Function Analysis of Sucrose Transporter OsSUT4 in Sucrose Transport in Rice

doi:10.16819/j.1001-7216.2020.0316

Abstract

Abstract:

【Objective】The transmembrane transport of sucrose is mainly mediated by sucrose transporters, among which five SUT coding genes have been identified inrice. However, the role of OsSUT4 in the sucrose transport is unclear. 【Method】CRISPR/Cas9 techniques were used to construct ossut4-deletedmutants. The ossut4 mutant was featured by lower plant height and grain yield as well as higher tiller number as compared withwildtype plants.【Result】Sucrose and starchaccumulationin the leaf of the ossut4 mutants lead to slower net photosynthetic rate; insufficient sugar supply results in the postponeof grain filling. Base on the subcellular and histochemical localization,OsSUT4 was located on the cytoplasmic membrane. Tissue localization showed that OsSUT4 was expressed in the embryo of germinating seed, vascular bundles of coleoptile, glume shell, anthers at flowering stage and aleurone layer of caryopsis atfilling stage. 【Conclusion】OsSUT4 plays an important role in sucrose transport.

Key words: rice, CRISPR/Cas9, gene editing, sucrose transport, sucrose transporter

摘要： 目的植物蔗糖的跨膜运输主要借助蔗糖转运蛋白（sucrose transporter,SUT）进行。在已鉴定出的5个水稻SUT编码基因中,OsSUT4的作用尚不清楚。方法采用CRISPR/Cas9基因编辑技术构建ossut4缺失变体,出现株高降低、分蘖数增加及产量下降等表型。结果通过进一步分析,发现ossut4突变体水稻叶片蔗糖、淀粉积累,反馈抑制光合速率;因源端光合同化物装载受阻,导致库端籽粒蔗糖供应不足,灌浆延迟。对OsSUT4进行亚细胞和组织化学定位,发现OsSUT4在细胞质膜上,且在萌发种子的胚、胚芽鞘的维管束、小穗颖壳、花药、颖果的糊粉层中都有表达,该现象表明OsSUT4具备转运蔗糖的特征。结论综上所述,OsSUT4在水稻蔗糖源端装载以及籽粒库端卸载等生理过程发挥重要作用。

关键词: 水稻, CRISPR/Cas9, 基因编辑, 蔗糖转运, 蔗糖转运蛋白

CLC Number:

Q786

Mengzhu LI, Gaopeng WANG, Yue WU, Yi REN, Ganghua LI, Zhenghui LIU, Yanfeng DING, Lin CHEN. Function Analysis of Sucrose Transporter OsSUT4 in Sucrose Transport in Rice[J]. Chinese Journal OF Rice Science, 2020, 34(6): 491-498.

李孟珠, 王高鹏, 巫月, 任怡, 李刚华, 刘正辉, 丁艳锋, 陈琳. 水稻蔗糖转运蛋白OsSUT4参与蔗糖转运的功能研究[J]. 中国水稻科学, 2020, 34(6): 491-498.

Figures/Tables 10

Fig.1. Results of sequence alignment between the ossut4 mutant lines and the wild-type.

W=	A	; 1)
W=	(1+Be^-kt)^1/N	; 1)
对方程(1)求导,得到灌浆速率R：
R=	AkBe^-kt	; 2)
R=	N(1+Be^-kt)^(N+1)/N	; 2)

Table 1 Primer sequences of RT-PCR.

基因名称 Gene name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer(5'→3')
Actin	CAATCGTGAGAAGATGACCC	GTCCATCAGGAAGCTCGTAGC
OsSUT1	GCTTTCAACCAGGGTGTCAG	ACTTTCCGGCACATTGGTTC
OsSUT3	ATCTGTGATCGTCATCGGGTTC	GCACGGTGTTGTTGGAGAAGT
OsSUT4	CTCGTGCCCTTTTAGCTGAC	AACGTTTCCAACAGCCATCC
OsSUT5	TTCGCCTTCATCTGTGGAGT	GCCTTTGATGGAGTTTCGCA

Fig. 2. Effects of OsSUT4 mutation on plant height in rice.

