# BCL2-ASSOCIATED ATHANOGENE4 Regulates the KAT1 Potassium Channel and Controls Stomatal Movement

Antonella Locascio, Maria Carmen Marqués, Guillermo García-Martínez, Claire Corratgé-Faillie, Nuria Andrés-Colás, Lourdes Rubio, José Antonio Fernández, Anne-Aliénor Véry, José Miguel Mulet, Lynne Yenush

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Antonella Locascio, Maria Carmen Marqués, Guillermo García-Martínez, Claire Corratgé-Faillie, Nuria Andrés-Colás, et al.. BCL2-ASSOCIATED ATHANOGENE4 Regulates the KAT1 Potas- sium Channel and Controls Stomatal Movement. Plant Physiology, 2019, 181 (3), pp.1277-1294. 10.1104/pp.19.00224 . hal-02349953

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1 **Short title: AtBAG4 is a KAT1 potassium channel regulator**

2 *Author for Contact details

3 Lynne Yenush

4 Instituto de Biología Molecular y Celular de Plantas, Universitat Politècnica de Valencia-

5 Consejo Superior de Investigaciones Científicas, 46022 Valencia, SPAIN

6 +34963879375

7 **Title: BCL2-ASSOCIATED ATHANOGENE4 Regulates the KAT1 Potassium Channel and**

8 **Controls Stomatal Movement**

10 **Author names and affiliations:** Antonella Locascio¹, Mª Carmen Marqués¹, Guillermo

11 García-Martínez¹, Claire Corratgé-Faillie², Nuria Andrés-Colás¹, Lourdes Rubio³, José

12 Antonio Fernández³, Anne-Aliénor Véry², José Miguel Mulet¹, Lynne Yenush¹*

13 ¹ Instituto de Biología Molecular y Celular de Plantas, Universitat Politécnica de Valencia-
14 Consejo Superior de Investigaciones Científicas, 46022 Valencia, SPAIN

15 ² Biochimie et Physiologie Moléculaire des Plantes, Univ Montpellier, CNRS, INRA, SupAgro
16 Montpellier, Campus SupAgro-INRA, 34060 Montpellier Cedex 2, France

17 ³ Facultad de Ciencias.Universidad de Málaga.Campus de Teatinos S/N 29010 Málaga, SPAIN

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22 **One Sentence summary:** The evolutionarily conserved Arabidopsis protein BAG4 regulates the KAT1 potassium channel and stomatal movement, and is a possible target for development of plants with increased water use efficiency.

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26 **Author Contributions:** AL, MM, GG-M, NA-C, CC-F and LY performed the experiments;
27 LY and JMM designed and supervised the project; LY, JMM, AL, LR, A-AV, JAF and CC-F
28 participated in the experimental design and writing of the manuscript.

29

30 **Funding Information:** This work was supported by the Spanish Ministry of Economy
31 and Competitiveness (BIO201677776-P and BIO2016-81957-REDT) and the Valencian
32 Government (AICO/2018/300).

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34 **Email address for Author for Contact:** lynne@ibmcp.upv.es

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40 **Abstract**

41 Potassium (K+) is a key monovalent cation necessary for multiple aspects of cell growth
42 and survival. In plants, this cation also plays a key role in the control of stomatal
43 movement. KAT1 and its homolog KAT2 are the main inward rectifying channels
44 present in guard cells, mediating K+ influx into these cells, resulting in stomatal
45 opening. To gain further insight into the regulation of these channels, we performed a
46 split-ubiquitin protein-protein interaction screen searching for KAT1 interactors in
47 Arabidopsis (Arabidopsis thaliana). We characterized one of these candidates, BCL2-
48 ASSOCIATED ATHANOGENE4 (BAG4), in detail using biochemical and genetic
49 approaches to confirm this interaction and its effect on KAT1 activity. We show that
50 BAG4 improves KAT1-mediated K+ transport in two heterologous systems and provide
51 evidence that in plants, BAG4 interacts with KAT1 and favors the arrival of KAT1 at the
52 plasma membrane. Importantly, lines lacking or overexpressing the BAG4 gene show
53 altered KAT1 plasma membrane accumulation and alterations in stomatal movement.
54 Our data allowed us to identify a KAT1 regulator and define a potential target for the
55 plant BAG family. The identification of physiologically relevant regulators of K+
56 channels will aid in the design of approaches that may impact drought tolerance and
57 pathogen susceptibility.
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59 **Key words:** Arabidopsis, BAG4, K⁺ channel regulator, KAT1, KAT2, stomata regulation
60 **Introduction**

61 Ion homeostasis is a dynamic process essential for the normal functioning of any
62 organism. Some minerals are required for biological processes, but their excess or
63 deficiency is deleterious. In addition, cells must discriminate between the
64 physiologically relevant ions and the toxic ions that may be chemically similar. For this
65 reason, all living organisms have developed efficient systems to capture and store ions
66 and complex mechanisms to maintain homeostatic concentrations. In plants, ion
67 homeostasis must provide the environment required to maintain all internal
68 processes, prevent toxicity and enable the response to environmental changes using
69 the minerals present in the soil.

70 Potassium is a key monovalent cation necessary for many aspects of growth and
71 survival, among them, compensation of the negative charges generated in processes
72 such as glycolysis, the maintenance of electroneutrality, turgor pressure and cell
73 volume, phloem loading, enzymatic activity, protein synthesis and the establishment of
74 proper membrane potential and an adequate intracellular pH (Rodríguez-Navarro,
75 2000).

76 In plant cells, potassium accumulates to relatively high concentrations in the plant cell
77 cytosol (about 100 mM) and in variable amounts in the vacuole (10-200 mM,
78 depending on the tissue and the environmental conditions), while other cations such
79 as sodium must be excluded to avoid toxicity (Pardo and Quintero, 2002). Potassium
80 homeostasis is essential for optimal water use efficiency, as potassium currents
81 participate in stomatal movement. Stomatal opening depends on potassium and anion
82 uptake coupled to increased proton efflux, while stomatal closing depends on
83 potassium and anion efflux (Lawson and Blatt, 2014). Understanding the molecular
84 mechanisms underlying potassium regulation in guard cells can provide valuable
85 information with applications to the development of new varieties of drought resistant
86 crops. In response to elevated CO2, drought may be among the main threats to world
87 food production because of its dramatic impact on agricultural productivity. Optimizing
88 water use efficiency of crops by improving the potassium regulation in the guard cell,
and therefore improving transpiration regulation, can directly affect food production under adverse conditions (Wang et al., 2014).

In the model plant *Arabidopsis thaliana*, there are three different families of plasma membrane potassium transport systems: the CPA2 subfamily including CHX and KEA $H^+/K^+$ antiporters (Mäser et al., 2001), the HAK/KUP/KT $K^+$ transporters (Gierth and Mäser, 2007), and the Shaker-type $K^+$ channels (Véry and Sentenac, 2003). The third family, Shaker channels, is present in animals, plants, yeast and bacteria. The genome of Arabidopsis contains 9 members that are classified into 5 different groups depending on their phylogeny and functional aspects (Pilot et al., 2003). Groups 1 and 2 contain 4 inward rectifying channels (AKT1, AKT6, KAT1 and KAT2, respectively), while group 3 contains a weak inward rectifier (AKT2). Group 4 contains a "Silent" channel (KC1) and group 5 consists of two outwardly rectifying channels (GORK and SKOR). This family of voltage-dependent channels selective for potassium is responsible for the $K^+$ conductance in the plasma membrane in most cell types. Based on the structural data obtained for bacterial and mammalian Shaker channels, it has been proposed that this family adopts a homo- or hetero-tetrameric structure that forms the potassium pore (Jiang et al., 2003; Long et al., 2005). For example, AKT1 has been proposed to form functional channels with KC1 and KAT2, while KAT1 and KAT2 can associate with each other and also with KC1 (and AKT2 in the case of KAT2) (Lebaudy et al., 2010; Jeanguenin et al., 2011). KC1 is considered a "silent" channel since it is only able to induce currents when it is part of heterotetramers (Dreyer et al., 1997; Duby et al., 2008; Jeanguenin et al., 2011). It is considered that this multiplicity in the composition of subunits confers different properties to the channel and this would be a reflection of different physiological functions (Ivashikina et al., 2001; Xicluna et al., 2007; Jeanguenin et al., 2011).

Two members of the Shaker family, KAT1 and KAT2 are major contributors to potassium influxes in guard cells (Nakamura et al., 1995; Szyroki et al., 2001; Lebaudy et al., 2008). As potassium homeostasis contributes to the regulation of stomatal movement, this process requires tight regulation, which allows fast activation and inactivation, to prevent excessive water loss, specifically under drought or saline conditions (Lebaudy et al., 2008). Therefore, the identification and characterization of
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the proteins interacting with the KAT1 potassium channel may provide new insights
into potassium homeostasis regulation and new ways to develop drought tolerant
plants.

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KAT1 is considered the prototype of inward rectifying potassium channels and plays an important role in potassium fluxes in the guard cell, as mentioned above (Anderson et al., 1992; Schachtman et al., 1992; Nakamura et al., 1995). Several proteins have been implicated in KAT1 regulation. For example, it has been described that fusicoccin can stabilize the interaction of KAT1 with 14-3-3 proteins and activate its transport activity (Saponaro et al., 2017), but the underlying mechanism of this regulation at the level of protein-protein interaction remains largely unexplored. Previous studies have shown that both the Ost1 (SnRK2.6) and the CPK13 kinases can phosphorylate KAT1, although the molecular mechanism by which these phosphorylation events regulate the channel are as yet undefined (Sato et al., 2009; Ronzier et al., 2014). In addition, work from the Blatt laboratory has shown that VAMP721 and SYP121 are important for KAT1 trafficking and gating of the channel (Sutter et al., 2006; Eisenach et al., 2012; Zhang et al., 2015; Zhang et al., 2017; Lefoulon et al., 2018). As these channels play an instrumental role in stomatal movement, their regulation is likely to be complex, involving several different classes of regulatory molecules.

