Identifier

etd-08182013-213856

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Late Embryogenesis Abundant (LEA) proteins are highly hydrophilic, intrinsically disordered proteins whose expression has been correlated with desiccation tolerance in anhydrobiotic organisms. Embryos of the brine shrimp, A. franciscana, contain high titers of group 3 LEA proteins during desiccation-tolerant stages such as diapause and pre-emergence development. Here I report the sequencing of three novel variants of AfrLEA3m mRNA (Afrlea3m_47, Afrlea3m_43 and Afrlea3m_29), whose deduced protein sequences are are predicted to localize to the mitochondrion. These mRNAs are very similar to Afrlea3m, but each has a stretch of sequence that is absent in at least one of the others. In addition Afrlea3m_43 has five single nucleotide changes scattered across its sequence, and Afrlea3m_47 and Afrlea3m_43 have three single nucleotide differences in the section of sequence shared only by these two variants. Protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29 is highest in diapause embryos and decreases throughout development to their lowest levels in desiccation-sensitive nauplius larvae. This pattern of protein expression is in agreement with previously reported mRNA expression for AfrLEA2 and AfrLEA3m and supports a role for LEA proteins in desiccation tolerance of embryos. When adjustment is made for mitochondria matrix volume, the effective concentrations of cytoplasmic versus mitochondrial group 3 LEA proteins are similar in vivo, and the values provide guidance for the design of in vitro functional studies with these proteins. Investigations of protein secondary structure show AfrLEA2 and AfrLEA3m to be intrinsically disordered in solution and that they gain structure during desiccation and in the presence of the solvents TFE and SDS. I also show that during drying recombinant AfrLEA2 and AfrLEA3m confer protection to three desiccation-sensitive enzymes (lactate dehydrogenase, phosphofructokinase and citrate synthase). The degree of protective ability was found to depend on the target enzyme chosen. The strongest degree of stabilization was observed when a given LEA protein was used in the presence of the stabilizing sugar trehalose, which is naturally accumulated by A. franciscana embryos. Finally, AfrLEA2 is shown by immunohistochemistry to reside in the cytoplasm and nucleus of embryonic cells of A. franciscana, and the AfrLEA3m proteins are localized to the mitochondrion. The presence of LEA proteins in multiple subcellular compartments suggests a requirement to protect biological structures in many areas of a cell in order for an organism to survive desiccation stress.

Date

2013

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Hand, Steven

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