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TurboID-based proteomic profiling of meiotic chromosome axes in Arabidopsis thaliana

Nature Plants volume 9, pages 616–630 (2023)Cite this article

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Abstract

During meiotic prophase I, sister chromatids are arranged in a loop-base array along a proteinaceous structure, called the meiotic chromosome axis. This structure is essential for synapsis and meiotic recombination progression and hence formation of genetically diverse gametes. Proteomic studies in plants aiming to unravel the composition and regulation of meiotic axes are constrained by limited meiotic cells embedded in floral organs. Here we report TurboID (TbID)-based proximity labelling (PL) in meiotic cells of Arabidopsis thaliana. TbID fusion to the two meiotic chromosome axis proteins ASY1 and ASY3 enabled the identification of their proximate ‘interactomes’ based on affinity purification coupled with mass spectrometry. We identified 39 ASY1 and/or ASY3 proximate candidates covering most known chromosome axis-related proteins. Functional studies of selected candidates demonstrate that not only known meiotic candidates but also new meiotic proteins were uncovered. Hence, TbID-based PL in meiotic cells enables the identification of chromosome axis proximate proteins in A. thaliana.

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Fig. 1: Phenotype of ASY1-eYFP-TbID and ASY3-eYFP-TbID plants.
Fig. 2: Biotinylation of meiotic chromosome axes in ASY1-eYFP-TbID and ASY3-eYFP-TbID plants.
Fig. 3: Exogenous biotin treatment enhances biotinylation in ASY1-eYFP-TbID plants.
Fig. 4: Identification of meiotic chromosome axis proximate candidates in ASY1-eYFP-TbID and ASY3-eYFP-TbID plants.
Fig. 5: Functional dissection of selected candidates.

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Data availability

All data supporting the findings of this research are presented in the main text, figures and supplementary information. Generated materials are available from the corresponding author upon reasonable request. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE76 partner repository with the dataset identifier PXD034241. The gene/protein sequences and accession codes of genes used in this study are found in the following databases: TAIR (https://www.arabidopsis.org/) and Ensembl Plants (http://plants.ensembl.org/index.html). Source data are provided with this paper.

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Acknowledgements

We thank F. Hartmann and M. Doelling (IPK Gatersleben) for their excellent technical assistance, D. Demidov (IPK Gatersleben) for advice on immunoblots, J. Paysan (Carl Zeiss GmbH) for providing FEP tubes, C. Franklin (University of Birmingham) for sharing antibodies, A. Houben (IPK Gatersleben) for critical reading of the paper, R. Imre (IMBA Vienna) for support in MS proteomics data submission, and all lab members for fruitful discussions. B.W. is a holder of a China Scholarship Council (CSC) fellowship (no. CSC202103250012). This work is funded by the Deutsche Forschungsgemeinschaft (DFG) within the framework of the ERA-CAPS MEIOREC (HE 7950/1-1) project to S.H. and by the Austrian Science Fund by ERA-CAPS I 3686-B25-MEIOREC international project to K.M.

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Authors and Affiliations

  1. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) OT Gatersleben, Seeland, Germany

    Chao Feng, Maria Cuacos, Jana Lorenz, Veit Schubert, Baicui Wang, Rui Wang & Stefan Heckmann

  2. Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria

    Elisabeth Roitinger, Otto Hudecz & Karl Mechtler

  3. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria

    Elisabeth Roitinger, Otto Hudecz & Karl Mechtler

  4. The Gregor Mendel Institute of Molecular Plant Biology of the Austrian Academy of Sciences (GMI), Vienna BioCenter (VBC), Vienna, Austria

    Elisabeth Roitinger, Otto Hudecz & Karl Mechtler

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  1. Chao Feng
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Contributions

C.F. supported by J.L., B.W. and R.W. conducted most of the research. O.H., E.R. and K.M. performed MS experiments and data analysis. M.C. performed LSFM, and V.S. performed SIM analysis. S.H. acquired funding. C.F. and S.H. analysed the data and wrote the paper. All authors approved the final paper.

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Correspondence to Stefan Heckmann.

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Extended data

Extended Data Fig. 1 Activity of mammalian and plant codon-optimized TbID in Arabidopsis protoplasts.

