Dataset Information

Co-immunoprecipitation of FAMA-interacting proteins in Arabidopsis thaliana seedlings

ABSTRACT: Identifying interaction partners and protein complex compositions for transcription factors (TFs) can produce valuable information on the mechanisms by which they regulate gene expression or are themselves regulated by signaling pathways in the cell. FAMA is a TF that is specifically expressed in the last stages of stomatal guard cell development in the epidermis of young leaves and other aerial tissues of higher plants. It is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. How this is achieved mechanistically is still unclear. We therefore isolated putative FAMA interaction partners from young Arabidopsis seedlings expressing FAMA-CFP under the FAMA promoter using proteasome inhibitor treatment and formaldehyde crosslinking, followed by a classical co-immunoprecipitation mass spectrometry approach. Plants expressing nuclear GFP under a stomatal lineage specific promoter were used as controls. In two independent experiments with different crosslinking and immunoprecipitation conditions, we found ICE1, a known heterodimerization partner of FAMA, to be enriched in FAMA-CFP versus control samples. Other proteins with known transcriptional regulator or co-regulator function were not identified, presumably due to the low abundance of the bait protein. The interaction of FAMA with BRXL2, another regulator of guard cell development, which was detected in one experiment, is likely an artifact since BRXL2 does not localize to the nucleus under normal conditions.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Root, Shoot

SUBMITTER: Shouling Xu

LAB HEAD: Shouling Xu

PROVIDER: PXD015212 | Pride | 2019-09-26

REPOSITORIES: Pride

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Publications

Proximity labeling of protein complexes and cell-type-specific organellar proteomes in <i>Arabidopsis</i> enabled by TurboID.

Mair Andrea A Xu Shou-Ling SL Branon Tess C TC Ting Alice Y AY Bergmann Dominique C DC

eLife 20190919

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to addre ...[more]

PMID: 31535972

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Project description:Identifying specific protein interactors and spatially or temporally restricted local proteomes contributes significantly to our understanding of cellular processes in virtually all aspects of life. Obtaining such data is challenging, especially when the protein, cell type or event of interest is rare. In recent years, different proximity labeling techniques have been developed that have greatly improved our ability to tackle these questions. However, while effective in mammalian systems, use in plants has been extremely limited due to technical challenges. Recent technological improvements in the form of two highly active versions of the biotin ligase BirA* (TurboID and miniTurboID), prompted us to test this new system on two challenging but widely asked questions in plants: what are interaction partners of low-abundant proteins and what are organellar proteomes in rare and transient cell types. To address the second question, we used nuclei of developing stomatal guard cells, which express the transcription factor FAMA, as our test case. FAMA is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. FAMA-expressing young guard cells are rare and restricted to the epidermis of developing aerial tissues, which makes them a good model system to test TurboID applicability under material-limiting conditions. For this experiment, young Arabidopsis seedlings expressing nuclear TurboID under the FAMA or UBIQUITIN10 (UBQ10) promoter were treated with biotin to label nuclear proteomes. Col-0 wild type was used as controls for unspecific binding of proteins to the beads. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. By targeting TurboID to the nucleus we were able to purify nuclear proteins with relatively high specificity. Enrichment of FAMA-stage specific proteins, when nuclear TurboID was driven by the FAMA compared to the UBQ10 promoter, supports general applicability of TurboID for identification of subcellular proteomes.

Project description:Identifying specific protein interactors and spatially or temporally restricted local proteomes contributes significantly to our understanding of cellular processes in virtually all aspects of life. Obtaining such data is challenging, especially when the protein, cell type or event of interest is rare. In recent years, different proximity labeling techniques have been developed that have greatly improved our ability to tackle these questions. However, while effective in mammalian systems, use in plants has been extremely limited due to technical challenges. Recent technological improvements in the form of two highly active versions of the biotin ligase BirA* (TurboID and miniTurboID), prompted us to test this new system on two challenging but widely asked questions in plants: what are interaction partners of low-abundant proteins and what are organellar proteomes in rare and transient cell types. To address the first question, we used the transcription factor FAMA as a test case. FAMA is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. FAMA-expressing young guard cells are rare and restricted to the epidermis of developing aerial tissues, which makes them a good model system to test TurboID applicability under material-limiting conditions. For this experiment, young Arabidopsis seedlings expressing FAMA-TurboID were treated with biotin to label FAMA complexes. Col-0 wild type and seedlings expressing nuclear TurboID under the FAMA promoter were used as controls for unspecific binding of proteins to the beads and stochastic labeling of nuclear proteins, respectively. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. Analysis of proteins labeled by FAMA-TurboID fusions revealed known interactors of this late stomatal lineage specific transcription factor, as well as novel proteins that could explain its dual function as an activator and repressor. Comparison with proteins obtained from classical co-immunoprecipitation approaches with FAMA showed that proximity labeling is superior for identification of meaningful interaction partners of low-abundant proteins.

Project description:Stomata are pores in the epidermis of plants that can open and close and allow for gas exchange vital for photosynthesis and regulate transpiration. Stomatal development is driven by a set of conserved bHLH transcription factors (SPCH, MUTE, FAMA, and their heterodimerization partners ICE1, SCRM2), that initiate and promote progression of cell fates in the stomatal lineage. Due to the shared ancestry of SPCH, MUTE and FAMA (subgroup Ia) and ICE1 and SCRM2 (subgroup IIIb) their DNA binding specificity is similar and there is some functional redundancy. However, individual bHLHs also have unique functions. For example, SPCH is required for initiation of the stomatal lineage, while FAMA is responsible for terminal differentiation of the guard cell pair. In grasses, the stomatal complex comprises the guard cell pair, and two flanking subsidiary cells. Recruitment of the latter from neighboring cell filed during development requires expression of MUTE. Remarkably, while MUTE is absolutely required for the promotion of guard mother cell fate in maize and rice, this is not the case in Brachypodium. This suggests that another TF can at least partially substitute for MUTE in this function. While different expression profiles of the bHLH dimers within the stomatal lineage may partially responsible for distinct functions of each pair, it is likely that each pair forms different transcriptional complexes and that interaction with other transcriptional regulators affects the dimer’s binding DNA binding and gene regulation properties. Given the differences in bHLH function between dicots and grasses and even within the grass family, we were interested in elucidating the protein interaction networks of the stomatal lineage regulators in Brachypodium. To this end, we performed co-immunoprecipitation coupled to LC-MS/MS of BdSPCH2-YFP, YFP-BdMUTE, YFP-BdFAMA, YFP-ICE1 and YFP-SCRM2 from the developmental zone of young B. distachyon leaves using GFP-Trap beads. All YFP-fusion proteins were expressed under the endogenous promoter in the Bd21-3 background. The only exception was BdICE1, which was expressed under the ZmUBI promoter, but was nevertheless mostly restricted to the stomatal lineage. As control lines we use the Bd21-3 wild type and a line expressing 3x YFPnls (nuclear YFP) under the MUTE promoter. Comparison of proteins enriched with the bHLH fusion proteins vs. the controls revealed overlapping and distinct putative interactors, which is in agreement with the assumption that these transcription factors have both shared and unique functions. Notably, in addition to the presumed hetero-dimerization partners, we found a number of other bHLH transcription factors that were identified with one or more of the bait proteins. This suggests the presence of a larger bHLH network acting in the stomatal lineage.

Dataset Information

Co-immunoprecipitation of FAMA-interacting proteins in Arabidopsis thaliana seedlings

Publications

Proximity labeling of protein complexes and cell-type-specific organellar proteomes in <i>Arabidopsis</i> enabled by TurboID.

Similar Datasets

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