Overexpression of OsRhoGDI1 Gene Regulates Grain Shape and Seed Vigor in Rice

doi:10.13560/j.cnki.biotech.bull.1985.2024-0405

Abstract

Abstract:

【Objective】 OsRhoGDI1, a rice RhoGDI gene, was isolated from young panicles through yeast two-hybrid screening, using Rho/Rop protein OsRac5 as bait. RhoGDIs genes encode Rho GDP dissociation inhibitors, which negatively regulate the activities of Rho proteins. Transgenic technology was applied to identify the functions of OsRhoGDI1, aiming to lay a foundation for further research on the functional relationship between OsRhoGDI1 and OsRac5 in rice growth and development. 【Method】 OsRhoGDI1-overexpressed vector was constructed, and rice were transformed to obtain positive transgenic plants. The grain shape and germination characteristics of transgenic rice was analyzed. The expressions of grain-shape-regulating genes in grain development were detected through RT-qPCR, and scanning electron microscopy was used to detect the characteristics of glume cells. The germination characteristics were compared between OsRhoGDI1-overexpressed rice and control, and the expressions of the genes involved in synthesis and metabolism of gibberellin（GA）and abscisic acid（ABA）were detected by RT-qPCR, as well as the expressions and activities of α-amylases. 【Result】 Compared with the control rice, the OsRhoGDI1-overexpressed rice showed a significant increase in grain length, decrease in grain width, thickness and thousand grain weight. The expressions of grain-shape-regulating gene GS2, GS5, GS6 and GLW7 in young panicles also reduced to varying degrees, while the expression of GW2 increased. The lengths of glume cells in the transgenic rice increased, while the width decreased. The germination of transgenic rice was earlier than that of control, and the expressions of GA synthesis genes, ABA catabolic genes, and α-amylase genes increased to varying degrees, while the expressions of GA catabolic genes and ABA synthesis genes decreased. α-amylase activities were also enhanced in the transgenic rice, compared to the control. 【Conclusion】 The overexpression of OsRhoGDI1 gene regulates rice grain shape by altering the expressions of grain shape genes, leading to elongation of glume epidermal cells, resulting in increase of grain length and decrease of grain width. Meanwhile, the overexpression of this gene also changes the expressions of germination-related genes, increase the activities of α-amylase, thus enhance seed vigor and promote germination.

Key words: rice, OsRhoGDI1, grain shape, germination, hormones, α-amylase

QI Wang, BI Yi-fan, XUAN Qiang-bing, ZHANG Xin, ZHOU Hui-gang, NIE Yuan-qing, CHENG Biao-biao, ZHANG Yu-shun, WANG Jun-jie, LIANG Wei-hong. Overexpression of OsRhoGDI1 Gene Regulates Grain Shape and Seed Vigor in Rice[J]. Biotechnology Bulletin, 2024, 40(11): 152-161.

