Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation

Report

Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation Highlights

Authors

d

Arabidopsis anion channels SLAH1 and SLAH3 co-localize in xylem-pole pericycle cells

d

SLAH1 and SLAH3 assemble to chloride-conducting heteromeric channels

Paloma Cubero-Font, Tobias Maierhofer, Justyna Jaslan, ..., Rainer Hedrich, Jose´ M. Colmenero-Flores, Dietmar Geiger

d

Soil salinity decreases SLAH1 expression, leading to Cl exclusion from the shoot

Correspondence

d

SLAH1 represents a promising candidate for engineering salinity-tolerant plants

[email protected] (J.M.C.-F.), [email protected] (D.G.)

In Brief Cubero-Font, Maierhofer, et al. report that plants adjust the ratio between Cl and NO3 in the shoot via heteromerization of Arabidopsis SLAH1/ SLAH3 anion channel subunits. Differential expression of SLAH1 controls Cl fluxes to the shoot without affecting the uptake of NO3 , making SLAH1 a promising target for engineering salinitytolerant plants.

Accession Numbers XM_003619199

Cubero-Font et al., 2016, Current Biology 26, 1–8 August 22, 2016 ª 2016 Elsevier Ltd. http://dx.doi.org/10.1016/j.cub.2016.06.045

Please cite this article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045

Current Biology

Report Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation Paloma Cubero-Font,1,4 Tobias Maierhofer,2,4 Justyna Jaslan,2 Miguel A. Rosales,1,5 Joaquı´n Espartero,1 Pablo Dı´az-Rueda,1 Heike M. Mu¨ller,2 Anna-Lena Hu¨rter,2 Khaled A.S. AL-Rasheid,3 Irene Marten,2 Rainer Hedrich,2 Jose´ M. Colmenero-Flores,1,* and Dietmar Geiger2,* 1Instituto de Recursos Naturales y Agrobiologı´a (IRNAS), Spanish National Research Council (CSIC), Avenida Reina Mercedes 10, 41012 Sevilla, Spain 2Institute for Molecular Plant Physiology and Biophysics, University of Wu ¨ rzburg, Julius-von-Sachs Platz 2, 97082 Wu¨rzburg, Germany 3Zoology Department, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia 4Co-first author 5Present address: Biochimie et Physiologie Mole ´ culaire des Plantes (BPMP), UMR 5004 (CNRS/INRA/SupAgro/UM), 2 Place Viala, 34060 Montpellier Cedex 2, France *Correspondence: [email protected] (J.M.C.-F.), [email protected] (D.G.) http://dx.doi.org/10.1016/j.cub.2016.06.045

SUMMARY

Higher plants take up nutrients via the roots and load them into xylem vessels for translocation to the shoot. After uptake, anions have to be channeled toward the root xylem vessels. Thereby, xylem parenchyma and pericycle cells control the anion composition of the root-shoot xylem sap [1–6]. The fact that salt-tolerant genotypes possess lower xylem-sap Cl contents compared to salt-sensitive genotypes [7–10] indicates that membrane transport proteins at the sites of xylem loading contribute to plant salinity tolerance via selective chloride exclusion. However, the molecular mechanism of xylem loading that lies behind the balance between NO3 and Cl loading remains largely unknown. Here we identify two root anion channels in Arabidopsis, SLAH1 and SLAH3, that control the shoot NO3 /Cl ratio. The AtSLAH1 gene is expressed in the root xylem-pole pericycle, where it co-localizes with AtSLAH3. Under high soil salinity, AtSLAH1 expression markedly declined and the chloride content of the xylem sap in AtSLAH1 loss-of-function mutants was half of the wild-type level only. SLAH3 anion channels are not active per se but require extracellular nitrate and phosphorylation by calcium-dependent kinases (CPKs) [11–13]. When co-expressed in Xenopus oocytes, however, the electrically silent SLAH1 subunit gates SLAH3 open even in the absence of nitrate- and calcium-dependent kinases. Apparently, SLAH1/SLAH3 heteromerization facilitates SLAH3-mediated chloride efflux from pericycle cells into the root xylem vessels. Our results indicate that under salt stress, plants adjust the distribution of NO3 and Cl between root and shoot via differential

expression and assembly of SLAH1/SLAH3 anion channel subunits. RESULTS Soil salinity restricts plant growth and thereby decreases the yield and quality of crops [14–16]. Besides accumulation of sodium, excessive accumulation of chloride ions within the shoot leads to the disturbance of growth and productivity of salt-sensitive plants [9, 14, 15, 17–19]. Thus, salt exclusion from the shoot, while sustaining the supply with essential nutrients such as potassium and nitrate, is believed to enhance plant salinity tolerance [6, 9, 14, 15, 20]. The negative plasma membrane potential of plant cells (above 150 mV) and the outward-directed nitrate and chloride concentration gradients suggest that the entry of NO3 and Cl into the xylem vessels is facilitated by anion efflux channels [21]. This notion is underpinned by NO3 and Cl currents of xylem parenchyma cells registered by early patch-clamp studies [1–3]. These currents are reminiscent of the electrical characteristics of anion channels from the SLAC/SLAH (slow-type) family [22, 23]. SLAC1 and its homolog anion channels SLAH3 and SLAH2 have been functionally characterized in Arabidopsis and Xenopus oocytes [11, 12, 24–29]. The physiological function of SLAH1 and SLAH4 remains, however, unexplored so far. SLAH1 and SLAH3 Co-express in Xylem-Pole Pericycle Cells To explore the tissue- and cell-specific expression of AtSLAH1 and AtSLAH3 genes, we obtained different transgenic lines expressing the chimeric GUS::GFP gene fusion under the control of the respective native promoters. Upon microscopic inspection, we were able to localize anion channel expression in the vascular cylinder with both AtSLAH1 and AtSLAH3 gene promoters (Figures 1A and 1E; cf. [22, 30]). Confocal microscopy analysis detected AtSLAH1-promoter-driven GFP fluorescence in cells adjacent to xylem vessels (Figure 1B). For AtSLAH3, Current Biology 26, 1–8, August 22, 2016 ª 2016 Elsevier Ltd. 1

