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 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.

Figure 1.

Suc dependence assay. Shown are hypocotyl lengths of 7-d etiolated seedlings (A) and root lengths of 7-d light-grown seedlings (B), both of which were grown on one-half-strength LS medium with or without 1% Suc. Data represent means ± se of three independent experiments. For each experiment, n ≥ 30. Student’s t test. *, Statistically significant difference of P < 0.01 from the wild type (Col-0, Col-3, or Ws-4); **, statistically significant difference of P < 0.001 from the wild type (Col-0, Col-3, or Ws-4); #, mutants in Ws-4 background; §, mutants in Col-3 background; arrows, mutants that show sugar dependence.

Figure 2.

2,4-DB response assay. Percentage of 7-d light-grown seedlings displaying resistance to 2,4-DB after growing on one-half-strength LS medium supplemented with 0.5% Suc and 0.4, 0.8, or 1 µm 2,4-DB is presented. Resistance was defined as having a root length with no statistically significant difference from that on medium without 2,4-DB. Data represent means ± se of three independent experiments. For each experiment, n ≥ 30. Student’s t test. *, Statistically significant difference of P < 0.01 from the wild type (Col-0, Col-3, or Ws-4); **, statistically significant difference of P < 0.001 from the wild type (Col-0, Col-3, or Ws-4); #, mutants in Ws-4 background; §, mutants in Col-3 background.

Figure 3.

Root response to OPDA. Relative root lengths (treated versus untreated) of 7-d seedlings grown on one-half-strength LS medium supplemented with 250 nm OPDA or 10 µm MeJA are shown. Data represent means ± se of three independent experiments. For each experiment, n = 50. Student’s t test. *, Statistically significant difference of P < 0.01 from the wild type (Col-0, Col-3, or Ws-4); **, statistically significant difference of P < 0.001 from the wild type (Col-0, Col-3, or Ws-4); #, mutants in Ws-4 background; §, mutants in Col-3 background.

Figure 4.

Seed germination in the mutants. Percentage of radicle emergence from seeds on plain agar (0.8% [w/v] agar) in the dark without or with 1 h of light pretreatment is presented. Data represent means ± se of three independent experiments. For each experiment, n = 50. Student’s t test. *, Statistically significant difference of P < 0.01 from the wild type (Col-0, Col-3, or Ws-4); **, statistically significant difference of P < 0.001 from the wild type (Col-0, Col-3, or Ws-4); #, mutants in Ws-4 background; §, mutants in Col-3 background.

Figure 5.

Seed germination in response to ABA. Percentage of radicle emergence from seeds grown on plain agar supplemented with 0, 2, or 5 µm ABA for 5 and 10 d is presented. Data represent relative means (treated versus untreated) ± se of three independent experiments. For each experiment, n = 100. Student’s t test. *, Statistically significant difference of P < 0.001 from the wild type (Col-0, Col-3, or Ws-4); #, mutants in Ws-4 background; §, mutants in Col-3 background.

Figure 6.

Confocal microscopic visualization of oil bodies in the hypocotyl of 7-d etiolated seedlings. Seedlings were grown on 0.8% (w/v) agar plate. Nile Red fluorescence images (top) and merged images (bottom) of bright-field and Nile Red fluorescence are shown. Area of fluorescence is the arbitrary value (assigned by Image J) within the 75-µm² area of the Nile Red fluorescent images shown. Bars = 10 µm.

Figure 7.

Fatty acid (FA) quantification in seedlings. Percentage of FAs remaining is shown for 3-d (A), 5-d (B), and 7-d (C) etiolated seedlings grown on 0.8% (w/v) agar plates compared with the level in seeds. The amount of FA in dry seeds was set as 100%. Data represent means ± se of three independent experiments. For each experiment, n ≥ 50. *, Statistically significant difference of P < 0.01 from the wild type (Col-0, Col-3, or Ws-4); **, statistically significant difference of P < 0.001 from the wild type (Col-0, Col-3, or Ws-4); #, mutants in Ws-4 background.