Comparative Proteomics Analysis and Identification of Phosphorylated Protein in Latex of Rubber Tree Clones PR107 and CATAS8-79

doi:10.3969/j.issn.1000-2561.2022.05.004

Abstract

Abstract:

Hevea brasiliensis is an important plant for producing natural rubber. RP107 and CATAS8-79 are two clones of H. brasiliensis with different properties of rubber production and expulsion. The study of protein function in the latex may help understand the regulatory mechanism related to the properties of rubber production and expulsion. This study aimed to compare and analyze the difference in latex protein between RP107 and CATAS8-79 at the level of protein accumulation and post-translational modification. Through the two-dimensional gel electrophoresis (2-DE) analysis, 65 proteins derived from 88 spots were found to be accumulated differently in the latex between the 2 clones. Among the proteins, 44 proteins had high accumulation in the latex of PR107 and 21 had high accumulation in the latex of CATAS8-79. The high-accumulation proteins (HAPs) in the latex of CATAS8-79 were involved in intracellular organelles, external encapsulating structure, and membrane-bound organelles in terms of cellular component, and most of them had drug-binding activity and hydrolase activity. Different from CATAS8-79, the HAPs in the latex of PR107 participated in a catalytic complex, nonmembrane-bound organelles, and apoplasts. Most of them had protein-binding and transferase activities. Furthermore, some proteins related to natural rubber synthesis and latex agglutination were found in DAPs. The rubber elongation factors (REFs) and small rubber particle proteins (SRPPs) were identified. The two classes of proteins played an important role in natural rubber biosynthesis in rubber trees. Some proteins mediating rubber particle aggregation (RPA) and participating in response to tapping were found in DAPs too. To determinate the phosphorylated proteins and amino acids in the latex of the two clones, the phosphopeptides were enriched using a Fe-NTA Phosphopeptide Enrichment Kit and shotgun analysis was performed through the high-throughput Tandem Mass Spectrometer (MS/MS). 74 phosphorylated amino acid residues derived from 31 phosphorylated proteins, as well as 166 phosphorylated amino acid residues derived from 80 phosphorylated proteins, were identified in PR107 and CATAS8-79, respectively. Among the phosphorylated proteins, 25 proteins of PR107 and 74 proteins of CATAS8-79 were specific in terms of the phosphorylation amino acids. Between the two clones, the members of REF/SRPP protein family, which regulate the synthesis of nature rubber (NR) in the latex, have high differentiation capacity at both protein accumulation and phosphorylation modification levels. The pro-hevein and hevamine proteins, which influence the process of rubber expulsion, also showed diversity at the phosphorylation modification level. The phosphorylation and dephosphorylation of an serine/threonine protein phosphatase kinase might play a regulatory role in NR synthesis. The results could provide a new theoretical basis for the study of the regulatory mechanism of NR biosynthesis.

Key words: Hevea brasiliensis, natural rubber biosynthesis, phosphoproteome, two-dimensional gel electrophoresis (2-DE)

CLC Number:

S794.1

WANG Dan, XU Bingqiang, SUN Yong, PENG Cunzhi, CHANG Lili, TONG Zheng. Comparative Proteomics Analysis and Identification of Phosphorylated Protein in Latex of Rubber Tree Clones PR107 and CATAS8-79[J]. Chinese Journal of Tropical Crops, 2022, 43(5): 904-914.

Figures/Tables 6

References 37

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点编号 Spots No.	蛋白名称 Protein name	得分 Score	等电点 pI	分子量MW/Da	差异比值 Relative V.R.	P值 P value
1	heat shock 70 kDa protein 8-like	63	5.38	62 933	0.2792	0.0021
13	rubber elongation factor protein-like isoform X1 (REF258)	351	5.21	25 299	0.4661	0.0148
14	rubber elongation factor protein-like isoform X1 (REF258)	218	5.21	25 299	0.2568	0.0001
18	rubber elongation factor protein-like isoform X1 (REF258)	379	5.21	25 299	0.2990	0.0028
19	rubber elongation factor protein-like (REF175)	48	5.28	19 612	0.4933	0.0226
24	rubber elongation factor protein-like isoform X1 (REF258)	205	5.21	25 299	0.4276	0.0211
26	heat shock cognate 70 kDa protein 2 isoform X1	109	5.14	71 451	2.3072	0.0411
28	heat shock cognate 70 kDa protein 2 isoform X1	236	5.14	71 451	2.7902	0.0047
41	acetyl-CoA acetyltransferase, cytosolic 1	598	6.01	41 646	2.1497	0.0026
43	acetyl-CoA acetyltransferase, cytosolic 1	525	6.01	41 646	2.5920	0.0210
62	REF/SRPP-like protein At3g05500 (SRPP243)	155	6.36	27 100	2.7989	0.0161
67	rubber elongation factor protein-like isoform X1 (REF258)	303	5.21	25 299	4.5117	0.0037
68	rubber elongation factor protein-like isoform X1 (REF258)	350	5.21	25 299	2.5995	0.0266
73	rubber elongation factor protein-like isoform X1 (REF258)	67	5.21	25 299	2.5393	0.0213
74	small rubber particle protein isoform X1 (SRPP204)	196	4.80	22 331	4.4673	0.0149
75	small rubber particle protein isoform X1 (SRPP204)	265	4.80	22 331	2.1722	0.0404
77	rubber elongation factor protein-like (REF175)	154	5.28	19 612	54.2933	0.0003
78	rubber elongation factor protein-like (REF175)	120	5.28	19 612	30.0239	0.0003
85	pro-hevein	48	5.89	23 154	3.0820	0.0030

