(A) big1,2,4 (big1 big2 big4), big2,3,4 (big2 big3 big4), big1,3,4 (big1 big3 big4) and big1,2,3/+,4 (big1 big2 big3/BIG3 big4) mutant plants without obvious phenotype but big1/+,2,3,4 (big1/BIG1 big2 big3 big4), big1,2/+,3,4 (big1 big2/BIG2 big3 big4) and big1,2,3 (big1 big2 big3) were dwarfed (yellow arrowheads). Scale bar, 2 cm. (B) F1 of reciprocal crosses between wild-type (Col) and big1 big2 big3/BIG3 big4 (1,2,3/+,4) mutants: 0% or 48% big3 heterozygous seedlings derived from mutant male or female gamete, respectively. (C) BFA inhibited primary root growth of big3 mutant seedlings with or without BFA-resistant GNOM (GNR big3). Numbers of analysed seedlings are indicated (B and C). (D-H) BFA treatment did not prevent seed germination in wild-type (Col; D) and BFA-resistant GN (GNR; G) but did so in big3 mutants without (E) or with BFA-resistant GNOM (GNR big3; H). This defect was suppressed by BFA-resistant BIG4 (UBQ10::BIG4R-YFP big3; F). Scale bar, 5 mm. (I-L) Live imaging of BIG4-YFP (I) and TGN marker VHA-a1-RFP (J) revealed co- localization (K; L, intensity–line profile). (M–P) Immunolocalization of BIG4 (UBQ10::BIG4-YFP; M) and Golgi-marker γCOP (N) indicated no co-localization (O; P, intensity–line profile). (I–K, M–O) Scale bar, 5 μm.