Statistical analysis

A one-tailed student T-test was performed using the Data Analysis ToolPak in Microsoft Excel 2016. Prior to this an F-Test was performed to compare whether the variance of each sample was equal or unequal. A one-tailed T-test was performed, assuming equal or unequal variance as appropriate. Significance levels were set at 0.05.

Single-factor ANOVA

Single-factor ANOVA was performed using IBM SPSS Statistics for Windows (Copyright ©IBM Corporation and its licensors 1989, 2016. Version 24.0) Ar- monk, NY:IBM Corp. The significance level was set to 0.05. The Levene statistic was used to assess the variance. From the ANOVA, the F critical value was com- pared to the F value and if smaller, the null hypothesis was rejected. If the null hypothesis was rejected, then a Tukey HSD post-hoc test was performed to assess the significance of pairwise differences.

Two-factor ANOVA

Univariate two-factor ANOVA was performed using IBM SPSS Statistics for Win- dows (Copyright ©IBM Corporation and its licensors 1989, 2016. Version 24.0) Armonk, NY:IBM Corp. The significance level was set to 0.05. From the ANOVA, the F critical value was compared to the F value and if smaller, the null hypoth- esis was rejected. If the null hypothesis was rejected, then multiple Tukey HSD post-hoc tests were performed to assess the significance of pairwise differences, both within groups and between groups.

Chapter 1

Introduction

1.1 The plant endoplasmic reticulum

Inside all eurkaryotic cells there are membrane-bound organelles that perform vital functions. The endoplasmic reticulum (ER) is the largest of these organelles (Chen et al., 2012a). It was first discovered in 1945, when a group identified “delicate lace-work” inside the cytoplasm of a fibroblast-like cell (Porter et al., 1945), although it was not given the name ‘ER’ until 1948 (Porter and Thompson, 1948).

The ER is the site of protein production for proteins that need to be secreted outside of the cell or for proteins that reside in membranes around the cell. Newly transcribed RNA leaves the nucleus through the nuclear pores and attaches to ribosomes for translation. For secretory and membrane proteins the ribosomes bind, via interaction of the protein’s signal peptide with SRP (signal recognition particle), to the SRP receptor integrated within the ER membrane. A translocon in the ER membrane allows the newly translated protein to enter into the ER lumen, where it is folded, or inserted into the ER membrane itself Lodish et al. (2007).

Protein production and assembly are assessed by the quality control pathway. When an increase in mis-folded or unfolded proteins is detected, the UPR (un- folded protein response) is activated (Fanata et al., 2013; Wan and Jiang, 2016; Bao and Howell, 2017). The UPR helps to protect the cell from external and internal stress, such as viral infection, lack of nutrients, genetic mutations and loss of calcium (Fanata et al., 2013). The ER is also the site of the production of lipids, which are used by other membranes in the cell (Chen et al., 2012a; Stefano et al., 2014a). Moreover, the ER stores calcium, used in signalling events, and plant hormones, such as auxin and ethylene (Stefano et al., 2014a).

lipids and calcium around the cell (Chen et al., 2012a). There are fast moving areas, in which active streaming takes place, and smaller, slower movements where tubules disconnect and reconnect with other areas of the network. In mammalian cells, ER movement is linked to microtubules. In plants however, ER movement is predominately due to the actin-myosin cytoskeleton (Runions et al., 2006; Sparkes et al., 2009a). Actin monomers form polymerised chains to create actin filaments, which can grow and shrink in length. InArabidopsis thaliana there are 17 myosin genes, (4 in class VIII and 13 in class XI) (Reddy and Day, 2001). Class XI myosins are implicated in organelle movement (Reddy and Day, 2001). Using ATP hydrolysis the mysosin heads can pull on the actin filament, thus moving along the filament. As the plant myosin XI is similar to mammalian myosin V Sparkes (2011), it is likely that the double-headed myosin ‘walks’ along the actin filament in a stepwise manner. The myosin attaches, through its tail domain, to the ER membrane via an intermediary protein Sparkes et al. (2009a); Sparkes (2011). The dependence on actin and myosin XI for ER movement was shown when actin chains were depolymerised (by the addition of lactrunculin B) and the ER stopped remodelling (Runions et al., 2006; Sparkes et al., 2009a). It has been shown however, that during mitosis, when the cells are dividing, that the ER movement is instead dependent on microtubules (Sparkes et al., 2009a). There are also stable points of the ER (Sparkes et al., 2009a), including spe- cific contact sites where the ER connects to the plasma membrane (PM). In mammalian and yeast cells this has been well investigated, and protein com- plexes at the contact sites have been discovered (Stefan et al., 2013). Though little work has been done on plant contact sites (Chen et al., 2012a), there are some proteins (such as VAPs (VAMP-associated protein, where VAMP stands for vesicle-associated membrane protein) and SYTs (synaptotagmins)) that have been implicated in ER-PM connections (Prez-Sancho et al., 2016).

Plants also have plasmodesmata, where ER (in the form of a desmotubule), PM and cytosol (as a cytoplasmic sleeve) pass through the cell walls into adjoining cells. These plasmodesmata allow the transfer of proteins, hormones and RNA molecules (William J. Lucas, 2009). The permeability of the plasmodesmata is controlled and dynamic, with three different states; the dilated state allows large molecules to pass though (subject to the size exclusion limit), the open state allows molecules smaller than 1 kDa to diffuse freely and the closed state will not allow any traffic through the plasmodesmata Sevilem et al. (2013). During the dilated state even a protein as large as GFP, at 27 kDa, has been proven to be able to pass between cells through the plasmodesmata Crawford and Zambryski (2000). Figure 1.1 highlights the organelles within a plant cell.

Figure 1.1: Plant cell with labelled organelles. The ER is continuous with the nuclear envelope and although not depicted here forms desmotubules which pass through the plasmodesmata. In most plant cells the vacuole occupies 70-90% of the space in the cell, pushing the ER to the edges of the cell. Image from figure 6.8c of Biology 8th Edition, Campbell, published by Pearson Benjamin Cummings, Copyright (2008). Labels added by thesis author.