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Fig. S1. Multiple evr alleles rescue shedding in nev flowers. Several mutant alleles of EVR rescue organ shedding in nev-3 flowers, including the evr-1 allele isolated from the suppressor screen (A), and the evr-3 and evr-4 T-DNA insertion alleles (J-L). The evr-2 mutation also rescues organ separation in nev-2 (C,D) and nev-6 (E,F) flowers (Liljegren et al., 2009), demonstrating that interactions between NEV and EVR are not allele-specific. As described for nev-3 evr-2 flowers (Fig. 1C), all nev evr double mutant flowers show collars of broken AZ cells at their bases (A,D,F,K,L). The morphology of AZ cells appears to be unaffected in each of the evr single mutants (B,G-I). Flowers (stage 17) of Ler (A-D), Col (E,G-L) and mixed Ler/Col (F) ecotypes are shown.
Fig. S2. Loss of EVR decreases the force required to remove petals from nev flowers. The force required to remove petals from wild-type, nev-3 and nev-3 evr-3 flowers (Col ecotype) was measured using a breakstrength meter constructed as described (Lease et al., 2006). Assays were conducted using the second open flower on an inflorescence through the sixth open flower (positions 2-6). If petals were already absent or shed during experimental manipulation, the force was recorded as zero. The breakstrength of wild-type petals (n≥7) gradually decreased from positions 2 to 4, and all organs were shed by position 5. For nev petals (n≥4), a significant decrease in breakstrength was not observed at the positions tested. The breakstrength of nev evr petals (n≥5) sharply decreased from positions 2 to 3, and all floral organs were shed by position 4.
Fig. S3. nev evr mutants show reduced growth and premature organ abscission. (A) Mutations in NEV cause a general decrease in plant size compared with wild type, and nev evr plants are typically smaller than nev plants of the same age. Other than reduced fruit growth (Fig. 2H), loss of EVR alone does not appear to affect plant size. (B) To track the shedding of floral organs with respect to time, we tagged individual flowers from wild-type and mutant plants at the time of bud opening (stage 13; n=5 per genotype). For each day, the percentage of flowers at each stage is shown. While wild-type and evr floral organs begin to shed on day 3, we observed nev evr floral organ shedding from stage 15* flowers beginning on day 2.
Fig. S4. Recombinant EVR can be dephosphorylated at tyrosine residues in vitro. (A) Gene diagram of EVR (At2g31880) and its neighboring genes. The protein coding regions are shaded dark gray or green, and the 5′ and 3′ UTRs are shaded light gray or green. The region selected for the EVR promoter begins 750 bp upstream of the EVR translational start site. The location of the small RNA cluster (AtsliRNA-1) encoded within the EVR/At2g31890 overlapping region is indicated by the arrowhead (Katiyar-Agarwal et al., 2007). (B) Wild-type (WT) and kinase-dead (K377E) EVR kinase domains (KDs) were treated with calf intestinal alkaline phosphatase (CIP; New England Biolabs, Ipswich, MA, USA) for 2 hours at 37°C to determine the extent of protein migration that is due to phosphorylation. CIP-treated EVR KDWT was observed to migrate at a position intermediate to that of the mock-treated KDWT and KDK377E, suggesting that the protein was not completely dephosphorylated. Duplicate blots were probed with antibodies specific for phosphorylated tyrosine, threonine and serine residues. While phosphate groups appear to be completely removed from EVR tyrosine residues by CIP treatment, dephosphorylation of both threonine and serine residues appears to be incomplete. As CIP is known to preferentially dephosphorylate tyrosine residues (Swarup et al., 1981), these results are not unexpected. Each of the phospho-antibodies recognizes both the CIP- and the mock-treated EVR KDWT. (C) EVR colocalizes with the lipophilic dye FM4-64 in epidermal root cells. The primary roots of pEVR::EVR-YFP T2 plants were stained with 10 µM FM4-64 (Invitrogen) for 5 minutes on ice, washed in MS liquid media, and imaged within 15 minutes. Imaging parameters were as follows: EVR-YFP (excitation, 488; emission, BP516-548) and FM4-64 (excitation, 543; emission, BP601-655). Scale bar: 10 µm.
Fig. S5. EVR shows a broad expression pattern during development. (A) RT-PCR analysis of EVR expression. EVR is expressed in mature cauline leaves and in flowers after the fruit begins to elongate and all floral organs are shed (stage 17), but is only weakly expressed in flowers prior to fruit elongation and shedding (stages 1-14). β-TUBULIN RT-PCR was used as a loading control. RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The reverse transcriptase reaction was performed on DNase-treated RNA using the Omniscript RT Kit (Qiagen). EVR was amplified with 5′-GAGTGATTCTTGGGATTCTGGTGATTG-3′ and 5′-AGATAGAAGCAAACAATACATATTGAAACAC-3′ for 30 cycles. β-TUBULIN was amplified with 5′-AGAGGTTGACGAGCAGATGA-3′ and 5′-ACCAATGAAAGTAGACGCCA-3′ for 35 cycles, using an annealing temperature of 55°C. (B) Global expression profiles of EVR, PEPR1 and At1g17750 (Schmid et al., 2005). Stage 12 sepals are indicated by the light gray arrow; stage 15 sepals by the dark gray arrow. (C) The regulatory region of EVR directs expression of β-glucuronidase (GUS) in the pedicels of the first true leaves of 5-day-old seedlings.
Fig. S6. PMB accumulation in nev, nev evr and evr AZs. (A-D) Transmission electron micrographs of longitudinal sections through wild-type (stage 16), nev (stage 16 non-shedding), nev evr (stage 15*) and evr (stage 16) flowers. Small PMBs (10-30 vesicles, blue dots) accumulate in sepal AZ cells of wild-type, nev, nev evr and evr flowers, whereas large PMBs (31 or more vesicles, yellow dots) were observed only in nev and evr AZ cells. Scale bars: 10 µm.