Starch Biosynthesis in Rice Grains: Natural Variation and Genetic Improvement

Starch Biosynthesis in Rice Grains

—— Natural Variation and Genetic Improvement

Qiao-quan Liu（刘巧泉）

College of Agriculture, Yangzhou University, Yangzhou, Jiangsu Province, China

E-mail: qqliu@yzu.edu.cn

Amylose Amylopectin

11th International Gluten Workshop

Outline

1. Allelic diversities in rice starch biosynthesis

and genetic network for rice grain quality

2. Genetic engineering of starch biosynthesis

for high resistant starch (RS) in rice

Determinants of rice grain quality

Milling quality

Appearance quality

Cooking & eating quality

Nutritional quality

Three key physicochemical properties determine

rice cooking and eating quality

Gelatinization

temperature

determines the

time required for

cooking the rice

Gel consistency

measures the

tendency of the

cooked rice to

harden on cooling.

High amylose content grains

cook dry, are less tender, and

become hard upon cooling.

GT GC

AC

Cultivar

type

Maturity

type No.

Amylose content

(%)

Gelatinization

Temperature (ASV)

Gel Consistency

(mm)

Range Mean Range Mean Range Mean

Indica

Early

Medium

Late

8

33

32

23.75-26.60

9.68-30.64

11.64-28.66

25.28

24.16

20.30

3.22-5.22

2.67-6.89

2.00-6.56

4.35

4.89

4.92

30-97

20-120

21-110

53.63

56.21

63.22

Japonica

Early

Medium

Late

13

25

5

10.54-23.09

11.34-18.00

15.64-22.16

15.25

14.77

18.35

5.94-6.91

3.32-7.00

6.00-6.94

6.42

5.98

6.38

51-95

38-108

24-85

71.77

75.64

59.20

Wide diversity of cooking and eating

qualities among rice cultivars

Amylose Amylopectin

SBE

SSS

DBE

Amylopectin Amylose

AGPase

ADPGlc

?

Starch, the major component in rice endosperm

Classification of key enzymes Gene Localization

ADP glucose pyrophosphorylase

(AGPase)

Large subunit 1 AGPL1 Chr 5

Large subunit 2 AGPL2 Chr 6

Small subunit AGPS Chr 9

Granule-bound starch synthase (GBSS)

GBSSI Wx Chr 6

GBSSII GBSSII Chr 7

Soluble starch synthase (SSS)

SSSI SSSI Chr 6

SSSII

SSSII-1 Chr 10

SSSII-2 Chr 2

SSSII-3 Chr 6

SSSIII SSSIII-1 Chr 4

SSSIII-2 Chr 8

SSSIV SSSIV-1 Chr 1

SSSIV-2 Chr 5

Starch branching enzyme (SBE)

SBEI Sbe1 Chr 6

SBEII Sbe3 Chr 2

Sbe4 Chr 4

Starch debranching enzyme (DBE)

Isoamylase (ISA) ISA Chr 8

Pullulanase (PUL) PUL Chr 4

Starch Synthesis Related Genes, SSRGs

1. Natural variation of starch synthesis

To search and identify the allelic

variation of SSRGs among different

rice ecotypes.

To find how these genes controlling

rice cooking and eating qualities.

(Cooperated with Prof. Jiayang Li, IGDB, CAS)

70 varieties with diverse grain qualities

Indica (33)

Japonica (37)

AC GC GT

AC 1.00 -0.91 a *

0.007 b

-0.46

0.779

GC 1.00 0.50

0.326

GT 1.00

a Correlation Coefficients b Pr > F

* The number marked in bold imply the according line and row quality are

correlated with each other

High correlation among AC, GC and GT

Tian et al., PNAS, 2009, 106: 21760-21765

Starch pasting curve of different rice cultivars

-500

500

1500

2500

3500

4500

5500

6500

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

Time (min)

Vis

cosi

ty (

cP

)

Tm TN1

LTP GCH

9311 WY7

CJ06 NIP

JZXN THN

SYN

Rapid Visco Analyser (RVA)

High AC

Low or intermediate AC

Very low or no AC

16 core varieties selected for sequence analysis of SSRGs

5„UTR

The Wx gDNA alignment among different varieties Varieties

Nipponbare ( japonica )

Chunjiang 06 ( japonica )

Wuyunjing 7 ( japonica )

Jiangzhouxiangnuo ( japonica - glutinous)

Suyunuo ( japonica - glutinous)

Taihunuo ( japonica - glutinous)

9308 ( indica )

