A B TR/ TR1.1 TR4/TR5. C ene name Promoter size (bp) Atg27 TR 251 At4g00 TR4 28 At4g0 TR5 2570 At5g90 TR1 81 TR TR1 TR4 TR5..1 D ene ID E Mw l-0 Co R R5 R4 R1 T T T T 55 ] - 5 25 Loading control Supplemental Figure 1. Characterization of fluorescently-labeled proteins (A) enomic organization of.1 (TR and TR1) and. (TR4 and TR5) genes used in this study. The genomic sequence was fused to appropriate tags and expressed in transgenic Arabidopsis plants under their endogenous promoters, as indicated in the main text and Experimental Procedures. (B) Size of promoters used to express each TR gene. (C) Absolute amount of endogenous and transgene expression. Copy number of each endogenous gene (grey bars) and each - transgene (dark green) was determined in wild type Col-0 and tagged lines, respectively. Data are the mean values ｱ s.d., n =. (D) Representative examples of the localization pattern of proteins used in this study in the root meristem nuclei of plants described in panel A. (E) Nuclear extract of Col-0 and transgenic plants expressing the indicated - proteins were fractionated and the - proteins detected with anti- antibodies. The line correspond to a line expressing alone. Note that the slight different mobility of - is due to the different vectors used that provide slightly different linkers between the and the moieties. 1
TR + TR5.1-.-mR A cell wall B B1 A1 D C1 D1.1- TR1 TR4.- TR5 Cortex TR C F I J K L M N O P QC plane Epidermis E Supplemental Figure 2. Expression pattern of various fluorescently labeled lines used in this study. (A-D) Embryos of plants expressing both.1 (TR-) and. (TR5-mR) at various stages: globular (A), early (B) and late (C) heart, and torpedo (D). Insets A1-D1 correspond to the sections highlighted in the upper panels. Arrowheads point to the QC. (E-P Examples of the patchy and constitutive expression patterns of.1(tr- and TR1-; E,F,I,J,M,N) and. (TR4- and TR5-;,,K,L,O,P), respectively, in the cortex (A-D), epidermis (I-L) and middle plane (M-P) of the root apical meristem. Note that panels I and L are also shown in Figs. 2A and 2B, respectively. 2
0 Atrichoblasts 0 Trichoblasts -0.5-0.5-1.0-1.0...1...1 Supplemental Figure. Relative position of mitotic figures in trichoblast and atrichoblast cell layers. Mitotic figures labeled with.(tr5-mr) only or with both.1(tr-) and.(tr5-mr) were scored along the RAM as described in Fig. 2F. Atrichoblasts, n=27; trichoblasts, n=26. The two populations were statistically different (p=0.05, t-test) in the case of atrichoblasts.
A differentiated wt ( -). (TR5- +).1 (TR1- +) 4 4 4 2 2 2 1 1 1 1 2 4 4 4 1 2 4 4 1 2 4 B 5 4 2 1 apical wt ( -). (TR4- +).1 (TR- +) 5 4 2 1 5 4 2 1 1 2 4 5 differentiated 4 5 1 2 4 5 4 5 1 2 4 5 4 5 5 wt ( -) 5 5 4 2 4 2 4 2 1 1 1 1 2 4 5 4 5 1 2 4 5 4 5 1 2 4 5 4 5 Supplemental Figure 4. Analysis of ploidy levels of -tagged nuclei (A) Analysis of ploidy level of wild type (Wt, ),.(TR5)- and.1(tr1)- nuclei of the differentiated part of the root (the entire root except the 5 mm apical part) by two-channel flow cytometry. was used for nuclear staining and determination of DNA content (2-16C). Wild type Col-0 and. (TR5)- nuclei were used to define the (left panel) and + (mid and right panels) gates, respectively. (B) Analysis of ploidy level of wild type (Wt, ),.(TR4)- and.1(tr)- nuclei of the 5 mm apical (upper panels) and differentiated (lower panels; as defined in panel a) parts of the root. was used for nuclear staining and determination of DNA content (2-16C). Wild type Col-0 and.(tr4)- nuclei were used to define the (left panels) and + (mid and right panels) gates, respectively. 4
