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17 Οκτ 2013 (πριν από 4 χρόνια και 21 μέρες)

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Supplementary Information


Control of final seed and organ size by the
DA1

gene
family in
Arabidopsis thaliana


Yunhai Li
1, 2, 3
, Leiying Zheng
1
, Fiona Corke
1
, Caroline Smith
1
, and Michael W.
Bevan
1
, 3


1

Department of Cell and Developmental Biology, J
ohn Innes Centre, Norwich NR4
7UH, UK.

2

The State Key Laboratory of Plant Cell and Chromosome Engineering,

Institute of
Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,
China.

3

Corresponding authors: E
-
MAIL:
michael.bevan@bbsrc.ac.uk

;
Tel
(44) 0
1603
450520; Fax

(44)

0
1603 450025 and
E
-
MAIL:
yhli@genetics.ac.cn
; Tel (86) 10
64807856; Fax: (86) 10 64854467



Running title
:
DA1,

cell prol
iferation and organ size



Key words
: DA1, EOD1/BB, seed and organ size, cell proliferation, Arabidopsis



Supplementary Methods and Materials

Plant materials and growth conditions.

Arabidopsis thaliana

Columbia (Col
-
0) was the wild type line used. All m
utants were
in the Col
-
0 background, except for
da1
-
1
Ler
,
bb
-
1
and

ant
-
5
, which was in Landsberg
erecta

(Ler).
da1
-
ko1

(Salk_126092),
da1
-
ko2

(SALK_110232),
da1
-
ko3

(SALK_
054295),
dar1
-
1

(SALK_067100),
dar2
-
1

(SALK_016122), and
eod1
-
2

(SALK_045169) were i
dentified in AtIDB (
www.atidb.org
) and obtained from the
Nottingham Arabidopsis Stock Centre (NASC). T
-
DNA insertions were confirmed

by
PCR and sequencing by using the primers described in Supplementary Table 4.
arf2
-
7
,
a
p2
-
7

and
ant
-
5

were obtained from the Nottingham Arabidopsis Stock Centre
(NASC) collection. Plants were grown on soil under standard conditions in a 16
-
hr
-
light/8
-
hr
-
dark cycle.

Seed size and seed mass analysis.

Average seed mass was determined by weighi
ng mature dry seeds in batches of 100
using an electronic analytical balance (Sartorius ME5, Germany). The weights of five
sample batches were measured for each seed lot. Size distributions of wild type and
mutant seed populations were analysed by separati
ng batches of seeds by using a
series of fine wire sieves, Sieve mesh sizes with exclusion sizes of 425, 355, 300 250
and 180µm (Fisher Scientific), respectively, were used for each analysis. Seeds
retained by each sieve were weighed and mass of each fract
ion was expressed as a
percent of the total mass of the seed sample analyzed.

Morphological and cellular analysis.

Area measurements of fully expanded cotyledons, petals (stage 14), and fifth leaves
collected at the different time points were made by fl
attening the organs, scanning to
produce a digital image, and then calculating area by using Image J software. Embryo
and petal cell sizes were measured on the adaxial sides of cotyledon and petals from
SEM images. Leaf cell sizes were measured from sub
-

e
pidermal palisade
parenchyma cells in the middle of the leaf.

Biomass accumulation in flowers (stage 14) and in leaves (from 1
st

to 7
th

) was
measured by weighing excised organs.

For analysis of whole
-
mount seeds, seeds were dissected from siliques and pl
aced in a
drop of clearing solution (8g chloral hydrate, 11ml water, 1ml glycerol). Samples
were photographed under a Nikon microscope with differential interference contrast
optics using a Nikon color camera.

For scanning electron microscopy (SEM), embryo
s and petals were frozen in liquid
nitrogen

slush at

190°C. Ice was sublimed at

90°C,

and the specimen was sputter
coated and examined on an XL 30

FEG (Philips, Eindhoven, The Netherlands)
cryoscanning electron

microscope fitted with a cold stage.

Petal
primordia were dissected from the open flower (stage 14) backward to the
youngest flower bud from which this was still manually possible. The time interval
between successive flowers was measured by either counting the number of silique
and open flower dur
ing one week or by marked a given floral primordium and
determining how long the marked primordium took to develop into an open flower.
Both methods of measurement gave essentially similar results.

