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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (6): 897-908.doi: 10.3724/SP.J.1006.2018.00897

• TILLAGE & CULTIVATION · PHYSIOLOGY & BIOCHEMISTRY • Previous Articles     Next Articles

Proteomic Analysis of the Effect of 2,4-Epibrassinolide on Rice Seedlings Response to Cold Stress

Dao-Ping WANG1,2,Jiang XU2,Yong-Ying MU2,3,Wen-Xiu YAN2,3,Meng-Jie ZHAO3,Bo MA3,Qun LI1,*(),Li-Na ZHANG2,3,Ying-Hong PAN2,3,*()   

  1. 1 College of Life Science and Technology, Xinjiang University, Urumqi 830046, Xinjiang, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3 National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2017-08-29 Accepted:2018-03-18 Online:2018-06-12 Published:2018-03-19
  • Contact: Qun LI,Ying-Hong PAN E-mail:214800653@qq.com;panyinghong@caas.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31571589);the National Basic Research Program of China (973 Program)(2015CB150401);the Agricultural Science and Technology Innovation Program(作物分子标记技术及其应用创新团队)

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Abstract:

As a plant growth regulator which functionally resembles a kind of plant hormone Brassinosteroids (BRs), 2,4-Epibrassinolide (EBR) has been widely studied and applied in different aspects. EBR can enhance plant’s cold tolerance effectively, but the proteomic characteristics of the effect of EBR on rice seedings response to cold stress are still unclear. In this study, the germinating seeds of rice Nipponbare were treated with 0.1 mg L -1 EBR and distilled water before they were cultivated at 4°C or 26°C, and then the total protein of each group of seedlings was extracted. Finally, proteomes of rice seedings were analyzed by label-free quantitative mass spectrometry, and some important proteins were verified by parallel reaction monitoring technique (PRM). A total of 5778 protein groups were identified by qualitative method and 4834 protein groups were accurately quantitated. Among them, 401 up-regulated and 220 down-regulated proteins were related to the effect of EBR on rice seedings response to cold stress. The up-regulated proteins were mainly related to molecular function of RNA binding and hydrolase activity, and mainly enriched in the pathways of carbon metabolism, folic acid synthesis and amino acid biosynthesis. The down-regulated proteins were mainly related to catalytic activity and oxidoreductase activity, and mainly enriched in the pathways of porphyrin and chlorophyll metabolism and other metabolic pathways. PRM validation and literature analysis showed that NADP-malic acidase, peroxidase, 3-phosphoglycerate dehydrogenase, enolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, which are distributed in the pathways of carbon metabolism and phenylpropanol metabolism and others, take part in the regulation of EBR on rice seedlings response to cold stress, suggesting that BRs can affect rice seedlings response to cold stress through a variety of pathways.

Key words: 2, 4-Epibrassinolide, cold stress, proteomics, rice

Fig. 1

Phenotypes under cold stress and of chlorophyll content in rice A: four groups of sample phenotype; B: comparison of chlorophyll a and chlorophyll b contents in four groups of samples; C: comparison of whole plant weight in four groups of samples, randomly selected 10 seedlings and weighed which was repeated three times and calculate the average value as the whole plant fresh weight of a group. 4B: samples treated with EBR then cultured at 4°C, 4: samples cultured at 4°C, 26B: samples treated with EBR then cultured at 26°C, 26: samples cultured at 26°C. Values within a group followed by a different small letter are significantly different at the 0.05 probability level in B and C."

Fig. 2

Qualitative and quantitative analysis of samples by mass spectrometry A: qualitatively identified proteins and common proteins between samples; B: Pearson correlation analysis between samples; C: signal intensity distribution of quantitative identified proteins."

Fig. 3

Comparison of quantitative protein A: proteins with intensity difference between every two samples; B: Venn diagram shows the 401 up-regulated proteins related to the effect of EBR on rice seedings response to cold stress; C: Venn diagram shows the 220 down-regulated proteins related to the effect of EBR on rice seeding response to cold stress. H means up regulated and L means down regulated."

