total leaf fatty acids (mol %) 16:0 16:1 16:2 16:3 18:0 · tmh1 tmh2 tmh2 (g41d) supplemental...
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fad6tgd5-1 fad6
WT
18:1
16:0
16:1
16:2
16:3
18:0
18:2
18:3
Tot
al le
af fa
tty a
cids
(mol
%)
010203040506070
A
B
WT fad6 tgd5-1 fad6
Supplemental Figure 1. Growth and Lipid Phenotypes of the tgd5 fad6 Double Mutant. (A) Morphology of 7-week-old plants grown in soil. (B) FA composition of leaf tissues. Data are the means of the three replicates with SE.
WT
tgd5-1
tgd5-1
(c)
tgd5-1
(c)
TGDG
DGDG
B
C
AW
T tgd
5-1
tgd5-1
(c)
tgd5-1
(c)
26619274
Supplemental Figure 2. Complementation of the tgd5-1 Mutation by the TGD5 cDNA Fused at the C Terminus with GFP. (A) Growth phenotype of the tgd5-1 mutant and two transgenic lines overexpressing the TGD5 cDNA-GFP construct in the tgd5-1 background (tgd5-1 (c)). (B) Lipid phenotype of the tgd5-1 mutant and two independent complementation lines. A thin-layer chromatogram stained for glycolipids is shown. (C) Genotyping at the TGD5 locus using a cleaved amplified polymorphism DNA marker. A point mutation in tgd5-1 creates an Hph1 site. The genomic DNA fragments flanking the Hph1 site were digested with Hph1 and separated on an agarose gel. Ethidium bromide-stained diagnostic DNA fragments are shown with their respective lengths in base pairs.
18:1
16:0
16:1
16:2
16:3
18:0
18:2
18:3
MGDG
DGDG
PC
PE
Fatty
aci
ds (m
ol %
)Fa
tty a
cids
(mol
%)
0102030405060
0
20
40
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80
0
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20
30
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0
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WTtgd5-1tgd5-2tgd5-3
WTtgd5-1tgd5-2tgd5-3
Supplemental Figure 3. Fatty Acid Composition of Major Membrane Lipids in the Leaves of tgd5 Mutants. Lipids were extracted from 4-week-old plants grown in soil. Values are means of three replicates with SE.
Fatty
aci
d co
mpo
sitio
n of
TA
G (m
ol %
)
18:116:0 16:1 16:2 16:3 18:0 18:2 18:3
WT
sdp1-4
tgd5-1
tgd5-2
tgd5-3
tgd5-3 sdp1-4
0
10
20
30
40
50
60
Supplemental Figure 4. Fatty Acid Composition of Leaf TAGs. Lipids were extracted from 5-week-old plants grown in soil. Values are means of three replicates with SE.
WT tgd5-3 tgd4-3 tgd5-3
tgd4-3 tgd5-3WT
WT tgd4-3 tgd5-3
A
B
C
Supplemental Figure 5. The Pollen Grain Germination Defect of the tgd4-3 tgd5-3 Double Mutant. (A) Pollen viability as determined by Alexander staining. Bars = 20 µm. (B) Flower morphology of the wild type (WT) and tgd4-3 tgd5-3. (C) Pollen germination in the transmitting tract of the wild-type style. Arrows indicate growing pollen tubes. Bars = 10 µm.
WTtgd4-3tgd5-3tgd4-3 tgd5-3
010203040506070
01020304050607080
Fatty
aci
ds (m
ol %
)
0
10
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0
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16:0
16:1
16:2
16:3
18:0
18:1
18:2
18:3
Fatty
aci
ds (m
ol %
)Fa
tty a
cids
(mol
%)
Fatty
aci
ds (m
ol %
) WTtgd4-3tgd5-3tgd4-3 tgd5-3
WTtgd4-3tgd5-3tgd4-3 tgd5-3
WTtgd4-3tgd5-3tgd4-3 tgd5-3
PC
PE
MGDG
DGDG
Supplemental Figure 6. Fatty Acid Composition of Major Membrane Lipids in Leaves of tgd4-3, tgd5-3 Single and tgd4-3 tgd5-3 Double Mutants. Lipids were extracted from 4-week-old plants grown in soil. Values are means of three replicates with SE.
