Phenotyping and Gene Cloning of a Floury Endosperm Mutant ws in Rice

doi:10.16819/j.1001-7216.2016.6048

Abstract

Abstract:

In this study, we obtained a stably inherited floury endosperm mutant ws from japonica variety cv. Dianjingyou 1 (DJY), with its 1000-grain weight, grain size, total starch content, and amylose content in mature seeds all decreased compared with wild-type, as well as the swelling power of starch. By observing the structure of mature and developing endosperm, we found that the starch granules in ws mutant were smaller and loosely packed. The WS locus was first mapped to a region near the centromere in chromosome 8 with 92 recessive individuals, and then was narrowed down to a 95 kb region by using 2025 recessive individuals. Sequence analysis revealed that the coding region of the small subunit of adenosine diphosphate glucose pyrophosphorylase (AGPS2) had a single nucleotide substitution, resulting in a change in the amino acid sequence. RT-PCR analysis showed no significant difference in the expression levels of genes encoding the AGPase subunits in the mutant endosperm, while immunoblot analysis revealed a reduced protein content of the AGPS2b. Meanwhile, the enzyme activity of AGPase in the mutant was decreased to half of the wild-type. These results showed that the mutation of OsAGPS2 caused the decreased activity of AGPase, thus affecting the starch biosynthesis in rice endosperm.

Key words: floury endosperm mutant, AGPase, starch, gene expression, physicochemical property, rice (Oryza sativa L.)

摘要：

从甲基亚硝基脲(1-Methyl-1-Nitrosourea, MNU)处理的粳稻品种滇粳优1号突变体库中,筛选到一个稳定遗传的胚乳粉质突变体ws,其籽粒的千粒重、籽粒大小、总淀粉含量、直链淀粉含量等指标均降低,淀粉在尿素溶液中的膨胀能力减弱。对成熟及发育中的胚乳淀粉结构进行观察,发现ws突变体的胚乳中产生大量小而不规则排布的单淀粉颗粒。利用F₂群体中分离出的92个隐性极端个体将突变基因连锁在第8染色体近着丝粒位置,随后共用2025个极端个体将目标基因定位于95 kb的区间。测序发现ws突变体中编码腺苷二磷酸葡萄糖焦磷酸化酶(Adenosine diphosphate glucose pyrophosphorylase, AGPase)小亚基S2的基因发生点突变,导致编码氨基酸的替换。基因表达分析发现,突变体胚乳中编码AGPase各亚基的相关基因表达量没有发生显著改变,而Western杂交分析显示突变体中AGPS2b的蛋白含量下降。同时,ws突变体的胚乳中AGPase活性下降为野生型的一半。研究结果表明,OsAGPS2的突变导致水稻胚乳中AGPase活性降低,从而影响了淀粉合成。

关键词: 胚乳粉质突变体, AGPase, 淀粉, 基因表达, 理化性质, 水稻

CLC Number:

Peng-yi PAN, Jian-ping ZHU, Yun-long WANG, Yuan-yuan HAO, Yue CAI, Wen-wei ZHANG, Ling JIANG, Yi-hua WANG, Jian-min WAN. Phenotyping and Gene Cloning of a Floury Endosperm Mutant ws in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(5): 447-457.

潘鹏屹, 朱建平, 王云龙, 郝媛媛, 蔡跃, 张文伟, 江玲, 王益华, 万建民. 水稻粉质胚乳突变体ws的表型分析及基因克隆[J]. 中国水稻科学, 2016, 30(5): 447-457.

Figures/Tables 9

Table 1 Markers for fine mapping of WS.

标记 Marker	正向引物Forward (5'→3')	反向引物Reverse (5'→3')
HY8-19	TTTGTTGCTTTTCTGATTC	ATGATAAAGCGATAAACCA
WQ8-28	GAGACGGACGGGTGTTGA	CAATGACATCCCAGCGTA
WZ8-17	TAAATCATGGTGGTGGGC	ACCGTCGTCTAGCAAGGAG
WZ8-3	ATTAAGATGATATGGGAAGT	ACATTGACCTGGTAGAAAC
HY8-24	ATTAAGATGATATGGGAAGT	ACATTGACCTGGTAGAAAC

Table 2 Primers used in real-time RT-PCR.

