Dissertation
Characterization of Arabidopsis long-chain Acyl-coenzyme A synthetases involved in cuticle development
Washington State University
Doctor of Philosophy (PhD), Washington State University
08/2009
DOI:
https://doi.org/10.7273/000006166
Abstract
The cuticle is a continuous extracellular layer deposited on the aerial surfaces of terrestrial plants. The important functions of the cuticle include reduction in non-stomatal water loss and protection against pathogen attacks. Cutin and cuticular wax, both synthesized in the epidermal cells, are major components of the cuticle. Long-chain acyl-CoA synthetase 2 (LACS2) has been proved to be required for cutin biosynthesis, by characterization of lacs2 null mutants. In this study, the subcellular localization and in vitro activity of LACS2 were investigated to better understand the function of LACS2. A green fluorescent protein-LACS2 fusion protein was localized to the endoplasmic reticulum. The recombinant hexahistidinetagged LACS2 protein showed high enzymatic activity using cutin monomers such as 16- hydroxypalmitate and three long-chain dicarboxylic acids. LACS1 is also highly transcribed in stem epidermal cells, as observed by a transcription profiling analysis. To study the function of LACS1 in vivo, a reverse genetics approach was used. lacs1 null mutants had the glossy stem phenotype typical of mutants with defects in cuticular wax biosynthesis. In lacs1 stems, the amounts of three predominant wax constituents - alkane, secondary alcohol and ketone with 29-carbon chains - were reduced by 36%, 45% and 37%, respectively, whereas the level of the precursor, a 30-carbon saturated fatty acid, was elevated over ten-fold. As such, LACS1 is an acyl-CoA synthetase that activates very-long-chain fatty acids for modification and use in cuticular wax biosynthesis. To assess the contribution of wax and cutin in cuticle development and function, lacs1 lacs2 double mutants were generated. lacs1 lacs2 plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set that neither lacs1 nor lacs2 single mutants had. lacs1 lacs2 had higher cuticular permeability and was more susceptible to drought stress than either of its parental lines. Taken together, these results extend the understanding of the function of LACS2 in cutin biosynthesis, provide evidence for the involvement of LACS1 in cuticular wax biosynthesis, and suggest that both cutin and cuticular wax are required for maintaining the integrity and functions of the cuticle.
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Details
- Title
- Characterization of Arabidopsis long-chain Acyl-coenzyme A synthetases involved in cuticle development
- Creators
- Hua Weng
- Contributors
- John Anthony Browse (Chair)Bernd M. Lange (Committee Member) - Washington State University, Institute of Biological ChemistryMICHAEL L. KAHN (Committee Member) - Washington State University, Institute of Biological ChemistryThomas W Okita (Committee Member) - Washington State University, Institute of Biological Chemistry
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 136
- Identifiers
- 99901055020801842
- Language
- English
- Resource Type
- Dissertation