Metabolism of γ-aminobutyrate and 2-acetyl-1-pyrroline Analyses at Various Grain Developmental Stages in Rice (Oryza sativa L.)

doi:10.16819/j.1001-7216.2021.0805

Abstract

Abstract:

【Objective】It is important to reveal the dynamic changes of metabolites and genes involved in γ-aminobutyric acid (GABA) and 2-acetyl-1-pyrroline (2AP) metabolic pathways at various grain developmental stages in scented and non-scented rice cultivars by metabolomes and transcriptomes. The purpose of this study is to lay a theoretical basis for high GABA and 2AP rice breeding. 【Method】The metabolomics and transcriptomics of grains including milky grains (8D, 8 days after flowering), dough grains (15D), mature grains (30D) and milled rice (40D) of scented rice cultivar Meixiangzhan 2(MXZ) along with non-scented rice cultivar Huanghuazhan (HHZ) were performed by LC-MS/MS and RNA-seq, respectively. Finally, the metabolites and transcript profiles of GABA and 2AP metabolic pathways were analyzed. 【Result】The LC-MS/MS-based metabolic PCA analysis illustrated that the 8D and 40D grains of both varieties were dispersedly distributed, while 15D and 30D grains were concentrated in a relatively narrow area. In a total of 623 metabolites were detected in all samples, among which 161 were different metabolites. The contents of nine metabolites, detected in the GABA and 2AP pathways, followed a similar changing trend in all samples. We found that putrescine was located in embryo and aleurone layer, while spermidine was specifically accumulated in the endosperm. About 33 000 genes were identified through the gene expression analysis, among which 14 genes involved in GABA pathway showed high transcriptional levels. These related genes had similar abundance patterns between two varieties, while the Badh2 was the only differentially expressed gene. GAD1, GABA-T1, DAO4 and PAO4 showed higher expression levels as compared with respective homologous genes. The L-arginine and putrescine profiles minimized in HHZ 5D grains, while the polyamine degradation had strong activity in whole MXZ grain development. There were all metabolites required for the 2AP synthesis in the grains, and both polyamine degradation pathway and the glutamate-proline pathway contributed to the accumulation of 2AP in grains. 【Conclusion】The GABA metabolite was mainly degraded, and GABA-T1 and GABA-T2 were responsible for GABA degradation. The metabolites involved in GABA pathway were mainly concentrated in the aleurone layer, and the metabolites and gene expression profiles had similar patterns in both rice varieties. A higher mitochondria activity was found in MXZ grains than HHZ.

Key words: γ-aminobutyric acid, 2-acetyl-1-pyrroline, grain development, fragrant rice, metabolomics

摘要：

【目的】结合代谢组和转录组技术,分析香稻和非香稻不同发育时期籽粒中γ-氨基丁酸（GABA）和2-乙酰-1-吡咯啉（2AP）代谢通路主要化合物和基因的动态变化,为高含量GABA和2AP的水稻育种提供理论依据。【方法】本研究收集优质香籼稻美香占2号和优质籼稻黄华占花后8 d（乳熟期）、花后15 d（蜡熟期）、30 d（完熟期）的脱壳籽粒和花后40 d收获的籽粒的精米,对各样本采用LC-MS/MS和RNA-seq技术进行代谢产物检测和转录表达分析,分析两个水稻品种GABA和2AP代谢通路相关化合物的空间分布以及含量变化特征。【结果】代谢组数据PCA分析表明两个水稻品种乳熟期籽粒和花后40 d收获的籽粒的精米明显分离,蜡熟期和完熟期籽粒分离不明显。代谢组共鉴定出623种代谢物,四个时期差异代谢物合计161个。GABA和2AP通路中9个代谢物被检出,两个水稻品种的相关代谢物变化趋势相似,其中腐胺主要定位于籽粒的糊粉层,而亚精胺在内胚乳特异累积。转录组共检出3.3万个左右基因,其中检出GABA通路18个酶中的14个酶对应的基因有较高的表达水平,除Badh2外其他基因均不是差异表达基因,同一基因在两份水稻材料中的表达水平和变化趋势相似,GAD1、GABA-T1、DAO4和PAO4在相应同源基因中表达水平较高。黄华占籽粒中L-精氨酸和腐胺的代谢在蜡熟期已基本结束,美香占2号在籽粒整个发育期具有持续较强的多胺降解水平。美香占2号籽粒具有合成2AP的物质基础,多胺降解途径和谷氨酸-脯氨酸转化通路同时有助于2AP的积累。【结论】水稻籽粒GABA代谢以降解为主,GABA-T1和GABA-T2共同调控GABA的降解。GABA相关代谢物主要集中在糊粉层,黄华占籽粒中代谢物分布和基因表达特征与美香占2号相似,美香占2号籽粒成熟过程中具有持续较高的线粒体活性。

