Molecular Characterization of Arabidopsis Plastid Rhomboid Proteins
MetadataShow full item record
Rhomboid protease genes have been found in the majority of sequenced genomes. Rhomboid proteins control various cellular mechanisms such as cellular development, quorum-sensing, immune system evasion, and mitochondrial membrane remodelling. Although there are no verified rhomboid-mediated mechanisms within plants, the emerging situation appears to be complex. Investigations into an Arabidopsis At1g74130 knockout line in Chapter 2 revealed a novel phenotype for proteolytically inactive rhomboids. The phenotype displayed lower chlorophyll content, under developed chloroplasts, and plants containing one less leaf throughout development. In chapter 3, the expression levels were initially investigated in order to understand the relationship with At1g74130 and Arabidopsis development. This study revealed evidence for alternative splicing, containing their own relative expression levels between young and mature leaf tissue, as well as in response to altered Tic40 expression. Each variant contained a unique relative ability to interact with Tic40 (the only verified plant rhomboid substrate) using protein pull-down methods, and demonstrated an ability to alter Mgm1 (a yeast rhomboid substrate) cleavage ratios when expressed in yeast. Chapter 4 was designed to further investigate protein-protein interactions between At1g74130 and known substrates. While investigating the ability of variant proteins to interact with Tic40 and Mgm1, an apparent physiological role within bacterial and yeast cells was uncovered. Bacterial At1g74130 expression assays revealed decreased β-lactimase secretion. With limited active β-lactimase protein, E. coli cells were observed to have increased sensitivity to ampicillin. Similarly, in S. cerevisiae cells, the expression of At1g74130 increased sensitivity to the fungicide Nystatin. The increased sensitivity within both bacterial and yeast cells were also observed through exogenously applied At1g74130 extract cocktails. Chapter 5 revealed that the Arabidopsis plastid rhomboid gene, At1g25290, also contains alternative splicing. The two transcripts that were discovered have altered relative expression in response to development and manipulated Tic40 levels. The variant At1g25290 protein contains an introduced RVL motif. Chapter 6 was designed to survey model species databases in order to uncover evidence for alternative splicing within rhomboid transcripts. This chapter suggests alternative splicing may serve a means to diversify the functions of rhomboid proteins. This thesis comprises the majority of known plant rhomboid-mediated processes.