Control of chloroplast movement in epidermal pavement cells is dependent on CHUP1 and the cytoskeleton

Abstract

Chloroplasts are a critical element for maintaining plant health. They are involved in countless cellular processes, from cellular stress responses, light-based responses, and even innate immunity. Additionally, chloroplasts have tubular extensions called stromules that have been linked to transmission of chloroplast generated signals. Despite stromules being implied to have a role in the transmission of vital chloroplast generated signals, they have not been heavily studied as to how they relate to chloroplast movement, or how stromules movement itself is regulated. In this dissertation, we found that stromules are significantly influenced by the cytoskeletal elements, and more specifically that their dynamism depends on the state of the microtubule filaments. Through stabilizing microtubules by silencing γ -Tubulin Complex Protein (GCP4), leading to bundling, we are able to observe a change in stromule dynamics that is directly tied to the changes in the microtubule filaments (MT). Additionally, we found that there is a clear connection between chloroplast movement and stromules by identifying a form of epidermal chloroplast movement called stromule directed movement (SDM), as well as establish a stromule analysis pipeline allowing for the collection of far greater and far more accurate measurements of stromule characteristics. ☐ Chloroplast unusual positioning 1 (CHUP1) is a chloroplast outer membrane protein that has been shown to be associated with chloroplast movement. Most significantly, CHUP1 has been shown to play a major role in chloroplasts binding to the plasma membrane, as well as being required for proper chloroplast positioning and light mediated movement in mesophyll cells. It is even thought that CHUP1 might be a source of motive force for chloroplast movement, through its interactions with chloroplastic actin (CP-actin). The hypothesis that was tested in the work of this dissertation was that CHUP1 acts as a negative regulator of chloroplast movement, and that silencing CHUP1-independently alters stromule and chloroplast movement dynamics. In addition, this work found that examination of epidermal chloroplasts allows for a broader and more complete understanding of chloroplast movement dynamics, stromule movement dynamics, as well as examining the function of proteins that have already been heavily studied in the mesophyll. This is supported by showing that in epidermal chloroplasts, CHUP1-silencing amplifies chloroplast movement, which is not the case in mesophyll chloroplasts. Additional work was done to show that CHUP1-silencing amplifies Reactive Oxygen Species (ROS), which induces stromules and amplifies effector-triggered immunity (ETI), but is not responsible for the changes in stromule or chloroplast movement dynamics caused by CHUP1-silencing. The results of this work show that CHUP1-silencing amplifies stromule and chloroplast responses that are associated with effector-triggered immunity. Furthermore, this works shows that that blue light mediated movement still occurs in epidermal chloroplasts, and shows that PHOTOTROPIN1 (PHOT1) might play a role in the regulation of stromules, while also functioning independently of CHUP1. ☐ With those results and more, I show that examining chloroplast dynamics only in the context of a single cell type leads to a limited understanding, and that by examining across various cell types, in which behaviors and cellular conditions might change, we are able to reveal new possibilities that will expand our understanding even further.