A dual role for the OsK5.2 ion channel in stomatal
movements and K+ loading into xylem sap.

Thanh Hao Nguyen, Shouguang Huang, Donaldo Meynard, Christian Chaine,
Rémy Michel, Rob Roelfsema, Emmanuel Guiderdoni, Herve Sentenac,
Anne-Aliénor Véry

<figure><img src="image_1.png" /></figure>

To cite this version:

Thanh Hao Nguyen, Shouguang Huang, Donaldo Meynard, Christian Chaine, Rémy Michel, et al. A dual role for the OsK5.2 ion channel in stomatal movements and K+ loading into xylem sap. Plant Physiology, 2017, 174 (4), pp.2409-2418. 10.1104/pp.17.00691. hal-01594830

HAL Id: hal-01594830

https://hal.science/hal-01594830

Submitted on 26 May 2020

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
1 **Short title:** OsK5.2 outward Shaker channel in rice stomata

2 **Corresponding author(s) details:** Anne-Aliénor VERY

3 Biochimie et Physiologie Moléculaire des Plantes, UMR 5004 CNRS/ 386 INRA/SupAgro-

4 M/UM, Campus SupAgro-M/INRA, 2 place Viala, 34060 Montpellier Cedex 2, France.

5 Phone: +33 499 612574 ; E-mail: very@supagro.inra.fr

6
7 A dual role for the OsK5.2 ion channel in stomatal movements and K+ loading into xylem sap
8

9
10 Thanh Hao Nguyen¹, Shouguang Huang², Donaldo Meynard³, Christian Chaine³, Rémy Michel³,
11 M Rob G Roelfsema², Emmanuel Guiderdoni³, Hervé Sentenac¹, Anne-Aliénor Véry¹,*

12 ¹Biochimie & Physiologie Moléculaire des Plantes, UMR 5004 CNRS/386 INRA/SupAgro
13 Montpellier/Université Montpellier, Campus SupAgro-INRA, 34060 Montpellier Cedex 2,
14 France

15 ²Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Biosciences,
16 Biocenter, University of Würzburg, Julius-von-Sachs-Platz 2, Würzburg D-97082, Germany

17 ³CIRAD, UMR AGAP, 34398 Montpellier Cedex 5, France

18
19 **One sentence summary:** The rice K+ channel OsK5.2 is necessary for rapid stomatal closure
20 and contributes to shoot K+ supply from the root, thus highlighting the differing functional
21 distribution of Shaker channels compared to Arabidopsis

22
23
24
25 **Footnotes:**

Footnotes:
26 **List of author contributions:** A.-A.V., H.S. and T.H.N. conceived the original research plans;

27 A.-A.V., E.G. and M.R.G.R. supervised the experiments; D.M., M.R.G.R., S.H. and T.H.N.

28 performed most of the experiments; C.C. and R.M. provided technical assistance to T.H.N.;

29 T.H.N. designed the experiments and analyzed the data; A.-A.V., H.S. and T.H.N. conceived the

30 project and wrote the article with contributions of all the authors; A.-A.V. and H.S. supervised

31 the writing.

32
33 **Funding information:** This work was supported in part by a doctoral fellowship from the French

34 Embassy in Vietnam (to TH.N.), by an ANR IA grant (ANR-11-RSNR-0005) (to E.G. and H.S.),

35 grants of the German Science Foundation to M.R.G.R. and a grant of the China Scholarship

36 Council to S.H.

37 **Corresponding author email:** very@supagro.inra.fr

38 The author responsible for distribution of materials integral to the findings presented in this

39 article in accordance with the policy described in the Instructions for Authors

40 (www.plantphysiol.org) is: Anne-Aliénor Véry (very@supagro.inra.fr).

41

42

43

44

45

46

47

48

49

50
51

52

53

54

ABSTRACT

55

56

57

58

59

60

61

62

63

64

65

66

67

68

The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis, but corresponding information is lacking outside this model species. Rice and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K⁺ channel gene OsK5.2.

Expression of the *OsK5.2* gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), in the root vasculature and root cortex. In stomata, loss of *OsK5.2* functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K⁺ translocation from the root system towards the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice.

69

70

INTRODUCTION

71

72

73

74

75

76

77

78

Since a waxy cuticle covers outer leaf tissues, water vapor diffusion into the atmosphere occurs mainly through the stomatal pores at the leaf surface. The size of the stomatal aperture is tightly regulated to optimize gas exchanges between the leaf inner tissues and the atmosphere, including CO₂ intake for photosynthesis and water loss by transpiration (Lawson and Blatt, 2014). This is achieved by fine tuning of the turgor pressure of the two guard cells which surround the stomatal pore, and involves a complex coordinated activity of transport systems at the guard cell plasma membrane and vacuolar membrane (Hedrich, 2012; Chen et al., 2012; Hills et al., 2012; Kollist et al., 2014). This control also affects long distance transport of mineral
nutrients from the roots, which take up these nutrients, to the aerial parts, to support plant growth
(Marschner et al., 1996).

Potassium ion ($K^+$), as a major inorganic constituent of the plant cells and the most abundant cation in the cytosol, is an essential macronutrient for growth and development. It is involved in various functions including electrical neutralization of negative charges, control of cell membrane polarization and osmoregulation (Clarkson and Hanson, 1980; Leigh and Wyn Jones, 1984). $K^+$ is thus the main cation absorbed by the roots and circulating within the plant at the cellular or long distance levels. In guard cells, it is well known as a major contributor, with $Cl^-$, $NO_3^-$ and malate, to the osmolarity (Raschke and Schnabl, 1978; Willner and Fricker, 1996). Stomatal opening is initiated by activation of plasma membrane proton pumps in guard cells, which promotes $K^+$ influx through voltage-gated inward $K^+$ channels, as well as anion uptake through $H^+$-anion symporters (Blatt, 1987a; Schroeder et al., 1987; Roelfsema and Prins, 1997; Talbott and Zeiger, 1998; Guo et al., 2003; Jezek and Blatt, 2017). Conversely, stomatal closure requires inhibition of proton pumping at the guard cell membrane and activation of both anion channels and voltage-gated outward $K^+$ channels.

The molecular mechanisms responsible for inward and outward $K^+$ fluxes across the plasma membrane have been extensively investigated in Arabidopsis. Shaker channel subunits, present as a 9-member family in Arabidopsis, have been shown to form the major pathways for these fluxes throughout the plant (Véry and Sentenac, 2003). In the Arabidopsis model species, four genes encoding Shaker channel subunits have been identified as playing a major role in root to shoot $K^+$ translocation and in stomatal movements. The SKOR subunit, which is expressed in root pericycle and xylem parenchyma, forms outwardly-rectifying channels involved in $K^+$ secretion into the xylem sap (Gaymard et al., 1998). In stomata, the inward Shaker channel subunits KAT1 and KAT2 are involved in guard cell $K^+$ uptake, and the outward Shaker channel GORK mediates guard cell $K^+$ release (Ache et al., 2000; Pilot et al., 2001; Szyroki et al., 2001; Hosy et al., 2003; Lebaudy et al., 2008). Whereas these Shaker subunits have been deeply characterized in Arabidopsis and the Shaker family, as a whole, can be considered as the best characterized family of plant membrane transport systems, little information at the molecular genetic level is yet available on this family outside Arabidopsis.

