Systematic phenotypic screen of Arabidopsis peroxisomal mutants identifies proteins involved in β -oxidation

One-sentence Summary: A systematic phenotypic analysis of mutants of recently discovered Arabidopsis peroxisomal proteins identified proteins involved in β –oxidation. ABSTRACT Peroxisomes are highly dynamic and multi-functional 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 through in silico and experimental proteomic analyses followed by in vivo protein targeting validations. To determine the functions of these proteins, we interrogated their T-DNA insertion mutants with a series of physiological, cytological and biochemical assays to reveal peroxisomal deficiencies. Sugar-dependence and 2,4-dichlorophenoxybutyric acid (2,4-DB) and 12-oxo-phytodienoic acid (OPDA) response assays uncovered statistically significant phenotypes in β -oxidation-related processes in mutants for 20 out of the 27 genes tested. Further 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 thioesterase 2 and Acyl-activating enzyme isoform 1 and 5, and proteins with functions previously unknown to be associated with β -oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein 1, Betaine aldehyde dehydrogenase, and Unknown protein 5. This multi-pronged 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. activation of short to medium-chain acid substrates ranging in length from C2 (acetate) to C14 (myristate) 2003). We have shown in this study the accumulation of oil bodies and delayed degradation of long-chain fatty acids in aae1-1 etiolated seedlings. In addition, aae1-1 is hypersensitive to ABA and OPDA, two hormones required for dormancy. These results suggest that during seed germination and early seedling development, AAE1 is involved in the peroxisomal activation of long-chain fatty acids before these substrates enter the β –oxidation cycle. the function of We shown that plants are resistant to 2,4-DB and accumulate oil bodies and long-chain fatty acids in germinated seedlings, suggesting B12D1’s involvement related processes during germination. The biochemical function of B12D1 remains to be determined.

of these proteins, we interrogated their T-DNA insertion mutants with a series of physiological, cytological and biochemical assays to reveal peroxisomal deficiencies. Sugar-dependence and 2,4-dichlorophenoxybutyric acid (2,4-DB) and 12-oxo-phytodienoic acid (OPDA) response assays uncovered statistically significant phenotypes in β -oxidation-related processes in mutants for 20 out of the 27 genes tested. Further 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 thioesterase 2 and Acyl-activating enzyme isoform 1 and 5, and proteins with functions previously unknown to be associated with β -oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein 1, Betaine aldehyde dehydrogenase, and Unknown protein 5. This multi-pronged 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.
β -oxidation of fatty acids and related metabolites is a major function of peroxisomes throughout the life cycle of a plant, from seed germination to senescence. Mobilization of seed oil reserves during seed germination and post-germinative growth requires peroxisomal β -oxidation and the glyoxylate cycle. In this process, fatty acids are transported into the peroxisome, where they are activated into fatty acyl-CoAs and later shortened by two carbons in each cycle of β -oxidation.
The product, acetyl-CoA, is converted to 4-carbon molecules by the glyoxylate cycle, whose products further undergo gluconeogenesis to provide energy for post-germinative development (Theodoulou and Eastmond, 2012). Using core β -oxidation enzymes as well as pathway-specific enzymes, 12-oxo-phytodienoic Acid (OPDA), the jasmonic acid (JA) precursor that enters the peroxisome after being synthesized in the chloroplast, is converted to JA (Acosta and Farmer, 2010), and indole 3-butyric acid (IBA) is converted to the principal form of auxin, indole 3acetic acid (IAA) (Strader and Bartel, 2011). Besides the core β -oxidation pathway, which metabolizes straight-chain saturated fatty acids, auxiliary β -oxidation pathways also occur in the peroxisome to metabolize unsaturated fatty acids, in which case accessory enzymes are required (Goepfert and Poirier, 2007;Graham, 2008).
To assess the full composition of this versatile organelle in plants, both in silico analysis and experimental proteomics have been employed to identify novel peroxisomal proteins.
Bioinformatic analysis of the Arabidopsis genome using Peroxisomal Target Signal type 1 (PTS1) and PTS2 sequences predicted a total of over 400 proteins to be potentially peroxisomal (Lingner et al., 2011). Experimental proteomics of Arabidopsis, spinach and soybean peroxisomes using different tissue/cell types and development stages together identified several dozens of novel peroxisomal proteins after in vivo targeting verification (Fukao et al., 2002;Fukao et al., 2003;Arai et al., 2008Arai et al., , 2008Eubel et al., 2008;Reumann et al., 2009;Babujee et al., 2010;Quan et al., 2013). Following 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 cysteine protease, RDL1 (RESPONSE TO DROUGHT21A-LIKE1), in seed germination, β oxidation, and stress response (Quan et al., 2013;Cassin-Ross and Hu, 2014). However, many other recently identified peroxisomal proteins have not been characterized with respect to their functions in peroxisomal physiology and plant development.
In this study, we interrogated the mutants of 27 recently identified peroxisomal genes with systematic phenotypic assays to analyze the function of the proteins in peroxisomal metabolism.
Mutants for 20 of the tested genes showed statistically significant phenotypes in at least one of the assays. Further analysis revealed that mutants for seven of the 20 genes displayed deficiencies in multiple assays, suggesting strongly that these seven proteins are involved in β oxidation-related processes. This multi-faceted screen enabled us to identify β -oxidation proteins that may not have been discovered otherwise by genetic screens based on single assays.

