Nature Plants 1, 15031 (2015); published online 23 March ... · SUPPLEMENTARY INFORMATION. DOI: 10.1038/NPLANTS.2015.31. NATURE PLANTS | . 1. 1 . SUPPLEMENTARY METHODS RT-PCR and

NATURE PLANTS | www.nature.com/natureplants

In the version of the Supplementary Information originally published several characters in Fig. 4 were rendered incorrectly. These errors have been corrected.

Regulation of organ straightening and plant posture by an actin–myosin XI cytoskeletonKeishi Okamoto, Haruko Ueda, Tomoo Shimada, Kentaro Tamura, Takehide Kato, Masao Tasaka, Miyo Terao Morita and Ikuko Hara-Nishimura

Nature Plants 1, 15031 (2015); published online 23 March 2015; corrected 14 April 2015.

ERRATUM

ARTICLESNATURE PLANTS DOI: 10.1038/NPLANTS2015.61

© 2015 Macmillan Publishers Limited. All rights reserved.


SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.31

NATURE PLANTS | www.nature.com/natureplants 1

1

SUPPLEMENTARY METHODS

RT-PCR and genotyping. Total RNA was isolated from inflorescence stems of 4 to

5-week-old plants with an RNeasy plant mini kit (Qiagen). Total RNA was subjected to

first-strand cDNA synthesis using Ready-To-Go RT-PCR Beads (GE Healthcare) and an

aliquot was subjected to PCR with Ex Taq polymerase (Takara). The gene-specific primers

used for RT-PCR and genotyping are listed in Supplementary Table 3.

Plasmid construction. We used the Gateway cloning system (Invitrogen) to construct

ProXIf:GUS, ProXIf:XIf, ProXIf:mCherry-ER, ProXIf:XIk-GFP, ProXIf:Lifeact-VENUS, and

ProXIf:GFP-RTN1. All primers used for cloning are listed in Supplementary Table 3. A

1,915-bp region upstream from the start codon of myosin XIf was selected as the promoter

region (ProXIf). This region was amplified by PCR using the primers proF-F3 and proF-2R

and then subcloned into pENTR/D-TOPO (Invitrogen). To express GUS under the control of

the myosin XIf promoter, the ProXIf fragment was introduced by the LR reaction into

pFAST-G04 plant expression vector for GFP-GUS37. To construct ProXIf:XIf for the

complementation assay, ProXIf was subcloned into pDONR P4-P1r (Invitrogen) using the

primers B4 XIFproF and B4 XIFproR. The genomic fragment of myosin XIf (from ATG to

the stop codon in At2g31900) was subcloned into pENTR/D-TOPO using the primers

CDSF-F and FallR. Then, the ProXIf fragment and the genomic fragment of myosin XIf were

introduced into the R4pGWB501 plant expression vector38 by the LR reaction. To construct

ProXIf:mCherry-ER, the sequences for the signal peptide (SP) of pumpkin 2S albumin,

mCherry, and the ER retention motif His-Asp-Glu-Leu (HDEL) were amplified and fused by

PCR using the following primers: SP-F, SP-R, mCherry-F, mCherry-R, HDEL-F, and

HDEL-R. This fragment was subcloned into pENTR/D-TOPO using the primers SP-F and

HDEL-R. The ProXIf fragment and the fused SP-mCherry-HDEL fragment were introduced

into the R4pGWB501 plant expression vector by the LR reaction. To construct

ProXIf:XIk-GFP, ProXIf, a genomic fragment of myosin XIk (from the ATG start codon to

just before the stop codon in At5g20490) and cDNA encoding synthetic GFP (S65T) were

amplified by PCR using the following primers: pXIf-LAV-IF-F, pXIf-XIk-R2, pXIf-XIk-F2,