Figures/Tables 10

References 43

[1]	Xie LX, Tan ZW, Zhou Y, et al. Identification and fine mapping of quantitative trait loci for seed vigor in germination and seedling establishment in rice[J]. J Integr Plant Biol, 2014, 56(8): 749-759.
[2]	He W, Wang L, Lin QL, et al. Rice seed storage proteins: Biosynthetic pathways and the effects of environmental factors[J]. J Integr Plant Biol, 2021, 63(12): 1999-2019.
[3]	Tappiban P, Ying YN, Xu FF, et al. Proteomics and post-translational modifications of starch biosynthesis-related proteins in developing seeds of rice[J]. Int J Mol Sci, 2021, 22(11): 5901.
[4]	Wang WJ, Huang RZ, Wu GW, et al. Transcriptomic and QTL analysis of seed germination vigor under low temperature in weedy rice WR04-6[J]. Plants, 2023, 12(4): 871.
[5]	Hou DP, Bi JG, Ma L, et al. Effects of soil moisture content on germination and physiological characteristics of rice seeds with different specific gravity[J]. Agronomy, 2022, 12(2): 500.
[6]	杨蓉兰, 朱明东, 余应弘. 水稻种子活力的分子遗传与调控机理研究进展[J]. 杂交水稻, 2023, 38(4): 1-11.
	Yang RL, Zhu MD, Yu YH. Research advances in molecular genetics and regulatory mechanisms of rice seed vigor[J]. Hybrid Rice, 2023, 38(4): 1-11.
[7]	Zhang H, Wang WQ, Liu SJ, et al. Proteome analysis of poplar seed vigor[J]. PLoS One, 2015, 10(7): e0132509.
[8]	Wang XM, Tang QY, Mo WW. Seed filling determines seed vigour of superior and inferior spikelets during hybrid rice(Oryza sativa)seed production[J]. Seed Sci Technol, 2020, 48(2): 143-152.
[9]	Huang M, Zhang RC, Chen JN, et al. Morphological and physiological traits of seeds and seedlings in two rice cultivars with contrasting early vigor[J]. Plant Prod Sci, 2017, 20(1): 95-101.
[10]	Park JR, Seo J, Park S, et al. Identification of potential QTLs related to grain size in rice[J]. Plants, 2023, 12(9): 1766.
[11]	Jiang HZ, Zhang AP, Liu XT, et al. Grain size associated genes and the molecular regulatory mechanism in rice[J]. Int J Mol Sci, 2022, 23(6): 3169.
[12]	Ren DY, Ding CQ, Qian Q, et al. Molecular bases of rice grain size and quality for optimized productivity[J]. Sci Bull, 2023, 68(3): 314-350.
[13]	刘迪, 冯连杰, 梁卫红. 水稻粒型调控相关信号通路的鉴定与解析[J]. 中国生物化学与分子生物学报, 2023, 39(2): 212-221.
	Liu D, Feng LJ, Liang WH. Identification and analysis of grain shape related regulation signal pathways in rice[J]. Chin J Biochem Mol Biol, 2023, 39(2): 212-221.
[14]	姚莎莎, 王晶晶, 王俊杰, 等. 植物激素信号通路调控水稻粒型的分子机制[J]. 生物技术通报, 2023, 39(8): 80-90.
	Yao SS, Wang JJ, Wang JJ, et al. Molecular mechanisms of rice grain size regulation related to plant hormone signaling pathways[J]. Biotechnol Bull, 2023, 39(8): 80-90.
[15]	Choi BS, Kim YJ, Markkandan K, et al. GW2 functions as an E3 ubiquitin ligase for rice expansin-like 1[J]. Int J Mol Sci, 2018, 19(7): 1904.
[16]	Wang SK, Li S, Liu Q, et al. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality[J]. Nat Genet, 2015, 47(8): 949-954.
[17]	Si LZ, Chen JY, Huang XH, et al. OsSPL13 controls grain size in cultivated rice[J]. Nat Genet, 2016, 48(4): 447-456.
[18]	Duan PG, Ni S, Wang JM, et al. Regulation of OsGRF4 by OsmiR396 controls grain size and yield in rice[J]. Nat Plants, 2015, 2: 15203.
[19]	Hu J, Wang YX, Fang YX, et al. A rare allele of GS2 enhances grain size and grain yield in rice[J]. Mol Plant, 2015, 8(10): 1455-1465.
[20]	Li YB, Fan CC, Xing YZ, et al. Natural variation in GS5 plays an important role in regulating grain size and yield in rice[J]. Nat Genet, 2011, 43(12): 1266-1269.
[21]	Sun LJ, Li XJ, Fu YC, et al. GS6, a member of the GRAS gene family, negatively regulates grain size in rice[J]. J Integr Plant Biol, 2013, 55(10): 938-949.
[22]	黎家, 李传友. 新中国成立70年来植物激素研究进展[J]. 中国科学: 生命科学, 2019, 49(10): 1227-1281.
	Li J, Li CY. Seventy-year major research progress in plant hormones by Chinese scholars[J]. Sci Sin Vitae, 2019, 49(10): 1227-1281.
[23]	Chen Y, Tan BC. New insight in the gibberellin biosynthesis and signal transduction[J]. Plant Signal Behav, 2015, 10(5): e1000140.
[24]	Liu Y, Fang J, Xu F, et al. Expression patterns of ABA and GA metabolism genes and hormone levels during rice seed development and imbibition: a comparison of dormant and non-dormant rice cultivars[J]. J Genet Genomics, 2014, 41(6): 327-338.
[25]	Huang YS, Song JW, Hao QX, et al. WEAK SEED DORMANCY 1, an aminotransferase protein, regulates seed dormancy in rice through the GA and ABA pathways[J]. Plant Physiol Biochem, 2023, 202: 107923.
[26]	He YG, Zhu MH, Li ZH, et al. IPA1 negatively regulates early rice seedling development by interfering with starch metabolism via the GA and WRKY pathways[J]. Int J Mol Sci, 2021, 22(12): 6605.
[27]	Liu HL, Huang JQ, Zhang XJ, et al. The RAC/ROP GTPase activator OsRopGEF10 functions in crown root development by regulating cytokinin signaling in rice[J]. Plant Cell, 2023, 35(1): 453-468.
[28]	Feng QN, Kang H, Song SJ, et al. Arabidopsis RhoGDIs are critical for cellular homeostasis of pollen tubes[J]. Plant Physiol, 2016, 170(2): 841-856.
[29]	梁卫红, 唐朝荣, 吴乃虎. 两种水稻GDP解离抑制蛋白基因的分离及特征分析[J]. 中国生物化学与分子生物学报, 2004, 20(6): 785-791.
	Liang WH, Tang CR, Wu NH. Isolation and characterization of two GDP dissociation inhibitor genes from Oryza sativa L.[J]. Chin J Biochem Mol Biol, 2004, 20(6): 785-791.
[30]	刘洪梅, 梁卫红, 毕佳佳. 2种水稻OsRhoGDIs基因在幼穗组织中的原位杂交分析[J]. 河南农业科学, 2009, 38(12): 22-25.
	Liu HM, Liang WH, Bi JJ. In situ hybridization analysis of two rice OsRhoGDIs genes in young panicle[J]. J Henan Agric Sci, 2009, 38(12): 22-25.
[31]	王凯婕, 安文静, 刘亚菲, 等. CRISPR/Cas9技术编辑OsRhoGDI2基因导致水稻半矮化[J]. 生物工程学报, 2020, 36(4): 707-715.
	Wang KJ, An WJ, Liu YF, et al. Disruption of OsRhoGDI2 by CRISPR/Cas9 technology leads to semi-dwarf in rice[J]. Chin J Biotechnol, 2020, 36(4): 707-715.
[32]	徐欢, 周涛, 孙悦, 等. 水稻颖壳类病斑突变体glmm1的鉴定与基因定位[J]. 中国水稻科学, 2023, 37(5): 497-506.
	Xu H, Zhou T, Sun Y, et al. Characterization and gene mapping of a glume lesion mimic mutant glmm1 in rice[J]. Chin J Rice Sci, 2023, 37(5): 497-506.
[33]	Cheng YC, Li G, Yin M, et al. Verification and dissection of one quantitative trait locus for grain size and weight on chromosome 1 in rice[J]. Sci Rep, 2021, 11(1): 18252.
[34]	You J, Chen WB, He ZF, et al. DEGENERATED LEMMA(DEL)regulates lemma development and affects rice grain yield[J]. Physiol Mol Biol Plants, 2023, 29(3): 335-347.
[35]	Wu XB, Liu JX, Li DQ, et al. Rice caryopsis development I: dynamic changes in different cell layers[J]. J Integr Plant Biol, 2016, 58(9): 772-785.
[36]	Zhao J, Li WJ, Sun S, et al. The rice small Auxin-up RNA gene OsSAUR33 regulates seed vigor via sugar pathway during early seed germination[J]. Int J Mol Sci, 2021, 22(4): 1562.
[37]	Hu CC, Wu C, Yang MY, et al. Catalase associated with antagonistic changes of abscisic acid and gibberellin response, biosynthesis and catabolism is involved in eugenol-inhibited seed germination in rice[J]. Plant Cell Rep, 2023, 43(1): 10.
[38]	Teshome S, Kebede M. Analysis of regulatory elements in GA2ox, GA3ox and GA20ox gene families in Arabidopsis thaliana: an important trait[J]. Biotechnol Biotechnol Equip, 2021, 35(1): 1603-1612.
[39]	Zhang YJ, Liu X, Su R, et al. 9-cis-epoxycarotenoid dioxygenase 1 confers heat stress tolerance in rice seedling plants[J]. Front Plant Sci, 2022, 13: 1092630.
[40]	Fu K, Song WH, Chen C, et al. Improving pre-harvest sprouting resistance in rice by editing OsABA8ox using CRISPR/Cas9[J]. Plant Cell Rep, 2022, 41(10): 2107-2110.
[41]	Yang B, Chen MM, Zhan CF, et al. Identification of OsPK5 involved in rice glycolytic metabolism and GA/ABA balance for improving seed germination via genome-wide association study[J]. J Exp Bot, 2022, 73(11): 3446-3461.
[42]	Damaris RN, Lin ZY, Yang PF, et al. The rice alpha-amylase, conserved regulator of seed maturation and germination[J]. Int J Mol Sci, 2019, 20(2): 450.
[43]	Liu L, Xia W, Li H, et al. Salinity inhibits rice seed germination by reducing α-amylase activities via decreased bioactive gibberellin content[J]. Front Plant Sci, 2018, 9: 275.

