Author ORCID Identifier

https://orcid.org/0000-0001-8659-7407

Date Available

5-31-2021

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Matthew Shawn Gentry

Abstract

Starch and glycogen are an essential component for the majority of species and have been developed to maintain homeostasis in response to environmental changes. Water-soluble glycogen is an excellent source of quick, short-term energy in response to energy demands. In contrast, plants and algae have developed the macromolecule starch that is elegantly suitable for their dependence on external circumstances. Semi-crystalline starch is water-insoluble and inaccessible to most amylolytic enzymes, thus plants and algae have developed a coordinated system so that these enzymes can gain access to the denser starch energy cache. Starch-like semi-crystalline polysaccharides are also found in red algae, termed floridean starch, and are located outside the plastid in the cytosol. Floridean starch resembles a unique class of polyglucans, intermediate of higher plant starch and mammalian glycogen. Reversible glucan phosphorylation is essential in facilitating normal degradation of starch in many higher plants. However, there is a knowledge gap in regards to this process in other starch-containing organisms such as algae. The relationship between phosphorylation and dephosphorylation activity on the structural consequences of starch are still in their infancy as well.

One such organism that produces floridean starch is the thermophilic red microalga, Cyanidioschyzon merolae, which has been rapidly advancing as a model organism. Several studies have shown that C. merolae contains a minimal set of genes required to metabolize a semi-crystalline carbohydrate called semi-amylopectin. Amongst this conservative set of genes, we identified a single glucan phosphatase (laforin) and a putative glucan dikinase (GWD), suggesting that reversible glucan phosphorylation may also be present in C. merolae as a means to metabolize their ‘floridean starch’. Therefore, we proposed that the genetically manipulatable C. merolae provides an excellent model organism to study the basic functions of enzymes involved in reversible glucan phosphorylation and how they affect the main constituent of starch.

Our work is the first to show specific effects of reversible glucan phosphorylation in a red algal system. In addition, a sole glucan dikinase (GWD) and phosphatase (laforin) are responsible for phosphorylation and dephosphorylation of semi-amylopectin type floridean starch in C. merolae. They both are highly specific to the C6-hydroxyl of glucose moieties of semi-amylopectin and the loss of either enzymatic activity significantly affects the fine structure of amylopectin and thus granule morphology. Loss of C6-phosphate content of semi-amylopectin in Dgwd lines results in suboptimal organization of semi-amylopectin indicating that C6-phosphate is required for proper synthesis and degradation in C. merolae. In the case of Dlaforin lines, without proper maintenance of C6- phosphate, too much C6-phosphate content can equally be as detrimental to amylopectin organization and thus plant vitality. Proper packing of amylopectin likely has direct biological effects in C. merolae as seen through prolonged energy deprivation. Loss of GWD or laforin in C. merolae resulted in excessive nutrient-scavenging which led to the depletion of critical photosynthetic pigments required to recover cell proliferation upon reintroduction of light.

These studies highlight the critical function and conservation of reversible glucan phosphorylation and its effect on starch structure in C. merolae.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.216

Funding Information

Max Steckler Award, University of Kentucky, 2018

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