ORCID

http://orcid.org/0000-0003-1283-868X

Date of Award

Winter 12-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Plant & Microbial Biosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Pollen, the male gametophyte of flowering plants, delivers the sperm cells to the ovule to carry out sexual reproduction. During this process, the pollen grain undergoes dramatic physical changes. Survival requires careful control of cell mechanics, particularly the balance between protoplast expansion and cell wall resistance. One control mechanism is the use of a mechanosensitive (MS) ion channel, MscS-Like (MSL)8. This pollen-specific protein was previously shown to be essential for pollen survival during hydration and was proposed to function as a tension-gated osmoregulator. However, direct proof of osmoregulation during initial hydration has not yet been found. In fact, studies of the role in plants have suggested that there are functions of MSL proteins beyond osmotic regulation, such as MSL10 which functions to promote programmed cell death signaling, particularly after a hypo-osmotic shock.In this work, mathematical modeling alongside in vitro experiments show that MSL8 is likely not acting via osmoregulation during early hydration. Instead, we conclude that channel function stiffens the cell wall, thus preventing overexpansion. This follows the previously published finding that there are changes in the cell wall (callose) of pollen tubes lacking MSL8. However, an initial survey performed here did not identify any cell wall-related mutants with an msl8-5-like hydration phenotype. Moreover, attempts to understand MSL8 protein interactions hit a roadblock due to strong induction of cell death when MSL8 is highly expressed ectopically. MSL10 – which is known to induce programmed cell death – did not rescue the reduced viability of msl8-5 pollen when expressed under the MSL8 promoter. This supports the idea that it is not a universal channel or signaling function inherent to MSLs that is maintaining pollen mechanics. In fact, it is likely the MSL8 channel function, not a separate signaling function, that is lethal to the plant. Ectopic expression of a previously published variant of MSL8 with a “blocked” channel does not result in strong induction of death and MSL8 with a constitutively open channel causes near-complete obliteration of male fertility. In the last chapter, the structure and function of the N-terminus of MSL8 was examined. We found that this domain of the protein, similar to other MSL proteins, is predicted to be intrinsically disordered. Purification of the MSL8 N-terminus found that it readily phase separates even in high salt and at low concentrations. This may allow for regulation of the protein in the pollen grain as it transitions from desiccated (high osmolyte concentration) to hydrated (low osmolyte concentration) conditions. Overall, these findings indicate that MSL8, contrary to our original hypothesis, may be functioning and regulated in pollen using mechanisms beyond tension-gated osmoregulation.

Language

English (en)

Chair and Committee

Elizabeth S Haswell

Committee Members

Anders Carlsson

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