Identifying mutants of recently discovered Arabidopsis peroxisomal genes
To investigate the role of the recently identified and uncharacterized peroxisomal proteins in peroxisomal physiology, we took a reverse genetic approach by searching the TAIR database (http://www.Arabidopsis.org) for 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. A few of the genes had been previously investigated in processes not directly related to β -oxidation, and a few others had been studied biochemically without mutant characterization.
Except for CPK1 and AACT1, 25 of the peroxisomal proteins analyzed in this study were initially identified from the leaf peroxisome proteome (Supplemental Table S2), indicating that they may be involved in photorespiration or general plant growth. Photorespiration is a major peroxisomal function in green tissue, where peroxisomes together with chloroplasts and mitochondria convert phosphoglycolate produced by the oxygenase activity of Ribulose-1,5bisphosphate-carboxylase/oxygenase (RuBisCO) into glycerate, a molecule that is re-used in the chloroplastic Calvin-Benson cycle (Foyer et al., 2009;Peterhansel et al., 2010). Apart from the control mutant pex14, which is defective in peroxisomal protein import, none of the mutants showed obvious difference in appearance from the wild type while growing under ambient air (CO 2 400 µL L -1 ) or decreased levels of CO 2 (80 µL L -1 ), the latter of which was expected to enhance the growth defect of photorespiratory mutants because of the higher demand for photorespiration under low CO 2 (Supplemental Figure S3). We concluded that these proteins are not essential for plant growth or photorespiration, or they play redundant roles with other proteins in these processes under our laboratory conditions. Since the majority of the peroxisomal proteome is dedicated to β -oxidation-related processes (Hu et al., 2012), we next employed parallel physiological assays to assess the efficiency of peroxisomal β -oxidation in these mutants.

Mutants for four genes were sugar-dependent in post-germinative growth
Prior to the establishment of photosynthesis, peroxisomal fatty acid β -oxidation is required to fuel early seedling growth. As a result, many β -oxidation mutants arrest or develop slowly after germination, a phenotype that can be rescued by adding to the growth medium a carbon source, such as sucrose (Suc) (Hu et al., 2012). To check for Suc dependence, we grew mutants for seven days on half-strength Linsmaier and Skoog (1/2 LS) medium in dark or light conditions with or without Suc, and quantified hypocotyl or root lengths. Only differences between mutants and wild-type plants with a p value <0.01 were considered statistically significant after an unpaired Student's t test.
As expected, pex14 displayed sugar-dependent growth in both dark and light conditions ( Figure   1). Although not showing sugar dependence, dhar1, icdh, atf2, b12d1 and cpk1 had statistically shorter or longer hypocotyls and/or roots than the wild-type plants regardless of the presence of Suc in dark or light conditions ( Figure 1). Since the size of these mutants was indistinguishable from that of the wild type as adults (Supplemental Figure S3), we reasoned that the developmental differences observed at the seedling stage were possibly overcome later.
In the dark, the absence of Suc resulted in a ~15% decrease in the hypocotyl length of wild-type Col-0 and most mutant seedlings, whereas the decrease was ~26% for icdh-1 and 21% for up5-1 ( Figure 1A, Supplemental Table S3). In light-grown seedlings, the absence of Suc caused a ~16% reduction of the primary root length in Col-0 and most mutants, whereas this reduction was ~24% for both inda-1 and up7-1 ( Figure 1B, Supplemental Table S3). These results indicated possible roles of ICDH and UP5, and INDA and UP7, in lipid mobilization during post-germinative growth in dark and light-grown conditions respectively.

