Phenotype of Rice Floury Endosperm Mutant flo7 and Fine Mapping of Mutated Gene

doi:10.1016/S1672-6308(14)60299-7

Abstract

Abstract:

The major storage substance in rice endosperm is starch, which accounts for 80% of dry matter weight. In this study, rice mutant flo7, selected from the progeny of Nipponbare’s tissue culture, displayed floury and opaque endosperm. Compared with its corresponding wild type (WT) Nipponbare, the mutant flo7 produced longer, narrower, thinner and lighter grains. The levels of glucose, fructose and sucrose in the mutant flo7 endosperm were higher than those in the WT endosperm, whereas the protein content was not affected. With respect to both amylose content and gel consistency, the mutant flo7 was lower than WT, but its alkali value was higher. Scanning electron microscopic examinations showed that the endosperm of the mutant flo7 contained irregular, loosely packed and compound starch granules. Genetic analysis indicated that the mutant phenotype was determined by a single recessive nuclear gene. The flo7 locus was mapped to a region on the long arm of chromosome 12, within a 95.1 kb interval defined by the markers C2-11 and C5-15. There are 13 open reading frames in the mapping interval. Transcription profiling of the developing grains showed that a number of genes involved in starch synthesis were affected differently in the mutant flo7.

Key words: flo7, floury endosperm, starch, fine mapping, rice, mutant

Zhong-hua Sheng, Peng-fei Fang, San-feng Li, Gui-ai Jiao, Li-hong Xie, Pei-song Hu, Shao-qing Tang, Xiang-jin Wei. Phenotype of Rice Floury Endosperm Mutant flo7 and Fine Mapping of Mutated Gene[J]. Rice Science, 2015, 22(4): 162-170.

