Supplemental Data. Wagner et al. Plant Cell (2015) … · 2015-10-23 · Mimulus guttatus A Mimulus...

DVDNDGLISFKEYDVDNDGLISFKEYDMNNDGLISFKEYDTNGDGLISFKEYDTDNDGLISFPEYDFDNDGLISFPEYDTNGDGLISFAEYDTNGDGVISFSEYDTNGDRLISFAEYDTNSDGLISFAEYDTNGDGLISFPEYDTDGDGLISFYEFDVDGDGLISYPEFDVDGDGLISYPEFTLGECGLISFSDYDLGDKGLISYTEYNLKEKGVISYTEY

DTDNNGEIDKEEFDTDNNGEIDKEEFDINNNGEIDKEEFDLDCNGKIDREEFDLDNNGEIDREEFDIDNDGGIDREEFDLDHSGEIDKEEFDLDHNGEIDKDEFDVDHSGEIDKEEFDVDHSGVIDREEFDLDENGMIDRAEFDLDGDGMIDRDEFDLDGNGQIDAEEFDLDGNGQIDRDEFDLNGDGEVDMEEFDTDGNEMIEKREFDTDGNEMVDKKEF

DSNQDGNLSVDEFDSNRDGNLSVEEFDMNDDGNLSSEELDANKDGRLSADEFDANRDGSLSLDEFDANKDGSLSSEEFDANRDGSLSADEFDANSDGNLSSEEFDTNHDGNLSSEEFDTNQDGNLSSEEFDTDADGLLTMEEFDTDGDGVLSMEEF-TDDKGNLDTATFD-DGQGGLDVQAFDCDGNGELSNKEFDLDGDECLSHEEFDVDKDDQLSYKEF

N

Homo sapiens MICU3

Homo sapiens MICU1

Homo sapiens MICU2

Volvox carteri

Chlamydomonas reinhardtii

Physcomitrella patens A

Physcomitrella patens B

Sorghum bicolor A

Oryza sativa A

Vitis vinifera A

Sorghum bicolor B

Oryza sativa B

Mimulus guttatus A

Mimulus guttatus B

Vitis vinifera B

Arabidopsis thaliana MICU.1

Capsella rubella

498 AAArabidopsis thaliana MICU.1

Homo sapiens MICU1476 AA

CEF1 EF2

N CEF3 EF2EF1

X Y Z -Z X Y Z -Z X Y Z -Z

Supplemental Figure 1. Predicted features of At-MICU. (A) Conservation of EF hands in plant MICU homologues. Sequences of human MICU family proteins and their homologues from representative plant species were aligned using MUSCLE (Edgar, 2004; Supplemental Data Set 1, Supplemental File 1). Conservation is shown for the Ca2+-coordinating loop of EF hand motifs present in human MICU1 (EF 1 & 2; Perocchi et al., 2010) and a third EF hand (EF3) predicted in all analysed land plant sequences. Side chains at positions X, Y, Z and -Z are critical for Ca2+ binding and boxed black if different from the canonical EF hand (Lewit-Bentley and Rety, 2000).