基因 Gene	正向引物 Forward (5'→3')	反向引物 Reverse (5'→3')
AGPL1	CATCAAGGACGGGAAGGTCA	ACTTCACTCGGGGCAGCTTA
AGPL2	CTGAGGAAGAGGTGCTTTGG	TCTTTCGGGAGGATTGTGTC
AGPS1	AGAATGCTCGTATTGGAGAAAATG	GGCAGCATGGAATAAACCAC
AGPS2a	ACTCCAAGAGCTCGCAGACC	GCCTGTAGTTGGCACCCAGA
AGPS2b	AACAATCGAAGCGCGAGAAA	GCCTGTAGTTGGCACCCAGA
UGPase1	CCATCACCGCCAAGTCA	GACCGTTGATGTCCTTGTTCT
Actin	CCCTCCTGAAAGGAAGTACAGTGT	GTCCGAAGAATTAGAAGCATTTCC

Fig. 1. Phenotype comparison of the wild-type and ws mutant. A, Comparison of wild-type and ws mutant seeds, bar = 3 mm; B, Seed cross-sections of wild-type and ws mutant, bar = 1 mm; C, Comparison of wild-type and ws mutant plants, bar = 20 cm. DJY, Dianjingyou 1(wild type).

Fig. 2. Comparison of 1000-grain weight, seed and major agronomic traits of wild-type and ws mutant. Values are mean ± SD (n = 10, except for 1000-grain weight, n = 3); t-test, ** P < 0.01. DJY, Dianjingyou 1(wild type).

Fig. 3. Physicochemical characteristics of wild-type and ws mature seeds. A, Comparison of chemical composition of wild-type and ws seeds, values are mean ± SD, n = 3; t-test, ** P < 0.01; B, Gelatinization properties of wild type and ws mutant seeds; C, Significant difference was observed at the urea concentration of 4 mol/L urea; D, The swollen volume of wild-type and ws starch in urea solutions of various concentrations (n = 3). DJY, Dianjingyou 1(wild type).

Fig. 4. Scanning electron microscopy observation of mature seeds of wild type and ws mutant. A, B, Endosperm of wild-type; C, D, Endosperm of ws mutant; A, C, Endosperm cross-section, Bars = 1 mm; B, D, Partial enlarged drawing picture, Bars = 10 μm.

Fig. 5. Semi-thin sections of wild type and ws mutant seeds. A, B, Peripheral part (A) and central part (B) of wild-type endosperm cells at 9 DAF (days after flowering); C, Central part of wild-type endosperm cells at 12 DAF; D, E, Peripheral part (D) and central part (E) of ws endosperm cells at 9 DAF; F, Central part of ws endosperm cells at 12 DAF. Bars = 100 μm in A and D, 50 μm in B, C, E, F.

Fig. 6. Fine-mapping of WS gene. A, WS was linked with markers HY8-19 and HY8-24; B, WS was located in a 95 kb region based on 2025 individuals; C, Candidate genes for WS; D, ws displayed a single nucleotide substitution in AGPS2 (in red),AGPS2 is composed of 10 exons (filled box) and 9 introns, the alternative use of exon 1a and 1b generates AGPS2a and AGPS2b transcripts, respectively.

Fig. 7. Expression of related genes and enzyme activity in wild type and ws mutant. A, Real-time RT-PCR analysis of the expression of AGPS2b in different organs in wild type; B, Real-time RT-PCR analysis of the expression of AGPS2b at different developing stages of endosperm in wild type; C, Real-time RT-PCR analysis of the expression of AGPS2a in leaves of the wild type and ws mutant seedlings at 7 days after germination; D, Real-time RT-PCR analysis of the expression of genes encoding AGPase in wild type and ws mutant endosperm; E, Immunoblot analysis of starch biosynthesis related proteins in wild type and ws mutant endosperm; F, AGPase and UGPase activities of wild-type and ws developing endosperm. For A, B, C, D and F, error bars show SD (n = 3).