The stomatal complex in rice (Oryza sativa L.), the present model cereal, is very different from that of Arabidopsis (Itoh et al., 2005; Franks and Farquhar, 2007; Roelfsema and Hedrich,
2009). In rice like in other cereals, it comprises two subsidiary cells in addition to the two guard cells. Substantial differences may thus exist between rice and Arabidopsis in stomatal functioning (Mumm et al., 2011). Rice possesses two putative outward Shaker channel subunits (Pilot et al., 2003; Véry et al., 2014), named OsK5.1 (or OsSKOR) and OsK5.2 (or OsGORK) (Pilot et al., 2003; Kim et al., 2015). OsK5.1 was reported to be mainly expressed in root vasculature, just as SKOR in Arabidopsis (Kim et al., 2015). In contrast, OsK5.2 was found to be expressed both in roots and shoots (Kim et al., 2015). The expression pattern of this gene at the tissue level has, however, not been described. Here, we investigate the expression pattern and role of this rice outward Shaker gene. OsK5.2 is shown to play two important roles in rice plants: it mediates K⁺ translocation into the xylem sap towards the shoots, and it is involved in K⁺ release from guard cells and stomatal movements.

121

122
123 RESULTS

124 The outward Shaker channel subfamily in Arabidopsis and rice

125 The Shaker group 5, which consists of the outward Shaker subunits, usually comprises 2 or 3
126 members in the plant genomes analyzed so far (Pilot et al., 2003; Véry et al., 2014). Figure 1
127 illustrates the phylogenetic relationships between the members of the Shaker group 5 present in
128 Arabidopsis and in 3 monocots, rice, maize (Zea mays) and Brachypodium distachyon. Based on
129 this phylogenetic tree, the rice OsK5.1 and OsK5.2 subunits appeared to belong to two different
130 subgroups within the Shaker group 5. The distance between these subgroups was larger than that
131 between GORK and SKOR from Arabidopsis.

132

133 Expression pattern of OsK5.2

134 Transgenic rice plants expressing GUS under the control of the OsK5.2 promoter region
135 (Fig. 2A) were generated to investigate the expression of this Shaker gene (Fig. 2, B-J). GUS
136 staining revealed that OsK5.2 was expressed in vascular tissue of root (Fig. 2, E-G), like OsK5.1
137 (Kim et al., 2015), as well as in the shoot vascular tissue (Fig. 2, B, D). In addition, the OsK5.2
138 promoter was active in root cortical cells (except at the root tip) (Fig. 2, E-G). Weak GUS
139 staining was also observed in flowers, in the pollen sacs and grains (Fig. 2, H-J). Analysis of
140 GUS staining in leaf epidermis revealed expression of OsK5.2 in stomata, the staining being
141 stronger in guard cells than in subsidiary cells (Fig. 2, B-D).

142 The following experiments were aimed at investigating the role of OsK5.2 in K⁺ translocation
143 towards the shoots and in stomatal movements.

144

145 Production of osk5.2 loss-of-function mutant plants

146 Genotyping experiments performed on the progeny of the mutant lines ASJA08 and ASHF06
147 (Supplemental Figure 1A) allowed us to identify osk5.2 homozygous mutant plants (Tos17
148 insertion alleles), as well as plants without insertion in the OsK5.2 gene, hereafter named wild-
149 type controls. The offspring of the selected plants (osk5.2 mutants and wild-type controls) was
150 used for in planta experiments. RT-PCR experiments on total RNA prepared from the osk5.2
<figure><img src="image_3.png" /></figure>

**Figure 1.** Phylogenetic analysis of outward Shaker channels in monocot species and *Arabidopsis thaliana*. The unrooted phylogenetic trees were generated with PhyML software (http://www.atgc-montpellier.fr/phyml/binaries.php) using the maximum-likelihood method and 1000 bootstrap replicates in Seaview application (http://doua.prabi.fr/software/seaview). The scale bar corresponds to a distance of 10 changes per 100 amino acid positions. The protein (GenBank) accession numbers are given in Supplemental Table S2. At, *Arabidopsis thaliana*; Bd, *Brachypodium distachyon*; Os, *Oryza sativa* (rice); Zm, *Zea mays* (maize).

151 mutants and the wild type control plants confirmed the loss of function of *OsK5.2* in the selected
152 *osk5.2* homozygous mutant lines (Supplemental Figure 1B).

153
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Expression pattern of *OsK5.2* outward Shaker channel gene in rice. A, *Promoter::GUS* construct (promoter region size: 2339 bp). B to J, GUS activity in transgenic rice plants, revealed by an overnight incubation in the presence of X-Gluc: leaf tissues (B), stomata (C), leaf cross-section (D), root (E), root tip (F), root cross-section (G), flower (H), anther (I), and pollen (J).</figcaption></figure>

154 Impaired $K^+$ transport into the xylem sap and $K^+$ accumulation in shoots in *osk5.2* mutant
155 plants
K⁺ was assayed in roots and shoots of 5 week-old plants hydroponically grown on standard Yoshida medium. Comparison of *osk5.2* mutant plants with the corresponding wild type plants revealed that the absence of *OsK5.2* functional expression did not affect K⁺ accumulation in roots, but resulted in significantly reduced K⁺ contents (by ~25%) in shoots (Fig. 3, A and B, left panels). The K⁺ concentration measurements, in xylem sap samples which were collected after leaf excision, revealed lower values, by 30 to 40%, in the mutant plants (Fig. 3, A and B, right panels). Thus, the whole set of data indicated that the absence of *OsK5.2* functional expression resulted in a decrease in K⁺ translocation from roots to shoots via the xylem sap.

**Increased transpirational water loss due to impaired stomatal movements in *osk5.2* mutant plants**

Water consumption (essentially due to transpirational water loss) was monitored in *osk5.2* mutant plants and the corresponding wild-type control plants hydroponically grown for 5 weeks on Yoshida medium using a multi-potometer (Supplemental Figure S2). Both mutant lines displayed larger steady state rates of transpirational water loss than the corresponding wild-type plants in the light, by about 25% (Fig. 4).

The transpiration rate is dependent on stomatal aperture (Pallas Jr, 1965; Hosy *et al.*, 2003). Light to dark transitions were used to provoke stomatal closure. The decrease in transpiration rate when light was switched off was slower in the mutant plants than in the corresponding wild-type control plants (Fig. 5, A and B, top panels), revealing a slower rate of dark-induced stomatal closure in absence of *OsK5.2* functional expression. Fitting the decrease in transpiration rates with exponential functions yielded a time constant of approximately 10 min for the wild-type control plants, and of 20-30 min for the mutants from the two *osk5.2* lines (Fig. 5, A and B, bottom panels).

The kinetics of the light-induced increase in transpiration rate was also studied (Fig. 6). In wild type plants, when light was switched on, the transpiration rate displayed a rapid increase, which was followed by a transient decrease. The *osk5.2* mutant plants also displayed a rapid increase in transpiration but the kinetics was slightly slower than in the wild type plants (Fig. 6, A and B, bottom panels), and no following transient decrease in transpiration rate was observed (Fig. 6).
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Tissue K⁺ contents and xylem sap K⁺ concentration in wild-type and *osk5.2* mutant plants. Five week-old plants were grown in standard Yoshida hydroponic solution. A and B, Root and shoot K⁺ contents (left) and xylem sap K⁺ concentration (right) in wild-type control (black bars) and *osk5.2* mutant (white bars) plants issued from the initial AJSA08 (A) or ASHF06 (B) lines. Assayed xylem sap exudates were collected in wild-type control and *osk5.2* mutant plants after 1-h exudation in the case of the plants issued from the AJSA08 line, and 2-h exudation in the case of the plants issued from the ASHF06 line. Data are means ± SE (n = 3 or 4 for wild-type control plants, and 4 or 5 for *osk5.2* mutant plants in A, and n = 4 or 6 for wild-type control plants, and 5 or 6 for *osk5.2* mutant plants in B). An asterisk indicates that the corresponding difference in K⁺ content or concentration between the wild-type control and *osk5.2* mutant plants were statistically significant (Student's t test, P < 0.05).</figcaption></figure>

