Abstract
Peroxisomes are highly dynamic and multifunctional organelles essential to development. Plant peroxisomes accommodate a multitude of metabolic reactions, many of which are related to the β-oxidation of fatty acids or fatty acid-related metabolites. Recently, several dozens of novel peroxisomal proteins have been identified from Arabidopsis (Arabidopsis thaliana) through in silico and experimental proteomic analyses followed by in vivo protein targeting validations. To determine the functions of these proteins, we interrogated their transfer DNA insertion mutants with a series of physiological, cytological, and biochemical assays to reveal peroxisomal deficiencies. Sugar dependence and 2,4-dichlorophenoxybutyric acid and 12-oxo-phytodienoic acid response assays uncovered statistically significant phenotypes in β-oxidation-related processes in mutants for 20 of 27 genes tested. Additional investigations uncovered a subset of these mutants with abnormal seed germination, accumulation of oil bodies, and delayed degradation of long-chain fatty acids during early seedling development. Mutants for seven genes exhibited deficiencies in multiple assays, strongly suggesting the involvement of their gene products in peroxisomal β-oxidation and initial seedling growth. Proteins identified included isoforms of enzymes related to β-oxidation, such as acyl-CoA thioesterase2, acyl-activating enzyme isoform1, and acyl-activating enzyme isoform5, and proteins with functions previously unknown to be associated with β-oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein1, Betaine aldehyde dehydrogenase, and Unknown protein5. This multipronged phenotypic screen allowed us to reveal β-oxidation proteins that have not been discovered by single assay-based mutant screens and enabled the functional dissection of different isoforms of multigene families involved in β-oxidation.
Highlights
Peroxisomes are highly dynamic and multifunctional organelles essential to development
To investigate the role of recently identified and uncharacterized peroxisomal proteins in peroxisomal physiology, we took a reverse genetic approach by searching The Arabidopsis Information Resource database for transfer DNA (T-DNA) insertion lines. Most of these proteins were identified from our own proteomic analyses of Arabidopsis peroxisomes from green leaves and etiolated seedlings (Reumann et al, 2009; Quan et al, 2013), and some were identified by other research groups
These included Calcium-dependent protein kinase1 (CPK1) and Hydroxy-acid oxidase isoform2 (HAOX2), which function in innate immunity (Coca and San Segundo, 2010; Rojas et al, 2012), 1,4-dihydroxy-2-naphthoyl (DHNA)-CoA thioesterase1 (DHNAT1), which is involved in DHNA-CoA hydrolysis in phylloquinone biosynthesis (Widhalm et al, 2012), Acetoacetyl CoA thiolase1 (AACT1), which is responsible for the condensation of two acetyl-CoA molecules to form acetoacetyl CoA (Jin et al, 2012), and Copper amine oxidase3 (CuAO3) in PA catabolism (Planas-Portell et al, 2013)
Summary
Peroxisomes are highly dynamic and multifunctional organelles essential to development. Proteins identified included isoforms of enzymes related to b-oxidation, such as acyl-CoA thioesterase, acyl-activating enzyme isoform, and acyl-activating enzyme isoform, and proteins with functions previously unknown to be associated with b-oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein, Betaine aldehyde dehydrogenase, and Unknown protein5 This multipronged phenotypic screen allowed us to reveal b-oxidation proteins that have not been discovered by single assay-based mutant screens and enabled the functional dissection of different isoforms of multigene families involved in b-oxidation. After the identification of peroxisomal proteins from etiolated Arabidopsis seedlings through proteomics and in vivo protein-targeting analysis, we used reverse genetics to analyze the mutants of five newly identified proteins and revealed the role of a Cys protease, RESPONSE TO DROUGHT21A-LIKE1, in seed germination, b-oxidation, and stress response (Quan et al, 2013; Cassin-Ross and Hu, 2014). Many other recently identified peroxisomal proteins have not been characterized with respect to their functions in peroxisomal physiology and plant development
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