点编号 Spots No.	蛋白名称 Protein name	得分 Score	等电点 pI	分子量MW/Da	差异比值 Relative V.R.	P值 P value
1	heat shock 70 kDa protein 8-like	63	5.38	62 933	0.2792	0.0021
13	rubber elongation factor protein-like isoform X1 (REF258)	351	5.21	25 299	0.4661	0.0148
14	rubber elongation factor protein-like isoform X1 (REF258)	218	5.21	25 299	0.2568	0.0001
18	rubber elongation factor protein-like isoform X1 (REF258)	379	5.21	25 299	0.2990	0.0028
19	rubber elongation factor protein-like (REF175)	48	5.28	19 612	0.4933	0.0226
24	rubber elongation factor protein-like isoform X1 (REF258)	205	5.21	25 299	0.4276	0.0211
26	heat shock cognate 70 kDa protein 2 isoform X1	109	5.14	71 451	2.3072	0.0411
28	heat shock cognate 70 kDa protein 2 isoform X1	236	5.14	71 451	2.7902	0.0047
41	acetyl-CoA acetyltransferase, cytosolic 1	598	6.01	41 646	2.1497	0.0026
43	acetyl-CoA acetyltransferase, cytosolic 1	525	6.01	41 646	2.5920	0.0210
62	REF/SRPP-like protein At3g05500 (SRPP243)	155	6.36	27 100	2.7989	0.0161
67	rubber elongation factor protein-like isoform X1 (REF258)	303	5.21	25 299	4.5117	0.0037
68	rubber elongation factor protein-like isoform X1 (REF258)	350	5.21	25 299	2.5995	0.0266
73	rubber elongation factor protein-like isoform X1 (REF258)	67	5.21	25 299	2.5393	0.0213
74	small rubber particle protein isoform X1 (SRPP204)	196	4.80	22 331	4.4673	0.0149
75	small rubber particle protein isoform X1 (SRPP204)	265	4.80	22 331	2.1722	0.0404
77	rubber elongation factor protein-like (REF175)	154	5.28	19 612	54.2933	0.0003
78	rubber elongation factor protein-like (REF175)	120	5.28	19 612	30.0239	0.0003
85	pro-hevein	48	5.89	23 154	3.0820	0.0030