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In the present report, we used a split-ubiquitin approach to identify proteins interacting with KAT1. We found that the BCL2-Associated Athanogene (BAG) 4 protein interacts with KAT1. BAG4 is a member of an evolutionarily conserved family defined by the presence of the BAG domain. This domain is approximately 110-125 amino acids long and is composed of three antiparallel α helices of 30-40 amino acids (Takayama and Reed, 2001). BAG family proteins have been extensively studied in mammalian systems where they have been shown to regulate several processes in many cases by recruiting co-chaperones and different chaperone systems, including the Heat shock protein 70 (Hsp70), which binds to helices 2 and 3 of the BAG domain (Takayama and Reed, 2001; Kabbage and Dickman, 2008). In plants, BAG proteins have been related to processes such as the unfolded protein response, pathogen resistance and abiotic stress and have been shown to conserve the ability to bind to Hsp70 (Doukhanina et al., 2006; Williams et al., 2010; Kabbage et al., 2016), although the molecular
mechanisms underlying their function are largely undefined. More specifically, overexpression of BAG4 is able to increase salinity tolerance in Arabidopsis and rice (Doukhanina et al., 2006; Hoang et al., 2015), and BAG1 and BAG6 have been implicated in the proteasomal degradation of plastid proteins and fungal resistance, respectively (Kabbage et al., 2016; Lee et al., 2016). In this report, we show that BAG4 expression increases KAT1 activity in both yeast and *Xenopus* oocytes. Moreover, we have confirmed the KAT1-BAG4 interaction in plants and provide evidence that BAG4 plays a role in the arrival of KAT1 at the plasma membrane in both gain- and loss-of-function experiments. In addition, mutants lacking or overexpressing the *BAG4* gene present alterations in stomatal opening dynamics, consistent with a physiological role in modulating potassium fluxes. Taken together, our data suggest that in plants BAG4 acts as a KAT1 regulator. Our work uncovers an important potential client for the plant BAG protein family.
164 **Results**

165 In order to gain further insight into the post-translational regulation of the KAT1
166 inward rectifying potassium channel, we carried out a high-throughput screening for
167 physical interactors using the Split-ubiquitin yeast two-hybrid assay with an
168 Arabidopsis cDNA library, as described in Materials and Methods. Previous reports
169 have shown that KAT1 interactions can be detected using this method (Obrdlik et al.,
170 2004). Using this approach, we identified BAG4 as a KAT1 interacting protein.

171 As a first step in the characterization of this interaction, we carried out a functional
172 complementation assay in yeast for selected candidates. We co-transformed KAT1
173 with BAG4 and two other candidate proteins into a yeast strain lacking its endogenous
174 high affinity potassium transporters (Trk1 and Trk2). This strain grows very poorly in
175 media with limiting amounts of potassium (12 µM) (Navarrete et al., 2010). However,
176 KAT1 expression functionally complements this phenotype. The plasmid containing the
177 KAT1 sequence is under control of the *MET25* promoter and in the presence of 0.75
178 mg/ml methionine the expression of *KAT1* is reduced to low levels (Mumberg et al.,
179 1994), providing a sensitive system to study KAT1 activity. In order to determine
180 whether BAG4 could functionally regulate KAT1, we performed growth assays in liquid
181 media under three conditions: 1) low KAT1 expression (methionine supplementation)
182 and low potassium (no KCl supplementation), 2) low KAT1 expression and high
183 potassium (50 mM KCl), and 3) high KAT1 expression and low potassium. As shown in
184 Fig. 1, co-expression of BAG4 with KAT1 improved growth under limiting potassium
185 conditions, whereas two other Arabidopsis proteins recovered in the screening, (PPI1
186 (Proton pump interacting protein 1) and RPT2 (Root phototropism 2)), had no
187 functional effect in this assay. Correct expression of the proteins was confirmed by
188 immunodetection (Fig. 1). As observed, both BAG4 and PPI1 accumulated to similar
189 levels, whereas RPT2 accumulated to lower levels in yeast. This result is consistent
190 with BAG4 improving KAT1 activity in this heterologous system. Based on this
191 phenotype, BAG4 was selected for further analysis. We next wanted to confirm that
192 the increase in growth in this assay was not due to increased expression of the KAT1
193 protein upon BAG4 overexpression. For this, we determined the levels of KAT1 in 6
194 control strains and 7 strains co-expressing BAG4. As shown in Figure 1, we observed
195 no change in KAT1 protein levels, suggesting that the effect of BAG4 is not due to
196 increased accumulation of KAT1, and so discards mechanisms based on transcriptional
197 regulation and protein turnover in this model system.

198 BAG4 belongs to a seven member family of proteins all containing a BAG domain
199 (Doukhanina et al., 2006). BAG1 and BAG7 have a domain structure similar to BAG4
200 and so were chosen for further analysis. Using the yeast assays described above, we
201 compared both the interaction and the functional complementation between BAG
202 family members. Since the original BAG4 clone recovered from the screening had a 13
203 amino acid N-terminal truncation, we cloned the full-length BAG4 gene and included it
204 in these assays. As observed in the split-ubiquitin protein-protein interaction assay
205 shown in Figure 2, we observed the strongest interaction between KAT1 and the BAG4
206 isoforms, as judged by the growth in selective media and the X-gal plate assay. A
207 moderate interaction was observed for BAG1, whereas the interaction between KAT1
208 and BAG7 was very weak. The data presented in Figure 2 confirms the proper
209 expression of each of the proteins. The same pattern of relative KAT1-BAG protein
210 interaction (BAG4>BAG1>>BAG7) was observed in the functional complementation
211 assay shown in Figure 2. The combinations between KAT1 and both versions of BAG4
212 show the highest growth in low potassium medium when KAT1 is limiting (black bars).
213 Some growth is detected under these conditions upon co-expression of BAG1, but in
214 the presence of BAG7 the level of growth is the same as the control. Thus, we provide
215 evidence for some level of specificity between KAT1 and BAG family members, lending
216 further support to the possible physiological relevance of the KAT1-BAG4 interaction.

217 In order to confirm that the observed improvement in growth of the KAT1_BAG4 strain
218 is due to improved potassium uptake, we analyzed potassium uptake using K⁺-specific
219 electrodes (see Materials and Methods for a complete description). As shown in Figure
220 3, co-expression of BAG4 increased both the total amount and the initial rate of
221 potassium uptake from the media. As an internal control, we measured the
222 acidification of the media (due to the H⁺-ATPase activity) and, as expected, observed
223 no changes in the presence or absence of BAG4 (Fig. 3). These experiments strongly
224 suggest that BAG4 favors KAT1 potassium transport activity, at least in yeast.
225 Xenopus oocytes have been extensively used to characterize potassium channels from
226 many organisms. We studied the effect of BAG4 co-expression on KAT1-mediated
227 currents in this model system. We observed an increase in KAT1 channel activity one
228 day after cRNA injection into the oocytes (Fig. 4 and Supplemental Fig. S1). At later
229 times (from Day 2), when reaching steady-state expression level, no differences in the
230 currents were observed (Supplemental Fig. S1). KAT1 current increase upon BAG4 co-
231 expression one day after oocyte injection was 50% to 100% in the different
232 experiments at all membrane voltages (Fig. 4 and Supplemental Fig. S1). No shift in
233 KAT1 voltage-dependence was observed under co-expression (Fig. 4). One
234 interpretation of these results contends that BAG4 co-expression favors the targeting
235 of active KAT1 channels at the oocyte cell membrane, consistent with what is observed
236 in the experiments described above in yeast.

237 We next wanted to confirm that the interaction between BAG4 and KAT1 also takes
238 place in plants. To this end, we performed both Bimolecular Fluorescence
239 Complementation (BiFC) and co-immunoprecipitation assays in Nicotiana benthamiana
240 infiltrated with Agrobacterium tumefaciens containing the appropriate plasmids. As a
241 positive control, we used the KAT1-KAT1 interaction (Fig. 5), observing a uniform
242 fluorescent signal at the plasma membrane. By contrast, we observed a punctate
243 signal corresponding to the KAT1-BAG4 interaction but observed no signal for the
244 corresponding control experiments (Fig. 5). In addition, to add experimental support
245 for this interaction in plants, we performed co-immunoprecipitation experiments upon
246 transient expression in N. benthamiana. We were able to efficiently recover BAG4
247 upon KAT1 immunoprecipitation performed in protein extracts obtained from N.
248 benthamiana leaves transiently expressing the two proteins (Fig. 5).
249

250 We next performed co-localization experiments to determine the subcellular
251 localization of the KAT1-BAG4 complex. We show that the signal corresponding to the
252 KAT1-BAG4 complex co-localizes with an ER marker protein (ChFP-KDEL), thus lending
253 support to the idea that BAG4 could be involved in KAT1 assembly at this organelle
254 (Fig. 6). Pearson and Mander coefficient analyses indicate a strong degree of co-
255 localization between the KAT1-BAG4 BiFC signal and the ER marker (0.75-0.97 and
0.88-0.99, respectively). By contrast, these same parameters for the KAT1-KAT1 BiFC interaction and the same ER marker indicated no co-localization, as expected (Pearson: -0.17-0.035 and Mander: 0.016-0.024). Since the signal for the complex is punctate and is not observed throughout the ER, we also performed co-localization experiments with the ER exit site (ERES) marker Sec24-mRFP and the transmembrane domain of the rat α-2,6-sialyltransferase fused to the Cherry fluorescent protein as a Golgi marker (STtmd-ChFP). We observed co-localization of the KAT1-BAG4 complex with the ERES marker (Pearson: 0.43-0.82 and Mander: 0.5-0.98), but not the Golgi marker (Pearson: -0.0039-0.042 and Mander: 0.1-0.5).

