+ All Categories
Home > Documents > Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech...

Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech...

Date post: 13-Jun-2020
Category:
Upload: others
View: 3 times
Download: 0 times
Share this document with a friend
10
B Col-0 gnl1-2 ech trs120-4 A Col-0 gnl1-1 rhd3-1 ech trs120-4 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 ȋȌǡ labels the secretory pathway and is secreted to the apoplast, labeled with propidium iodide ȋȌǡ ϐ Ǧ Ǥ trs120-4, gnl1, and ech ǡ Ǧ epidermal cells, indicating a defect in protein secretion (B). Scale bars represent 10 µm.
Transcript
Page 1: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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.

Page 2: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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.

Page 3: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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

Page 4: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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�

Page 5: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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.

Page 6: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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.

Page 7: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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.

Page 8: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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

Page 9: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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!

Page 10: Ð á æ á á Ð æ ä trs120-4 gnl1 ech á æ...2014/01/27  · cer2-5 gnl1-1 ech CER2pro::CER2-GFP Supplemental Figure 7: The CER2 wax biosynthetic enzyme is localized to abnormal

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.


Recommended