186

187 Absence of Shaker-type outwardly rectifying K⁺ channel activity in guard cells of *osk5.2*

188 mutant plants
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Steady-state transpiration rates in wild-type control and *osk5.2* mutant plants. Five week-old plants issued from the initial ASJA08 line (above) or ASHF06 line (below) were used for steady-state transpiration rate analyses in dark (ca. 5 h after light was switched off) and in light (ca. 3 h after light was switched on) conditions. The steady-state transpiration rate (in mL/cm²/h) was determined by dividing the average speed of water loss at steady-state (mean of 3 values) by the total area of the plant aerial parts. Black and white bars represent wild-type control and *osk5.2* mutant plants, respectively. Data are means ± SE; *n* = 6 for wild-type control and 13 for *osk5.2* mutant plants in the case of the plants issued from the ASJA08 line, and *n* = 7 for wild-type control and 9 for *osk5.2* plant plants in the case of the plants issued from the ASHF06 line. An asterisk indicates that the corresponding difference between the wild-type control and mutant plants was statistically significant (Student's t test, P < 0.05).</figcaption></figure>

The consequences of *OsK5.2* loss of function mutation on the K⁺ conductance of the guard cell membrane were analyzed *in planta*. Guard cells from two-week-old plants were impaled with double-barreled electrodes (Blatt, 1990; Thiel et al., 1992; Roelfsema et al., 2001) and inward
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 5.** Kinetics of the decrease in transpiration rate upon dark-induced stomatal closure in wild-type and *osk5.2* mutant plants. A and B, Kinetics (top) and time constant (bottom) of the dark-induced decrease in transpiration rate in wild-type control (●,■) and *osk5.2* mutant (○,□) plants issued from the initial ASJA08 (A) or ASHF06 (B) lines in standard Yoshida hydroponic solution. The light was switched off after 30 min of recording under light condition as indicated by the change from white to black in the bar below the graph. Transpiration rates were normalized by dividing absolute transpiration rates by the steady-state transpiration rate in light condition, and are presented as percentages (%). Time constants were derived by fitting the experimental dark-induced transpiration rate decrease kinetics with an exponential function (least-squares fitting). Black and white bars correspond to wild-type control and *osk5.2* mutant plants, respectively. Data are means ± SE; *n* = 6 for wild-type control and 13 for *osk5.2* mutant plants in A, and *n* = 7 for wild-type control and 9 for *osk5.2* mutant plants in B. The asterisks indicate that the corresponding differences between the wild-type control and mutant plants were statistically significant (Student's t test, P < 0.05).</figcaption></figure>

192 and outward currents through the membrane were elicited by voltage clamp pulses (Fig. 7A). In 193 wild-type guard cells, depolarizing voltage pulses elicited slowly activating outward currents 194 (Fig. 7B, left panel, and Fig. 7C), clearly reminiscent of those mediated by the outwardly-
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Kinetics of the increase in transpiration rate upon light-induced stomatal opening after a long period of darkness in wild-type and *osk5.2* mutant plants. A and B, Kinetics of light-induced increase in transpiration rate (top) and time of the corresponding half-increase in transpiration rate (bottom) in wild-type control (●, ■) and *osk5.2* mutant (○, □) plants from the initial ASJA08 (A) and ASHF06 (B) lines in standard Yoshida hydroponic solution. The change from black to white in the bar below the curves indicates the time at which light was switched on after the dark period. The normalized transpiration rate (%) was determined as described in Figure 5. Times of half light-induced increase in transpiration rate were derived from the fit of the experimental data up to the end of the initial rapid phase of light-induced transpiration rate increase to a sigmoidal equation (least-squares fitting). Data are means ± SE; *n* = 6 for wild-type control and 13 for *osk5.2* mutant plants in A, and *n* = 7 for wild-type control and 9 for *osk5.2* mutant plants in B). An asterisk indicates that the corresponding difference between the wild-type control and mutant plants in time of half-increase in transpiration rate was statistically significant (Student's t test, P < 0.05).</figcaption></figure>

rectifying K⁺ channels from the Shaker family (Gaymard et al., 1998; Hosy et al., 2003; Hedrich, 1996; 2012; Véry et al., 2014). Slowly activating outward currents were lacking in *osk5.2* mutant plants (Fig. 7, D and E). In contrast, the inward conductance appeared to be very similar in *osk5.2*.
<figure><img src="image_11.png" /><figcaption>B WT guard cells</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>D *osk5.2* guard cells</figcaption></figure>

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

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

<figure><img src="image_15.png" /><figcaption>**Figure 7.** Voltage-clamp experiments with guard cells in intact rice plants. A, Voltage-clamp protocols used to record either outward (left) or inward (right) currents in rice guard cells. Starting at a holding value of -100 mV, the voltage was shifted in 20 mV steps to either +20 mV, or -220 mV. B and D, Representative outward (left) and inward (right) current traces recorded in guard cells from ASJA08 line wild-type control (B) or *osk5.2* mutant (D) plants. C and E, Average current-voltage relationships obtained in wild-type control (C) or mutant (E) guard cells. Data are means ± SE (n = 6 in C, and n = 4 in E).</figcaption></figure>

mutant and wild type guard cells (Fig. 7, B and D, right panels). This suggested that, although *in planta* voltage-clamp analyses can give rise to experimental drawbacks (Blatt, 1987b; Roelfsema et al., 2001), comparison between wild type and mutant guard cell currents was valid in our
conditions, and thus that OsK5.2 encodes slowly-activating outwardly rectifying K⁺ channels in rice guard cells. It was also observed that the absence of OsK5.2 channel activity caused a positive shift of the free running membrane potential (cross point with X-axis in the current-voltage relationship), by about 50 mV (Fig. 7, C and E).

205

206
208 Comparison of the expression patterns of the outward Shaker genes in rice and Arabidopsis

209 In rice, as in Arabidopsis, the outward Shaker channel group comprises two members.
210 However, based on the phylogenetic tree displayed in Figure 1, OsK5.1 and OsK5.2 cannot be
211 simply considered as the orthologs of the Arabidopsis outward Shaker channels SKOR and
212 GORK. OsK5.2 and OsK5.1 belong to two separate subgroups within the group of outward
213 Shaker channels from cereal plant species (Fig. 1).
214

215 In Arabidopsis, *SKOR* has been reported to be essentially expressed in the root vasculature
216 (Gaymard et al. 1998), and *GORK* in root periphery cells (except in the root apex) and in stomata
217 (Ache et al., 2000; Ivashikina et al., 2001). The expression pattern of *OsK5.1* in rice is
218 reminiscent of that of *SKOR* in Arabidopsis, with strong expression in root vasculature (Kim et
219 al., 2015). In contrast, *OsK5.2* displays high expression levels in both the root vasculature (Fig. 2
220 E-G) and the stomata (Fig. 2 B-D), and thus its expression pattern clearly encompasses the
221 patterns reported for both *SKOR* and *GORK* in Arabidopsis (Gaymard et al., 1998; Ache et al.,
222 2000; Ivashikina et al., 2001). It is not yet known whether the expression patterns of *OsK5.1* and
223 *OsK5.2* are shared by genes belonging to the same outward Shaker subgroups from other
224 monocots. The expression pattern of only one maize gene from *OsK5.2* subgroup has been
225 partially characterized so far, by qRT-PCR analyses (Büchsenschütz et al., 2005). Like *OsK5.2*,
226 this maize outward Shaker gene is expressed in both stomata and roots, and thus its expression
227 pattern may be similar to that of *OsK5.2*.
228

228 Functional properties of OsK5.2 channels

229 Members of Shaker group 5 have been cloned in several plant species and successfully
230 expressed in heterologous systems (*Xenopus* oocyte essentially) for electrophysiological analysis
231 of their functional properties (Véry et al., 2014). They all behave as slowly-activating outwardly-
232 rectifying K⁺-selective channels. However, so far, all the channels for which such functional
233 analyses have been reported belong to dicotyledonous species. We attempted to express OsK5.2
234 in *Xenopus* oocytes, but no exogenous current in the oocytes was detected using the classical
235 two-electrode voltage-clamp technique (Véry et al., 1995; Tounsi et al., 2016). This might
indicate that these outward $K^+$ channels have to be co-expressed with regulatory protein partners to be active at the cell membrane, as reported for number of members of the inwardly rectifying Shaker channel group 1 from dicots (Xu et al., 2006; Honsbein et al., 2009; Cuéllar et al., 2010; Cuéllar et al., 2013) and monocots (Boscari et al., 2009; Geiger et al., 2009).