Table 2 Plant height and yield components ofossut4 mutant and wild type.

株系名称 Name of line	株高 Plant height	分蘖数 Tiller number	每穗粒数 Grain number per panicle	结实率 Seed setting rate/%	一次枝梗数 Number of primary rachis branches	二次枝梗数 Number of secondary rachis branches	千粒重 1000-grain weight
WT	87.5 a	12.2 b	87.6 a	84.0a	11.0a	10.0a	25.6a
sut4-15-1	82.4bc	12.4 a	66.9 b	73.3 b	8.8b	7.4c	24.7 b
sut4-15-2	81.8 c	12.2 a	68.4 b	65.8 c	8.9 b	7.8 c	24.8b
sut4-46	83.3 b	12.0 a	68.5 b	72.8b	8.8b	8.9b	23.5 c

Fig. 3. Effects of OsSUT4 mutation on the relative expression level of sucrose transporters in rice leaves.

Fig.4. Effects ofOsSUT4mutation on sucrose, starch contents, photosynthetic rate and SPAD value in rice leaves. A, Sucrose content at the end of day and night; B, Starch content at the end of day and night; C, Net photosynthetic rate; D, SPAD value. Values are Mean±SE (n=3). Different letters above bars indicate significant difference(P<0.05) among the lines (Duncan’s test).

Fig. 5. Effects of OsSUT4 mutation on pollen vigor in rice. A, Pollen staining; B, Pollen dyeability rate. Values are Mean±SE(n=5). Different letters above bars indicate significant difference(P<0.05) among the lines(Duncan’s test).

Fig.6. Effects of OsSUT4 mutation on grain filling in rice.

Fig.7. Sub-cellular localization(A-D) of OsSUT4 and tissue expression(E-K) of pOsSUT4:GUS transgenic rice. A, cDNA-GFP; B, Bright field; C, Membrane marker OSMCA1-RFP; D, Merged; E, Germinated seed; F, Leaf; G, Leaf sheath; H, Root; I&J, Spikelet; K, Caryopsis.