Mutants for 14 genes showed abnormal 2,4-DB response
IBA is converted to IAA through peroxisomal β -oxidation (Strader and Bartel, 2011). To check whether any mutants showed reduced response to the inhibitory effect of IBA on primary root elongation, root lengths of 7d seedlings grown on 1/2 LS medium supplemented with IBA or IAA were measured. For wild-type and mutants, control treatment with 100 nM IAA resulted in similar degrees of decreases in the primary root length coupled with excessive root hair growth (Supplemental Figure S4). The pex14 mutant seedlings exhibited strong IBA resistance, yet all the other mutants showed IBA response similar to that of the wild type (Supplemental Figure S4 and S5) with no statistically significant differences observed at tested IBA concentrations (Student's t test, p<0.01).

Mutants of nine genes showed deficiency in OPDA metabolism
Methyl-JA (MeJA) inhibits primary root elongation (Staswick et al., 1992). We reasoned that the JA precursor 12-oxo-phytodienoic acid (OPDA), which is converted to JA through peroxisomal β -oxidation, would similarly inhibit primary root elongation. Based on this assumption, we previously developed a simple assay to identify new peroxisomal proteins involved in OPDA metabolism (Cassin-Ross and Hu, 2014). The same assay was applied in this study to assess the function of the tested proteins in OPDA metabolism.

Most proteins with β -oxidation-related phenotypes are involved in seed germination
Our sucrose dependence and 2,4-DB/OPDA response assays together identified 31 mutants for 20 peroxisomal genes that showed a phenotype in at least one of the assays; 10 of the genes had at least two alleles with similar phenotypes. These data suggested the potential roles of AACT1, Fresh seeds from all 30 mutants germinated normally, as quantified by radicle emergence from seeds grown on plain agar (0.8% agar) or 1/2 LS medium (Supplemental Figure S8). We then tested seed germination in response to factors known to influence this process. Light is a positive regulator of seed germination (Lau and Deng, 2010), so we compared the germination rate of mutant seeds that were placed in total darkness with those that were subjected to 1h light treatment before germination. Quantification of radicle emergence from 5d seedlings revealed that, while wild-type Col-0, Col-3 and the positive control kat2-3 showed no light dependence, wild-type Ws-4 appeared to depend more on the light treatment for germination ( Figure 4).
Together with acx4-1, mutants for 19 of the 20 genes tested showed statistically lower germination rate in comparison with their respective wild-type controls when germinated in total darkness (Student's t test, p<0.01), and the 1h light pre-treatment could rescue the germination potential for all these mutants except for inda-1, b12d1-1, and elt1-1 ( Figure 4).
We also quantified seed germination rate on medium supplemented with the phytohormone abscisic acid (ABA), which acts synergistically with OPDA to inhibit germination (Vanstraelen and Benkova, 2012). Radicle emergence was quantified on seeds sown on plain agar supplemented with 2 or 5 µM ABA ( Figure 5). As previously reported, kat2-3 exhibited insensitivity to ABA regardless the concentration (Jiang et al., 2011). However, germination rate of acx4-1 was statistically lower than the wild type, and similarly, most of the 5d or 10d mutant seedlings showed statistically significant hypersensitivity to 2 µM ABA in comparison with the wild type (Student's t test, p<0.001). At 5 µM ABA, all the 5d seeds except b12d1-1 showed hypersensitive response, whereas at 10d the germination rate for ach2, up6, dhar1, cuao3, cpk1 and aae5 were comparable to the wild type ( Figure 5).
Based on results from these two germination assays, we concluded that most of these 20 proteins have potential positive roles in initial seed germination and the role for INDA and B12D1 seemed more prominent given the stronger phenotypes of their mutants. To determine whether the seed germination phenotypes correlated with gene expression pattern, we analyzed the expression of the 20 genes during seed maturation and germination. Using data from the publicly

