Identification and characterisation of MS1 putative interacting proteins and regulatory targets in Arabidopsis

Yu, Suyang (2015) Identification and characterisation of MS1 putative interacting proteins and regulatory targets in Arabidopsis. PhD thesis, University of Nottingham.

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The Arabidopsis thaliana MALE STERILITY1 (MS1) gene, encodes a plant homeodomain (PHD) transcription factor critical for viable pollen formation (Wilson et al., 2001). In the ms1 mutant, there are alterations in the production of pollen wall materials, as well as a failure of tapetal programmed cell death (PCD) (Vizcay-Barrena and Wilson, 2006). This ultimately results in the failure to produce viable pollen. Large numbers of genes are down-regulated in the ms1 mutant indicating that MS1 plays a key role in regulating late tapetal expression and pollen wall deposition (Ito et al., 2007; Yang et al., 2007).

Two putative MS1 interacting proteins At1g58210/NET2A (termed as Y2H54) and AT2G46260/ LRB1 (termed as POB2) were identified from a previous Arabidopsis stamen specific yeast-2-hybrid screen, using a truncated version of the MS1 protein without the PHD motif. POB2 and MS1 were found co-localised in the nucleus, while Y2H54 was specifically located at the plasma membrane. Further confirmation of the interaction using Förster resonance energy transfer (FRET) assay methods showed that POB2 failed to interact with MS1 in planta, however, the association between the two proteins occurred in vitro, as confirmed by protein pull-down assays. Additionally, enhanced general plant growth and floral development were seen in the overexpression lines of Y2H54. However, no significant phenotypes were observed in the RNAi silencing lines.

Chromatin Immunoprecipitation (ChIP) analysis uncovered that MS1 directly regulated the expression of MYB DOMAIN PROTEIN 99 (MYB99) by binding to its promoter. Other putative MS1 direct targets identified by ChIP include 3-KETOACYL-COA SYNTHASE 7 (KCS7), 3-KETOACYL-COA SYNTHASE 15 (KCS15), SPERMIDINE HYDROXYCINNAMOYL TRANSFERASE (SHT) and TAPETUM-SPECIFIC METHYLTRANSFERASE 1 (TSM1). Histone extraction and western blotting assays suggest a role for MS1 in facilitating detrimethylation of H3 marks. H3K36me3 deposition was enhanced at MYB99 in ms1 compared with the wild type, suggesting that MS1 may regulate MYB99 via H3K36me3. A new model for the MS1 regulatory network in pollen wall formation has therefore been proposed.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Wilson, Z.A.
Subjects: Q Science > QK Botany > QK457 Spermatophyta. Phanerogams
Q Science > QK Botany > QK640 Plant anatomy
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 28024
Depositing User: Yu, Suyang
Date Deposited: 21 Aug 2015 11:13
Last Modified: 13 Sep 2016 16:52

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