Please cite this article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045

A

B

C

E C

*

E

··

E C * **

·

* *

*

F

** *

*

··

E

··

·

*

*

* *

* ·· * H

C* * * * * * * * * * * **

C

a similar expression pattern was confirmed by histochemistry using GUS gene fusion (Figure 1F). Inspection of transversal sections of roots expressing GUS under control of AtSLAH1 (Figures 1C and 1D) and AtSLAH3 promoter (Figures 1G and 1H) localized both S-type anion channel isoforms in the xylem pole pericycle. SLAH1, Together with SLAH3, Feeds Chloride into the Root Xylem Given that SLAH3 conducts both NO3 and Cl currents and colocalizes with its homolog, SLAH1, in cells facing xylem vessels, one would predict that the S-type anion channel pair does feed the xylem sap with anions ascending from root to shoot. To test this assumption, we inspected an AtSLAH1 loss-of-function mutant (slah1-2) and two independent slah1-2 complementation lines (P1-12 and P1-31) expressing the wild-type (WT) AtSLAH1 gene under the control of its native promoter (Figure S1A). Moreover, we crossed the slah1-2 knockout (KO) mutant with the slah3-1 KO mutant and named the resulting double-KO mutant slah1-2/slah3-1. For phenotype analyses, the mutant lines slah1-2 and slah1-2/slah3-1 (homozygous for the transfer DNA [T-DNA] insertions) were compared with their respective azygous segregant lines, which were assigned as WT plants in this work. When grown under low (70 mM) or high (5 mM) chloride, the slah1-2 KO mutant line accumulated less Cl in the shoot than the WT, independent from the feeding regime (Figures S1B and S1C). Given that AtSLAH1 and AtSLAH3 expression is localized in the xylem-pole facing the xylem vessels in the root, the pericycle cells should contribute to the anion composition of the ascending xylem sap. We therefore collected xylem sap samples [31] and quantified the content of Cl and NO3 colorimetrically [32, 33]. In xylem sap samples from slah1-2, slah3-1, and slah3-4 mutant lines the chloride, but not the nitrate, content was reduced by 50% compared to their respective WT lines (Figures 2A–2D, S1F, S1H, S1K, and S1M; Table S1). To confirm the slah1-2 chloride phenotype, we inspected the xylem sap of the slah1 complementation lines, P1-12 and P1-31 (Figures 2A and 2B). In line with the assumed role of SLAH1 in Cl feeding, the complemented lines reached chloride levels similar to the wildtype. Reduction of Cl content in the xylem sap of plants lacking functional copies of AtSLAH1 or AtSLAH3 genes was a conse2 Current Biology 26, 1–8, August 22, 2016

*

*

G

C

Figure 1. AtSLAH1 and AtSLAH3 Are Co-expressed in the Xylem-Pole Pericycle

D

* *

* *

* ··

*

*

*

· ·

* *

* ·

* · ·· *

*

*

* * **

Localization of AtSLAH1 (A–D) and AtSLAH3 (E–H) gene expression in transgenic Arabidopsis plants expressing the GFP::GUS or GUS reporter genes under the control of the AtSLAH1 and AtSLAH3 promoter regions, respectively. Tissue- and cellspecific expression of AtSLAH1 gene (A, C, and D) or AtSLAH3 gene (E–H) was obtained through histochemical localization of GUS activity. Localization of SLAH1-regulated GFP fluorescence in pericycle cells was observed through confocal microscopy (B). E, epidermis; C, cortex; asterisk, endodermis; red spot, protoxylem; blue spot, metaxylem. Scale bars, 100 mm (A and E), 20 mm (C and G) and 10 mm (B, D, F, and H).

quence of a lower root-to-shoot Cl translocation rate (Figures S1G and S1L), whereas the NO3 translocation rate was not significantly altered (Figures S1I and S1N). Interestingly, the analysis of the xylem sap of the double mutant slah1-2/slah3-1 revealed a reduction of the chloride content similar to the respective single mutants (Figure 2C), whereas the nitrate concentration remained unchanged (Figure 2D) Given the participation of SLAH1 in facilitating root-to-shoot transfer of Cl , this channel unit may serve as a pathway for shoot chloride accumulation under salt stress conditions. To test this assumption, we treated slah1-2 mutant and WT plant roots with increasing concentrations of Cl salt (15, 30, 60 and 120 mM Cl ). From the root sap samples collected 3 hr after salt exposure (Figure 2E), it was evident that slah1-2 mutants in the xylem sap accumulated less chloride than WT plants, a difference that became even more pronounced when roots were exposed to increasing salt concentrations. Stress Downregulates SLAH1 Expression To prevent non-physiological chloride levels reaching the shoot on one side and nitrate depletion on the other, the entry of Cl relative to NO3 has to be well controlled. To further explore the regulatory properties of AtSLAH1 and its potential interaction with AtSLAH3, we tested whether salt (NaCl) stress feeds back on root SLAH1 and SLAH3 expression. Upon an increase in the salt content of the soil, the water potential drops. Soil salinity and water deficit cause the level of the stress hormone ABA to rise. Given that the dehydrin AtLEA-M is strongly induced in response to osmotic stress and ABA [34], in our experiments we used AtLEA-M as a salt stress marker (Figure S2B). To differentiate between osmotic shocks caused by either salt stress or water deficit, we alternatively exposed roots to NaCl (150 mM) or to the non-permeable osmolyte PEG-8000 (287 g L 1). Twenty-four hours after NaCl treatment, root transcript levels of the stress marker AtLEA-M increased (Figure S2B), whereas those of AtSLAH1 and AtSLAH3 dropped by about ten and five times, respectively (Figures 2F and S2A). When water deficit was applied by transfer of the plants to PEG-8000, transcripts for AtLEA-M also increased (Figure S2B) and levels for AtSLAH1 and AtSLAH3 decreased by 60 and five times, respectively (Figures 2F and S2A). As ABA is downstream of salt and water

