International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012

1. 11th International Gluten Workshop
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

2. 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

3. Determinants of rice grain quality
Milling quality
Appearance quality
Cooking & eating quality
Nutritional quality

4. Three key physicochemical properties determine
rice cooking and eating quality
GT GC
Gelatinization Gel consistency
temperature measures the
determines the tendency of the
time required for cooked rice to
cooking the rice harden on cooling.
AC
High amylose content grains
cook dry, are less tender, and
become hard upon cooling.

5. Wide diversity of cooking and eating
qualities among rice cultivars
Amylose content Gelatinization Gel Consistency
(%) Temperature (ASV) (mm)
Cultivar Maturity
No.
type type
Range Mean Range Mean Range Mean
Early 8 23.75-26.60 25.28 3.22-5.22 4.35 30-97 53.63
Indica Medium 33 9.68-30.64 24.16 2.67-6.89 4.89 20-120 56.21
Late 32 11.64-28.66 20.30 2.00-6.56 4.92 21-110 63.22
Early 13 10.54-23.09 15.25 5.94-6.91 6.42 51-95 71.77
Japonica Medium 25 11.34-18.00 14.77 3.32-7.00 5.98 38-108 75.64
Late 5 15.64-22.16 18.35 6.00-6.94 6.38 24-85 59.20

6. Starch, the major component in rice endosperm
Amylose Amylopectin
Amylopectin Amylose
DBE
SBE
SSS
?
AGPase
ADPGlc

7. Starch Synthesis Related Genes, SSRGs
Classification of key enzymes Gene Localization
Large subunit 1 AGPL1 Chr 5
ADP glucose pyrophosphorylase
Large subunit 2 AGPL2 Chr 6
(AGPase)
Small subunit AGPS Chr 9
Granule-bound starch synthase GBSSI Wx Chr 6
(GBSS) GBSSII GBSSII Chr 7
SSSI SSSI Chr 6
SSSII-1 Chr 10
SSSII SSSII-2 Chr 2
Soluble starch synthase SSSII-3 Chr 6
(SSS) SSSIII-1 Chr 4
SSSIII
SSSIII-2 Chr 8
SSSIV-1 Chr 1
SSSIV
SSSIV-2 Chr 5
SBEI Sbe1 Chr 6
Starch branching enzyme
(SBE) Sbe3 Chr 2
SBEII
Sbe4 Chr 4
Starch debranching enzyme Isoamylase (ISA) ISA Chr 8
(DBE) Pullulanase (PUL) PUL Chr 4

8. 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)

9. 70 varieties with diverse grain qualities
Indica (33)
Japonica (37)

10. High correlation among AC, GC and GT
AC GC GT
-0.91 a * -0.46
AC 1.00
0.007 b 0.779
0.50
GC 1.00
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
Tian et al., PNAS, 2009, 106: 21760-21765

11. Starch pasting curve of different rice cultivars
6500
Tm TN1
LTP GCH
5500
9311 WY7 High AC
CJ06 NIP
4500
Viscosity (cP)
JZXN THN
SYN
3500
Low or intermediate AC
2500
1500
Very low or no AC
500
-500
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0
Time (min)
Rapid Visco Analyser (RVA)