关键词: γ-氨基丁酸, 2-乙酰-1-吡咯啉, 籽粒发育, 香稻, 代谢组学

Yangyang PAN, Yibo CHEN, Chongrong WANG, Hong LI, Daoqiang HUANG, Degui ZHOU, Zhidong WANG, Lei ZHAO, Rong GONG, Shaochuan ZHOU. Metabolism of γ-aminobutyrate and 2-acetyl-1-pyrroline Analyses at Various Grain Developmental Stages in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2021, 35(2): 121-129.

潘阳阳, 陈宜波, 王重荣, 李宏, 黄道强, 周德贵, 王志东, 赵雷, 龚蓉, 周少川. γ-氨基丁酸和2-乙酰-1-吡咯啉代谢通路在水稻籽粒发育过程中的变化分析[J]. 中国水稻科学, 2021, 35(2): 121-129.

Figures/Tables 6

References 42

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基因名称 Gene name	基因缩写 Gene symbol	基因登录号 Locus ID	HHZ8D	HHZ15D	HHZ30D	MXZ8D	MXZ15D	MXZ30D
谷氨酸脱羧酶	GAD1	LOC_Os08g36320	10.65±0.67	64.70 ±9.21	60.76±3.22	10.41±0.96	48.13±6.90	70.77±18.90
Glutamate decarboxylase	GAD2	LOC_Os04g37500	1.92±0.20	7.98±1.07	6.11±0.67	1.30±0.38	5.86±1.68	7.06±2.38
	GAD3	LOC_Os03g13300	0.29±0.11	0.71±0.04	0.55±0.15	0.16±0.03	0.98±0.92	0.81±0.22
	GAD4	LOC_Os03g51080	3.88±0.41	5.55±1.07	5.06±0.74	5.43±1.03	3.77±0.05	4.50±0.36
	GAD5	LOC_Os04g37460	0.46±0.28	0.12±0.07	0.16±0.13	0.32±0.16	0.25±0.03	0.37±0.30
γ-氨基丁酸转氨酶	GABA-T1	LOC_Os04g52450	44.71±2.12	79.40±4.99	79.46±1.66	31.79±3.80	65.98±6.65	69.10±20.38
γ-aminobutyrate	GABA-T2	LOC_Os08g10510	28.11±0.90	9.01±1.69^*	7.83±1.83	25.10±2.85	21.29±2.84	12.73±1.14
transaminase	GABA-T3	LOC_Os04g52440	0.83±0.20	1.42±0.77	0.41±0.10	1.47±0.34	1.11±0.48	0.59±0.07
	GABA-T4	LOC_Os02g02210	N/A	N/A	N/A	N/A	N/A	N/A
谷氨酸脱氢酶	GDH1	LOC_Os03g58040	17.13±0.80	6.87±0.35	6.22±0.84	13.65±0.85	8.15±1.12	9.07±0.94
Glutamate dehydrogenase	GDH2	LOC_Os04g45970	58.97±2.54	17.06±2.58	20.12±1.04	43.85±1.93	19.51±5.50	13.24±1.93
琥珀酸半醛脱氢酶 Succinic semialdehyde dehydrogenase	SSADH	LOC_Os02g07760	92.65±8.12	105.31±16.3	100.52±3.45	96.75±9.28	102.63±6.72	167.60±46.58
甜菜碱醛脱氢酶2 Betaine aldehyde dehydrogenase 2	BADH2	LOC_Os08g32870	77.12±3.91^*	64.64±3.54^*	62.18±4.62^*	10.32±0.84	9.21±2.19	8.41±1.17
二胺氧化酶	DAO1	LOC_Os06g23114	0.22±0.10	0.17±0.05	0.26±0.09	0.11±0.04	0.10±0.04	0.12±0.07
