Fig. 2: Cytokinin response correlates with enrichment of intracellular trans-zeatin content and is enhanced by inhibition of cytokinin turnover, but bioactive extracellular cytokinins are also present.

a Ratio of cytokinin metabolite concentration between GFP⁺ and GFP⁻ protoplasts from 9-day-old TCSn::GFP seedlings roots, treated with or without 20 μM INCYDE during protoplasting. Cytokinins were quantified as fmol per 100,000 protoplasts. Colours represent different cytokinin metabolite groups: free bases (yellow), ribosides (blue) and glucosides (green). Darker bars are INCYDE-treated samples and lighter colours are corresponding mock treatment. Whiskers represent the entire range of values, boxes indicate first and third quartiles, centre line is median, dots are individual values; n = 9 for mock and n = 6 for treated samples. Black asterisks indicate statistically significant differences in cytokinin concentration between GFP⁺ and GFP⁻ cells of TCSn::GFP treated or mock samples (paired sample t-test). *p < 0.05; **p < 0.01; ***p < 0.001. Grey asterisks above brackets denote statistically significant difference in cytokinin ratios between mock and treated experiment (one-way ANOVA and Tukey’s test, significance levels as above). b Confocal imaging of TCSn::GFP 6-day-old roots with or without 10 μM INCYDE treatment for 6 h. Insets show the respective root vasculature in 16-colour LUT (Look-Up Tables; https://imagej.net/) highlighting the gradations of fluorescence intensity. Scale bar is 35 μm. c Detection of extracellular cytokinins: ratios of cytokinin concentrations in symplastic/apoplastic fluid extracted from 9-day-old Arabidopsis wild-type roots. Ratios were derived from cytokinin levels calculated as fmol (g fresh weight)⁻¹ of original tissue and are shown as scatter plot with bar (mean with s.e.m). n = 3 pools of at least 1500 roots; colour coding as in a. Grey asterisks or dots indicate statistically significant differences in cytokinin concentration between symplast and apoplast by paired sample t-test. Significance levels are: dot, p < 0.1; *p < 0.05; **p < 0.01.