Arabidopsis MS1 functions as a hub in the transcriptional regulatory network of late tapetum development

YIN, WENZHE (2017) Arabidopsis MS1 functions as a hub in the transcriptional regulatory network of late tapetum development. PhD thesis, University of Nottingham.

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Abstract

The development of the pollen grains within the anther locule relies upon the nourishing and secretory properties of a tissue layer termed tapetum. The transition of the post-meiotic phase of tapetum development depends on the MALE STERILITY 1. In the ms1 mutant tapetum development is arrested post-meiosis and lacks subsequent biological processes, such as biosynthesis and secretion of pollen wall/coat components and the tissue programmed cell death process. MS1 exhibits a transient expression pattern, which is tightly regulated and critical for tapetum development and viable pollen formation. Therefore, understanding the genetic control of MS1 is key to uncover the regulation of post-meiotic tapetum development. During this project, three regulation levels of MS1 were studied: (i) transcriptional activation, (ii) auto-repression and (iii) post-translational proteolysis. Phylogenetic footprinting analysis and molecular promoter dissection was used to investigate the transcriptional control of expression and a distal upstream sequence (−2900 to −2066 bp) was found to be essential for the activation of MS1. Three evolutionarily conserved non-coding sequences (CNS), enriched with unusually long consensus motifs, and binding site combinations of MS1 upstream transcription factors (TFs) were found within the −2 kb MS1 upstream sequence. These may serve as essential cis-regulatory elements (CREs) for MS1 expression. ChIP experiments were used to investigate MS1 autorepression; the MS1 protein was shown to bind to the second exon of its genomic locus and to repress its own expression. Post-translational proteolysis was investigated using a triple mutant of the MS1 interacting gene that encodes for an E3 ubiquitin ligase LRB1 and its two paralogs LRB2 and LRB3; which exhibits a novel tapetum phenotype that may be induced by altered removal of MS1 protein in the lrb123 tapetum.

The MS1 protein possesses a Plant Homeotic Domain (PHD) finger and belongs to a plant-specific C-terminal PHD contained protein (CPCP) family. Although extensive research has been carried out on the tapetum regulation role of MS1, very little is known about the underlying molecular mechanism. A series in-silico comparative analysis of the CPCP sequences in this thesis found that this family originated from green algae. Besides the PHD, two evolutionarily conserved domains, termed MS1/MMD1 Associated Domain 1 (MAD1) and MAD2, were identified in the protein. Molecular modelling of the MS1 PHD domain predicted a histone reader role with high affinity to H3K4me2/3 histone peptides. Super-resolution STED confocal observation showed that subnuclear localisation of the MS1 protein is distinctive with canonical TFs and aggregates at rounded speckles that resemble Polycomb bodies. A meta-data-analysis of MS1 microarrays found that most MS1 immediately responding genes are repressed by MS1, which is on the contrary to the previously proposed activator role of MS1. MS1 may therefore be a unique plant-specific histone reader family protein that participates in gene repression as a co-repressor.

MYB99 has previously been hypothesised to be a direct target of MS1, regulating late tapetum development. Comprehensive phenotyping was carried out on two MYB99 null alleles; however, no defects were identified, probably due to high function redundancy among the MYB family TFs. In addition, no evidence of direct activation by MS1 was observed by yeast one-hybrid and ChIP analysis. Interestingly, a PCD indicator gene was down-regulated in the myb99 mutant, suggesting a tapetal PCD regulatory role for MYB99.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Wilson, Z.A.
Subjects: Q Science > QK Botany > QK710 Plant physiology
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 43214
Depositing User: Yin, Wenzhe
Date Deposited: 03 Nov 2017 14:35
Last Modified: 03 Nov 2017 14:37
URI: http://eprints.nottingham.ac.uk/id/eprint/43214

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