Table S1 : Partial lethality of the tsn1tsn2 mutation. Genotypes of representative progenies of tsn1tsn2/TSN1tsn2 or tsn1tsn2/tsn1TSN2 autopollinating plants, established by PCR at the seedling stage (details in Materials and Methods). Observed and expected frequencies of genotypes at the segregating TSN1 or TSN2 loci are shown. In each family, the double mutation (-/-) is significantly under- represented.

Figure S1.

tsn1 (Ws)tsn2 (Col)x Isolation of tsn1tsn2 plants Transformation with TSN1 or TSN2 genomic locus Complemented lines : double mutant carrying a TSN ectopic allele (homozygous, single copy) Nearly isogenic double mutants : co-segregating double mutant with no TSN ectopic allele “TSNEC”“tsn1tsn2” Figure S2. Obtention of tsn1tsn2 mutants, and strategy employed to generate nearly isogenic pairs of complemented and double mutant lines. tsn1tsn2 plants were isolated in the F2 of a tsn2 x tsn1 cross. Their descendants (F3) were transformed with TSN1 or TSN2 genomic loci. T3 plants (F6 with respect to the initial cross) expressing a single copy of the ectopic allele of either the TSN1 or TSN2 transgenic locus were isolated (TSN1 EC or TSN2 EC plants, EC : ectopic). In parallel, co-segregating and nearly isogenic double mutants were re-isolated for each of the TSN EC independent line in the T3(F6) and T4(F7) generations, respectively.Double mutant and TSN EC plants were propagated thereafter as pools. F1 F2 F3 F4 / T1 F5 / T2 F6 / T3 Sister line carrying a TSN ectopic allele (single copy and heterozygous) F6 / T3 F7 / T4 pool F6:7 pool F7:8 Nearly isogenic pair

TSN1 EC tsn1tsn2 Figure S3 Cell elongation defect in tsn1tsn2 roots of 5 days old seedlings. Length of the first epidermal cell with visible root hair bulge (LEH; REFF Le et al, 2001) was measured (n ~120) on two independent nearly isogenic pairs (A and A13.3.3) of tsn1tsn2 and TSN1 EC lines. Standard error bars are indicated. *, ** Significant difference : t-test P value <0,001. TSN1 EC tsn1tsn2 A13.3.2A ** TSN1 EC tsn1tsn2

Figure S4 Organ distribution of TSN1 and TSN2 transcripts in publicly available transcriptomic data (

Figure S5 Abiotic stress and ABA independence of TSN expression. (A-D) ABA or salt treatments do not modulate TSN accumulation in 7 days old seedlings. Seedlings were transferred onto plates containing 100mM NaCl or 10µM ABA for 4h (A, B) or 20h (C, D). tsn1 and tsn2 single mutants allow the exclusive detection of TSN2 and TSN1 proteins, respectively. Detection of N- myristoyltransferase (NMT) serves as control. (E) Effect of abiotic stresses of TSN1 and TSN2 transcripts in AtGenExpress root transcriptome data (Schmid et al., 2005), visualized at Fold repression/induction is expressed as ratio in log2 units, shown at the top. Blue and red dots, TSN1 and TSN2 RNA levels, respectively. No significant regulation of TSN transcripts by other stimuli/hormones was detected in public transcriptomic data. Coltsn2-1WStsn1-1 NaCl TSN NMT TSN NMT A CD B GM NaCl ABA GMNaCl ABA GM ABA GM ABA E TSN NMT TSN NMT

Figure S6 tsn1tsn2 is hypersensitive to mannitol stress (A) Fresh weight (FW) measurements of 21 days old plants grown in the presence of 180mM mannitol, reported as percentage of FW on control plates. Mutant plants are significantly smaller (Mann & Whitney test P <0,001, n=9 samples containing 50 plants in total). (B) Germination in the presence increasing concentration of mannitol. Hypersensitivity of tsn1tsn2 becomes apparent at 270mM mannitol. ** chi-2 independence test : P< 0,001 AB TSN1 EC tsn1tsn2 **