XIk-linkerGFP-R2, XIk-linkerGFP-F2, and GFP-IF-R. The resulting fragment was subcloned

into pENTR using an In-Fusion Advantage PCR cloning kit (Takara). The amino acid linker

between myosin XIk and GFP was Gly-Gly-Gly-Gly-Gly-Ala. The ProXIf:XIk-GFP fragment

was introduced into the pGWB601 plant expression vector39 by the LR reaction. To construct

ProXIf:Lifeact-VENUS, DNA fragments of ProXIf and Lifeact-VENUS40 were amplified by

PCR using the following primers: pXIf-LAV-IF-F, pXIf-LA-R, pXIf-LA-F, and

Regulation of organ straightening and plant posture by an actin–myosin XI cytoskeleton



http://dx.doi.org/10.1038/nplants.2015.31

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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.31

2

pXIf-LAV-IF-R. The resulting fragment was subcloned into pENTR using an In-Fusion

Advantage PCR cloning kit. The ProXIf:Lifeact-VENUS fragment was introduced into the

pGWB601 plant expression vector39 by the LR reaction. To construct ProXIf:GFP-RTN1,

ProXIf, cDNA encoding synthetic GFP (S65T), and a genomic fragment of Reticulon-like

Protein B1 (RTN1) (from ATG to the stop codon and 535 bp downstream in At4g23630) were

amplified by PCR using the following primers: pXIf-LAV-IF-F, pXIf-GFP-R, pXIf-GFP-F,

G-RTN1-R, G-RTN1-F, and RTN1-IF-R. The resulting fragment was subcloned into pENTR

using an In-Fusion Advantage PCR cloning kit. The amino acid linker between GFP and

RTN1 was Gly-Gly-Gly. The ProXIf:GFP-RTN1 fragment was introduced into the pBGW

plant expression vector (Plant System Biology) by the LR reaction. With these vectors, plants

were stably transformed using standard protocols for Agrobacterium (strain

GV3101)-mediated transformation41.

SDS-PAGE and immunoblotting. SDS-PAGE and immunoblotting were performed as

described previously42. Antibodies used in this study were anti-myosin XIk (diluted

2,000-fold)33 and anti-GFP (diluted 2,000-fold; JL-8, Clontech).

Transmission electron microscopy. Ultrastructural analysis was conducted by the Tokai

Electron Microscopy service. Briefly, hypocotyls and roots of the wild type and the

ProXIf:GFP-RTN1 plants were cut into slices with a razor blade and fixed with 4%

paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. After

dehydration, the samples were infiltrated with LR White resin (London Resin Co.Ltd.) and

polymerised. The blocks were ultrathin sectioned at 80 nm using an ultramicrotome

(ULTRACUT UCT, Leica). The ultrathin sections were treated with antibodies against GFP

(rabbit anti-GFP polyclonal antibody) and the sections were examined using a transmission

electron microscope (JEM-1400Plus, JEOL) at 80 kV.

Analysis of lignification in fibre cells. Basal segments of primary inflorescence stems of

5-week-old plants were embedded in 5% agar and cut with a vibrating blade microtome

(Leica Microsystems) into 100-µm-thick sections. Samples were stained with 0.02% toluidine

blue, and observed under an upright microscope (Axioskop 2, Zeiss).

Measurement of mechanical properties of the inflorescence stem. A compression test was

performed using a creep meter (RE33005, Yamaden) as described previously43. Briefly, the

basal part of the 20 cm inflorescence stem was cut into 5-mm sections that were placed

horizontally between the plunger and the stage of the creep meter. The stem was compressed

in the horizontal axis until 2 N of force was applied. The force applied to the stem was

monitored by the load-cell connected to the plunger, and the force deformation curve was






3

recorded. The deformation percentage was calculated when the applied force reached 2 N and

represented the deformation distance per the initial stem diameter. The stem diameter was

measured using the same creep meter and represented the distance between the surfaces of the

plunger and the stage when 0.01 N of force is detected.

33 Peremyslov, V. V., Prokhnevsky, A. I., Avisar, D. & Dolja, V. V. Two class XI myosins

function in organelle trafficking and root hair development in Arabidopsis. Plant Physiol. 146, 1109-1116 (2008).

