Systematic Phenotypic Screen of Arabidopsis Peroxisomal Mutants Identifies Proteins Involved in β-Oxidation    

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 b-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 b-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 b-oxidation and initial seedling growth. Proteins identified included isoforms of enzymes related to b-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 b-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 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.

Plant peroxisomes: A nitro-oxidative cocktail

“…Thus, fatty acids enter peroxisomes via ATP-binding cassette (ABC) transporters of subfamily D. They are then oxidized to fatty acetyl-CoA in peroxisomes and shortened by two carbons in each β-oxidation cycle. Finally, acetyl-CoA is converted to four-carbon molecules by the glyoxylate cycle, which then undergo gluconeogenesis in the mitochondrion and cytosol to provide energy for seedling development [14], [5], [78]. However, H 2 O 2 , which acts as a priming factor involving specific changes at the proteome, transcriptome and hormonal levels, is not simply a byproduct of peroxisomal β-oxidation [114], [6], [83].…”

Plant peroxisomes: recent discoveries in functional complexity, organelle homeostasis, and morphological dynamics

“…Systematic phenotypic screening has revealed β-oxidation-related functions for several newly identified peroxisomal proteins [36,37]. Extending mutant screens to abiotic stress conditions combined with co-expression analyses uncovered Arabidopsis peroxisomal proteins involved in drought response, including the LON2 protease (see below) and peroxisomal hydroxypyruvate reductase, which is involved in photorespiration [38].…”

The Arabidopsis E3 Ubiquitin Ligase SP1 Targets to Chloroplasts, Peroxisomes, and Mitochondria

“…On the other hand, our measurements were done in the presence of 2,4-dichlorophenoxybutryic acid (2,4-DB), an analog of proauxin that is converted to the auxin analog 2,4-D by b-oxidation, a pathway occurring exclusively in the peroxisome in plants. The 2,4-DB assay is not a general assessment of plant growth, but instead is specifically used to evaluate peroxisome function and has been widely used by the plant peroxisome research community and our own lab (Cassin-Ross and Hu, 2014;Pan et al, 2016). Without 2,4-DB, the root or hypocotyl lengths of sp1 mutants are actually similar to that of the wild type.…”