Our voltage-clamp measurements with intact guard cells from wild type plants and *osk5.2* mutants (Fig. 7) indicate that OsK5.2 displays functional features reminiscent of group 5 Shaker channels as determined upon expression in heterologous systems (Ache et al., 2000; Langer et al., 2002; Johansson et al., 2006; Sano et al., 2007), especially a strong outward rectification, a slow activation upon membrane depolarization and slow deactivation of the so-called tail currents when the membrane is repolarized. Finally, the fact that these Shaker-type outward currents were no longer observed in guard cells from the *osk5.2* mutants indicates that OsK5.2 is the major contributor to the expression of such a channel activity at the guard cell membrane.

## Comparison of the roles of OsK5.2 and SKOR in $K^+$ translocation towards the shoots

Both ASJA08 and ASHF06 *osk5.2* loss-of-function mutations caused reduced leaf $K^+$ contents, by 21 to 24% (Fig. 3). Taking into account that *OsK5.2* displays expression in root vascular tissues, the simplest explanation is that *OsK5.2*-encoded channel activity contributes to $K^+$ secretion into the xylem sap in roots, like SKOR in Arabidopsis, and thereby in $K^+$ translocation towards the shoots. This hypothesis received support from the assay of $K^+$ in xylem sap exudates from excised roots (Fig. 3), which revealed a marked concentration decrease (by >30%) in the *osk5.2* loss-of-function mutants.

In Arabidopsis, the absence of SKOR channel activity due to a loss-of-function mutation has been shown to lead to a decrease in the concentration of $K^+$ in the xylem sap by about 40%, and to a decrease in the accumulation of $K^+$ in shoots by about 50% (Gaymard et al., 1998). Thus, the contribution of SKOR to the control of $K^+$ translocation and accumulation in leaves seems a bit higher than that of OsK5.2 in rice. Since *SKOR* is the only outward Shaker channel gene expressed in the root stele, $K^+$-permeable channels belonging to other families and/or other types of transport systems (e.g., from the KUP/HAK family; Han et al., 2016), have to significantly contribute to $K^+$ secretion into the xylem sap in Arabidopsis. The situation may be different in rice, where both outward Shaker channel genes *OsK5.1* and *OsK5.2* are expressed in the plant
vasculature (Fig. 2; Kim et al., 2015). In other words, besides OsK5.2, another outward Shaker channel, OsK5.1, could contribute to K⁺ translocation towards the shoots.

## Comparison of the roles of OsK5.2 and GORK in control of stomatal aperture and transpirational water loss

Altogether, the present data indicate that OsK5.2 enhances the rate of stomatal closure by mediating K⁺ efflux through the guard cell membrane, just as its counterpart GORK in Arabidopsis (Hosy et al., 2003). The kinetics of dark-induced decrease in transpiration rate indicated that stomata can close in absence of this outwardly rectifying Shaker-type channel activity in rice, like in Arabidopsis, but the closure kinetics is slowed down. It is likely that poorly selective cation channels (Hedrich, 2012) substitute for Shaker channels as K⁺ efflux systems, but stomatal closure is then less efficient. The defect in stomatal closure in *osk5.2* mutant plants is also likely to result from the large difference in free running membrane potential, by approximately 50 mV, between *osk5.2* and wild type guard cells (Fig. 7). Indeed, in *osk5.2* guard cells, the free running voltage across the plasma membrane was shifted towards the reversal potential of anion channels, which was approximately +25 mV (as in Kollist et al., 2014). Thus, the driving force for anion efflux (difference between the free running membrane potential and reversal potential of anion channels) was reduced in *osk5.2* guard cells. This is expected to cause a decrease in anion efflux, and thus in the rate of stomatal closure.

Besides its impact on the rate of dark-induced stomatal closure and steady-state stomatal aperture in darkness, the absence of Shaker-type outward K⁺ channel activity in rice is also likely to result in larger steady-state stomatal aperture in light, based on the observation that the steady state transpiration rate was increased (Fig. 4). This would mean that OsK5.2 outwardly-rectifying channel activity displays an inhibitory effect on steady-state stomatal aperture in light. The fact that the leaf K⁺ content was lower in the *osk5.2* mutant plants (Fig. 3) is likely to lead to a decrease in turgor of the leaf cells. A decrease in the turgor of epidermal and subsidiary cells more important than that of guard cells could result in reduced back-pressure on stomatal guard cells and, thus, in increased steady state stomatal aperture in the light. The impact of OsK5.2 activity on steady-state transpiration rate in light could be understood within the framework of this latter hypothesis.
296

297 **CONCLUSION**

298 By controlling the transpirational water loss at leaf surface, stomata also regulate the flux of
299 water from roots to shoots within the xylem vasculature, and thereby contribute to the regulation
300 of nutrient ion translocation towards the shoots. In Arabidopsis, various ion transport systems
301 play a role in both ion translocation into the xylem sap towards the shoots and ion transport
302 across the guard cell membrane and control of stomatal aperture. This is the case, for example, of
303 the anion channel SLAH3 (Geiger et al., 2011; Cubero-Font et al., 2016), the nitrate transporter
304 NRT1.1 (Guo et al., 2003; Wang et al., 2012) and the phosphate transporter PHO1 (Hamburger *et
305 al.*, 2002; Zimmerli *et al.*, 2012). In contrast, regarding K⁺, some kind of specialization has
306 occurred within the Shaker group 5 in Arabidopsis since K⁺ release from guard cells for stomatal
307 closure has been strictly assigned to GORK, and K⁺ secretion into the xylem sap to SKOR. In
308 rice, OsK5.2 contributes to both K⁺ release by guard cells for stomatal closure and K⁺
309 translocation towards the shoots. The physiological significance of such evolutionary differences
310 would be interesting to clarify. The organization and the roles of the Shaker family are strongly
311 conserved in plants, allowing one to assume functional equivalence between orthologs, but the
312 present data clearly indicate that the biological variability deserves to be investigated since
313 differences between plant species in the use of this gene family are likely to play a role in major
314 physiological functions and in adaptation to environmental constraints.
315

315

316 **MATERIALS AND METHODS**

317 **Plant material and growth conditions**

318 Rice Nipponbare cultivar (Oryza sativa L. var. Nipponbare) was used in all the experiments.
319 For transpiration analysis, seeds were germinated in Petri dishes containing deionized water.
320 One-week-old plants were then transferred onto Yoshida hydroponic solution in 10 L containers
321 (0.7 mM KNO3, 0.8 mM KH2PO4, 1.2 mM Ca(NO3)2, 0.5 mM (NH4)2SO4, 1.6 mM MgSO4, 60
322 µM Na2FeEDTA, 45 µM H3BO3, 20 µM MnSO4, 1.6 µM CuSO4, 1.4 µM ZnSO4, and 0.3 µM
323 (NH4)6Mo7O24., pH adjusted to 5.5). Plants were grown in growth chambers (day/night 12 h/12 h
324 photoperiod, 28 °C/25 °C, photon flux density 500 µmol.m⁻².s⁻¹, 80% relative humidity).
325