34 Nelson, B. K., Cai, X. & Nebenfuhr, A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51, 1126-1136 (2007).

35 Saito, C., Morita, M. T., Kato, T. & Tasaka, M. Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem. Plant Cell 17, 548-558 (2005).

36 Ye, Z. H., Freshour, G., Hahn, M. G., Burk, D. H. & Zhong, R. Vascular development in Arabidopsis. Int. Rev. Cytol. 220, 225-256 (2002).

37 Shimada, T. L., Shimada, T. & Hara-Nishimura, I. A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J. 61, 519-528 (2010).

38 Nakagawa, T. et al. Development of R4 gateway binary vectors (R4pGWB) enabling high-throughput promoter swapping for plant research. Biosci. Biotechnol. Biochem. 72, 624-629 (2008).

39 Nakamura, S. et al. Gateway binary vectors with the bialaphos resistance gene, bar, as a selection marker for plant transformation. Biosci Biotechnol Biochem 74, 1315-1319 (2010).

40 Era, A. et al. Application of Lifeact reveals F-actin dynamics in Arabidopsis thaliana and the liverwort, Marchantia polymorpha. Plant Cell Physiol 50, 1041-1048 (2009).

41 Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743 (1998).

42 Shimada, T. et al. Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana. J Biol Chem 278, 32292-32299 (2003).

43 Hongo, S., Sato, K., Yokoyama, R. & Nishitani, K. Demethylesterification of the primary wall by PECTIN METHYLESTERASE35 provides mechanical support to the Arabidopsis stem. Plant Cell 24, 2624-2634 (2012).






Supplementary Figure 1. Myosin XIf gene structure and phenotypes of certain myosin xi mutants. (a) Schematic representation of myosin XIf gene. Arrows indicate position of each T-DNA insertion in myosin xif-1 and myosin xif-2. Arrowheads show positions of primers. Filled boxes, exons; solid lines, introns. (b) Transcript levels of myosin XIf mRNA in inflorescence stems, as assessed by RT-PCR with primers myosin xif-2 F and myosin xif-1 R. ACTIN2 (ACT2; At3g18780) was used as a control. Transcripts of myosin XIf were undetectable in myosin xif-1 and myosin xif-2 mutants. (c) Myosin XI-deficient mutants. (d) Plants grown upright, as in Fig. 1a.

XIf

ACT2

wild type xif-1 xif-2ba

xif-1 Rxif-1 Fxif-2 Rxif-2 F

myosin XIf (At2g31900)

xif-1 (SALK_094787)

xif-2 (SAIL_514_H11)

1000 bp

c

wild type xif xikxif xik

xi2 xif xik

xi2 xif xig xik

xi1 xi2 xib xif xik

xi1 xi2 xib xif xig xik

xi1 xi2 xib xik

30-d

ay-o

ld37

-day

-old

dwild type ProXIf:XIf

in xif-1 xik-2 ProXIk:XIk-YFP

in xif-1 xik-2 ProXIf:XIk-GFP

in xif-1 xik-2 ProXIf:XIk-GFP

in xik-2 xif-1 xik-2

Hara-Nishimura, Supplementary Figure 1© 2015 Macmillan Publishers Limited. All rights reserved.





Supplementary Figure 2. Transcript levels of myosins XIf and XIk in various tissues. Myosin XIf (magenta) and myosin XIk (green). Data were obtained from the public Arabidopsis eFP Browser.

0 50 100 150 200 250 300

Gene expression

Hara-Nishimura, Supplementary Figure 2 © 2015 Macmillan Publishers Limited. All rights reserved.