引物名称 Primer name	引物序列 Primer sequence（5'-3'）		用途 Purpose
OsRhoGDI1-CDS	F1: GAGGTACCCGGGGATCC TCTAGA ATGTCGTCGGCGGTGGATG	R1: ATTAAAGCAGGGCATGC CTGCAG TCAGCTCAACGCCGGCCACTCTC	OsRhoGDI1过表达载体构建及转基因水稻鉴定引物 Primers for the vector construction of OsRhoGDI1 overexpression and for validation of transgenic rice
Hyg	F2:ACGGTGTCGTCCATCACAGT TTGCC	R2:TTCCGGAAGTGCTTGACATT GGGGA
OsRhoGDI1-PCR	F3: GCCATTGCTGGATCCAACC	R3: CCTCCTACGGAGTTCAAATCCA
OsRhoGDI1-qPCR	F4: TCAAGGAGGGCTCCCTCTAC	R4: GAGCATCTCCTTGTGGCTGT
OsAct1-qPCR	F5: CATGCTATCCCTCGTCTCGACCT	R5: CGCACTTCATGATGGAGTTGTAT
GW2	F6: TTTTCAGTGCCGTCATACC	R6: TTTTCAGTGCCGTCATACC	粒型调控基因表达水平检测引物 Primers for detecting the expressions of grain-shape-regulating genes
GW8	F7: AGGAGTTTGATGAGGCCAAG	R7: GCGTGTAGTATGGGCTCTCC
GS2	F8: TGCGTCCCTTCTTTGATGAGT	R8: ACAGTTGGGTGCCTGAGAATG
GS5	F9: TTTGGCTGAGTATGCCTGGAGCA TCTGCACA	R9: ATTTGCGAAGAATGCACGAT TATGCTGGAA
GS6	F10: TGCGGATACTCAACGCCATCA	R10: ACTCGCCGACTCCGGTGATC
GLW7	F11: TATCCCTTTCAACCTTTTCCA	R11: GACGACGAGCTAGTGCTACTGT
OsGA20ox1	F12: TTCTTCCTCTGCCCGGAGAT	R12: CATGTCGGCCCTGTAGTGG	GA、ABA代谢基因表达水平检测引物 Primers for detecting the expressions of metabolic gene GA and ABA
OsGA3ox2	F13: CTTCTGTGACGTGATGGAGGAG	R13:CTCAAGAACAACCTCAGCAACTC
OsGA2ox3	F14: TCGTTGCAGGTTCTGACCAA	R14: TGGCAATGGTGCAATCCTCT
OsGA2ox8	F15: GCATGAATCGCAGGAGATCG	R15: CCACGTCTTGTGCTGGCTAT
OsNCED5	F16:ACATCCGAGCTCCTCGTCGTGAA	R16:TTGGAAGGTGTTTTGGAATGAACCA
OsABA8ox2	F17: CTACTGCTGATGGTGGCTGA	R17: CCCATGGCCTTTGCTTTAT
OsABA1	F18: GAGTTGGTGGGAGATTCTTCAT	R18: CAGCTTAACGGTCTTCCTTCT
OsAmy1A	F19: TTTCGGTCCTCATCGTCCTCC	R19: TCCACGACTCCCAGTTGAATC	α-amylase基因表达水平检测引物 Primers for detecting the expression of α-amylase genes
OsAmy2A	F20: CAGGGGTTCAACTGGGAGTC	R20: CATGTACCCTTGCGTGGAGA
OsAmy3A	F21: CTCTTCCAGGGTTTTAACTGGGA	R21: GCATGCTACAAAGAGAAGCGT
OsAmy3E	F22: TCACCCTGTGTTGTGTCGTT	R22: AAAGTTGTACCACCCGCCTT