326

Construction of reporter gene constructs and transgenic plants

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

OsK5.2 promoter region (2339 bp) was amplified by PCR from rice Nipponbare genomic DNA using the primers listed in table S1. The PCR product was then cloned into pGemTeasy vector for sequencing before sub-cloning into destination vector. The promoter was then transferred upstream the GUS reporter gene in pCAMBIA1301 vector, where it replaced CAMV35S promoter. The resulting plasmid was introduced into Agrobacterium tumefaciens EHA105 strain for rice transformation, as described by Sallaud et al. (2002). Briefly, dehulked rice seeds were rinsed in ethanol 70% for 90 seconds and treated with disinfection solution (50 g/L of sodium hypochlorite) for 30 minutes. After sterilization, the seeds were transferred to a callus induction medium leading to calluses development from the scutellum region. Rice grains with developing calluses were incubated with bacteria suspension for 90 to 180 seconds and co-cultured during 3 days. After the co-culture period, developing calluses were washed with carbenicillin (500 mg/L), separated, divided into small pieces and transferred to shoot induction medium containing hygromycin for transgenic tissue selection. Shoots regenerated from calluses were separated and transferred to rooting medium. Four-week-old plantlets were transferred to soil and grown in greenhouse for multiplication and selection of homozygous plants in the next generation using antibiotics leaf painting method (Cotsaftis et al., 2002).

345

346

347

348

349

350

351

352

Histochemical analysis of GUS activity

Leaf samples of 4 week-old plants and flowers were collected from homozygous plants grown in a greenhouse. Root samples were collected from 1 week-old plants grown in standard Yoshida hydroponic medium. All samples were submerged in GUS solution (50 mM Phosphate Buffer pH 7, 2.5 mM ferricyanide, 2.5 mM ferrocyanide, 0.05% v/v Triton X100 and 1 mM X-Gluc) contained in a 24-well plate, the leaves having been previously quickly divided into 1x1 cm pieces. The plate was placed under vacuum for 30 minutes to facilitate the penetration of GUS solution into the tissues, then the samples were incubated overnight at 37°C. After the incubation step, green samples were washed with 70% ethanol solution to remove chlorophyll. Leaf samples
353 were furthermore treated overnight with chloral hydrate (2.5 mg/L in 30% glycerol) before being
354 observed under microscope (BH2, Olympus) for GUS activity detection.

355 Thin (8-µm thick) sections were prepared, using a RM 2165 microtome (Leica, Germany),
356 from GUS-stained fixed samples (pre-fixation in 50 mM phosphate buffer pH 7, 1.5%
357 formaldehyde, 0.05% Triton X100; fixation in 75 mM phosphate buffer, 2% paraformaldehyde,
358 0.5% glutaraldehyde) embedded in Technovit 7100 resin (Kulzer, Wehrheim, Germany).

359
360 Selection of KO mutant and wild type control plants

361 *Tos17* insertion mutant lines (cv Nipponbare background) displaying insertion in *OsK5.2* gene
362 were ordered from OTL collection (CIRAD-Genoscope, France; www.oryzatagline.cirad.fr;
363 Piffanelli et al., 2007). Homozygous mutant plants as well as “wild-type” plants for *OsK5.2* gene
364 were selected by PCR on genomic DNA extracted from plantlets issued from received seeds,
365 using Phire® Plant Direct PCR Kit (Thermo Fischer Scientific) and specific primers hybridizing
366 on the target gene and on the *Tos17*. Total RNA of 2 plants selected in the progeny of a
367 homozygous mutant, or of a plant displaying a wild-type genotype for *OsK5.2* (hereafter
368 generically named wild-type control plants), were isolated using RNeasy kit (QIAGEN) and used
369 as templates to synthesize total cDNA. PCR experiments, using primers hybridizing downstream
370 from the *Tos17* insertion sites (listed in Supplemental Table S1), allowed us to check the
371 presence (in the wild-type control plants) or absence (in the *osk5.2* mutants) of *OsK5.2*
372 transcripts.

373
374 Voltage-clamp recordings on intact plant guard cells

375 Two-electrode voltage-clamp experiments were performed on intact guard cells (Roelfsema et
376 al., 2001; Mumm et al., 2011) of intact plants grown on standard Yoshida hydroponic solution for
377 two weeks. A plant (wild type control or homozygous *osk5.2* mutant) was transferred into a 15
378 mL tube (Falcon type) containing about 5 mL of standard Yoshida solution, the tube being
379 inclined so that the root system was fully immersed in the solution. Outside of the tube, the
380 youngest fully developed leaf was attached to a Plexiglas block with double-sided adhesive tape,
381 the plant remaining intact. The whole plant, attached with its leaf to the plexiglass, was carefully
mounted onto the table of an upright microscope (Axoiskop 2FS, Zeiss). About 0.5 to 1 mL of bath solution (5 mM potassium citrate, 5 mM KCl, 0.1 mM $CaCl_2$, pH 5.0) was placed between the leaf and the water immersion objective (Achroplan 40/0.80W, Zeiss), used to visualize the stomata. Stomatal opening was induced by light provided by the microscope lamp. Guard cells were impaled using double-barreled electrodes prepared from 1-mm-diameter glass capillaries (1403547, Hilgenberg, Germany), filled with 300 mM KCl. A glass capillary filled with 300 mM KCl and plugged with 300 mM KCl in 2% agarose was placed in the drop of solution between the leaf and the objective and served as a reference electrode. The series resistance present between leaf apoplast and reference electrode was estimated by measuring the resistance between two drops of bath solution placed on the leaf surface, at a distance of approximately 1 cm. This approach revealed a series resistance (which was not different between wild type and *osk5.2* mutant plants) of 1.3 ± 0.1 (n=5) MOhm at the abaxial leaf face. With guard cell currents lower than 1 nA (Fig. 7), such a series resistance leads to an overestimation of the applied voltage of less than 2 mV. The potential difference between the bath solution and guard cell apoplast ("surface" potential) was -10 ± 2 mV (n = 6). Because the series resistance and potential difference between the external solution and the apoplast appeared as low, no attempt was made to correct their impact on current-voltage plots. The Win WCP V5.1.6 software (J. Dempster, Strathclyde University, Glasgow, UK) was used to setup the protocol and record applied voltage-and current traces, using an ITC-16 interface (Instrutech Corp., Elmont, NY) and a GeneClamp 500 (Axon Instruments) amplifier.

402

## Analysis of stomatal movements induced by light or darkness

Wild type control and homozygous *osk5.2* mutant plants were grown in parallel in standard Yoshida hydroponic medium for five weeks. One day before the experiment, plants were transferred into a multi-potometer device (Supplemental Figure S2), the root system bathed in standard Yoshida hydroponic solution. On the day of the experiment, transpiration rates started to be recorded after 2 h under light condition. A dark treatment was imposed after 30 minutes of recording and was maintained for 4 h before switching back to light condition.

410
411 **Analysis of K⁺ content in tissues and xylem sap**

412 Root and shoot K⁺ contents were assayed in plants grown as described above for the analysis
413 of stomatal movements, i.e., hydroponic conditions for 5 weeks on standard Yoshida solution.
414 After excision, roots and shoots were weighed, dried (60°C during 2 days) and weighed again.
415 Ions were extracted from the tissues with 0.1 N HCl during 2 days. K⁺ content in the extracts was
416 determined by flame spectrophotometry (SpectrAA 220FS, Varian).