Supplemental Data. Wagner et al. Plant Cell (2015) 10.1105/tpc.15.00509

100

70

55

kDa

9766

45

30

kDa

preimmune serum

0.2 20.2 2

µg purified MICU

B C

116

35

45

25

kDa

67

18

14

flow

-thr

ough

A

lysi

sbu

ffer

[imidazole]100 mM 500 mM

Elution with lysis buffer+ increasing concentrations of imidazole

lysi

sbu

ffer

+ 1

M N

aCl

lysi

sbu

ffer

+ 1

5 m

Mim

idaz

ole

Wash with

ΔMICU

Supplemental Figure 2. Purification of ΔMICU and immunoblot with At-MICU antibody. (A) Purification of recombinant ΔMICU. E. coli ArcticExpress DE3 (Agilent Technologies) or E. coli Rosetta(DE3) cells expressing His:ΔMICU were lysed in lysis buffer (25 mM Tris, 150 mM NaCl, 5 % (v/v) glycerol, 0.1 mM DTT, pH 7.5) and soluble proteins were loaded onto a Ni2+ chromatography column (GE Healthcare) following centrifugal separation of cell debris. The flow-through was collected for SDS-PAGE analysis. The column was rigorously washed with lysis buffer (± 1 M NaCl and 15 mM imidazole). Proteins were eluted with a 100-500 mM imidazole gradient in lysis buffer. Wash and elution fractions were analysed by SDS-PAGE. The band representing ΔMICU is indicated by an arrow. (B) 0.2 and 2 µg of purified ΔMICU were transferred to a PVDF membrane. The membrane was blocked and probed with rabbit serum before and after rabbit immunization with ΔMICU. A secondary antibody conjugated with horse radish peroxidase was used to allow chemiluminescent detection of ΔMICU. (C) 20 µg of mitochondrial protein isolated from 35S:MICU.1:GFP,Col-0, micu-1 and micu-2 seedlings according to Sweetlove et al., 2007 and Schwarzländer et al., 2011 were separated by SDS-PAGE and MICU was immunodetected as described in (B) using the purified MICU primary antibody instead of serum. Mitochondrially processed MICU and MICU.1:GFP were expected to have molecular masses of ~51 and ~81 kDa, respectively. The MICU.1:GFP band is indicated by an arrow.


GFP MitoTracker chlorophyll brightfieldMICU.1:GFP/MitoTracker

GFP:MICU.1

MICU.1:GFP

Supplemental Figure 3. MICU.1:GFP and GFP:MICU.1 localisation in wild tobacco leaf epidermis. Nicotianabenthamiana leaf epidermal cells transiently expressing GFP:MICU.1 and MICU.1:GFP stained with MitoTracker Orange and imaged by CLSM. GFP, green; MitoTracker, magenta. Scale bar = 10 µm.


Leaf

Root

Hypocotyl

MICU.1:GFP MitoTracker chlorophyll brightfieldMICU.1:GFP/MitoTracker

Supplemental Figure 4. Localisation of MICU.1:GFP in different Arabidopsis tissues. Arabidopsis seedlings stably expressing MICU.1:GFP and stained with MitoTracker Orange were imaged by CLSM. Representative images are shown for epidermal cells from the Arabidopsis leaf, root and hypocotyl. GFP, green; MitoTracker, magenta. Scale bar = 50 µm.


TMRM cpVenus

Col-0

micu-1

micu-2

0

1

2

TM

RM

/cpV

enus ns ns

A

B

Supplemental Figure 5. Assessment of mitochondrial membrane potential in root tips. (A) Root tips expressing 4mt-YC3.6 equilibrated in 50 nM TMRM. Representative confocal images of TMRM (magenta) and cpVenus fluorescence intensity (green). Scale bar = 50 µm. (B) TMRM fluorescence intensity normalized over cpVenus fluorescence intensity from the ROI defined in Figure 5C is shown for the Col-0 (black), micu-1 (purple) and micu-2 (green) backgrounds. n = 5; error bars = SD. The pairwise comparisons (micu vs. wild-type; t test) indicated no significant (P ≤ 0.05) change (ns).


3

5

7

9

0 500 10003

5

7

9

0 500 1000 1500

3.5

4

4.5

0 500 10003

5

7

0 500 1000 1500

time (s)

FR

ET

rat

io

A B

C DATPNAA

ATPNAA

4mt-YC3.6

NES-YC3.6

3

5

7

9

0 500 1000 1500

2 mM ATP

0.1 mM ATP

0.1 mM ATP

10 µM NAA

10 µM NAA

Supplemental Figure 6. Reproducibility of Ca2+ transients. FRET ratio traces of individual time lapse experiments of Col-0 seedlings expressing 4mt-YC3.6 (A and B) or NES-YC3.6 (C and D). Ca2+ transients were induced by the application of 10 µM NAA (A and C) or 0.1 mM ATP (B and D) at 175 s. The inset in (D) shows response traces for application of 2 mM ATP under otherwise identical conditions as for the main graph in (D). NAA and ATP were removed after another 180 s. (A) n = 11; (B) n = 11; (C) n = 9; (D) n = 8; insert in (D) n = 7.


