doi:1.138/nture1794 BR EPFs BRI1? ERECTA TMM BSKs YDA PP2A BSU1 BIN2 pbzr1/2 BZR1/2 MKK4/5/7/9 MPK3/6 SPCH Cell growth Stomtl production Supplementry Figure 1. The model of BR nd stomtl signling pthwys. () nd () show previously defined BR nd stomtl development pthwys, respectively. This study shows tht BR regultes stomtl development through BIN2 phosphoryltion of YDA (red line), nd provides genetic evidence for importnt roles of BSUs nd BIN2/GSK3s downstrem of ERECTA in regulting stomtl development (dshed lue lines). Arrows indicte ctivtion nd r ends show inhiitory effects. Solid lines indicte direct iochemicl regultion nd dotted lines, possile regultion inferred from genetic dt or hypothesized sed on indirect evidence.! WWW.NATURE.COM/NATURE 1
RESEARCH SUPPLEMENTARY INFORMATION 5 4 Pvement cell Meristemoid + Gurd mother cell Gurd cell pirs Numer of cells (per mm 2 ) 3 2 1 Supplementry Figure 2. Quntifiction of stomt nd precursors in xil cotyledons of BR-relted mutnts. The totl numers of ech cell type in BR-relted mutnts were counted to clculte the frequency of cell type shown in Figure 1U. Ws ws used s wild-type control for the in2-3;il1;il2 mutnt. Error rs = ±SE.! 2 WWW.NATURE.COM/NATURE
RESEARCH Wild type 2 dpg Wild type 3 dpg su-q 2 dpg su-q 3 dpg Supplementry Figure 3. Genertion of excess meristemoids, gurd mother cells nd gurd cells in su-q. DIC imges of wild-type Col- () nd su-q () xil cotyledons t 2 (left pnels) nd 3 (right pnels) dyspost-germintion (dpg). Exmples of meristemoids re noted with red sterisks. Scle rs, 5 μm.! WWW.NATURE.COM/NATURE 3
RESEARCH SUPPLEMENTARY INFORMATION wild-type su-q Supplementry Figure 4. The su-q mutnt seedlings develop incresed numers of clustered stomt on hypocotyls. DIC imges of hypocotyls of wildtype () nd the su-q mutnt () seedlings grown under continuous light for 7 dys. Red sterisks nd rckets indicte single nd clustered stomt, respectively. Scle rs=1 μm. 4 WWW.NATURE.COM/NATURE
RESEARCH ri1-116 ri1-116;zr1-1d Supplementry Figure 5. The zr1-1d muttion suppresses the dwrf phenotype of ri1-116. Phenotypes of 4-week-old ri1-116 nd ri1-116;zr1-1d mutnt plnts. Scle r = 1 cm. WWW.NATURE.COM/NATURE 5
RESEARCH SUPPLEMENTARY INFORMATION Numer of cells (mm 2 ) 15 12 9 6 3 PC M + GMC GC (%) 3 %M + GMC %GC 2 1 Supplementry Figure 6. Quntifiction of stomtl linege cell types cross entire xil cotyledons of 8-dpg plnts overexpressing BSU1 fmily memers. () Numer of cells of ech epiderml clss. () frequency of cell types clculted from (). PC, pvement cell; M, meristemoid; GMC, gurd mother cell; GC, gurd cell pir. Error rs = ±SE. Sttisticl significnces compred to WT were clculted y using student s t-test. P<.2, P<.1. 6 WWW.NATURE.COM/NATURE
RESEARCH er;erl1(+/-);erl2 er;erl1(+/-);erl2 - ik + ik Supplementry Figure 7. Bikinin effects on stomtl development in ERf mutnts. The er;erl1(+/-);erl2 mutnt plnts were grown on MS medium contining mock (DMSO, -ik) or 3 μm ikinin (+ik) for 8 dys. Scle rs = 5 µm. WWW.NATURE.COM/NATURE 7
