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Functional analysis of the novel 190kDa microtubule and actin binding protein MAP190 in plant development

Reference	BB/D521430/1
Principal Investigator / Supervisor	Professor P Hussey
Co-Investigators / Co-Supervisors	Professor Keith Lindsey
Institution	Durham University
Department	Biological and Biomedical Sciences
Funding type	Research
Value (£)	222,662
Status	Completed
Type	Research Grant
Start date	01/11/2005
End date	31/10/2008
Duration	36 months

Abstract

Plant cell morphogenesis is governed by the plant microtubule and actin cytoskeleton and whole plant morphogenesis is controlled by cellular patterning and division through the activity of meristems. We have a cytoskeletal protein that is not essential for plant cell morphogenesis but is essential for normal whole plant morphogenesis. This protein MAP190, a novel 190kDa microtubule and actin binding protein that is not required for cell growth in suspension cultures (determined through the knockout of this protein by RNAi) but is required for normal meristem activity (determined by the analysis of MAP190 insertion mutants). The map190 mutants are embryo defective/lethal. The seeds that do germinate result in plants with short roots in which the radial patterning generated or maintained by the root meristem is severely disrupted. The aim of this proposal is to determine how MAP190 can regulate root meristem activity. To this end we will localise MAP190 through embryo and root development and determine if there is any differential localisation of MAP190 protein both intra and intercellularly. This will be done by complementing the map190 mutant with a chimeric gene construct harbouring MAP190 fused to YFP. In addition, any defects in the cytoskeleton of map190 root cells will be assessed by crossing map190 with Arabidopsis lines harbouring GFP constructs that can decorate the microtubule and actin networks. Any defects in cell cycle will be assessed by crosses with cyclin promoter: GUS lines. As cellular patterning is controlled by plant hormones in signal pathways still to be fully realised, and as it is known that plant hormones can affect cytoskeletal organisation and also require cytoskeletal activity for their correct functioning, it is reasonable to assess whether the MAP190 mutations affect hormone signalling. We will carry out relevant crosses to rest these possibilities. Indeed, we have found that the suppression of ethylene signalling in map190 seedlings bysilver leads to partial restoration of root meristem activity, contrasting with the inhibitory effect of silver on wild type root growth. Forward genetics has always been a powerful approach for identifying previously uncharacterised components of biochemical and developmental pathways and we intend to carry out mutagenic screens for enhancers and suppressors of the map190 phenotype. We will use this approach to identify novel genetic interactions that will help us to understand how MAP190 exerts its function in regulating meristem activity. Finally we will examine the functional sites and domains that are essential for MAP190 activity in regulating meristem activity. Using bioinformatics, we have identified several motifs and novel conserved domains. These will be mutated and constructs expressing the muated MAP190 fused to GFP will be used to complement the map190 phenotype. Failure of the mutant proteins to complement will be indicative of essential domains and analysis of their localisation by GFP will give an indication as to whether the temporal and spatial cellular localisation (nucleus, spindle, phragmoplast) is governed by any of the identified domains. TILLING is a procedure that will allow us to identify an allelic series of induced point mutations covering the entire MAP190 gene. Analysis of point mutations in such a series will have the added advantage of identifying other essential amino acids or domains e.g. microtubule/acting binding domain, not governed or directed by our bioinformatics analysis and complementation assay. In conclusion, in this programme, we aim to analyse the role played by MAP190 in controlling the normal function of the root meristem, using the available tools afforded by the close collaboration of two complementary laboratories: PJH, cell and molecular biology of the plant cytoskeleton in plant cell morphogenesis; KL, the molecular genetics of whole plant morphogenesis, cellular patterning and meristem activity.

Summary

Each cell within a plant has an internal skeleton which is made up of two filament systems named microtubules and actin, and a plethora of associated proteins that anchor, crosslink or regulate this network within the cell. These filament systems and their associated proteins constitute the Cytoskeleton. The cytoskeleton is very dynamic and is able to break down and/or reorganise into different structures or arrays, all of which play key roles in plant cell shape and cell division. There are four key cytoskeletal arrays. One appears in Interphase, the interphase cortical array which controls the direction of cell expansion, and three appear successively in the Mitotic phase, the preprophase band which decides where the new cross-wall will form during cell division, the spindle that separates the daughter chromosomes and the phragmoplast array that forms the new crosswall that separates the daughter cells. The cytoskeleton is controlled by signals which tell it when to reorganise to change the form of the cell, when to divide, and in which direction it should divide. We know that developmental cues must activate these signals and we know that some signalling molecules such as plant hormones require an active cytoskeleton for their activity and can also cause the cytoskeleton to reorganise. MAP190 is a novel plant cytoskeletal associated protein that binds both microtubules and actin. Unusually, this cytoskeletal protein is not involved in regulating the cytoskeletal network in rapidly growing and dividing plant cells that are grown in liquid culture. However, it is involved in promoting organised cell growth in the growing points (meristems) of seedling roots. Organised cell growth from meristems is known to be controlled by certain plant hormones. So the question we have is how does this cytoskeletal associated protein direct organised cell growth in a multicellular plant? We can address this question using molecular cell biology and genetic techniques. Firstly, wecan find out when and where the protein is localised within the root cells and in which root cells. Secondly, we can assess whether there are any defects in the cytoskeleton of root cells where MAP190 expression is abolished, i.e. in the map190-1 mutant. These experiments will give a dynamic picture of the localisation of MAP190 in multicellular growth, and we will be able to correlate this with any cytoskeletal defects observed in the cells of the map190-1 mutant. Thirdly, we will determine whether the absence of MAP190 has any effect on normal auxin and ethylene signalling, hormones known to control cellular patterning from the meristem. In order to understand how MAP190 exerts its function in regulating meristem patterning, it is essential that we find out the molecular environment in which MAP190 works. So this leads to the fourth experiment where we will mutate the map190-1 mutant once again and look for new mutations that increase (enhancers) or decrease (suppressors) the severity of the appearance of the map190-1 mutant, and also identify any new map190 mutants. These experiments will allow us to identify and configure the MAP190 protein into a series of domains which are either essential or non-essential for its normal function. In all we have outlined a series of experiments that will help us to understand the role of MAP190 in regulating the activity of the root meristem in particular. In doing these experiments we have combined the expertise of two laboratories in Durham, one (PJH) whose interest is in the structure and regulation of the cytoskeleton and another (KL) whose interest is in how meristems function.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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