12. 16 core varieties selected for sequence analysis of SSRGs

13. Wx gene alignment
The Wx gDNA alignment among different varieties
Varieties 175 298 495 528 771-785 841 926 987 1056 1083 1088
Nipponbare ( japonica ) C C A C CT ( 18 ) T G AATT(6) A C A
Chunjiang 06 ( japonica ) C C A C CT ( 17 ) T G AATT(6) A C A
Wuyunjing 7 ( japonica ) C C A C CT ( 17 ) T G AATT(6) A C A
Jiangzhouxiangnuo ( japonica - glutinous) C C A C CT ( 16 ) T G AATT(6) A C A
Suyunuo ( japonica - glutinous) C C A C CT ( 16 ) T G AATT(6) A C A
Taihunuo ( japonica - glutinous) C C A C CT ( 16 ) T G AATT(6) A C A
9308 ( indica ) C C A C CT ( 18 ) T G AATT(6) A C G
9311 ( indica ) C C A C CT ( 18 ) T G AATT(6) A C A
Guichao 2( indica ) C C G T CT ( 11 ) G A AATT(5) G T A
Longtepu ( indica ) C T G T CT ( 11 ) G A AATT(5) G T A
Minghui 63 ( indica ) C C A C CT ( 18 ) T G AATT(6) A C A
Taizhongbendi 1( indica ) C C G T CT ( 11 ) G A AATT(5) G T A
Zhenshan97B ( indica ) A C G T CT ( 11 ) G A AATT(5) G T A
5„UTR
Varieties 2111-2112 3019 3097 3804 4078 4211 4235 4244-4246 4282 4285
Nipponbare ( japonica ) ------------------------ C C T C G G ATA A G
Chunjiang 06 ( japonica ) ------------------------ C C T C G G ATA A G
Wuyunjing 7 ( japonica ) ------------------------ C C T C G G ATA A G
Jiangzhouxiangnuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
Suyunuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
Taihunuo ( japonica - glutinous) ACGGGTTCCAGGGCCTCAAGCCC C C T C G G ATA A G
9308 ( indica ) ------------------------ C C T C G G ATA A G
9311 ( indica ) ------------------------ C C T C G G ATA A G
Guichao 2( indica ) ------------------------ - T C T A A --- G A
Longtepu ( indica ) ------------------------ - T C T A A --- G A
Minghui 63 ( indica ) ------------------------ C C T C G G ATA A G
Taizhongbendi 1( indica ) ------------------------ - T C T A A --- G A
Zhenshan97B ( indica ) ------------------------ - T C T A A --- G A
Exon 2 intron exon intron

14. Wx gene alignment
The cDNA Alignment Among Different Varieties
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
Stop
Guichao2( indica ) ---------------------------------------- C T codon
Longtepu ( indica ) ---------------------------------------- C T
Minghui 63 ( indica ) ---------------------------------------- T C
Taizhongbendi 1( indica ) ---------------------------------------- C T
Zhenshan97B ( indica ) ---------------------------------------- C T
• 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

15. 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
? …

16. Association analysis
— e.g. Who control AC?
2111-2112 3019 3097 3804 4078 4211 4235 4244-4246 4282 4285
------------------------ C C T C G G ATA A G
Wx II ------------------------ 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
Wx III 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
Wx I ------------------------ 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 III Wx II Wx III Wx II

17. Association analysis
— e.g. Who control AC?
30.00
A Major
25.00
Amylose content (%)
20.00
15.00
10.00
5.00
0.00
Wx I Wx II Wx III

18. Association analysis
— e.g. Who control AC?
30.00 Minor Minor Minor
A Major B C D
25.00
Amylose content (%)
20.00
15.00
10.00
5.00
0.00
Wx I Wx II Wx III SBE3 I SBE3 II SSII-3 I SSII-3 II SSIII-2 I SSIII-2 II
30.00 Minor Interaction
E F
Amylose content (%)
25.00
20.00
15.00
10.00
5.00
0.00
SSIV-2 I SSIV-2 II SBE3 I SBE3 II SBE3 I SBE3 II SBE3 I SBE3 II
Wx I Wx II Wx III
Tian et al., PNAS, 2009, 106: 21760-21765

19. SSRGs form a network controlling rice cooking and eating quality
 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
Tian et al., PNAS, 2009, 106: 21760-21765

20. Verification of SSRGs
Transgenic tests Near-isogenic lines
Receptor╳ Donor (s)
F1 ╳ Receptor
MAS
BCnF1
BCnF2(3)

21. Verification of the major gene for AC, Wx
(Transgenic)
Down-regulation Over-expression

22. Verification of the minor gene for AC, SBE3
(Transgenic)

23. Verification of the minor gene, SSSI
(Near-isogenic lines)
3500
SSSI i
SSSI j LTF
3000 ( SSSI i )
LTF × 9311
2500 NILs
F1 × LTF ( SSSI j )
Viscosity (cP)
2000
1500
BC1F1
1000
500
BC6F1 BC6F3 0
0 200 400 600 800
LTF-NIL-SSSI j
Time(Sec)
Breeding of NILs RVA profiles of NILs

24. The starch quality of RNAi transgenic lines
containing different SSSI allele
Nipponbare (SSSI j) LTF (SSSI i)
WT RNAi lines WT RNAi lines
4000 4000
3500 3500 LTF (SSSI i)
3000
Nipponbare (SSSI j)
3000
Viscosity(cP)
Viscosity(cP)
2500 2500
2000 2000
1500 1500
1000 RNAi 1000
RNAi
500 500
0 0
0 200 400 600 800 0 200 400 600 800
Time(sec) Time(sec)