0

2

4

6

8

10

0.001 0.01 0.1 1 10

matrix

cytosol

FR

ET

rat

io

[Ca2+] (µM)

00.5

11.5

22.5

3

0 500 1000

0.10.20.30.40.50.6

0 500 1000

00.5

11.5

22.5

3

0 500 1000 1500

00.5

11.5

22.5

3

0 500 10000

0.51

1.52

2.53

0 500 1000 1500

0.14

0.16

0.18

0 500 10000.15

0.2

0.25

0.3

0 500 1000 1500

NAA ATP

NAA ATP

time (s)

[Ca2

+] (

µM

)

4mt-YC3.6

NES-YC3.6

B C

D E

0.10.20.30.40.50.6

0 500 1000 1500

A

Col-0micu-1micu-2

Supplemental Figure 7. Considerations on absolute free Ca2+ concentrations. (A) The YC3.6 sensor was calibrated based on the assumption of a matrix Ca2+ resting value of 208 nM in Col-0 (Logan and Knight, 2003) and in vitro sensor behaviour (K’d: 250 nM, Hill coefficient: 1.7; Nagai et al., 2004; Palmer and Tsien, 2006) at pH 8.0 (assuming a dynamic range of 5.32) and pH 7.4 (dynamic range: 5.43) reflecting that of the mitochondrial matrix and the cytosol, respectively. Based on this approximation, free resting Ca2+ in the mitochondrial matrix of the micu lines is increased by > 35 % from 208 ± 22 nM in Col-0 to 287 ± 31 nM on average (B,C). The NAA-induced transients peak is >45% higher reaching 390 ± 58 nM in micu-1 and 451±57 nM in micu-2 as compared to 266 ± 52 nM in Col-0 (B). The eATP-induced maximum is even more strongly increased reaching 758 ± 576 nM in micu-1 and 1438 ± 1575 nM in micu-2 as compared to 410 ± 82 nM in Col-0 (C). By comparison, cytosolic free Ca2+ can be estimated with 154 ± 9 nM at baseline and 155 ± 8 or 249 ± 98 nM at the NAA or eATP transient maximum in all lines. All estimates, with exception of the eATP-induced maxima in the micu matrix, are within the linear range of the sensor response. Non-linear effects are introduced by the relationship between sensor FRET ratio and free Ca2+ concentration, which is particularly pronounced when the sensor population is close to empty or fully occupied with Ca2+ (here: FRET ratio ranges < ~2.5 and > ~8.5). In that response region small changes in ratio are linked to large changes in free Ca2+ concentrations and the calculation of absolute free Ca2+ concentrations is particularly prone to error. By contrast, good correlation around the sensor K’d (250 nM) validates quantitative assessment based on the FRET ratio data.


Col-0 micu-1 Col-3

Roo

t ape

xR

oot e

long

atio

nzo

ne

micu-2

B

A

Root apex

***ns

0

10

20

30

40

% o

fm

itoch

ondr

iaw

ithtr

ansl

ucen

tm

atrix

Supplemental Figure 8. Mitochondrial ultrastructure in cells of the root elongation zone and apex. (A) Mitochondrial ultrastructure in cells of the root apex and the elongation zone of Arabidopsisseedlings. Exemplary electron micrographs of micu-1, micu-2 and their respective wild types (Col-0 and Col-3). Scale bars = 0.5 µm. Percentages of root apex mitochondria scored by matrix appearance are shown as a bar graph. ≥ 167 mitochondria from 3 different seedlings were scored per line. The analysis was repeated twice with consistent observations. ***P ≤ 0.001, ns: P > 0.01 (Chi-square test; Supplemental Table 2).


130

150

170

190

210

230

250

270

0 10 20 30 40

050

100

150200

250

O2

cons

umpt

ion

rate

(nm

oles

/min

/mg

prot

ein)

0

50

100

150

200

250

0

50

100

150

200

250

050

100

150200

250

0

1

2

3

0

1

2

0

1

2

3

0

1

2

RCR RCR ADP:O ratioADP:O ratio

Succinate Pyruvate/malate

130

150

170

190

210

230

250

270

0 10 20 30 40 50

mito

chon

dria

Col-0 micu-1 Col-0 micu-1

succ

inat

e

AD

P

AD

P

AD

P

CC

CP

KC

N pGal

mito

chon

dria

pyru

vate

/mal

ate

AD

P

AD

P

AD

P

CC

CP

KC

N pGal

AD

P (

Col

-0 o

nly)

