Limits...
A Quantitative Proteomic Analysis of Brassinosteroid-induced Protein Phosphorylation in Rice ( Oryza sativa L.)

View Article: PubMed Central - PubMed

ABSTRACT

The group of polyhydroxysteroid phytohormones referred to as the brassinosteroids (BRs) is known to act on plant development and the stress response. BR signal transduction relies largely on protein phosphorylation. By employing a label-free, MS (Mass Spectrometry)-based phosphoproteomic approach, we report here the largest profiling of 4,034 phosphosites on 1,900 phosphoproteins from rice young seedlings and their dynamic response to BR. 1,821 proteins, including kinases, transcription factors and core components of BR and other hormone signaling pathways, were found to be differentially phosphorylated during the BR treatment. A Western blot analysis verified the differential phosphorylation of five of these proteins, implying that the MS-based phosphoproteomic data were robust. It is proposed that the dephosphorylation of gibberellin (GA) signaling components could represent an important mechanism for the BR-regulated antagonism to GA, and that BR influences the plant architecture of rice by regulating cellulose synthesis via phosphorylation.

No MeSH data available.


Western-blot analysis of five selected proteins to verify the MS identified phosphorylation pattern at 0, 3, and 12 h. Red triangle indicates the target band in original size; Red asterisk indicates the phosphorylated target protein band. Anti-tubulin was used as an internal control for normalization. The values above the phosphorylated bands represent the normalized, relative band intensities by setting 0 h into 1.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383725&req=5

Figure 5: Western-blot analysis of five selected proteins to verify the MS identified phosphorylation pattern at 0, 3, and 12 h. Red triangle indicates the target band in original size; Red asterisk indicates the phosphorylated target protein band. Anti-tubulin was used as an internal control for normalization. The values above the phosphorylated bands represent the normalized, relative band intensities by setting 0 h into 1.

Mentions: To validate the MS identified phosphorylation status, we performed Western-blot analysis for seven selected DP protein in the time course of BR treatment, including BZR1 (LOC_Os07g39220), D1 (LOC_Os05g26890), GID2 (LOC_Os02g36974), SAPK9 (LOC_Os12g39630), and SMG1 (LOC_Os02g54600). Due to the phosphates attachment, phosphorylated protein bands migrate more slowly through the gel than the unmodified proteins. By using β-tubulin as an internal control, the intensity of each phosphorylated band was semi-quantified. Fit well with our expectations, all the five DP proteins showed similar phosphorylation tendencies as the phosphoproteomic data indicated, though the extent of phosphorylation may vary from each other (Figure 5). The reduced phosphorylation status of BZR1, D1, GID2, and SMG1 in response to the BR treatment was confirmed by the Western blot result, as was the induced increase in the level of phosphorylation for SAPK9. Moreover, the absence of the slower-migrating bands following treatment with CIAP (Calf Intestine Alkaline Phosphatase) confirmed their identity as phosphorylated proteins. The results above strongly suggested that our phosphoproteomic data is highly reliable and such a MS-based quantification strategy could be applied for the phosphorylation dynamic detections in other biological processes.


A Quantitative Proteomic Analysis of Brassinosteroid-induced Protein Phosphorylation in Rice ( Oryza sativa L.)
Western-blot analysis of five selected proteins to verify the MS identified phosphorylation pattern at 0, 3, and 12 h. Red triangle indicates the target band in original size; Red asterisk indicates the phosphorylated target protein band. Anti-tubulin was used as an internal control for normalization. The values above the phosphorylated bands represent the normalized, relative band intensities by setting 0 h into 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383725&req=5

Figure 5: Western-blot analysis of five selected proteins to verify the MS identified phosphorylation pattern at 0, 3, and 12 h. Red triangle indicates the target band in original size; Red asterisk indicates the phosphorylated target protein band. Anti-tubulin was used as an internal control for normalization. The values above the phosphorylated bands represent the normalized, relative band intensities by setting 0 h into 1.
Mentions: To validate the MS identified phosphorylation status, we performed Western-blot analysis for seven selected DP protein in the time course of BR treatment, including BZR1 (LOC_Os07g39220), D1 (LOC_Os05g26890), GID2 (LOC_Os02g36974), SAPK9 (LOC_Os12g39630), and SMG1 (LOC_Os02g54600). Due to the phosphates attachment, phosphorylated protein bands migrate more slowly through the gel than the unmodified proteins. By using β-tubulin as an internal control, the intensity of each phosphorylated band was semi-quantified. Fit well with our expectations, all the five DP proteins showed similar phosphorylation tendencies as the phosphoproteomic data indicated, though the extent of phosphorylation may vary from each other (Figure 5). The reduced phosphorylation status of BZR1, D1, GID2, and SMG1 in response to the BR treatment was confirmed by the Western blot result, as was the induced increase in the level of phosphorylation for SAPK9. Moreover, the absence of the slower-migrating bands following treatment with CIAP (Calf Intestine Alkaline Phosphatase) confirmed their identity as phosphorylated proteins. The results above strongly suggested that our phosphoproteomic data is highly reliable and such a MS-based quantification strategy could be applied for the phosphorylation dynamic detections in other biological processes.

View Article: PubMed Central - PubMed

ABSTRACT

The group of polyhydroxysteroid phytohormones referred to as the brassinosteroids (BRs) is known to act on plant development and the stress response. BR signal transduction relies largely on protein phosphorylation. By employing a label-free, MS (Mass Spectrometry)-based phosphoproteomic approach, we report here the largest profiling of 4,034 phosphosites on 1,900 phosphoproteins from rice young seedlings and their dynamic response to BR. 1,821 proteins, including kinases, transcription factors and core components of BR and other hormone signaling pathways, were found to be differentially phosphorylated during the BR treatment. A Western blot analysis verified the differential phosphorylation of five of these proteins, implying that the MS-based phosphoproteomic data were robust. It is proposed that the dephosphorylation of gibberellin (GA) signaling components could represent an important mechanism for the BR-regulated antagonism to GA, and that BR influences the plant architecture of rice by regulating cellulose synthesis via phosphorylation.

No MeSH data available.