Supplementary Figure 3. Deficiency of myosins XIf and XIk enhances organ bending in response to gravity stimulus. (a) Maximum intensity projections of time-lapse images during gravitropism of inflorescence stems. Left, wild type; right, myosin xif-1 xik-2. These images are related to Fig. 1c, d and Supplementary Movie 1. (b) Comparison of hypocotyl gravitropism among myosin XI-deficient genotypes. Etiolated seedlings were placed horizontally and curvature was measured after 9 h. Error bars indicate SD. *, P < 1.0 × 10-15 (Student’s t test). Absolute values and statistical analysis of the data are shown in a table. n, number of data points in the data set. (c) Comparison of root gravitropism among myosin XI-deficient genotypes. Etiolated seedlings were placed horizontally and curvature was measured after 9 h. Error bars indicate SD. *, P < 1.0 × 10-15 (Student’s t test). Absolute values and statistical analysis of the data are shown in a table. n, number of data points in the data set. (d) Gravitropism of petioles. Each number shows the same leaf. Note: myosin xif xik shows enhanced gravitropic bending, making leaf no. 7 face downwards. Bars = 1 cm.

a

wild type

1

1

2

2

3

3

4

4

g

5

5

6

6

7

7

8

8

0 h

24 h

xif-1 xik-2d

bwild type

xif-1xif-2xik-2xik-3

xif-1 xik-2xif-1 xik-3xif-2 xik-2xif-2 xik-3

0 30 60 90Hypocotyl curvature (deg)

Hypocotyl curvature (gravi-stimulation)

wild type xif-1xif-2xik-2xik-3

xif-1 xik-2xif-1 xik-3xif-2 xik-2xif-2 xik-3

Root curvature (deg)0 30 60 90 120

c Root curvature (gravi-stimulation)

Hara-Nishimura, Supplementary Figure 3

curvature (deg)n P value

average SDwild type 37.9 5.50 32

xif-1 36.0 8.13 31 2.99E-01xif-2 35.1 6.27 25 7.96E-02xik-2 33.6 8.44 30 2.27E-02xik-3 39.6 7.00 22 3.13E-01

xif-1 xik-2 66.4 12.71 35 1.12E-17xif-1 xik-3 76.0 7.18 21 1.49E-27xif-2 xik-2 72.9 10.04 23 1.13E-22xif-2 xik-3 66.7 8.40 30 1.98E-23

curvature (deg)n P value

average SDwild type 45.0 5.65 32

xif-1 54.7 12.02 31 1.08E-04xif-2 36.5 6.28 25 1.78E-06xik-2 45.7 9.29 30 7.17E-01xik-3 38.8 12.93 22 2.03E-02

xif-1 xik-2 84.4 15.75 35 2.30E-20xif-1 xik-3 82.4 8.10 21 1.32E-25xif-2 xik-2 78.6 10.48 23 3.76E-21xif-2 xik-3 77.7 10.28 30 6.68E-23






Supplementary Figure 4. A horizontal clinostat test showing that myosins XIf and XIk and intact actin are required for straightening of inflorescence stems. (a) An experimental system of clinostat test for straightening. Two plants with 4- to 8-cm-long primary stems were set symmetrically on each clinostat disc with a 7-cm radius and were gravistimulated (1 g) for 45 min by placing horizontally. After gravistimulation, the plants were rotated on a horizontal clinostat at 5 rpm to neutralize earth’s unilateral gravitational pull for 3-10 h. The clinorotation-induced centrifugal force (~0.002 g) was too small to affect the straightening behavior of the plants. The angles of stem bending were measured at 1 and 3 h of clinorotation. Magenta solid line, clinorotation axis; magenta dashed line, plant axis. (b, c) Representative images of wild type and myosin xif-1 xik-2 during 3-h clinorotation. (d) Comparison of straightening among myosin XI-deficient genotypes and transgenic lines expressing ProXIf:XIf (lines #2 and #3) or ProXIk:XIk-YFP in myosin xif-1 xik-2 background. (e) Representative image of actin8 (fiz1/+) during 3-h clinorotation. g indicates the gravity direction.


