Overexpression of RGG2, a Heterotrimeric G Protein γ Subunit-Encoding Gene, Improves Drought Tolerance in Rice

doi:10.16819/j.1001-7216.2024.230907

Abstract

Abstract:

【Objective】To investigate the role of RGG2, a heterotrimeric G protein γ subunit, in improving drought tolerance in rice.【Methods】The interaction between RGG2 and RGB1 was characterized using the yeast two-hybrid system and split luciferase complementation assays. The expression level of RGG2 and seed germination rates of transgenic lines were examined under exogenous ABA treatment conditions to determine whether RGG2 was involved in ABA response. The role of RGG2 in rice drought tolerance was analyzed by comparing the water loss rates of detached leaves and plant survival rates between wild-type and overexpression lines of RGG2 after drought treatment.【Results】RGG2 physically interacts with RGB1. The expression level of RGG2 was significantly induced by ABA, PEG-6000, and drought treatments. Overexpression lines of RGG2 in the backgrounds of Nipponbare and Wuyunjing 7 exhibited significantly lower seed germination and root length than the wild-type under ABA treatment condition, suggesting that RGG2 positively regulates ABA response. Compared with the wild-type, overexpression lines of RGG2 showed lower water loss of detached leaves and a higher survival ratio under drought treatment. After drought treatment, the expression levels of several ABA- and drought stress-related genes in the overexpression lines of RGG2 were higher than that in the wild-type.【Conclusion】RGG2 positively regulates ABA and drought stress responses, and overexpression of RGG2 improves drought tolerance in rice.

Key words: rice, heterotrimeric G protein, RGG2, ABA responses, drought stress

摘要：

【目的】探究水稻异三聚体G蛋白γ亚基RGG2在提高水稻抗旱性中的作用。【方法】利用酵母双杂交和荧光素酶互补实验，鉴定RGG2与RGB1的相互作用。施加外源ABA，检测RGG2的表达水平和RGG2过量表达系的种子萌发率，用于阐明RGG2是否参与ABA响应。通过比较野生型和RGG2过量表达系的离体叶片失水率和干旱处理后的植株存活率，解析RGG2在干旱胁迫响应中的作用。【结果】RGG2与RGB1之间存在相互作用。RGG2的表达水平可以被ABA、PEG-6000和干旱处理显著诱导。ABA处理条件下，日本晴和武运粳7号背景下RGG2过量表达系种子萌发率和根长均明显下降，且显著低于野生型，表明RGG2正调控ABA响应。过量表达系离体叶片的失水率低于野生型，但干旱条件下的植株存活率高于野生型。干旱处理条件下，多个ABA和干旱胁迫响应相关基因的表达被诱导，并且在过量表达系中的表达水平明显高于野生型。【结论】RGG2正调控ABA和干旱胁迫响应，过量表达RGG2可以提高水稻抗旱性。

关键词: 水稻, 异三聚体G蛋白, RGG2, ABA响应, 干旱胁迫

MIAO Jun, RAN Jinhui, XU Mengbin, BO Liubing, WANG Ping, LIANG Guohua, ZHOU Yong. Overexpression of RGG2, a Heterotrimeric G Protein γ Subunit-Encoding Gene, Improves Drought Tolerance in Rice[J]. Chinese Journal OF Rice Science, 2024, 38(3): 246-255.

缪军, 冉金晖, 徐梦彬, 卜柳冰, 王平, 梁国华, 周勇. 过量表达异三聚体G蛋白γ亚基基因RGG2提高水稻抗旱性[J]. 中国水稻科学, 2024, 38(3): 246-255.

Figures/Tables 7

Table 1. Primers used in this study

引物名称	前引物	后引物
Primer name	Forward (5’-3’)	Reverse (5’-3’)
RGG2-BD	AAAGAATTCATGAGGGGGGAGGCGAACGGGGAGG	AAAGGATCCCTAGGAAAAATCTGAGCCTTTGGATGCC
RGG2-AD	gccatggaggccagtgaattcATGAGGGGGGAGGCGAAC	cagctcgagctcgatggatccCTAGGAAAAATCTGAGCCTTTGGA
RGB1-AD	AAACCCGGGTATGGCGTCCGTGGCGGAGCTCA	AAAGGATCCTCAAACTATTTTCCGGTGTCCGCTGAA
RGB1-BD	atggccatggaggccgaattcATGGCGTCCGTGGCGGAG	ccgctgcaggtcgacggatccTCAAACTATTTTCCGGTGTCCG
RGG2-cLUC	tacgcgtcccggggcggtaccATGAGGGGGGAGGCGAAC	acgaaagctctgcaggtcgacCTAGGAAAAATCTGAGCCTTTGGA
RGB1-nLUC	acgggggacgagctcggtaccATGGCGTCCGTGGCGGAG	cgcgtacgagatctggtcgacAACTATTTTCCGGTGTCCGCT
OsActin-qPCR	GATGACCCAGATCATGTTTG	GGGCGATGTAGGAAAGC
RGG2-qPCR	GCAGGATGAACTGAACGAGC	GGATGCCCACCATTTGTTA
OsLEA3-qPCR	GCCGTGAATGATTTCCCTTTG	CACACCCGTCAGAAATCCTCC
OsRAB16A-qPCR	CATGGACAAGATCAAGGAGAAGC	CTTATTATTCAGGAAGGTGACGTGG
OsNCED4-qPCR	GATTGCACGGCACCTTCATT	CTCTGTAATTTGATTTTTCACTGGCTAAT
OsLIP9-qPCR	TGGAATTTGGAAGTGTTTGGC	CCCACACGAAACACAAACTTC
OsNAC6-qPCR	CGAGAAGACCAACTGGAT	CAACCTGAGGCTGTTCTT
OsABIL3-qPCR	GAGCGGGCAAGGATT	CCGTGGAACGACCATAAC

