Activities of Several Starch Synthesis Enzymes in Filling Grains for Rice Sugary Endosperm Mutant (Sug-11)and Its Relation to Starch Quality

doi:10.3969/j.issn.1001-7216.2015.01.009

Abstract

Abstract:

With the rice sugary endosperm mutant (Sug-11) and its wild-type (Zhonghua 11) as materials, the total soluble sugar, sucrose contents, starch accumulation in filling grains and the starch physicochemical properties were compared to further reveal the temporal patterns of sugar content, several key enzymes activities and their isoform genes transcriptional expression level during the grain filling so as to analyze the physiological mechanism for Sug-11 whose sugars contents change and 1000-grain weight decline from the point of starch biosynthesis pathway in rice grains. The result showed that the differences in soluble sugar and sucrose contents between Sug-11 and its wild-type were not obvious at the initial stage of grain filling, but with the filling process it gradually became apparent. Secondly, the amylose(Am) content and BV(Am) of Sug-11 were significantly decreased while the starch solubility and BV(amylopectin, Ap) were increased compared with Zhonghua 11, indicating that the physicochemical properties of grain starch were significantly affected in the sugary endosperm mutant. Finally, among the key enzymes in starch synthetic metabolism, debranching enzyme(DBE), existed in Sug-11 seeds, were quite different from its wild type in activity and dynamic in the process of grain filling, and PUL transcriptional expression level and DBE activity strikingly decreased at the middle-late stage, resulting in the declined starch accumulation and increased sugar content in Sug-11. However the poor filling and 1000-grain weight decline were, to some extent, associated with dropped ADPGase activity at the early grain filling stage.

Key words: rice, sugary endosperm mutant, starch synthesis, enzymatic activity, starch quality

摘要：

以水稻糖质胚乳突变体Sug-11与其野生型对照中花11为材料,通过对两者籽粒中可溶性总糖、蔗糖含量和淀粉含量以及有关淀粉品质理化指标的比较,结合籽粒灌浆过程中糖类物质含量、淀粉合成代谢关键酶活性和相关同工型基因转录表达水平的动态测定,从籽粒淀粉合成代谢角度,对水稻糖质突变体Sug-11的籽粒糖类含量变化和千粒重下降的生理原因进行了分析。结果表明,Sug-11糖质突变体与其野生型在灌浆初期的可溶性糖和蔗糖含量差异并不明显,随着籽粒灌浆进程,两者间的籽粒糖分含量差异在灌浆中后期逐步趋于明显;与野生型相比,Sug-11糖质胚乳突变体的稻米直链淀粉含量和直链淀粉碘蓝值显著下降,而淀粉溶解度和支链淀粉碘蓝值则显著升高,糖质胚乳突变对稻米淀粉的理化特性也产生了明显的影响;在籽粒淀粉合成代谢的几个关键酶中,Sug-11糖质突变体籽粒中的DBE活性及其在灌浆过程中的动态变化与其野生型存在明显差异,揭示了胚乳糖质突变体Sug-11籽粒中淀粉积累减少、糖分含量增加主要是由籽粒灌浆中后期的PUL转录表达水平和DBE活性的大幅下降所引起的,而Sug-11的籽粒灌浆不良和千粒重下降等现象,则与其ADPGase活性在籽粒灌浆前期的显著下降存在一定的联系。

关键词: 水稻, 糖质突变, 淀粉合成, 酶活性, 淀粉品质

CLC Number:

Hua ZHAO, Jun-min WANG, Qi-fang ZHANG, Qian ZHAO, Shu-fang MEI, Xiang-lei LIU, Fang-min CHENG. Activities of Several Starch Synthesis Enzymes in Filling Grains for Rice Sugary Endosperm Mutant (Sug-11)and Its Relation to Starch Quality[J]. Chinese Journal OF Rice Science, 2015, 29(1): 73-81.

赵华, 王俊敏, 张其芳, 赵倩, 梅淑芳, 刘向蕾, 程方民. 水稻糖质胚乳突变体Sug-11籽粒灌浆过程的淀粉合成关键酶活性及其与淀粉理化特性关系[J]. 中国水稻科学, 2015, 29(1): 73-81.