Interestingly, KAT1 was previously shown to interact with Sec24 through its di-acidic ER export signal motif (Sieben et al., 2008). Thus, our data confirm the interaction between BAG4 and KAT1 in a plant model system and show that the KAT1-BAG4 interaction likely takes place at ERES, possibly facilitating its incorporation in coat protein complex II (COPII) vesicles. This is not unexpected as KAT1, like essentially all multi-span plasma membrane proteins, before arriving to the cell surface transits through the ER, where the protein is thought to be assembled into tetramers to form a functional channel that will be inserted into the plasma membrane via the secretory pathway. Our data suggest that BAG4 interacts with KAT1 as it transits through this organelle on its way to the plasma membrane. This model is consistent with that proposed for mammalian BAG proteins that are involved in the regulation of potassium and chloride channels that also act at the ER in cooperation with Hsp70 (Knapp et al., 2014; Hantouche et al., 2017).

Our results indicate that BAG4 favors KAT1 activity in yeast and oocytes and that the interaction appears to take place at the ER exit sites. We hypothesized that BAG4 acts to facilitate KAT1 transit out of the ER and thus would promote the arrival of active KAT1 channels at the plasma membrane. In order to test this model, we examined whether BAG4 influences the arrival of this channel to the plasma membrane. Our first approach was to observe the time course of KAT1 plasma membrane accumulation in the presence and absence of co-expression of BAG4 in *N. benthamiana*. As shown in Figure 7, when BAG4 is co-expressed, a higher percentage of KAT1 protein is present at
the plasma membrane on day 1 after infiltration, whereas in the absence of BAG4, KAT1 accumulation at the plasma membrane is not comparable to that observed for KAT1\_BAG4 until day 3. Figure 7 shows representative images on each day with sufficient signal to clearly visualize KAT1 distribution and the quantification of the percentage of the total KAT1-YFP signal present at the plasma membrane for each condition and time point (n = 10 cells). This result is consistent with ectopic BAG4 expression facilitating the assembly of KAT1 containing tetramers and/or their delivery to the plasma membrane, which would in turn favor the accumulation of active channels at the plasma membrane. Since both the yeast and oocyte experiments suggest that BAG4 does not affect overall KAT1 protein accumulation, we tested whether this was also the case in this plant model system. As described in materials and methods, for these transient expression experiments we constructed vectors containing multiple transcriptional units, including an internal control for infiltration efficiency (dsRED containing an HA tag) within the same plasmid. Plants were agroinfiltrated with strains containing KAT1-YFP:dsRED-HA alone or KAT1-YFP:dsRED-HA:BAG4-myc. We analyzed the amount of KAT1-YFP and the internal control (dsRED-HA) in the infiltrated areas using the same time course. As shown in Figure 7, BAG4 co-expression does not increase the steady state amount of KAT1 protein. So, taken together, the data presented in Figures 7 clearly suggest that expression of BAG4 promotes KAT1-YFP arrival at the plasma membrane.

In order to corroborate these observations, we carried out the opposite approach. We investigated the localization of KAT1 in Col-0 wild type plants and in *bag4* mutant lines using transient expression in *A. thaliana* (Fig. 7). Employing the AGROBEST transient transformation protocol (Wu et al., 2014), we observed KAT1 accumulation at the plasma membrane in wild type control plants. However, under the same conditions, in *bag4* mutants, the KAT1 signal observed at the cell surface was markedly decreased and an accumulation of punctate staining was observed (Fig. 7). We could complement this defect of KAT1 plasma membrane targeting observed in the *bag4* mutant by employing vectors co-expressing BAG4 with KAT1. Figure 7 also shows the quantification of the percentage of the total KAT1 signal present at the plasma membrane for each condition tested (n = 10). We observed a much lower percentage
320 of KAT1-YFP at the plasma membrane in *bag4* mutants, as compared to the Col-0
321 control. Moreover, KAT1-YFP plasma membrane localization is recovered when we
322 functionally complement the *bag4* mutant. These results support the previous
323 experiments and suggest that the presence of BAG4 promotes the arrival of KAT1 at
324 the plasma membrane, possibly by facilitating its assembly and/or delivery to the cell
325 surface possibly through their physical interaction at the ER exit sites.

326
327 In order to provide additional evidence showing that BAG4 is a physiologically relevant
328 KAT1 regulator, we analyzed phenotypes related to KAT1 activity in Arabidopsis lines
329 lacking or overexpressing the *BAG4* gene. Two independent *bag4* mutant lines and two
330 Col-0 lines and one *kat1* line overexpressing *BAG4* were tested for stomatal opening
331 dynamics. The *kat1* and *kat2* single mutants and the *kat1 kat2* double mutant were
332 included for comparison. As shown in Figure 8, two mutant lines lacking the *BAG4* gene
333 show a delay in stomatal opening under all conditions tested. We also observed an
334 initial delay in stomatal opening in the *kat1* and *kat2* mutant lines in response to light
335 treatment, but not potassium-containing opening buffer. Therefore, at high potassium
336 concentrations, the simple mutants are able to open their stomata, likely due to the
337 redundancy of inward rectifying potassium channels. This idea is supported by the
338 phenotype observed for the *kat1 kat2* double mutant, which shows a marked delay in
339 both light and opening buffer. On the other hand, we observed that the
340 overexpression of *BAG4* in Col-0 leads to an increase in stomatal aperture and this
341 response is attenuated in the *kat1* mutant overexpressing *BAG4* (Figure 8). The levels
342 of expression of the BAG4 protein are shown in Supplemental Figure S2.

343
344 As a complementary approach, we measured the temperature of the different mutants
345 and BAG4 gain and loss-of-function lines using infrared thermography (Figure 9).
346 Several studies have shown that this technique can be used for analyzing mutants with
347 altered stomatal function as a relationship exists between the temperature of the
348 leaves and variations in stomatal conductance (Jones, 1999; Merlot et al., 2002; Wang
349 et al., 2004). We observed the expected increase in temperature in the lines that
350 showed delayed stomatal aperture dynamics and a decrease in temperature in the Col-
351 0 lines overexpressing BAG4 (Figure 9). When these results are considered together,
352 they strongly suggest that BAG4 plays a physiologically relevant role in regulating
353 potassium fluxes in stomata and possibly other cells. Importantly, the *BAG4* gene has
354 been reported to be expressed in guard cells, which is a prerequisite for a
355 physiologically relevant KAT1 regulator (Yang et al., 2008).
356

357 As discussed above, in the stomatal response assay, we observed additional
358 phenotypes in the *bag4* mutant lines, as compared to the *kat1* or *kat2* simple mutants.
359 These data suggest that BAG4 may regulate proteins in addition to KAT1, including
360 other potassium channels, like KAT2. Lebaudy and collaborators showed the
361 importance of the guard cell membrane inward K+ channel (GCK<sub>in</sub>) activity in stomatal
362 movement (Lebaudy et al., 2008), identifying KAT1 and KAT2 as the major
363 contributors. Therefore, we studied whether BAG4 could interact with KAT2 in a BiFC
364 assay in *N. benthamiana*. As shown in Figure 10, we observed a pattern of fluorescence
365 very similar to that observed for the KAT1-BAG4 interaction, but observed no signal in
366 the control combinations. We used the KAT2-KAT1 interaction as a positive control for
367 these assays, showing a uniform interaction at the plasma membrane, similar to what
368 we observed with the KAT1-KAT1 interaction (Figure 5), confirming the functionality of
369 the KAT2 BiFC fusion. Although further studies are required to characterize the
370 molecular details of these interactions and ascertain whether there are additional
371 targets, our data suggest that BAG4 may act as a regulator of at least these two
372 potassium channels and provide a plausible explanation for the results obtained in the
373 stomatal response assays described.
374

375 Discussion

376 The regulation of ion fluxes in guard cells is crucial for stomatal movement, which is an
377 important determinant of the plant's response to fluctuating environmental conditions
378 (Lebaudy et al., 2008). Inward rectifying potassium channels are known to play
379 important roles in this process. As such, these channels are predicted to be highly
380 regulated and as expected, several proteins have been identified as regulators of the
381 KAT1 channel (Sottocornola et al., 2006; Sottocornola et al., 2008; Sato et al., 2009;
382 Eisenach et al., 2012; Ronzier et al., 2014; Zhang et al., 2015; Saponaro et al., 2017). In
this report, we describe the identification and initial characterization of a KAT1 regulator that we recovered in a split-ubiquitin screening in yeast. The BAG4 protein was found to physically interact with KAT1 and also to increase potassium uptake in yeast. In oocytes, a similar phenomenon was observed, as increased KAT1 currents were observed one day after injection. Thus, our data clearly indicate that in two heterologous systems, BAG4 co-expression increases KAT1 transport activity, likely by increasing the number of active channels at the membrane. It is very unlikely that this regulation is at the transcriptional level in these model organisms, since we could show that the total amount of KAT1 does not change upon BAG4 co-expression in yeast and in the oocyte experiments, the same amount of KAT1 cRNA is injected in both cases.

