B
Col-0 gnl1-2 echtrs120-4
A
Col-0 gnl1-1rhd3-1 echtrs120-4
sec-GFP & propodium iodide
Supplemental Figure 1: trs120-4, gnl1-1, and ech are defective in secretion. All mutants in
this study are defective in cell elongation to
varying degrees as indicated by their reduced
growth after six weeks, compared to wild type
(A). In stem epidermal cells, the secreted
ϐ������������������ǡ����Ǧ��ȋ�����Ȍǡ���������labels the secretory pathway and is secreted to
the apoplast, labeled with propidium iodide
ȋ�������Ȍǡ�������ϐ�������������������������������Ǧ����������Ǥ�����trs120-4, gnl1, and ech
�������ǡ����Ǧ��������������������������epidermal cells, indicating a defect in protein
secretion (B). Scale bars represent 10 µm.
ABCG12pro::GFP-ABCG12
abcg12-2 gnl1-1 ech
Supplemental Figure 2: ABCG12 is correctly localized to the plasma membrane in gnl1-1 and ech. In abcg12-2 stem epidermal cells
complemented with GFP-ACBG12, the
transporter is localized to the plasma
membrane. Despite their secretion defects, the
majority of the GFP-ABCG12 signal is localized
to the plasma membrane in gnl1-1 and ech
mutants. Scale bars represent 10 µm.
Col-0 mah1-1 gnl1-1 ech
secondary alcohol
ketone
free fatty acid
aldehydes
primary alcohols
alkanes
surf
ace w
ax (
µg/c
m )2
16
12
8
4
0mah1-1 echmah1-1 gnl1-1
Supplemental Figure 3: gnl1-1 and ech wax accumulation phenotypes cannot be rescued by altering the chemical composition of surface waxes. Changing the chemical
composition of gnl1-1 and ech wax by introducing the mah1-1 mutation, which eliminates
secondary alcohols and ketones (green) and increases alkane levels (blue), fails to increase
the amount of total wax secreted in these mutants. Values are means of 3 experiments of at
������͵����������������������������ȋ�Ǥ�Ǥ���η�ͻȌǡ������������������Ǥ�����������������������������������ϐ�����������������������������������������������������mah1-1 double mutants (t-test,
���η�ͲǤͲͷȌǤ
Col-0 gnl1-1 echechtrs120-4 rhd3-1
ech
Col-0
trs120-4
gnl1-1
ech
rhd3-1
A
B C
ech + YFP-ABCG11abcg12-2
abcg12-2
Supplemental Figure 4: Waxes do not substantially aggregate in trs120-4, rhd3-1, gnl1-1, or ech mutants. In wild-type stem epidermal cells, the lipophillic dye, Nile red, does
���������ϐ��������������������������������������ȋ�Ȍǡ��������������������������������ǡ������������������������������������������������ȋ�ȌǤ�����������ǡ�������������������������������������������������in trs120-4, gnl1-1, ech, or rhd3-1, compared to abcg12-2�ȋ�Ȍǡ��������������������������������������������������������������������������������������Ǧ���ȋ�ȌǤ��ech ��������������������������������������������������������������������ȋ�ǡ�������������������Ȍǡ����������������������������ǡ����������������������������������������������������������Ǧ���ͳͳ�ȋ�ǡ������Ǧ��������������ȌǤ�����������������������ͳͲ�ρ�Ǥ�
Supplemental Figure 5: Waxes areassociated with intracellular membranes. The average levels of fatty acid methyl esters
(FAMEs) from membrane fractions and the
major cuticular wax component (i.e. 29-alkane)
was determined relative to the total protein
content in microsomal and mitochondrial
fractions from wild-type seedlings using GC-
MS. The values represent means ± SD of three
biological replicates.
Col-0 g
DN
A
Col-0
gnl1
-1
rhd3-1
ech
trs120-4
no tem
pla
te
UBC10
CER6
CER10
CER2
MAH1
ABCG11
ABCG12
Supplemental Figure 6: Transcript levels of wax biosynthetic genes are not affected in gnl1-1, rhd3-1, ech, or trs120-4 mutants. Semi-quantitative RT-PCR of wax biosynthesis
genes (CER6, CER10, CER2, MAH1) and wax
transport genes (ABCG11, ABCG12) reveals no
changes to transcript levels in gnl1-1, rhd3-1,
ech, or trs120-4, stems relative to wild type.
cDNA levels were normalized to transcript
levels of the gene encoding an ubiquitin
conjugating enzyme, UBC10; genomic DNA
����������������������������ǡ����������ϐ��������without template DNA served as a negative
control.
cer2-5 gnl1-1 ech
CER2pro::CER2-GFP
Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal ER membranes in gnl1-1 and ech mutants. In cer2-5 stem epidermal cells
complemented with GFP-CER2, the enzyme is
primarily localized to ER membranes. GFP-
CER2 is also primarily localized to ER
membranes in gnl1-1 and ech mutants and
reveals defects in ER morphology, relative to
wild type. Scale bars represent 10 µm.