gravistimulation for 45 min

clinorotation for 1 h

clinorotation for 2 h

Gravitational force on plants

1 g�

1 g�

1 g�

1 g�

0.002 g�

0.002 g�

0.002 g�

0.002 g�

Centrifugal force on plants

��

��

��

0 1 3

a1

a3

0 20 40 8060 100Frequency of plants (%)

wild type

xif-1 xif-2

xik-1xik-2xik-3

xif-1 xik-2

xik-4

xif-2 xik-3

ProXIf:XIf xif-1 xik-2 (#2) ProXIf:XIf xif-1 xik-2 (#3)

ProXIk:XIk-YFP xif-1 xik-2

d

a1 = a3

a1 > a3 straightening

a1 < a3 bending

e

Tim

e on

clin

osta

t (h)

1

3

0

actin8 (fiz1/+)

g

a

0

0.5

1.0

1.5

2.0

Tim

e on

clin

osta

t (h)

b wild type xif-1 xik-2c

g






Supplementary Figure 5. Promoter activity of myosin XIf in fibre cells. (a) Immunoblot analysis of extracts prepared from upper part of stem using anti-myosin XIk and anti-GFP antibodies. (b-d) Vertical sections of upper part of inflorescence stem in a transgenic plant expressing ProXIf:mCherry-ER and ProXIk:XIk-YFP (b, d) and non-transgenic plant (c). Images were taken with same conditions between b and c. Note that cortex layers show strong autofluorescence (asterisks).


a

anti-GFP

220

12010080

6050

40

30

kDa

anti-XIk

XIk-GFP/XIk-YFPXIk

epidermal cellscortex cell layersendodermal cells

fiber cells

cb

100 µm

ProX

Ik:

XIk-

YFP

ProX

If:

mC

herr

y-ER

m

erge

brig

ht fi

eld

co cofc fc

* *

* *

ProXIk: XIk-YFP

ProXIf: mCherry-ER merge

5 µm

d

non-transgenic (control)

brig

ht fi

eld

fluor

esce

nce

co co

* *

100 µm






Supplementary Figure 5. Promoter activity of myosin XIf in fibre cells. (a) Immunoblot analysis of extracts prepared from upper part of stem using anti-myosin XIk and anti-GFP antibodies. (b-d) Vertical sections of upper part of inflorescence stem in a transgenic plant expressing ProXIf:mCherry-ER and ProXIk:XIk-YFP (b, d) and non-transgenic plant (c). Images were taken with same conditions between b and c. Note that cortex layers show strong autofluorescence (asterisks).


a

anti-GFP

220

12010080

6050

40

30

kDa

anti-XIk

XIk-GFP/XIk-YFPXIk

epidermal cellscortex cell layersendodermal cells

fiber cells

cb

100 µm

ProX

Ik:

XIk-

YFP

ProX

If:

mC

herr

y-ER

m

erge

brig

ht fi

eld

co cofc fc

* *

* *

ProXIk: XIk-YFP

ProXIf: mCherry-ER merge

5 µm

d

non-transgenic (control)

brig

ht fi

eld

fluor

esce

nce

co co

* *

100 µm

Supplementary Figure 6. Comparison of inflorescence stems between double mutant myosin xif-1 xik-2 and wild type. (a) Lignification in inflorescence stems. Cross sections of upper and basal parts of inflorescence stems from 4- to 5-week-old plants were stained with 0.02% toluidine blue, which stains heavily lignified cell walls deep blue. (b) Diameter of inflorescence stem of 4- to 5-week-old plants. Bars plot mean ± SE of 15 individuals. *, P < 0.05 (Student’s t test). (c) Deformation rate of inflorescence stem as determined in a compression assay. Same stems as those measured in b were assayed. Bars plot mean ± SE of 15 individuals.

