Protein Phosphorylation and Phosphoproteome: An Overview of Rice

doi:10.1016/j.rsci.2020.04.003

Abstract

Abstract:

Protein phosphorylation, one of the major post-translational modifications, plays a crucial role in cell signaling, DNA replication, gene expression and differentiation; and alters enzyme activity and other biological activities; and regulates cell proliferation and enlargement, phytohormone biosynthesis and signaling, plant disease resistance, and grain filling and quality during rice seed development. Research work on protein phosphorylation started in the 1950s with the discovery of phosphorylase a and phosphorylase b which are phospho and dephospho forms of the same enzyme. Over the last decade, rice proteomics has accomplished tremendous progress in setting up techniques to proteome nearly all tissues, organs and organelles. The progress made in this field is evident in number of research works. However, research on rice protein phosphorylation is still at its infancy and there are still many unanswered questions. In this review, the general description of protein phosphorylation, including history, structure, frequency of occurrence and function, are discussed. This work also elucidates the different methods for identification, qualification and finally, the progress in rice phosphoproteome research and perspectives.

Key words: phosphoproteome, protein phosphorylation, post-translational modification, rice

Adijat Ajadi Abolore, Cisse Amara, Ahmad Shakeel, Yifeng Wang, Yazhou Shu, Shufan Li, Xixi Liu, Kazeem Bello Babatunde, Muhammad Tajo Sani, Xiaohong Tong, Jian Zhang. Protein Phosphorylation and Phosphoproteome: An Overview of Rice[J]. Rice Science, 2020, 27(3): 184-200.

