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Characterization of Auxin Receptors and Auxin-Ethylene Interaction During Pea Fruit Development
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- Author / Creator
- Jayasinghege, Charitha P. A.
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Normal development of a pea (Pisum sativum L.) fruit requires the presence of seeds within the fruit. Removal of seeds from young pea fruits results in slowing of pericarp (fruit) growth and subsequent abscission. Indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA) are two types of naturally occurring auxins in pea. 4-Cl-IAA can mimic the presence of seeds by stimulating deseeded pericarp growth, but IAA does not. Differential interactions of the two auxins with gibberellin and ethylene are at least partially responsible for their differential action on pericarp growth, but the underlying mechanisms are not completely understood. In this thesis, experiments were designed to further understand seed and auxin regulation of pericarp ethylene biosynthesis and signaling pathways and to examine the relationship between auxin (IAA and 4-Cl-IAA) effects on pericarp growth and the auxin receptors within this tissue. Effects of seeds and the two auxins on pericarp ethylene biosynthesis were evaluated by analyzing the 1-aminocyclopropane-1-carboxylic acid synthase (PsACS1, PsACS2 and PsACS4; enzyme converts S-adenosyl-Met to ACC) and ACC oxidase (PsACO1, PsACO2 and PsACO3; enzyme converts ACC to ethylene) gene transcript abundances, the ethylene precursor ACC level, and the ACO enzyme activity in deseeded pericarps and deseeded pericarps treated with IAA or 4-Cl-IAA. The results showed that the two auxins regulate pericarp ethylene biosynthesis in distinctly different manners. 4-Cl-IAA strongly stimulated pericarp PsACS1 and PsACO1 transcript levels and mimicked the role of seeds in maintaining low levels of pericarp PsACS4, PsACO2 and PsACO3 transcripts. IAA had low or no effect on modulating pericarp PsACS or PsACO transcript levels compared to 4-Cl-IAA. Associated with these PsACS and PsACO transcript abundance patterns, IAA and 4-Cl-IAA differentially modulated pericarp ACC level and ACO enzyme activity. Transcript abundance of the genes that code for the ethylene receptors PsERS1 and PsETR2, and signaling-related proteins PsEBF1 and PsEBF2, which act as negative regulators of ethylene signaling, increased in response to ethylene. Application of 4-Cl-IAA, but not IAA, increased the transcript abundance of those genes indicating that the suppression of ethylene signaling through the stimulation of ethylene receptor or EBF gene transcript abundance could be associated with the ethylene action inhibitory effect of 4-Cl-IAA. The relationship between the auxin (IAA and 4-Cl-IAA) effects on pericarp growth and the auxin receptors within the pericarp tissue was examined by functionally characterizing selected pea auxin receptors in Arabidopsis and relating the transcript abundance patterns of pea TIR1/AFB family members to pollination status, seed removal and to auxin and ethylene treatments. Functional characterization of PsTIR1a and PsTIR1b in Arabidopsis tir1-10 and Attir1-10 afb2-3 auxin receptor mutants indicates that they code for functional auxin receptors and that they mediate the comparatively stronger auxin effect of 4-Cl-IAA compared to that of IAA. Expression of PsAFB2 in Attir1-10 afb2-3 double mutants indicates that PsAFB2 is also a functional auxin receptor. The transcript profiles of five pea TIR1/AFB family gene members (PsTIR1a, PSTIR1b, PsAFB2, PsAFB4 and PsAFB6) in fruit tissues during early development suggested that the expression of these auxin receptors is developmentally regulated. Expression of PsTIR1b and PsAFB6 was hormone regulated, where developing seeds and 4-Cl-IAA suppressed PsTIR1b and PsAFB6 transcript accumulation, and ethylene stimulated PsAFB6 transcript accumulation in the pericarps. Expression of the auxin-responsive DR5::GUS reporter construct and auxin content in the pericarps was related to auxin receptor transcript abundance, which suggested that the modulation of pericarp auxin level and the auxin sensitivity could be important in regulating the auxin action in developing pea fruit. Overall, the ability of 4-Cl-IAA but not IAA to mimic the presence of seeds in stimulating pericarp development is associated with their differential effects on ethylene biosynthesis, ethylene signaling, and the auxin receptor abundance. Common receptors likely perceive the two auxins, but the modulation of auxin receptor abundance and the possible differences in IAA and 4-Cl-IAA affinities to those receptors may initiate differential auxin effects.
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- Graduation date
- Spring 2017
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.