To detect the influence of
da1
-
1

mutation on cell prolife
ration, a
pCyclinB::GUS

reporter gene was introgressed into the
da1
-
1

mutant. The total cell number and the
number of cells with GUS activity in petals were counted and expressed as a mitotic
index (% of cells with GUS activity/ number of total cells). Eac
h value represents
measurements from at least 10 petals.

Map
-
based cloning and plant transformation.
F
2

mapping populations were
generated from a single cross of Ler/
da1
-
1
, Ler/
sod1
-
3da1
-
1
, and
da1
-
1
Ler
/
eod1
-
1da1
-
1

plants. The
DA1, SOD1
and

EOD1
genes were

mapped by using simple
sequence

length polymorphic and cleaved
-
amplified

polymorphic sequence markers
and fine mapped using specific markers (Supplementary Table 2). A genomic DNA
fragment containing the entire
da1
-
1

coding

region, the ~2.5kb upstream seq
uence,
and 400bp of downstream

sequence was inserted into the binary vector pGreen to

generate the transformation plasmid DA1COM for complementation.
The
DA1 CDS

and
DA1
R358K

CDS
w
ere

subcloned

into

BamHI
site of
the binary vector pGreen
-
35S

to

generate t
he transformation plasmid
35S::DA1

and 35S::

DA1
R358K

, respectively.

The specific primers for the
DA1

CDS

and
DA1
R358K

CDS
are DA1cds
BamH1
-
F and
DA1cds
BamH1
-
R (Supplementary Table 5)
.
The plasmids were introduced into
plants using
Agrobacterium tumefacien
s

GV3101 and transformants selected on
kanamycin (50
µ
g/mL) medium
-
containing medium.

The
DA1
promoter::GUS
construct w
as

made using a PCR
-
based

Gateway system. The specific primers for the
DA1

promoter are TOPODA1PROM
-
F and TOPODA1PROM
-
R (Supplementary
T
able 5). PCR products were subcloned into pCR8/GW/TOPO TA cloning vector
(Invitrogen) using TOPO enzyme

and sequenced. The
DA1

promoter w
as
then
subcloned into Gateway Binary Vector
pGWB3

containing the

GUS reporter gene,
respectively. The specific primers

for the
EOD1/BB

gene were EOD1cds
-
F and
EOD1cds
-
R (Supplementary Table 5).

PCR products were subcloned into
pCR8/GW/TOPO TA cloning vector (invitrogen) using TOPO enzyme

and
sequenced. The
EOD1

gene was then subcloned into Gateway Binary Vector
(
pMDC32
) c
ontaining the 35S promoter
(Curtis and Grossniklaus 2003)
. The
plasmids were introduced into plants using
Agrobacterium tumefaciens

GV3101 and
transformants selected on hygromycin (30µg/ml)
-
containing medium.

GUS staining.

S
amples (
pDA1::GUS
) were

stained in a solution of 1 m
M

X
-
gluc, 50 m
M

NaPO
4

buffer, 0.4

m
M

each K
3
Fe(CN)6/K
4
Fe(CN)6, 0.1% (v/v) Triton X
-
100 and incubated

at
37°C for 10 to 24 h. After GUS staining chlorophyll was

removed using 70% ethanol.

Protein expressio
n and Ubiquitin
-
binding assays
. The UIM1+2 domain from DA1
(residues 63

127)
, DA1 and DA1
R358K

w
ere

cloned into a pGEX
-
4T
-
2 vector
(Amersham
-
Pharmacia) using
BamHI

and
XhoI

restriction sites and expressed in
E
.
coli

BL21 (DE3) in rich (LB) medium as a fusi
on with N
-
terminal GST tag. The
specific primers for the UIM1+2 domain were UIM
BamHI
-
XhoI