Please cite this article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045

B a

a

a

E 120

a

a

a

100

100 80

b

60

-

40 20 0

12

80 60 40 20 0

slah1-2 P1-12 P1-31 slah1/SLAH1

WT

*

14

a

slah1-2 P1-12 P1-31 slah1/SLAH1

WT

Xylem Sap [Cl-] (mM)

[Cl ] relative to WT (%)

120

[NO3-] relative to WT (%)

A

10

*

8 6 4 2

D

S W

3-

4

1

x

x 2 1-

ah

1-

2

C

x

ol

-0

3-

ah sl

T W

3-

1

S

sl

x -0

ol

T W

T

sla h1 -2

W

T

sla h1 -2

W

T

sla h1 -2

W

30 60 Cl- Treatment (mM)

120

CTR NaCl

sl

sl

G

control

100

20

ah

C

40

0 ah

W

3-

4

1 3-

ah sl

W

sl

ah

T

0

F

60

1

20

15

3-

40

a

80

sl

60

a

a

ah

b

b

a

100

sl

b

80

sla h1 -2

a

120

100

-

0

140

a

ah

a

[NO3-] relative to WT (%)

[Cl ] relative to WT (%)

120

Relative expression

C

75

ABA 50

25

0

+ABA

PEG

3h

24h AtSLAH1

3h

24h AtSLAH3

Figure 2. AtSLAH1 and AtSLAH3 Regulate Root-to-Shoot Translocation of Cl– (A and B) Percentage of Cl (A) or NO3 (B) xylem sap concentration, relative to the wild-type (WT; 100%), in the slah1-2 mutant line and in the two slah1-2/SLAH1 complemented lines (P1-12 and P1-31). For the slah1-2 line, values are the average (±SE) of 12 biological replicates obtained from 12 independent experiments. For slah1-complemented lines, values are the average (±SE) of four biological replicates obtained from four independent experiments. Different letters indicate statistically significant differences (p < 0.05, Student’s t test). (C and D) Percentage of Cl (C) or NO3 (D) xylem sap concentration relative to the WT line (100%) in the slah3-1 and slah3-4 mutant lines. For the slah3-1 line, values are the average (±SE) of nine biological replicates from three independent experiments. For the slah3-4 line, values are the average (±SE) of three biological replicates from one experiment. Different letters indicate statistically significant differences (p < 0.05, Student’s t test). (E) Chloride concentration in the xylem sap of the slah1-2 mutant line and the WT lines 3 hr after high salt load treatments (15, 30, 60, and 120 mM Cl ). Values are the average (±SE) of three biological replicates from one experiment. Asterisks indicate statistically significant differences (p < 0.05, Student’s t test). (F) Quantification of the abundance of AtSLAH1 and AtSLAH3 transcripts 3 hr and 24 hr after treatments. Transcript abundance was quantified in plants growing under control conditions (CTR) and 3 hr and 24 hr after the application of abiotic stress treatments consisting of salinity induced with NaCl 150 mM (NaCl), water deficit induced with PEG-8000 287 g L 1 (PEG), or exogenous application of 100 mM abscisic acid (ABA). Transcript abundance was quantified with standard curves calculated for the individual PCR products and normalized with the housekeeping translation initiation factor AteIF4A1 gene (At3g13920). Results are the mean (±SE) of three biological replicates. (G) Histochemical localization of AtSLAH1 expression in transgenic Arabidopsis plants expressing the GUS reporter genes under the control of the AtSLAH1 promoter (PSLAH1-GUS) either treated with 100 mM ABA or without the phytohormone (control). See also Figures S1 and S2 and Table S1.

stress, in a subsequent experiment we treated the mutant and WT plants with the stress hormone (Figures 2F, 2G, and S2A). Application of ABA strongly increased AtLEA-M (Figure S2B), but, unlike AtSLAH3, it caused a particularly strong repression (4003) of AtSLAH1 expression (Figures 2F and S2A). This response is well in line with the disappearance of AtSLAH1 promoter activity in the vascular cylinder of PSLAH1-GFP::GUS plants after 15 hr pre-incubation with ABA (Figure 2G).

SLAH1 Represents a Silent Anion Channel Subunit To understand how the downregulation of SLAH1 affects root xylem loading, we studied functional properties of SLAH1 and SLAH3 with the two-electrode voltage clamp (TEVC) technique in the heterologous expression system of Xenopus oocytes. When we injected SLAH1 cRNA into Xenopus leavis oocytes, however, anion currents were not observed in chloride- and nitrate-based buffers (Figures S3A–S3C). A similar situation has Current Biology 26, 1–8, August 22, 2016 3

