0 10 　 25 　 50 　 　 100

+ Suc + ABA 　- Suc + ABA

ABA (M)

OsAGPL3

rRNA

Con

trol

0 10 　 25 50 　 　 100

Fig1.Accumulation of OsAGPL3 mRNA in response to ABAPre-cultured cells were transfer to the medium supplemented with various concentrations of ABA as indicated. After 24 h of cultured in the dark, suspension cells were collected and total RNAs were isolated. OsAGPL3 (accession no. AK100910) mRNA levels were measured by northern blot analysis.

0 1 3 6 12 24 （ h ） 　　OsAGPL3

Fig2. Time course of OsAGPL3 mRNA accumulation in response to sucrose and ABA. Pre-cultured cells were transfer to medium supplemented with 3%(w/v) sucrose + 50 M ABA. 　 Expression of  OsAGPL3  at different time points was assessed by Northern blot analysis.

Time after transfer

rRNA

Fig3. Synergistic effect of sucrose and ABA on OsAGPL3 mRNA accumulation. Pre-cultured suspension cell was used as control (control). Pre-cultured cells were transfer to medium supplemented with either 50-M 　 ABA (+ABA) or 3% (w/v) sucrose (+Suc) alone, or 3%(w/v) sucrose plus 50M 　 ABA (+Suc+ABA). After 6 h of culture, suspension cells were collected and total RNAs were isolated. OsAGPL3 mRNA levels were measured by Northern blot analysis.

contr

ol+ A

BA+ Suc+ Suc

+ ABA

OsAGPL3

rRNA

0

20

40

60

80

100

120

Rel

ativ

e tr

ansc

ript

s le

vel (

%)

120

100

80

60

40

20

0

0

2

4

6

8

10

121系列2系列

Star

ch c

onte

nts

mg/

100m

g D

W

+Suc +Suc+ABA+ABAControl

Fig4. Synergistic Effect of sugars and ABA on starch content in rice suspension cell. Pre-cultured cell was used as control (control). Pre-cultured cells were transfer to medium supplemented with either 50 M 　 ABA (+ABA) or 3%(w/v) sucrose (+Suc) alone, or 3% (w/v) sucrose plus 50 M 　 ABA (+Suc+ABA). After 6 h of culture, cultured cell were collected and starch contents were determined.

6 h12h

Cont

rol

+ABA

+Suc

+Suc

+AB

ACo

ntro

l+A

BA+S

uc+S

uc+A

BACo

ntro

l+A

BA+S

uc+S

uc+

ABA

Cont

rol

+ABA

+Suc

+Suc

+ABA

Cont

rol

+ABA

+Suc

+Suc

+AB

ACo

ntro

l+A

BA+S

uc+S

uc+A

BACo

ntro

l+A

BA+S

uc+S

uc+

ABA

Cont

rol

+ABA

+Suc

+Suc

+ABA

9799 63

321

189

66

213

94

The number of bandsCo

ntro

l +A

BA +S +S

+ABA

(A) (B)

Fig5 cDNA-AFLP display of gene expression in rice cultured cell.(A) A typical autoradiogram showing the TDFs pattern of rice cultured cells. RNAs were isolated from pre-cultured cell (control), 50 M 　 ABA (+ABA) or 3% (w/v) sucrose (+S) alone, or 3% (w/v) sucrose plus 50 M 　 ABA (+S+ABA) treated cells. Primer combinations for restriction endonucleases AvaII and TaqI are shown at the top of the cDNA-AFLP autoradiogram.(B) Classification of TDFs by their induction patterns. Total 11099 TDFs were identified. In total, 9799 TDFs were not different between each samples. 63, 321, 66 and 94 TDFs were increased in the cultured cells grown in the +S+ABA, + ABA, +S and +S+ABA medium, respectively.

AvaII TC TC TC TC TC TC TC TC TaqI AA AT AC AG GA GT GC GG

TDF38

TDF41

TDF54

TDF59

TDF64

TDF65

TDF69

TDF1

TDF5

TDF9

TDF10

TDF21

TDF56

TDF67

TDF6

TDF22

TDF24

TDF28

TDF29

TDF32

TDF80

TDF79

TDF23

TDF15

TDF47

TDF75

+AB

A

+Suc

+AB

A

+Suc

Con

trol

Fig6 Northern blot analysis of 26 genes which is identified by cDNA-AFLP as synergistically induced by sucrose and ABA.Rice cultured cell were cultured in the medium supplement with 50 M ABA (+ABA) , 3% (w/v) Sucrose ( +Suc) and 3% (w/v) Sucrose and 50 M ABA (+Suc+ABA) for 6h. Pre-cultured cell was used as control (Control). Ten g of total RNAs from cultured cells were loaded in each lane, The blot were hybridized with 32P-labeled DNA probes.

1 2 3 4

5 6 7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

(A) (B)

+AB

A

+Suc

+AB

A

+Suc

Con

trol

INV

Suc

Hexose HXT Hexose

Hexose-P

ADPG

Hexose-P

ADPG

Starch

OsAGPL3

GPT

AGPase

Pi?

PlastidCytosol

ChloroplastAcetate

Acetate-CoA C16:0 ACP C18:0 ACP C18:1 ACP

fab2

C18:1 COA

Endoplasmic reticulum

PA

18:1 18:1

DAGPC

P-EA P-MEA P-DEA

P-cholinePEAMT

18:118:118:118:1

PCPC

18:218:218:318:3

PC

fad2

chloroplast

AcetateAcetate-CoA C16:0 ACP C18:0 ACP C18:1 ACP

C18:1 GLC18:2 GLfad6

fab2

C18:1 COA

Endplasmic reticulum

PA

18:1 18:1

DAG

18:1 18:1

PC

18:1 18:1

PC

18:2 18:2

PC

18:3 18:3

fad3 fad2

P-AI

18:1 18:1

P-EA

18:1 18:1

P-EA P-MEA P-DEA

P-choline

CDP-choline

phosphoethanolamine N-methyltransferase

*1; Analyzable cDNAs, which containing both AvaII and TaqI site.*2; Unanalyzable cDNAs , which dose not containing either AvaII or TaqI or Both site.

*1*2

32127240818046

70824

32127183771375136590

256 128Total number of full length cDNAsAnalyzable cDNAsUnanalyzable cDNAsTotal number of TDF ｓ

Table1 In silico cDNA-AFLP analysis of Oryza sativaprimers combinations