Extended Data Fig. 7. Cytokinin controls PEAR expression.

a, Expression of auxin (pIAA2::GFP-GUS) and cytokinin (pARR5::RFPer) response genes (n=15). Auxin response is restricted to the xylem cells at initial stage (a’) and maintained during development (a”, a”’). High cytokinin response is activated initially and maintained in PSE and its neighbouring cell (a’, aa’), and later becomes concentrated into the intervening procambial cells flanking xylem cells (a”’). b, Exogenous cytokinin application rapidly promotes the transcript level of PEAR genes, including PEAR2, DOF6, and TMO6. Asterisks indicate significant (p<0.05) upregulation as determined by a two-sided t-test on three biological replicates. Bar graphs represent mean. Error bars are s.e.m. For individual P values, see Supplementary Table 3. c, Sustained cytokinin application leads to ectopic transcription of PEAR genes. The optical cross section images are obtained after 4 days (for PEAR1, PEAR2 and HCA2) or 1-day (DOF6 and OBP2) treatment of 1μM of BA. d, Conditional induction of CRE1 expression restores TMO6 transcription in wol root. In the absence of cytokinin response, TMO6 transcription is increased in pericycle and attenuated in the vascular tissue (Control, hashtags), and is restored in the vascular tissue after CRE1 induction (Est). e-l, Expression pattern of auxin (pIAA2::GFP-GUS) and cytokinin (pARR5::RFPer) response reporters during embryogenesis in wild type (e-h, n=29, 13, 13, 11, respectively) and wol (i-l, n=12, 8, 6, respectively). At globular stage, auxin response is activated among provascular cells both in wild type (e) and in wol (i). At early heart stage, cytokinin response is activated in cells positioned below shoot apical meristem (f, ult, arrowheads), and the stripe of cytokinin response domain is formed by mid heart stage (g, ult, arrowheads), simultaneously auxin response becomes concentrated in the cells proximal to the cotyledon (g, ult, asterisks), resulting in the bisymmetric hormonal response pattern. During the torpedo stage, cytokinin response domain reaches to llt (h, llt, arrowheads). In wol embryos, activation of cytokinin response in vascular tissue does not occur and a radial auxin response pattern is maintained (i-l). m-r, Expression of ARR5 and PEAR1 during embryogenesis. In the wild-type embryo (m-o, n=17, 25, 10, respectively), PEAR1 is broadly transcribed among provascular cells both in ult and llt with radial symmetric pattern at the globular stage (m). At the heart stage, PEAR1 transcription is enhanced in ult cells underneath the shoot apical meristem, which correlated with the activation of cytokinin response in this domain (n, arrowheads), and expression of both ARR5 and PEAR1 extends rootward and reaches to llt, becoming more concentrated within the cell files where phloem is specified post-embryonically (o, arrowheads). In wol embryos (p-r, n=19, 13, 13, respectively), PEAR1 transcription is initiated among provascular cells at the globular embryo stage (p) similar to wild type (m), but neither cytokinin response nor PEAR1 transcription occurs in ult at the heart stage (q), and PEAR1 expression is gradually attenuated by the torpedo stage (r). ult and llt represent upper- and lower tier, respectively. In a, e-r, n represents independent biological samples. In c and d, number in each panel indicates samples with similar results of the total independent biological samples analysed. Scale bars represent 25 μm.