Please cite this article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045

been observed when SLAC1, SLAH2, or SLAH3 has been expressed in the absence of anion-channel-activating protein kinases [11, 26–28]. SLAH3 requires extracellular nitrate and Ca2+-dependent kinases such as CPK21 to conduct NO3 and Cl anions (Figures S3B and S3C) [11, 12]. The presence of the protein kinases that were effective with SLAH3 or SLAC1, however, did not render SLAH1 active—independent from the presence of extracellular nitrate (Figures S3A–S3C). Chen and colleagues crystallized the bacterial homolog of SLAC1 from Haemophilus influenza (HiTeaH [35]) and found Phe450 in the pore region of SLAC1 linked to the anion gate [35]. Replacement of this pore phenylalanine by alanine turned AtSLAC1 into a constitutively open anion channel. We have shown that this open-gate mutation shifts also AtSLAH2 [28] and AtSLAH3 (F517A; Figure S3D) into an anion-conducting state. When the respective phenylalanine residue in SLAH1 (Phe307) was mutated to alanine, however, the putative anion channel remained electrically silent (Figure S3D). SLAH1 Substitutes for Kinase- and Nitrate-Dependent Activation of SLAH3 The 3D model of HiTeaH revealed a trimeric structure with three subunits, each forming an individual pore [35]. As mentioned before, in kinase-free oocytes, or in the absence of nitrate, SLAH3 is as electrically silent as SLAH1 (Figures 3A, S3B, and S3C). When SLAH1 and SLAH3 were co-expressed, however, macroscopic anion currents could be measured without the co-expression of a kinase and in the absence of extracellular nitrate (Figures 3A 3B, S4A, and S4B). The chloride and nitrate current amplitude of the SLAH3/SLAH1 complex was thus similar to the currents of SLAH3 when activated by CPK21 in nitrate-based buffers (Figure 3A, S4A, and S4B). Co-expression of kinases and/or application of nitrate did not further increase the current amplitude associated with constitutively active SLAH1/SLAH3 complexes (Figures S4A and S4B). To elucidate whether the effect of SLAH1 is SLAH3 specific, we individually co-expressed SLAH1 together with each member of the Arabidopsis SLAC/SLAH anion channel family. Interestingly, SLAH1 could only activate SLAH3, and not SLAC1, SLAH2, or SLAH4 (Figures 3B and S4A). In contrast, SLAC1 and SLAH2 remained silent when co-expressed with SLAH3 in the absence of the activating kinase (Figure S4B). To further substantiate the preference of SLAH1 for SLAH3-type anion channels, we co-expressed SLAH1 with SLAH3-type anion channels from Medicago trunculata (Mt), Dionaea muscipula (Dm), and Populus tremula 3 P. tremuloides (Ptt). Similar to the SLAH3activating role of SLAH1 in Arabidopsis, AtSLAH1 was capable of activating DmSLAH3, PttSLAH3, and MtSLAH3, even in the absence of extracellular nitrate and without co-expressing an activating kinase (Figure S4C). These findings indicate that SLAH1 specifically activates SLAH3-type anion channels and that this function is conserved across species borders. Silent SLAH1 and Anion Conducting SLAH3 Form Heteromeric Anion Channels To further substantiate that AtSLAH1 and AtSLAH3 assemble to functional complexes, we expressed TSapphire:SLAH3 (TS:SLAH3) fusion constructs either alone or together with SLAH1:mOrange (SLAH1:mO) in Nicotiana benthamiana leaves. 4 Current Biology 26, 1–8, August 22, 2016

Using a combination of fluorescence resonance energy transfer and fluorescence lifetime imaging (FRET-FLIM) [36], we tested physical interaction between SLAH1 and SLAH3. Expression of constructs was verified via confocal laser scanning microscopy prior to FRET-FLIM experiments (Figures S3E and S3F). Expression of TS:SLAH3 alone revealed an average TSapphire lifetime of 2.70 ± 0.03 ns (Figures 3C and 3D). Upon co-expression with SLAH1:mO, the fluorescence lifetime of TS:SLAH3 decreased significantly to 2.45 ± 0.03 ns (ANOVA, p < 0.01; Figures 3C and 3D), which is equivalent to a FRET efficiency of 9%—a value well in line with FRET efficiencies reported for other known plant interaction partners (cf. [36, 37]). Thus, the activation of SLAH3 through SLAH1 in Xenopus oocytes results from the physical interaction of these anion channel subunits. In contrast to monocots [1–3], the stelar cells of dicots such as Arabidopsis are not directly accessible for protoplasting and patch clamping. However, guard cells express SLAH3 and SLAC1 anion channels [11] and so represent a plant cell system that is accessible for functional patch-clamp studies. Guard cells lacking SLAC1 anion channels cannot release chloride [22] and do not express SLAH1 but do express wild-type levels of SLAH3 transcripts [11] (Figure 3E). Interestingly, when the slac1-2 phenotype is complemented with SLAH1 under the control of the SLAC1 promoter (slac1-2/SLAH1), the mutant phenotype is revived [22]. Via qRT-PCR experiments, we quantified the expression of SLAH1–SLAH4 in both the slac1-2 and the slac1-2/ SLAH1 mutant plants (Figure 3E). As expected, SLAH1 expression was only detectable in the slac1-2/SLAH1 mutant line. Thus, these lines represent ideal in planta expression systems for studying the electrical behavior of SLAH3 when either expressed alone (slac1-2) or when co-expressed with SLAH1 (slac1-2/SLAH1) [1–3]. In line with previous findings, our wholecell patch-clamp analysis revealed only weak S-type chloride currents in slac1-2 guard cells (Figures 3F and 3G; cf. [22, 23, 26]). When SLAH1 was expressed in slac1-2 guard cells, however, S-type chloride currents were found restored (Figures 3F and 3G). This finding in guard cells, as well as the activation of SLAH3 by SLAH1 in the oocyte system (Figures 3A and 3B), explains the reduced root xylem sap chloride levels of the SLAH1 and SLAH3 loss-of-function mutants. The kinase- and nitrate-independent activity of the heteromeric channel complex between SLAH1 and SLAH3 seen in oocyte experiments could result from the addition of a SLAH1 intrinsic chloride conductance superimposing the NO3 /Cl conductance of SLAH3. Alternatively, SLAH1 might represent a silent subunit that modulates the properties of SLAH3. To test the contribution of each interaction partner to the anion currents of the heteromeric complex, we created inactive SLAH3 and SLAH1 channel mutants. Chen et al. [35] could show that the point mutations G194D and F450L in AtSLAC1 lead to a nonfunctional channel. Based on homology models of SLAH1 and SLAH3 to the crystal structure from HiTeaH [35], one would predict that exchange of Gly194 to a negatively charged aspartate and the substitution of Phe450 by leucine block the pores of SLAH3 and SLAH1, similar to SLAC1. When we generated the respective SLAH3 mutants (G264D and F517L) and expressed the mutants in Xenopus oocytes, neither the co-expression with CPK21DEF (Figure S4D) nor co-expression with SLAH1 (Figure 4A) could elicit macroscopic currents. Note that the