We provide experimental evidence for the physical interaction between KAT1 and BAG4 in plants using two complementary approaches, BiFC and co-immunoprecipitation. The signal corresponding to the KAT1-BAG4 complex co-localizes with a general ER marker and with an ER exit site marker, which supports the notion that BAG4 could be involved in KAT1 assembly at this organelle. BAG4 is a member of a highly conserved family of proteins that all contain a characteristic BAG domain. This domain has been shown to interact with the Hsp70 chaperone in both mammals and plants (Takayama and Reed, 2001; Doukhanina et al., 2006; Kabbage and Dickman, 2008; Lee et al., 2016). On the other hand, Hsp70 has been shown to be required for the assembly of the mammalian potassium channel, hERG1 at the ER (Li et al., 2017). Given the role for the BAG proteins documented in mammals and our observations in yeast and oocytes, we hypothesized that BAG4 may be implicated in the arrival of KAT1 to the plasma membrane, likely at the level of protein folding, tetramer assembly and/or trafficking. We provide experimental evidence supporting this idea using both gain- and loss-of-function experiments, where we observe an improvement in KAT1 plasma membrane arrival upon BAG4 expression and a delay in its accumulation at the plasma membrane in lines lacking the *BAG4* gene. Importantly, we show that the total amount of KAT1 is not affected by BAG4 co-expression, but the percentage of KAT1 that arrives at the plasma membrane is increased. Taken together, these data suggest that the modulation of a step required for KAT1 channel assembly, ER exit and/or plasma membrane delivery may be one of the functions of the BAG4 protein.
Several studies have addressed KAT1 trafficking and its regulation. For example, efficient transport of KAT1 to the plasma membrane is mediated by a di-acidic ER export signal in the C-terminus of the protein, which binds to the Sec24 component of coat protein complex II (COPII) (Hurst et al., 2004; Meckel et al., 2004; Sieben et al., 2008). Here, we observed the co-localization of the KAT1-BAG4 complex with Sec24, which has been described as a marker of ER exit sites (reviewed in (Matheson et al., 2006; Langhans et al., 2012)). Therefore, BAG4 may regulate this step of KAT1 processing as it moves out of the ER through the secretory pathway towards the plasma membrane. It has also been shown that abscisic acid stimulates the endocytosis of KAT1 in both epidermal and guard cells, which can then recycle back to the plasma membrane when the levels of the hormone decrease (Sutter et al., 2007). In addition, two trafficking related proteins, SYP121 and VAMP721 are involved in regulating KAT1 delivery and recycling, and more recently have been shown to regulate channel gating (Sutter et al., 2006; Eisenach et al., 2012; Zhang et al., 2015; Zhang et al., 2017; Lefoulon et al., 2018). Whether BAG4 is related to any of these known regulatory mechanisms is an interesting question for future studies.

In plants, there are seven members of the BAG family and different phenotypes have been attributed to the *bag1*, *bag4* and *bag6* loss-of-function mutants, suggesting that each family member may carry out distinct functions (Doukhanina et al., 2006; Kabbage et al., 2016; Lee et al., 2016). Moreover, high throughput studies have indicated that, for example, both *BAG4* and *BAG1* are expressed in guard cells, whereas *BAG7* expression is low in this cell type (Winter et al., 2007). In order to study the specificity of the KAT1-BAG4 interaction, we used the split-ubiquitin and functional complementation assays to test whether two other BAG family members that share similar domain architecture, BAG1 and BAG7, interacted with KAT1. Our results show that the BAG protein with a higher level of conservation as compared to BAG4, BAG1 showed a lower, but detectable level of interaction and regulation of KAT1 in yeast. However, a negligible level of interaction was observed for the more distantly related BAG7 protein, thus suggesting a considerable level of specificity for the KAT1-BAG4 interaction.
In order to begin to establish BAG4 as a bona fide KAT1 regulator, we tested whether BAG4 may play a role in a physiological response in which this channel is implicated. Indeed, we observed a delay in stomatal aperture in two independent *bag4* mutant lines in response to external potassium and light. We observed a similar delay in stomatal aperture in response to light in the *kat1* and *kat2* mutant lines. Our data regarding the *kat1* mutant is in contrast to a previous report (Szyroki et al., 2001), but may be explained by differences in the time course studied and/or the mutant lines used. The line used here contains a T-DNA insertion in the first exon. In addition, we observed an increase in stomatal aperture in Col-0 lines overexpressing *BAG4* and this response was reduced when the gene was overexpressed in a *kat1* mutant line. Thus, we were able to show that both loss- and gain-of-function of *BAG4* affects stomatal aperture dynamics and that in the case of *BAG4* overexpression, *KAT1* is required to observe the full response.

We corroborated these results by measuring the temperature of the plants as an indirect measure of the transpiration rate. If the stomatal aperture is decreased in *bag4* mutants, so is the rate of transpiration and therefore, the temperature of the leaves of these plants would be expected to be higher. Our results show that lines lacking the *BAG4* gene display an increased temperature, again adding support to its role in regulating ion fluxes important for stomatal movement. As expected, double mutants lacking both the *KAT1* and *KAT2* genes have a more pronounced phenotype. Moreover, Col-0 lines overexpressing *BAG4* display lower leaf temperatures, which is in agreement with the data regarding stomatal aperture. Taken together, our results establish a role for *BAG4* in the regulation of stomatal aperture dynamics through the regulation of the *KAT1* inward rectifying potassium channel.

It is interesting to note that the two *bag4* mutant lines showed a longer delay in stomatal opening in response to opening buffer, which was not observed in the *kat1* and *kat2* single mutants but was observed in the *kat1 kat2* double mutant. BAG proteins are very likely to regulate many different proteins and in terms of stomatal aperture, other inward rectifying potassium channels, like KAT2, AKT1, and AKT2/3 are known to contribute to this response and our genetic data suggest a role for KAT2. Thus, we tested whether BAG4 could also interact with KAT2 in plants. Our results
475 using the BiFC assay show a KAT2-BAG4 interaction. Thus, it appears that BAG4
476 regulates both KAT1 and KAT2, but we cannot rule out other channels as additional
477 targets at this stage.

478 In mammalian systems, although BAG proteins have been related to many cellular
479 processes, a specific role for BAG proteins in the regulation of ion channels has been
480 reported. Both BAG1 and BAG2 have been shown to regulate the Cystic Fibrosis
481 transmembrane conductance regulator (CFTR) chloride channel and BAG1 was more
482 recently implicated in human Ether-à-go-go-related gene (hERG) potassium channel
483 regulation (Young, 2014; Hantouche et al., 2017). In both cases, it appears that the
484 BAG proteins mediate the misfolded protein response, thus likely playing a role in
485 channel degradation, not plasma membrane delivery. Further experiments will be
486 required to clarify the molecular mechanisms responsible for this apparent difference
487 observed here for the plant BAG4 protein. The Arabidopsis BAG1 protein has been
488 reported to be a co-factor in Hsc70-mediated proteasomal degradation of unimported
489 plastid proteins, and so may have a function more analogous to its mammalian
490 counterparts (Lee et al., 2016). However, our results identify BAG4 as a positive
491 regulator of the KAT1 (and likely KAT2) potassium channel in plants and thus opens a
492 novel line of investigation by linking the plant BAG family to the regulation of
493 potassium channels and stomatal movement.

494

495 **Conclusions**

496 Taken together, the data presented here suggest that one main role for BAG4 in plants
497 may be related to the post-translational regulation of ion channels and, consequently
498 stomatal movement. Thus, BAG4 may constitute a novel target for the design of
499 engineered crops with altered potassium fluxes, and concomitantly, improved water
500 use efficiency and drought tolerance.

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## 508 Materials and Methods

509 *Plant materials and plant growth.* The following Arabidopsis lines were employed: *kat1* mutant (SALK 093506, carrying the T-DNA insertion in the 1st exon), *kat2* mutant (SALK 025933) and two *bag4* mutant lines SAIL_144_A10 (carrying the T-DNA insertion in the 1st exon) and SALK_033845 (carrying the T-DNA insertion in the 2nd exon) (obtained from the Salk Institute Genomic Analysis Laboratory and (Sessions et al., 2002)).
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514 Arabidopsis plants were grown under an 8h light: 16h dark photoperiod at 22 °C in MS media supplemented with 1% sucrose. For stomatal movement and leaf temperature assays, after 2 weeks, plants were transplanted to soil and analyzed at 4-6 weeks.
515
516
517 *Nicotiana benthamiana* plants were grown for 4-5 weeks in soil under an 16h light: 8h dark photoperiod at 24 °C.
518

519 *Yeast strains and plasmids.* The Split-ubiquitin KAT1 bait vector used for the screening was derived from the KAT1-pMetYCgate vector (ABRC CD3-815, (Obrdlik et al., 2004)).
520
521 In order to carry out the screening, the AMPr marker was substituted with KANr by recombination in yeast as follows: the kanamycin resistance gene was amplified with the primers Kan-F 5'-ttcttgaagacgaaagggcctcgtgatacgcctattTCCAGTTCGATTTATTC AACAAAG-3' and Kan-R 5'-taaagtatatatgagtaaaacttggtctgacagttacGATCGATCCTAG TAAGCCACGTTG-3' and was co-transformed in the PLY240 strain (MATa his3Δ200 leu2-3,112 trp1Δ901 ura3-52 suc2Δ9 trk1Δ51 trk2Δ50::lox-KanMX-lox; (Bertl et al., 2003) together with the KAT1-pMETYCgate vector linearized with AatII. Clones growing in media lacking leucine were tested for growth in media containing 50 mM LiCl to select strains expressing KAT1. Plasmids were recovered from these yeast strains and transformed into DH5α and kanamycin resistant colonies were selected. The resulting plasmid was confirmed by sequencing. The cDNA library from 6-day-old *Arabidopsis thaliana* seedlings in the pDSL-Nx vector was obtained from Dualsystems Biotech AG. The screening was carried out in the THY.AP4 strain (MATa, ura3, leu2, lexA::lacZ::trp1,
lexA::HIS3, lexA::ADE2; (Paumi et al., 2007)). The BAG4 clone recovered in the screening lacks the sequence encoding the first 13 amino acids. The BAG1, BAG7 and full-length BAG4 plasmids were constructed by recombination into the pDSL-Nx vector in yeast using PCR products amplified from a cDNA library made from Arabidopsis seedlings.

