Dataset Information

LC-MSMS of Arabidopsis thaliana flower bud samples (TurboID-based proximity labeling of meiotic chromosome axes)

ABSTRACT: The meiotic chromosome axis is a proteinaceous structure along which sister chromatids are arranged in a loop-base array during meiotic prophase I. In diverse species including plants, chromosome axis components have been shown to be critical for synapsis and meiotic recombination and hence essential for assuring the fidelity of meiosis. In Arabidopsis thaliana, the limited number of meiotic cells embedded in floral organs constrains proteomic approaches for the identification of new axis components. Therefore, we employed TurboID (TbID) based proximity labeling (PL) in meiotic cells of A. thaliana. The two axis proteins ASY1 and ASY3 were selected and ASY1-TbID and ASY3-TbID transgenic plants were generated to enable specific targeted biotin labeling of meiotic chromosome axis. ASY1-eYFP and nucTbID (TbID driven by UBQ10 promoter) were generated and used as controls. These lines were either treated or not with exogenous biotin followed by streptavidin affinity-purification coupled with mass spectrometry to identify their proximate candidate proteins. By stringent filtering a final candidates list was established which covered most of the known meiotic chromosome axis-related proteins and new proteins without a reported meiotic function. Functional validation of selected candidates and comparison with ASY1 AP-MS data (in Brassica oleracea) suggest the TbID based PL is a robust tool for identification of proximate proteins in meiotic cells of A. thaliana.

INSTRUMENT(S): Q Exactive HF

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

TISSUE(S): Flower Bud

SUBMITTER: Richard Imre

LAB HEAD: Stefan Heckmann

PROVIDER: PXD034241 | Pride | 2023-03-08

REPOSITORIES: Pride

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			Action	DRS
	20201204_QExHFX3_RSLC10_Feng_Heckmann_EXT_onbead_dig_30per_control_C1_2.raw	Raw
	20201204_QExHFX3_RSLC10_Feng_Heckmann_EXT_onbead_dig_30per_control_C2_3.raw	Raw
	20201204_QExHFX3_RSLC10_Feng_Heckmann_EXT_onbead_dig_30per_sample_S3_1.raw	Raw
	20201204_QExHFX3_RSLC10_Feng_Heckmann_EXT_onbead_dig_30per_sample_S4_2.raw	Raw
	20210205_QExHFX3_RSLC10_Feng_Heckmann_EXT_onbead_dig_30_per_control_Asy1_50mM_bio_C2_4.raw	Raw

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Project description:The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication, and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis, and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not directly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes, and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms. Multiple studies of meiotic chromosomes were undertaken. To study DNA replication, the locations of replicative helicase (Mcm2-7) were mapped in pre-meiotic and pre-mitotic cells, and DNA replication profiles were created for pre-meiotic S (meiS) and pre-mitotic S (mitS) phases. Early origins were mapped in hydroxyurea for wild-type cells in mitS + 200mM HU, and meiS +20mM HU for wild-type, sml1, rec8 and spo11 deletion cells. Rec8, Hop1 and Red1 binding to meiotic chromosomes was evaluated using ChIP-chip in wild-type cells with and without 20 mM HU, and in cdc6-mn and clb5 clb6 delete cells. Finally, meiotic DNA double-strand breaks (DSBs) were mapped in cdc6-mn dmc1 delete cells by measuring the ssDNA that accumulates at DSB hotspots. This SuperSeries is composed of the following subset Series: GSE35658: Chromatin IP for Mcm2-7, Rec8, Hop1 and Red1 GSE35662: S phase and HU profiles in wild-type and mutant cells GSE35666: DSB formation in replication compromised cells

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LC-MSMS of Arabidopsis thaliana flower bud samples (TurboID-based proximity labeling of meiotic chromosome axes)

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