A. thaliana 1 MVLSDFTGVGVGFGFGVGCGFGVGWGFGG---------MPMNILGVGAGGGCGVGLGLGW E. salsugineum 1 MVLSDFTGVGVGFGFGVGCGFGVGWGFGG---------MPLNVLGVGVGGGCGVGLGLGW A. alpina 1 MVLSDFTGVGVGFGFGVGCGFGVGWGFGG---------MPLNVLGVGVGGGCGVGLGLGW S. lycopersicum 1 MVMTNFTGTGIGVGAGIGCGFGVGWGFGG---------MPLNFLGLGVGGGCGIGVGLGW B. napus 1 MVRSDFTGVGVGLGLGVGCGFGLGWVFGGSFTMNLVPRMPLNVLGVGVGGGCGMGLGLGW O. sativa 1 MVVNNFTGPGIGLGFGIGCGFGVGWGFGG---------MPLNMFGLGIGGGCGVGLGLGW S. tuberosum 1 MVMTNFTGTGIGFGAGIGCGFGVGWGFGG---------MPLNFLGLGVGGGCGVGVGLGW B. distachyon 1 MVVSTFSGPGIGLGFGVGCGFGIGWGFGG---------MPLNAFGLGIGGGCGVGLGLGW Z. mays 1 MVVSTFSGPGIGLGFGVGCGFGIGWGFGG---------MPLNAFGLGIGGGCGVGLGLGW G. max 1 MVITTFNGTGVGFGFGVGCGFGIGWGFGG---------MPLNLLGLGAGGGCGVGVGLGW T. urartu 1 MVVSTFSGPGIGVGFGVGCGFGVGWGFGG---------MPLNVFGLGIGGGCGVGLGLGW M. truncatula 1 MVITNFNGAGIGFGFGVGCGFGVGWGFGG---------MPLNFLGLGAGGGCGVGVGLGW A. thaliana 52 GFGTAFGSHYRSSRLTFQGIELETADKREEVVANMSKNST------- E. salsugineum 52 GFGSAFGSHYRSSRLTFQGIELEKTDKHEVKVDNMSKNT-------- S. alpina 52 GFGSAFGSHYRSSRLTFQGIELEKTDKHEVKVDNMSKNT-------- S. lycopersicum 52 GFGSAFGSQYRNSRVTFDGTDFINKERSEERDLKDPAKGTGKALSSQ B. napus 61 GFGTAFGSHYRSSRLTFQGIQLGKAD-------NMSQNT-------- O. sativa 52 GFGKAYGCQYRSSRVQFQGIEFQKKTEGDEASSLVSPERVEKSRPYG S. tuberosum 52 GFGSAFGSQYRNSRVTFDGTDFINKEHSEERDSKDLAKGTRKAHSSQ B. distachyon 52 GFGNGYGCQYRSSKVQFQGIEFQKKSEGDDSPKLISSGLVEKSRPYG Z. mays 52 GFGNGYGCQYRSSKVQFQGIEFQKKSEGDDSPKLISSGLVEKSRPYG G. max 52 GFGTAFGSKYRSSRVTFQGVDFDSKEKV---NSTELSKPRTEVGGAR T. urartu 52 GFGNAYGCQYRSSRVQFQGIEFQKKSEGDEAPK-----LVEKSRPYG M. truncatula 52 GFGSAYGSRYRLSRITFQGVEFDSKEKS---DSMELSKSSPEVRNSR
*
TMH1 TMH2
TMH2(G41D)
Supplemental Figure 7. Comparison of the Predicted Arabidopsis TGD5 Protein and Its Homologs in Different Plant Species. Proteins are from Arabidopsis thaliana, Eutrema salsugineum, Arabis alpina, Solanum lycopersicum, Brassica napus, Oryza sativa, Solanum tuberosum, Brachypodium distachyon, Zea mays, Glycine max, Triticum urartu, Medicago truncatula. Black boxes indicate identical and gray boxes conserved amino acids. Predicted transmembrane domains (THMs) are indicated above the sequence with bars. An amino acid substitution in the Arabidopsis tgd5-1 mutant allele is shown below the sequence. The asterisk indicates the mutated residue in tgd5-1.