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, 9 (1), 12282

A Ca 2+/CaM-regulated Transcriptional Switch Modulates Stomatal Development in Response to Water Deficit

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A Ca 2+/CaM-regulated Transcriptional Switch Modulates Stomatal Development in Response to Water Deficit

Chan Yul Yoo et al. Sci Rep.

Abstract

Calcium (Ca2+) signals are decoded by the Ca2+-sensor protein calmodulin (CaM) and are transduced to Ca2+/CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca2+/CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca2+/CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca2+/CaM interaction with the 2nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca2+/CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca2+/CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
CaM binds to the N-terminal DNA-binding domain of GTL1 in a Ca2+-dependent manner. (a) Schematic illustration of GTL1 domain topology with N- and C-terminal trihelical domains. The primary and secondary structure of GTL1N (residue number 60~126) consists of three α-helices (α1 – grey, α2 – green, and α3 – orange) with α2 containing a predicted CaM-binding domain. (b) The helical wheel projection of the α2 helix shows a canonical amphipathic helix with hydrophobic (yellow) and hydrophilic (basic – blue and acidic – red) residues. (c) GTL1N interaction with Ca2+/CaM was performed by in vitro pull-down assay using Escherichia coli expressed MBP-GTL1N or MBP to pull down in vitro-translated HA-AtCaM2 that was detected by immunoblots using anti-HA antibodies (upper panel). Immobilized MBP and MBP-GTL1N fusion proteins are shown in the Coomassie blue-stained SDS-PAGE gel (lower panel). (d) Schematic illustration of the bait and prey proteins used in the pull-down assays. Ca2+-dependent CaM interaction with GTL1N or GTL1C were performed by pull-down assay using HA-AtCaM2. Bound and 10% of input HA-AtCaM2 fractions were detected by immunoblot using anti-HA antibodies. The MBP-GTL1N (Nt) and MBP-GTL1C (Ct) fusion protein bands are indicated by red arrows. Full-length blots and gels for c and d are presented in Supplementary Fig. S10.
Figure 2
Figure 2
Ca2+/CaM binds to the α2 helix of GTL1N. (a) Schematic illustration of two protein fragments (GTL1N and GTL1N∆del) fused to maltose-binding protein (MBP). In vitro pull-down assay was performed using MBP-fusion proteins with Ca2+/HA-AtCaM2. Bound and 10% of input HA-AtCaM2 fractions were detected by immunoblot using anti-HA antibodies. The MBP-GTL1N and MBP- GTL1N∆del fusion protein bands are indicated by red arrows. (b) Sequence alignment of the α2 helix of the N-terminal (Nt) or C-terminal (Ct) trihelical domain from GT2 family proteins, including GTL1. AtGTL1_Nt and AtGT2L_Ct have CaM-binding activity. *Indicates the residue that distinguishes CaM-binding and non-CaM-binding groups. (c) An in vitro pull-down assay was performed using MBP-GTL1N or MBP-GTL1N[H94E] fusion proteins with Ca2+/HA-AtCaM2. Bound and 10% of input HA-AtCaM2 fractions were detected by immunoblot using anti-HA antibodies. The MBP-GTL1N and MBP-GTL1N[H94E] fusion protein bands are indicated by red arrows. Full-length gels for a and c are presented in Supplementary Fig. S10.
Figure 3
Figure 3
The GTL1 N-terminal DNA-binding domain forms a trihelical tertiary structure that is stabilized by hydrophobic residues. (a) Schematic illustration of GTL1 N-terminal trihelical domain. The primary and secondary structure of GTL1N (Residue Number 60–126) consists of three helices with α2 (green) and α3 (orange) helices containing CaM-binding and DNA-recognition sequence motifs, respectively. The three-dimensional GTL1N structure is predicted to be stabilized by a hydrophobic core (grey dot area) that is formed by hydrophobic residues shown in black, green, and orange distributed in the three helices. Blue residues indicate charged residues necessary for DNA recognition (α3) and CaM binding (α2). (b) DNA-binding activity of recombinant proteins (5 or 10 μg) of MBP, GTL1N (WT), or GTL1N mutations ([V95D], [W92R], and [L91R]) with biotin-labeled SDD1 promoter fragments was performed by electrophoretic mobility shift assay (EMSA). (c) GTL1N (WT) or H94E proteins were used to determine binding activity with a biotin-labeled SDD1 promoter fragment (left panel) or rice PHYA promoter fragment (right panel). Free promoter and protein-promoter complexes are indicated by an asterisk and arrows, respectively.