Figures/Tables 5

References 121

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Treatment/Sample type	Proteomic approach; Subcellular fraction	Number of phosphoproteins and number of sites identified	Reference
GAs, BL, 2,4-D, ABA, cold or salt treatment of rice seedlings	Total protein extracts from leaf sheaths, leaf blades and roots, in vitro ³²P labeling, 2DE; ESI-Q-TOF-MS/MS; MALDI-TOF-MS	42 phosphoproteins identified, among them 13 were stress/hormonal responsive; 8 sites of phosphorylation in 6 phosphoproteins	Khan et al, 2005
Salt treatment of rice roots	Total protein extracts; 2DE; Pro-Q Diamond, SYPRO Ruby; MALDI-TOF/TOF PMF and MS/MS	17 differentially up-regulated and 11 down-regulated phosphoproteins for Pro-Q Diamond dye; 26 up-regulated and 5 down-regulated phosphoproteins for SYPRO Ruby	Chitteti and Peng, 2007
Protoplast isolation from rice cell suspension	Nucleus and chromatin isolation; Total protein extracts; 2D-PAGE; Pro-Q Diamond; MALDI-TOF/TOF MS and LC/LC-MS/MS	972 distinct protein spots and 509 proteins identified, which correspond to 269 unique proteins	Tan et al, 2007
ABA treatment of rice seedlings	Total protein; 2DE; Immunodetection of phospho-Ser and phospho-Thr; MALDI-TOF/TOF MS and MS/MS	6 ABA responsive phosphoproteins out of 28 proteins	He and Li, 2008
Cd stress in leaves and root of rice seedlings	Total protein extracts; 2DE; Colloidal coomassie blue; MALDI-TOF/TOF PMF and MS/MS	13 proteins in the roots and 12 proteins in the leaves	Ge et al, 2009
Drought treatment of rice seedlings	Total protein; 2DE; Immunodetection of phospho-Ser and phospho-Thr; MALDI-TOF/TOF MS and MS/MS	3 drought-responsive differentially expressed proteins and 10 differentially phosphorylated (dp) phosphoproteins	Ke et al, 2009
Unfractionated whole-cell lysates of rice and Arabidopsis cells	Total protein extract; SCX prefractionation; Ti-HAMMOC; Zr-HAMMOC; Fe-IMAC using Phos-select; Nano-LC-MS/MS	6 919 phosphopeptides from 3 393 proteins	Nakagami et al, 2010
Salinity, cold and drought stress	Genome sequences; Microarray gene expression analysis; Online databases and in silico search tools; RT-PCR	132 proteins; 46 phosphatases were differentially expressed in 3 abiotic stresses	Singh et al, 2010
Heat (at 42 ºC) of rice seedlings	Total protein; 2DE; Pro-Q Diamond and coomassie, MALDI-TOF/TOF MS and MS/MS; Immunoblotting of ATP-β	10 dp proteins	Chen et al, 2011
High salinity treatment of A. thaliana and rice plants	Microsomal fractions; Protein digestion; Enrichment using Zr⁴⁺-IMAC and TiO₂ magnetic beads; Q-TOF Nano LC-MS/MS; Label-free quantification on Q-TOF, LTQ-FT and Q-Trap	3 membrane phosphoproteins and 4 nonmembrane phosphoproteins; 15 phosphopeptides from rice microsomes	Chang et al, 2012
Cold stress (6 ºC and 15 ºC) of rice seedlings	Total protein extracts; 2DE; Coomassie brilliant blue; Pro-Q Diamond; MALDI-TOF/TOF MS	12 up-regulated and 1 down-regulated phosphoprotein among 13 phosphoproteins	Chen et al, 2012
ABA treatment of rice inferior spiklets at 20 d after flowering	Total protein extracts; 2DE; Coomassie brilliant blue; Pro-Q Diamond; MALDI-TOF/TOF MS	16 up-regulated and 15 down-regulated among 31 differentially regulated phosphoproteins	Zhang et al, 2012
Rice embryo germination	Total protein extracts; 2DE; Pro-Q Diamond; Coomassie brilliant blue and MALDI-TOF/TOF MS	About 500 phosphorylated protein spots were detected on each gel	Han et al, 2014a
Poor grain-filling of rice inferior spikelets	Total protein extracts; 2DE; Coomassie brilliant blue; Pro-Q Diamond; MALDI-TOF/TOF MS and LC-ESI-MS/MS	123 proteins in abundance and 43 phosphoproteins	Zhang et al, 2014
Rice pistils	Total protein; Fe³⁺-IMAC protein enrichment; HILIC and Nano-LC-MS/MS	2 347 phosphorylation sites and 1 588 phosphoproteins	Wang K et al, 2014
Rice leaves in response to bacterial blight	Total protein; TiO₂ MOAC and LC-MS/MS	2 367 and 2 223 phosphosites on 1 334 and 1 297 proteins in 0 and 24 h after Xoo infection, respectively	Hou et al, 2015
Early stage of seed germination	Isolation of nuclei and extraction of nuclear proteins; Total protein; PolyMAC-Ti and MS/MS	3 467 phosphopeptides and 102 nuclear phosphorproteins from rice embryos; 115 nuclear phosphoproteins identified after 24 h of imbibition	Li M et al, 2015
Early seed development	Total protein; TiO₂ protein enrichment and LC-MS/MS	Pistils and seeds at 3 and 7 d after pollination resulted in 38 854 313 and 4 135 phosphopeptides, respectively. A total of 2487 proteins were dp	Qiu et al, 2015
Magnaporthe oryzae treatment of susceptible and resistant rice cultivars	Total protein extracts; Al(OH)3-MOAC; 2DE; Pro-Q Diamond; MALDI-TOF/TOF PMF and Nano LC-MS/MS	1 522 unique peptides, of which 1 365 were dp	Li Y F et al, 2015
Developing rice anthers	Total protein; TiO2 MOAC and Nano UHPLC-MS/MS	4 984 proteins and 3 203 phosphoproteins with 8 973 unique phosphorylation sites	Ye et al, 2015
Brassinosteroid treatment in rice	Total protein; TiO2 MOAC and LC-MS/MS	4 034 phosphosites on 1 900 phosphoproteins and 1 821 brassinosteroid-responsive proteins	Hou et al, 2017
Callus, leaf, root, shoot meristem, young panicle, and mature panicle	Total protein extracts; Pro-Q diamond; TiO2 enrichment and LC-MS/MS	7 171 unique phosphosites in 4 792 phosphopeptides from 2 657 identified phosphoproteins	Wang et al, 2017
Cd stress in rice seedlings	Total protein; iTRAQ labeling; TiO2 enrichment and LC-MS/MS	2 454 phosphosites associated with 1 244 proteins, among them, 482 were dp	Zhong et al, 2017
Response to Cd stress in rice		32 up-regulated and 1 down-regulated Cd tolerant, and 15 up-regulated and 4 down-regulated Cd sensitive among 53 differentially expressed phosphoproteins	Fang et al, 2019

