A proteomics study of brassinosteroid response in Arabidopsis

Abstract

The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we performed a proteomics study of BR-regulated proteins in Arabidopsis using two-dimensional DIGE coupled with LC-MS/MS. We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR-insensitive mutant bri1-116 and BR-hypersensitive mutant bzr1-1D identified five proteins (PATL1, PATL2, THI1, AtMDAR3, and NADP-ME2) affected both by BR treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knock-out mutants or immunoblotting. Interestingly about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed gene-specific kinetic relationships between RNA and protein responses. Furthermore BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in two-dimensional DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses.

Figure 1. BL induces seedling growth

(A) Representatives of Arabidopsis det2 seedling treated with 100 nm BL (right) or mock solution (left) for 24 hr. (B and C) Kinetics of seedling growth measured by change of fresh weight per seedling (B) and hypocotyl length (C) during the course of BL treatment. Results are shown as means (±standard deviation).

Figure 2. 2-D DIGE analysis of BR-regulated proteins

Proteins of det2 seedlings treated with 100 nM BL for 24 hr (Cy5, red) were compared with those of mock treated samples (Cy3, green) by 2-D DIGE using 24 cm pH 4–7 IPG strips and 12.5% SDS-PAGE gel. Proteins increased by BL appear red, and those repressed by BR appear green, while those unaffected show yellow. Protein spots of interest are marked with spot numbers and their identities are shown on Table 1.

Figure 3. Proteomic changes in the bri1-116 or bzr1-1D mutant

(A) Representative proteins regulated by both BL treatment and bri1-116 or bzr1-1D mutations. Protein samples from bri1-116 or a transgenic line expressing mBZR1-CFP (bzr1-1D) (Cy5 labeled, red) were compared with wild type samples (Cy3, green) by 2-D DIGE. The representative images showing the affected spots and the relative ratios of protein abundance are listed. In the superimposed 2-D DIGE images, protein spots up regulated by BL, bri1-116, or bzr1-1D appear red, those down regulated appear green, and those unaffected are yellow. (B) Venn diagram of protein spots regulated by BL treatment, the bri1-116 mutation, or bzr1-1D mutation.

Figure 4. Immunoblot analysis of PATL1 and PATL2 expression in response to BL treatment

Immunoblot of mock- and BR-treated samples was probed first with anti-PATL1 antibody, striped, and then probed with anti-PATL2 antibody. A duplicate SDS-PAGE gel stained by Commassie blue R-250 is shown. The graph shows the PATL1 and PATL2 protein levels quantified using either immunoblots or 2-D DIGE. Immunoblot data were quantitated using ImageQuant 5.2 and 2-D DIGE data were quantitated using the DeCyder 6.5 software.

Figure 5. Confirmation of protein identity by 2-D DIGE analyses of T-DNA knockout mutant of thi1 (A–C), and single and double mutants of 14-3-3λ and 14-3-3κ (D–F)

In each panel, the top image shows the Cy3 (green) and Cy5 (red) overlay, in the middle are the individual Cy3 (left) or Cy5 (right) images, and at the bottom are the 3D view of the middle images. As expected, spots 2898 (red arrow) and 2957 are absent in the thi1 mutant compared to the wild type (A). BL treatment reduced the signal levels of both spots in wild type (B) but not in thi1 (C). Disappearance of spots in the T-DNA mutants confirms the spots 3062 (white arrow) and 3108 (red arrow) as 14-3-3λ (D, F), and spot 3115 as 14-3-3κ (E, F).

Figure 6. Comparison between changes of protein and mRNA levels caused by BR mutants (A, B) or BR treatment (C–F)

The 2-D DIGE data of Table 1 and microarray data were used to compare the ratios (log2) of protein versus mRNA expression levels between det2 (A) or bzr1-1D (B) and wild type. (C to F) The ratios of RNA (open bars) or protein (closed bars) abundance between samples treated with BL for various time and un-treated samples are shown for THI1 (panel C), ACC oxidase (ACO, panel D), allene oxide cyclase (AOC, panel E), and S-adenosylmethionine synthetase (SAMS, panel F). Protein levels were determined by 2-D DIGE analysis, and the RNA levels were quantified using real-time RT-PCR. Error bars indicate standard deviation.

Figure 7. BR regulates BiP2 phosphorylation

BR treatment and the bzr1-1D mutation affect phosphorylation of BiP2. From top panel to bottom in: Overlaid 2-D DIGE images of BR treated (Cy5, red) compared with untreated (Cy3, green) det2 samples, 2-D DIGE image of transgenic line expressing mBZR1-CFP (Cy5, red) compared with wild type (Cy3), and Pro-Q Diamond stain and Cy5 images from the same 2-DE gel, and the table showing the quantified data. A higher ratio of Pro-Q diamond to Cy5 signal indicates increased phosphorylation. The Pro-Q/Cy5 ratio was normalized to spot 3.