chambers at 22°C, under long days (16-hr light/8-hr dark), 100 μE light, and were used at 5 days after shift to room temperature. For microarray, transpiration, hydraulic conductivity, and ionomic analysis in Lausanne (May 2012) assays, plant were germinated on hydroponic conditions after 2 days in dark at 4°C. Nutrient solution was changed weekly and contained 0.5 mM KNO₃, 0.25 mM Ca(NO₃)₂, 1 mM KH₂PO₄, 1 mM MgSO₄, FeNH₄-EDTA 0.1 mM, KCl 50 μM, H₃BO₃ 30 μM, MnSO₄ 5 μM, ZnSO₄ 1 μM, CuSO₄ 1 μM, (NH₄)₆Mo₇O₂₄ 0.1 μM. Light/dark cycle was 16 hr/8 hr, 23°C/18°C, 100 μE light.

For xylem sap and guttation assays, plants were germinated after 2 days in dark at 4°C on soil in short day condition and irrigated with water. Light/dark cycle was 10 hr/14 hr, 22°C/18°C, 100 μE light.

For phenotypic assays at different light intensities (30, 90, 150, or 300 μE), temperatures (17, 21, or 25°C), or day lengths (8, 16 or 24 hr), plants were germinated after 2 days in dark at 4°C on soil and irrigated with water. The standard condition was: light/dark cycle 16 hr/8 hr, 21°C/19°C with light intensity of 150 μE. Temperature difference between day and night was always kept at 2°C.

For ionomic assays plants were grown in different laboratories as indicated. For the Hokkaido experiments plants were grown hydroponically as described in Takano et al. (2001), with slight modifications. Environmental parameters in a growth chamber are as follows: 10-hr/14-hr light/dark cycle, 22°C under fluorescent lamps, 70% humidity. The seeds were sown on rockwool and grown supplied with hydroponic media (Fujiwara et al., 1992) supplemented with 50 µM Fe-EDTA. After 15 days, the plants were additionally supplied with 3 µM CsCl and 10 µM SrCl₂. The media were changed twice a week. For the Aberdeen experiment plants were grown in short day conditions, in Bulrush multi-purpose compost (http://www.bulrush.co.uk/retail-range/all-purpose-composts.html) spiked with various elements as detailed in Lahner et al. (2003), and bottom watered using ¼ Hoagland solution containing Fe as 10 μM Fe-HBED as described in Baxter et al. (2008). 1–2 adult rosette leaves were harvested 36 days after planting. For the Lausanne experiment in soil (March 2011), plants were grown in long days 16-hr/8-hr light/dark, 23°C/19°C, and leaves were harvested after 5 weeks.

For potassium deficiency assay plants were germinated after 2 days in dark at 4°C on a poor gravel-like substrate (OIL DRI US-Special) watered with a ½ Hoagland based solution containing : 1.5 mM NH₄NO₃ or 1.5 mM KNO₃ (−K or +K).

Confocal laser scanning microscopy experiments were performed either on a Leica SP2, a Zeiss LSM 700, a Zeiss LSM 710, or a Zeiss LSM 710 NLO 2-Photon microscope. Excitation and detection windows were set as follows: Leica SP2: GFP 488 nm, 500–600 nm; propidium iodide 488 nm, 600–700. Zeiss LSM 700: GFP/mVenus 488 nm, 490–555 nm; mCherry/propidium iodide 555 nm, SP 640. LSM 710: GFP/mVenus 488 nm, 495–554 nm; mCherry/propidium iodide 561 nm, 573–681 nm. Zeiss LSM 710 NLO 2-Photon: GFP/mVenus, 960 nm, 500–550 nm (NDD). For visualization of the apoplastic barrier, seedlings were incubated in the dark for 10 min in a fresh solution of 15 mM (10 mg/ml) Propidium Iodide (PI) and rinsed two times in water (Alassimone et al., 2010, Naseer et al., 2012). For quantification, ‘onset of elongation’ was defined as the point where an endodermal cell in a median, longitudinal section reached a length more than twice its width. From this point, cells in the file were counted until the PI signal was blocked in the endodermal cells. For plants grown in hydroponic and in soil, roots of 13 and 14 days were used, respectively.

Casparian strip autofluorescence after clearing was visualized as described (Alassimone et al., 2010, Naseer et al., 2012).

Fluorescence recovery after photobleaching (FRAP) was performed with 5-day-old seedlings and imaged with a Leica SP2 confocal microscope as described (Roppolo et al., 2011).

Plasmolysis was induced by incubating 5-day-old seedlings for 1 hr in 0.8 M mannitol and then mounted in the same solution. Plasmolyzed cells were imaged at 15 cells after the onset of CASP1 expression.

For time-lapse imaging of CASP1::CASP1-GFP in Col-0 and sgn3-3, 5-day-old seedlings were into a Lab-Tek II chambered coverglass (Nunc) and covered with a small block of agar to prevent drying. Subsequently, slides were mounted and imaged on an upright Zeiss LSM710 NLO confocal microscope. Excitation was provided by a Ti-Sapphire Chameleon II Ultra (Coherent) with 960 nm and fluorescence was detected using non-descanned detection (NDD) between 500 and 550 nm. Image stacks were taken every 15 min for a total time of 16 hr. Confocal images were analyzed and contrast and brightness were adjusted with the FIJI package (http://fiji.sc/Fiji) and Adobe Photoshop CS5.