Figures/Tables 11

References 36

1	Agricultural Industry Standard of the People’s Republic of China. 2002. The Rice Quality Measurement Method (NY147-88). Beijing, China: China Standards Press. (in Chinese)
2	Chavrier P, Goud B.1999. The role of ARF and Rab GTPases in membrane transport.Curr Opin Cell Biol, 11(4): 466-475.
3	Fu F F, Xue H W.2010. Coexpression analysis identifies Rice Starch Regulator 1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator.Plant Physiol, 154(2): 927-938.
4	Fukuda M, Wen L Y, Satoh-Cruz M, Kawagoe Y, Nagamura Y, Okita T W, Washida H, Sugino A, Ishino S, Ishino Y, Ogawa M, Sunada M, Ueda T, Kumamaru T.2013. A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.Plant Physiol, 162(2): 663-674.
5	Fujita N, Yoshida M, Kondo T, Saito K, Utsumi Y, Tokunaga T, Nishi A, Satoh H, Park J H, Jane J L, Miyao A, Hirochika H, Nakamura Y.2007. Characterization of SSIIIa-deficient mutants of rice: The function of SSIIIa and pleiotropic effects by SSIIIa deficiency in the rice endosperm.Plant Physiol, 144(4): 2009-2023.
6	Gao M, Fisher D K, Kim K N, Shannon J C, Guiltinan M J.1996. Evolutionary conservation and expression patterns of maize starch branching enzyme I and IIb genes suggests isoform specialization.Plant Mol Biol, 30(6): 1223-1232.
7	Hamada S, Nozaki K, Ito H, Yoshimoto Y, Yoshida H, Hiraga S, Onodera S, Honma M, Takeda Y, Matsui H.2001. Two starch-branching-enzyme isoforms occur in different fractions of developing seeds of kidney bean.Biochem J, 359: 23-34.
8	Hanashiro I, Itoh K, Kuratomi Y, Yamazaki M, Igarashi T, Matsugasako J I, Takeda Y.2008. Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice.Plant Cell Physiol, 49(6): 925-933.
9	Hanzal-Bayer M, Renault L, Roversi P, Wittinghofer A, Hillig R C.2002. The complex of Arl2-GTP and PDEσ: From structure to function. EMBO J, 21(9): 2095-2106.
10	Hirose T, Terao T.2004. A comprehensive expression analysis of the starch synthase gene family in rice (Oryza sativa L.).Planta, 220(1): 9-16.
11	Jin T Y, Li H, Guo T, Liu X L, Su N, Wu F Q.2010. Analysis of physiological and biochemical characteristics of six mutants with stable high percentage of chalkiness in rice grains.Acta Agron Sin, 36(1): 121-132. (in Chinese with English abstract)
12	Kang H G, Park S, Matsuoka M, An G.2005. White-core endosperm floury endosperm 4 in rice is generated by knockout mutations in the C4-type pyruvate orthophosphate dikinase gene (OsPPDKB).Plant J, 42(6): 901-911.
13	Kang H Q, Cheng H Y.2007. Study on chalkiness characters and endosperm structures of the main parents’ kernel of hybrid rice.Chin Agric Sci Bull, 23(4): 180-185. (in Chinese with English abstract)
14	Kawagoe Y, Kubo A, Satoh H, Takaiwa F, Nakamjura Y.2005. Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm.Plant J, 42(2): 164-174.
15	Kawasaki T, Mizuno K, Baba T, Shimada H.1993. Molecular analysis of the gene encoding a rice starch branching enzyme.Mol Gen Genet, 237(1/2): 10-16.
16	Li Z, Trick H N.2005. Rapid method for high-quality RNA isolation from seed endosperm containing high levels of starch.Bio-Techniques, 38(6): 872-876.
17	Liu F, Ren Y L, Wang Y H, Peng C, Zhou K N, Lv J, Guo X P, Zhang X, Zhong M S, Zhao S L, Jiang L, Wang H Y, Bao Y Q, Wan J M.2013. OsVPS9A functions cooperatively with OsRAB5A to regulate post-Golgi dense vesicle-mediated storage protein trafficking to the protein storage vacuole in rice endosperm cells.Mol Plant, 6(6): 1918-1932.
18	Myers A M, Morell M K, James M G, Ball S G.2000. Recent progress toward understanding biosynthesis of the amylopectin crystal.Plant Physiol, 122(4): 989-998.
19	Nishi A, Nakamura Y, Tanaka N, Satoh H.2001. Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm.Plant Physiol, 127(2): 459-472.
20	Nishio T, Iida S.1993. Mutants having a low content of 16-kDa allergenic protein in rice (Oryza sativa L.).Theor Appl Genet, 86(2/3): 317-321.
21	Peng C, Wang Y H, Liu F, Ren Y L, Zhou K N, Lv J, Zheng M, Zhao S L, Zhang L, Wang C M, Jiang L, Zhang X, Guo X P, Bao Y Q, Wan J M.2014. FLOURY ENDOSPERM6 encodes a CBM48 domain-containing protein involved in compound granule formation and starch synthesis in rice endosperm.Plant J, 77(6): 917-930.
22	Ren Y L, Wang Y H, Liu F, Zhou K N, Ding Y, Zhou F, Wang Y, Liu K, Gan L, Ma W W, Han X H, Zhang X, Guo X P, Wu F Q, Cheng Z J, Wang J L, Lei C L, Lin Q B, Jiang L, Wu C Y, Bao Y Q, Wang H Y, Wan J M.2014. GLUTELIN PRECURSOR ACCUMULATION3 encodes a regulator of post-Golgi vesicular traffic essential for vacuolar protein sorting in rice endosperm.Plant Cell, 26(1): 410-425.
23	Qu Y, Jin Z X, Liu H Y, Xu Z H, Zhu L N, Zheng G L, Zhu F X, Zhang Z C.2014. Analysis of expression characters of soluble starch synthase and isoform genes involved in japonica hybrid progeny.Chin J Rice Sci, 28(1): 23-31. (in Chinese with English abstract)
24	Ryoo N, Yu C, Park C S, Baik M Y, Park I M, Cho M H, Bhoo S H, An G, Hahn T R, Jeon J S.2007. Knockout of a starch synthase gene OsSSIIIa/Flo5 causes white-core floury endosperm in rice (Oryza sativa L.).Plant Cell Rep, 26(7): 1083-1095.
25	Satoh H, Omura T.1981. New endosperm mutations induced by chemical mutagens in rice Oryza sativa L.Jpn J Breeding, 31(3): 316-326.
26	Schmittgen T D, Livak K J.2008. Analyzing real-time PCR data by the comparative CT method.Nat Protoc, 3: 1101-1108.
27	She K C, Kusano H, Koizumi K, Yamakawa H, Hakata M, Imamura T, Fukuda M, Naito N, Tsurumaki Y, Yaeshima M, Tsuqe T, Matsumoto K, Kudoh M, Itoh E, Kikuchi S, Kishimoto N, Yazaki J, Ando T, Yano M, Aoyama T, Sasaki T, Satoh H, Shimada H.2010. A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality.Plant Cell, 22(10): 3280-3294.
28	Shimada H, Tada Y, Kawasaki T, Fujimura T.1993. Antisense regulation of the rice waxy gene expression using a PCR-amplified fragment of the rice genome reduces the amylose content in grain starch.Theor Appl Genet, 86(6): 665-672.
29	Steegmans M, Iliaens S, Hoebregs H.2004. Enzymatic, spectro- photometric determination of glucose, fructose, sucrose, and inulin/oligofructose in foods. J AOAC Int, 87(5): 1200-1207.
30	Sun C X, Duan B W, Xie L H, Chen N.2006. Determination of several quality characteristics of brown rice by near infrared transmission spectroscopy.Chin J Rice Sci, 20(4): 451-454. (in Chinese with English abstract)
31	Wang J C, Xu H, Zhu Y, Liu Q Q, Cai X L.2013.OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm.J Exp Bot, 64(11): 3453-3466.
32	Wang Y H, Ren Y L, Liu X, Jiang L, Chen L M, Han X H, Jin M N, Liu S J, Liu F, Lv J, Zhou K N, Su N, Bao Y Q, Wan J M.2010. OsRab5a regulates endomembrane organization and storage protein trafficking in rice endosperm cells. Plant J, 64(5): 812-824.
33	Woo M O, Ham T H, Ji H S, Choi M S, Jiang W Z, Chu S H, Piao R H, Chin J H, Kim J A, Park B S, Seo H S, Jwa N S, McCouch S, Koh H J.2008. Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.). Plant J, 54(2): 190-204.
34	Wu K S, Tanksley S D.1993. Abundance, polymorphism and genetic mapping of microsatellites in rice.Mol Gen Genet, 241(1/2): 225-235.
35	Yao D S, Song R T.2013. Research progress of maize opaque endosperm mutants.Chin J Nat, 35(2): 105-111. (in Chinese with English abstract)
36	(Managing Editor: Li Guan)

