Takuji Ichino, Kentaro Fuji, Haruko Ueda, Hideyuki Takahashi, Yasuko Koumoto,
Junpei Takagi, Kentaro Tamura, Ryosuke Sasaki, Koh Aoki, Tomoo Shimada, and
Ikuko Hara-Nishimura
Supplemental Inventory
Figure S1.
Characterization of the gfs9-2 mutation and of N-terminal conserved domain within GFS9.
Figure S2.
Quantification of flavonoids in the gfs9-3 mutant.
Figure S3.
Both GFS9-GFP and GFP-GFS9 can rescue the gfs9 phenotypes.
Figure S4.
The intracellular distributions of GFS9-GFP and GFP-GFS9.
Figure S5.
The gfs9 cells exhibit defective vacuolar morphology in seeds.
Figure S6.
The gfs9 cells have many non-degraded membrane structures in vacuoles.
Table S1.
Flavonol derivatives in the L er and Col-0 wild-type seeds detected by LC-
MS.
Table S2.
Plant materials used in this study.
Table S3.
Primers used in this study.
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Figure S1.
Characterization of the gfs9-2 mutation and of N-terminal conserved domain within GFS9.
(a) RT-PCR analysis of the GFS9 transcript in the seeds of wild type (WT) and the gfs9-2 mutant. Total RNA was extracted from siliques containing developing seeds and subjected to 40 cycles of RT-PCR. The full length coding sequence of GFS9 is
2514 base pairs (bps) in the WT. Ex1-Ex3, which was amplified using primers
At3g28430-ex1-F and At3g28430-ex3-R (see Table S3) of GFS9 mRNA, is 115 bps in the WT and 99 bps in gfs9-2 . Actin2 ( ACT2 ) served as a loading control.
(b) The second exon is deleted in the gfs9-2 mutant. The alignment of the genomic sequence of the GFS9 gene in the WT with the mRNA sequence of the GFS9 gene in the gfs9-2 mutant. Upper case in GFS9 genomic, exon; lower case in GFS9 genomic, intron; green boxes, sequence identities; blue-highlighted sequences, the sequences of At3g28430-ex1-F primer (upper) and At3g28430-ex3-R primer (lower); red-highlighted 481st g, a point mutation site of the gfs9-2 mutant.
(c) The N-terminal 34 residues of GFS9 protein are deleted in the gfs9-2 mutant. The alignment of the GFS9 protein sequence in the WT with that of the gfs9-2 mutant is shown. Red boxes, sequence identities.
(d) Testing the allelism of gfs9 and tt9 . tt9 was crossed with gfs9-3 . The seed color of the F1 progeny was examined by light microscopy. All F1 progeny had the transparent testa phenotype in their seed coats. Bar = 1 mm.
(e) The alignment of the protein sequence of the N-terminal conserved domain of
Arabidopsis thaliana GFS9, Caenorhabditis elegans gop-1, Drosophila melanogaster
Ema, and Homo sapiens Clec16A. Blue letters, acidic amino acids; red letters, basic amino acids; green letters, polar amino acids; asterisks, sequence identities; periods, sequence similarities.
Figure S2.
Quantification of flavonoids in the gfs9-3 mutant.
(a) Levels of soluble and insoluble proanthocyanidins in the wild-type (WT) and gfs9-
3 seeds after acid-catalyzed hydrolysis. The averages and standard errors of three independent measurements are shown.
(b) Flavonol composition of WT and gfs9-3 seeds detected by LC-MS. The flavonol derivatives are listed in Table S1. The averages and standard errors of three
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independent measurements are shown. K, kaempferol; Q, quercetin; I, isorhamnetin;
†, not detected in the WT or gfs9-3 .
Figure S3.
Both GFS9-GFP and GFP-GFS9 can rescue the gfs9 phenotypes.
