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Curr Biol
2018 May 07;289:1370-1379.e5. doi: 10.1016/j.cub.2018.03.027.
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A Tandem Amino Acid Residue Motif in Guard Cell SLAC1 Anion Channel of Grasses Allows for the Control of Stomatal Aperture by Nitrate.
Schäfer N
,
Maierhofer T
,
Herrmann J
,
Jørgensen ME
,
Lind C
,
von Meyer K
,
Lautner S
,
Fromm J
,
Felder M
,
Hetherington AM
,
Ache P
,
Geiger D
,
Hedrich R
.
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The latest major group of plants to evolve were the grasses. These became important in the mid-Paleogene about 40 million years ago. During evolution, leaf CO2 uptake and transpirational water loss were optimized by the acquisition of grass-specific stomatal complexes. In contrast to the kidney-shaped guard cells (GCs) typical of the dicots such as Arabidopsis, in the grasses and agronomically important cereals, the GCs are dumbbell shaped and are associated with morphologically distinct subsidiary cells (SCs). We studied the molecular basis of GC action in the major cereal crop barley. Upon feeding ABA to xylem sap of an intact barley leaf, stomata closed in a nitrate-dependent manner. This process was initiated by activation of GC SLAC-type anion channel currents. HvSLAC1 expressed in Xenopus oocytes gave rise to S-type anion currents that increased several-fold upon stimulation with >3 mM nitrate. We identified a tandem amino acid residue motif that within the SLAC1 channels differs fundamentally between monocots and dicots. When the motif of nitrate-insensitive dicot Arabidopsis SLAC1 was replaced by the monocot signature, AtSLAC1 converted into a grass-type like nitrate-sensitive channel. Our work reveals a fundamental difference between monocot and dicot GCs and prompts questions into the selective pressures during evolution that resulted in fundamental changes in the regulation of SLAC1 function.
Figure 1
ABA-Induced Stomatal Closure Requires NO3–
Barley stomatal movement measured by infrared gas exchange. Barley stomata were opened in the light (400 μE) at ambient CO2 (400 ppm).
(A) Excised leaves were supplied with water and ABA via the transpiration stream. Note, stomatal closure in the presence of ABA was remarkably slow and largely incomplete.
(B) Leaves were pre-incubated with 5 mM nitrate. Under this condition, leaves started to close their stomata 5 min after ABA application. This process was largely completed within 10 min and finished in less than 15 min. Data were normalized to their open value 10 min before the application of ABA (indicated by the arrow). n ≥ 5 means ± SE.
(C) EDX analysis of barley stomatal complexes. The respective changes in K (left) and Cl contents (right) are shown after the transition from open to closed stomata in guard cells (GC) and subsidiary cells (SC). During stomatal opening, potassium and chloride ions shuttle from SCs to GCs, while ions move in the opposite direction when stomata close. n = 11, means ± SE.
Figure 2
Nitrate-Dependent Activation of HvSLAC1
(A) Whole-oocyte currents of Xenopus oocytes expressing HvSLAC1 alone or co-expressing either AtOST1 or AtCPK6 were measured in response to the standard voltage protocol. Currents were recorded in standard buffers containing either 3 mM chloride, 30 mM chloride, or 30 mM nitrate. Representative cells of 2 independent experiments with n = 3 oocytes are shown.
(B) Whole-oocyte currents of oocytes expressing HvSLAC1 equipped with the C-terminal half of YFP (HvSLAC1:YC) either expressed alone or together with WT OST1, OST1 D140A, or WT OST1 and ABI1. Both OST1 versions were fused to the N-terminal half of YFP (OST1:YN, OST1 D140A:YN). Currents were recorded in nitrate-based buffers (30 mM). Representative cells are shown. Co-expression of HvSLAC1 and OST1 or OST1 D140A was confirmed by detection of YFP fluorescence. Quarter of representative oocytes of 2 independent experiments with n = 4 oocytes are shown.
(C) Statistical analysis of the steady-state currents at −100 mV derived from the experiment described in (B) (n = 4 experiments, mean ± SD).
(D) Chord conductance recorded at a membrane potential of −120 mV of oocytes co-expressing AtOST1 with SLAC1 from different plant species indicated in the figure. Chord conductance was calculated from instantaneous currents recorded in chloride- or nitrate-based buffers (100 mM). Chord conductance in nitrate was set to 1 (n ≥ 4 from 2 independent experiments, mean ± SD).