Please cite this article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045

A

B

NO3-

-12 Iinst (μA) at -100 mV

Cl-

SLAH1

-10

10 μA

SLAH3 CPK21ΔEF 4s

SLAH3 SLAH1

chloride nitrate

-8 -6 -4 -2

TS:SLAH3

1 C

4 +S

LA

H LA +S

LA

TS:SLAH3 +SLAH1:mO

D

TS:SLAH3 +SLAH1:mO

TS:SLAH3

normalized frequency

C

+S

+S

LA

H

H

2

3

0

1.0

0.5

2.0

F

5000 4000

slac1-2

slac1-2 /SLAH1

n.d.

SLAH3

SLAH1

n.d.

SLAH3

0

n.d.

500

n.d.

1500 1000

2.0 2.2 2.4 2.6 2.8 3.0 fluorescence lifetime (ns)

G slac1-2

SLAH1 SLAH2 SLAH3 SLAH4

slac1-2/SLAH1

20

-50

50 V (mV) -10 -20

1s

-30

A

0 -40 -20

3.2

10 -100

40 pA/pF

6000

n.d.

Relative expression

E

0.0 1.8

3.0

lifetime (ns)

I/Cm (pA/pF)

40 μm

-40 -50 -60

slac1-2 slac1-2/ SLAH1

Figure 3. Activation of S-Type Anion Currents via Co-expression of SLAH3 and SLAH1 in Oocytes and Guard Cell Protoplasts (A) Whole-oocyte currents of Xenopus oocytes in response to the standard voltage protocol co-expressing either SLAH3 with CPK21DEF or SLAH3 with SLAH1 were recorded in chloride- or nitrate-based buffers (30 mM). Representative cells are shown. (B) Instantaneous currents (Iinst) of oocytes co-expressing SLAH1 with different members of the Arabidopsis S-type anion channel family recorded at 100 mV in the presence of either 100 mM chloride or 100 mM nitrate (n R 4; mean ± SE). (C) Pixel-by-pixel FRET-FLIM analysis of TSapphire:SLAH3 (TS:SLAH3) in the presence (left) or in the absence (right) of SLAH1:mOrange (SLAH1:mO) expressed in epidermal cells of N. benthamiana. The false color code depicts the lifetime window from 2 ns (blue) to 3 ns (red). Representative images are shown. (D) TS:SLAH3 lifetime histogram in the presence (Gaussian fit, solid line; n = 9 experiments) or absence (Gaussian fit, dotted line; n = 11 experiments) of SLAH1:mO (mean ± SE). (E) Relative Expression of the SLAC1 homologs in guard cells of slac1-2 compared to the complementation line slac1-2/SLAH1. Transcript numbers per 10,000 act2/8 molecules are presented as mean ± SE (n R 4 experiments; n. d., not detectable). (F) Representative macroscopic currents monitored from slac1-2 and slac1-2/SLAH1 in response to the standard patch-clamp voltage protocol. (G) Steady-state current densities (Iss/Cm) are shown as a function of the clamped voltage (slac1-2, n = 7 from four independent experiments; slac1-2/SLAH1, n = 6 independent experiments; mean ± SE). See also Figure S3.

background currents of the mutant SLAH3 F517L, when co-expressed with CPK21DEF, SLAH1 WT, or SLAH1 mutants (Figure 4A, Figure S4D), result from the residual activity of the SLAH3 mutant, rather than from a conductivity of SLAH1. In contrast, the mutations at the equivalent position in SLAH1 (R47D and F307L) did not significantly affect the anion currents mediated by the SLAH3/SLAH1 channel complex (Figure 4A).

These findings indicate that SLAH1 modifies the electrical properties of SLAH3, rather than being a conductive subunit within the heteromeric complex itself. The chloride currents of the SLAH3/SLAH1 complex were markedly increased compared to the currents of SLAH3 homomeric channels activated by CPK21 (Figures 3A and S4A). When we tested the relative permeability of SLAH3 in Current Biology 26, 1–8, August 22, 2016 5