awild type xif-1 xik-2

Upp

er p

art o

f ste

mB

asal

par

t of

stem

Deformation rate (%)302520151050

Stem diameter (mm)1.00.80.60.40.20

*

*

Upper

Basal

wild type

wild type

xif-1 xik-2

xif-1 xik-2

Upper

Basal

wild type

wild type

xif-1 xik-2

xif-1 xik-2

cb

100 µm 100 µm

100 µm 100 µm







Supplementary Figure 7. Activity of myosin XIf promoter. (a-c) GUS expression driven by myosin XIf promoter in hypocotyl (a), mature region of root (b), and root tip (c) of 3-day-old seedlings. (d,e) Immunoelectron micrographs of seedlings that expressed a GFP fusion with the ER membrane protein RTN1 under the control of myosin XIf promoter. GFP-RTN1 was detected with anti-GFP antibody. (d) Hypocotyl of 3-day-old etiolated seedling. (e) Root of 12-day-old seedling. Right panels are enlarged images of cells in a left panel indicated by arrows. Magenta asterisks indicate immuno-positive cells; blue asterisks indicate immuno-negative cells. ep, epidermal cells; co, cortex cells; en, endodermal cells; xy, xylem; ph, phloem.


b

100 µm

root

a

100 µm

hypocotyld

100 µm

root tip

c

positive negative

e

ep

co

enen

en

coep

co

pe

pepe

xyxy

pe

pepe

20 µm

0.5 µm

0.5 µm

root

xyxy

en

en

co

co

coen

hypocotyl

20 µm






Supplementary Table 1. Absolute values and statistical analysis of the data presented in Fig. 1. n, number of data points in the data set; SD, Standard Deviation.

Gravitropism of the inflorescence stems. (Related to Fig. 1c)

Time (min)

wild type xif-1 xik-2 P value by t-test (wild type versus xif-1 xik-2) curvature (deg) n curvature (deg) n average SD average SD

0 -10.0 6.8 11 -9.8 8.6 8 9.36E-01 20 -8.1 7.3 11 -12.0 10.6 8 3.45E-01 40 35.7 13.2 11 23.0 13.6 8 5.52E-02 60 74.0 18.3 11 55.6 17.5 8 4.21E-02 80 99.7 15.4 11 88.2 22.0 8 1.96E-01

100 111.6 14.3 11 110.0 24.9 8 8.63E-01 120 112.9 13.4 11 126.4 29.7 8 1.98E-01 140 106.1 16.5 11 139.8 27.5 8 3.81E-03 160 98.5 14.8 11 144.9 25.3 8 1.02E-04 180 93.5 11.3 11 141.0 27.3 8 6.92E-05 200 89.8 7.4 11 134.0 30.2 8 1.99E-04 220 86.5 5.2 11 125.1 31.2 8 7.89E-04 240 85.4 5.0 11 116.9 30.2 8 3.15E-03 260 87.8 5.6 11 106.4 32.1 8 7.30E-02 280 89.1 6.0 11 99.2 34.4 8 3.51E-01 300 90.1 7.0 11 92.3 30.2 8 8.15E-01 320 89.1 6.6 11 84.9 23.7 8 5.75E-01 340 89.1 6.3 11 78.2 20.7 8 1.18E-01 360 89.6 5.5 11 73.8 17.7 8 1.19E-02 380 89.5 6.2 11 71.8 18.7 8 8.88E-03 400 89.7 5.3 11 72.3 17.3 8 5.65E-03 420 88.7 4.3 11 69.4 16.2 8 1.44E-03 440 88.4 3.9 11 69.2 18.9 8 4.09E-03 460 88.7 5.1 11 73.3 18.6 8 1.73E-02 480 89.1 5.2 11 77.4 18.3 8 5.18E-02

Gravitropism of hypocotyls. (Related to Fig. 1e)

Time (min)

wild type xif-1 xik-2 P value by t-test

(wild type versus xif-1 xik-2) curvature (deg) n curvature (deg) n average SD average SD 0 -0.74 5.09 35 -0.24 7.92 35 7.56E-01