For the percentage quantification of Casparian strips, autofluorescence after clearing was performed on ten seedlings for each genotype investigated. For each individual seedling, Casparian strips were imaged by confocal microscopy by doing one z-stack and measurements were done using ImageJ. The percentage of Casparian strip was the portion where autofluorescence was visible along a line showing Casparian strip signal. A minimum of 3.5 mm of Casparian strip was quantified in each genotype.

Suberin lamella was observed in 5-day-old roots after Fluorol Yellow staining as described in Naseer et al. (2012). Seedlings were incubated in Fluorol Yellow 088 (0.01% wt/vol, lactic acid) at 70°C for 30 min, rinsed with water, and counterstained with aniline blue (0.5% wt/vol, water) at RT for 30 min in darkness, washed, mounted on slides with glycerol and observed with epifluorescence microscope. Suberin pattern were observed and counted from the hypocotyl junction to the onset of endodermal cell elongation. Three distinct patterns were considered: continuous suberin lamellae, discontinuous suberin lamellae (corresponding to the area where suberin lamellae establish), and non- suberized (corresponding to the young part of the root). Experiments were repeated at least four times.

For ionomic assays plants were grown in different laboratories as indicated. For the Hokkaido and Lausanne experiments (hydroponic conditions), the shoots of plants were harvested, dried in an air incubator at 60°C for more than 60 hr, and the dry weights were measured. The tissues were digested with 3 ml of 61% HNO₃ (for boron determination; Wako Pure Chemicals, Osaka, Japan) in a tube at 110°C in a DigiPREP apparatus (SCP Science, Quebec, Canada) until complete dryness. The residues were dissolved in 10 ml of 2% HNO₃ and analyzed for elements by inductively coupled plasma mass spectrometry (ICP-MS) (ELAN, DRC-e; Perkin–Elmer, Waltham, MA, USA).

For Aberdeen assays, the leaves were cleaned by rinsing with ultrapure water and placed into Pyrex digestion tubes. Samples were dried in an oven at 88°C for 20 hr. After cooling, seven reference samples from each planted block were weighed. The samples together with blank controls were digested with 0.90 ml concentrated nitric acid (Baker Instra-Analyzed; Avantor Performance Materials) and diluted to 10.0 ml with ultrapure water (18.2 MΩcm). The internal standard Indium (In) was added to the acid prior to digestion for monitoring technical errors and plasma stability in the ICP-MS instrument. After samples and controls were prepared, elemental analysis was performed with an ICP-MS (NexION 300D; PerkinElmer) coupled to Apex desolvation system and SC-4 DX autosampler (Elemental Scientific Inc., Omaha, NE, USA), monitoring these elements: B, Na, Mg, P, K, Ca, Mn, Fe, Co, Cu, Zn, Sr, and Mo. All samples were normalized to calculated weights, as determined with a heuristic algorithm using the best-measured elements, the weights of the seven weighed samples and the solution concentrations (Lahner et al., 2003), detailed at www.ionomicshub.org.

For ionic chromatography, for each sample one rosette leaf of a 5-week-old plant was harvested, put in 30 ml Nanopure water and incubated at 80°C for 1 hr. Each sample was then filtered with a 0.45-µm non-pyrogenic sterile filter (Sarstedt, Germany). The samples were then diluted to reach a final concentration of 0.83 mg fresh weight per 1 ml. Ion concentrations were obtained with an ion chromatography system (ICS-1100/2100, Dionex-Thermo Fischer).

All statistical analyses were done in the R environment (http://www.R-project.org/). Binary comparisons between wild type and sgn3 were performed using Student's t-test. Multiple comparisons between wild type, sgn3-3, and sgn3-4 mutants, one-way ANOVA was performed, and Tukey's test was subsequently used as a multiple comparison procedure.

The different features were predicted using the following web resources. Signal peptide, Leucine-rich repeats, transmembrane domain, and kinase domain: http://www.uniprot.org/uniprot/C0LGQ5; LRR N-terminal domain: http://pfam.sanger.ac.uk/search/sequence; kinase sub-domains were defined with reference to Hanks and Hunter (1995) and by aligning to the BRI1 kinase domain (Vert et al., 2005). Borders between kinase sub-domains were set manually by comparison of amino acids in the respective border regions for PKA C alpha and BRI1. In case of conflict, the BRI1 annotation was considered as more meaningful because of its plant origin.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank the Genomic Technologies Facility (GTF) and the Central Imaging Facility (CIF) of the University of Lausanne for expert technical support. We thank Valérie Dénervaud Tendon, Guillaume Germion, Deborah Mühlemann, and Kayo Konishi for technical assistance and John Danku and Véronique Vacchina for ICP-MS analysis. This work was funded by grants from the Swiss National Science Foundation (SNSF), the European Research Council (ERC) to NG and a Human Frontiers Science Program (HFSP) grant to JT and NG. GL and CM were supported by the Agropolis foundation (Rhizopolis) and the Agence Nationale de la Recherche (HydroRoot; ANR-11-BSV6-018). MB was supported by a EMBO long-term postdoctoral fellowship, JEMV by a Marie Curie IEF fellowship and TK by the Japan Society for the Promotion of Sciences (JSPS).

© 2014, Pfister et al.

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