Identification of Proteins Associated with Spatial-Specific Phytochrome- Mediated Light Signaling in Arabidopsis thaliana by Liquid Chromatography-Tandem Mass Spectrometry

Photosensory phytochromes perceive mainly red and far-red light and utilize a linear tetrapyrrole chromophore, phytochromobilin, for their photoactivity in plants. Although phytochromes have been extensively studied for light-dependent regulation of numerous developmental processes, our understanding of the molecular mechanisms responsible for distinct organand tissue-specific phytochrome responses is still limited. Recent studies using transgenic Arabidopsis thaliana plants expressing a gene that encodes the biliverdin IXα reductase (BVR) enzyme, which reduces the biosynthesis and accumulation of phytochromobilin, and thus inactivates phytochromes, have led to advances in probing tissue-specific roles of phytochromes in plant development. We performed one-dimensional SDS-PAGE, followed by protein identification and peptide quantification using liquid chromatography-tandem mass spectrometry (LC/MSMS) to identify proteins that accumulate differentially in transgenic Arabidopsis lines with mesophyllspecific phytochrome deficiencies (i.e., CAB3::pBVR2 plants) compared to wild-type (WT). We identified the large subunit of Rubisco (RbcL) and small subunit of Rubisco (RbcS), which accumulated to lower levels in CAB3::pBVR2 relative to WT under continuous far-red light. We found that Beta-glucosidase proteins (BGLUs) accumulated highly in the CAB3::pBVR2 line under these conditions. RT-PCR and microarray analyses showed a positive correlation between the expression of the target genes and the accumulation of their products in BVR lines. We conclude that RbcL, RbcS, and BGLU18 are targets of mesophyll-specific phytochrome-mediated light signaling under far-red conditions.