1.5 3.11 3.97 32 4.81 6.74 32 2.25E-01 3.0 14.86 4.19 35 21.39 9.72 32 5.74E-04 6.0 26.66 7.23 33 49.66 9.05 35 1.79E-17 9.0 37.85 5.50 32 66.35 12.71 35 1.12E-17

16.0 52.20 8.03 30 70.12 9.89 35 4.73E-11 24.0 61.54 7.58 33 81.46 8.59 35 4.75E-15

Gravitropism of roots. (Related to Fig. 1f)

Time (min)


(wild type versus xif-1 xik-2) curvature (deg) n

curvature (deg) n average SD average SD

0 -0.55 5.08 35 -0.29 15.01 35 9.21E-01 1.5 0.42 7.70 32 0.43 16.66 32 9.98E-01 3.0 14.14 10.92 35 19.40 13.87 32 8.81E-02 6.0 33.68 8.32 30 59.09 14.03 35 2.27E-12 9.0 44.96 5.65 32 84.42 15.75 35 2.30E-20

16.0 61.60 12.64 30 90.75 12.99 35 3.80E-13 24.0 76.27 12.37 33 96.23 10.76 35 1.03E-09






Phototropism of hypocotyls. (Related to Fig. 1g)

Time (min)


(wild type versus xif-1 xik-2) curvature (deg) n curvature (deg) n average SD average SD 0 1.78 3.99 7 -1.19 6.79 7 3.38E-01

40 2.29 2.90 7 -0.32 5.50 7 2.87E-01 80 12.51 5.90 15 9.98 7.75 18 3.08E-01

120 49.94 9.15 20 58.79 11.85 20 1.19E-02 160 83.77 9.28 25 111.04 13.87 24 1.70E-10 200 90.10 10.74 8 107.85 9.21 7 4.67E-03 240 87.08 6.57 10 100.31 11.85 15 3.94E-03






Supplementary Table 2. Absolute values and statistical analysis of the data presented in Fig. 4. n, number of data points in the data set; SD, Standard Deviation; SE, Standard Error.

Maximal velocity of plastids in the fiber cells of myosin xi mutants. (Related to Fig. 4b)

maximal velocities

(µm/sec) n P value by t-test (wild type versus each myosin xi mutant) average SD SE

wild type 8.60 1.85 0.34 30 xif-1 7.28 1.35 0.25 30 2.58E-03 xik-2 8.21 2.30 0.42 30 4.74E-01

xif-1 xik-2 1.72 0.73 0.14 29 6.60E-26

Gravitropism of the actin8 (fiz1/+) inflorescence stem. (Related to Fig. 4c)

Time (min)

wild type actin8 (fiz1/+) P value by t-test (wild type versus fiz1/+) curvature (deg) n curvature (deg) n average SD average SD

0 -5.9 5.5 8 -5.9 6.9 11 9.91E-01 30 14.0 6.8 8 -13.2 8.5 11 9.34E-07 60 77.8 15.7 8 50.8 20.9 11 7.10E-03 90 108.0 21.9 8 87.5 28.2 11 1.06E-01

120 94.1 12.2 8 116.3 26.8 11 4.48E-02 150 65.6 12.4 8 151.5 23.3 11 3.55E-08 180 78.1 13.0 8 154.0 25.3 11 5.73E-07 210 77.6 11.9 8 143.0 29.9 11 2.04E-05 360 78.1 3.4 8 96.1 46.6 11 2.95E-01

Maximal velocity of plastids in the fiber cells of actin8 (fiz1/+). (Related to Fig. 4e)

maximal velocities

(µm/sec) n P value by t-test (wild type versus fiz1/+)