Fig. 1. Interaction analysis between RGG2 and RGB1 proteins A, Yeast two-hybrid analysis of RGG2 and RGB1. RGG2 was cloned into pGBKT7 (BD) or pGADT7 (AD) vectors, respectively. RGB1 was cloned into AD and BD vectors, respectively. Yeast was grown on the medium lacking Trp, Leu, His, and Ade to analyze protein interactions; B, Split-LUC analysis of RGG2 and RGB1. RGG2 was linked to the C-terminal fragment of luciferase (cLUC) and RGB1 was linked to the N-terminal fragment of luciferase (nLUC), respectively. Luciferase activity was detected after infestation of N. benthamiana leaves for 2 days.

Fig. 2. ABA, PEG-6000 and drought treatment induce the expression of RGG2 A, The expression levels of RGG2 after the treatment with ABA at various concentrations. 2-week-old Nipponbare seedlings were treated with 10 and 100 μmol/L ABA, then the samples were collected at 6 h after treatment for expression analysis; B, The expression levels of RGG2 at different times after treatment with and without 100 μmol/L ABA. C, The expression levels of RGG2 at different times after treatment with and without 20% PEG-6000. D, The expression levels of RGG2 at different times after drought treatment. Values represent the mean ± SD (n=3). t-test: ns, Not significant; *, P ≤ 0.05;**, P ≤ 0.01.

Fig. 3. Overexpression of RGG2 in the Nipponbare background increases rice sensitivity to ABA A, Seed germination of NIP and RGG2 overexpression lines exposed to different concentrations of ABA. Scale bars = 1 cm. B-F, Seed germination rates of NIP and RGG2 overexpression lines under 0 μmol/L (B), 1 μmol/L (C), 2 μmol/L (D), 5 μmol/L (E), 10 μmol/L (F) ABA treatment. G, Seed germination rates of NIP and RGG2 overexpression lines with various ABA concentrations treatment for 84 h. H, Root length of NIP and RGG2 overexpression lines under different concentrations of ABA. Values are mean ± SD (n = 4). t-test: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01. NIP, Nipponbare. NIP-OE1 and NIP-OE2 represent the overexpression lines of RGG2 in the background of Nipponbare.

Fig. 4. Overexpression of RGG2 in the Wuyunjing 7 background increases rice sensitivity to ABA A, Seed germination of WYJ7 and RGG2 overexpression lines (WYJ7-OE1, WYJ7-OE2) at different concentrations of ABA. Scale bars = 1 cm. B-F, Seed germination rates of WYJ7 and RGG2 overexpression lines under 0 μmol/L (B), 1 μmol/L (C), 2 μmol/L (D), 5 μmol/L (E), 10 μmol/L (F) ABA treatment. G, Seed germination rates of WYJ7 and RGG2 overexpression lines under various ABA concentrations treatment for 84h. H, Root length of WYJ7 and RGG2 overexpression lines at different concentrations of ABA. Values represent the mean ± SD (n = 4). t-test: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01. WYJ7, Wuyunjing 7. WYJ7-OE1 and WYJ7-OE2 represent the overexpression lines of RGG2 in the background of Wuyunjing 7.

Fig. 5. Overexpression of RGG2 increases the tolerance to drought in rice A, The 3-week-old plants of NIP and overexpression lines of RGG2 grown under normal conditions. Scale bar = 5 cm. B, The plants of NIP and overexpression lines of RGG2 after drought treatment for 4 days. Scale bar = 5 cm. C, The plants of NIP and overexpression lines of RGG2 after drought treatment for 6 days and rewatering for 1 day. Scale bar = 5 cm. D, Survival rates of NIP and overexpression lines of RGG2 after drought treatment and rewatering. Values represent mean ± SD (n = 3). E, Water loss rates of detached leaves of NIP and overexpression lines of RGG2. Values represent mean ± SD (n = 10). t-test: **, P ≤ 0.01. NIP, Nipponbare. NIP-OE1 and NIP-OE2 represent the overexpression lines of RGG2 in the background of Nipponbare.

Fig. 6. RGG2 promotes the expression of ABA- and drought stress-related genes The expression levels of OsLEA3, OsRAB16A, OsNCED4, OsLIP9, OsNAC6, and OsABIL3 were detected in NIP and overexpression lines of RGG2 under drought treatment for 0, 4, and 6 days, respectively. Values represent mean ± SD (n = 3). t-test: ns, not significant; **, P ≤ 0.01. NIP, Nipponbare. NIP-OE1 and NIP-OE2 represent the overexpression lines of RGG2 in the background of Nipponbare.

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