Mutants for seven peroxisomal proteins retain oil bodies and accumulate fatty acids in early seedling development
Previous studies have shown that the inability to break down triacylglycerol (TAG) leads to prolonged presence of oil bodies in β -oxidation mutants (Graham, 2008). To assess lipid mobilization defects in the peroxisomal mutants, we used the lipophilic stain nile red (Greenspan et al., 1985) to detect the presence of lipid bodies in hypocotyls of 5d and 7d etiolated seedlings.
To confirm the oil body accumulation phenotype in these seven mutants, we quantified longchain fatty acid species and compared their levels in 3d, 5d, and 7d etiolated seedlings against seeds. At 3d and 5d after germination, the amount of C20:1, which is a marker for TAG (Lemieux et al., 1990), remained at a statistically higher level in virtually all the mutants than in wild-type seedlings ( Figure 7A and Figure 7B). Similarly, higher accumulation of other longchain fatty acids, i.e. C16:0, C18:0, C18:1. C18:2, C18:3, C20:2, and C20:3, was observed in nearly all the mutants at 3d and in all the mutants at 5d ( Figure 7A and Figure 7B; Student's t test, p<0.01). Consistent with the strongest retention of oil bodies found in this study ( Figure 6), kat2-3 showed the highest accumulation of all long-chain fatty acids analyzed (Figure 7). At 7d after germination, the level of long-chain fatty acids was still higher in kat2-3 and inda-1 but comparable or even lower in other mutants in comparison with the wild type ( Figure 7C). These data support the notion that ach2-3, aae1-1, inda-1, b12d1-1, badh-1, aae5-1 and up5-1 had a reduced rate of β -oxidation, which caused delayed degradation of the fatty acid substrates.

Multipronged phenotypic screen allows the identification of additional peroxisomal proteins potentially involved in β -oxidation
Forward genetic screens for mutants with sugar dependence or resistance to 2,4-DB/IBA identified key enzymes in β -oxidation and proteins involved in peroxisome biogenesis (Hu et al., 2012). However, these screens rely on single assays and tend to isolate mutants with strong visual phenotypes. In this study, we used multiple assays to simultaneously screen mutants of 27 uncharacterized peroxisomal genes in a quantitative manner. Mutants of 20 genes showed statistically significant phenotypes in β -oxidation-related processes, and further investigations discovered a subset of them 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 β -oxidation-based assays, strongly suggesting the involvement of these proteins in peroxisomal β -oxidation. ACH2, AAE1 and AAE5 are isoforms of enzymes known to be related to β -oxidation, whereas INDA, B12D1, BADH, and UP5 belong to functional categories previously unknown to be associated with β -oxidation, and their roles in this pathway could be direct or indirect. This screen provided a complementary approach to previous genetic screens in the identification of β -oxidation proteins that may have not been uncovered by single-assay-based screens. In addition, although β -oxidation occurs throughout the life cycle of a plant, many mutants in this pathway do not show obvious phenotypes at mature stages (Hu et al., 2012). Therefore, using assays aimed at dissecting β -oxidation in initial plant development, this study was able to unmask phenotypes for several mutants and will help us understand the β -oxidation network in more depth.

ACH2, an acyl-CoA thioesterase involved in the hydrolysis of long-chain fatty acyl-CoAs
Acyl-CoA thioesterases (ACOTs) hydrolyse fatty acyl-CoAs, yielding free fatty acids and coenzyme A (CoASH). The complex role of these enzymes in lipid metabolism has been previously documented, in particular in animals (Hunt et al., 2012). Mammalian ACOT8, the closest homologue to the two Arabidopsis peroxisomal ACOTs (ACH1 and ACH2), showed high activities toward a broad range of acyl-CoA substrates and was strongly inhibited by CoASH, suggesting that ACOT8 is involved in the regulation of the intracellular levels of acyl-CoAs, free fatty acids and CoASH (Hunt et al., 2002;Ofman et al., 2002).
ACH2 was the first acyl-CoA thioesterase to be cloned from plants, and its recombinant protein showed high levels of acyl-CoA thioesterase activity against both medium and long-chain fatty acyl-CoAs, with the highest activity toward long-chain unsaturated fatty acyl-CoAs (Tilton et al., 2000;Tilton et al., 2004). We have shown in this study that loss-of-function ach2 mutants have higher accumulation of long-chain fatty acids in germinated seedlings and partial resistance to 2,4-DB and OPDA, supporting ACH2's positive role in β -oxidation,. Interestingly, the ACH2 knock-down allele ach2-3 displayed a stronger seed germination phenotype than the null alleles, possibly due to functional compensation among multigene family members in the null alleles.
Our study has expanded the list of potential substrates for ACH2, suggesting that it may be involved in the regulation of the intracellular level of acyl-CoAs, acid-CoAs (OPDA and 2,4-DB), free fatty acids, acids and CoASH. However, ACH2's high expression in mature tissues such as young leaves and flowers and low expression in germinating seedlings (Tilton et al., 2004), the relatively weak phenotypes observed in ach2 mutants in seed germination, and the fact that ach2 mutant was never isolated from previous forward genetic screens, together suggest that ACH2 may not play a major role during early seedling development.