average SD SE WT 7.26 1.07 0.38 8

fiz1/+ 2.18 0.67 0.19 12 1.21E-10






Supplementary Table 3. Primers used for cloning and genotyping. Cloning primers

primer sequence proF-F3 CACCAGTGGACAAATCTTAAATTTTTTAT proF-2R AACTCAAGCTCTTCCAAGCTCTTACACTGA B4 XIFproF GGGGACAACTTTGTATAGAAAAGTTGAGTGGACAAATCTTAAATT B4 XIFproR GGGGACTGCTTTTTTGTACAAACTTGAACTCAAGCTCTTCCAAGC CDSF-F CACCATGGGGACTCCTGTAAATATCACAC FallR TTATTCCGGCAATGTCTGGAATAAG SP-F CACCATGAAG GTACAGGAGGGTTTG SP-R TGCTCACCATGGATCCGCACTCCTTGCGAG mCherry-F GTGCGGATCCATGGTGAGCAAGGGCGAGGA mCherry-R TCCATGCCGCCGGTGGAGTGGCGGCCCTCG HDEL-F CACTCCACCGGCGGCATGGACGAGCTGTAC HDEL-R TTACAGCTCGTCATGAGATC pXIf-LAV-IF-F GCAGGCTCCGCGGCCGCTAAAGTGGACAAATCTTAAATTTTTTATCA pXIf-LA-R GACACCCATAACTCAAGCTCTTCCAAGCTC pXIf-LA-F GCTTGAGTTATGGGTGTCGCAGATTTGATC pXIf-LAV-IF-R AGCTGGGTCGGCGCGCCTTACTTGTACAGCTCGTCCATGCCGAGA pXIf-XIk-R2 TTTTACCATAACTCAAGCTCTTCCAAGC pXIf-XIk-F2 TGAGTTATGGTAAAATCATAGATATCATCA XIk-linkerGFP-R2 ACCTCCACCACCTCCCGATGTACTGCCTTC XIk-linkerGFP-F2 GGAGGTGGTGGAGGTGCTATGGTGAGCAAG GFP-IF-R AGCTGGGTCGGCGCGCCTCACTTGTACAGC pXIf-GFP-R GCTCACCATAACTCAAGCTCTTCCAAGCTC pXIf-GFP-F GCTTGAGTTATGGTGAGCAAGGGCGAGGAG G-RTN1-R CGCCATTCCTCCTCCGAGATCTCCCTTGTA G-RTN1-F ATCTCGGAGGAGGAATGGCGGAAGAACATA RTN1-IF-R AGCTGGGTCGGCGCGCCTTCTGCTTAATACTCTACTCTGTGTAGTTT

Genotyping primers

primer sequence myosin xif-1 F GAATGATAGCAGGGCAAGTGT myosin xif-1 R ACACTATCCAAGCTGCCACAG myosin xif-2 F ATCCAATTTGACAAGCGTGGGAAAA myosin xif-2 R CCTATTGAACTGTTGATCTTATCTA myosin xik-2 F ATCGCAAACGTATTTCCAAAGGATACAGAG myosin xik-2 R CTCCCACAGTTGTCAAACTGAAGCTCGACA myosin xik-3 F CACAGGACATGACTCTTGCTGTACG myosin xik-3 R GCACTTTGAGGAGATCCCCTTAATCC myosin xi1-2 F CTGCAATTGTTTTGCAATCCTTCCTACGAG myosin xi1-2 R CTCGAGCCGCCTCCTGTTCCTTTACGACCA myosin xi2-2 F AAAGATTACGTTATTGCCGAGCATCAGGCA myosin xi2-2 R GTGATCTTATCCATTAATTCCTGATCAACC myosin xib-1 F TGGTCAGATCTTTGTCTTATCACAG myosin xib-1 R CAGAAGCAACCCACTTCAGTC myosin xig-1 F ATATAAAGGAGCCGCTTTGGG myosin xig-1 R AGAAGCATCAGGACATAGTTTC LBa1 TGGTTCACGTAGTGGGCCATCG LB1 GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC




Documents

Nature Plants 1, 15031 (2015); published online 23 March ... · SUPPLEMENTARY INFORMATION. DOI: 10.1038/NPLANTS.2015.31. NATURE PLANTS | . 1. 1 . SUPPLEMENTARY METHODS RT-PCR and