Mori, H. Nagoya University

Apical Dominance is controlled by Interaction between Cytokinin

biosynthesis/degradation and Auxin in Stem.

Mori, H.Nagoya University

3days after decapitation intact

auxin

axillary buddormant

decapitation

Apical Dominance

cytokinin

outgrowth

G2 check point replication cell size environment

G1 check point cell size environment

Quiescence (G0)Terminal Differentiation

CellCycle

S

G2

M

G1

cyclinD1, 2

cyclinD3

PCNA cyclinB

histoneH4

G1 S G2 M

cdc2

mR

NA

cdc2

CycD3;1

CycB1 ;2

histoneH4

PCNA

0 2 4 86 10 12 14 16 18 20 22 24Time after decapitation (hours)

EtBr

Northern Blot Analyses of Cell Cycle Related Genes

1cm 2cm4cm

6cm

Time after decapitation (hr)

0 2 4 6

Rela

tive A

cit

ivit

y

0

1 cm

2 cm

4 cm

6 cm

2000

Expresson of PCNA mRNA

axillary buddormant decapitation

auxin

cytokinin

outgrowth

・ Auxin derived from terminal bud is transported basipetally.

・ When auxin applied to the cut stump after decapitation,

Auxin can repress axillary bud outgrowth.

・ When auxin directly applied to axillary bud after decapitation,

Auxin cannot repress axillary bud outgrowth.

Auxin is basipetally transported in stem.

IAAaxillary bud

Scale leaf at 2nd node

The basipetal transport of auxin is essential for dormancy of axillary bud.

Auxin acts at node or in stem, not in axillary bud.

The basipetal transport of IAA is essentialfor axillary buds dormancy

intact

IAA

0 hr

decapitated

IAA

1cm

1 hr 3 hr

auxin transport rate: 1cm/hr

Subtracted cDNA clones

0hr-3hr

3hr-0hr

Ps-IAA4/5, IAA7, Aux28, Aux22, GH3, F-box, cullin, auxin efflux carrier protein(PIN1 ),RD29B, LEA4, zeaxanthin epoxidase

IAA-Ala hydrolase, GA2 oxidase,HMG-CoA synthase, squalene synthase,Isopentenyltransferase (IPT )

Current model of cytokinin metabolism in higher plants

LOG

(revised)

LOG LOG LOG

1cm2nd node

0.5 4321 5 6 9 15 18 24120

PsIPT1

PsIPT2

rRNA

time after decapitation (hr)

Expression of PsIPTs at the 2nd Node after Decapitation

excise the segmentsfrom intact seedlingsIPT is NOT expressed

incubate in MES bufferwith or without IAA

Is IPT expressed?

excise the segments3hr after decapitation

IPT is expressed


Is IPT expression repressed ?

1cm

1 2 3 4 50

IAA Free

1 2 3 4 50

10-5 M IAA

PsIPT1

PsIPT2

rRNA

incubation time (hr)

PsIPTs Expression was Repressed by IAA

excise the segmentsfrom intact seedlingsIPT is NOT expressed


Is IPT expressed?

excise the segmentsafter 3hr decapitation

IPT is expressed


Is IPT expression repressed?

1cm

PsIPTs Expression was Induced in IAA Free Buffer

IAA Free 10-5 M IAA

0 1 65432

incubation time (hr)incubation time (hr)

0 1 65432 987

PsIPT1

PsIPT2

rRNA

excise the segmentsfrom intact seedlingsNot IPT expression


IPT express?

excise the segmentsafter 3hr decapitation

IPT is expressed


Repress IPT expression?

1cm

1 2 3 4 50

IAA Free

1 2 3 4 50

10-5 M IAA

IAA Free 10-5 M IAA

0 1 654320 1 65432 987

PsIPTs Expression was Repressed by IAA Transported Basipetally in the Stem

PsIPT1

PsIPT2

PsPIN1

＋IAA

Lanoline paste w or w/o IAA after decapitation

3hr


1cm

pPsIPT2::GUS in Arabidopsis

pPsIPT2::GUS (2,4-D treated for 1 week)

pPsIPT2::GUS (2,4-D free for 3 days)

IAA Free 10-5M IAA

10 2 3 4 5 10 2 3 4 5

incubation time (hr)

GUS Transcripts Deceased by Auxinin Transformants harboring pPsIPT2::GUS

Cytokinin Levels in the Stems and Axillary Buds after Decapitation

Cytokinin Levels in the Excised Segments treated with/without IAA

0

100

200

300

iPMP iPA iP t-ZMP t-ZR t-zeatin

0h3h6h

-IAA

(p m

ol/

gF

W)

0

100

200

300

iPMP iPA iP t-ZMP t-ZR t-zeatin

0h3h6h

(p m

ol/

gF

W)

+IAA

excised the segmentsput on the agar plate

± 1%(w/v) IAA treatment

Conclusion

One role of auxin in apical dominance is to repress the PsIPTs

expression; local cytokinin biosynthesis in the nodal stem is

negatively regulated by auxin through the control of IPT

expression.