磷酸化蛋白编号 The Phosphorylated protein number	序列登陆号 Accession #	蛋白名称 Protein name	磷酸化氨基酸位点 Phosphorylation amino acid sites
磷酸化蛋白编号 The Phosphorylated protein number	序列登陆号 Accession #	蛋白名称 Protein name	PR107	CATAS8-79
1	ref\|XP_021671430.1	hevamine-A-like	157 T	157 T
2	ref\|XP_021641666.1	hsp70-Hsp90 organizing protein 3-like	120 S, 122 S	122 S
3	ref\|XP_021653585.1	stress-related protein-like	3 S, 8 S, 10 T, 18 T, 20T, 21 S	8 S, 10 T, 20 T, 21 S
4	ref\|XP_021653593.1	rubber elongation factor protein-like isoform X1 (REF 258)	9 S, 49 S, 71 S, 74 S	3 S, 9 S, 71 S, 74 S
5	ref\|XP_021653597.1	small rubber particle protein isoform X1 (SRPP204)	184 S, 185 S	26 S, 184 S, 185 S
磷酸化蛋白编号 The Phosphorylated protein number	序列登陆号 Accession #	蛋白名称 Protein name	磷酸化氨基酸位点 Phosphorylation amino acid sites
磷酸化蛋白编号 The Phosphorylated protein number	序列登陆号 Accession #	蛋白名称 Protein name	PR107	CATAS8-79
6	ref\|XP_021653602.1	rubber elongation factor protein (REF138)	4 D, 6 D, 80 D, 82 T, 100 D, 102 S, 107 S, 118 S, 121 S, 122 S, 127 T	4 D, 6 D, 82 T, 102 S, 118 S, 121 S, 122 S, 127 T
7	ref\|XP_021672285.1	pro-hevein-like	155 D, 158 S, 196 S	149 T, 155 D, 158 S
8	ref\|XP_021682526.1	pleckstrin homology domain-containing protein 1-like	130 S	130 S, 132 T
9	ref\|XP_021690193.1	SNF1-related protein kinase regulatory subunit gamma-1	19 S, 43 S	-
10	ref\|XP_021653583.1	stress-related protein-like	-	27 S
11	ref\|XP_021653604.1	small rubber particle protein-like isoform X2 (SRPP117)	-	45 S
12	ref\|XP_021659098.1	pleckstrin homology domain-containing protein 1-like	-	130 S, 132 T
13	ref\|XP_021659443.1	serine/threonine-protein kinase tricorner-like isoform X1	-	34 S, 157 S, 312 S, 323 T
14	ref\|XP_021662186.1	REF/SRPP-like protein At3g05500 (SRPP243)	-	109 S
15	ref\|XP_021684968.1	serine/threonine protein phosphatase 2A regulatory subunit B''beta-like	-	118 S
16	ref\|XP_021686529.1	3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR)	-	20 S

磷酸化蛋白编号 The Phosphorylated protein number	序列登陆号 Accession #	蛋白名称 Protein name	磷酸化氨基酸位点 Phosphorylation amino acid sites
磷酸化蛋白编号 The Phosphorylated protein number	序列登陆号 Accession #	蛋白名称 Protein name	PR107	CATAS8-79
1	ref\|XP_021671430.1	hevamine-A-like	157 T	157 T
2	ref\|XP_021641666.1	hsp70-Hsp90 organizing protein 3-like	120 S, 122 S	122 S
3	ref\|XP_021653585.1	stress-related protein-like	3 S, 8 S, 10 T, 18 T, 20T, 21 S	8 S, 10 T, 20 T, 21 S
4	ref\|XP_021653593.1	rubber elongation factor protein-like isoform X1 (REF 258)	9 S, 49 S, 71 S, 74 S	3 S, 9 S, 71 S, 74 S
5	ref\|XP_021653597.1	small rubber particle protein isoform X1 (SRPP204)	184 S, 185 S	26 S, 184 S, 185 S
磷酸化蛋白编号 The Phosphorylated protein number	序列登陆号 Accession #	蛋白名称 Protein name	磷酸化氨基酸位点 Phosphorylation amino acid sites
磷酸化蛋白编号 The Phosphorylated protein number	序列登陆号 Accession #	蛋白名称 Protein name	PR107	CATAS8-79
6	ref\|XP_021653602.1	rubber elongation factor protein (REF138)	4 D, 6 D, 80 D, 82 T, 100 D, 102 S, 107 S, 118 S, 121 S, 122 S, 127 T	4 D, 6 D, 82 T, 102 S, 118 S, 121 S, 122 S, 127 T
7	ref\|XP_021672285.1	pro-hevein-like	155 D, 158 S, 196 S	149 T, 155 D, 158 S
8	ref\|XP_021682526.1	pleckstrin homology domain-containing protein 1-like	130 S	130 S, 132 T
9	ref\|XP_021690193.1	SNF1-related protein kinase regulatory subunit gamma-1	19 S, 43 S	-
10	ref\|XP_021653583.1	stress-related protein-like	-	27 S
11	ref\|XP_021653604.1	small rubber particle protein-like isoform X2 (SRPP117)	-	45 S
12	ref\|XP_021659098.1	pleckstrin homology domain-containing protein 1-like	-	130 S, 132 T
13	ref\|XP_021659443.1	serine/threonine-protein kinase tricorner-like isoform X1	-	34 S, 157 S, 312 S, 323 T
14	ref\|XP_021662186.1	REF/SRPP-like protein At3g05500 (SRPP243)	-	109 S
15	ref\|XP_021684968.1	serine/threonine protein phosphatase 2A regulatory subunit B''beta-like	-	118 S
16	ref\|XP_021686529.1	3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR)	-	20 S