RESEARCH SUPPLEMENTARY INFORMATION PC M + GMC GC Numer of cells (mm 2 ) 5 4 2 1 (%) 1 %PC %M + GMC %GC 8 6 4 2 Supplementry Figure 8. Quntifiction of ikinin effect on stomtl development of 8-dpg stomt-relted mutnts (s shown in Figure 2). () Numer of epiderml cells of mutnts grown on MS medium treted with DMSO (mock, unleled on xis) or 3 um ikinin. () frequency of cell types clculted from (). C24 ws used s wild-type for the yd mutnt. PC, pvement cell; M, meristemoid; GMC, gurd mother cell; GC, gurd cell pir. Error rs = ±SE.! 8 WWW.NATURE.COM/NATURE
RESEARCH 8 PC M + GMC GC Numer of cells (mm 2 ) 6 4 2 (%) 1 %GC %M + GMC 8 P<.1 6 4 2 Supplementry Figure 9. Quntifiction of rssinzole effect on stomtl linege development of 8-dpg er;erl1;erl2 nd tmm mutnts. Epiderml cell numers () nd frequency of cell types () in the er;erl1;erl2 nd tmm mutnts grown on MS medium without BRZ or with BRZ. PC, pvement cell; M, meristemoid; GMC, gurd mother cell; GC, gurd cell pir. Error rs = ±SE. P vlue denotes significnt difference etween BRZ-treted nd untreted smples (Pired student s t-test. P<.1).! WWW.NATURE.COM/NATURE 9
RESEARCH SUPPLEMENTARY INFORMATION Supplementry Figure 1. Amino cid sequence of YDA protein. Puttive GSK3 phosphoryltion sites re shown in red. Ctlytic domin of YDA is highlighted in lue, nd region deleted in CA-YDA, in yellow. 1 WWW.NATURE.COM/NATURE
RESEARCH MBP + - - + MBP-YDA - + + - GST-BIN2 + + - - 32 P-YDA none + MBP-YDA MBP-BZR1 MBP-BSL1 MBP-BSL2 GST-PP2A B β 32 P-BIN2 Autordiogrph MBP-YDA GST-BIN2 CBB MBP Supplementry Figure 11. BIN2 phosphoryltes YDA in vitro. GST-BIN2 phosphoryltes MBP-YDA ut not MBP. YDA does not phosphorylte other BR signling components BZR1, BSL1, BSL2 or PP2AB β. CBB, coomssie rillint lue stining. WWW.NATURE.COM/NATURE 11
RESEARCH SUPPLEMENTARY INFORMATION GST-mBIN2 GST-BIN2 MBP-YDA Bikinin GST-mMKK4 32 P-mMKK4 GST-mMKK4 + + - - - + - - + + + - - + + + + + + + 3.1 3.1 1.6 1. MBP-YDA Supplementry Figure 12. BIN2 phosphoryltion of YDA inhiits YDA phosphoryltion of MKK4. Replicte of in vitro kinse ssy shown in Figure 3h. YDA phosphorylted y BIN2 or mbin2 (kinse-inctive mutnt) ws purified, nd then incuted with mmkk4 nd 32 P-γ-ATP. Bikinin ws dded to prevent BIN2 phosphoryltion of mmkk4 nd to confirm ikinin does not inhiit YDA kinse ctivity. Numers indicte reltive signl level of 32 P-mMKK4 normlized to totl GST-mMKK4.! 12 WWW.NATURE.COM/NATURE
RESEARCH Col- er;erl1;erl2 BL - + - + Anti-BZR1 Ponceu S er;erl1;erl2 Col- ri1-116 in2-1 Reltive gene expression 3 2 1 er;erl1;erl2 + BL Col- + BL ri1-116 + BL in2-1 + BL SAUR-Ac1 IAA19 DWF4 Supplementry Figure 13. BR signling does not require ER fmily receptors () Immunolot nlysis of BZR1 in wild-type nd the er;erl1;erl2 mutnt indicting tht BZR1 phosphoryltion sttus in response to BL does not require ER fmily receptors. Seedlings grown on MS medium for 7d were treted with mock or 1 nm BL for 3 min. Immunolot ws proed y nti-bzr1 ntiody. () Quntittive PCR nlysis of BR-regulted genes indictes norml response to BL tretment in er;erl1;erl2 mutnts (similr to Col, SAUR-Ac1 nd IAA19 re induced nd DWF4 repressed upon tretment) vs BL insensitive ri1 nd in2 mutnts. Seedlings were grown on MS medium without BL or with BL (1 nm) for 7d.! WWW.NATURE.COM/NATURE 13