Date of Award

12-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Andreas Nebenführ

Committee Members

Bruce D. McKee, Beth Mullin, Albrecht G. von Arnim, Joseph Williams

Abstract

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which non-motile sperm cells are delivered to ovules thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth depends on actin filaments. Plants encode class VIII and class XI myosins as actin-based motor proteins, of which class XI myosins are required for cell expansion in vegetative tissues.

In Arabidopsis thaliana, 6 of 13 myosin XI genes are expressed in pollen: XIA, XIB, XIC, XID, XIE, and XIJ. Initially, two artificial microRNA constructs were designed to target multiple class XI myosins; however, plants expressing the artificial microRNAs had no reduction in overall fertility and only a slight reduction in root hair elongation. Therefore, to explore the functions of individual pollen myosins, homozygous T-DNA insertion mutants were isolated for all six pollen myosin genes. Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, xic xie pollen tubes grew more slowly than WT pollen tubes, which resulted in reduced fitness compared to WT and a drastic reduction in seed set. Organelle motility was significantly reduced in xic xie pollen tubes; however, vesicle accumulation and actin filament dynamics were not altered in the double mutant. Thus, it remains unclear how reduced organelle motility in xic xie pollen tubes leads to a slower growth rate.

A novel role of myosin XI in pollen germination was also revealed in this study. Pollen from mutants of XIJ, the only short-tailed myosin XI in Arabidopsis, germinated poorly in vitro. This in vitro pollen germination defect was rescued by the addition of diffusible components from female tissues. Interestingly in pollen grains, YFP-XIJ and YFP-XIA localized to the future site of germination, suggesting a role in pollen germination for multiple myosin XI isoforms. In summary, this study provided the first direct evidence that class XI myosins are involved in pollen tube growth and pollen germination.

Movie 2.1.avi (42038 kB)
Example gravitropism experiment with WT grown on dexamethasone

Movie 4.1.mov (38 kB)
YFP-XIA localized to the tip of growing pollen tubes

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YFP-XIJ localized to the tip of growing pollen tubes

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YFP-XIA localization during 100 nM latrunculin B treatment

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YFP-XIA localization during 1 μM latrunculin B treatment

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YFP-XIJ localization during 100 nM latrunculin B treatment

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YFP-XIJ localization during 1 μM latrunculin B treatment

Movie 4.7.mov (236 kB)
YFP-XIA localization during mock DMSO treatment

Movie 4.8.mov (420 kB)
YFP-XIJ localization during mock DMSO treatment

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Growing pollen tubes expressing CFP-RabA4d and YFP-XIA

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Growing pollen tubes expressing CFP-RabA4d and YFP-XIJ

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YFP-XIA localization during brefeldin A treatment

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YFP-XIJ localization during brefeldin A treatment

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YFP-XIA localization during mock ethanol treatment

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YFP-XIJ localization during mock ethanol treatment

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Accumulation of YFP-RabA4d in growing WT pollen tubes

Movie 4.16.mov (1151 kB)
Accumulation of YFP-RabA4d in growing xic-1 xie-1 pollen tubes

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Accumulation of YFP-RabA4d in growing xij-1 pollen tubes

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Peroxisome movements in WT pollen tubes

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Golgi stack movements in WT pollen tubes

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Peroxisome movements in xic-1 xie-1 pollen tubes

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Golgi stack movements in xic-1 xie-1 pollen tubes

Movie 4.22.mov (969 kB)
Peroxisome movements in xij-1 pollen tubes

Movie 4.23.mov (1054 kB)
Golgi stack movements in xij-1 pollen tubes

Movie 4.24.mov (572 kB)
YFP-FABD2 labeled actin filaments in WT pollen tubes

Movie 4.25.mov (1069 kB)
YFP-FABD2 labeled actin filaments in xic-1 xie-1 pollen tubes

Movie 4.26.mov (846 kB)
YFP-FABD2 labeled actin filaments in xij-1 pollen tubes

Movie A.1.mov (13484 kB)
Tobacco BY-2 cells expressing ST-CFP and Man1-YFP during monensin treatment

Movie A.2.mov (2024 kB)
Tobacco BY-2 cells expressing ST-CFP and XT-YFP during monensin treatment

Movie A.3.mov (1576 kB)
Tobacco BY-2 cells expressing ST-CFP and Man1-YFP during simultaneous image capture

Movie A.4.mov (227 kB)
Tobacco BY-2 cells expressing ST-CFP and Man1-YFP during brefeldin A treatment

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