Supplemental Figure 8: gnl1-1, ech, and rhd3-1 are defective in ER morphology. In wild-
type (Col-0) and trs120-4 stem epidermal cells, the ER lumen marker GFP-HDEL is localized
primarily to a reticulate cortical network in both single optical sections and (A) maximum
projections (B), while gnl-1, ech, and rhd3-1 mutants have defects in GFP-HDEL distribution.
In gnl1-1 mutants, puncta are present in the cortical cytoplasm. In ech, under the same
imaging conditions, a central bright aggregate dominates the cell, alongside reticulate ER
(insert shows these aggregations when not saturated). In rhd3-1 mutants, GFP-HDEL
distribution shows cable-like bundle of ER. Scale bars represent 10 µm.
A
Col-0 gnl1-1 echtrs120-4 rhd3-1
B
Col-0 gnl1-1 echtrs120-4 rhd3-1
Supplem
ental*T
able*I:!Cu
ticular*w
ax*extracted*from
*the*p
lant*su
rface*compared
*to*total*stem*wax*in
*vesicle*traffic*mutan
ts*of*Arab
idopsis.!!Average!w
ax!content!(µg/cm2)!±!standard!error!of!trs120'4,!rhd3'1,!gnl1'1,!and!ech!com
pared!to!Col;0!w
ild!type.!
Free Fatty A
cids
Alkanes
Secondary
Alcohol
Ketone
Aldehydes
Prim
ary Alcohols
Total
C25
C27
C29
C31
C29
C29
C26
C28
C30
C24
C26
C28
C30
Col-0 surface w
ax 0.020 ±0.003
0.012 ±0.002
0.115 ±0.032
8.200 ±0.665
0.2018 ±0.019
2.396 ±0.126
5.269 ±0.275
0.038 ±0.004
0.136 ±0.016
0.477 ±0.085
0.039 ±0.006
0.335 ±0.030
0.708 ±0.051
0.332 ±0.027
18.295 ±
1.158
Col-0 total w
ax 0.066 ±0.016
d 0.026 ±0.006
d 0.133 ±0.012
8.070 ±0.707
0.245 ±0.021
2.461 ±0.132
5.369 ±0.132
0.079 ±0.021
0.192 ±0.041
0.734 ±0.245
0.075 ±0.010
c 0.491 ±0.067
d 1.168 ±0.081
c 0.550 ±0.041
c 19.812 ±
1.657
trs120-4 surface wax
0.017 ±0.003
0.011 ±0.000
0.198 ±0.012
a 9.015 ±0.509
0.147 ±0.008
b 2.142 ±0.103
4.346 ±0.287
b 0.050 ±0.002
b 0.222 ±0.027
b 0.575 ±0.090
0.029 ±0.001
0.233 ±0.008
b 0.544 ±0.012
b 0.186 ±0.006
a 17.720 ±
1.012
trs120-4 total wax
0.049 ±0.009
c 0.017 ±0.002
d 0.209 ±0.009
10.03 ±0.527
0.170 ±0.003
a,d 2.264 ±0.131
4.444 ±0.334
0.076 ±0.006
c 0.217 ±0.035
0.657 ±0.099
a 0.070 ±0.009
c 0.522 ±0.060
c 1.049 ±0.122
c 0.353 ±0.044
a,c 19.090 ±
1.318
gnl1-1 surface wax
0.007 ±0.001
a 0.012 ±0.001
0.098 ±0.007
4.461 ±0.222
a 0.114 ±0.006
a 1.102 ±0.046
a 2.140 ±0.157
a 0.029 ±0.002
0.070 ±0.007
a 0.149 ±0.019
a 0.028 ±0.003
0.387 ±0.034
0.702 ±0.062
0.184 ±0.018
a 9.482 ±
0.541a
gnl1-1 total wax
0,031 ±0.008
c 0.021 ±0.005
0.111 ±0.009
5.013 ±1.056
a 0.167 ±0.024
b,d 1.290 ±0.099
a 2.863 ±0.221
a,d 0.070 ±0.011
c 0.167 ±0.100
d 0.714 ±0.150
b,c 0.045 ±0.016
0.499 ±0.042
d 0.999 ±0.089
d 0.336 ±0.035
a,c 12.269 ±
1.111a,d
ech surface wax
0.005 ±0.001
a 0.029 ±0.005
a 0.194 ±0.035
b 1.791 ±0.990
a 0.022 ±0.006
a 0.290 ±0.037
a 0.201 ±0.068
a 0.040 ±0.005
0.075 ±0.044
0.031 ±0.013
a 0.013 ±0.002
a 0.235 ±0.044
b 0.263 ±0.056
a 0.020 ±0.005
a 3.210 ±
0.595a
ech total wax
0.016 ±0.005
a,d 0.037 ±0.008
0.246 ±0.025
a 2.851 ±0.215
a,d 0.135 ±0.022
a,c 0.319 ±0.049
a 0.524 ±0.052
a,c 0.081 ±0.010
c 0.354 ±0.086
c 0.331 ±0.057
c 0.021 ±0.004
a 0.478 ±0.041
c 0.712 ±0.061
a,c 0.091 ±0.008
a,c 6.179 ±
0.639a,c
rhd3-1 surface wax
0.009 ±0.002
a 0.018 ±0.002
0.137 ±0.009
3.391 ±0.301
a 0.077 ±0.004
a 0.611 ±0.055
a 1.545 ±0.147
a 0.031 ±0.002
0.097 ±0.007
b 0.146 ±0.010
a 0.026 ±0.003
0.503 ±0.028
a 0.760 ±0.038
0.095 ±0.004
a 7.445 ±
1.572a
rhd3-1 total wax
0.038 ±0.006
c 0.021 ±0.004
0.140 ±0.008
3.360 ±0.214
a 0.131 ±0.044
b 0.678 ±0.075
a 1.703 ±0.135
a 0.075 ±0.012
c 0.172 ±0.035
c 0.302 ±0.041
c 0.028 ±0.015
a 0.631 ±0.238
0.973 ±0.336
0.125 ±0.019
a 8.341 ±
0.783a
a!statistically!significant!difference!between!m
utant!and!wild!type,!t;test,!p<0.01!
