1. NRGA1, a Putative Mitochondrial Pyruvate Carrier, Mediates ABA Regulation of Guard Cell Ion Channels and Drought Stress Responses in Arabidopsis 
Chun-Long Li, Mei Wang, Xiao-Yan Ma, Wei Zhang 
Molecular Plant 
Volume 7, Issue 10, Pages (October 2014)
DOI: /mp/ssu061
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Homologs of NRGA1 in the Indicated Species.
(A) Phylogenetic analysis of NRGA1 (starred) and other homologous proteins. The amino acid sequences of these proteins were aligned by CLUSTALX, and the phylogenetic tree was constructed using the neighbor-joining method with the following parameters: bootstrap (replicates 1000), Poisson model, and complete deletion. The numbers at the nodes indicate the bootstrap values.
(B) Protein sequence alignment of NRGA1 and MPC2 homologs in the indicated species. Accession numbers are: NRGA1: NP_ ; Arabidopsis thaliana MPC2: NP_ ; Homo sapiens MPC2: NP_ ; Mus musculus MPC2: NP_ ; Caenorhabditis elegans MPC2: NP_ ; Drosophila melanogaster MPC2: NP_ ; Schizosaccharomyces pombe MPC2: NP_ ; Saccharomyces cerevisiae MPC2: NP_
Molecular Plant 2014 7, DOI: ( /mp/ssu061)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
NRGA1 and AtMPC1 Combine to Complement the Loss of Pyruvate Carrier Activity in Yeast Deletion Mutants.
(A) Spot assay of the indicated yeast strains transformed with EV or AtMPC1.
(B) Spot assay of the indicated strains transformed with EV or NRGA1.
Serial 10-fold dilutions in (A) and (B) were spotted on YPAD and SC-Ura-Leu plates and cultured at 30°C for 48h before photographing. WT, wild-type; EV, empty vector.
Molecular Plant 2014 7, DOI: ( /mp/ssu061)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Transcription Behavior of NRGA1 and Subcellular Localization of Its Product.
Real-time PCR of NRGA1 transcript under ABA (A) and drought conditions (B) were analyzed. Two-week-old seedlings (Col-0) were treated with 50 μM ABA or exposed to drought stress for 1h, 4h, 8h, 16h, and 24h before RNA isolation. Transcript levels relative to that of ACTIN2 are presented for each treatment. Each value represents the mean ± SE of three independent measurements.
(C, D) qPCR-based assessment of NRGA1 transcription in various organs of the plant. * and ** represent significant difference determined by the Student's t-test at P < 0.05 and P < 0.01, respectively. Leaves of 2-week-old proNRGA1::GUS seedlings ((C) bar = 1mm), guard cells ((D) bar = 10 μm), root ((E) bar = 2 μm), flower ((F) bar = 1mm), and silique ((G) bar = 2mm) were stained with X-Gluc. Real-time PCR of NRGA1 expression in different organs (H, I) were analyzed. Leaf denotes rosette leaf in (I).
(J) The GFP vector control 35S::GFP was transiently expressed in protoplasts of Arabidopsis leaves.
(K) The protoplasts expressed with 35S::NRGA1–GFP were observed with a confocal laser microscope.
(L) Co-localization of GFP-tagged NRGA1 and RFP-tagged mitochondria marker protein F1-ATPase-γ. The yellow signal indicates areas of overlap between the green and red signals. Bars =10 μm.
Molecular Plant 2014 7, DOI: ( /mp/ssu061)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Loss of NRGA1 Resulted in ABA Hypersensitivity of Stomatal Movement and Enhanced Drought Tolerance.
(A) ABA inhibition of stomatal opening in Col-0 and the nrga1 mutant.
(B) ABA promotion of stomatal closure in Col-0 and the nrga1 mutant. Error bars represent mean ± SE from three independent experiments. At least 60 stomata were measured for each genotype per replication. * means differ significantly at P < 0.05.