9311 ( indica )

Guichao 2( indica )

Longtepu ( indica )

Minghui 63 ( indica )

Taizhongbendi 1( indica )

Zhenshan97B ( indica )

175 298 495 528 771-785 841 926 987 1056 1083 1088

C C A C CT ( 18 ) T G AATT(6) A C A

C C A C CT ( 17 ) T G AATT(6) A C A

C C A C CT ( 17 ) T G AATT(6) A C A

C C A C CT ( 16 ) T G AATT(6) A C A

C C A C CT ( 16 ) T G AATT(6) A C A

C C A C CT ( 16 ) T G AATT(6) A C A

C C A C CT ( 18 ) T G AATT(6) A C G

C C A C CT ( 18 ) T G AATT(6) A C A

C C G T CT ( 11 ) G A AATT(5) G T A

C T G T CT ( 11 ) G A AATT(5) G T A

C C A C CT ( 18 ) T G AATT(6) A C A

C C G T CT ( 11 ) G A AATT(5) G T A

A C G T CT ( 11 ) G A AATT(5) G T A

Exon 2 intron exon intron

Varieties

Nipponbare ( japonica )

Chunjiang 06 ( japonica )

Wuyunjing 7 ( japonica )

Jiangzhouxiangnuo ( japonica - glutinous)

Suyunuo ( japonica - glutinous)

Taihunuo ( japonica - glutinous)

9308 ( indica )

9311 ( indica )

Guichao 2( indica )

Longtepu ( indica )

Minghui 63 ( indica )

Taizhongbendi 1( indica )

Zhenshan97B ( indica )

2111-2112 3019 3097 3804 4078 4211 4235 4244-4246 4282 4285

------------------------ C C T C G G ATA A G

------------------------ C C T C G G ATA A G

------------------------ C C T C G G ATA A G

ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G

ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G

ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G

------------------------ C C T C G G ATA A G

------------------------ C C T C G G ATA A G

------------------------ - T C T A A --- G A

------------------------ - T C T A A --- G A

------------------------ C C T C G G ATA A G

------------------------ - T C T A A --- G A

------------------------ - T C T A A --- G A

Wx gene alignment

Varieties 111-112 172 1086 1243

Nipponbare ( japonica ) ---------------------------------------- T C

Chunjiang 06 ( japonica ) ---------------------------------------- T C

Wuyunjing 7 ( japonica ) ---------------------------------------- T C

Jiangzhouxiangnuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC TGA

Suyunuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC TGA

Taihunuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC TGA

9308 ( indica ) ---------------------------------------- T C

9311 ( indica ) ---------------------------------------- T C

Guichao2( indica ) ---------------------------------------- C T

Longtepu ( indica ) ---------------------------------------- C T

Minghui 63 ( indica ) ---------------------------------------- T C

Taizhongbendi 1( indica ) ---------------------------------------- C T

Zhenshan97B ( indica ) ---------------------------------------- C T

Wx gene alignment The cDNA Alignment Among Different Varieties

Stop

codon

• The diversities of the coding sequences were much

lower than those of whole genes in all SSRGs.

• The diversities of the nonsynonymous substitution

were lower than the synonymous.

• This result suggested that these SSRGs had likely

undergone artificial selection during domestication

Tian et al., PNAS, 2009, 106: 21760-21765

Association analysis

? How many major and minor genes control grain

cooking and eating quality

? Are AC, GC, and/or GT controlled by one or

multiple genes

? What is the relationship among these genes

? …

— e.g. Who control AC? Association analysis

2111-2112 3019 3097 3804 4078 4211 4235 4244-4246 4282 4285

------------------------ C C T C G G ATA A G

------------------------ C C T C G G ATA A G

------------------------ C C T C G G ATA A G

ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G

ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G

ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G

------------------------ C C T C G G ATA A G

------------------------ C C T C G G ATA A G

------------------------ - T C T A A --- G A

------------------------ - T C T A A --- G A

------------------------ C C T C G G ATA A G

------------------------ - T C T A A --- G A

------------------------ - T C T A A --- G A

Wx I

Wx II

Wx III

Wx I

Wx III Wx II Wx II Wx III

0.00

5.00

10.00

15.00

20.00

25.00

30.00

Wx I Wx II Wx III

Am

ylo

se c

on

ten

t (%

)

A

— e.g. Who control AC? Association analysis

Major

0.00

5.00

10.00

15.00

20.00

25.00

30.00

Wx I Wx II Wx III SBE3 I SBE3 II

Am

ylo

se c

on

ten

t (%

)