Stage I Stage IV Supplemental Figure 5..1- labeling during initiation of lateral roots. Cell cycle reactivation of lateral root founder cells is associated with incorporation of.1, since stage I. At later stages, e.g. stage IV, nuclei show different levels of.1- labeling, indicative of cells at different phases of the cell cycle. Note that, frequently, cells of central cylinder appear -labeled. The exact nature of these nuclei remains to be determined. 5
Supplemental Table 1. Chromatin and cell cycle genes downregulated from slice 1 + 2 to slice 4 (fold change fc > 1.5) ene ID AT4g0 AT5g19 AT4g08 ATg226 AT122 AT177470 AT161 AT AT11 AT275 AT4299 AT558 AT2167 AT259 AT2164 AT5491 AT116970 AT5488 AT21270 AT2000 AT244950 AT427 AT84 AT561 AT507 AT185 AT48 AT15150 AT2451 AT154690 AT518 AT2550 AT0 AT5146 AT5254 AT5167 AT11 AT190 AT1765 AT5649 ene name SWINER SUV4 SD4 RDM1 RBR RFC RFC2 MCM7 ORC4 ORC2 ORC5 MSI1 MSI2 MAD2 MCM4 MET1 KU70 ICK6 CDT1A OBBIT UB1 TA2 DA08 DA6 D2C DA15 D1 PY2 AM1 AMMA-2AX FZR ET1 DEL/E2FF DRM2 DNMT2 DOT2/MDF CDKD1; CDKB2;2 CDKB2;1 CDKC2 AT50070 AT5672 AT11 AT176 AT456 AT2176 AT144 AT2267 AT115 AT170 AT1441 AT169770 AT24 AT519 AT1156 AT016 AT670 AT166750 AT509790 AT48150 AT178770 AT218290 AT5 AT54990 AT1496 AT248 AT170 AT7790 AT2195 AT55090 AT1447 AT8 AT8590 AT1578 AT59550 CYCD; CYCD;2 CYCB;1 CYCB2;4 CYCB2;2 CYCB2;1 CYCB1;5 CYCB1;4 CYCB1; CYCA2;4 CYCA1;1 CMT CR CR2 CENP-C CDC48C CDC48B CAK4 ATXR5 APC8 APC6 APC ARP6 SUVR2 ICK5 ICK TR12 TB1 MSI4 ISTONE ISTONE CAPERONE DOMAIN ISTONE CAPERONE DOMAIN VIM6 VIM1 VIM 6
Chromatin and cell cycle genes downregulated from slice 2 + to slice 4 (fold change fc > 1.5) ene ID AT59550 AT428190 AT5049 AT5159 AT8 AT122 AT5546 AT224490 AT161 AT AT11 AT275 AT1268 AT4299 AT558 AT45050 AT5491 AT116970 AT21270 AT427 AT15 AT185 AT5228 AT5567 AT1798 AT154690 AT0 AT5167 AT5649 AT47 AT5 AT25 AT52 AT218290 AT170 AT7790 AT88 AT55090 AT400 AT50 ene name VIM ULT1 SUV1 SMC5 SD4 RBR TA1 RPA2 RFC2 MCM7 ORC4 ORC2 ORC6 ORC5 MSI1 MSI MET1 KU70 CDT1A TA2 TA DA15 TB2 A2 L2 AMMA 2AX DEL/E2FF DOT2/MDF CDKC2 CYCD5;1 CYCA;1 CDC45 ATXR6 APC TR12 TB1 TA5 ISTONE ISTONE ISTONE AT590 AT27470 AT4590 AT27 ISTONE ISTONE ISTONE ISTONE 7
Supplemental Table 2. Primers used in this study Cloning of TR genes TR genes were cloned using the following primers: TR-W-F: 5 -ACAATTTTACAAAAAACACTATCCATCTCTTACTTTCCTA- TR-W-R: 5 -ACCACTTTTACAAAAACTTCACCCTCTCACCTCTAATCC- TR4-W-F: 5 -ACAATTTTACAAAAAACACTAAACTCCCTTTCCAA- TR4-W-R: 5 -ACCACTTTTACAAAAACTTCACCTTCACCTCTATAC- TR5-W-F: 5 -ACAATTTTACAAAAAACACTCATTTACTCAAAACAATA- TR5-W-R: 5 -ACCACTTTTACAAAAACTTCACACTTCTCCTCTATCCT- TR1-W-F: 5 -ACAATTTTACAAAAAACACTATTCTTCTTCATCTCCAT- TR1-W-R: 5 -ACCACTTTTACAAAAACTTCACTCTTTCTCCTCTATTCTCC- qpcr measurements The expression of the TR genes in Col-0 plants was monitored using the following primers: TR-endo-F 5 -TCAAACACACATCTCAACAA- TR-endo-R 5 -CTACTTTCCTACAATCT- TR4-endo-F 5 -CCTTAACCAACCAAAAT- TR4-endo-R 5 -ATTAAAAAATTCCAACACAC- TR5-endo-F 5 -CTTAATTTATTAA- TR5-endo-R 5 -CACCATTTTTCCAATCTCC- TR1-endo-F 5 -CAAAATCAAAA- TR1-endo-R 5 -CCAAATCAAAATCCAAAA- The expression of the TR- tagged genes was measured using the following primers: TR--F 5 -TATCATCCACTCTTC- TR--R 5 -TTTTACAAAAACTTCA- TR4--F 5 -CTAATTCTCAAATTTCA- TR4--R 5 -TTTTACAAAAACTTCA- TR5/1--F 5 -ACAACAAAAAACCATC- TR5/1--R 5 -AAACAATTTTAC- 8