Fig. 2: SnRK2.2/2.3 are N-glycosylated during prolonged ABA signalling.
From: N-glycosylation of SnRK2s affects NADPH maintenance in peroxisomes during prolonged ABA signalling
a Y2H assays showed specific interactions between GIIβ and SnRK2.2/2.3/2.6. b Co-IP assays showed the interactions between GIIβ and SnRK2s in vivo. Proteins were extracted from 35 S::GFP and 35 S::GIIβ-GFP transgenic plants. c LCI assays showed the interactions between SnRK2.2/2.3/2.6 and STT3A, GIIα, GIIβ, CGL1 and FUT11 with ABA-dependent manner. d, e N-glycosylation assay of SnRK2.3. SnRK2.3-His was coexpressed with STT3A-mCherry in the tobacco leaves with or without ABA treatment for 48 h. Western blots revealed a larger band shift (d), and Endo H and PNG F treatments confirmed that the band shift is due to N-glycosylation (e). N-SnRK2.3-His, N-glycosylated SnRK2.3-His. f N-glycosylation assay of SnRK2.2. SnRK2.2-His was coexpressed with STT3A-mCherry in the tobacco leaves with or without ABA treatment for 48 h. g N-glycosylation of SnRK2.6. SnRK2.6-His was coexpressed with STT3A-mCherry in the tobacco leaves with ABA treatment for 48 h. h, i N-glycosylation assay of endogenous SnRK2.2/2.3 in the snrk2.6 mutant background (snrk2.2/2.3/2.6 as a negative control). j N323 site mutation of SnRK2.3 (SnRK2.3N323A) disrupts its subcellular change. SnRK2.3-GFP and STT3A-mCherry were transiently coexpressed in N.benthamiana leaves. CFP-HDEL (CFP channel) was used as an ER marker. Scale bars, 10 μm. The experiment was repeated 3 times. k N323 site mutation blocks the N-glycosylation of SnRK2.3. SnRK2.3N323A-His and SnRK2.3N288A-His (negative control) were coexpressed with STT3A-mCherry in the tobacco leaves with or without ABA treatment for 48 h. Repeated results were displayed in the Source Data file for (b, d–k). l, m SnRK2.2/2.3 are epistatic to STT3A in ABA signalling. snrk2.2/2.3 double mutant suppressed the hypersensitivity of stt3a-2 in terms of primary root length (l). Quantification of the relative length of primary roots (ABA + /-) as shown in (m). The ratio (ABA + /-) of Col-0 was designated as 1, and the relative ratio to Col-0 was shown for double mutant and triple mutants. Data are means ± s.d. n = 5 plants. Asterisks indicate significant differences (***P-value < 0.001), as determined by two-tailed paired t tests.
