Fig. 2: The thermosensor function of TWA1.
From: The temperature sensor TWA1 is required for thermotolerance in Arabidopsis
a–c, TWA1 and TWA1 variants inhibit ABA-responsive pRD29B:LUC expression in leaf protoplasts. a, Protoplasts of WT, twa1-1 and twa1-2 plants were transfected with a TWA1or twa1-1 expression cassette (shown as µg DNA per 105 protoplasts), incubated for 16 h at 25 °C with 10 µM ABA. Relative light units were normalized to fluorescence units of a co-transfected control (RFU). The dashed line indicates expression of mutant twa1 product in twa1-2 protoplasts. b, TWA1 action at different exogenous ABA levels. c, The action of TWA1 and variants at different incubation temperatures with 0.3 µg effector DNA per 105 twa1-2 protoplasts. The variants included TWA1(ΔN), A. lyrata AlTWA1, mustard SaTWA1 and TWA1(AlHVR). d, Top, GFP–tagged TWA1 and twa1 product in yeast nuclei and temperature-dependent phase separation. Bottom, nuclear subdomain formation of TWA1(AlHVR) at 22 °C. Insets: 1.5-fold magnifications. Scale bar, 10 μm. See the ‘Statistics and reproducibility’ section of the Methods. e, Temperature-induced changes in the proximity of TWA1 amino- and carboxy-terminal domains as revealed by intramolecular FRET–FLIM analysis. Top, false-colour images of GFP FLs of mCherry–TWA1–GFP (TWA1) in yeast nuclei. Scale bar, 2 µm. Bottom, GFP FL of TWA1 and mCherry–GFP. n = 10–15 per datapoint. Statistical analysis was performed using Student’s t-tests; *****P = 10−5. For the box plots, the centre line shows the median, the box limits show the upper (25th percentile) and lower (75th percentile) quartiles, and the whiskers show 1.5× the interquartile range. f,g, The temperature dependence of JAM2 binding to TWA1 and variants including the twa1 product (f) and binding to orthologues (g) was analysed using a Y2H growth assay. The growth rate µ was calculated on the basis of cell density increase within 24 h. JAM2–JAM2 dimerization served as the control (Extended Data Fig. 7). For a–c,f,g, n = 3 per datapoint. Statistical significance details are provided as Source Data.