Figures/Tables 7

References 31

[1]	Jeon J S, Ryoo N, Hahn T R, et al.Starch biosynthesis in cereal endosperm.Plant Physiol Biochem, 2010, 48:383-392.
[2]	Ball S, Guan H P, James M, et al.From glycogen to amylopectin: A model for the biogenesis of the plant starch granule.Cell, 1996, 86:349-352.
[3]	左晓旭,郑涛,舒小丽,等.水稻胚乳突变体筛选与特性研究进展.核农学报,2006,20(2):118-122.
[4]	Yano M, Isono Y, Satoh H, et al.Gene analysis of sugary and shrunken mutants of rice, Oryza sativa L.Jpn J Breed,1984:34(1):43-49
[5]	Satoh H, Nishi A, Fujita N, et al.Isolation and characterization of starch mutants in rice.J Appl Glycosci, 2003, 50: 225-230.
[6]	Kubo A, Rahman S, Utsumi Y, et a1. Complementation of sugary-1 phenotype in rice endosperm with the wheat isoamylasel gene supports a direct role for isoamylasel in amylopectin biosynthesis.Plant Physiol, 2005,137:43-56.
[7]	Kawagoe Y, Kubo A, Satoh H, et al.Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm.Plant J, 2005,42:164-174.
[8]	Vandeputte G E, Delcour J A.From sucrose to starch granule to starch physical behavior: A focus on rice starch.Carbohydrate Polymer, 2004,58: 245-266.
[9]	Wong K S, Kubo A, Jane J L, et al.Structure and properties of amylopectin and phytoglycogen in the endosperm of sugary-1 mutants of rice.J Cereal Sci, 2003,37: 139-149.
[10]	Geigenberger P.Regulation of starch biosynthesis in response to a fluctuating environment.Plant Physiol, 2011, 155:1566-1577.
[11]	程方民, 蒋德安, 吴平, 等.早籼稻籽粒灌浆过程中淀粉合成酶的变化及温度效应特征.作物学报, 2001, 27(2): 201-206.
[12]	Nakamura Y, Yuki K, Park S Y, et al.Carbohydrate metabolism in the developing endosperm of rice grains.Plant Cell Physiol,1989, 30: 833-839.
[13]	Livak J K, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method.Methods, 2001,25: 402-408.
[14]	中国科学院上海植物生理研究所,上海市植物生理学会. 现代植物生理学实验指南. 北京:科学出版社, 1999.
[15]	邹琦. 植物生理学实验指导. 北京:中国农业出版社,2000.
[16]	钟连进,程方民.水稻籽粒灌浆过程直链淀粉积累及其相关酶的品种类型间差异.作物学报,2003,29(3): 93-498.
[17]	蔡一霞,王维,朱智伟,等.不同类型水稻支链淀粉理化特性及其与米粉糊化特征的关系.中国农业科学,2006, 9(6): 122-1129.
[18]	Mizuno K, Kawasaki T, Shimada H, et al.Alteration of the structural properties of starch components by the lack of an isoform of starch branching enzyme in rice seeds.J Biol Chem, 1993, 268:19084-19091.
[19]	Hussain H, Mant A, Seale R, et al.Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans.Plant Cell, 2003,15:133-149.
[20]	Li Q F, Zhang G Y, Dong Z W, et al.Characterization of expression of the OsPUL gene encoding a pullulanase-type debranching enzyme during seed development and germination in rice.Plant Physiol Biochem, 2009, 47:351-358.
[21]	Wattebled F, Dong Y, Dumez S, et al.Mutants of Arabidopsis lacking a chloroplastic isoamylase accumulate phytoglycogen and an abnormal form of amylopectin.Plant Physiol, 2005, 138:184-195.
[22]	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: 2131-2145.
[23]	Thitisaksakul M, Jiménez R C, Arias M C, et al.Effects of environmental factors on cereal starch biosynthesis and composition,J Cereal Sci, 2012, 56: 67-80.
[24]	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: 399-410.
[25]	李钱峰,张桂云,于恒秀,等.水稻异淀粉酶基因ISA1及其启动子的表达特性分析.中国水稻科学,2009,23(1):21-18.
[26]	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, 56:3229-3244.
[27]	Nakamura Y, Umemoto T, Ogata N, et al.Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: Purification, cDNA and chromosomal localization of the gene.Planta, 1996,199:209-218.
[28]	Utsumi Y, Nakamura Y.Structural and enzymatic characterization of the isoamylase1 homo-oligomer and the isoamylase1-isoamylase2 hetero-oligomer from rice endosperm.Planta, 2006, 225:75-87.
[29]	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, 133:1111-1121.
[30]	Fujita N, Kubo A, Suh D S, et al.Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm.Plant Cell Physiol,2003,44: 607-618.
[31]	Fujita N, Toyosawa Y, Utsumi Y, 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 Exp Bot, 2009, 60:1009-1023.