*Split-ubiquitin screening.* The THY.AP4 strain containing the modified KAT1-Cub (KAT1-CubKAN') plasmid was transformed with 20 micrograms of the *A. thaliana* cDNA library in the pDLSN-X vector (Dual Systems). Approximately 8.6 x 10⁶ transformants were analyzed for growth in selective media. The first 125 colonies growing in selective media were analyzed. Plasmids were recovered, re-transformed, tested for growth in selective media, classified by restriction analysis and subjected to specificity tests using the Ost3-Cub vector encoding a yeast oligosaccharyl transferase (Yan et al., 2005). The clones that passed these tests were then sequenced and analysed in the yeast functional complementation assay.

*Functional complementation assay in yeast.* The PLY240 strain was co-transformed with the KAT1-CubKAN' and the indicated pDLSN-X vectors and the empty vector corresponding controls. Pre-cultures were grown to saturation in SD media supplemented with auxotrophic requirements, 0.75 mg/mL methionine (to reduce KAT1 expression) and 0.1 M KCl. Fresh cultures (1:20 dilution) were grown for 16 hours in low potassium Translucent media supplemented with requirements and methionine (ForMedium™ UK; (Navarrete et al., 2010)) and diluted to OD₆₀₀ 0.2 in three different conditions of Translucent media supplemented with auxotrophic requirements: 1) low potassium, no methionine (low KCl, high KAT1 expression); low potassium, 0.75 mg/mL methionine (low KCl, low KAT1 expression) and 3) 0.1 M KCl, no methionine (high KCl, high KAT1 expression). The Translucent media contains 12 µM K⁺. The optical density was determined periodically for 72 hours using a BioscreenC system (Oy Growth Curves Ab Ltd). Triplicate determinations were performed for at least 3 independent transformants for each plasmid combination. Similar results were observed in all experiments. Correct expression of each fusion protein was confirmed by immunoblot analysis of the corresponding whole cell extracts of cultures grown in Translucent media supplemented with 0.1 M KCl and 0.75 mg/mL methionine using anti-HA and
anti-LexA antibodies (Covance and Abcam, respectively), the corresponding anti-mouse or anti-rabbit HRP secondary antibodies and ECL detection system (GE Healthcare). Several independent clones were used for the quantification of KAT1 protein expression in the absence and presence of BAG4. ImageJ was used to quantify the bands corresponding to the LexA signal and the loading control to calculate normalized KAT1 expression.

*Potassium consumption uptake and external acidification in yeast.* The indicated plasmids were transformed in the PLY240 background and grown as described above in the functional complementation assay to mid-log phase OD600 = 0.5-0.6 in Translucent media supplemented with auxotrophic requirements and 0.75 mg/mL methionine. Cells were collected by centrifugation, washed, resuspended in sterile water and incubated for 30 minutes at room temperature. External K⁺ was monitored using external K⁺-selective mini-electrodes. Single-barrelled borosilicate glass capillaries, 7 cm long and of 1.5 mm of external diameter, were pulled in a patch clamp puller to get an open tip. Then, capillaries were sylanized and back-filled with K⁺ ionophore I sensor (cocktail B, cat. No. 60398; Fluka, now part of Sigma) dissolved in a mixture of polyvinyl-chloride/tetrahydrofuran (40 mg/mL) at a ratio of 30:70 (v/v), as was previously described (Planes et al., 2015). For the assays, approximately 20 mg of cells (wet weight) were resuspended in 3 mL of 10 mM MES pH 4.0 and loaded in a temperature controlled (20 °C) plexiglass cylinder chamber under continuous stirring. K⁺-selective and reference electrode tips were placed in the assay medium and connected to a high-impedance differential amplifier (FD223; World Precision Instruments). In addition, a single glass pH electrode (model 5209, Crison) was also submerged into the assay medium in order to measure the external acidification to check the activation of the proton ATPase. After steady readings of both pH and external potassium K⁺ signals were obtained, potassium chloride was added to a final concentration of 1 mM and then, after stable readings values were again attained, 20 mM of glucose were added to activate the proton ATPase, Pma1 and energize potassium uptake. Both the external pH and the potassium concentration were recorded for 45 minutes after glucose addition. The K⁺ electrode signal was calibrated before and after experiments by adding potassium chloride to final concentrations of
0.1, 0.5, 1 and 10 mM in the assay media. Calibration curves render slopes around 45 mV per pK⁺ unit. Both the total uptake (after 45 minutes) and the initial rate of K⁺ uptake (slope of the curve representing the initial uptake; 5-8 minutes) were determined for three independent clones of each plasmid combination indicated (Figures 3A and B, respectively).

*Two-electrode voltage clamp in Xenopus oocytes.* KAT1 and BAG4 coding regions were cloned into a modified pGEM-HE vector (D. Becker, University of Würzburg, Germany). cRNA were synthesized from 1 µg of linearized vector using the HiScribe™ T7 ARCA mRNA (with tailing) kit (NEB, http://www.NEB.com). Oocytes were obtained and prepared as previously described (Véry et al., 1995) and were injected with 30 ng of KAT1 cRNA or co-injected with 30 ng of KAT1 cRNA and 22.5 ng of BAG4 cRNA using a pneumatic injector. One day after oocyte injection, currents from whole oocytes bathed in K100 medium (100 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES/Tris pH 6) were recorded using the two-electrode voltage clamp technique. Data acquisition and analyses were performed as previously described (Corratgé-Faillie et al., 2017). Voltage drops resulting from the series resistance of the bath were corrected by using two external electrodes connected to a bath probe (VG-2A x100 Virtual-ground bath clamp; Axon Instruments). KAT1 currents were obtained by subtraction of mean currents recorded in water-injected oocytes from the same oocyte batch. The percentage of KAT1 current increase with BAG4 = Mean current in the presence of BAG4 - Mean current in its absence / Mean current in its absence.

*BiFC and co-immunoprecipitation assays in Nicotiana benthamiana.* All KAT1 and BAG4 plasmids used for the BiFC and co-immunoprecipitation experiments and to generate overexpression lines were constructed using the GoldenBraid system (Sarrion-Perdigones et al., 2013). For BiFC assays, we used pUPD2 vectors containing the YFN or YFC sequences from the GoldenBraid collection. Then, we cloned KAT1 and BAG4 versions, compatible with the cloning system into the pUPD2 vector. Alpha vectors containing the indicated fusion proteins and the 35S promoter and Tnos terminator were assembled and combined to generate the omega level plasmids that were transformed into the *Agrobacterium tumifaciens* strain C58Ci. For indicated experiments, alpha level plasmids were constructed from these omega plasmids to
generate constructs containing 3 or 4 transcriptional units. For plant infiltration, we used 4- to 5-week-old *N. benthamiana* plants grown at 24 °C under a 16h light: 8h dark photoperiod. For co-localization experiments used to determine the subcellular localization of the complex, the *KAT1-BAG4* interacting combination was co-infiltrated with a second plasmid containing either the ER marker (calreticulin targeting sequence-Cherry fluorescent protein-KDEL retention sequence (ChFP-KDEL)), the Sec24-RFP ER exit site (ERES) marker (both kindly provided by V. Pallás, IBMCP, Spain), or the transmembrane domain of the rat α-2,6-sialyltransferase enzyme fused to the Cherry fluorescent protein as a Golgi marker (STtmd-ChFP) (Prokhnevsky et al., 2005). An agrobacterium strain (C58Ci) transformed with the plasmid encoding the P19 Tomato Bushy Stunt Virus silencing suppressor was also used in all the infiltrations (Sarrion-Perdigones et al., 2013). The images were acquired using a Zeiss fluorescence confocal microscope with the following settings: YFP was excited with an Argon laser (514 nm) and detected at 516-548 nm. Cherry, RFP and dsRED fluorescent proteins were excited using the DPSS 561-10 laser (561 nm) and detected at 580-650 nm. Chloroplast autofluorescence was detected between 675 and 760 nm. The statistical analyses for KAT1-BAG4 BiFC and the organelle makers were determined by calculating the Pearson and Mander coefficients (corresponding to the range of values obtained in 4 independent images) (Dunn et al., 2011). For co-IP assays, we used a pUPD2 4x-*c-myc* sequence from the Addgene collection, to create the *c-myc-BAG4* fusion. We combined the *KAT1-YFP* and *c-myc-BAG4* alpha vectors to build the omega plasmid containing both fusion proteins and infiltrated *N. benthamiana* leaves. Extraction of total proteins was carried out in modified PBS buffer (140 mM NaCl, 8 mM Na₂HPO₄·7H₂O, 2 mM KH₂PO₄, 10 mM KCl, Proteinase Inhibitor, pH 7.4). The KAT1-YFP protein was purified using the GFP-trap (GFP-Trap®_MA, Chromotech) according to the manufacturer's instructions. Samples of the protein extracts, the unbound material and the KAT1 purification were separated on 8% SDS-PAGE gels, transferred to nitrocellulose membranes and probed with an anti-GFP antibody (clone 7.1 and 13.1 mixture, Roche) and an anti-*c-myc* antibody (clone 9E10, Roche) to detect BAG4. Immune complexes were visualized using the corresponding anti-mouse or anti-rabbit HRP secondary antibodies and ECL detection system (GE Healthcare).