Treatment/Sample type	Proteomic approach; Subcellular fraction	Number of phosphoproteins and number of sites identified	Reference
GAs, BL, 2,4-D, ABA, cold or salt treatment of rice seedlings	Total protein extracts from leaf sheaths, leaf blades and roots, in vitro ³²P labeling, 2DE; ESI-Q-TOF-MS/MS; MALDI-TOF-MS	42 phosphoproteins identified, among them 13 were stress/hormonal responsive; 8 sites of phosphorylation in 6 phosphoproteins	Khan et al, 2005
Salt treatment of rice roots	Total protein extracts; 2DE; Pro-Q Diamond, SYPRO Ruby; MALDI-TOF/TOF PMF and MS/MS	17 differentially up-regulated and 11 down-regulated phosphoproteins for Pro-Q Diamond dye; 26 up-regulated and 5 down-regulated phosphoproteins for SYPRO Ruby	Chitteti and Peng, 2007
Protoplast isolation from rice cell suspension	Nucleus and chromatin isolation; Total protein extracts; 2D-PAGE; Pro-Q Diamond; MALDI-TOF/TOF MS and LC/LC-MS/MS	972 distinct protein spots and 509 proteins identified, which correspond to 269 unique proteins	Tan et al, 2007
ABA treatment of rice seedlings	Total protein; 2DE; Immunodetection of phospho-Ser and phospho-Thr; MALDI-TOF/TOF MS and MS/MS	6 ABA responsive phosphoproteins out of 28 proteins	He and Li, 2008
Cd stress in leaves and root of rice seedlings	Total protein extracts; 2DE; Colloidal coomassie blue; MALDI-TOF/TOF PMF and MS/MS	13 proteins in the roots and 12 proteins in the leaves	Ge et al, 2009
Drought treatment of rice seedlings	Total protein; 2DE; Immunodetection of phospho-Ser and phospho-Thr; MALDI-TOF/TOF MS and MS/MS	3 drought-responsive differentially expressed proteins and 10 differentially phosphorylated (dp) phosphoproteins	Ke et al, 2009
Unfractionated whole-cell lysates of rice and Arabidopsis cells	Total protein extract; SCX prefractionation; Ti-HAMMOC; Zr-HAMMOC; Fe-IMAC using Phos-select; Nano-LC-MS/MS	6 919 phosphopeptides from 3 393 proteins	Nakagami et al, 2010
Salinity, cold and drought stress	Genome sequences; Microarray gene expression analysis; Online databases and in silico search tools; RT-PCR	132 proteins; 46 phosphatases were differentially expressed in 3 abiotic stresses	Singh et al, 2010
Heat (at 42 ºC) of rice seedlings	Total protein; 2DE; Pro-Q Diamond and coomassie, MALDI-TOF/TOF MS and MS/MS; Immunoblotting of ATP-β	10 dp proteins	Chen et al, 2011
High salinity treatment of A. thaliana and rice plants	Microsomal fractions; Protein digestion; Enrichment using Zr⁴⁺-IMAC and TiO₂ magnetic beads; Q-TOF Nano LC-MS/MS; Label-free quantification on Q-TOF, LTQ-FT and Q-Trap	3 membrane phosphoproteins and 4 nonmembrane phosphoproteins; 15 phosphopeptides from rice microsomes	Chang et al, 2012
Cold stress (6 ºC and 15 ºC) of rice seedlings	Total protein extracts; 2DE; Coomassie brilliant blue; Pro-Q Diamond; MALDI-TOF/TOF MS	12 up-regulated and 1 down-regulated phosphoprotein among 13 phosphoproteins	Chen et al, 2012
ABA treatment of rice inferior spiklets at 20 d after flowering	Total protein extracts; 2DE; Coomassie brilliant blue; Pro-Q Diamond; MALDI-TOF/TOF MS	16 up-regulated and 15 down-regulated among 31 differentially regulated phosphoproteins	Zhang et al, 2012
Rice embryo germination	Total protein extracts; 2DE; Pro-Q Diamond; Coomassie brilliant blue and MALDI-TOF/TOF MS	About 500 phosphorylated protein spots were detected on each gel	Han et al, 2014a
Poor grain-filling of rice inferior spikelets	Total protein extracts; 2DE; Coomassie brilliant blue; Pro-Q Diamond; MALDI-TOF/TOF MS and LC-ESI-MS/MS	123 proteins in abundance and 43 phosphoproteins	Zhang et al, 2014
Rice pistils	Total protein; Fe³⁺-IMAC protein enrichment; HILIC and Nano-LC-MS/MS	2 347 phosphorylation sites and 1 588 phosphoproteins	Wang K et al, 2014
Rice leaves in response to bacterial blight	Total protein; TiO₂ MOAC and LC-MS/MS	2 367 and 2 223 phosphosites on 1 334 and 1 297 proteins in 0 and 24 h after Xoo infection, respectively	Hou et al, 2015
Early stage of seed germination	Isolation of nuclei and extraction of nuclear proteins; Total protein; PolyMAC-Ti and MS/MS	3 467 phosphopeptides and 102 nuclear phosphorproteins from rice embryos; 115 nuclear phosphoproteins identified after 24 h of imbibition	Li M et al, 2015
Early seed development	Total protein; TiO₂ protein enrichment and LC-MS/MS	Pistils and seeds at 3 and 7 d after pollination resulted in 38 854 313 and 4 135 phosphopeptides, respectively. A total of 2487 proteins were dp	Qiu et al, 2015
Magnaporthe oryzae treatment of susceptible and resistant rice cultivars	Total protein extracts; Al(OH)3-MOAC; 2DE; Pro-Q Diamond; MALDI-TOF/TOF PMF and Nano LC-MS/MS	1 522 unique peptides, of which 1 365 were dp	Li Y F et al, 2015
Developing rice anthers	Total protein; TiO2 MOAC and Nano UHPLC-MS/MS	4 984 proteins and 3 203 phosphoproteins with 8 973 unique phosphorylation sites	Ye et al, 2015
Brassinosteroid treatment in rice	Total protein; TiO2 MOAC and LC-MS/MS	4 034 phosphosites on 1 900 phosphoproteins and 1 821 brassinosteroid-responsive proteins	Hou et al, 2017
Callus, leaf, root, shoot meristem, young panicle, and mature panicle	Total protein extracts; Pro-Q diamond; TiO2 enrichment and LC-MS/MS	7 171 unique phosphosites in 4 792 phosphopeptides from 2 657 identified phosphoproteins	Wang et al, 2017
Cd stress in rice seedlings	Total protein; iTRAQ labeling; TiO2 enrichment and LC-MS/MS	2 454 phosphosites associated with 1 244 proteins, among them, 482 were dp	Zhong et al, 2017
Response to Cd stress in rice		32 up-regulated and 1 down-regulated Cd tolerant, and 15 up-regulated and 4 down-regulated Cd sensitive among 53 differentially expressed phosphoproteins	Fang et al, 2019

