Expression of GPDHc1 under Standard Growth Conditions and in Response to ABA and Stress Conditions.
(A) Tissue-specific expression of GPDHc1. Top panel, blot of total RNA extracted from Arabidopsis roots, 2-week-old seedlings, and fully expanded leaves, flowers, and siliques of various developmental stages, hybridized with GPDHc1 cDNA probe. Bottom panel, ethidium bromide staining of rRNA.
(B) ABA-induced expression of GPDHc1. Arabidopsis seedlings were sprayed with 50 μM ABA, and total RNA was extracted from material harvested at 0, 0.5, 3, 6, and 12 h after treatment. Top panel, RNA gel blot hybridized with GPDHc1 cDNA probe; middle panel, a duplicate RNA gel blot hybridized with At GPDHp cDNA probe; bottom panel, ethidium bromide staining of rRNA.
(C) Expression of GPDHc1 under salinity stress. Arabidopsis seedlings were treated with 50 mM NaCl, and total RNA was extracted from material harvested at 0, 0.5, 3, 6, and 12 h after treatment. Top panel, RNA gel blot hybridized with GPDHc1 cDNA probe; middle panel, duplicate RNA gel blot hybridized with At GPDHp cDNA probe; bottom panel, ethidium bromide staining of rRNA.
(D) Expression of GPDHc1 under dehydration. Arabidopsis seedlings were dehydrated in open Petri dishes, and total RNA was extracted from material harvested at 0, 0.5, 6, and 12 h after treatment. Top panel, RNA gel blot hybridized with GPDHc1 cDNA probe; middle panel, duplicate RNA gel blot hybridized with At GPDHp cDNA probe; bottom panel, ethidium bromide staining of rRNA.
(E) Expression of GPDHc1 under hypoxia. Arabidopsis seedlings were submerged under water, and total RNA was extracted from materials harvested at 0, 0.5, 6, and 18 h after treatment. Top panel, RNA gel blot hybridized with GPDHc1 cDNA probe; bottom panel, ethidium bromide staining of rRNA.
(F) Expression of GPDHc1 under anoxic condition. Arabidopsis seedlings were under an N₂ stream, and total RNA was extracted from material harvested at 0, 0.5, 6, and 18 h after treatment. Top panel, RNA gel blot hybridized with GPDHc1 cDNA probe; bottom panel, ethidium bromide staining of rRNA.

Molecular Characterization of the gpdhc1 Mutants.
(A) Genomic organization of the gpdhc1-1 and gpdhc1-2 loci. The T-DNA insertions are not drawn to scale.
(B) GPDHc1 transcript analysis. RT-PCR of the GPDHc1 transcript in seedlings of the gpdhc1 mutants and their respective wild-type lines.
(C) Seedling RNA gel blot of the gpdhc1 mutants and their respective wild-type lines hybridized with the GPDHc1 cDNA probe (top panel); ethidium bromide staining of rRNA (bottom panel).

GPDH Activities and G-3-P Contents of the gpdhc1 Mutants and Wild-Type Lines.
(A) GPDH activities in total leaf and root extracts as well as in subcellular fractions of leaf tissues. GPDH activities in total leaf extract (L), in chloroplastic (Chl), mitochondrial (Mit), and microsomal (Mic) fractions, and in cellular root extract (R) of gpdhc1-1 and gpdhc1-2 mutants and their respective wild-type lines, Ws and Col. Values are expressed as means ± sd (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. FW, fresh weight.
(B) G-3-P contents in seedlings and roots. Left panel, G-3-P contents in seedlings of gpdhc1-1 and gpdhc1-2 mutants compared with Ws and Col, respectively; right panel, G-3-P contents in roots of gpdhc1-1 and gpdhc1-2 mutants compared with Ws and Col, respectively. Values are expressed as means ± sd (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Metabolic Impact of GPDHc1 Deficiency on NADH/NAD⁺ and Pyruvate/Lactate.
(A) NADH contents, (B) NAD⁺ contents, (C) NADH/NAD⁺ ratios, (D) pyruvate contents, (E) lactate contents, and (F) the calculated cytosolic NADH/NAD⁺ ratios of gpdhc1-1 and gpdhc1-2 mutants compared with Ws and Col, respectively. Values are expressed as means ± sd (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effect of ABA Treatment on NADH/NAD⁺ Ratios in gpdhc1 Mutants and Wild-Type Lines.
(A) Time courses of NADH contents, (B) NAD⁺ contents, and (C) the NADH/NAD⁺ ratios after ABA treatment of seedlings of gpdhc1-1 and gpdhc1-2 mutants and the wild-type lines, Ws and Col. Values are expressed as means ± sd (n = 3).