658
659 Kinetics of plasma membrane accumulation in N. benthamiana and A. thaliana

660 For transient expression in *N. benthamiana*, leaves were infiltrated, treated and
661 imaged as described for BiFC and Co-IP. However, in this case P19 silencing suppressor
662 was not added to the infection mixture. The ImageJ program was used to quantify the
663 percentage of KAT1-YFP present at the plasma membrane as follows: the free draw
664 tool was used to trace the contour of individual cells, directly adjacent to the cell
665 boarder (inside cell fluorescence). These forms were digitally enlarged by 7 pixel units
666 using the ImageJ tool to quantify the whole cell fluorescence. Fluorescence levels in
667 ellipses adjacent to the cells were also quantified and used as the background
668 measurement. The corrected total cell fluorescence (CTCF) was calculated using the
669 following formula: Integrated Density – (area of selected cell × mean fluorescence of
670 background readings). The percentage of fluorescence signal at the plasma membrane
671 was then calculated using the following formula: % fluorescence PM = 100 –
672 ($\frac{\text{CTF inside fluorescence}}{\text{CTF whole cell fluorescence}} * 100$). This procedure was carried out for 10 individual cells
673 from each of the indicated conditions deriving from experiments done on different
674 days. In order to quantify the amount of KAT1 protein with and without BAG4 co-
675 expression, an alpha1 level plasmid was constructed to combine the KAT1-YFP_myc-
676 BAG4 omega construct (or the control) with the dsRED protein which incorporates an
677 HA epitope tag (dsRED-HA) in the same vector. In this way, the amount of dsRED can
678 be used as an internal control monitoring the level of transient expression in each case
679 because all transcriptional units are in the same plasmid. Infected plants were
680 observed on days 1, 2 and 3 post-infiltration by fluorescence confocal microscopy and
681 whole cell protein extracts were generated from the infiltrated material by grinding 2
682 cm leaf discs in SDS-PAGE loading buffer. Samples were separated on SDS-PAGE,
683 transferred and KAT1, BAG4 and dsRED were detected using anti-GFP, anti-c-myc and
684 anti-HA antibodies.

685 For transient expression in Arabidopsis, the Col-0 control and the *bag4* mutant lines
686 (SALK_033845 and SAIL_144_A10) were agroinfiltrated following the AGROBEST
687 protocol (Wu et al., 2014). The plasmid used was the alpha level plasmid containing
688 KAT1-VENUS<sub>intron</sub> which included an intron in the Venus sequence (kindly provided by
689 Stan Gelvin, Purdue University, USA). This was required to avoid expression of the
KAT1 fusion protein in the agrobacterium strain. An omega vector combining the KAT1- VENUS<sub>intron</sub> and myc-BAG4 transcriptional units was used for the complementation assays. Confocal images were acquired as described above using the 514 laser and an emission wavelength between 515 nm and 530 nm. The quantification of the amount of KAT1-YFP at the plasma membrane was calculated as described above for 10 individual cells from each condition indicated.

Stomatal movement assays. The following lines were employed: *kat1* mutant (SALK 093506, carrying the T-DNA insertion in the 1st exon), *kat2* mutant (SALK 025933) and the two *bag4* mutant lines SAIL_144_A10 (carrying the T-DNA insertion in the 1st exon) and SALK_033845 (carrying the T-DNA insertion in the 2nd exon) (obtained from the Salk Institute Genomic Analysis Laboratory and (Sessions et al., 2002), two independent transformants that are homozygous for the *BAG4* insertion in the Col-0 and *kat1* mutant (SALK 093506, carrying the T-DNA insertion in the 1st exon) lines generated by the floral dipping method using an omega level plasmid containing the c-myc-BAG4 fusion and the BASTA resistance gene. The double *kat1 kat2* mutant was generated by crossing the single mutants listed above. Plants were grown 4-6 weeks under an 8h light: 16h dark photoperiod at 22 °C. Five leaves (each taken from different plants) from each genotype and condition were analysed. For the dark conditions, leaves were harvested 1 hour before the lights switched on and the leaf was split into two halves: one was maintained in the dark in MES solution, while the other was incubated in stomatal opening buffer (10 mM KCl, 7.5 mM iminodiacetic acid; 10 mM MES/Tris pH 6.2), for 2.5 hours. For light treatment, the leaf was split into two halves and both were kept in 10 mM MES (pH 5.6). One half was kept in the dark and the other exposed to light and observed 2.5 hours later. To take stomatal images, the tissue was mounted on a microscope slide and immersed in the respective solution. The stomatal aperture was defined as the ratio between the length of the stomatal aperture from the point of junction of the inner lips and the maximal width between the inner cuticular lips. For each condition, between 60 and 100 stomata were analyzed in five different plants. The entire assay was repeated three times.
721 Leaf temperature measurements. The same lines used in the stomatal movement
722 assays were grown under an 8h light: 16h dark photoperiod at 22 °C. The
723 thermographic camera Bosch GTC 400 C was used to generate the infrared images of
724 the plants 1.5 hours after the lights turned on. The plants were sown in individual pots
725 such that the rosette leaves were not touching the walls of the pot, the tray or other
726 plants. For imaging, each plant was moved to a tray partially filled with water, to
727 create a uniform environmental temperature around the pot. Leaf temperature was
728 calculated using the GTC Transfer Software. A total of 6 plants were measured for each
729 genotype. Ten points were measured for each plant (60 data points/genotype). Data
730 from each genotype were pooled together and statistically analyzed (Student t-test)
731 using the Graph Prism6 software.

732

733 Accession Numbers

734 Sequence data from this article can be found in the GenBank data libraries under
735 accession numbers: AtKAT1, At5g46240; AtKAT2, At4g18290; AtBAG4, At3g51780.
736 Mutants used in this article can be obtained from the Arabidopsis Biological Resource
737 Center under the following accession numbers: *kat1* mutant (SALK 093506), *kat2*
738 mutant (SALK 025933), and *bag4* mutant lines (SAIL_144_A10) and (SALK_033845).

739

740 Supplemental Data

741 Supplemental Figure S1. BAG4 co-expression favors early activity of KAT1 in Xenopus
742 oocytes.

743 Supplemental Figure S2. Immunodetection of myc-BAG4 protein in homozygous
744 transgenic lines.

745

746 Acknowledgements

747 The authors would like to thank Elena Moreno, María José Falaguer, Alejandro Mossi,
748 Sara Aljama, José Antonio Navarro, Vicente Pallás, Daniel Franco-Aragón, Jorge Lozano,
749 and Stan Gelvin for assistance in the completion of this work and for providing
750 reagents.

751
753 **Figure Legends**

754 **Figure 1. Effect of the co-expression of interacting proteins on the functional complementation by KAT1 in yeast.** A) The indicated plasmids were co-transformed in the *trk1 trk2* mutant strain (PLY240) and the growth of the strains was assayed as described in Materials and Methods using the Translucent media (which contains 12 µM potassium) with or without methionine supplementation to decrease the expression of KAT1 (under control of the *MET25* promoter). The graph shows the average value of the optical density at 72 hours for triplicate determinations and the experiment was done with at least 3 independent transformants for each plasmid combination. (Translucent media with no KCl supplementation = Low KCl (12 µM K⁺); Translucent media containing 0.75 mg/mL methionine = Low KAT1; Translucent media + 50 mM KCl added = High KCl; Translucent media -/+ KCl without methionine = High KAT1). Data presented are the mean ±SD and similar results were observed for three independent transformants. Asterisks (**) indicate statistical significance (Student *t*-test) with a p value < 0.01. B) Immunoblot analysis of protein extracts from the indicated strains showing the correct expression of each of the fusion proteins. The KAT1 bait vector protein is detected with the anti-LexA antibody and the prey proteins with the anti-HA antibody. Results for a representative clone are shown. The long exposure is included to visualize the expression of the RPT2 prey protein, which accumulates less than the other two prey proteins. C) The KAT1 signal and the corresponding loading control were quantified using the ImageJ software and the average values of KAT1/loading control (Normalized KAT1) was calculated for 6 control strains (KAT1_empty vector) and 7 KAT1_BAG4 strains. Data presented are the mean ±SD.

777

778 **Figure 2. Study of the specificity of the interaction between KAT1 and other BAG family members.** A) The indicated plasmids were transformed into the THY.AP4 strain and grown to saturation in selective media. Serial dilutions were spotted onto media with the indicated composition to test for the protein-protein interaction between KAT1 and the indicated BAG family proteins. Identical results were observed for four independent clones. B) Immunoblot analysis of protein extracts from the indicated

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784 strains showing the correct expression of each of the fusion proteins. The KAT1 bait
785 protein is detected with the anti-LexA antibody and the prey proteins with the anti-HA
786 antibody. Results for a representative clone are shown. C) The indicated plasmids were
787 co-transformed in the *trk1 trk2* mutant strain (PLY240) and the growth of the strains
788 was assayed as described in Figure 1A. The average value of triplicate determinations
789 of the optical density of the growth normalized to the potassium-supplemented media
790 is shown for each strain. Data presented are the mean ±SD and similar results were
791 observed for 3 independent clones of each plasmid combination. Asterisks (***)
792 indicate statistical significance (Student t-test) with a p value < 0.001 compared to the
793 KAT1_Ø control strain.
794

795 **Figure 3. Potassium uptake and external acidification.** The indicated plasmids were
796 co-transformed in the *trk1 trk2* mutant strain (PLY240) and the strains were grown in
797 low KCl and low KAT1 conditions and processed as described in Materials and
798 Methods. A) The bars represent the average value for three independent experiments
799 for the total potassium uptake for 45 minutes expressed as µmol K⁺/mg cells ±SD. B)
800 The bars represent the average value for three independent experiments for the initial
801 rate of potassium uptake expressed as µmol K⁺/min*mg cells calculated using the
802 slope of the depletion curve (first 5-18 minutes) normalized to the wet weight of the
803 cells ±SD. C) The bars represent the average value for three independent experiments
804 of the change in the pH value measured in parallel with the potassium consumption
805 ±SD. The change in the external pH was determined during the first 15 minutes after
806 glucose addition and was normalized to the wet weight of the cells. Asterisks (*)
807 indicate statistical significance (Student t-test) with a p value < 0.05.