A B

SSII-3 I SSII-3 II

C

SSIII-2 I SSIII-2 II

D

SSIV-2 I SSIV-2 II

E

Am

ylo

se c

on

ten

t (%

)

Wx I

SBE3 I SBE3 II

Wx II

SBE3 I SBE3 II

Wx III

SBE3 I SBE3 II

F

0.00

5.00

10.00

15.00

20.00

25.00

30.00

A

— e.g. Who control AC? Association analysis

Major Minor Minor Minor

Minor Interaction

Tian et al., PNAS, 2009, 106: 21760-21765

SSRGs form a network controlling rice cooking and eating quality

Tian et al., PNAS, 2009, 106: 21760-21765

Wx and SSII-3 are central in determining

grain quality by affecting all three properties

Ttwo genes affect two properties

simultaneously, both ISA and SBE3 affect

GC and GT.

Several minor genes are specific for single

properties, SSIII-2, AGPlar, PUL, and SSI

for AC, AGPiso for GC, and SSIV-2 for GT.

The correlations among AC, GC, and GT

were caused by the joint action of these

associated genes and unequal haplotype

combination.

Fig. Summary of genes

controlling rice grain quality

Verification of SSRGs

Donor (s) ╳

F1 Receptor ╳

BCnF1

MAS

Receptor

BCnF2(3)

Transgenic tests Near-isogenic lines

Down-regulation Over-expression

Verification of the major gene for AC, Wx

(Transgenic)

Verification of the minor gene for AC, SBE3

(Transgenic)

Breeding of NILs

BC6F3

LTF × 9311

F1 × LTF

BC1F1

BC6F1

SSSI i SSSI j

0

500

1000

1500

2000

2500

3000

3500

0 200 400 600 800

Time(Sec)

Vis

cosi

ty (

cP

)

NILs

( SSSI j )

LTF

( SSSI i )

RVA profiles of NILs

LTF-NIL-SSSI j

Verification of the minor gene, SSSI

(Near-isogenic lines)

0

500

1000

1500

2000

2500

3000

3500

4000

0 200 400 600 800

Time(sec)

Vis

cosi

ty(c

P)

LTF (SSSI i)

RNAi

0

500

1000

1500

2000

2500

3000

3500

4000

0 200 400 600 800

Time(sec)

Vis

co

sity

(cP

)

Nipponbare (SSSI j)

RNAi

WT RNAi lines

LTF (SSSI i)

WT RNAi lines

Nipponbare (SSSI j)

The starch quality of RNAi transgenic lines

containing different SSSI allele

0

2

4

6

8

10

12

WXJ9 GLXN ZS97 LTP

Ex

press

ion

lev

el

rela

tiv

e t

o A

cti

n

SSSI j SSSI i

SSSI

13193 bp

TGASSS IATG

GUS

SSSI

13193 bp

TGASSS IATG

GUS

SSSI j-GUS

SSSI i-GUS 0

1

0 100 200 300 400 500

GUS activityGUS activity in developing seeds of transgenic rice

Q-RT-PCR analysis in developing rice seeds

The transcriptional level of

SSSI j allele is much lower

than that of SSSI i allele in

rice endosperm

Liu et al., unpublished

GOI Ter Promoter

Transgenic regulation

BC6F3

Receptor × Donor

F1 × Receptor

BC1F1

BC6F1

Allele i Allele j

Marker-assisted selection (MAS)

MAS

Molecular improvement of rice grain/starch quality

Functional SSRGs‟ markers for MAS

M Nip LTF 9311 9308 SYN

MAS

Tian et al., Chinese Sci Bull., 2010. 55: 3768-3777

Improvement of cooking and eating quality

of the female line Longtefu by MAS

Line Wx allele AC

(%)

GC

(cm)

GT

(ASV)

LTF Wxa Wxa 27.81 6.00 7.00

LTF-TT-1 Wxb Wxb 15.30 11.75 2.50

LTF-TT-3 Wxb Wxb 17.91 11.05 3.00

LTF-TT-5 Wxb Wxb 15.56 10.35 5.00

MAS

Liu et al., Crop Science, 2006; Yu et al., J Cereal Sci, 2009

Wxb J1 J3 J4 J5

Wxa I1 I5 I6 wx

Down of AC by transformation of antisense Wx gene

Northern blot

0

5

10

15

20

25

30

Am

ylo

se c

on

ten

t (%

)

WY7 WY8 WX LTF QLZ TQ

Japonica Indica

Wild type

Transgenic

Liu et al., Mol Breed, 2005; Yu et al., J Cereal Sci, 2009

Summary

Rice grain cooking and eating qualities are

regulated by starch synthesis related genes

(SSRGs) in a network.

