Figure 7A, Co-immunoprecipitation of TSN and SnRK1α1 and SnRK1α2 in protein extracts prepared from 10-d-old Arabidopsis seedlings expressing ProUBQ:GFP-SnRK1α1 or ProUBQ:GFP-SnRK1α2 and exposed to HS (39ºC for 60 min). The GFP-expressing line was used as a negative control. Endogenous TSN was detected in the total fractions (Input) and in the fractions co-immunoprecipitated (Co-Ip) with SnRK1α1 or SnRK1α2 but not with free GFP. Input and Co-Ip fractions were analyzed by immunoblotting using α-TSN and α-GFP. B, Co-immunoprecipitation of TSN and SnRK1α1 in protein extracts prepared from 10-d-old Arabidopsis seedlings expressing ProUBQ:GFP-SnRK1α1 and grown under NS (23ºC) conditions or subjected to HS (39ºC for 60 min). Endogenous TSN was detected in the total fractions (Input) and in the fractions co-immunoprecipitated (Co-Ip) with SnRK1α1 under both NS and HS conditions. Input and Co-Ip fractions were analyzed by immunoblotting using α-TSN and α-GFP. C, FRET assay of the indicated protein combinations using CFP-YFP pair in N. benthamiana leaves under HS (39ºC for 40 min). EV, empty vector (negative control). Upper and lower box boundaries represent the first and third quantiles, respectively; horizontal lines mark the median of at least eight replicate measurements, and whiskers mark the highest and lowest values. The experiment was repeated three times with similar results. P values denote statistically significant differences for comparisons to plants expressing EV (two-tailed t-test). D, Localization of GFP-SnRK1α1 and GFP-SnRK1α2 in root cells of 5-d-old Arabidopsis WT and tsn1 tsn2 seedlings grown under 23°C (NS) or incubated at 39°C for 60 min (HS). Insets show enlarged areas inside dashed rectangles. Scale bars = 10 μm. E, F, Number (E) and size (F) of SnRK1α1- and SnRK1α2-foci in root tip cells of WT and tsn1 tsn2 seedling expressing ProUBQ:GFP-SnRK1α1 or ProUBQ:GFP-SnRK1α2, respectively, after HS (60 min at 39ºC). Data represent means ± SD of at least 16 replicate measurements from three independent experiments. P values denote statistically significant differences for comparisons to WT plants (two-tailed t-test). G, H, Signal recovery rate (t1/2; G) and proportion of the initial signal recovered (%; H) of GFP-tagged isoforms of SnRK1α in root tip cells of WT and tsn1 tsn2 seedlings expressing ProUBQ:GFP-SnRK1α1 and ProUBQ:GFP-SnRK1α2 after HS (60 min at 39°C). nd, not detected. Upper and lower box boundaries represent the first and third quantiles, respectively; horizontal lines mark the median of at least seven replicate measurements, and whiskers mark the highest and lowest values. The experiment was repeated three times with similar results. P values denote statistically significant differences for comparisons to WT plants (two-tailed t-test). I, Schematic diagram of SnRK1α protein structure showing catalytic (CD) and regulatory (RD) domains. The CD includes the phosphorylated T-loop region. RD includes both kinase-associated 1 (KA1) and ubiquitin-associated (UBA) subdomains. J, Colocalization of GFP-SnRK1α1, GFP-SnRK1α1CD, or GFP-SnRK1α1RD with RFP-RBP47 in N. benthamiana protoplasts subjected to HS (40 min at 39°C). For colocalization analysis under NS conditions see Fig. EV5B. For CHX treatment, protoplasts were incubated with 200 ng μL-1 CHX for 30 min at 23ºC before HS. GFP and RFP fusion proteins were expressed under the control of the UBQ and 35S promoter, respectively. Scale bars = 5 μm.