O2

conc

entr

atio

n(µ

M)

Time (min) Time (min)

Col-0 micu-1

micu-2Col-3

A

B

C

**

Supplemental Figure 9. Analysis of respiratory parameters in purified mitochondria. (A) Representative polarographic traces illustrating the oxygen consumption assays on purified mitochondria isolated from Arabidopsis seedlings, exemplified for Col-0 and micu-1 energized with succinate or pyruvate/malate respectively. Additions are marked by arrows. Succinate: 10 mM succinate, 0.25 mM ATP; pyruvate/malate: 10 mM pyruvate, 10 mM malate, 0.3 mM NAD and 0.1 mM thiamine pyrophosphate; ADP: 50 µM ADP; CCCP: 10 µM carbonyl cyanide m-chlorophenylhydrazone; KCN: 1 mM KCN, 10 mM DTT, 2 mM pyruvate; pGal 0.2 mM propylgallate. (B) Oxygen consumption rates for purified mitochondria from Col-0 (black), micu-1 (purple), Col-3 (grey), micu-2(green) energized with succinate or pyruvate/malate in response to the successive treatments indicated in (A). Outer membrane intactness was >90% for all preparations as measured by cytochrome c latency. (C) Respiratory control ratio (RCR; state IV rate/ state III rate) and ADP:O ratio (ADP or phosphate molecules consumed, or ATP molecules synthesized per consumed oxygen atom). n = 3 biological; error bars = SD. The statistical analysis (two way ANOVA with post hoc Holm-Sidak comparisons for micu vs. wild-type pairs) indicated no significant (P ≤ 0.05) change, except in one case; **P ≤ 0.01.


≥1 silique≥1 open flowerboltedflower bud

-sucrose

150 mM NaCl

60 µM menadione

300 mM mannitol

1 µM IAA

1 mM DTT

control

100 µM CdCl2

20 µM antimycin A

Col-0 micu-1 Col-0 micu-1 Col-0 micu-1

C

D

A

0

0.2

0.4

0.6

0.8

1

5 8 11 14 17 20 23 26 290

20

40

60

80

5 8 11 14 17 20 23 26 29

Ros

ette

sol

idity

Days after stratification

Maj

or r

oset

teax

is(m

m)

Days after stratification

B

Col-0micu-1Col-3micu-2

Supplemental Figure 10. Experimental design of micu phenotyping. Plants grown under controlled conditions were photographically documented in a standardised way using a Canon EOS 1000 D DSLR camera. For soil-grown rosettes the Leaf Lab tool (Version 1.41 b) was used to automatically detect rosette leaves and to quantify leaf area (Fig. 8C), solidity (area of leaves in the rosette/ area of convex hull enclosing the rosette) and the length of the major rosette axis (A, B). (C) Representative images of plants in growth stages as classified in Fig. 8D. (D) Representative photographs of Col-0 and micu-1 seedlings grown in the presence of a variety of additives and used for quantification of primary root length in Fig. 8E.


Supplemental Data. Wagner et al. Plant Cell (2015) 10.1105/tpc.15.00509 Supplemental Table 1. Kinetic parameters of cytosolic calcium transients in Col-0 and micu lines after stimulus with either NAA or eATP. n ≥ 7; ± SD.

NAA stimulus eATP stimulus

Col-0 micu-1 micu-2 Col-0 micu-1 micu-2

Accumulation rate (ratio change s-1) (Fig. 7C,H)

0.004

±0.001

0.005

±0.002

0.003

±0.002

0.04

±0.01

0.03

±0.01

0.04

±0.01

Maximal FRET ratio (Fig. 7D,I)