Cytokinin, which is thought to be derived from the roots so far,

is locally biosynthesized in the stem rather than in the roots

after decapitation.

PsIPTs Expression was Repressed by Basipetally Transported IAA in the Stem

PsIPT1

PsIPT2

PsPIN1

＋IAA

1% (w/v) IAAafter decapitation

3hr


1cm

Effect of IAA Applied to the Stump on PsPIN1 Expression

PsPIN1

actin

0 1 2 (hours)4

(cm)1 2 3 4 1 2 3 4 1 2 3 41 2 3 4

1 2 3 4IAA

Can IAA from Axillary Buds induce PsPIN1?

intact

0hr 9hr

decapitated

3hr

IAA

IAA

IAA Biosynthesis

Time after decapitation ( hours )

PsPIN1

actin

0 2 4 6 8 10 12 0 2 4 6 8 10 12

IAA

×

Upper Stem

IAA

Lower Stem

0.5 4321 5 6 9 15 18 24120

PsIPT1

PsIPT2

rRNA

time after decapitation (hr)

Expression of PsIPTs at the 2nd Node after Decapitation

IAA

81.7CK

2.9

IAA

44.0CK

9.1

IAA

19.3CK

135.5

IAA 20.8

CK105.

5

IAA

60.5CK

3.6

IAA

35.4CK

175.2

IAA175.

3CK

47.6

IAA

79.5CK

1.9

IAA

50.5CK

6.3

IAA

27.7CK

76.6

IAA

33.6CK

158.0

IAA

34.6

CK

94.8

IAA

39.9CK

50.7

IAA

55.2

CK

23.8

IAA 72.2

CK 7.8

0 1 3 12 18 24 36 48

(p mol/gFW)

node

upperstem

lowerstem

PsCKX1 ： was constantly expressed in stem before and after decapitation, was not induced by auxin.

PsCKX2 ： was increased in stem after decapitation was induced by auxin.

Cytokinin oxidase cDNAs were isolated from the stem 12 hr after decapitation.

Time after treatment (hr)0 10 20 300

40

80

120

decapitation

TIBA treatment

Rel

ativ

e am

ou

nts

of

mR

NA

Expression of PsCKX2 in Pea Stem

Time after treatment (hr)0 10 20

0

10

30

40

(x 103)

20

Rel

ativ

e am

ou

nts

of

mR

NA

+NAA

-NAA

PsCKX2 Expression in Excised Pea stem by Auxin


- NAA40

0

80

120

PsCKX2 Expression in Pea stem

Rel

ativ

e am

ou

nts

of

mR

NA


0

10

30

40

(x 103)

20

Rel

ativ

e am

ou

nts

of

mR

NA

+NAA

-NAA

PsCKX2 Expression in Excised Pea stem by Auxin

IAA

81.7CK

2.9

IAA

44.0CK

9.1

IAA

19.3CK

135.5

IAA 20.8

CK105.

5

IAA

60.5CK

3.6

IAA

35.4CK

175.2

IAA175.

3CK

47.6

IAA

79.5CK

1.9

IAA

50.5CK

6.3

IAA

27.7CK

76.6

IAA

33.6CK

158.0

IAA

34.6

CK

94.8

IAA

39.9CK

50.7

IAA

55.2

CK

23.8

IAA 72.2

CK 7.8

0 1 3 12 18 24 36 48

(p mol/gFW)

node

upperstem

lowerstem

Time after decapitation (hr)

0 10 20 300

40

80

120

PsCKX2

PsPIN1

PsIPT2

Rel

ativ

e am

ou

nts

of

mR

NA

Expression of PsCKX2, PsPIN1, PsIPT2 in stem

Model of Interaction Between Auxin and Cytokinin in Apical Dominance

Effect of Striolactone GR24 on outgrowth of axillary bud after decapitation

GR24 control

Apical dominance

Nagoya University

・ Mina TANAKA

・ Sae SATO-SHIMIZU

・ Aiko ISHIWATA

・ Yuka MADOKA

RIKEN

・ Mikiko KOJIMA

・ Hitoshi SAKAKIBARA

Mori, H. Nagoya University

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