Approach	Method	Advantage	Disadvantage
	2D-DIGE	Deliver a visual map of all the proteins and allow the fractionation of thousands of proteins in a complex mixture	Relatively small scale, low sensitivity, poor detection of low-abundance phosphoproteins, and insufficient collected proteins for downstream enrichment methods
Stable isotope labeling quantification	SILAC	Lower experimental error	Allow up to only three samples
	HILEP, SILIP, SILIA	Applicable to entire plant and other plant tissues	Only be used in auxotrophic cells
	iTRAQ and TMT	Allow multiplex up to 8 (iTARQ) and 10 (TMT); Labeling agent doesn’t affect overall peptide behavior thus minimizes experimental error	Reduced identification efficiency as much as 50% in multi-stage activation
	Chemical derivatization (DiLeu)	Higher protein coverage and quantification efficiency; Cheaper than iTRAQ and TMT; Provide higher reproducibility in quantitation
Label-free quantification	Signal intensity and spectral counting	Allow as many samples as possible	Time-consuming; Less degree of reproducibility in quantitation
Absolute quantification	AQUA	Allow quantification of isoforms in low abundance and stoichiometry; Achieve accurate and sensitive quantification
	AQUIP	Can determine the absolute amount of protein
2D-DIGE, Two-dimensional differential gel electrophoresis; SILAC, Stable isotope labeling by amino acids in culture; HILEP, Hydroponic isotope labeling of entire plants; SILIP, Stable isotope labeling in planta; SILIA, Stable isotope labeling in Arabidopsis; iTRAQ, Isobaric tags for relative and absolute quantification; TMT, Tandem mass tags; AQUA, Absolute quantification of proteins; AQUIP, Absolute quantitation of isoforms of post-translationally modified proteins.