417 K⁺ concentration in xylem sap was analyzed in 5 week-old plants grown in standard Yoshida
418 hydroponic solution. Excision at 1 cm above the crown allowed collection of the xylem sap.
419 Exudation duration before collection (identical in control and *osk5.2* mutant plants) lasted for 1 to
420 2 h. Collected samples were diluted in 0.1 N HCl and the concentration of K⁺ was determined
421 like that of tissue extracts.

422

423 **Accession numbers**

424 Sequence data for *OsK5.2* Shaker gene can be found in the GenBank library under accession
425 number Os06g0250600.

426

427 **ACKNOWLEDGMENTS**

428 We are grateful to Nathalie Leonhardt, Thierry Simonneau and Alain Vavasseur for fruitful
429 discussions, to Sonia Mohamed and Manuel Nieves-Cordones for help in genotyping, and to
430 Thanh Danh Nguyen, Thi Xuan Vo and Binh Anh Thu Nguyen for help in analysis of raw
431 transpiration rate data.

432

433 **FIGURE LEGENDS**

434 **Figure 1.** Phylogenetic analysis of outward Shaker channels in monocot species and *Arabidopsis*
435 *thaliana*. The unrooted phylogenetic trees were generated with PhyML software
436 (http://www.atgc-montpellier.fr/phyml/binaries.php) using the maximum-likelihood method and
437 1000 bootstrap replicates in Seaview application (http://doua.prabi.fr/software/seaview). The
scale bar corresponds to a distance of 10 changes per 100 amino acid positions. The protein
(GenBank) accession numbers are given in Supplemental Table S2. At, *Arabidopsis thaliana*; Bd,
*Brachypodium distachyon*; Os, *Oryza sativa* (rice); Zm, *Zea mays* (maize).

**Figure 2.** Expression pattern of OsK5.2 outward Shaker channel gene in rice. A, *Promoter::GUS* construct (promoter region size: 2339 bp). B to J, GUS activity in transgenic rice plants, revealed by an overnight incubation in the presence of X-Gluc: leaf tissues (B), stomata (C), leaf cross-section (D), root (E), root tip (F), root cross-section (G), flower (H), anther (I), and pollen (J).

**Figure 3.** Tissue K⁺ contents and xylem sap K⁺ concentration in wild-type and *osk5.2* mutant plants. Five week-old plants were grown in standard Yoshida hydroponic solution. A and B, Root and shoot K⁺ contents (left) and xylem sap K⁺ concentration (right) in wild-type control (black bars) and *osk5.2* mutant (white bars) plants issued from the initial AJSA08 (A) or ASHF06 (B) lines. Assayed xylem sap exudates were collected in wild-type control and *osk5.2* mutant plants after 1-h exudation in the case of the plants issued from the AJSA08 line, and 2-h exudation in the case of the plants issued from the ASHF06 line. Data are means ± SE (n = 3 or 4 for wild-type control plants, and 4 or 5 for *osk5.2* mutant plants in A, and n = 4 or 6 for wild-type control plants, and 5 or 6 for *osk5.2* mutant plants in B). An asterisk indicates that the corresponding difference in K⁺ content or concentration between the wild-type control and *osk5.2* mutant plants were statistically significant (Student's t test, P < 0.05).

**Figure 4.** Steady-state transpiration rates in wild-type control and *osk5.2* mutant plants. Five week-old plants issued from the initial ASJA08 line (above) or ASHF06 line (below) were used for steady-state transpiration rate analyses in dark (ca. 5 h after light was switched off) and in light (ca. 3 h after light was switched on) conditions. The steady-state transpiration rate (in mL/cm²/h) was determined by dividing the average speed of water loss at steady-state (mean of 3 values) by the total area of the plant aerial parts. Black and white bars represent wild-type control and *osk5.2* mutant plants, respectively. Data are means ± SE; n = 6 for wild-type control and 13 for *osk5.2* mutant plants in the case of the plants issued from the ASJA08 line, and n = 7 for wild-type control and 9 for *osk5.2* plant plants in the case of the plants issued from the ASHF06 line. An asterisk indicates that the corresponding difference between the wild-type control and mutant plants was statistically significant (Student's t test, P < 0.05).
**Figure 5.** Kinetics of the decrease in transpiration rate upon dark-induced stomatal closure in wild-type and *osk5.2* mutant plants. A and B, Kinetics (top) and time constant (bottom) of the dark-induced decrease in transpiration rate in wild-type control (●, ■) and *osk5.2* mutant (○, □) plants issued from the initial ASJA08 (A) or ASHF06 (B) lines in standard Yoshida hydroponic solution. The light was switched off after 30 min of recording under light condition as indicated by the change from white to black in the bar below the graph. Transpiration rates were normalized by dividing absolute transpiration rates by the steady-state transpiration rate in light condition, and are presented as percentages (%). Time constants were derived by fitting the experimental dark-induced transpiration rate decrease kinetics with an exponential function (least-squares fitting). Black and white bars correspond to wild-type control and *osk5.2* mutant plants, respectively. Data are means ± SE; *n* = 6 for wild-type control and 13 for *osk5.2* mutant plants in A, and *n* = 7 for wild-type control and 9 for *osk5.2* mutant plants in B. The asterisks indicate that the corresponding differences between the wild-type control and mutant plants were statistically significant (Student's t test, P < 0.05).

**Figure 6.** Kinetics of the increase in transpiration rate upon light-induced stomatal opening after a long period of darkness in wild-type and *osk5.2* mutant plants. A and B, Kinetics of light-induced increase in transpiration rate (top) and time of the corresponding half-increase in transpiration rate (bottom) in wild-type control (●, ■) and *osk5.2* mutant (○, □) plants from the initial ASJA08 (A) and ASHF06 (B) lines in standard Yoshida hydroponic solution. The change from black to white in the bar below the curves indicates the time at which light was switched on after the dark period. The normalized transpiration rate (%) was determined as described in Figure 5. Times of half light-induced increase in transpiration rate were derived from the fit of the experimental data up to the end of the initial rapid phase of light-induced transpiration rate increase to a sigmoidal equation (least-squares fitting). Data are means ± SE; *n* = 6 for wild-type control and 13 for *osk5.2* mutant plants in A, and *n* = 7 for wild-type control and 9 for *osk5.2* mutant plants in B). An asterisk indicates that the corresponding difference between the wild-type control and mutant plants in time of half-increase in transpiration rate was statistically significant (Student's t test, P < 0.05).

**Figure 7.** Voltage-clamp experiments with guard cells in intact rice plants. A, Voltage-clamp protocols used to record either outward (left) or inward (right) currents in rice guard cells.
Starting at a holding value of -100 mV, the voltage was shifted in 20 mV steps to either +20 mV, or -220 mV. B and D, Representative outward (left) and inward (right) current traces recorded in guard cells from ASJA08 line wild-type control (B) or *osk5.2* mutant (D) plants. C and E, Average current-voltage relationships obtained in wild-type control (C) or mutant (E) guard cells. Data are means ± SE (n = 6 in C, and n = 4 in E).

502

## 503 Supplemental Material

**504 Supplemental Figure S1.** Selection of knock-out mutant plants for *OsK5.2* gene.

**505 Supplemental Figure S2.** Potometer device. The multi-potometer system used for plant transpiration rate monitoring.

**507 Supplemental Table S1.** Primer sequences used for cloning, genotyping, and verification of KO *osk5.2* mutant plants.