引物名称 Primer name	引物序列 Primer sequence（5'-3'）		用途 Purpose
OsRhoGDI1-CDS	F1: GAGGTACCCGGGGATCC TCTAGA ATGTCGTCGGCGGTGGATG	R1: ATTAAAGCAGGGCATGC CTGCAG TCAGCTCAACGCCGGCCACTCTC	OsRhoGDI1过表达载体构建及转基因水稻鉴定引物 Primers for the vector construction of OsRhoGDI1 overexpression and for validation of transgenic rice
Hyg	F2:ACGGTGTCGTCCATCACAGT TTGCC	R2:TTCCGGAAGTGCTTGACATT GGGGA
OsRhoGDI1-PCR	F3: GCCATTGCTGGATCCAACC	R3: CCTCCTACGGAGTTCAAATCCA
OsRhoGDI1-qPCR	F4: TCAAGGAGGGCTCCCTCTAC	R4: GAGCATCTCCTTGTGGCTGT
OsAct1-qPCR	F5: CATGCTATCCCTCGTCTCGACCT	R5: CGCACTTCATGATGGAGTTGTAT
GW2	F6: TTTTCAGTGCCGTCATACC	R6: TTTTCAGTGCCGTCATACC	粒型调控基因表达水平检测引物 Primers for detecting the expressions of grain-shape-regulating genes
GW8	F7: AGGAGTTTGATGAGGCCAAG	R7: GCGTGTAGTATGGGCTCTCC
GS2	F8: TGCGTCCCTTCTTTGATGAGT	R8: ACAGTTGGGTGCCTGAGAATG
GS5	F9: TTTGGCTGAGTATGCCTGGAGCA TCTGCACA	R9: ATTTGCGAAGAATGCACGAT TATGCTGGAA
GS6	F10: TGCGGATACTCAACGCCATCA	R10: ACTCGCCGACTCCGGTGATC
GLW7	F11: TATCCCTTTCAACCTTTTCCA	R11: GACGACGAGCTAGTGCTACTGT
OsGA20ox1	F12: TTCTTCCTCTGCCCGGAGAT	R12: CATGTCGGCCCTGTAGTGG	GA、ABA代谢基因表达水平检测引物 Primers for detecting the expressions of metabolic gene GA and ABA
OsGA3ox2	F13: CTTCTGTGACGTGATGGAGGAG	R13:CTCAAGAACAACCTCAGCAACTC
OsGA2ox3	F14: TCGTTGCAGGTTCTGACCAA	R14: TGGCAATGGTGCAATCCTCT
OsGA2ox8	F15: GCATGAATCGCAGGAGATCG	R15: CCACGTCTTGTGCTGGCTAT
OsNCED5	F16:ACATCCGAGCTCCTCGTCGTGAA	R16:TTGGAAGGTGTTTTGGAATGAACCA
OsABA8ox2	F17: CTACTGCTGATGGTGGCTGA	R17: CCCATGGCCTTTGCTTTAT
OsABA1	F18: GAGTTGGTGGGAGATTCTTCAT	R18: CAGCTTAACGGTCTTCCTTCT
OsAmy1A	F19: TTTCGGTCCTCATCGTCCTCC	R19: TCCACGACTCCCAGTTGAATC	α-amylase基因表达水平检测引物 Primers for detecting the expression of α-amylase genes
OsAmy2A	F20: CAGGGGTTCAACTGGGAGTC	R20: CATGTACCCTTGCGTGGAGA
OsAmy3A	F21: CTCTTCCAGGGTTTTAACTGGGA	R21: GCATGCTACAAAGAGAAGCGT
OsAmy3E	F22: TCACCCTGTGTTGTGTCGTT	R22: AAAGTTGTACCACCCGCCTT