Complementation assay of the gfs9 phenotypes with either promGFS9:GFS9-GFP
(a  c) or promUBQ10:GFP-GFS9 (d  f) constructs. (a, d) Photographs of the seeds of the wild type (WT), gfs9-3 mutant, and transgenic gfs9-3 or WT containing either promGFS9:GFS9-GFP (a) or promUBQ10:GFP-GFS9 (d). (b, e) p -
Dimethylaminocinnamaldehyde staining using the seeds of WT, gfs9-3 mutant, and transgenic gfs9-3 or the WT containing either promGFS9:GFS9-GFP (b) or promUBQ10:GFP-GFS9 (e). To compare the seed colors, all photographs were acquired under the same conditions. Bars = 1 mm. (c, f) Immunoblot analyses of dry seeds of WT, gfs9-3 mutant, and transgenic gfs9-3 containing either promGFS9:GFS9-GFP (c) or promUBQ10:GFP-GFS9 (f) with the anti-12S globulin antibody. 12S, mature forms of 12S globulin; p12S, precursor forms of 12S globulin; asterisks, not complemented T2 seeds. Molecular masses are given on the left. All
T2 seeds of the gfs9-3 transformants showed that GFP-fused GFS9 could rescue the transparent testa phenotype, the defect in flavonoid accumulation, and the defect of protein sorting into vacuoles in gfs9-3 .
Figure S4.
The intracellular distributions of GFS9-GFP and GFP-GFS9.
(a  d) Co-localization analysis of GFS9-GFP with either Golgi apparatus or trans
-
Golgi network (TGN) markers. Each left panel shows the intracellular distribution of
GFS9-GFP. Each middle panel shows Golgi apparatus markers [mCherry-SYP32 (a) and mCherry-MEMB12 (b)] and TGN markers [VHA-a1-RFP (c) and mCherry-VTI12
(d)]. Each right panel shows a merged image. Bars = 5 μm.
(e) Intracellular distribution of GFP-GFS9 in root, hypocotyl, and cotyledon cells of transgenic Arabidopsis seedlings containing promUBQ10:GFP-GFS9 . Bars = 10 μm.
(f, g) Co-localization analysis of GFP-GFS9 with either Golgi apparatus or TGN markers. Each left panel shows the intracellular distribution of GFP-GFS9. Each middle panel shows a Golgi apparatus marker [mCherry-SYP32 (f)] and a TGN marker [VHA-a1-RFP (g)]. Each right panel shows a merged image. Bars = 5 μm.
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(h) Co-localization analysis of GFP-GFS9 (left panel) with FM4-64 dye for 6 min
(middle panel). GFS9 puncta are associated with FM4-64-labelled puncta (right panel). Bar = 5 μm.
(i) GFP-GFS9 puncta are included brefeldin A (BFA) bodies. Cells expressing GFP-
GFS9 (left panel) after treatment with BFA for 160 min. Co-staining with FM4-64
(middle panel) and a merged image (left panel) are shown. Bar = 10 μm.
Figure S5.
The gfs9 cells exhibit defective vacuolar morphology in seeds.
(a) Protein storage vacuole (PSV) autofluorescence in the cotyledon, embryo axis, and root cells of wild type (WT) and gfs9-4 / tt9 . The embryos were inspected with a confocal laser scanning microscope immediately after the seed coat of the dry seeds was peeled off. Bars = 10 μm.
(b, c) Thin sections from dry WT (Col-0 and L er ) and gfs9 seeds were stained with toluidine blue and examined by light microscopy. The cotyledon cells (b) and endosperm cells (c) are shown. The dark blue structures correspond to PSVs
(arrowheads). Bars = 10 μm.
Figure S6.
The gfs9 cells have many non-degraded membrane structures in vacuoles.
Ultrastructure of heart-staged embryo cells of the wild type (WT) and gfs9-4 / tt9 mutant. gfs9-4 / tt9 cells abnormally accumulate autophagic vacuoles, whereas WT cells only have a few autophagic vacuoles. V, vacuole; N, nucleus; M, mitochondrion;
CW, cell wall. Bars = 1 μm.
Table S1.
Flavonol derivatives in the L er and Col-0 wild-type seeds detected by LC-
MS.
Table S2.
Plant materials used in this study.
Table S3.
Primers used in this study.
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