forward primer and
UIM
BamHI
-
XhoI

reverse primer (Supplementary Figure 5). T
he specific primers for
the

DA1 and DA1
R358K

were DA1
BamH1
-
XhoI

forward primer and DA1
BamH
1
-
XhoI

reverse primer (Supplementary Figure 5).
Bacterial lysates expressing GST
,

GST
-
UIM1+2
, GST
-
DA1 and GST
-
DA1
R358K

were prepared from
E
.
coli

BL21 induced
with 1 mM IPTG for 3 hr. Bacteria were lysed in TGH lysis buffer (50 mM HEPES
[pH 7.5], 150 mM Na
Cl, 1.5 mM MgCl
2
, 1 mM EGTA, 1% Triton X
-
100, 10%
glycerol, 1

mM phenylmethylsulfonyl fluoride [PMSF], 1 mM sodium vanadate, 10
µg/ml aprotinin, and 10 µg/ml leupeptin) and sonicated, and the lysates were cleared
by ultracentrifugation. GST and GST
-
UIM1+2
fusion proteins immobilized on
glutathione Sepharose 4B beads (GE Healthcare, UK) were incubated with 10 μg of
ubiquitin (Boston Biochem, USA) for 2 hrs at 4°C. GST
-
DA1

and GST
-

DA1
R358K

fusion proteins were incubated with ubiquitin
agarose
(
Santa

Cruz Bio
technology,
USA
) for 2 hrs at 4°C. The beads were washed extensively with TGH buffer, and
proteins remaining on the beads were resolved by SDS
-
PAGE and identified by
immunoblotting using an antibody to DA1.


RT
-
PCR
,
Quantitative real
-
time RT
-
PCR

and North
ern blot

analysis.

Total RNA was extracted from Arabidopsis seedlings using an RNeasy Plant Mini kit
(Qiagen).
R
everse transcription (RT)
-
PCR

was

performed as described

(Li et al.
2006)
. cDNA samples were standardized on

actin transcript amount using the primers
ACTIN
-
F and ACTIN
-
R (Supplementary Table 3). Quantitative real
-
time RT
-
PCR
analy
sis was performed with an Opticon 2 DNA engine (MJ Research) using the
SYBR Green JumpStart Taq readyMix (Sigma).
TUB6

mRNA was used

as an internal
control, and relative amounts of mRNA were calculated

using the comparative
threshold cycle method. The prim
ers used for RT
-
PCR and
Real
-
Time

RT
-
PCR are
described in Supplementary Table 3.

RNA gel
-
blot analysis was performed

as
described
(Rook et al. 2001; Li et al. 2004)
. Gene specific probes were

digoxigenin
-
labeled (DIG
-
dUTP) by RT
-
PCR with PCR DIG Labeling

Mix (Roche Diagnostics,
Lewes, UK) and gene specific primers.

The gene specific primers

are
RT
-
DA1
F and
RT
-
DA1R (Supplement
ary Table 3).


Hybridization was as described
(Rook et al.
2001)
; washes were

with 0.2x SSC, 0.1% SDS at 65°C, twice for 15 min. Detection

used antidigoxigenin
-
A, Fab fragments, and the chemiluminescent

substrate CSPD,
according to t
he manufacturer's instructions

(Roche, Mannheim, Germany).


Supplementary references

Curtis, M.D. and Grossniklaus, U. 2003. A gateway cloning vector set for high
-
throughput functional analysis of genes in planta.
Plant physiology

133
(2
):
462
-
469.

Li, Y., Lee, K.K., Walsh, S., Smith, C., Hadingham, S., Sorefan, K., Cawley, G., and
Bevan, M.W. 2006. Establishing glucose
-

and ABA
-
regulated transcription
networks in Arabidopsis by microarray analysis and promoter classification
using a Rele
vance Vector Machine.
Genome Res

16
(3): 414
-
427.

Li, Y., Sorefan, K., Hemmann, G., and Bevan, M.W. 2004. Arabidopsis NAP and PIR
regulate actin
-
based cell morphogenesis and multiple developmental
processes.
Plant physiology

136
(3): 3616
-
3627.

Rook, F., Cor
ke, F., Card, R., Munz, G., Smith, C., and Bevan, M.W. 2001. Impaired
sucrose
-
induction mutants reveal the modulation of sugar
-
induced starch
biosynthetic gene expression by abscisic acid signalling.
Plant J

26
(4): 421
-
433.