Extended Data Fig. 9: SCREW–NUT regulates leaf water loss and ABA responses.
From: Phytocytokine signalling reopens stomata in plant immunity and water loss
a, Transgenic plants carrying p35S::SCREW1 or p35S::SCREW2 exhibit curled leaves and increased sensitivity to dehydration stress. Leaves of five-week-old soil-grown plants were detached and imaged at 0 and 6 h after detachment. Scale bar, 1 cm. b, Increased water-loss rate in transgenic plants carrying p35S::SCREW1 or p35S::SCREW2. The rates of cumulative water loss from rosette leaves of five-week-old plants were measured at 6 h post-detachment. Data are shown as means ± s.d. (n = 6, biologically independent samples). c, Reduced water-loss rate in nut and screw1/2. The rate of cumulative water loss from detached leaves of four-week-old plants was measured at the indicated time points after detachment. Data are shown as means ± s.d. (n = 6, biologically independent samples). d, Enhanced resistance to mannitol treatment in nut mutants. Seedlings were grown on 1/2MS plates with 0, 50, 100, 150, or 200 mM mannitol for 15 days. Scale bar, 2 mm. e, Cuticle permeability of nut and screw1/2 seedlings is similar to that of WT. Three-week-old plate-grown plants were soaked with 0.05% toluidine blue for 15 min and washed with ddH2O before imaging. Scale bar, 1 cm. At least six seedlings for each genotype were analysed for the presence of the blue-coloured patches, which indicate an increased permeability of the stain into the leaf through the cuticle. No apparent differences were observed between WT and mutants. f, Leaf cuticle permeability of nut and screw1/2 is similar to that of WT. Leaves of four-week-old soil-grown plants were drop-stained with 0.05% toluidine blue on the adaxial surface for the indicated time. The red circles and rectangles indicate the sites of inoculation. Inserts show zoomed-in areas. No blue-coloured patches were observed, indicating the intact cuticle for each genotype. Scale bar, 20 µm. Scale bar, 1 cm. Ten leaves for each genotype were analysed. g, Leaf cuticle layers of nut and screw1/2 are similar to those of WT. Three-week-old plate-grown plant leaves were examined by transmission electron microscopy from the adaxial side. Red arrows indicate cuticles observed as a thin (~80–100 nm) electron-dense layer on the surface of the cell wall. Scale bar, 1 µM. Four leaves of each genotype were analysed. No apparent differences in thickness were detected among different genotypes. h, The nut and screw1/2 mutants are more sensitive to ABA treatment than WT plants. Seedlings were grown on 1/2MS plates without (Ctrl) or with 1 μM ABA for seven days (left). Cotyledon greening rates are shown as means ± s.d. (right, n = 4, biologically independent repeats). i, SCREW1 and SCREW2 suppress ABA-induced expression of RAB18 and RD29A in plants. Ten-day-old plate-grown WT seedlings were treated with H2O, 10 µM ABA, or combinations of 10 μM ABA and 1 μM SCREW1 or SCREW2 for 3 h. Transcript levels of RAB18 and RD29A normalized to UBQ10 were determined via RT–qPCR. Data are shown as mean ± s.d. (n = 4, biologically independent samples). j, NUT is upregulated by ABA, mannitol, and drought treatments. The expression data were extracted from Genevestigator V3 and shown as histograms. Grey squares indicate no data available. k, SCREWs and NUT are up-regulated after ABA treatments. Ten-day-old plate-grown WT seedlings were treated with 100 μM ABA for 0, 3, and 6 h. Transcript levels of SCREWs and NUT normalized to UBQ10 were determined via RT–qPCR. Data are shown as mean ± s.d. (n = 3, biologically independent samples). Experiments were repeated three times with similar results. Data were analysed by one-way (b) or two-way (c, h, i, k) ANOVA followed by Tukey’s test. Exact P values are provided in the graphs and Supplementary Table 3.