(C) Water loss rates from detached leaves of the Col-0 and the nrga1 mutant. Leaves at similar developmental stages were excised and weighed at the indicated time after detachment. The proportion of fresh weight losses was calculated on the basis of the initial weight of the leaves. Each data point represents the mean with SD (n = 30).
(D) Enhanced tolerance to drought in the nrga1 mutant. Water was withheld from 4-week-old plants for 2 weeks before the photograph was taken, and the rehydration photographs were taken 3 d after re-watering. The experiments were repeated three times, with similar results.
Molecular Plant 2014 7, DOI: ( /mp/ssu061)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Loss of NRGA1 Resulted in ABA Hypersensitivity of Inward K+ Channel and Slow Anion Channel Activity.
(A) Patch-clamp whole-cell recordings of the inward K+ currents in guard cell protoplasts isolated from Col-0 and the nrga1 mutant with/without 50 μM ABA in the bath solution. Time and voltage scales are as shown.
(B) The current/voltage relationship of time-activated whole-cell inward K+ currents as illustrated in (A). The number of guard cells measured was as follows: Col-0 (12), Col-0-ABA (12), nrga1 (12), and nrga1-ABA (9). Error bars represent mean ± SE.
(C) Activation by ABA (50 μM) of slow anion currents in Col-0 and nrga1 guard cell protoplasts. Time and voltage scales are as shown.
(D) The current/voltage relationship of time-activated whole-cell slow anion currents as illustrated in (C). The number of guard cells measured was as follows: Col-0 (8), Col-0-ABA (10), nrga1 (8), and nrga1-ABA (11). Error bars represent mean ± SE.
Molecular Plant 2014 7, DOI: ( /mp/ssu061)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
NRGA1 Overexpression Resulted in ABA Hyposensitivity of Guard Cell Movement and Decreased Drought Tolerance.
(A) qPCR analysis of NRGA1 transcription in Col-0, the nrga1 mutant, empty vector control 1 (EV1), overexpression lines 5 and 7 (NRGA1-OE-5, NRGA1-OE-7), empty vector control 2 (EV2), and complementation lines 1 and 7 (NRGA1-C-1, NRGA1-C-7). The ACTIN2 was used as an internal control.
(B) The inhibitory effect of 50 μM ABA on stomatal opening in Col-0, NRGA1 OE, and complementation transgenic lines.
(C) The promotive effect of 50 μM ABA on stomatal closure in Col-0, NRGA1 OE, and complementation lines. Error bars represent mean ± SE from three independent experiments. At least 60 stomata were measured for each genotype per replication. * means differ significantly at P < 0.05.
(D) The rate of water loss from detached leaves of the EV1, EV2, NRGA1-OE-5, NRGA1-OE-7, NRGA1-C-1, and NRGA1-C-7 plants. The proportion of fresh weight losses was calculated on the basis of the initial weight of the leaves.
(E) The phenotype of NRGA1 OE and complementation lines under drought stress conditions. The experiments were repeated three times, with similar results.
Molecular Plant 2014 7, DOI: ( /mp/ssu061)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Guard Cell Inward K+ Currents and Slow Anion Channels in Col-0, NRGA1 OE, and Complementation Lines.
(A) Patch-clamp whole-cell recordings of the inward K+ currents in guard cell protoplasts with/without 50 μM ABA in the bath solution. Time and voltage scales are as shown.
(B) The current/voltage relationship of time-activated whole-cell inward K+ currents as illustrated in (A). The numbers of guard cells measured were Col-0 (12), Col-0-ABA (12), NRGA1-OE-5 (10), NRGA1-OE-5-ABA (7), NRGA1-C-1 (9), and NRGA1-C-1-ABA (11). Error bars represent mean ± SE.
(C) Patch-clamp whole-cell recordings of the slow anion currents in guard cell protoplasts with or without 50 μM ABA. Time and voltage scales are as shown.
Molecular Plant 2014 7, DOI: ( /mp/ssu061)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

9. Figure 8
Proposed Model of NRGA1 in the ABA-Regulated Ion Channel Activity and the Plant Response to Drought Stress.
Molecular Plant 2014 7, DOI: ( /mp/ssu061)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions