Fig. 4: SnRK2-GPT1 module regulates NADPH homoeostasis during prolonged ABA signalling.

a, d Measurement of H₂O₂ and NADPH/NADP⁺. After 4 days of growth, the Col-0 and stt3a-2 were treated with 3 μM ABA for the corresponding time periods. SnRK2.3^WT/snrk2.2/2.3 transgenic Arabidopsis (SnRK2.3^WT) and SnRK2.3^N323A/snrk2.2/2.3 (SnRK2.3^N323A) were treated with 1 μM ABA. H₂O₂ and NADPH/NADP⁺ were measured in roots. b Co-IP assays showed the interactions between GPT1 and SnRK2s. Proteins were extracted from 35 S::YFP and pGPT1::GPT1-YFP transgenic plants treated with 50 μM ABA for different time periods. c BiFC assays demonstrated the interactions of GPT1 and SnRK2.3 on the peroxisomal membrane. CD3-959 was used as ER marker and dsRed-mPTS^PEX26 was used as peroxisome membrane marker. Scale bars, 5 μm. n = 4 tobacco leaves. d Measurement of NADPH/NADP⁺. The subfigure highlights two phases of NADPH accumulation induced by short-term ABA (dark blue) and by prolonged ABA (light blue). e Phosphorylation analysis of GPT1. GST-GPT1^N, GPT1 N-terminus 1–96 amino acids. For the biological replicate, see the Source Data file. f AmiRNA for silencing endogenous GPT1, but not GPT1^WT, GPT1^S32A, GPT1^S32D (in which the amiR-GPT1-1 binding site was replaced with synonymous codons). g, h Measurement of NADPH/NADP⁺ (g) and H₂O₂ (h). GPT1^WT, GPT1^S32A, and GPT1^S32D were coexpressed with amiR-GPT1-1 (for endogenous GPT1 silencing) and amiRNA vector (EV, as negative controls) in Arabidopsis hairy roots, respectively. Both GPT1^WT, GPT1^S32A and GPT1^S32D were driven by native promoter (1000 bp upstream sequences of the GPT1 coding region). i and j Measurement of NADPH/NADP⁺ (i) and H₂O₂ (j). SnRK2.3^WT-GFP, SnRK2.3-NLS-GFP and SnRK2.3-GFP-mPTS^PEX26 were coexpressed in snrk2.2/2.3 hairy roots, respectively. Both SnRK2.3^WT-GFP, SnRK2.3-NLS-GFP and SnRK2.3-GFP-mPTS^PEX26 were driven by the native promoter (2020 bp upstream sequences of the SnRK2.3 coding region). EV (pMDC107 vector) was used as a negative control. Data are presented as means ± s.d., and asterisks indicate significant differences (*P-value < 0.05, **P-value < 0.01, ***P-value < 0.001), as determined by two-tailed paired t tests. “ns” means no significant difference. For quantification of H₂O₂ level and NADPH/NADP⁺ ratio, three biological replicates were carried out in each time-point during ABA application in (a, d, g–j).