Fig. 4

Gene ontology analysis of proteins related to the effect of EBR on rice seeding response to cold stress A: gene ontology analysis of 401 up-regulated proteins; B: gene ontology analysis of 220 down-regulated proteins."

Fig. 5

Analysis of KEGG pathways A: KEGG pathways analysis of 401 up-regulated proteins; B: KEGG pathways analysis of 220 down-regulated proteins."

Table 1

Important pathways and enriched proteins related to the effect of EBR on rice seedings response to cold stress"

通路和蛋白
Pathway name and protein IDs		 注解
Annotation		 信号强度Intensity
26 26B 4 4B
碳代谢 Carbon metabolism
LOC_Os01g54030.1 Nadp-dependent malic enzyme 0.00E+00 0.00E+00 0.00E+00 1.78E+07
LOC_Os02g38200.1 Dehydrogenase 3.97E+08 1.78E+08 9.81E+07 2.64E+08
LOC_Os03g15050.2 Phosphoenolpyruvate carboxykinase 1.22E+08 5.70E+07 4.21E+07 1.26E+08
LOC_Os04g24140.1 Ribose-5-phosphate isomerase a 1.10E+08 8.97E+06 1.79E+07 7.07E+07
LOC_Os06g04510.1 Enolase 5.68E+07 1.08E+07 0.00E+00 2.00E+07
LOC_Os06g05700.1 Cysteine synthase 0.00E+00 0.00E+00 0.00E+00 8.73E+06
LOC_Os06g45590.1 Glyceraldehyde-3-phosphate dehydrogenase 0.00E+00 0.00E+00 0.00E+00 3.28E+07
LOC_Os07g09890.1 Hexokinase 2.82E+07 0.00E+00 0.00E+00 2.09E+07
LOC_Os08g02700.1 Fructose-bisphospate aldolase isozyme 3.73E+07 0.00E+00 0.00E+00 8.83E+06
LOC_Os09g24910.2 Phosphofructokinase 0.00E+00 0.00E+00 0.00E+00 2.38E+07
LOC_Os11g10980.1 Pyruvate kinase 0.00E+00 0.00E+00 0.00E+00 1.06E+07
LOC_Os11g41160.3 Phosphoserine phosphatase 2.13E+07 1.00E+07 0.00E+00 2.83E+07
LOC_Os12g05110.1 Pyruvate kinase 2.37E+08 1.29E+08 5.51E+07 2.95E+08
LOC_Os06g35540.1 Aminotransferase 5.93E+07 1.63E+08 1.61E+08 7.89E+07
LOC_Os06g44460.1 D-3-phosphoglycerate dehydrogenase 9.76E+06 7.49E+07 2.85E+07 3.37E+07
LOC_Os05g49760.1 Dehydrogenase 0.00E+00 1.20E+08 1.00E+08 3.93E+07
苯丙素生物合成 Phenylpropanoid biosynthesis
LOC_Os10g17650.1 Os10bglu34—beta-glucosidase homologue 1.02E+08 0.00E+00 7.90E+07 4.76E+08
LOC_Os01g32364.1 Os1bglu1—beta-mannosidase/glucosidase homologue 0.00E+00 0.00E+00 0.00E+00 8.51E+06
LOC_Os01g73200.1 Peroxidase 0.00E+00 0.00E+00 0.00E+00 2.12E+07
LOC_Os02g41680.1 Phenylalanine ammonia-lyase 7.94E+06 0.00E+00 0.00E+00 6.33E+06
LOC_Os04g56180.1 Peroxidase 1.25E+08 2.86E+07 5.14E+07 7.05E+07
LOC_Os03g11420.1
 Os3bglu6—beta-glucosidase/beta-fucosidase/beta-galactosidase 0.00E+00
 2.25E+07
 3.03E+07
 6.87E+06
