Supplemental Figure 1

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Figure 1 : Sequence logo representation of the six motifs defined from the 34

TRM proteins

The overall height of the stack of residues indicates the sequence conservation at each

position, while the height of residues within the stack indicates the relative frequency of each

amino or nucleic acid at that position (bits = information contents in bits). Amino acids are

colored according to their chemical properties: polar amino acids (G, S, T, Y, C) are green,

amid (Q, N) are purple, basic (K, R, H) are blue, acidic (D, E) are red and hydrophobic (A,V,

L, I, P, W, F, M) are black.

The significance of each motif is indicated by its E-value (from E-value = 1.6 e-24

for the M6

motif to E-value = 5.4 e-127

for the M1 motif). The MEME tool defines a ungapped 25 residue

M2 motif, but a tryptophan residue is very often found at its N-terminal, separated from the

"core" motif by a variable gap of 0 to 3 residues ; a corrected, gapped version is presented

here. To define the M1-M6 motifs on the 34 Arabidopsis TRMs, the MEME tool (version

3.5.7) was used with the following parameters : -protein -mod anr -nmotifs 6 -minsites 17 -

minw 10 -maxw 100.

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Figure 2

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Figure 2 : Gene expression analysis of the TRM super family in Arabidopsis organs

using the Genevestigator tool (Zimmermann et al., 2004).

For each anatomy category, the figure displays the average expression value calculated from all

Arabidopsis Affymetrix arrays available. Results are displayed in linear scale as a percentage of

expression potential for each gene, calculated as the mean of the top 1% signals, from no or low

expression (white) to high expression (dark blue). No Affymetrix probes are available for the TRM5,

TRM11, TRM18, TRM27, TRM30 and TRM33 genes.

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Figure 3

A

B

Supplemental Figure 3 : Analysis of the ProTRM1:GFP-TRM1 transformed lines

(A) The ProTRM1:GFP-TRM1 construct complements the trm1 mutant phenotype.

In order to check the functionality of the ProTRM1:GFP-TRM1 fusion, we assessed its ability

to complement the trm1 phenotype. The trm1 mutant phenotype is rather mild (compare wild-

type Col-0 to trm1), and complementation was easier to assess in a trm2 mutant background

(compare trm2 to trm1 trm2). Therefore, we introduced the ProTRM1:GFP-TRM1 construct

in a trm1 trm2 double mutant line (lng2-1/SALK_034645 lng1-3/Salk_135585; (Lee et al,

2006)) and assessed its ability to revert a trm1 trm2 phenotype to a trm2 one. Out of 26

transgenic lines obtained, 20 displayed a phenotype similar to the trm2 mutant regarding

seedling and rosette morphology or length and width of silique, demonstrating the

functionality of the ProTRM1:GFP-TRM1 construct.

(B) Immunoblot analysis of GFP-TRM1 expression in Arabidopsis transgenic lines

expressing the pTRM1:GFP-TRM1 construct.

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Out of 26 Arabidopsis independent lines stably transformed with the pTRM1:GFP-TRM1, 14

displayed a fluorescence signal decorating microtubules arrays in petal cells (See Figure 3).

Six were chosen for further analysis of TRM1 protein expression and localization studies.

Western blot analysis using the anti-TRM1 antibody and flower buds tissues showed that

GFP-TRM1 (apparent molecular weight of ~150 kDa) expression level in transgenic lines is

similar to the endogenous TRM1 protein level in untransformed Col-0 plant (apparent

molecular weight of ~120 kDa).

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Figure 4

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Figure 4 : Charge plot analysis of TRM proteins.

The charge plot is shown in black, points above the protein representing basic domains and

the ones below, acidic domains. The eight groups of TRM proteins obtained by multiple

alignment procedures followed by neighbour-joining phylogenetic analysis and bootstrap

validation (1000 trials) are indicated on the left.

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Figure 5

Supplemental Figure 5 : Sequence alignment of M2 motifs in Arabidopsis TRM proteins

and CAP350 homologues. Identities and similarities are boxed. Identities are shaded in dark

grey and similarities in light grey.

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Accession numbers : Apis mellifera XP_393557.3; Strongylocentrotus purpuratus

XP_782826; Ciona intestinalis ENSCINP00000028718; Danio rerio XP_001343753.2;

