Kara M. Soule

Date of Award

12-2009

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Mary E. Rumpho

Second Committee Member

Carol Kim

Third Committee Member

Robert Gundersen

Abstract

Photosynthesis is comprised of tightly coupled reactions and therefore requires strict matrices of regulation, particularly involving alterations in gene expression and enzyme activity within the nucleus and plastid. Extensive research has been carried out on these light-regulated mechanisms in plants and green algae, however, much less is known in the red algal lineage, including heterokonts. The goal of this study was to investigate the influence of light on photosynthetic gene expression and select enzyme activity in the heterokont alga Vaucheria litorea and its symbiotic partner Elysia chlorotica, a photosynthetic sacoglossan mollusc (sea slug). Elysia chlorotica harbors V. litorea plastids intracellularly in its digestive epithelia. The plastids remain intact and carry out photosynthesis for several months despite the absence of any algal nuclei in the sea slug. This is remarkable considering that greater than ninety percent of the plastid proteome, the metabolic and regulatory components of the plastid, is encoded by the nuclear genome. In this study, the nuclear-encoded plastid-localized Calvin cycle enzyme phosphoribulokinase (PRK) was targeted for characterization because of the evidence supporting horizontal gene transfer of prk from the alga to the sea slug. PRK was demonstrated, in vivo, to react differently to redox conditions in crude extracts from light- vs. dark-treated V. litorea but not in E. chlorotica. However, redox regulation of PRK, in vitro, was observed in crude extracts of both organisms following treatment with various oxidizing and reducing agents. This study additionally characterized light regulation of gene expression in V. litorea and E. chlorotica through the quantitative analysis of nuclear and plastid gene expression. In V. litorea, nuclear (prk) and plastid (psaA, rbcL, psbA) transcript levels were quantified over a 12 hr light: 12 hr dark photoperiod. Maximal plastid- and nuclear-encoded transcript levels were observed in response to light, 2 hr and 6 hr post-illumination, respectively. Nuclear (prk) and plastid (psaA, rbcL) genes in E. chlorotica were quantified 1 hr before and 1 hr after illumination. In contrast to V. litorea, expression of the nuclear gene prk was down-regulated in E. chlorotica in response to illumination. Plastid gene transcript levels, however, were similarly up-regulated by light in the sea slug and alga, but on a much smaller scale in the sea slug. Overall these results imply that the nuclear-encoded factors responsible for redox regulation of nuclear-encoded proteins, such as PRK, in V. litorea are probably not present in the sea slug. This could be due to the absence of homologous proteins encoded by the sea slug and/or the lack of horizontal gene transfer from the alga. The factors regulating chloroplast gene expression, however, appear to be present and functional in the sea slug. These findings are discussed relative to the differences reported for light regulation of PRK gene expression and activity in the green vs. red lineages of algae and in the context of the sea slug-algal plastid symbiotic association. Additionally, these studies represent the first molecular analysis of photosynthetic gene expression and regulation in a photosynthetic mollusc.

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