**509 Supplemental Table S2.** GenBank accession numbers of Shaker polypeptides present in phylogenetic trees

511

**512 Supplemental Figure S1.** Selection of knock-out mutant plants for *OsK5.2* gene. A, *Tos17* insertion sites in the *osk5.2* mutant ASJA08 (FST accession CU316012) and ASHF06 (FST accession CU316066) lines. B, Lack of *OsK5.2* transcripts in homozygous *osk5.2* mutants issued from ASJA08 and ASHF06 lines. RT-PCR experiments were performed for *OsK5.2* and *eEF1-β* (internal control) using total mRNA isolated from roots of 5 week-old wild-type control (WT) or homozygous mutant (ho) plants issued from each insertion line.

518

**519 Supplemental Figure S2.** Potometer device. The multi-potometer system used for plant transpiration rate monitoring consisted of twenty 50 mL syringes (Terumo corporation, Philippines) vertically fixed into a polystyrene plaque laid over an empty container, and connected to graduated 1 mL serological plastic pipettes ("1 mL Stripette", Costar Corporation, Cambridge, MA) via thin silicone tubes. A camera allowed to record the water level in the
pipettes over time. Four to five week-old plants were inserted through the black rubber plunger of the syringes via a 4 mm hole at the plunger center, and sealed by dental polyvinylsiloxane-based elastomer (Coltène® PRESIDENT light body). After polymerization of the elastomer, the plungers were placed to the syringes filled with experimental solution (Yoshida medium). The pipettes and connecting tubes were filled with the same solution, ensuring that no air bubble was present in the system. The transpiration rate was calculated from the decrease in level of the solution in the pipettes recorded over time.

531

532 **Supplemental Table S1.** Primer sequences used for cloning, genotyping, and verification of KO
533 *osk5.2* mutant plants.

534

535 **Supplemental Table S2.** GenBank accession numbers of Shaker polypeptides present in
536 phylogenetic trees

537

538

539
## Parsed Citations

**Ache P, Becker D, Ivashikina N, Dietrich P, Roelfsema MRG, Hedrich R (2000) GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K+-selective, K+-sensing ion channel. FEBS lett 486: 93-98**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Boscari A, Clement M, Volkov V, Golldack D, Hybiak J, Miller AJ, Amtmann A, Fricke W (2009) Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development. Plant, cell & environ 32: 1761-1777**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Blatt MR (1987a) Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent "electrogenic" transport revealed by current-voltage and difference-current-voltage analysis. J Membr Biol 98: 257-74**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Blatt MR (1987b) Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chloride leakage from microelectrodes. Planta 170: 272-287**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Blatt MR (1990) Potassium channel currents in intact stomatal guard cells: rapid enhancement by abscisic acid. Planta 180: 445-455**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Büchsenschütz K, Marten I, Becker D, Philippar K, Ache P, Hedrich R (2005) Differential expression of K+ channels between guard cells and subsidiary cells within the maize stomatal complex. Planta 222: 968-976**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Chen Z-H, Hills A, Bätz U, Amtmann A, Lew VL, Blatt MR (2012) Systems dynamic modeling of the stomatal guard cell predicts emergent behaviors in transport, signaling, and volume control. Plant Physiol 159: 1235-1251**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Clarkson DT, Hanson JB (1980) The mineral nutrition of higher plants. Annu Rev Plant Physiol 31: 239-298**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Cotsaftis O, Sallaud C, Breitler JC, Meynard D, Greco R, Pereira A, Guiderdoni E (2002) Transposon-mediated generation of T-DNA-and marker-free rice plants expressing a Bt endotoxin gene. Mol Breeding 10: 165-180**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Cubero-Font P, Maierhofer T, Jaslan J, Rosales MA, Espartero J, Díaz-Rueda P, Müller HM, Hürter A-L, AL-Rasheid KA, Marten I (2016) Silent S-Type anion channel subunit SLAH1 gates SLAH3 open for chloride root-to-shoot translocation. Curr Biol 26: 2213-2220**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Cuéllar T, Azeem F, Andrianteranagna M, Pascaud F, Verdeil JL, Sentenac H, Zimmermann S, Gaillard I (2013) Potassium transport in developing fleshy fruits: the grapevine inward K+ channel VvK1. 2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells. Plant J 73: 1006-1018**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Cuéllar T, Pascaud F, Verdeil JL, Torregrosa L, Adam-Blondon AF, Thibaud JB, Sentenac H, Gaillard I (2010) A grapevine Shaker inward K+ channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions. Plant J 61: 58-69**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only Title Only Author and Title](#)

**Franks PJ, Farquhar GD (2007) The mechanical diversity of stomata and its significance in gas-exchange control. Plant physiol 143: 78-87**

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)
Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J, Michaux-Ferriere N, Thibaud J-B, 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

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Geiger D, Becker D, Vosloh D, Gambale F, Palme K, Rehers M, Anschuetz U, Dreyer I, Kudla J, Hedrich R (2009) Heteromeric AtKC1.AKT1 channels in Arabidopsis roots facilitate growth under K⁺-limiting conditions. *J Biol Chem* **284**: 21288-21295

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Geiger D, Maierhofer T, AL-Rasheid KA, Scherzer S, Mumm P, Liese A, Ache P, Wellmann C, Marten I, Grill E (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-ra32

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Guo F-Q, Young J, Crawford NM (2003) The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis. *Plant Cell* **15**: 107-117

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Hamburger D, Rezzonico E, Petétot JM-C, Somerville C, Poirier Y (2002) Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem. *Plant Cell* **14**: 889-902

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Han M, Wu W, Wu W-H, Wang Y (2016) Potassium transporter KUP7 is involved in K⁺ acquisition and translocation in Arabidopsis root under K⁺-limited conditions. *Mol Plant* **9**: 437-446

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Hedrich R (2012) Ion channels in plants. *Physiol Rev* **92**: 1777-1811

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Hills A, Chen Z-H, Amtmann A, Blatt MR, Lew VL (2012) OnGuard, a computational platform for quantitative kinetic modeling of guard cell physiology. *Plant physiol* **159**: 1026-1042

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Honsbein A, Sokolovski S, Grefen C, Campanoni P, Pratelli R, Paneque M, Chen Z, Johansson I, Blatt MR (2009) A tripartite SNARE-K⁺ channel complex mediates in channel-dependent K⁺ nutrition in Arabidopsis. *Plant Cell* **21**: 2859-2877

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Hosy E, Vavasseur A, Mouline K, Dreyer I, Gaymard F, Porée F, Boucherez J, Lebaudy A, Bouchez D, Véry A-A (2003) The Arabidopsis outward K⁺ channel GORK is involved in regulation of stomatal movements and plant transpiration. *Proc Natl Acad Sci USA* **100**: 5549-5554

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Itoh J-I, Nonomura K-I, Ikeda K, Yamaki S, Inukai Y, Yamagishi H, Kitano H, Nagato Y (2005) Rice plant development: from zygote to spikelet. *Plant Cell Physiol* **46**: 23-47

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Ivashikina N, Becker D, Ache P, Meyerhoff O, Felle HH, Hedrich R (2001) K⁺ channel profile and electrical properties of Arabidopsis root hairs. *FEBS lett* **508**: 463-469

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)

Jezek M, Blatt MR (2017) Guard cell photosynthesis is critical for stomatal turgor production, yet does not directly mediate CO₂- and ABA-induced stomatal closing. *Plant Physiol* **174**: 487-519

Pubmed: [Author and Title](#)

CrossRef: [Author and Title](#)

Google Scholar: [Author Only](#) [Title Only](#) [Author and Title](#)
Johansson I, Wulfetange K, Porée F, Michard E, Gajdanowicz P, Lacombe B, Sentenac H, Thibaud JB, Mueller-Roeber B, Blatt MR (2006) External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism. Plant J 46: 269-281