LOC_Os01g22249.1 Peroxidase 1.24E+07 5.91E+07 2.05E+08 0.00E+00
LOC_Os05g04500.1 Peroxidase 8.07E+06 1.92E+08 5.61E+07 0.00E+00
LOC_Os07g01410.1 Peroxidase 2.01E+07 5.70E+06 8.45E+07 0.00E+00
LOC_Os08g34280.1 Cinnamoyl-coa reductase 0.00E+00 6.70E+06 6.25E+07 0.00E+00
LOC_Os09g33680.1 Os9bglu31—beta-glucosidase, dhurrinase 0.00E+00 4.08E+07 1.92E+07 1.48E+07
卟啉和叶绿素代谢 Porphyrin and chlorophyll metabolism
LOC_Os03g22780.1 DVR 8.72E+07 0.00E+00 4.27E+07 9.48E+07
LOC_Os01g16520.1 Glutamyl-tRNA synthetase 0.00E+00 1.59E+07 1.40E+07 0.00E+00
LOC_Os01g57460.1 Frataxin, putative, expressed 4.04E+07 9.91E+07 1.15E+08 0.00E+00
LOC_Os10g37210.1
 FAD dependent oxidoreductase domain containing
Protein		 2.01E+07
 3.51E+06
 5.45E+07
 0.00E+00
LOC_Os04g41260.1 Amine oxidase 0.00E+00 2.87E+06 1.70E+07 0.00E+00
叶酸生物合成 Folate biosynthesis
LOC_Os11g29390.1
 Bifunctional dihydrofolate reductase-thymidylate
synthase		 4.47E+06
 1.31E+07
 3.66E+06
 0.00E+00
LOC_Os09g38759.1 Dihydroneopterin aldolase 2.42E+07 0.00E+00 5.66E+06 2.25E+07
LOC_Os04g38950.1 Class I glutamine amidotransferase 6.99E+07 0.00E+00 2.89E+07 2.37E+07
LOC_Os03g02030.2 Folylpolyglutamate synthase 0.00E+00 0.00E+00 0.00E+00 2.04E+07
LOC_Os02g35200.1 Vp15 0.00E+00 0.00E+00 0.00E+00 7.22E+06
通路和蛋白
Pathway name and protein IDs		 注解
Annotation		 信号强度Intensity
26 26B 4 4B
不饱和脂肪酸生物合成 Biosynthesis of unsaturated fatty acids
LOC_Os01g65830.1 Acyl-desaturase 6.12E+07 0.00E+00 0.00E+00 8.06E+06
LOC_Os02g48560.6 Fatty acid desaturase 4.69E+08 7.57E+07 8.14E+07 3.83E+08
LOC_Os08g10010.1 Acyl-desaturase 1.38E+08 2.72E+07 4.92E+07 6.52E+07
LOC_Os11g39220.2 Acyl-coenzyme A oxidase 0.00E+00 4.07E+07 3.18E+07 0.00E+00
脂肪酸生物合成 Fatty acid biosynthesis
LOC_Os01g65830.1 Acyl-desaturase 6.12E+07 0.00E+00 0.00E+00 8.06E+06
LOC_Os08g10010.1 Acyl-desaturase 1.38E+08 2.72E+07 4.92E+07 6.52E+07
LOC_Os03g28420.1 3-oxoacyl-synthase 8.02E+07 3.53E+07 0.00E+00 1.01E+08
LOC_Os01g48910.2 Long-chain acyl-coa synthetase 3.27E+07 2.61E+07 4.38E+06 5.34E+07
LOC_Os12g04990.3 Long-chain acyl-coa synthetase 1.80E+07 2.67E+07 6.15E+07 9.72E+06

Fig. 6

Targeted PRM quantification of some proteins with intensity difference A: 3-phosphoglycerate dehydrogenase; B: enolase; C: NADP-dependent malic enzyme; D: glyceraldehyde-3-phosphate dehydrogenase; E: pyruvate kinase; F: peroxidase."

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