Rosette size (cm) Seed set (mg) Dry weight (mg) Seeds per silique Inflorescence length (cm) TSN1 EC tsn1tsn2 Silique filling Figure S7 : Growth and fitness of tsn1tsn2 and TSN1 EC plants in the greenhouse. (A-C) Growth traits : (A),rosette size; (B), main inflorescence length; (C), dry weight of aerial parts. (D-G) Fitness traits : (D),silique filling; (E),seeds per silique; (F), seed set. (G), pictures of representative siliques. Size difference has not been quantified in these conditions. Clear gaps in the siliques correspond to “missing seeds” employed to evaluate silique filling. *, ** Significant difference : t-test P values <0,05 and <0,001 respectively. ABC DEF G TSN1 EC tsn1tsn2 A A ** *

TSN2 EC Col tsn1 tsn2 Ws TSN1 EC tsn1tsn2 * * (cm) tsn1tsn2WsCol AB TSN1 EC TSN2 EC tsn1tsn2 TSN1 EC tsn1tsn2 C D Seeds per silique E Silique length (cm) Number of stems I + IIIII I FG Siliques cm -1 Figure S8. Growth and fitness of tsn1tsn2 and TSN1 EC plants under optimal conditions. (A), rosette morphology (no significant differences were measured); (B), main inflorescence length. (C) Cartoon depicting the positions of (I), primary; (II) secondary; (III), tertiary stems considered for branching analyisis. (D-G) Fitness traits: (D), branching; (E) silique density on the main inflorescence (+); (F), seeds per silique; (G), and silique length. *, ** Significant difference : t-test P values <0,05 and <0,001 respectively. ** * THIS PLANT TOO SMALL

Root length (cm) Germination (%) 200mM NaCl AB Figure S9. Complementation of the root elongation and germination phenotypes of tsn1tsn2 by TSNpro-GFP-TSN constructs. (A,B) Root length of 5 days old seedlings. (C,D) Relative germination frequency on medium supplemented with 200mM NaCl Identities of individual lines and genotypes are indicated at the bottom. All diiferences within pairs of tsn1tsn2 and GFP lines are statistically significant ( t-test P value <0,001 for A, B; chi-2 P value <0,001 for C,D) AJ1.1AJ1.8AJ4.3AJ4.16 AK22AK5AK21AK13 TSN1pro-GFP-TSN1 tsn1tsn2 CD scales and axes TSN1pro-GFP-TSN1TSN2pro-GFP-TSN2

TSN1 EC tsn1tsn2 TSN2 EC tsn1tsn2 Full-length ARF10 mRNA 3’ fragment A B Figure S10. RISC directed mRNA cleavage activity in tsn1tsn2. (A) Detection of the 3’ product generated by miR160-directed cleavage of ARF10 mRNA in roots by Northern blot. The probe hybridizes downstream of the cleavage site and allow the recognition of full length and cleavage products. Similar amounts of the 3’ cleavage product are present in tsn1tsn2 and TSNEC lines. (B) Quantification of well characterized miRNA targets in 7 days old roots by QPCR. A representative experiment is shown. Levels in TSN1EC (white bars) and tsn1tsn2 (black bars) roots under standard conditions (left panel) or transferred for 2 days on media containing 100mM NaCl (right panel), are expressed in actin units (a.u.). Oligonucleotides surrounded the cleavage sites or were placed downstream of it (ARF10-3’ and ARF17-3’). POINT TO TABLE with IDENTITIES AGI, REFF OLIGOS +100mM NaCl a.u. Standard medium TSN1 EC tsn1tsn2 C

Figure S11. tsn1tsn2 downregulated transcripts are targets of the Unfolded Protein Response. Levels of the five most strongly and robustly downregulated transcripts in tsn1tsn2 were determined by RT-qPCR of root RNA,after chemical induction of the UPR with Tunicamycin (T) or DTT (D). All values are expressed in actin units (a.u.). TSN1 EC tsn1tsn2

Figure S12. Effect of abiotic stresses on the abundance of tsn1tsn2-dependent transcripts. AtGenExpress root transccriptome data ( Schmid et al., 2005) visualized at visualized at Fold repression/induction is expressed as ratios in log2 units, shown at the top of each panel. Levels of (A), downregulated, and (B) upregulated transcripts ( cf. Table 1) are particularly responsive to salt and osmotic stress. Stimuli are ordered from the lowest to the highest ratio of transcripts AT3G53980 and AT5G23020 (boxed in blue). A B