b!statistically!significant!difference!between!m
utant!and!wild!type,!t;test,!p<0.05!
c!statistically!significant!difference!between!surface!w
ax!and!total!wax!w
ithin!a!genotype,!t;test,!p<0.01!d!statistically!significant!difference!betw
een!surface!wax!and!total!w
ax!within!a!genotype,!t;test,!p<0.05!
Name
ECH.P1
ECH.P1
GNL1.P1
GNL1.P2
GNL1.P3
TRS120.P1
TRS120.P2
ABCG11.P1
ABCG11.P2
ABCG12.P1
ABCG12.P2
MAH1.P1
MAH1.P2
CER2.P1
CER2.P2
UBC10.P1
UBC10.P2
CER2.P3
CER2.P4
CER6.P1
CER6.P2
CER10.P1
CER10.P2
MAH1.P3
MAH1.P4
ABCG11.P3
ABCG11.P4
ABCG12.P3
ABCG12.P4
Primer Sequence (5` -> 3`)
TCATTGATCTCGTTCCACCA
CCACAACGGAGAAGGTGTTT
GGAATGGCGAAATCAAGGCAT
GCGGTGGCGTCCATGATTA
TCGGATCCCAGTCCACTGTTC
AAACCGATCAACGATTTCCTC
CACCAAGCACAAATTTGAACC
TCGAATGCAGTTCTTTGCAC
AACAGATTGTCCCGGAGATG
GTGAAATTCCGTCCTGGTGT
GCTTTGCTTGTATCGCCTTC
TCAAATCACGCCCTATTCCA
ATCTGGCTTCGCAGGAGACT
GCTGGAAACGCAACGTTATT
CAAACTCCGGCACTCTCTTC
GGTAGCATTTGCCTCGACAT
CTTCGTGCAGTGGACTCGTA
CCAACGTCGAAGAGTTCACA
ATGACCCGATACCAGCTGTC
CACGTTGGCATCAACTTGTC
CTGCGTGAATGCAAAAGTGT
TCTGCAAGAAGCGTTTCATAA
TGTGCGGTAGGAAACTTGTG
GTTGTGGGAGGAGATGAGGA
CTCCACAAGATCACCGGTTT
CCAAATGACATCCCAAAACC
CGATGCAAGTCGATCTGAAA
CGATTTCCTTCATGAGTTATGGT
GCTTTGCTTGTATCGCCTTC
Purpose
genotyping ech mutants
genotyping ech mutants
genotyping gnl1-1 mutants
genotyping gnl1-1 mutants
genotyping gnl1-1 mutants
genotyping trs120-4 mutants
genotyping trs120-4 mutants
genotyping abcg11-3 mutants
genotyping abcg11-3 mutants
genotyping abcg12-2 mutants
genotyping abcg12-2 mutants
genotyping mah1-1 mutants
genotyping mah1-1 mutants
genotyping cer2-5 mutants
genotyping cer2-5 mutants
RT-PCR for UBC10 (At5g53300)
RT-PCR for UBC10 (At5g53300)
RT-PCR for CER2 (At4g24510)
RT-PCR for CER2 (At4g24510)
RT-PCR for CER6 (At1g68530)
RT-PCR for CER6 (At1g68530)
RT-PCR for CER10 (At3g55360)
RT-PCR for CER10 (At3g55360)
RT-PCR for MAH1 (At1g57750)
RT-PCR for MAH1 (At1g57750)
RT-PCR for ABCG11 (At1g17840)
RT-PCR for ABCG11 (At1g17840)
RT-PCR for ABCG12 (At1g51500)
RT-PCR for ABCG12 (At1g51500)
Supplemental Table II: A list of primers employed in this study.