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Kim HY, Choi E-H, Min MK, Hwang H, Moon S-J, Yoon I, Byun M-O, Kim B-G (2015) Differential gene expression of two outward rectifying shaker-like potassium channels OsSKOR and OsGORK in rice. J Plant Biol 58: 230-235

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Kollist H, Nuhkat M, Roelfsema MRG (2014) Closing gaps: linking elements that control stomatal movement. New Phytol 203: 44-62

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Langer K, Ache P, Geiger D, Stinzing A, Arend M, Wind C, Regan S, Fromm J, Hedrich R (2002) Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis. Plant J 32: 997-1009

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Lawson T, Blatt MR (2014) Stomatal size, speed, and responsiveness impact on photosynthesis and water use efficiency. Plant physiol 164: 1556-1570

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Lebaudy A, Vavasseur A, Hosy E, Dreyer I, Leonhardt N, Thibaud J-B, Véry A-A, Simonneau T, Sentenac H (2008) Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels. Proc Natl Acad Sci USA 105: 5271-5276

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Leigh R, Wyn Jones R (1984) A hypothesis relating critical potassium concentrations for growth to the distribution and functions of this ion in the plant cell. New Phytol 97: 1-13

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Marschner H, Kirkby E, Cakmak I (1996) Effect of mineral nutritional status on shoot-root partitioning of photoassimilates and cycling of mineral nutrients. J Exp Bot 47: 1255-1263

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Mumm P, Wolf T, Fromm J, Roelfsema MRG, Marten I (2011) Cell type-specific regulation of ion channels within the maize stomatal complex. Plant Cell Physiol 52: 1365-1375

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Piffanelli P, Droc G, Mieulet D, Lanau N, Bes M, Bourgeois E, Rouvière C, Gavory F, Cruaud C, Ghesquière A, Guiderdoni E (2007) Large-scale characterization of Tos17 insertion sites in a rice T-DNA mutant library. Plant Mol Biol 65: 587-601. Pallas Jr JE (1965) Transpiration and stomatal opening with changes in carbon dioxide content of the air. Science 147: 171-173

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Pilot G, Lacombe B, Gaymard F, Cherel I, Boucherez J, Thibaud JB, Sentenac H (2001) Guard cell inward K+ channel activity in Arabidopsis involves expression of the twin channel subunits KAT1 and KAT2. J Biol Chem 276: 3215-3221

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Pilot G, Pratelli R, Gaymard F, Meyer Y, Sentenac H (2003) Five-group distribution of the Shaker-like K+ channel family in higher plants. J Mol Evol 56: 418-434

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]

Raschke K, Schnabl H (1978) Availability of chloride affects the balance between potassium chloride and potassium malate in guard cells of Vicia faba L. Plant Physiol 62: 84-87

Pubmed: [Author and Title]

CrossRef: [Author and Title]

Google Scholar: [Author Only Title Only Author and Title]
### Roelfsema MRG, Hedrich R (2009) Stomata. eLS

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1186/1748-7302-1-1)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1186/1748-7302-1-2)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Roelfsema MR, Prins HB (1997) Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh. Planta 202: 18-27

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-007-0018-6)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-007-0019-7)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Roelfsema MRG, Steinmeyer R, Staal M, Hedrich R (2001) Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane. Plant J 26: 1-13

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/S0029-1745(00)00012-6)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/S0029-1745(00)00013-5)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Sallaud C, Meynard D, Van Boxtel J, Gay C, Bes M, Brizard J-P, Larmande P, Ortega D, Raynal M, Portefaix M (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106: 1396-1408

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-003-0042-6)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-004-0046-8)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Sano T, Becker D, Ivashikina N, Wegner LH, Zimmermann U, Roelfsema MRG, Nagata T, Hedrich R (2007) Plant cells must pass a K+ threshold to re-enter the cell cycle. Plant J 50: 401-413

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/S0029-1745(06)00107-9)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/S0029-1745(06)00108-8)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Szyroki A, Ivashikina N, Dietrich P, Roelfsema MRG, Ache P, Reintanz B, Deeken R, Godde M, Felle H, Steinmeyer R (2001) KAT1 is not essential for stomatal opening. Proc Natl Acad Sci USA 98: 2917-2921

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1073/pnas.0103106100)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1073/pnas.0103106100-1)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Talbott L, Zeiger E (1998) The role of sucrose in guard cell osmoregulation. J Exp Bot 49: 329-337

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1093/jxb/98-3-337)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1093/jxb/98-3-338)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Thiel G, MacRobbie E, Blatt M (1992) Membrane transport in stomatal guard cells: the importance of voltage control. J Membr Biol 126: 1-18

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-002-0031-6)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-003-0032-7)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Tounsi S, Ben Amar S, Masmoudi K, Sentenac H, Brini F, Véry A-A (2016) Characterization of two HKT1; 4 transporters from Triticum monococcum to elucidate the determinants of the wheat salt tolerance Nax1 QTL. Plant Cell Physiol 57: 2047-2057

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-015-0013-6)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-015-0014-7)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Véry A-A, Nieves-Cordones M, Daly M, Khan I, Fizames C, Sentenac H (2014) Molecular biology of K+ transport across the plant cell membrane: what do we learn from comparison between plant species? J Plant Physiol 171: 748-769

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-015-0021-6)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1007/s00148-015-0022-7)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Very A-A, Sentenac H (2003) Molecular mechanisms and regulation of K+ transport in higher plants. Annu Rev Plant Biol 54: 575-603

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1146/annrdb/1003100)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1146/annrdb/1003101)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Véry A-A, Sentenac H (2002) Cation channels in the Arabidopsis plasma membrane. Trends Plant Sci 7: 168-175

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/S0029-1745(01)00017-9)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/S0029-1745(01)00018-8)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Véry AA, Gaymard F, Bosseux C, Sentenac H, Thibaud JB (1995) Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents. Plant J 7: 321-332

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/S0029-1745(94)00017-9)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/S0029-1745(94)00018-8)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

### Wang Y-Y, Hsu P-K, Tsay Y-F (2012) Uptake, allocation and signaling of nitrate. Trends Plant Sci 17: 458-467

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/j.tps.2011.11.001)

CrossRef: [Author and Title](https://www.ncbi.nlm.nih.gov/nlncs/doi/abs/10.1016/j.tps.2011.11.002)

Google Scholar: [Author Only Title Only](https://www.google.com/search?q=Author Only Title Only Author and Title)

Willmer C, Fricker M (1996) Topics in plant functional biology: 2- Stomata, M Black & B Charlwood eds (2nd ed), Chapman and Hall
publishers, London, UK, 375p
Xu J, Li H-D, Chen L-Q, Wang Y, Liu L-L, He L, Wu W-H (2006) A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell 125: 1347-1360

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlp/nqg/)

CrossRef: [Author and Title](https://wwwCrossRef.com)

Google Scholar: [Author Only](https://scholar.google.com) [Title Only](https://scholar.google.com/search?q=arabidopsis) [Author and Title](https://scholar.google.com/search?q=arabidopsis&hl=zh-TW)

Zimmerli C, Ribot C, Vavasseur A, Bauer H, Hedrich R, Poirier Y (2012) PHO1 expression in guard cells mediates the stomatal response to abscisic acid in Arabidopsis. Plant J 72: 199-211

Pubmed: [Author and Title](https://www.ncbi.nlm.nih.gov/nlp/nqg/)

CrossRef: [Author and Title](https://wwwCrossRef.com)

Google Scholar: [Author Only](https://scholar.google.com) [Title Only](https://scholar.google.com/search?q=arabidopsis) [Author and Title](https://scholar.google.com/search?q=arabidopsis&hl=zh-TW)