25. Q-RT-PCR analysis in developing rice seeds
12
Expression level relative to Actin
The transcriptional level of 10
8
SSSI j allele is much lower
6
than that of SSSI i allele in 4
rice endosperm 2
0
WXJ9 GLXN ZS97 LTP
SSSI j SSSI i
SSSI j-GUS GUS activity in developing seeds of transgenic rice
GUS activity
ATG TGA
SSS I
GUS
1
ATG TGA
SSS I
GUS
SSSI i-GUS SSSI
0
0 100 200 300 400 500
13193 bp
SSSI Liu et al., unpublished

26. Molecular improvement of rice grain/starch quality
Marker-assisted selection (MAS) Transgenic regulation
Promoter GOI Ter
Allele i Allele j
Receptor × Donor
F1 × Receptor
MAS BC1F1
BC6F1 BC6F3

27. MAS
Functional SSRGs‟ markers for MAS
M Nip LTF 9311 9308 SYN
Tian et al., Chinese Sci Bull., 2010. 55: 3768-3777

28. MAS
Improvement of cooking and eating quality
of the female line Longtefu by MAS
AC GC GT
Line Wx allele
(%) (cm) (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
Liu et al., Crop Science, 2006; Yu et al., J Cereal Sci, 2009

29. Transgenic
Down of AC by transformation of antisense Wx gene
Wxb J1 J3 J4 J5
30
Wild type
25
Amylose content (%)
20
15
Wxa I1 I5 I6 wx 10
5
0
WY7 WY8 WX LTF QLZ TQ
Japonica Indica
Northern blot
Liu et al., Mol Breed, 2005; Yu et al., J Cereal Sci, 2009

30. 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.

31. 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

32. Resistant Starch (RS)
 Starch that escapes degradation in the small intestine,
and, therefore, is available for bacterial fermentation in the
large intestine.
 Butyrate production
 Prebiotic-stimulate growth
 Inhibit cancer
 Boost immune system
 Reduce glycemic response
(slower insulin release)
 Low calorie intake
Christer Jansson, Bioproducts, Nov. 2008

33. Content of resistant starch in different starch sources
Source Resistant Non-Resistant
starch starch
Potato
Oat
Corn
Wheat
Pea
Taro
Millet
Buck wheat
Rice
Bean
Sweet potato
Resistant starch

34. High amylose content is a source of
Resistant starch (%)
resistant starch (RS)
Zhu et al., Carbohydrate Polymers, 2011, 86: 1751-1759

35. Effects of regulation of different SSRGs
on high-amylose production
Zhu et al., Plant Biotech J, 2012, 10: 353-362

36. Very-high-amylose rice grain with a high
level of RS and total dietary fiber
(Wild type: Indica, high AC)
Zhu et al., Plant Biotech J, 2012, 10: 353-362

37. Starch granule morphology of RS-rich rice
WT RS
Polygonal granules with sharp Irregularly large voluminous starch granules and
angles and edges sausage-like elongated small starch granules
WT RS
J Agri & Food Chem, 2010, 58: 1224; 2010, 58:11946

38. Fine structure of starches from RS-rich rice
WT (High-amylose)
RS
(Increase of B-chains)
RS-WT
Zhu et al., Plant Biotech J, 2012, 10: 353

39. RS-rich rice highly resistant to alkali
digestion and gelatinization
Regular rice High-resistant starch rice
(Intact milled rice soaked in 5% KOH solution for 16 hours)
Wei et al., J Agri Food Chem, 2010, 2011

40. WT RS
50 oC Resistant to
gelatinization
during heating
70 oC in water
75 oC
80 oC
90 oC Wei et al., Food Chemistry,
2011, 128: 645-652

41. Improvement in indices of animal health
in rats by RS-rich rice meal
360
Regular rice group
320
Body weight (g)
280
RS rice group
240
200
1 3 5 7 9 11 13 15 17 19 21 23
Feeding time (d)
Zhu et al., Plant Biotech J, 2012, 10: 353-362

42. Improvement in indices of animal health
in rats by RS-rich rice meal
250
200
WT RS
Content (umole/g)
150
100
50
0
Acetic
乙酸 Propionic
丙酸 Butyric
丁酸 Total
短链脂肪酸
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
Zhu et al., Plant Biotech J, 2012, 10: 353-362

43. Reduce of blood glucose response in diabetic
Zucker fatty rats fed the RS-rich rice starch
16.0
14.0 WT
Glucose level
12.0 RS
10.0
8.0
6.0
4.0
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)
Acute oral rice tolerance test (ORTT) in type II diabetic rats
Zhu et al., Plant Biotech J, 2012, 10: 353-362

44. 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.

45. 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

46. Thank you !