Diamine oxidase	DAO2	LOC_Os04g20164	3.99±0.75	27.83±4.11	31.22±0.37	2.21±0.38	19.15±0.56	23.76±3.57
	DAO3	LOC_Os04g04950	N/A	N/A	N/A	N/A	N/A	N/A
	DAO4	LOC_Os04g40040	71.02±9.02	159.52±13.5	200.30±5.59	57.67±6.26	116.76±13.03	124.73±13.74
	DAO5	LOC_Os02g37990	0.00	N/A	0.00	0.21±0.12	N/A	0.20±0.26
多胺氧化酶	PAO1	LOC_Os01g51320	1.35±0.26	0.22±0.01	0.20±0.16	1.03±0.38	0.24±0.08	0.66±0.43
Polyamine oxidase	PAO2	LOC_Os03g09810	0.35±0.06	0.08±0.07	0.34±0.24	0.36±0.18	0.26±0.14	0.32±0.20
	PAO3	LOC_Os04g53190	11.90±1.27	28.45±1.63	26.83±1.56	11.26±0.69	22.94±0.81	19.37±4.34
	PAO4	LOC_Os04g57550	60.57±3.81	96.12±6.23	106.53±6.95	55.90±2.42	55.71±3.12	89.17±6.59
	PAO5	LOC_Os04g57560	29.95±0.50	24.23±1.45	26.50±1.55	28.07±0.50	22.78±1.35	25.90±1.94
亚精胺合成酶	SpdS1	LOC_Os07g22600	65.33±7.15	57.42±4.88	41.89±3.78	77.27±11.83	44.77±3.59	45.24±1.23
Spermidine synthase	SpdS2	LOC_Os06g33710	14.53±2.97	37.87±4.49	29.55±3.53	11.78±0.65	25.93±2.67	35.42±11.54
精胺合成酶 Spermine synthase	SpmS2	LOC_Os02g15550	31.20±1.47	32.68±1.65	30.30±1.81	32.98±2.50	32.87±2.56	33.11±2.66
精氨酸酶 Arginase	ARG	LOC_Os04g01590	96.36±4.27	78.83±3.01	80.27±3.07	102.45±3.68	85.64±4.11	76.70±6.07
鸟氨酸脱羧酶 Ornithine decarboxylase	ODC	LOC_Os02g28110	N/A	N/A	N/A	N/A	N/A	N/A
脯氨酸脱氢酶 Proline dehydrogenase	ProDH	LOC_Os10g40360	23.89±5.83	66.88±7.54	78.81±7.47^*	15.76±4.19	49.54±8.94	25.74±4.81
1-吡咯啉-5-羧酸合成酶 1-pyrrolidine-5-carboxylate synthetase	P5CS1	LOC_Os05g38150	43.21±2.08	31.28±1.56	25.46±2.29	41.61±2.96	42.14±2.06	39.42±3.01
1-吡咯啉-5-羧酸还原酶 1-pyrrolidine-5-carboxylic reductase	P5CR	LOC_Os01g71990	9.00±0.43	3.72±0.61	3.59±0.74	10.00±1.01	3.16±0.36	6.13±1.36
1-吡咯啉-5-羧酸脱氢酶 1-pyrrolidine-5-carboxylic dehydrogenase	P5CDH	Os05g0536400	N/A	N/A	N/A	N/A	N/A	N/A
鸟氨酸转氨酶 Ornithine aminotransferase	OAT	LOC_Os03g44150	52.54±0.78	32.10±7.61	25.48±4.34	57.42±4.49	33.33±1.53	38.72±6.02
精氨酸脱羧酶 Arginine decarboxylase	ADC1	LOC_Os06g04070	13.55±1.72	15.43±1.21	18.36±1.78	15.41±3.11	14.40±0.84	19.42±6.76
鲱精胺酶 Agmatine amidinohydrolase	AgmAH	LOC_Os09g37200	1.78±0.11	5.52±1.75	5.33±0.98	3.17±0.32	8.65±0.21	3.78±0.15

