Fig. S1. Early markers of floral organ boundaries are not affected in nev flowers. β-Glucuronidase expression of the 2870, YJ44, 5311 and 11411 markers in Arabidopsis inflorescences. The temporal and spatial patterns of each of these markers of the proximal regions of the outer differentiating floral organs are not affected in nev mutant flowers as compared with wild type. The YJ44 enhancer trap line was kindly provided by Yuval Eshed and John Bowman and was generated by Agrobacterium-mediated transformation with the plasmid pOCA-28-15-991 (Eshed et al., 1999). The 2870, 5311, 11411 enhancer trap lines were kindly provided by Tom Jack and are from the Jack collection described previously (Campisi et al., 1999).

Fig. S2. Mutations in NEV affect fruit growth. (A) Mature nev fruit (late stage 17) are significantly shorter than wild type. The average length of nev-3 fruit (7.7±0.5 mm; n=50) was 61% of that of wild-type fruit (12.7±0.5 mm; n=50). (B) Measurement of wild-type and nev-3 fruit length throughout development, from the youngest open flower (position 1; stage 13/14) to mature fruit (position 25; late stage 17) on primary inflorescences. A significant decrease in length is apparent in nev fruit (position 8) just prior to the initiation of a rapid growth phase in wild-type fruit (position 9). For each position on an inflorescence n=6.

Fig. S3. Identification of a candidate NEV ortholog in rice. Sequence alignment of NEV and a related protein from rice (Oryza sativa). Amino acids conserved between NEV and other proteins are shaded, and a line above the sequences indicates the region corresponding to the ARF-GAP domain.

Fig. S4. Developmental expression profile of NEV. NEV is expressed ubiquitously, in contrast to the dynamic expression profiles of HAE and IDA. Substantially higher levels of HAE and IDA transcripts are apparent in sepals just before abscission (stage 15, arrowhead) compared with sepals in unfertilized flowers (stage 12, arrow), whereas levels of NEV increase only slightly. AtGenExpress data (Schmid et al., 2005) http://jsp.weigelworld.org/resources.

Fig. S5. NEV co-localizes with a subset of endosomal compartments. Immunofluorescent localization of NEV and endomembrane markers in primary root epidermal cells of transgenic marker plants. Compartments tested for co-localization included the prevacuolar compartment (PVC)/late endosome (YFP-RabF2a), the endoplasmic reticulum (ER) (YFP-NIP1;1) and the plasma membrane (PM) (YFP-NPSN12). NEV staining is distinct from both the PVC/late endosome (A) and the ER (B); however, frequent association is seen between NEV and the PM marker NPSN12 (arrows, C). Partial overlap is also seen between NEV and YFP-RabD2a, a marker that is predicted to associate with Golgi and endosomal compartments (arrows, D). Scale bars: 5 m.

Fig. S6. VTI12 co-localizes with VHA-a1 at the trans-Golgi network. Immunofluorescent detection of the YFP-VTI12 and VHA-a1-RFP trans-Golgi/early endosome markers in primary root epidermal cells of transgenic plants carrying both markers. Nearly complete association is seen between these markers. Scale bar: 5 m.

Fig. S7. NEV localizes to BFA-sensitive endosomal compartments. Immunofluorescent detection of NEV in wild-type primary root epidermal cells treated with (+BFA, 100 M) or without (−BFA) BFA suggests that NEV is associated with endosomes undergoing BFA-sensitive sorting. NEV antigen condenses into structures resembling BFA bodies derived from TGN/early endosomal membranes. Scale bars: 5 m.

Fig. S8. Mutations in NEV alter the location of the trans-Golgi network. Transmission electron micrographs of cells at the base of wild-type and nev sepals at the time of shedding (stage 16). Whereas vesicular-tubular clusters of the trans-Golgi network (t, TGN) are frequently observed (84%, n=19) near Golgi stacks (g) in wild-type cells (A-C), these compartments are not readily apparent near the circularized multilamellar structures (cg) characteristic of nev cells (D-F). Occasionally, structures that could be TGN vesicles (arrow) are found within these circularized structures (F; 12%, n=17). Scale bars: 0.5 m.

Fig. S9. The fruit and stems of nev flowers also show membrane trafficking defects. Low-magnification transmission electron micrographs of cells in the fruit wall and pedicel (stem) of wild-type and nev flowers (stage 16). Paramural vesicles are found at a higher frequency in nev cells (B,D) as compared with wild-type cell (A,C). Analyzed cells are colored aquamarine, and sites with more than 30 vesicles or 10-30 vesicles are indicated by red and blue circles, respectively. Scale bars: 10 m.