Please cite this article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045

A 120

B

SLAH1 +

Iinst (%) at -100 mV

100 80

SLAH3 chloride nitrate

0.1

0.08

60

0.06

0.04

40

0.02

20

0 SL A H 1 SL W A T H 1 SL R 4 A 7D H 1 F3 07 L SL no A ne H 3 SL G2 64 A H D 3 F5 17 L

0

+CPK21ΔEF

no ne

C

0.12

chord conductance (mS) at -120 mV

SLAH3 +

SLAH3 +CPK21ΔEF 3 Cl10 Cl-30 Cl 30 NO3 [mM]

rel.PO

D 1.0 0.8 0.6

SLAH3 +SLAH1 3 Cl10 Cl-30 Cl 30 NO3 [mM]

rel.PO

+SLAH1

1.0

Figure 4. SLAH1 Modifies the Activation Properties of SLAH3 (A) Instantaneous currents (Iinst) recorded at 100 mV in nitrate-based solutions (100 mM). Currents were recorded from oocytes expressing different combinations of SLAH1 WT or mutants and SLAH3 WT or mutants as indicated in the figure. (B) Chord conductance of Xenopus oocytes coexpressing SLAH3 either with CPK21DEF or with SLAH1. Standard bath solution contained 100 mM chloride or nitrate (n = 4 experiments; mean ± SE). (C and D) Relative voltage-dependent open probabilities (rel. PO) derived from oocytes co-expressing SLAH3 with CPK21DEF (C) or SLAH3 with SLAH1 (D) in the presence of varying Cl concentrations (black) or 30 mM NO3 (red). Data points were fitted by a Boltzmann equation (continuous line) (n = 4 experiments; mean ± SE). See also Figure S4.

0.8 0.6

and maize (Zea mays) [1, 2, 21] conform closely with the characteristics of SLAC/SLAH anion channels expressed 0.2 0.2 in Xenopus oocytes, and the transcriptional downregulation of SLAH1 -200 -150 -100 -50 V (mV) 50 -200 -150 -100 -50 V (mV) 50 by ABA (Figures 2F and 2G) provides strong evidence for involvement of S-type channels in anion xylem loading comparison to that of the SLAH3/SLAH1 complex, it became (for details, see the Supplemental Experimental Procedures). apparent that the permeability for chloride relative to nitrate Although we measured the salt stress and ABA-dependent was unaltered between the homomer and the heteromer (Fig- downregulation of SLAH1 and SLAH3 on the transcript level, ure S4F). However, the chloride conductance was seven times there is evidence from plant ion channel studies [38–40] that higher with oocytes expressing SLAH3/SLAH1 heteromeric ion channel protein abundance directly follows the respective channels compared to the kinase-activated SLAH3 homomeric transcript level with delay times of tens of minutes to a few anion channels, whereas the conductance for nitrate remained hours only. unaltered (Figure 4B). To investigate the molecular mechanism SLAH1/SLAH3 co-expression in oocytes and guard cells, as underlying the enhanced chloride current amplitudes of the well as Arabidopsis mutant phenotypes, indicates that SLAH1, SLAH3/SLAH1 complex, we analyzed the nitrate-dependent via formation of SLAH1/SLAH3 heteromers, facilitates Cl gating properties of the kinase-activated SLAH3 compared to efflux by rendering SLAH3 nitrate and phosphorylation indethe SLAH3/SLAH1 heteromer. Whereas CPK21-activated pendent. SLAH1/SLAH3 heteromer formation is further SLAH3 required extracellular nitrate to gate open at physiolog- undepinned by the fact that just like the double mutant ical membrane potentials (Figure 4C; cf. [11]), the SLAH3/ (slah1-2/slah3-1), each of the single mutants (slah1-2, SLAH1 heteromer appeared to be already open in the absence slah3-1, and slah3-4) exhibited the identical phenotype of of nitrate (Figure 4D). It became obvious that the elevated chlo- reduced xylem sap chloride content (Figures 2A–2D). Since ride currents of the SLAH3/SLAH1 heteromeric channel (Figures SLAH1 abundance determines a significant contribution to 3A and 3B) result from the nitrate-independent activation of the degree of Cl exclusion without significantly affecting SLAH3 and thus from a massive increase of its chloride conduc- the inclusion of NO3 , the SLAH1 gene arises as a novel tance, rather than from an increased chloride permeability biotechnological tool with great potential for engineering salinity-tolerant plants. (Figures 4B–4D). 0.4

0.4

DISCUSSION

EXPERIMENTAL PROCEDURES

We have shown that both AtSLAH1 and AtSLAH3 genes coexpress in the root pericycle (Figure 1, cf. [22, 30]). According to previous studies, this cell type is involved in loading the root xylem vessels with nutrient anions and cations [5, 28, 31]. The electrical properties of anion conductances found in xylem parenchyma protoplasts of barley (Hordeum vulgare)

See the Supplemental Experimental Procedures.

6 Current Biology 26, 1–8, August 22, 2016

ACCESSION NUMBERS Newly reported accession numbers are as follows: DmSLAH3, TBro1.0.0: comp228551_c0 (http://tbro.carnivorom.com/tbro/details/byId/184091); MtSLAH3, GenBank: XM_003619199.

Please cite this article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045

SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.cub.2016.06.045. AUTHOR CONTRIBUTIONS P.C.-F., T.M., J.J., M.A.R., J.E., P.D.-R., H.M.M., A-L.H., J.M.C.-F., and D.G. conducted the experiments and analyzed the data. P.C.-F., T.M., K.A.S.A-R., I.M., R.H., J.M.C.-F., and D.G. designed the experiments and wrote the paper. ACKNOWLEDGMENTS We thank Tracey A. Cuin for critical reading of the manuscript and the Nottingham Arabidopsis Stock Centre and the Institute Jean-Pierre Bourgin for distributing the T-DNA insertion mutant seeds, and we acknowledge Dr. Sheng Luan for providing the homozygous slah3-4 mutant line SALK_111623 [30] and Dr. Koh Iba for providing the slac1-2/SLAH1 and PSLAH3-GUS lines [22]. J.M.C.-F. was supported by the Spanish Ministry of Science and Innovation FEDER grants AGL2009-08339/AGR and AGL2015-71386-R. R.H. and D.G. were supported by the German Research Foundation (DFG) within the SFB/TR166 ‘‘ReceptorLight’’ project B8. P.C.-F. had fellowship support from the Spanish National Research Council (CSIC) and the German Academic Exchange Service (DAAD). R.H. and K.A.S.A.-R. were further supported by the International Research Group Program (project IRG14-08) of the Deanship of Scientific Research, King Saud University. We appreciate J.D. Franco-Navarro, Inmaculada Flores, and F.J. Dura´n for the technical assistance provided to this work. Investigations of plant ion channels in Xenopus oocytes adhere to the regulations and provisions of the Animal Protection Act and the Experimental Animals Ordinance. Permission for keeping African clawfrog Xenopus laevis and using Xenopus oocytes exists at the Julius-von-Sachs Institute, University Wu¨rzburg and is registered and oversight at/from the district government of Unterfranken, Germany. Received: April 21, 2016 Revised: June 5, 2016 Accepted: June 21, 2016 Published: July 7, 2016 REFERENCES 1. Gilliham, M., and Tester, M. (2005). The regulation of anion loading to the maize root xylem. Plant Physiol. 137, 819–828.