基因名称 Gene name	基因缩写 Gene symbol	基因登录号 Locus ID	HHZ8D	HHZ15D	HHZ30D	MXZ8D	MXZ15D	MXZ30D
谷氨酸脱羧酶	GAD1	LOC_Os08g36320	10.65±0.67	64.70 ±9.21	60.76±3.22	10.41±0.96	48.13±6.90	70.77±18.90
Glutamate decarboxylase	GAD2	LOC_Os04g37500	1.92±0.20	7.98±1.07	6.11±0.67	1.30±0.38	5.86±1.68	7.06±2.38
	GAD3	LOC_Os03g13300	0.29±0.11	0.71±0.04	0.55±0.15	0.16±0.03	0.98±0.92	0.81±0.22
	GAD4	LOC_Os03g51080	3.88±0.41	5.55±1.07	5.06±0.74	5.43±1.03	3.77±0.05	4.50±0.36
	GAD5	LOC_Os04g37460	0.46±0.28	0.12±0.07	0.16±0.13	0.32±0.16	0.25±0.03	0.37±0.30
γ-氨基丁酸转氨酶	GABA-T1	LOC_Os04g52450	44.71±2.12	79.40±4.99	79.46±1.66	31.79±3.80	65.98±6.65	69.10±20.38
γ-aminobutyrate	GABA-T2	LOC_Os08g10510	28.11±0.90	9.01±1.69^*	7.83±1.83	25.10±2.85	21.29±2.84	12.73±1.14
transaminase	GABA-T3	LOC_Os04g52440	0.83±0.20	1.42±0.77	0.41±0.10	1.47±0.34	1.11±0.48	0.59±0.07
	GABA-T4	LOC_Os02g02210	N/A	N/A	N/A	N/A	N/A	N/A
谷氨酸脱氢酶	GDH1	LOC_Os03g58040	17.13±0.80	6.87±0.35	6.22±0.84	13.65±0.85	8.15±1.12	9.07±0.94
Glutamate dehydrogenase	GDH2	LOC_Os04g45970	58.97±2.54	17.06±2.58	20.12±1.04	43.85±1.93	19.51±5.50	13.24±1.93
琥珀酸半醛脱氢酶 Succinic semialdehyde dehydrogenase	SSADH	LOC_Os02g07760	92.65±8.12	105.31±16.3	100.52±3.45	96.75±9.28	102.63±6.72	167.60±46.58
甜菜碱醛脱氢酶2 Betaine aldehyde dehydrogenase 2	BADH2	LOC_Os08g32870	77.12±3.91^*	64.64±3.54^*	62.18±4.62^*	10.32±0.84	9.21±2.19	8.41±1.17
二胺氧化酶	DAO1	LOC_Os06g23114	0.22±0.10	0.17±0.05	0.26±0.09	0.11±0.04	0.10±0.04	0.12±0.07
Diamine oxidase	DAO2	LOC_Os04g20164	3.99±0.75	27.83±4.11	31.22±0.37	2.21±0.38	19.15±0.56	23.76±3.57
	DAO3	LOC_Os04g04950	N/A	N/A	N/A	N/A	N/A	N/A
	DAO4	LOC_Os04g40040	71.02±9.02	159.52±13.5	200.30±5.59	57.67±6.26	116.76±13.03	124.73±13.74
	DAO5	LOC_Os02g37990	0.00	N/A	0.00	0.21±0.12	N/A	0.20±0.26