Transgenic and near-isogenic studies with

selected major and/or minor SSRGs have

verified the above results, and which shown that

genetic modification with SSRGs will improve

rice grain qualities as desired.

Outline

1. Allelic diversities in rice starch biosynthesis

and genetic network for rice grain quality

2. Genetic engineering of starch biosynthesis

for high resistant starch (RS) in rice

Resistant Starch (RS)

Butyrate production

Prebiotic-stimulate growth

Inhibit cancer

Boost immune system

Reduce glycemic response

(slower insulin release)

Low calorie intake

Starch that escapes degradation in the small intestine,

and, therefore, is available for bacterial fermentation in the

large intestine.

Christer Jansson, Bioproducts, Nov. 2008

Potato

Oat

Corn

Wheat

Pea

Taro

Millet

Buck wheat

Rice

Bean

Sweet potato

Resistant starch

Source Resistant Non-Resistant

starch starch

Content of resistant starch in different starch sources

High amylose content is a source of

resistant starch (RS) R

es

ista

nt

sta

rch

(%

)

Zhu et al., Carbohydrate Polymers, 2011, 86: 1751-1759

Effects of regulation of different SSRGs on high-amylose production

Zhu et al., Plant Biotech J, 2012, 10: 353-362

Very-high-amylose rice grain with a high

level of RS and total dietary fiber

Zhu et al., Plant Biotech J, 2012, 10: 353-362

(Wild type: Indica, high AC)

J Agri & Food Chem, 2010, 58: 1224; 2010, 58:11946

WT

WT

RS

RS

Starch granule morphology of RS-rich rice

Polygonal granules with sharp

angles and edges

Irregularly large voluminous starch granules and

sausage-like elongated small starch granules

Fine structure of starches from RS-rich rice

Zhu et al., Plant Biotech J, 2012, 10: 353

(Increase of B-chains)

WT

RS

(High-amylose)

RS-WT

High-resistant starch rice Regular rice

RS-rich rice highly resistant to alkali

digestion and gelatinization

Wei et al., J Agri Food Chem, 2010, 2011

(Intact milled rice soaked in 5% KOH solution for 16 hours)

50 oC

70 oC

75 oC

80 oC

90 oC

RS WT

Wei et al., Food Chemistry,

2011, 128: 645-652

Resistant to

gelatinization

during heating

in water

200

240

280

320

360

1 3 5 7 9 11 13 15 17 19 21 23

Feeding time (d)

Bo

dy

wei

gh

t (g

)

Regular rice group

RS rice group

Zhu et al., Plant Biotech J, 2012, 10: 353-362

Improvement in indices of animal health

in rats by RS-rich rice meal

0

50

100

150

200

250

乙酸 丙酸 丁酸 短链脂肪酸

Con

ten

t (u

mole

/g)

WT RS

Acetic Propionic Butyric Total

acid acid acid SCFA

The rats consuming the RS-rich rice excreted more total short

chain fatty acids (SCFAs) than those fed the regular rice

Improvement in indices of animal health

in rats by RS-rich rice meal

Zhu et al., Plant Biotech J, 2012, 10: 353-362

Reduce of blood glucose response in diabetic

Zucker fatty rats fed the RS-rich rice starch

4.0

6.0

8.0

10.0

12.0

14.0

16.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Time (h)

Glu

cose

lev

el

WT

RS

Acute oral rice tolerance test (ORTT) in type II diabetic rats

Zhu et al., Plant Biotech J, 2012, 10: 353-362

Summary

A high-amylose (64.8%) rice enriched with resistant

starch (14.6%) was developed by transgenic regulation

of starch biosynthesis.

RS-rich rice starches highly resistant to digestion and

gelatinization

Consumption of the RS-rich rice had improved in

indices of animal health in both normal and diabetic rats.

Acknowledgements

Collaborators：

Prof. Jiayang Li (Inst. Genet. Develop. Biol., CAS)

Prof. Mengming Hong (Shanghai Inst. Plant Physiol. Eco., CAS)

Prof. Qian Qian (Chinese Rice Research Institute)

Prof. Yongcheng Shi (Kansas State University, USA)

……

Supported by: National Natural Science Foundation of China (NSFC)

National Key Basic Research Projects (“973” project)

National Major Projects for Transgenic Research

Thank you !