Primer	Forward (5'-3')	Reverse (5'-3')	Product size (bp)
RM6411	GGGTATTGTCGGTGTTCAGG	GAAGGCTGTACCATCCAACG	140
RM1227	CATGGTAGCACACACCCTTG	CATCGACATGTGGACCACTC	137
RM28746	GAAGAAAGAAGACGCCAAGAAACG	CATTCCATTCCCTTCCTCTTCG	158
C5-2	GCTGTCAGATCGGTAGGCA	ACCACATCAACCCAGAAAA	90
C5-15	TGTTTGTGCCTTCAGATAA	TGAGATAGGGGAAGTAGAT	87
C2-6	AGATTCTTTTCCAAACAAC	AATCATCATATTGCCGAAA	81
C2-11	TGCATCATCCAATCTGTCTTGG	TCGACAAGCCAATCTACTCTTCC	139
C7-3	CTACGTGCATCCATCGAGAA	TGAATTGTTTACCGGAACCC	153
C7-5	TCTTCTACTCCTCGTCTTCGTTCG	CATGCAGAGCAGAGACTTCTTGG	143
C7-12	TGCTGGACACAGCAAGTTTT	TAGATGGTGGGGGTGATAGG	185
C7-15	GCGTTGATTGATGATGATGG	CGACCCGACGTAAAAGAGAA	113

Primer	Forward (5'-3')	Reverse (5'-3')	Product size (bp)
RM6411	GGGTATTGTCGGTGTTCAGG	GAAGGCTGTACCATCCAACG	140
RM1227	CATGGTAGCACACACCCTTG	CATCGACATGTGGACCACTC	137
RM28746	GAAGAAAGAAGACGCCAAGAAACG	CATTCCATTCCCTTCCTCTTCG	158
C5-2	GCTGTCAGATCGGTAGGCA	ACCACATCAACCCAGAAAA	90
C5-15	TGTTTGTGCCTTCAGATAA	TGAGATAGGGGAAGTAGAT	87
C2-6	AGATTCTTTTCCAAACAAC	AATCATCATATTGCCGAAA	81
C2-11	TGCATCATCCAATCTGTCTTGG	TCGACAAGCCAATCTACTCTTCC	139
C7-3	CTACGTGCATCCATCGAGAA	TGAATTGTTTACCGGAACCC	153
C7-5	TCTTCTACTCCTCGTCTTCGTTCG	CATGCAGAGCAGAGACTTCTTGG	143
C7-12	TGCTGGACACAGCAAGTTTT	TAGATGGTGGGGGTGATAGG	185
C7-15	GCGTTGATTGATGATGATGG	CGACCCGACGTAAAAGAGAA	113