多胺氧化酶	PAO1	LOC_Os01g51320	1.35±0.26	0.22±0.01	0.20±0.16	1.03±0.38	0.24±0.08	0.66±0.43
Polyamine oxidase	PAO2	LOC_Os03g09810	0.35±0.06	0.08±0.07	0.34±0.24	0.36±0.18	0.26±0.14	0.32±0.20
	PAO3	LOC_Os04g53190	11.90±1.27	28.45±1.63	26.83±1.56	11.26±0.69	22.94±0.81	19.37±4.34
	PAO4	LOC_Os04g57550	60.57±3.81	96.12±6.23	106.53±6.95	55.90±2.42	55.71±3.12	89.17±6.59
	PAO5	LOC_Os04g57560	29.95±0.50	24.23±1.45	26.50±1.55	28.07±0.50	22.78±1.35	25.90±1.94
亚精胺合成酶	SpdS1	LOC_Os07g22600	65.33±7.15	57.42±4.88	41.89±3.78	77.27±11.83	44.77±3.59	45.24±1.23
Spermidine synthase	SpdS2	LOC_Os06g33710	14.53±2.97	37.87±4.49	29.55±3.53	11.78±0.65	25.93±2.67	35.42±11.54
精胺合成酶 Spermine synthase	SpmS2	LOC_Os02g15550	31.20±1.47	32.68±1.65	30.30±1.81	32.98±2.50	32.87±2.56	33.11±2.66
精氨酸酶 Arginase	ARG	LOC_Os04g01590	96.36±4.27	78.83±3.01	80.27±3.07	102.45±3.68	85.64±4.11	76.70±6.07
鸟氨酸脱羧酶 Ornithine decarboxylase	ODC	LOC_Os02g28110	N/A	N/A	N/A	N/A	N/A	N/A
脯氨酸脱氢酶 Proline dehydrogenase	ProDH	LOC_Os10g40360	23.89±5.83	66.88±7.54	78.81±7.47^*	15.76±4.19	49.54±8.94	25.74±4.81
1-吡咯啉-5-羧酸合成酶 1-pyrrolidine-5-carboxylate synthetase	P5CS1	LOC_Os05g38150	43.21±2.08	31.28±1.56	25.46±2.29	41.61±2.96	42.14±2.06	39.42±3.01
1-吡咯啉-5-羧酸还原酶 1-pyrrolidine-5-carboxylic reductase	P5CR	LOC_Os01g71990	9.00±0.43	3.72±0.61	3.59±0.74	10.00±1.01	3.16±0.36	6.13±1.36
1-吡咯啉-5-羧酸脱氢酶 1-pyrrolidine-5-carboxylic dehydrogenase	P5CDH	Os05g0536400	N/A	N/A	N/A	N/A	N/A	N/A
鸟氨酸转氨酶 Ornithine aminotransferase	OAT	LOC_Os03g44150	52.54±0.78	32.10±7.61	25.48±4.34	57.42±4.49	33.33±1.53	38.72±6.02
精氨酸脱羧酶 Arginine decarboxylase	ADC1	LOC_Os06g04070	13.55±1.72	15.43±1.21	18.36±1.78	15.41±3.11	14.40±0.84	19.42±6.76
鲱精胺酶 Agmatine amidinohydrolase	AgmAH	LOC_Os09g37200	1.78±0.11	5.52±1.75	5.33±0.98	3.17±0.32	8.65±0.21	3.78±0.15