(a) Immunoblots (IB) of protein extracts from protoplasts transformed with mammalian or plant codon-optimized TbID and treated with 0.05 mM biotin for 2 hrs. Anti-HA antibody and Streptavidin (SA) used for blotting. Protein extract from non-transformed protoplasts used as control. The experiment was repeated three times independently with similar results. (b) DNA sequence of custom synthesized TbID (plant codon-optimized).

Source data

Extended Data Fig. 2 Phenotype of ASY1-eYFP-TbID and ASY3-eYFP-TbID transgenic lines.

(a) Flowering plants of Col-0, ASY1-eYFP (asy1), UBQeYFP-TbID (Col-0), ASY1-eYFP-TbID (asy1), ASY3-eYFP-TbID (asy3), asy1 and asy3. Scale bar = 5 cm. (b) Pollen viability in plant lines as indicated in (a) revealed by Alexander red staining. Scale bar = 50 μm. (c) Seed setting in fruit pods of Col-0, ASY1-eYFP, ASY1-eYFP-TbID and asy1. Scale bar = 0.5 cm. (d) Localization of ASY1-eYFP and ASY1-eYFP-TbID fusion proteins in female meiotic cells. Pistils containing cells undergoing female meiosis squashed in H2O. Scale bar = 50 μm. (e) Female meiotic chromosome spread analysis in ASY1-eYFP-TbID and ASY3-eYFP-TbID. Scale bar = 5 μm. The experiments in b-e were repeated at least three times with similar results.

Extended Data Fig. 3 ASY1-eYFP-mTb plants: Phenotype and biotinylation.

(a) Siliques (Scale bar = 1 cm), (b) seeds per silique (ASY1-eYFP-mTb (48.65 ± 3.80) vs. Col-0 (51.63 ± 3.91), p = 1.04 ×10-3; two-sided Student’s t test; n = 40; *, P < 0.01), (c) pollen viability assessed by Alexander staining (Scale bar = 50 μm), (d) male meiotic chromosomes from pachytene to tetrads (Scale bar = 10 μm; DNA stained with DAPI in gray), and (e) minimum chiasmata number (MCN) in WT (n = 28) and ASY1-eYFP-mTb (n = 27); two-sided Student’s t test; N.S., not significant. Biotinylation in ASY1-eYFP, ASY1-eYFP-mTb and ASY1-eYFP-TbID plants treated with 0.5 mM of exogenous biotin based on (f) indirect immunolocalization of fluorophore-conjugated streptavidin in male meiocytes (Scale bar = 5 μm) and (g) immunoblot analysis. Streptavidin (SA) used for blotting, Coomassie brilliant blue (CBB) stained protein gel as loading control. The experiments in f-g were repeated three times independently with similar results.

Source data

Extended Data Fig. 4 Meiosis-specific ASY1-eYFP-TbID and ASY3-eYFP-TbID fusion protein expression.

(a) Fusion protein expression in flower buds of UBQeYFP-TbID, ASY1-eYFP, ASY1-eYFP-TbID, ASY3-eYFP and ASY3-eYFP-TbID plants revealed using light sheet fluorescence microscopy (LSFM). Scale bar = 50 μm. (b) ASY1-eYFP-TbID and ASY3-eYFP-TbID fusion proteins show similar dynamics in meiotic nuclei when compared with ASY1- and ASY3-eYFP, respectively. ASY1-eYFP duration in meiotic nucleus as example (left) from initial signal appearance until nuclear envelop break down (star) and average time duration determined (right) for ASY1-eYFP-TbID (30.21 ± 1.52)/ASY3-eYFP-TbID (29.55 ± 1.64) and ASY1-eYFP (29.85 ± 1.56)/ASY3-eYFP (30.50 ± 1.18) fusion proteins (n = 10 nuclei). N.S., not significant (p = 0.4262; one-way ANOVA). Immunolocalization of ASY1 (magenta) and ZYP1 (green) during (c) meiosis in WT (Scale bar = 5 μm) as well as (d) early zygotene and pachytene in ASY1-eYFP-TbID, ASY3-eYFP-TbID and WT plants (Scale bar = 10 μm). Note, ASY1 and ZYP1 presence in WT tetrad nuclei in (c). The experiments in a, c and d were repeated at least three times with similar results.

