A Tandem Amino Acid Residue Motif in Guard Cell SLAC1 Anion Channel of Grasses Allows for the Control of Stomatal Aperture by Nitrate

Abstract

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 CO 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.

Form, development and function of grass stomata

“…Similarly in maize, zmslac1 mutants exhibited ineffective stomatal closure in response to different cues, and it was suggested that ZmSLAC1 mediates the efflux of nitrate rather than Cl − (Qi et al , ). In barley, it was shown that a tandem amino acid residue motif in monocot SLAC1 channels accounts for nitrate‐dependent gating of grass SLAC1 prior to anion (both nitrate and Cl − ) efflux and ABA‐induced stomatal closure (Schäfer et al , ). In Arabidopsis, AtSLAC1 is nitrate‐insensitive while in grasses SLAC1 shows nitrate‐sensitive gating, and it was suggested that nitrate‐dependent gating of SLAC1 might be the ancestral state (Schäfer et al , ).…”

“…In barley, it was shown that a tandem amino acid residue motif in monocot SLAC1 channels accounts for nitrate‐dependent gating of grass SLAC1 prior to anion (both nitrate and Cl − ) efflux and ABA‐induced stomatal closure (Schäfer et al , ). In Arabidopsis, AtSLAC1 is nitrate‐insensitive while in grasses SLAC1 shows nitrate‐sensitive gating, and it was suggested that nitrate‐dependent gating of SLAC1 might be the ancestral state (Schäfer et al , ). Interestingly, the replacement of the dicot tandem signature in the transmembrane domain 3 of AtSLAC1 with the monocot signature (VV‐>IA) converted AtSLAC1 into a nitrate‐sensitive channel such as the one described in grass species (Kusumi et al , ; Qi et al , ; Schäfer et al , ).…”

“…In Arabidopsis, AtSLAC1 is nitrate‐insensitive while in grasses SLAC1 shows nitrate‐sensitive gating, and it was suggested that nitrate‐dependent gating of SLAC1 might be the ancestral state (Schäfer et al , ). Interestingly, the replacement of the dicot tandem signature in the transmembrane domain 3 of AtSLAC1 with the monocot signature (VV‐>IA) converted AtSLAC1 into a nitrate‐sensitive channel such as the one described in grass species (Kusumi et al , ; Qi et al , ; Schäfer et al , ). However, the reciprocal experiment did not abolish nitrate‐dependent gating in HvSLAC1 but rather impaired anion gating altogether, suggesting that additional moieties in monocot SLAC1 are required for nitrate‐dependent gating (Schäfer et al , ).…”

“…Interestingly, the replacement of the dicot tandem signature in the transmembrane domain 3 of AtSLAC1 with the monocot signature (VV‐>IA) converted AtSLAC1 into a nitrate‐sensitive channel such as the one described in grass species (Kusumi et al , ; Qi et al , ; Schäfer et al , ). However, the reciprocal experiment did not abolish nitrate‐dependent gating in HvSLAC1 but rather impaired anion gating altogether, suggesting that additional moieties in monocot SLAC1 are required for nitrate‐dependent gating (Schäfer et al , ). Cell‐type‐specific RNA sequencing suggested that HvSLAC1, HvABI1 and HvOST1.1 were strongly expressed in GCs only rather than in both stomatal cell types, suggesting a bias of the ABA‐related signalling module towards GCs (Figure b; Schäfer et al , ).…”

“…However, the reciprocal experiment did not abolish nitrate‐dependent gating in HvSLAC1 but rather impaired anion gating altogether, suggesting that additional moieties in monocot SLAC1 are required for nitrate‐dependent gating (Schäfer et al , ). Cell‐type‐specific RNA sequencing suggested that HvSLAC1, HvABI1 and HvOST1.1 were strongly expressed in GCs only rather than in both stomatal cell types, suggesting a bias of the ABA‐related signalling module towards GCs (Figure b; Schäfer et al , ). Next, it was shown that RNAi constructs downregulating the R‐type anion channel HvALMT1 (aluminium‐activated malate transporter 1) strongly impaired stomatal closure (Xu et al , ).…”