School of BioSciences - Theses

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    The Role of DEFECTIVE KERNEL1 (DEK1) in control of mechano-sensitive growth in Arabidopsis thaliana
    Amanda, Dhika ( 2016)
    During growth, plants experience mechanical stress from external and internal stimuli (Jaffe et al., 2002). These mechanical stimuli have been shown to play a crucial role in plant morphogenesis (Monshausen and Gilroy, 2009). For example, when plants are exposed to external mechanical stimuli, such as, wind, touching or rubbing, they tend to produce shorter and thicker stems, more roots, and increased rigidity of stems and roots (Liu et al., 2007). As plant cells are joined together by their cell walls, cells also experience internal mechanical pressure from their neighbours during growth. There is evidence that plants are able to perceive mechanical stimuli, transduce signals into cells, and respond to modify the cell wall (Braam, 2005). This is thought to be important to maintain coordinated growth and structural integrity during cell expansion. A putative cell wall integrity sensor is the plant specific phytocalpain, DEFECTIVE KERNEL1 (DEK1). DEK1 is a 240kDa modular protein located at the plasma membrane, with a large integral membrane region and a cytoplasmic calpain domain (Lid et al., 2002). In animals, calpains are a family of calcium-dependent cytosolic cysteine proteinases and have been reported to be involved in cell proliferation, apoptosis, differentiation, signal transduction, and also have been implicated in endocytosis, exocytosis, and intracellular membrane fusion (Wang et al., 2003). Previously it has been shown that overexpression of the CALPAIN domain of DEK1 results in plants with increased expression of cell wall-related genes and changes in plant morphology (Johnson et al., 2008). Reduced levels of DEK1 result in loss of adhesion between epidermal cells (Galletti et al., 2015). These features have led to the proposal that DEK1 acts as a putative cell wall mechano-sensor to promote cell wall re-modelling during growth. The general aim of this study was to investigate the signalling events downstream of DEK1 that result in changes to the cell wall and plant growth. Results of this research indicate that DEK1 acts as a novel regulator of plant growth and cell wall re-modelling in rosette leaves (Chapter 3) and stems (Chapter 4). In plants with increased levels of calpain (calpain oe), the timing of the transition from cell division to expansion was altered in rosette leaves, followed by a longer expansion phase resulting in enlarged cells. In contrast, an early exit from cell division occurs in plants with reduced levels of DEK1 (amiRNA-DEK1) contributes to smaller organ size. Changes in cell division and expansion also occurred in the stems of plants with altered DEK1. Increased number and size of cells in the cortex and pith were observed in calpain oe stems whereas a decrease in cortex and pith cell size was seen in amiRNA-DEK1 lines. Investigation of cellular morphology of the epidermal layer revealed defects in cell-cell contact in both calpain oe and amiRNA-DEK1lines. The altered contact zones most likely occur due to irregular size and shape of epidermal cells and changes in cell wall composition. Transmission electron microscopy (TEM) studies of walls in epidermal cells from rosette leaves and stems show increased and decreased wall thickness in calpain oe and amiRNA-DEK1, respectively. These results support previous findings by Johnson et al. (2008) that DEK1 is involved in regulating cell wall-related genes. A detailed analysis of cell wall composition was undertaken to determine if changes occur in calpain oe and amiRNA-DEK1 plants compared to wildtype (Chapter 3 and Appendix 1). No obvious changes were observed in monosaccharide and polysaccharide composition of walls whereas immunofluorescence, light and TEM studies revealed changes in the distribution and proportion of cell wall polysaccharides in the epidermal layer. Cellulose and pectin are the cell wall components most influenced by changes in DEK1, both in leaves and stem and suggest a role for DEK1 in the regulation of primary wall growth. Gene expression studies using an inducible calpain overexpression (ioex-calpain) construct showed genes involved in cell wall loosening and pectin biosynthesis were up-regulated. Based on these results, we suggest that the altered phenotypes observed in DEK1 mutants most likely arise from changes in cell wall composition that impact upon their mechanical properties.