Figure 3
Nitrate Activates HvSLAC1 by Shifting Its Relative Open Probability to Hyperpolarized Voltages
(A) Nitrate dependence of steady-state currents (ISS) of oocytes co-expressing HvSLAC1 and AtCPK6 are plotted as a function of the applied membrane potential (n = 4 from 2 independent experiments, mean ± SD).
(B) The relative open probability (relative PO) measured in different NO3– concentrations of HvSLAC1/AtCPK6-expressing oocytes was plotted against the membrane potential. Data points were fitted with a single Boltzmann equation (solid lines, n = 4 from 2 independent experiments, mean ± SD).
(C) Steady-state currents (ISS) of oocytes co-expressing HvSLAC1 and AtCPK6 recorded in the presence of different external chloride concentrations or 30 mM nitrate (n = 4 from 2 independent experiments, mean ± SD).
(D) The relative open probability (relative PO) of HvSLAC1 in the presence of different Cl– concentrations or 30 mM NO3– was plotted against the membrane potential. Data points were fitted with a single Boltzmann equation (solid lines, n = 4 from 2 independent experiments, mean ± SD).
(E) The half-maximal PO (V1/2) calculated from the data in (B) was plotted against the nitrate concentration. A Michaelis-Menten equation was used to calculate a K0.5 of 10.9 mM NO3– (n = 4, mean ± SD).
(F) Steady-state currents of HvSLAC1 and AtCPK6 co-expressing oocytes in the presence of different Cl–/NO3–-ratios were plotted against the applied voltage (n = 4 from 2 independent experiments, mean ± SD).
(G) The relative open probability (relative PO) of HvSLAC1 in different Cl–/NO3–-ratios was plotted against the membrane potential.
Figure 4
IA-Motif on TMD3 Coevolved with Residues on TMD1 and 2 to Provide Monocot SLAC1 Anion Channels with a Nitrate-Depending Gating Mechanism
(A) Frequency logos of transmembrane three (TMD3) from SLAC1 anion channels of different monocot or dicot species. The respective sequence alignment is shown in Figure S3. The most prominent difference (IA motif in monocots versus VV/IV motif in dicots) is marked with a red box.
(B) Chord conductance at −120 mV of AtSLAC1 WT and AtSLACa1 V272I V273A compared to HvSLAC1 WT and HvSLAC1 I286V A287V or PdSLAC1 WT and PdSLAC1 I285V A286V. All channels and mutants thereof were co-expressed with CPK6. Currents were recorded in nitrate- or chloride-based buffers. (n = 4 from 2 independent experiments, mean ± SD). Note, the phenotypes of HvSLAC1 and AtSLAC1 anion channels were highly reproducible showing nitrate-independent gating properties of AtSLAC1 and nitrate-dependent gating of HvSLAC1 expressing oocytes. In contrast, with the mutant AtSLAC1 V272I V273A we observed a conditional phenotype strongly dependent on the investigated oocyte batch. In 30% of the tested oocyte batches, the mutations in the selectivity signature converted AtSLAC1 into a HvSLAC1-type nitrate-dependent anion channel (these data are shown in this study), whereas the remaining oocyte batches revealed a AtSLAC1 WT behavior.
(C) Evolutionary coupling analysis. The top 50 amino acid residues (see also Table S3) that showed evolutionary coupling to AtSLAC1-V272 and V273 (purple spheres) were highlighted in red on previously generated homology models [18, 36] using VMD [37]. Note, some of the highlighted residues co-evolved with both V272 and V273. The sphere size of co-evolved residues does not relate to the evolutionary coupling strength but reflects the side chain size. TMD1 is depicted in light gray, TMD2 in green, and TMD3 in dark gray. The remaining TMDs are shown in transparent orange.
(D) Chord conductance of oocytes co-expressing AtCPK6 with AtSLAC1, HvSLAC1, or one of the indicated chimeras. Currents were recorded in nitrate or chloride-based buffers. Chord conductance for nitrate was set to 1 (n = 4 from 2 independent experiments, mean ± SD). See also Figure S4B.
(E and F) Relative open probability (relative PO) of (E) AtSLAC1 and the chimera AtSLAC1(HvTMD1–3) or (F) HvSLAC1 and HvSLAC1(AtTMD1–3) in the presence of 30 mM chloride or nitrate (n = 4 from 2 independent experiments, mean ± SD).