基因名 Gene name	登录号 Accession No.	引物 Primer pairs	产物大小 Product size/ bp
Actin	XM_469569	F:5’-CAGCACATTCCAGCAGATGT	198
		R:5’-TAGGCCGGTTGAAAACTTTG
ISA1	AB093426	F:5’-GTTGGAACCGTGTGGTGGAT	112
		R:5’-AGCAGAATGGAAGAGTATGGAGC
ISA2	AC132483	F:5’-CAGTGAGTGCTGCCTTGC	106
		R:5’-TTGGGATTAGATTCGGTTG
ISA3	AP005574	F:5’-CGCAGACTTCCCAACAGC	144
		R:5’-CCTTTCGTTTCATCTTTCGTG
PUL	AB012915	F:5’-ACCTTTCTTCCATGCTGG	202
		R:5’-CAAAGGTCTGAAAGATGGG

基因名 Gene name	登录号 Accession No.	引物 Primer pairs	产物大小 Product size/ bp
Actin	XM_469569	F:5’-CAGCACATTCCAGCAGATGT	198
		R:5’-TAGGCCGGTTGAAAACTTTG
ISA1	AB093426	F:5’-GTTGGAACCGTGTGGTGGAT	112
		R:5’-AGCAGAATGGAAGAGTATGGAGC
ISA2	AC132483	F:5’-CAGTGAGTGCTGCCTTGC	106
		R:5’-TTGGGATTAGATTCGGTTG
ISA3	AP005574	F:5’-CGCAGACTTCCCAACAGC	144
		R:5’-CCTTTCGTTTCATCTTTCGTG
PUL	AB012915	F:5’-ACCTTTCTTCCATGCTGG	202
		R:5’-CAAAGGTCTGAAAGATGGG

年份与基因型 Year and genotype	株高 Plant height/cm	每穗实粒数 Number of filled grains per panicle	千粒重 Grain weight/g	结实率 Setting-rate /%	蔗糖含量 Sucrose content/%	可溶性总糖含量 Total soluble sugar contents/%	淀粉含量 Starch content/%
2011
中花11 Zhonghua 11	90.6±4.6	132.6±8.2	24.4±0.5	83.1±3.9	1.73±0.15	1.99±0.13	76.83±1.87
Sug-11	86.9±3.9	107.3±6.6	20.6±0.3	75.9±6.7	5.40±0.49	5.87±0.22	71.35±0.89
差值Difference	3.7	25.3^**	3.8^**	7.2^*	-3.67^**	-3.88^**	5.48^**
2012
中花11 Zhonghua 11	97.6±4.9	127.2±7.7	25.5±0.3	88.7±4.6	1.63±0.41	1.82±0.54	75.16±0.91
Sug-11	95.5±4.0	119.3±10.2	19.6±0.6	83.3±5.5	5.76±0.53	5.91±0.61	72.51±1.02
差值 Difference	2.1	8.8	5.9^**	5.4	-4.13^**	4.09^**	2.65^*

年份与基因型 Year and genotype	株高 Plant height/cm	每穗实粒数 Number of filled grains per panicle	千粒重 Grain weight/g	结实率 Setting-rate /%	蔗糖含量 Sucrose content/%	可溶性总糖含量 Total soluble sugar contents/%	淀粉含量 Starch content/%
2011
中花11 Zhonghua 11	90.6±4.6	132.6±8.2	24.4±0.5	83.1±3.9	1.73±0.15	1.99±0.13	76.83±1.87
Sug-11	86.9±3.9	107.3±6.6	20.6±0.3	75.9±6.7	5.40±0.49	5.87±0.22	71.35±0.89
差值Difference	3.7	25.3^**	3.8^**	7.2^*	-3.67^**	-3.88^**	5.48^**
2012
中花11 Zhonghua 11	97.6±4.9	127.2±7.7	25.5±0.3	88.7±4.6	1.63±0.41	1.82±0.54	75.16±0.91
Sug-11	95.5±4.0	119.3±10.2	19.6±0.6	83.3±5.5	5.76±0.53	5.91±0.61	72.51±1.02
差值 Difference	2.1	8.8	5.9^**	5.4	-4.13^**	4.09^**	2.65^*

淀粉指标 Starch parameters	中花11 Zhonghua 11			Sug-11			差值 Difference
直链淀粉含量 Amylose content/%	17.74	±	0.68	10.46	±	0.61	7.28^**
淀粉膨胀势 Swelling power/(g·g^-1)	13.68	±	0.31	9.26	±	0.57	4.45^**
淀粉溶解度 Starch solubility/%	4.06	±	0.51	24.95	±	0.58	-20.89^**
直链淀粉最大吸收波长 λ_max.Am	620.2	±	2.0	608.0	±	1.3	12.0^**
直链淀粉碘蓝值 BV_Am	0.39	±	0.01	0.35	±	0.03	0.04^*
支链淀粉最大吸收波长λ_max.Ap	589.3	±	2.1	600.4	±	2.2	-11.1^**
支链淀粉碘蓝值 BV_Ap	0.09	±	0.01	0.12	±	0.04	-0.03