7. La¨uchli, A., James, R.A., Huang, C.X., McCully, M., and Munns, R. (2008). Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion. Plant Cell Environ. 31, 1565–1574. 8. Moya, J.L., Go´mez-Cadenas, A., Primo-Millo, E., and Talon, M. (2003). Chloride absorption in salt-sensitive Carrizo citrange and salt-tolerant Cleopatra mandarin citrus rootstocks is linked to water use. J. Exp. Bot. 54, 825–833. 9. Teakle, N.L., and Tyerman, S.D. (2010). Mechanisms of Cl(-) transport contributing to salt tolerance. Plant Cell Environ. 33, 566–589. 10. Brumo´s, J., Talo´n, M., Bouhlal, R., and Colmenero-Flores, J.M. (2010). Cl homeostasis in includer and excluder citrus rootstocks: transport mechanisms and identification of candidate genes. Plant Cell Environ. 33, 2012–2027. 11. Geiger, D., Maierhofer, T., Al-Rasheid, K.A.S., Scherzer, S., Mumm, P., Liese, A., Ache, P., Wellmann, C., Marten, I., Grill, E., et al. (2011). Stomatal closure by fast abscisic acid signaling is mediated by the guard cell anion channel SLAH3 and the receptor RCAR1. Sci. Signal. 4, ra32. 12. Maierhofer, T., Diekmann, M., Offenborn, J.N., Lind, C., Bauer, H., Hashimoto, K., S Al-Rasheid, K.A., Luan, S., Kudla, J., Geiger, D., and Hedrich, R. (2014). Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid. Sci. Signal. 7, ra86. 13. Scherzer, S., Maierhofer, T., Al-Rasheid, K.A.S., Geiger, D., and Hedrich, R. (2012). Multiple calcium-dependent kinases modulate ABA-activated guard cell anion channels. Mol. Plant 5, 1409–1412. 14. Munns, R. (2002). Comparative physiology of salt and water stress. Plant Cell Environ. 25, 239–250. 15. Munns, R., and Tester, M. (2008). Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651–681. 16. Roy, S.J., Negra˜o, S., and Tester, M. (2014). Salt resistant crop plants. Curr. Opin. Biotechnol. 26, 115–124. 17. Brumo´s, J., Colmenero-Flores, J.M., Conesa, A., Izquierdo, P., Sa´nchez, G., Iglesias, D.J., Lo´pez-Climent, M.F., Go´mez-Cadenas, A., and Talo´n, M. (2009). Membrane transporters and carbon metabolism implicated in chloride homeostasis differentiate salt stress responses in tolerant and sensitive Citrus rootstocks. Funct. Integr. Genomics 9, 293–309. 18. Go´mez-Cadenas, A., Iglesias, D.J., Arbona, V., Colmenero-Flores, J.M., Primo-Millo, E., and Talo´n, M. (2003). Physiological and molecular responses of citrus to salinity. Recent Res. Dev. Plant Mol. Biol. 1, 281–298. 19. Storey, R., and Walker, R.R. (1999). Citrus and salinity. Sci. Hortic. (Amsterdam) 78, 39–81. 20. Tester, M., and Davenport, R. (2003). Na+ tolerance and Na+ transport in higher plants. Ann. Bot. (Lond.) 91, 503–527.

2. Ko¨hler, B., and Raschke, K. (2000). The delivery of salts to the xylem. Three types of anion conductance in the plasmalemma of the xylem parenchyma of roots of barley. Plant Physiol. 122, 243–254.

21. Roberts, S.K. (2006). Plasma membrane anion channels in higher plants and their putative functions in roots. New Phytol. 169, 647–666.

3. Ko¨hler, B., Wegner, L.H., Osipov, V., and Raschke, K. (2002). Loading of nitrate into the xylem: apoplastic nitrate controls the voltage dependence of X-QUAC, the main anion conductance in xylem-parenchyma cells of barley roots. Plant J. 30, 133–142.

22. Negi, J., Matsuda, O., Nagasawa, T., Oba, Y., Takahashi, H., KawaiYamada, M., Uchimiya, H., Hashimoto, M., and Iba, K. (2008). CO2 regulator SLAC1 and its homologues are essential for anion homeostasis in plant cells. Nature 452, 483–486.

4. Li, B., Byrt, C., Qiu, J., Baumann, U., Hrmova, M., Evrard, A., Johnson, A.A.T., Birnbaum, K.D., Mayo, G.M., Jha, D., et al. (2016). Identification of a stelar-localized transport protein that facilitates root-to-shoot transfer of chloride in Arabidopsis. Plant Physiol. 170, 1014–1029.