808 **Figure 4. BAG4 co-expression increases KAT1 current in *Xenopus* oocytes.** A) Voltage-
809 clamp protocol and representative current traces recorded by two-electrode voltage
810 clamp in the presence of 100 mM KCl on oocytes co-expressing KAT1 or KAT1 and
811 BAG4. B) KAT1 current (I) –voltage (V) relationships of oocytes co-expressing KAT1 and
812 BAG4 (white circles) or expressing KAT1 alone (black circles). Data are means ± SE (n=7
813 for KAT1, n=11 for KAT1 + BAG4), and are representative of three experiments
814 performed on different oocyte batches. C) Increase of mean KAT1 current upon BAG4
co-expression, at -110 mV (black) and at -155 mV (grey). Mean currents are from B). D) Voltage-dependence of KAT1-current activation in the presence of BAG4 (white circles) or with KAT1 expressed alone (black circles). Gating parameters (z, gating charge, and Ea50, half-activation potential) were estimated by performing fits to the KAT1 I-V relationships (in the presence or absence of BAG4) with a Boltzmann function coupled to a linear relation (Lebaudy et al., 2010). G, KAT1 macroscopic conductance (G = I/V); $G_{max}$, KAT1 macroscopic conductance at infinitely negative voltage.

**Figure 5. Confirmation of the interaction between KAT1 and BAG4 in N. benthamiana.** Agrobacterium strains harbouring the indicated plasmids were used to infiltrate *N. benthamiana* leaves and images were obtained using fluorescence confocal microscopy 72 hours post-infiltration. A) Representative BiFC images for the KAT1-KAT1 interaction and control plasmids. The overlay of the grey scale and BiFC fluorescence is shown. Leaf epithelial cells and stomata are visible. B) Representative BiFC images for the KAT1-BAG4 interaction. Representative images of the experiments performed with the control plasmids are shown below. The red signal corresponds to chloroplast autofluorescence. For panels A and B, similar results were observed in at least four independent experiments performed on different days. (Bar = 20µm) C) Agrobacterium strains expressing the indicated plasmids were used to infiltrate *N. benthamiana* leaves. Samples were taken at 72 hours post-infiltration and processed for protein extraction and co-immunoprecipitation as described in Materials and Methods. The figure shows the results of the immunodetection using antibodies that recognize the BAG4 and KAT1 proteins. The amount of BAG4 recovered in the KAT1 purification is shown in the first lane on the left. Similar results were observed in two independent experiments performed on different days. (YFN = N-terminal part of YFP; YFC=C-terminal part of YFP; IP=immunoprecipitation; FT=flow through).

**Figure 6. The KAT1-BAG4 complex co-localizes with the ER and ERES markers.** A) A plasmid containing the KAT1-BAG4 BiFC interaction and the ER marker ChFP-KDEL were infiltrated as described. The BiFC signal (right), ChFP signal (center), and the overlay image of the grey scale, BiFC fluorescence and the ChFP signals (left) are shown. Leaf epithelial cells and stomata are visible. B and C) The same co-localization
analysis of the KAT1-BAG4 complex was performed with the ER exit site marker, Sec24 fused to RFP (Sec24-RFP) (B) and the STtmd-ChFP Golgi marker (C). The yellow arrows indicate the points of co-localization with the ER marker. Chloroplast autofluorescence is shown in blue. (Bar = 20µm). (YFN = N-terminal part of YFP; YFC=C-terminal part of YFP; ChFP=Cherry Fluorescent Protein; RFP=Red Fluorescent Protein; STtmd-ChFP= rat α-2,6-sialyltransferase transmembrane domain fused to the Cherry fluorescent protein).

**Figure 7. BAG4 expression effects the subcellular localization of KAT1.** A) Plasmids containing KAT1-YFP or both KAT1-YFP and BAG4 were transiently expressed in *N. benthamiana* using agro-infiltration. The fluorescence signal was analysed by confocal microscopy 1-, 2- and 3-days post-infiltration. Representative images are shown. Leaf epithelial cells and stomata are visible. Similar results were observed in three independent experiments performed on different days. (Bar = 20µm) B) The percentage of the fluorescence signal present in the plasma membrane (PM) was quantified using ImageJ, as described in Materials and Methods. The graph shows the average values ±SD for 10 cells for each condition tested. C) Similar experiments as described in panel A were performed, but using GoldenBraid plasmids containing KAT1-YFP:dsRED-HA or KAT1-YFP:myc-BAG4:dsRED-HA. Whole cell extracts were prepared from infiltrated areas (previously confirmed to express KAT1-YFP) and proteins were processed for immunodetection. The dsRED-HA protein serves as an internal control for the efficiency of transient expression. (DAY PI= Day post-infiltration). D) The KAT1-VENUS<sub>intron</sub> (KAT1-VENUS<sub>i</sub>, top row) or the KAT1-VENUS<sub>intron_BAG4</sub> (bottom row) plasmids were transiently expressed in the Col-0 and the *bag4* mutant Arabidopsis lines using agro-infiltration. The fluorescence signal was analyzed by confocal microscopy 2 days after infiltration. Representative images are shown and similar results were observed in three independent experiments. Leaf epithelial cells and stomata are visible. (Bar = 20µm). E) The percentage of the fluorescence signal present in the plasma membrane (PM) was quantified using ImageJ, as described in Materials and Methods. The graph shows the average values ±SD for 10 cells for each condition tested. (Student *t*-test) (**: p value <0.01).
**Figure 8.** Effect of BAG4 loss and gain-of-function on stomatal aperture. Stomata from the indicated Arabidopsis lines were analyzed as described in Materials and Methods. The data show the average ratio for 60-100 stomata ±SD. A) The width/length ratio of stomata of the different mutant lines were determined in leaves from the indicated lines harvested 1 hour before the lights turned on (Dark), incubated for 2.5 hours in opening buffer in the dark (Opening buffer, O.B.) or 2.5 hours after the lights turned on (Light). Similar results were observed in three independent experiments. B) The width/length ratio of stomata of control lines and Col-0 and *kat1* homozygous for 35S:BAG4 transgene were determined as described in panel A. For both experiments, the asterisks indicate statistical significance as compared to the Col-0 dark control (Student *t*-test) (**: p value <0.01; **: p value < 0.001; ***: p value < 0.0001). The length/width ratio was also significantly increased in BAG4 overexpressing lines in both light and opening buffer (# indicates statistical comparison with Col-0 O.B. and ▪ indicates statistical comparison with Col-0 light).

892

**Figure 9.** Effect of BAG4 loss and gain-of-function on leaf temperature. The same lines described in Figure 8 were analyzed for leaf temperature using an infrared thermography, as described in Materials and Methods (Panel A mutant lines, Panel B 35S:BAG4 lines). Each symbol represents an individual measurement and the bar represents the average value for 10 measurements of 6 different plants for each genotype. The error bars represent the standard deviation. For both experiments, the asterisks indicate statistical significance as compared to the Col-0 control or the comparisons indicated by the brackets above the graphs (Student *t*-test) (**: p value <0.01; **: p value < 0.001; ***: p value < 0.0001).

902

**Figure 10.** BAG4 interacts with KAT2 in BiFC assays in *N. benthamiana*. Interaction assays were carried out and analyzed as described in the legend to Figure 5. Leaf epithelial cells and stomata are visible. As shown, a similar pattern of interaction is observed for KAT2 when tested with BAG4 (compare with Figure 5B). The BiFC signals corresponding to the KAT2-KAT1 and KAT2-BAG4 interactions are shown in green and the chloroplast autofluorescence is shown in red. (YFN = N-terminal part of YFP; YFC=C-terminal part of YFP). (Bar = 20µm).

903

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909
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<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_5.png" /></figure>

Figure 1. Effect of the co-expression of interacting proteins on the functional complementation by KAT1 in yeast. A) The indicated plasmids were co-transformed in the trk1 trk2 mutant strain (PLY240) and the growth of the strains was assayed as described in Materials and Methods using the Translucent media (which contains 12 µM potassium) with or without methionine supplementation to decrease the expression of KAT1 (under c ontrol of the MET25 promoter). The graph shows the average value of the optical density at 72 hours for triplicate determinations and the experiment was done with at least 3 independent transformants for each plasmid combination. (Translucent media with no KCI supplementation = Low KCI (12 µM K+);

Translucent media containing 0.75 mg/mL methionine = Low KAT1; Translucent media + 50 mM KCI added = High KCI; Translucent media +/- KCI without methionine = High KAT1). B) Immunoblot analysis of protein extracts from the indicated strains showing the correct expression of each of the fusion proteins.

The KAT1 bait vector protein is detected with the anti-LexA antibody and the prey proteins with the anti-HA antibody. Results for a representative clone are shown.

The long exposure is included to visualize the expression of the RPT2 prey protein, which accumulates less than the other two prey proteins.

Data presented are the mean ±SD and similar results were observed for three independent transformants. Asterisks (**) indicate statistical significance (Student t test) with a p value < 0.01. C) The KAT1 signal and the corresponding loading control were quantified using the ImageJ software and the average values of KAT1/loading control (Normalized KAT1) was calculated for 6 control strains (KAT1_empty vector) and 7 KAT1_BAG4 strains.

Data presented are the mean ±SD.

