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Regulation of nuclear morphology in plants results from signaling and physical interactions that span the nuclear envelope. A key regulatory component is a family of Nuclear Matrix Constituent Proteins (NMCPs) that form the plant nuclear lamina. Arabidopsis thaliana contains four NMCP-like proteins, called CROWDED NUCLEI (CRWN). CRWN proteins are necessary for nuclear size and shape maintenance in differentiated cells, where CRWN1 acts as the chief regulator followed by CRWN4. However, the coordination of activities among these paralogous proteins remains unclear. I surveyed nuclear CRWN abundance within crwn single and double mutants using protein immunoblots. I found that plants lacking either CRWN1 or CRWN3 showed a decrease in CRWN4 abundance. Further, loss of CRWN1 along with either CRWN2 or CRWN3 led to a significant reduction in CRWN4 protein levels in the nucleus. Based on these data, the stability of CRWN4 in the nucleus is proposed to depend on an interaction with CRWN1, CRWN2 and CRWN3. Additionally, a functional nuclear localization sequence was uncovered in CRWN4 based on the discovery of the crwn4-2 missense allele. A suppressor mutation within IMPORTIN-alpha (IMPA-1) restores the level of CRWN4 in crwn4-2 nuclei, demonstrating that CRWN4 uses canonical nuclear import machinery. To expand our knowledge of CRWN function in a species with a larger, more complex genome, I utilized CRISPR/Cas9 technology to knock out the two CRWN1 and one CRWN4 orthologs in tomato. I recovered viable homozygous mutants for both CRWN1 orthologs that exhibited smaller, spherical nuclei compared to wild type. Conversely, a homozygote for the CRWN4 ortholog was not viable. These results confirm the importance of CRWN proteins for nuclear architecture in plants, and demonstrate that there are differences among species in their response to a deficiency in NMCP2 function. Finally, to investigate the effects of nuclear structure on various stress response pathways, I moved back to Arabidopsis to assess germination rates of nuclear morphology mutants (crwn and kaku) in response to different types of abiotic stresses. The kaku mutants were found to be more sensitive to salt and osmotic stresses than the crwn single mutants. Also, in response to exogenous ABA, the phytohormone abscisic acid, I was able to replicate previous data presenting the hypersensitivity of the crwn1 crwn3 double mutant. However, crwn3 and two kaku mutants tested (kaku1 and kaku4) also exhibited sensitivity to ABA, albeit less than the double mutant. Taken together, my research sheds light on how CRWN proteins cooperate to form the plant nuclear lamina, and highlight some important whole-plant responses to defects in nuclear structure.

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Genetics; Molecular biology; CRWN/NMCP; nuclear lamina; nuclear morphology; plant nucleus; Plant sciences
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Richards, Eric Jean
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Liu, Jun
Roeder, Adrienne H.K.
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Genetics, Genomics and Development
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Ph.D., Genetics, Genomics and Development
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Doctor of Philosophy
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Government Document
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dissertation or thesis
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