Primer	Accession number	Forward (5'-3')	Reverse (5'-3')
Actin	X16280	CATGCTATCCCTCGTCTCGACCT	CGCACTTCATGATGGAGTTGTAT
OsAGPS1	AK073146	AGAATGCTCGTATTGGAGAAAATG	GGCAGCATGGAATAAACCAC
OsAGPS2a	AK071826	ACTCCAAGAGCTCGCAGACC	GCCTGTAGTTGGCACCCAGA
OsAGPS2b	AK103906	AACAATCGAAGCGCGAGAAA	GCCTGTAGTTGGCACCCAGA
OsAGPL1	D50317	GGAAGACGGATGATCGAGAAAG	CACATGAGATGCACCAACGA
OsAGPL3	AK069296	AAGCCAGCCATGACCATTTG	CACACGGTAGATTCACGAGACAA
OsAGPL4	AK121036	TCAACGTCGATGCAGCAAAT	ATCCCTCAGTTCCTAGCCTCATT
OsSSI	D16202	GGGCCTTCATGGATCAACC	CCGCTTCAAGCATCCTCATC
OsSSIIa	AF419099	GCTTCCGGTTTGTGTGTTCA	CTTAATACTCCCTCAACTCCACCAT
OsSSIIIa	AY100469	GCCTGCCCTGGACTACATTG	GCAAACATATGTACACGGTTCTGG
OsBEIIb	D16201	ATGCTAGAGTTTGACCGC	AGTGTGATGGATCCTGCC
GBSSI	AK070431	TCCGAGAGGTTCAGGTCATC	ATGAGCTCCTCGGCGTAGTA
OsISA1	AB093426	TGCTCAGCTACTCCTCCATCATC	AGGACCGCACAACTTCAACATA
OsISA2	AC132483	TAGAGGTCCTCTTGGAGG	AATCAGCTTCTGAGTCACCG
OsISA3	AP005574	ACAGCTTGAGACACTGGGTTGAG	GCATCAAGAGGACAACCATCTG
OsPUL	AB012915	ACCTTTCTTCCATGCTGG	CAAAGGTCTGAAAGATGGG
OsPHOL	AK063766	TTGGCAGGAAGGTTTCGCT	CGAAGCCTGAAGTGAACTTGCT

Primer	Accession number	Forward (5'-3')	Reverse (5'-3')
Actin	X16280	CATGCTATCCCTCGTCTCGACCT	CGCACTTCATGATGGAGTTGTAT
OsAGPS1	AK073146	AGAATGCTCGTATTGGAGAAAATG	GGCAGCATGGAATAAACCAC
OsAGPS2a	AK071826	ACTCCAAGAGCTCGCAGACC	GCCTGTAGTTGGCACCCAGA
OsAGPS2b	AK103906	AACAATCGAAGCGCGAGAAA	GCCTGTAGTTGGCACCCAGA
OsAGPL1	D50317	GGAAGACGGATGATCGAGAAAG	CACATGAGATGCACCAACGA
OsAGPL3	AK069296	AAGCCAGCCATGACCATTTG	CACACGGTAGATTCACGAGACAA
OsAGPL4	AK121036	TCAACGTCGATGCAGCAAAT	ATCCCTCAGTTCCTAGCCTCATT
OsSSI	D16202	GGGCCTTCATGGATCAACC	CCGCTTCAAGCATCCTCATC
OsSSIIa	AF419099	GCTTCCGGTTTGTGTGTTCA	CTTAATACTCCCTCAACTCCACCAT
OsSSIIIa	AY100469	GCCTGCCCTGGACTACATTG	GCAAACATATGTACACGGTTCTGG
OsBEIIb	D16201	ATGCTAGAGTTTGACCGC	AGTGTGATGGATCCTGCC
GBSSI	AK070431	TCCGAGAGGTTCAGGTCATC	ATGAGCTCCTCGGCGTAGTA
OsISA1	AB093426	TGCTCAGCTACTCCTCCATCATC	AGGACCGCACAACTTCAACATA
OsISA2	AC132483	TAGAGGTCCTCTTGGAGG	AATCAGCTTCTGAGTCACCG
OsISA3	AP005574	ACAGCTTGAGACACTGGGTTGAG	GCATCAAGAGGACAACCATCTG
OsPUL	AB012915	ACCTTTCTTCCATGCTGG	CAAAGGTCTGAAAGATGGG
OsPHOL	AK063766	TTGGCAGGAAGGTTTCGCT	CGAAGCCTGAAGTGAACTTGCT

Material	Plant height (cm)	Main panicle length (cm)	Effective tiller number per plant	No. of grains per panicle	Seed-setting rate (%)
WT	67.25 ± 4.25	16.88 ± 2.32	26.25 ± 2.75	60.00 ± 5.80	66.25 ± 8.32
flo7	74.20 ± 3.50	17.22 ± 1.80	26.75 ± 4.68	61.75 ± 5.25	82.20 ± 9.33
P value (t test)	0.003	0.190	0.150	0.850	0.036