ANALYSES OF CHLOROPLAST NUCLEOID AND CHLOROPLAST DNA-BINDING PROTEINS

Abstract

The organisation of chloroplast genes into nucleoids and chloroplast DNA (cpDNA) copy number are among the main factors influencing the expression, inheritance and function of cpDNA. Abnormal chloroplast nucleoids and non-optimal levels of cpDNA in the chloroplast result in an interruption of chloroplast development and function. The influence of chloroplast nucleoid proteins towards chloroplast DNA copy number, chloroplast nucleoid structure and chloroplast developmental biology is not fully understood. Research carried out in this thesis involved the study of the impact of reduction and overexpression of chloroplast nucleoid proteins on plant phenotype, cpDNA copy number and chloroplast nucleoid morphology. I studied plants in which various chloroplast nucleoid mRNAs were down-regulated via RNA interference (RNAi), namely the MAR-filament matrix protein 1 (MFP1), chloroplast DNA gyrase B (Gyrase B), RecA, MutS homolog 1 (MSH1), chloroplast DNA polymerase IB (NtPolI-like-1) and Whirly1, while the overexpressed genes were RecA fused with GFP and NtPolI-like-1 (transiently overexpressed). These proteins are actively involved in the DNA replication, repair and replication maintenance pathways of cpDNA. The implication of the down-regulation of these proteins is reported in the Chapter 3. The down-regulation of MFP1 in Nicotiana tabacum var. W38 gave rise to a wild-type like phenotype, suggesting a functional redundancy of MFP1. The down-regulation of MFP1 resulted in a lower cpDNA copy number, while down-regulation of Gyrase B and Whirly1 gave rise in between 0.5 to one fold higher than WT cpDNA copy number level. In order to combine the MFP1, Gyrase B and Whirly1 down-regulated genes, thirty crosses were made and the progenies were analysed. The progeny phenotypes were classified into 1) wild-type like phenotype, 2) severe phenotype and 3) variegated phenotype. The resistance of the down-regulated lines were tested against UV-C light stress. No significant changes in all irradiated lines were found except that a RecA deficient transgenic line was hypersensitive towards UV-C irradiation stress by forming extensive variegation on leaves. Transient overexpression of the chloroplast NtPolI-like-1 protein in Nicotiana benthamiana agroinfected leaf presented in Chapter 5 led to NtPolI-like-1 expression levels of 1.25% of total plant protein. The overexpression of NtPolI-like-1 in N. benthamiana leaves resulted in an increase of cpDNA copy number that was 1.5 fold higher than wild-type levels after three days post-agroinfection. The overexpression of NtPolI-like-1 showed that the protein levels of Whirly1, Gyrase B and Topoisomerase 2 were affected to various degrees. The attempt to isolate NtPolI-like-1 and interacted proteins was not successful due to the avidity and suitability of the primary antibody. To examine chloroplast nucleoid morphology and distribution in transgenic lines with down-regulated or overexpressing chloroplast nucleoid proteins, the chloroplast nucleoids were stained with 4′,6-diamidino-2-phenylindole (DAPI) and subjected to confocal laser scanning microscopy (CLSM). The CLSM results of the DAPI-stained chloroplast nucleoids are presented in Chapter 4. CLSM revealed variation in chloroplast nucleoid morphology and abnormalities in the down-regulated lines. The modification of chloroplast nucleoid DNA binding proteins affected chloroplast nucleoid morphology in terms of its structure and distribution. Lack of RecA in the chloroplast reduced the resistance of RecA deficient chloroplasts to UV-C genotoxicity effects, as the chloroplasts exhibited an aberrant shape and highly damaged nucleoid structure. To conclude, the results in this thesis indicate that the dynamicity of the chloroplast nucleoids is influenced by the chloroplast nucleoid protein levels. The cpDNA copy number level and chloroplast nucleoid topology are suggested to play important roles for optimum chloroplast and plant developmental biology.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anil Day (Supervisor) & Giles Johnson (Supervisor)