Figure 4
Figure 4
Hyperosmotic stress-induced Ca2+/CaM binding to the α2 helix inhibits DNA-binding activity of GTL1 to the SDD1 promoter. (a–c) EMSA was performed using GTL1N or GTL1N[H94E], and a biotin-labeled SDD1 promoter fragment that includes the GT3 box. (a) GTL1N (1 or 5 mg) was pre-incubated with human CaM (100 ng) without or with 2 mM CaCl2 or 10 mM EGTA, and then incubated with the biotin-labeled SDD1 promoter fragment (250 ng). (b) GTL1N (2.5 or 5 mg) was pre-incubated with the SDD1 promoter fragment without or with 2 mM CaCl2, and then with human CaM (100 ng). (c) GTL1N or GTL1N[H94E] was pre-incubated with the SDD1 promoter fragment without or with 2 mM CaCl2 and then with human CaM (50 or 100 ng). (d) ChIP assays using anti-HA antibody were performed using protoplasts isolated from gtl1-4 plants, transformed with HA-GTL1 or HA-GTL1[H94E], and incubated with 0 or 200 mM mannitol for 1 h. Input is the total isolated chromatin before immunoprecipitation. Mouse IgG was the negative control for immunoprecipitation. The SDD1 promoter region including the GT3 box was amplified by SDD1 promoter-specific primers.
Figure 5
Figure 5
Hyperosmotic stress induces SDD1 expression through Ca2+/CaM-dependent inhibition of the GTL1 transrepressor. (a) Transcript abundance of SDD1, DREB2A, and UBC was determined in total RNA extracted from 5-week-old Col-0 plants that were well-watered or water-deficit stressed. (b) Schematic diagram of the reporter and internal control constructs. The reporter construct included the 2 kb SDD1promoter fused with firefly luciferase (LUC) gene, and nopaline synthase termination signal (NOS-t). The internal control construct included the 35Spromoter, Renilla reniformis LUC (RrLUC), and NOS-t. SDD1-LUC activity relative to RrLUC activity (% of SDD1-LUC to RrLUC activity) was measured from protoplasts isolated from Col-0 (wild type), gtl1-4, and gtl1-4 expressing GTL1 (GTL1/gtl1-4). (c) Relative SDD1-LUC activities were determined in Col-0 and gtl1-4 protoplasts that were incubated with or without 1 mM GdCl3, 600 mM W7, or GdCl3 and W7 combined prior to addition of mannitol to a final concentration of 200 mM. (d) SDD1- LUC activities of Col-0, gtl1-4, or HA-GTL1 or HA-GTL1[H94E] protoplasts were determined after incubation with 0 or 200 mM mannitol for 1 h. All results shown are mean ± SEM (n = 3). Columns with the same letters above are not significantly different from each other based on Tukey’s Honestly Significant Difference (HSD) test (P < 0.05) (One-way ANOVA).
Figure 6
Figure 6
Stomatal development is repressed under severe water-deficit conditions through Ca2+/CaM and the GTL1 transrepressor. (a) Leaf area of Col-0 and gtl1-4 plants grown under well-watered (WW), mild water-deficit (50% media water content, MWC), and severe water-deficit (30% MWC) conditions. These leaves were used to quantify stomatal length (b), stomatal index (c), and stomatal precursor index (d) in adaxial and abaxial leaf surfaces of Col-0 and gtl1-4 plants. Adaxial (upper case) and abaxial (lower case) leaves were analyzed separately for the statistical comparisons. (e) Stomatal index of the abaxial surface was quantified in Col-0 and gtl1-4 plants grown under 0 and 200 mM mannitol conditions with or without W7 (50 and 100 μM) and GdCl3 (85 and 170 μM). Col-0 (left panel) and gtl1-4 (right panel) were analyzed separately for the statistical comparisons. Data shown are the means with SD for 8–12 replicates (ad) and 7 replicates (e). Columns with the same letters above are not significantly different from each other based on Tukey’s Honestly Significant Difference (HSD) test (P < 0.05) (Two-way ANOVA).
Figure 7
Figure 7
A Ca2+/CaM-regulated GTL1 transrepressor is a transcriptional switch to control stomatal development, transpiration, and water-use efficiency in plants. Proposed model for Ca2+/CaM-regulated transcriptional switch to repress stomatal development and to improve water-use efficiency and drought tolerance through GTL1.

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