Takifugu rubripes ENSTRUP00000024375; Gallus gallus XP_422260.2; Ornithorhynchus

anatinus XP_001515795.1; Homo sapiens Q5VT06; Branchiostoma floridae

B6N2E1_BRAFL ; Nematostella vectensis jgi|Nemve1|87242|; Lottia gigantea

jgi|Lotgi1|140328; Trichoplax adhaerens XP_002109308.1; Paramecium tetraurelia

GSPATP00002266001; Tetrahymena thermophila TTHERM_00474890

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Table 1

TRM #

AGI name

Growth

on MM

leu-

lacZ

% of

total

clones

Independent

clones

Predicted

protein

size

N-terminal residue

of clones

TRM1 At3g02170.1 ++ ++ 11 4 905 P756, R795, A822, K826

TRM2 At5g15580.1 ++ +++ 10 3 927 P535, E613, N789

TRM3 At1g18620.1 + ++ 3 2 978 T804, F855

TRM7 At3g58650.1 ++ +++ 7 1 820 P172

TRM11 At4g23020.1 ++ ++ 3 1 425 K175

TRM14 At3g61380.1 ++ ++ 5 1 718 R471

TRM19 At3g53540.1 +++ ++ 3 1 924 P594, L811

TRM20 At4g28760.1 ++ +++ 5 2 924 S677, R818

TRM21 At5g43880.1 ++ + 7 1 836 L667

TRM25 At5g62170.1 +++ - 4 1 703 L616

TRM22 At1g67040.1 ++ ++ 3 1 826 S604

TRM26 At2g17550.1 +++ +++ 5 3 779 P445, G594, E698

Supplemental Table 1 : TON1 two-hybrid interactants

Among 14 major families of clones identified in a two-hybrid screen using TON1b as a bait, one

corresponded to CEN1, an Arabidopsis centrin (Azimzadeh et al., 2008), one to a proteasome

subunit. The twelve other yeast interactants are summarized here. For each clone isolated in the

primary screen, growth on minimal medium without leucine, as well as activation of the lacZ reporter

gene were assayed and noted visually on a "-" to "+++" scale. The percentage of clones originating

from each gene is indicated, as well as the number of independent clones of different length and the N-

terminal starting residues of those.

Supplemental Data. Drevensek et al. (2012). Plant Cell 10.1105/tpc.111.089748

Supplemental Table 2

Primer name Construct Sequence TRM1 (+1) GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGTCTGCAAAACTTCT TRM1 (-133) GGGGACCACTTTGTACAAGAAAGCTGGGTCACCATTGGGCTGTTCTC TRM1 (+134) GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGGGCTTGATGATGCCGTAT TRM1 (-341) GGGGACCACTTTGTACAAGAAAGCTGGGTCTGCTTTCATTTGCTTCCA TRM1 (+342) GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGGGAGACTCTGCACTGACG TRM1 (-586) GGGGACCACTTTGTACAAGAAAGCTGGGTTTATGTTGCTGTCAGACC TRM1 (+587) GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGAGCTTGGGATCGAATGTT TRM1 (-827) GGGGACCACTTTGTACAAGAAAGCTGGGTATTTTTTAAGTGGATTTGC TRM1 (+828) GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGTTGGAGAAAATATCAAA TRM1 (+871) GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGAGTCAAAGCATGAACTTG TRM1 (-905)

Entry vectors of TRM1 and

truncated versions of TRM1

GGGGACCACTTTGTACAAGAAAGCTGGGTAGCAGAAGCAAACTTCAT ProTRM1-NheI TTTGCGCTAGCATCTTCTTCCTCTACTTAAC ProTRM1-AvrII

ProTRM1:GFP-TRM1 ACACCTAGGTTAAGCAACCGACTAATTGT

TRM2-GW1 GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGTCGGCGAAGCTTTTG TRM2-GW2

TRM2 entry vector GGGGACCACTTTGTACAAGAAAGCTGGGTAGCAATGAAAAAGCTGCC

TRM8GW1 GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGGAGGTTGTTGAGAGG TRM8GW2

TRM8 entry vector GGGGACCACTTTGTACAAGAAAGCTGGGTAGAATGCCGAGAGAAGAT

TRM20GW1 GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGAATGAACTCCGAGG TRM20GW2

TRM20 entry vector GGGGACCACTTTGTACAAGAAAGCTGGGTATCGTGTAAGATCGATAA

TRM25GW1 GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGGGAAGAGACTGG TRM25GW2

TRM25 entry vector GGGGACCACTTTGTACAAGAAAGCTGGGTATCCACGTGGAAGCCTTA

TRM26GW1 GGGGACAAGTTTGTACAAAAAAGCAGGCTACAAAATGGGAGGATTATTGC TRM26GW2

TRM26 entry vector GGGGACCACTTTGTACAAGAAAGCTGGGTAACATATTTGTCTTCTTA

CAP350-M2-GW1 GGGGACAAGTTTGTACAAAAAAGCAGGCTGGGAGGAGGCACAGTGGGTG CAP350-M2-GW2

CAP350 entry vector GGGGACCACTTTGTACAAGAAAGCTGGGTTCACACAAGTAGCATTCTCC

G-TON1A-P1 GTCGACCTGTAATTTTAAAATAGTAAACAA G-TON1A-P2 TCCATGGCAAAATCAAATCAAAGTAAGGAA G-TON1A-P3 GCCATGGACGATTATACAAGAGAGATGATG G-TON1A-P4 CCCATGGATTTGAAGGTAAATAAAGCAATC G-TON1A-P5 TCCATGGGCAGGCCGGTTTCTGCATCTCAA G-TON1A-P6 CAGCTGAGAAGGTACAATTTTTTCACAGAC GFP-PscI-U GGGGACATGTTGAGCAAGGGCGAGGAGCTG GFP-NcoI-L

GFP-gTON1a

CCCCCCATGGGTTTGTACAAACTTGTGATG

Supplemental Table 2 : List of the primers used in this study