Approach	Method	Advantage	Disadvantage
	2D-DIGE	Deliver a visual map of all the proteins and allow the fractionation of thousands of proteins in a complex mixture	Relatively small scale, low sensitivity, poor detection of low-abundance phosphoproteins, and insufficient collected proteins for downstream enrichment methods
Stable isotope labeling quantification	SILAC	Lower experimental error	Allow up to only three samples
	HILEP, SILIP, SILIA	Applicable to entire plant and other plant tissues	Only be used in auxotrophic cells
	iTRAQ and TMT	Allow multiplex up to 8 (iTARQ) and 10 (TMT); Labeling agent doesn’t affect overall peptide behavior thus minimizes experimental error	Reduced identification efficiency as much as 50% in multi-stage activation
	Chemical derivatization (DiLeu)	Higher protein coverage and quantification efficiency; Cheaper than iTRAQ and TMT; Provide higher reproducibility in quantitation
Label-free quantification	Signal intensity and spectral counting	Allow as many samples as possible	Time-consuming; Less degree of reproducibility in quantitation
Absolute quantification	AQUA	Allow quantification of isoforms in low abundance and stoichiometry; Achieve accurate and sensitive quantification
	AQUIP	Can determine the absolute amount of protein
2D-DIGE, Two-dimensional differential gel electrophoresis; SILAC, Stable isotope labeling by amino acids in culture; HILEP, Hydroponic isotope labeling of entire plants; SILIP, Stable isotope labeling in planta; SILIA, Stable isotope labeling in Arabidopsis; iTRAQ, Isobaric tags for relative and absolute quantification; TMT, Tandem mass tags; AQUA, Absolute quantification of proteins; AQUIP, Absolute quantitation of isoforms of post-translationally modified proteins.

Rice tissue	Distribution of single (S) : double (D) : triple (T) : multiple (M) phosphorylated protein	Frequency of occurrence ratio (pS : pT : pY)	Reference
Unfractionated whole-cell lysates of rice cells		84.8 : 12.3 : 2.9	Nakagami et al, 2010
Leaves from150 mmol/L NaCl treated rice roots		75 : 25 : 0	Shen et al, 2012
Pistils	98 : 2 : 0 (S : D : M)	87 : 12 : 1	Wang et al, 2014a
Magnaporthe oryzae infected seedlings	86 : 14 : 0 (S : D : M)		Li Y F et al, 2015
Rice nucleus during the early stage of seed germination	65 : 30 : 5 (S : D : M)	64 : 31 : 5	Li M et al, 2015
Developing rice anthers	39 : 23 :14 : 24 (S : D : T : M)	81.33: 17.06: 1.61	Ye et al, 2015
Rice leaves in response to bacterial blight	(88.3‒89.9) : (11.0‒9.6) : (0.7‒0.5) (S : D : M)	(88.8‒89.2) : (10.6‒10.1) : (0.6‒0.7)	Hou et al, 2015
Pistils and seeds	95.5 : 4.4 : 0.1 (S : D : M)	90.93 : 8:84 : 0.23	Qiu et al, 2015
ABA induced rice seedlings	93.2 : 6.7 : 0.1 (S : D : M)	90:93 : 8.84 : 0.23	Qiu et al, 2017
Brassinosteroid treatment on rice seedlings	(92.9‒93.3) : (6.5‒6.8) : (> 0.3) (S : D : M)	89.7 : 9.9 : 0.4	Hou et al, 2017
Callus, leaf, root, shoot meristem, young panicle, and mature panicle	64.82 : 24.83 : 7.29 : 3.06 (S : D : T : M)	86.0 : 13.0 : 1.0	Wang et al, 2017