Source data

Extended Data Fig. 5 Assessment of endogenous biotin(ylation), turnover of biotinylation (on meiotic chromatin) and impact of exogenous biotin treatment.

(a) In WT anthers, immunolocalization of biotin (magenta) in organelles of mitotic cells. Scale bar = 5 μm. (b) Male meiotic chromosome spreads from leptotene to tetrad stage from flower buds of Col-0, ASY1-eYFP-TbID and ASY3-eYFP-TbID plants treated with 0.5 mM of exogenous biotin for 20 hrs and immunostained for biotinylation (magenta). DNA counterstained with DAPI in blue. Scale bar = 5 μm. (c) Scatter plots depicting increased protein intensities after biotin treatment in ASY1-eYFP-TbID. Statistical significance of differentially expressed proteins was determined using limma. The experiments in a-b were repeated at least three times with similar results.

Extended Data Fig. 6 Peptide coverage of ASY1 and ASY3 and in planta phosphorylated residues.

(a, b) Blue highlighted are combined peptide coverage for ASY1 and ASY3 by MS analysis. Residues (bold and underlined) identified being phosphorylated by MS analysis within (a) ASY1 (in total six samples, triplicate of both ASY1-eYFP-TbID with and without biotin treatment) and (b) ASY3 (three samples, triplicate of ASY3-eYFP-TbID with biotin treatment), respectively. Indicated above each site in how many samples a given residue was covered (denominator) and in how many of these the residue was found being phosphorylated (numerator) by MS analysis.

Extended Data Fig. 7 ASY1-eYFP-TbID and ASY3-eYFP-TbID proximate candidates after the first filtering step versus ASY1-eYFP.

(a) Venn diagram shows candidates identified for ASY1-eYFP-TbID and/or ASY3-eYFP-TbID (versus ASY1-eYFP). See also Supplemental Table 1. (b) Proteins with reported meiotic function. (c-f) Cellular component and Molecular function of candidates identified from ASY1-eYFP-TbID (c, e) or ASY3-eYFP-TbID (d, f) by gene ontology classification. GO analysis performed by PANTHER (https://www.arabidopsis.org/tools/go_term_enrichment.jsp).

Extended Data Fig. 8 Functional dissection of putative ASY1 phosphoresidues in planta.

(a) Phosphorylation sites within ASY1: six predicted ATM/ATR (blue) and two predicted CDK sites (red). (b) Seeds per silique (Col-0 (48.67 ± 4.96, n = 24), ASY1hexa-A (46.68 ± 5.29, n = 40) and ASY1365A+382A (47.13 ± 1.55, n = 40)), (c), male meiotic chromosomes and (d) MCN (minimum chiasmata number) in ASY1hexa-A (six predicted ATM/ATR sites (S/T) modified to A, n = 22), ASY1365A+382A (two predicted CDK sites modified to A, n = 29) and WT (Col-0, n = 28) plants. N.S., not significant (p = 0.4512 in b, p = 0.0711 in d; one-way ANOVA). Scale bar = 10 μm.

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Extended Data Fig. 9 Expression analysis of selected candidate T-DNA mutants by reverse transcription-PCR and functional dissection of ATC5.

(a) Full-length transcripts (CDS) of candidate genes (ATC3, ATC5, ATC8, ATC21, ATC22, ATC23 and ATC28) are present in Col-0, while absent in their respective homozygous T-DNA insertion mutants. For ATC3 and ATC21, two T-DNA insertion alleles are tested. ACTIN used as a positive control. (b) Seed setting in fruit pods of Col-0 and atc5. Scale bar = 0.2 cm. (c) ATC5-HA-mRuby2 fluorescence (RFP, magenta) in squashed anther (at tetrad stage) compared with Col-0 as control. Scale bar = 50 μm. The experiments in a, c were repeated three times independently with similar results.

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Supplementary information

Reporting Summary

Supplementary Data 1

Detailed data for Extended Data Fig. 7 and Table 1and list of oligonucleotides used in this study.

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Feng, C., Roitinger, E., Hudecz, O. et al. TurboID-based proteomic profiling of meiotic chromosome axes in Arabidopsis thaliana. Nat. Plants 9, 616–630 (2023). https://doi.org/10.1038/s41477-023-01371-7

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