Agronomic traits	WT	T₁-2	T₁-8	T₁-24
株高Plant height/cm	94.68±0.97	92.76±0.96	89.59±0.80**	92.89±0.61
穗长Panicle length/cm	20.27±0.68	20.67±0.73	20.43±0.75	19.47±0.38
一级枝梗数Number of primary branches	10.33±0.88	10.67±0.88	10.33±1.20	10.33±1.45
粒长Grain length/mm	7.49±0.02	7.63±0.02**	7.61±0.01**	7.65±0.01**
粒宽Grain width/mm	3.38±0.01	3.28±0.01**	3.22±0.01**	3.27±0.01**
粒厚Grain thickness/mm	2.27±0.01	2.21±0.02**	2.16±0.01**	2.19±0.02**
千粒重1000-grain weight/g	26.88±0.12	25.25±0.02**	24.37±0.05**	25.64±0.3**

Agronomic traits	WT	T₁-2	T₁-8	T₁-24
株高Plant height/cm	94.68±0.97	92.76±0.96	89.59±0.80**	92.89±0.61
穗长Panicle length/cm	20.27±0.68	20.67±0.73	20.43±0.75	19.47±0.38
一级枝梗数Number of primary branches	10.33±0.88	10.67±0.88	10.33±1.20	10.33±1.45
粒长Grain length/mm	7.49±0.02	7.63±0.02**	7.61±0.01**	7.65±0.01**
粒宽Grain width/mm	3.38±0.01	3.28±0.01**	3.22±0.01**	3.27±0.01**
粒厚Grain thickness/mm	2.27±0.01	2.21±0.02**	2.16±0.01**	2.19±0.02**
千粒重1000-grain weight/g	26.88±0.12	25.25±0.02**	24.37±0.05**	25.64±0.3**