23. Vahisalu, T., Kollist, H., Wang, Y.F., Nishimura, N., Chan, W.Y., Valerio, G., Lamminma¨ki, A., Brosche´, M., Moldau, H., Desikan, R., et al. (2008). SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling. Nature 452, 487–491.

5. Lin, S.H., Kuo, H.F., Canivenc, G., Lin, C.S., Lepetit, M., Hsu, P.K., Tillard, P., Lin, H.L., Wang, Y.Y., Tsai, C.B., et al. (2008). Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell 20, 2514–2528.

24. Brandt, B., Brodsky, D.E., Xue, S., Negi, J., Iba, K., Kangasja¨rvi, J., Ghassemian, M., Stephan, A.B., Hu, H., and Schroeder, J.I. (2012). Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proc. Natl. Acad. Sci. USA 109, 10593–10598.

6. Taochy, C., Gaillard, I., Ipotesi, E., Oomen, R., Leonhardt, N., Zimmermann, S., Peltier, J.B., Szponarski, W., Simonneau, T., Sentenac, H., et al. (2015). The Arabidopsis root stele transporter NPF2.3 contributes to nitrate translocation to shoots under salt stress. Plant J. 83, 466–479.

25. Brandt, B., Munemasa, S., Wang, C., Nguyen, D., Yong, T., Yang, P.G., Poretsky, E., Belknap, T.F., Waadt, R., Aleman, F., et al. (2015). Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells. eLife 4, e03599.

Current Biology 26, 1–8, August 22, 2016 7

Please cite this article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045

26. Geiger, D., Scherzer, S., Mumm, P., Marten, I., Ache, P., Matschi, S., Liese, A., Wellmann, C., Al-Rasheid, K.A.S., Grill, E., et al. (2010). Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities. Proc. Natl. Acad. Sci. USA 107, 8023–8028. 27. Geiger, D., Scherzer, S., Mumm, P., Stange, A., Marten, I., Bauer, H., Ache, P., Matschi, S., Liese, A., Al-Rasheid, K.A.S., et al. (2009). Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proc. Natl. Acad. Sci. USA 106, 21425–21430. 28. Maierhofer, T., Lind, C., Hu¨ttl, S., Scherzer, S., Papenfuß, M., Simon, J., Al-Rasheid, K.A.S., Ache, P., Rennenberg, H., Hedrich, R., et al. (2014). A single-pore residue renders the Arabidopsis root anion channel SLAH2 highly nitrate selective. Plant Cell 26, 2554–2567. 29. Lee, S.C., Lan, W., Buchanan, B.B., and Luan, S. (2009). A protein kinasephosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells. Proc. Natl. Acad. Sci. USA 106, 21419–21424. 30. Zheng, X., He, K., Kleist, T., Chen, F., and Luan, S. (2015). Anion channel SLAH3 functions in nitrate-dependent alleviation of ammonium toxicity in Arabidopsis. Plant Cell Environ. 38, 474–486. 31. Gaymard, F., Pilot, G., Lacombe, B., Bouchez, D., Bruneau, D., Boucherez, J., Michaux-Ferrie`re, N., Thibaud, J.B., and Sentenac, H. (1998). Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap. Cell 94, 647–655.

R.H. Loeppert, eds. (Soil Science Society of America, American Society of Agronomy), pp. 833–867. 34. Colmenero-Flores, J.M., Campos, F., Garciarrubio, A., and Covarrubias, A.A. (1997). Characterization of Phaseolus vulgaris cDNA clones responsive to water deficit: identification of a novel late embryogenesis abundant-like protein. Plant Mol. Biol. 35, 393–405. 35. Chen, Y.H., Hu, L., Punta, M., Bruni, R., Hillerich, B., Kloss, B., Rost, B., Love, J., Siegelbaum, S.A., and Hendrickson, W.A. (2010). Homologue structure of the SLAC1 anion channel for closing stomata in leaves. Nature 467, 1074–1080. 36. Bayle, V., Nussaume, L., and Bhat, R.A. (2008). Combination of novel green fluorescent protein mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay. Plant Physiol. 148, 51–60. 37. Singh, S., Katzer, K., Lambert, J., Cerri, M., and Parniske, M. (2014). CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development. Cell Host Microbe 15, 139–152. 38. Bemm, F., Becker, D., Larisch, C., Kreuzer, I., Escalante-Perez, M., Schulze, W.X., Ankenbrand, M., Van de Weyer, A.L., Krol, E., Al-Rasheid, K.A., et al. (2016). Venus flytrap carnivorous lifestyle builds on herbivore defense strategies. Genome Res. 26, 812–825.

32. Cataldo, D.A., Maroon, M., Schrader, L.E., and Youngs, V.L. (1975). Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid 1. Commun. Soil Sci. Plant Anal. 6, 71–80.

39. Philippar, K., Fuchs, I., Luthen, H., Hoth, S., Bauer, C.S., Haga, K., Thiel, G., Ljung, K., Sandberg, G., Bottger, M., et al. (1999). Auxin-induced K+ channel expression represents an essential step in coleoptile growth and gravitropism. Proc. Natl. Acad. Sci. USA 96, 12186–12191.

33. Frankenberger, W.T., Jr., Tabatabai, M.A., Adriano, D.C., and Doner, H. (1996). Bromine, chlorine, and fluorine. In Methods of Soil Analysis, Part 3: Chemical Methods, D.L. Sparks, A.L. Page, P.A. Helmke, and

40. Sano, T., Becker, D., Ivashikina, N., Wegner, L.H., Zimmermann, U., Roelfsema, M.R., Nagata, T., and Hedrich, R. (2007). Plant cells must pass a K+ threshold to re-enter the cell cycle. Plant J. 50, 401–413.

8 Current Biology 26, 1–8, August 22, 2016