References 42

[1]	Buléon A, Colonna P, Planchot V, et al.Starch granules: Structure and biosynthesis.Int J Biol Macr, 1998, 23(2): 85-112.
[2]	Hirose T, Terao T.A comprehensive expression analysis of the starch synthase gene family in rice (Oryza sativa L.) .Planta, 2004, 220(1): 9-16.
[3]	Ohdan T, Francisco P B, Sawada T, et al.Expression profiling of genes involved in starch synthesis in sink and source organs of rice.J Exp Bot, 2005, 56(422): 3229-3244.
[4]	Tetlow I J, Morell M K, Emes M J.Recent developments in understanding the regulation of starch metabolism in higher plants.J Exp Bot, 2004, 55(406): 2131-2145.
[5]	Fujita N, Satoh R, Hayashi A, et al.Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa.J Exp Bot, 2011, 62(14): 4819-4831.
[6]	Hanashiro I, Itoh K, Kuratomi Y, et al.Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice.Plant & Cell Physiol, 2008, 49(6): 925-933.
[7]	Liu L, Ma X, Liu S, et al.Identification and characterization of a novel Waxy allele from a Yunnan rice landrace.Plant Mol Biol, 2009, 71(6): 609-626.
[8]	Nishi A, Nakamura Y, Tanaka N, et al.Biochemical and genetic analysis of the effects of Amylose-Extender mutation in rice endosperm.Plant Physiol, 2001, 127(2): 459-472.
[9]	Satoh H, Nishi A, Yamashita K, et al.Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm.Plant Physiol, 2003, 133(3): 1111-1121.
[10]	Kubo A, Fujita N, Harada K, et al.The starch-debranching enzymes isoamylase and pullulanase are both involved in amylopectin biosynthesis in rice endosperm.Plant Physiol, 1999, 121(2): 399-410.
[11]	Fujita N, Toyosawa Y Y, Higuchi T, et al.Characterization of pullulanase (PUL)-deficient mutants of rice (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm.J Nano Res, 2008, 16(1): 43-48.
[12]	Preiss J.Bacterial glycogen synthesis and its regulation.Ann Rev Microbiol, 1984, 38(1):419-458.
[13]	Villand P, Olsen O A, Kleczkowski L A.Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana.Plant Mol Biol, 1993, 23(6): 1279-1284.
[14]	Okita T W, Nakata P A, Anderson J M, et al.The subunit structure of potato tuber ADPglucose pyrophosphorylase.Plant Physiol, 1990, 93(2): 785-790.
[15]	Takashi A, Kouichi M, Tatsuhito F.Gene expression of ADP-glucose pyrophosphorylase and starch contents in rice cultured cells are cooperatively regulated by sucrose and ABA.Plant & Cell Physiol, 2005, 46(6): 937-946.
[16]	Binquan H, Hennen-Bierwagen T A, Myers A M. Functions of multiple genes encoding ADP-glucose pyrophosphorylase subunits in maize endosperm, embryo, and leaf.Plant Physiol, 2014, 164(2): 596-611.
[17]	Sikka V K, Choi S B, Kavakli I H, et al.Subcellular compartmentation and allosteric regulation of the rice endosperm ADP-glucose pyrophosphorylase.Plant Sci, 2001, 161(3):461-468.
[18]	Tetlow I J, Davies E J, Vardy K A, et al.Subcellular localization of ADP-glucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform.J Exp Bot, 2003, 54(383):715-725.
[19]	Kawagoe Y, Kubo A, Satoh H, et al.Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm.Plant J, 2005, 42(2): 164-174.
[20]	Lee S K, Hwang S K, Han M, et al.Identification of the ADP-glucose pyrophosphorylase isoforms essential for starch synthesis in the leaf and seed endosperm of rice (Oryza sativa L.).Plant Mol Biol, 2007, 65(4): 531-546.
[21]	Johnson P E, Patron N J, Bottrill A R, et al.A low-starch barley mutant, risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase, reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit.Plant Physiol, 2003, 131(2):81-99.
[22]	Sakulsingharoj C, Choi S, Hwang S, et al.Engineering starch biosynthesis for increasing rice seed weight: The role of the cytoplasmic ADP-glucose pyrophosphorylase.Plant Sci, 2004, 167(6):1323-1333.