References 19

[1]	Pal S K, Liput M, Piques M, Ishihara H, Obata T, Martins M C,Sulpice R,van Dongen J T,Fernie A R,Yadav U P,Lunn J E,Usadel B,Stitt M.Diurnal changes of polysome loading track sucrose content in the rosette of wild-type Arabidopsis and the starchlesspgm mutant[J].Plant Physiology,2013,162(3):1246-1265.
[2]	张志兴, 李忠, 陈军, 李奇松, 陈龙怀, 陈鸿飞, 黄锦文, 林文雄. 氮肥运筹对大穗型水稻品种金恢809灌浆期叶片蛋白质表达的影响[J]. 作物学报, 2011, 37(5): 842-854.
	Zhang Z X, Li Z, Chen J, Li J S, Chen L H, Chen H F, Huang J W, Lin W X.Effects of nitrogen management on protein expression of flag leaves during grain-filling period in large panicle rice[J].ActaAgronomica Sinica, 2011, 37(5): 842-854.(in Chinese with English abstract)
[3]	李国辉, 崔克辉. 水稻蔗糖转运及其与产量形成的关系[J]. 植物生理学报, 2014, 50(6): 735-740.
	Li G H, Cui K H.Sucrose translocation and its relationship with grain yield formation in rice[J].Plant Physiology Communications, 2014, 50(6): 735-740.(in Chinese with English abstract)
[4]	Liesche J, Schulz A.In vivo quantification of cell coupling in plants with different phloem-loading strategies.Plant Physiology,2012,159(1):355-365.
[5]	Braun DM, Wang L, Ruan YL.Understanding and manipulating sucrose phloem loading, unloading, metabolism, and signaling to enhance crop[J]. Journal of Experimental Botany, 2014, 65:1713-1735.
[6]	Regmi K C, Zhang S J, Gaxiola R A.Apoplasmic loading in the rice phloem supported by the presence of sucrose synthase and plasma membrane-localized proton pyrophosphatase.AnnualBotany,2016,117(2):257-268.
[7]	孙学武, 谭炎宁, 孙志忠, 袁定阳, 段美娟. 水稻蔗糖转运蛋白研究进展[J]. 生命科学研究, 2014, 18(2): 157-161.
	Sun X W, Tan Y N, Sun Z Z, Yuan D Y, Duan M J.Advances in sucrose transporters in rice[J].Life Science Research, 2014, 18(2): 157-161.(in Chinese with English abstract)
[8]	Aoki N, Hirose T, Scofield G N, Whitfeld P R, Furbank R T.The sucrose transporter gene family in rice[J].Plant Cell Physiology,2003,44(3):223-232.
[9]	Scofield G N, Aoki N, Hirose T, Takano M, Jenkins C L, Furbank R T.The role of the sucrose transporter, OsSUT1, in germination and early seedling growth and development of rice plants[J].Journal of Experimental Botany,2007,58(3):483-495.
[10]	Scofield G N, Hirose T, Gaudron J A, Furbank R T, Upadhyaya N M, Ohsugi R.Antisense suppression of the rice transporter gene, OsSUT1, leads to impaired grain filling and germination but does not affect photosynthesis[J].Functional Plant Biology,2002,29(7):815-826.
[11]	Scofield G N, Hirose T, Aoki N, Furbank R T.Involvement of the sucrose transporter, OsSUT1, in the long-distance pathway for assimilate transport in rice[J].Journal of Experimental Botany,2007,58(12):3155-3169.
[12]	Siao W, Chen J Y, Hui-Hsin H, Chung P, Shu-Jen W.Characterization of OsSUT2 expression and regulation in germinating embryos of rice seeds[J].Rice,2011,4(2):39-49.
[13]	Eom J S, Cho J I, Reinders A, Lee S W, Yoo Y, Tuan P Q, Choi S B, Bang G, Park Y I, Cho M H, Bhoo S H, An G, Hahn T R, Ward J M, Jeon J S.Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth[J].Plant Physiology,2011,157(1):109-119.
[14]	Kurusu T, Nishikawa D, Yamazaki Y, Gotoh M, Nakano M, Hamada H, Yamanaka T, Iida K, Nakagawa Y, Saji H, Shinozaki K, Iida H, Kuchitsu K.Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+ influx and modulates Generation of reactive Oxygen species in cultured rice cells[J/OL].BMC Plant Biology,2012,12:11.
[15]	钟英健, 王春台, 刘学群, 徐鑫. 水稻OsVDAC2基因的克隆及亚细胞定位分析[J]. 中国农学通报, 2015, 31(24): 168-172.
	Zhong Y J, Wang C T, Liu X Q, Xu X.Cloning and subcellular localization analysis of OsVDAC2 gene[J].Chinese Agricultural Science Bulletin, 2015, 31(24): 168-172.(in Chinese with English abstract)
[16]	Richards F J.A flexible growth function for empirical use[J].Journal of Experimental Botany,1959,10(2):290-301.
[17]	Zhang C, Han L, Slewinski T L, Sun J L, Zhang J, Wang Z Y, Turgeon R.Symplastic phloem loading in poplar[J].Plant Physiology,2014,166(1):306-313.
[18]	Slewinski T L, Meeley R, Braun D M.Sucrose transporter1 functions in phloem loading in maize leaves[J].Journal of Experimental Botany,2009,60(3):881-892.
[19]	Eom J S, Choi S B, Ward J M, Jeon J S.The mechanism of phloem loading in rice (Oryza sativa)[J].Molecular Cells,2012,33(5):431-438.