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Proximity labeling – LC/MS-MS with SCRM-TbID in A. thaliana seedlings

ABSTRACT: Stomata are pores in the epidermis of plants that can open and close, allowing for gas exchange with the environment, vital for photosynthesis, and regulating transpiration. They are formed from protodermal cells by a series of asymmetric and symmetric divisions and differentiation steps. Initiation into and progression within the stomatal lineage are driven by the consecutive expression and action of three sets of bHLH transcription factor dimers (SPCH, MUTE, FAMA plus their heterodimer partners SCRM or SCRM2) and is accompanied by massive changes in the transcriptome as well as the chromatin landscape. Strong shifts in chromatin accessibility have been observed specifically at the initiation stage and at the transition from division to differentiation. In line with this, FAMA, which promotes the differentiation of stomata, has been shown to interact with RBR, which can recruit the repressive PRC2 complex, to shut down the early stomatal lineage transcriptional program. Interestingly, several of the stomatal lineage bHLHs bind chromatin regions that are closed/inaccessible in a primordial state (the shoot apical meristem). This suggests that the stomatal lineage bHLHs could be able to access closed chromatin and that they might also be directly involved in opening (and closing) of chromatin at a subset of their target regions. In order to test this hypothesis and identify chromatin modifiers that associate with the stomatal lineage bHLH dimers, we did proximity labeling with TurboID fused to SCRM, which is expressed throughout the stomatal lineage and is the main heterodimerization partner of SPCH, MUTE and FAMA. We identified a number of proteins acting as chromatin modifiers and remodelers, including the histone acetyl transferase HAC1, which deposits activating marks on nucleosomes, and multiple subunits of the ATPase-dependent chromatin remodeling SWI/SNF complex. Together, these data support a role of SCRM and its partners in regulating target gene accessibility to impose lasting changes in gene expression within the stomatal lineage.

INSTRUMENT(S): Q Exactive HF

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

TISSUE(S): Plant Cell, Whole Body

SUBMITTER: Shouling Xu

LAB HEAD: Shouling Xu

PROVIDER: PXD040690 | Pride | 2024-06-14

REPOSITORIES: Pride

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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.

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: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.

			Action	DRS
	Q20191225_05.pep.xml	Pepxml
	Q20191225_05.raw	Raw
	Q20191225_06.pep.xml	Pepxml
	Q20191225_06.raw	Raw
	Q20191225_07.pep.xml	Pepxml

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Proximity labeling – LC/MS-MS with SCRM-TbID in A. thaliana seedlings

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