[23]	Curtis L H, Brandon F, James B, et al.A shrunken-2 transgene increases maize yield by acting in maternal tissues to increase the frequency of seed development.Plant Cell, 2012, 24(6):2352-2363.
[24]	Satoh H, Omura T.New endosperm mutations induced by chemical mutagens in rice Oryza sativa L.Jpn J Breeding, 1981, 31(3):316-326.
[25]	She K C, Kusano H, Koizumi K, et al.A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality.Plant Cell, 2010, 22(10): 3280-3294.
[26]	Nishio T, Iida S.Mutants having a low content of 16-kDa allergenic protein in rice (Oryza sativa L.).Theor Appl Genet, 1993, 86(2-3): 317-321.
[27]	Kang H G, Park S, Matsuoka M, et al.White-core endosperm floury endosperm-4 in rice is generated by knockout mutations in the C-type pyruvate orthophosphate dikinase gene (OsPPDKB).Plant J, 2005, 42(6): 901-911.
[28]	Ryoo N, Yu C, Park C S, et al.Knockout of a starch synthase gene OsSSIIIa/Flo5 causes white-core floury endosperm in rice (Oryza sativa L.).Plant Cell Rep, 2007, 26(7):1083-1095.
[29]	Peng C, Wang Y, Liu F, et al.FLOURY ENDOSPERM6 encodes a CBM48 domain-containing protein involved in compound granule formation and starch synthesis in rice endosperm.Plant J, 2014, 77(6): 917-930.
[30]	Zhang L, Ren Y, Lu B, et al.FLOURY ENDOSPERM7 encodes a regulator of starch synthesis and amyloplast development essential for peripheral endosperm development in rice.J Exp Bot, 2015, 67(3):633-647.
[31]	Wu K S, Tanksley S D.Abundance, polymorphism and genetic mapping of microsatellites in rice.Mol General Genet, 1993, 241(1-2): 225-235.
[32]	李永庚, 于振文, 姜东, 等. 冬小麦旗叶蔗糖和籽粒淀粉合成动态及与其有关的酶活性的研究. 作物学报, 2001, 27(5): 658-664.
	Li Y G, Yu Z, Jiang D, et al.Studies on the dynamic changes of the synthesis of sucrose in the flag leaf and starch in the grain and related enzymes of high-yield wheat.Acta Agron Sin, 2001, 27(5): 658-664. (in Chinese with English abstract)
[33]	Denyer K, Dunlap F, ThorbjoRnsen T, et al. The major form of ADP-glucose pyrophosphorylase in maize endosperm is extra-plastidial.Plant Physiol, 1996, 112(2): 779-785.
[34]	Thorbjornsen T, Villand P, Denyer K, et al.Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm.Plant J, 1996, 10(2): 243-250
[35]	Hannah L C, Shaw J R, Giroux M J, et al.Maize genes encoding the small subunit of ADP-glucose pyrophosphorylase.Plant Physiol, 2001, 127(1): 173-183.
[36]	Sikka V K, Choi S B, Kavakli I H, et al.Subcellular compartmentation and allosteric regulation of the rice endosperm ADPglucose pyrophosphorylase.Plant Sci, 2001, 161(3): 461-468.
[37]	Seon-Kap H, Yasuko N, Dongwook K, et al.Direct appraisal of the potato tuber ADP-glucose pyrophosphorylase large subunit in enzyme function by study of a novel mutant form.J Biol Chem, 2008, 283(11): 6640-6647.
[38]	Laughlin M J, Chantler S E, Okita T W.N- and C-terminal peptide sequences are essential for enzyme assembly, allosteric, and/or catalytic properties of ADP-glucose pyrophosphorylase.Plant J, 1998, 14(2): 159-168.
[39]	Pedro C, Ballicora M A, Angel M, et al.The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme.J Biol Chem, 2003, 278(31): 28508-28515.
[40]	Petreikov M, Eisenstein M, Yeselson Y, et al.Characterization of the AGPase large subunit isoforms from tomato indicates that the recombinant L3 subunit is active as a monomer.Biochem J, 2010, 428(2): 201-212.
[41]	Ventriglia T, Kuhn M L, Ruiz M T, et al.Two Arabidopsis ADP-glucose pyrophosphorylase large subunits (APL1 and APL2) are catalytic.Plant Physiol, 2008, 148(1): 65-76.
[42]	Aytug T, Joe K, Yasuharu I, et al.The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme.Plant Cell Physiol, 2014, 55(6): 1169-1183.