Alteration of Levels of Metabolites in the gpdhc1 Mutants.
(A) Glucose-6-phosphate (Glc-6-P), (B) fructose-6-phosphate (Fru-6-P), (C) glucose-1-phosphate (Glc-1-P), (D) 3-PGA, (E) glyceraldehyde-3-phosphate (glyceraldehyde-3-P), (F) DHAP, (G) PEP, (H) malate, (I) citrate, (J) 2-OG, (K) Glu, (L) Asp, and (M) OAA. Glc-6-P, Fru-6-P, Glc-1-P, 3-PGA, glyceraldehyde-3-P, DHAP, and PEP are measured in nmol·g⁻¹ FW. Citrate, OAA, malate, 2-OG, Glu, and Asp are measured in μmol·g⁻¹ FW. The results are expressed as means ± se (n = 3). *, P < 0.05; **, P < 0.01. OAA content was calculated from the Glu/OAA aminotransferase equilibrium reaction.

Elevated ROS Levels in gpdhc1 Mutants.
(A) ROS staining of leaves of the gpdhc1 mutants and wild-type lines. Top panel, 3,3′-diaminobenzidine stain forms a brown polymerization product upon reaction with H₂O₂; bottom panel, NBT stain forms an insoluble blue formazan deposit upon reaction with superoxide.
(B) Intracellular ROS levels measured using H₂DCF-DA in cultured roots of gpdhc1-1 and gpdhc1-2 compared with Ws and Col, respectively. AU denotes arbitrary units. Values are expressed as means ± sd (n = 3). ***, P < 0.001.
(C) Quantification of H₂O₂ in ABA-treated seedlings of gpdhc1-1 and gpdhc1-2 mutants compared with Ws and Col, respectively, at 0, 0.5, 3, 6, 12, and 24 h after treatment. Values are expressed as means ± sd (n = 3).

Compensatory Responses of Antioxidant Enzyme Activities in gpdhc1 Mutants.
(A) SOD activity, (B) CAT activity, (C) GR activity, (D) APX activity, (E) MDHAR activity, (F) GSH and GSSG contents, (G) GSH/GSSG ratio, (H) reduced and oxidized ascorbate (AsA and DHA, respectively) contents, and (I) AsA/DHA ratio in seedlings of gpdhc1-1 and gpdhc1-2 mutants compared with Ws and Col, respectively. Values are expressed as means ± sd (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Altered Respiration Characteristics and AOX Protein Contents in the gpdhc 1 Mutants.
(A) Respiration rates of fully expanded leaves of the gpdhc1 mutants and the wild-type lines (left panel) and AOX capacity (right panel).
(B) Respiration rates of cultured roots of the gpdhc1 mutants and the wild-type lines (left panel) and AOX capacity (right panel). Values are expressed as means ± sd (n = 4). *, P < 0.05.
(C) Immunoblot analysis of AOX protein. Immunoblots of leaf protein (top panel) and root protein (bottom panel) of the gpdhc1 mutants and wild-type lines probed with a monoclonal antibody raised to AOX. The AOX protein was visualized by a chemiluminescence method. Numbers on the left indicate the molecular masses of the standard proteins (data not shown) in kilodaltons. Approximately 50 μg of protein was loaded per lane.

Mutant Phenotype of GPDHc1 in Response to Stress.
(A) Two-week-old seedlings of the wild-type and gpdhc1 mutants lines grown in Murashige and Skoog (MS) (Murashige and Skoog, 1962) medium with (right) or without (left) 1 μM ABA.
(B) Germination of wild-type (closed circles and squares) and gpdhc1 mutants (open circles and squares) in the presence of different concentrations of ABA at day 5 after imbibition. Data represent means ± sd of three independent experiments (>100 seeds per point). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
(C) Three-week-old seedlings of wild-type and gpdhc1 mutant lines grown in half-strength MS medium with (bottom panel) or without (top panel) 100 mM NaCl.
(D) Fresh weight of 3-week-old seedlings of wild-type (closed circles and squares) and gpdhc1 mutant (open circles and squares) seedlings in the presence of different concentrations of NaCl. Fresh weights of seedlings grown without NaCl were 0.0556 ± 0.009, 0.0552 ± 0.01, 0.065 ± 0.005, and 0.063 ± 0.008 (g/seedling) for Col, gpdhc1-2, Ws, and gpdhc1-1, respectively. Values are expressed as means ± sd (n = 20). *, P < 0.05.

Model for the Involvement of G-3-P Shuttle in Redox Regulation.
The mitochondrial G-3-P shuttle consists of two components: GPDHc1 and FAD-GPDH. The linkage of cytosolic G-3-P metabolism to mitochondria is cyclical for G-3-P oxidoreduction but irreversible for the oxidation of NADH and regeneration of NAD⁺ in the cytosol. The shuttle donates electrons to ubiquinone and bypasses complex I. Higher shuttle activity is in demand in developmental stages or growth conditions where increased adjustments of cytoplasmic NADH/NAD⁺ ratio are required. By modulating the NADH/NAD⁺ ratio, the shuttle also operates to ensure that malate/OAA in the cytosol is maintained in a proper ratio. A deficiency of the G-3-P shuttle causes an imbalance of NADH/NAD⁺ ratio in the cytosol and results in metabolic disturbance of pyruvate and citrate, which are important for regulation of AOX capacity. The shuttle presents a route energy dissipation to avoid radical formation upon oxidative stress. Q, ubiquinone pool.