4.0 ±0.1 4.1 ±0.2 4.1 ±0.2 5.6 ±0.2 5.6 ±0.3 5.6 ±0.2

t½ up (s) 26 ±5 23 ±4 24 ±9 21 ±4 25 ±7 24 ±7

t½ down (s) (Fig. 7E,J) 516 ±45 486 ±64 471 ±133 329 ±8 348 ±15 347 ±25


Supplemental Table 2. Analysis of mitochondrial ultrastructure

Genotype Root zone Number of mitochondria Percentage of mitochondria with

translucent (abnormal) matrix

p-value χ² test

scored with dense

(normal) matrix

with translucent (abnormal)

matrix

Col-0 Root apex 274 265 9 3.3 micu-1 Root apex 255 254 1 0.4 0.015 Col-3 Root apex 268 257 11 4.1

micu-2 Root apex 167 139 28 16.8 6.94E-06 Col-0 Elongation zone 235 202 33 14.0

micu-1 Elongation zone 234 155 79 33.8 5.50E-07 Col-3 Elongation zone 185 142 43 23.2

micu-2 Elongation zone 206 128 78 37.9 0.002

Supplemental Data. Wagner et al. Plant Cell (2015) 10.1105/tpc.15.00509 Supplemental Table 3. List of primers

Name Primer sequence Purpose LBb1.3 ATTTTGCCGATTTCGGAAC micu-1 genotyping P3054 GACAAGAATTAGGCAACAGG micu-1 genotyping P3055 TAGACAAGGAGTTGGCCAC micu-1 genotyping LB1mod ATGGATAAATAGCCTTGCTTCC micu-2 genotyping P3057 GATATGTCTGATACCGCGG micu-2 genotyping P3058 TCCGAAGATGAATCCAGAGC micu-2 genotyping P364 CAACCGGTATTGTGCTCGATTC PCR reference gene ACTIN7 P436 AACCTCAGGACAACGGAATCTC PCR reference gene ACTIN7 P2886 CGTCCGTTGACCGATCATTC Validation of MICU knock-out

through PCR P2893 TCACGATGAGGAACAGTTCTTAG Validation of MICU knock-out

through PCR P2455 CCATATTGCAAGAAGTTTGCGCGTCTG qRT-PCR reference gene

SAND FAMILIY PROTEIN P2456 GCAAGTCATCGGGATGGAGAGACG qRT-PCR reference gene

SAND FAMILIY PROTEIN P3529 GACCGATCATTCCTTCTCATC Validation of MICU knock-out

through qRT-PCR P3530 CCAGAGGACCAATAGAGAAACC Validation of MICU knock-out

through qRT-PCR P3389 GGGACATGCGTATCTCACTG Validation of MICU knock-out

through qRT-PCR P3390 GCGGCTCGTTTGAAATCTT Validation of MICU knock-out

through qRT-PCR ΔMICU_fw TTTGCTAGCTCGTTTATTCCGAAATTGTC Cloning of ΔMICU; NheI site

is underlined ΔMICU_rv AAACTCGAGTCACGATGAGGAACAGTTCTTA Cloning of ΔMICU; XhoI site

is underlined


SUPPLEMENTAL REFERENCES

Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32, 1792-1797.

Lewit-Bentley, A., and Rety, S. (2000). EF-hand calcium-binding proteins. Curr Opin Struct Biol 10, 637-643.

Logan, D.C., and Knight, M.R. (2003). Mitochondrial and cytosolic calcium dynamics are differentially regulated in plants. Plant Physiol. 133, 21-24.

Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M., and Miyawaki, A. (2004). Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101, 10554-10559.

Palmer, A.E., and Tsien, R.Y. (2006). Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc 1, 1057-1065.

Perocchi, F., Gohil, V.M., Girgis, H.S., Bao, X.R., McCombs, J.E., Palmer, A.E., and Mootha, V.K. (2010). MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake. Nature 467, 291-296.

Schwarzländer, M., Logan, D.C., Fricker, M.D., and Sweetlove, L.J. (2011). The circularly permuted yellow fluorescent protein cpYFP that has been used as a superoxide probe is highly responsive to pH but not superoxide in mitochondria: implications for the existence of superoxide 'flashes'. Biochem J 437, 381-387.

Sweetlove, L.J., Taylor, N.L., and Leaver, C.J. (2007). Isolation of intact, functional mitochondria from the model plant Arabidopsis thaliana. Methods Mol Biol 372, 125-136.

Supplemental Data. Wagner et al. Plant Cell (2015) … · 2015-10-23 · Mimulus guttatus A Mimulus...

Documents

Transcript of Supplemental Data. Wagner et al. Plant Cell (2015) … · 2015-10-23 · Mimulus guttatus A Mimulus...