<caption>Figure 1</caption>
<figure><img src="image_6.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_8.png" /></figure>

Figure 2. Study of the specificity of the interaction between KAT1 and other BAG family members. A) The indicated plasmids were transformed into the THY.AP4 strain and grown to saturation in selective media. Serial dilutions were spotted onto media with the indicated composition to test for the protein-protein interaction between KAT1 and the indicated BAG family proteins. Identical results were observed for four independent clones. B) Immunoblot analysis of protein extracts from the indicated strains showing the correct expression of each of the fusion proteins. The KAT1 bait protein is detected with the anti-LexA antibody and the prey proteins with the anti-HA antibody. Results for a representative clone are shown. C) The indicated plasmids were co-transformed in the *trk1 trk2* mutant strain (PLY240) and the growth of the strains was assayed as described in Figure 1A. The average value of triplicate determinations of the optical density of the growth normalized to the potassium concentration used is shown for each strain. Similar results were observed for 3 independent clones. **Czechoslovakian Askeritskiy Biopirniki** (Student t test) with a p value < 0.001 compared to the *KAT1_Ø* strain.

<caption>Figure 2</caption>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 3. Potassium uptake and external acidification. The indicated plasmids were co-transformed in the *trk1 trk2* mutant strain (PLY240) and the strains were grown in low KCl and low KAT1 conditions and processed as described in Materials and Methods. A) The bars represent the average value for three independent experiments for the total potassium uptake for 45 minutes expressed as µmol K+/mg cells. B) The bars represent the average value for three independent experiments for the rate of potassium uptake expressed as µmol K+/min*mg cells calculated using the slope of the depletion curve normalized to the wet weight of the cells. C) The bars represent the average value for three independent experiments of the change in the pH value measured in parallel with the potassium consumption. The change in the external pH was determined during the first 15 minutes after glucose addition and was normalized to the wet weight of the cells. Asterisks (*) indicate statistical significance (Student t test) with a p value < 0.05.</figcaption></figure>
A

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

KAT1

<figure><img src="image_14.png" /></figure>

KAT1+BAG4

B

<figure><img src="image_15.png" /></figure>

C

<figure><img src="image_16.png" /></figure>

D

<figure><img src="image_17.png" /></figure>

Figure 4. BAG4 co-expression increases KAT1 current in Xenopus oocytes. A) Voltage-clamp protocol and representative current traces recorded by two-electrode voltage clamp in the presence of 100 mM KCl on oocytes co-expressing KAT1 or KAT1 and BAG4.

B) KAT1 current (I) –voltage (V) relationships of oocytes co-expressing KAT1 and BAG4 (white circles) or expressing KAT1 alone (black circles).

Data are means +/- SE (n=7 for KAT1, n=11 for KAT1 + BAG4), and are representative of two experiments performed on different oocyte batches.

C) Increase of mean KAT1 current upon BAG4 co-expression, at -110 mV (black) and at -155 mV (grey). Mean currents are from B). D) Voltage-dependence of KAT1-current activation in the presence of BAG4 (white circles) or with KAT1 expressed alone (black circles). Gating parameters

(z, gating charge, and Ea50, half-activation potential) were estimated by performing fits to the KAT1 I-V relationships

Downloaded from www.plantphysiol.org at University of California, Berkeley on August 29, 2019, published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

(in the presence or absence of 100 nM BAG4).

Copyright © 2019 American Society of Plant Biologists. All rights reserved.

G, KAT1 macroscopic conductance (G = I/V); Gmax, KAT1 macroscopic conductance at infinitely negative voltage.

<caption>Figure 4</caption>
**Figure 5. Confirmation of the interaction between KAT1 and BAG4 in N. benthamiana.** Agrobacterium strains harbouring the indicated plasmids were used to infiltrate N. benthamiana leaves and images were obtained using fluorescence confocal microscopy 72 hours post-infiltration. A) Representative BiFC images for the KAT1-KAT1 interaction and control plasmids. The overlay of the grey scale and BiFC fluorescence is shown. Leaf epithelial cells and stomata are visible. B) Representative BiFC images for the KAT1-BAG4 interaction. Representative images of the experiments performed with the control plasmids are shown below. The red signal corresponds to chloroplast autofluorescence. For panels A and B, similar results were observed in at least four independent experiments performed on different days. (Bar = 20µm) C) Agrobacterium strains expressing the indicated plasmids were used to infiltrate N. benthamiana leaves. Samples were taken at 72 hours post-infiltration and processed for protein extraction and co-immunoprecipitation as described in Materials and Methods. The figure shows the results of the immunodetection using antibodies that recognize the BAG4 and KAT1 proteins. The amount of BAG4 recovered in the KAT1 purification is shown in the first lane on the left. Similar results were observed in two independent experiments performed on different days. (YFN = N-terminal part of YFP; YFC=C-terminal part of YFP; IP=immunoprecipitation; FT=flow through).

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

Downloaded from www.plantphysiol.org
Copyright © 2019 American Society of Plant Biologists. All rights reserved.

<caption>Figure 5</caption>
<figure><img src="image_23.png" /><figcaption>Figure 6. The KAT1-BAG4 complex co-localizes with the ER and ERES markers.</figcaption></figure>

A) A plasmid containing the KAT1-BAG4 BiFC interaction and the ER marker ChFP-KDEL were infiltrated as described. The BiFC signal (right), ChFP signal (center), and the overlay image of the grey scale, BiFC fluorescence and the ChFP signals (left) are shown. Leaf epithelial cells and stomata are visible. B and C) The same co-localization analysis of the KAT1-BAG4 complex was performed with the ER exit site marker, Sec24 fused to RFP (Sec24-RFP) (B) and the STtmd-ChFP Golgi marker (C).

The yellow arrows indicate the points of co-localization with the ER marker. Chloroplast autofluorescence is shown in blue. (Bar = 20µm). (YFN = N-terminal part of YFP; YFC=C-terminal part of YFP; ChFP= Cherry Fluorescent Protein; RFP=Red Fluorescent Protein; STtmd-ChFP= rat α-2,6-sialyltransferase transmembrane domain fused to the Cherry fluorescent protein).
<figure><img src="image_24.png" /></figure>

Figure 7. BAG4 expression affects the subcellular localization of KAT1. A) Plasmids containing KAT1-YFP or both KAT1-YFP and BAG4 were transiently expressed in *N. benthamiana* using agro-infiltration. The fluorescence signal was analysed by confocal microscopy days 1, 2 and 3 days post-infiltration. Representative images are shown. Similar results were observed in three independent experiments. B) The percentage of the fluorescence signal present in the plasma membrane was quantified using ImageJ, as described in Materials and Methods. The graph shows the average values ±SD for 10 cells for each condition tested. C) Similar experiments as described in part a) were performed, but using GoldenBraid plasmids containing KAT1-YFP:dsRED-HA or KAT1-YFP:myc-BAG4:dsRED-HA. Whole cell extracts were prepared from infiltrated areas (previously confirmed to express KAT1-YFP) and proteins were processed for immunodetection. The dsRED-HA protein serves as an internal control for the efficiency of transient expression. D) The KAT1-VENUSintron (KAT1-VENUSi, top row) or the KAT1-VENUSintron_BAG4 (bottom row) plasmids were transiently expressed in the Col-0 and the bag4 mutant Arabidopsis lines using agro-infiltration. The fluorescence signal was analyzed by confocal microscopy 2 days after infiltration. Representative images are shown and similar results were observed in three independent experiments. (Bar = 20 µm) E) The percentage of the fluorescence signal present in the plasma membrane was quantified using ImageJ, as described in Materials and Methods and downloaded from https://www average published by www.plantphysiol.org. Copyright © 2019 American Society of Plant Biologists. All rights reserved.
<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

Figure 8. Effect of BAG4 loss and gain-of-function on stomatal aperture. Stomata from the indicated Arabidopsis lines were analyzed as described in Materials and Methods. The data show the average ratio for 60-100 stomata and the error bars represent the standard deviation. A) The width/length ratio of stomata of the different mutant lines were determined in leaves from the indicated lines harvested 1 hour before the lights turned on (Dark), incubated for 2.5 hours in opening buffer in the dark (Opening buffer, O.B.) or 2.5 hours after the lights turned on (Light). Similar results were observed in three independent experiments. B) The width/length ratio of stomata of control lines and Col-0 and kat1 homozygous for 35S:BAG4 transgene were determined as described in A. For both experiments, the asterisks indicate statistical significance as compared to the Col-0 dark control (Student t test) (**: p value <0.01; **: p value < 0.001; ***: p value < 0.0001).

The length/width ratio was also significantly increased in BAG4 overexpressing lines in both light and opening buffer (# indicates statistical comparison with Col-0 O.B. and • indicates statistical comparison with Col-0 light).
<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>Figure 9. Effect of BAG4 loss and gain-of-function on leaf temperature. The same lines described in Figure 7 were analyzed for leaf temperature using an infrared thermography, as described in Materials and Methods (Panel A mutant lines, Panel B control and 35S:BAG4 lines). Each symbol represents an individual measurement and the bar represents the average value for 10 measurements of 6 different plants for each genotype. The error bars represent the standard deviation. For both experiments, the asterisks indicate statistical significance as compared to the Col-0 control or the comparisons indicated by the brackets above the graphs (Student t-test) (**: p value <0.01; **: p value < 0.001; ****: p value < 0.0001).</figcaption></figure>
KAT2-YFN_KAT1-YFC

<figure><img src="image_29.png" /></figure>

KAT2-YFN_YFC-BAG4

<figure><img src="image_30.png" /></figure>

KAT2-YFN_YFC

Figure 10. BAG4 interacts with KAT2 in BiFC assays in *N. benthamiana*. Interaction assays were carried out and analyzed as described in the legend to Figure 5. As shown, a similar pattern of interaction is observed for KAT2 when tested with BAG4 (compare with Figure 5B). In this image, the BiFC signals corresponding to the KAT2-KAT1 and KAT2-BAG4 interactions are shown in green and the chloroplast autofluorescence is shown in red. (Bar = 20µm)
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