Two additional isoforms of acyl-activating enzymes involved in early seedling development
Arabidopsis AAE1 and AAE5 belong to a 14-member plant specific clade in the acyl-activating It was reported that AAE1 is not involved in the activation of short to medium-chain acid substrates ranging in length from C2 (acetate) to C14 (myristate) (Shockey et al., 2003). We have shown in this study the accumulation of oil bodies and delayed degradation of long-chain fatty acids in aae1-1 etiolated seedlings. In addition, aae1-1 is hypersensitive to ABA and OPDA, two hormones required for dormancy. These results suggest that during seed germination and early seedling development, AAE1 is involved in the peroxisomal activation of long-chain fatty acids before these substrates enter the β -oxidation cycle.
In our study, loss-of-function mutant of AAE5 showed a weaker phenotype than that of AAE1, where aae5-1 exhibited partial resistance to 2,4-DB, and weak accumulation of oil bodies and delayed degradation of long-chain fatty acids in etiolated seedlings. These results suggest that AAE5 may be involved in the activation of long-chain fatty acids in Arabidopsis but its role is not as prominent as AAE1 in germination and early seedling development. AAE1 is ubiquitously expressed throughout the plant whereas the expression of AAE5 is limited to developing seeds and roots (Shockey et al., 2003). In light of its high expression in developing seeds, a tissue in which the flow of fatty acids can be adjusted via β -oxidation (Poirier et al., 1999), we speculate that AAE5 may play a stronger role in activating long-chain fatty acids in developing seeds.
Taken together, our assays enabled us to distinguish the function of AAE1 and AAE5, two proteins that encode enzymes with the same biochemical activity and had been considered functionally redundant. We have demonstrated that plants lacking BADH are hypersensitive to ABA's inhibitory effect on germination, accumulate higher amounts of long-chain fatty acids and oil bodies days after germination, and exhibit 2,4-DB resistance. These data point to a positive role for BADH in β oxidation and seed germination. BADH's role in β -oxidation remains to be determined.

B12D1 in β -oxidation
The expression of the barley B12D gene (HvB12Dg1) is high in the aleurone layer and in the embryo of developing seeds, diminishes towards seed maturity, and reappears in germinating seeds (Aalen et al., 1994). Consistent with this, the expression of HvB12Dg1 was shown to be regulated by two key hormones that control seed germination: up-regulation by gibberellic acid (GA3) and down-regulation by ABA (Steinum et al., 1998). Similarly, Arabidopsis B12D1 is preferentially expressed during seed maturation and germination, causing poor seed germination in its absence. In cereal seeds, proteins localized in aleurone and embryo are involved in the synthesis and accumulation of lipid bodies, desiccation tolerance and dormancy (Aalen et al., 1994), yet the precise function of HvB12D1 is still unclear. We have shown that plants lacking B12D1 are resistant to 2,4-DB and accumulate oil bodies and long-chain fatty acids in germinated seedlings, suggesting B12D1's involvement β -oxidation related processes during germination. The biochemical function of B12D1 remains to be determined.

Conclusions
Our systematic and quantitative analysis of mutants of recently discovered Arabidopsis peroxisomal genes identified additional peroxisomal proteins with varying degrees of contribution to β -oxidation. This study has taken a step forward towards completely dissecting the plant β -oxidation network, and provides a framework for future investigations to integrate genetics and physiology with biochemical and metabolic assays to identify substrates for all enzymes in β -oxidation.

Resource Center, the Nottingham Arabidopsis Stock Centre and the INRA-Versailles Genomic
Resource Center (Table 1 and Supplemental Table S1). Surface sterilized seeds were plated on  Supplemental Table S5.

RNA analysis
For RT-PCR analysis of the mutants, total RNA was isolated from 7d seedlings grown on medium with 1% (w/v) sucrose as described previously (Mallory et al., 2001 For germination assays, seeds harvested from plants that had been grown simultaneously were sown on 0.8% agar (w/v) plates (i.e. plain agar plate) or 1/2 LS media solidified with 0.8% agar.
The plates were kept at 4˚C for 4 d before being transferred to growth chamber with continuous low light intensity (plates covered with mesh) or darkness. An additional set of plates was exposed to light for 1 h before being placed in the dark in growth chamber. After 5d, radicle emergence was scored. For seed germination in response to ABA, seeds were sown on plain agar plates supplemented with ABA (Sigma-Aldrich). After 4d stratification at 4 °C, plates were transferred to growth chamber and grown in the light for 5d or 10d before radicle emergence was scored. All the data are representative of at least three independent experiments. For each experiment, n=50.
For sucrose dependence analysis, seeds were placed on plates supplemented with or without 1% (w/v) sucrose and stratified at 4 °C for 2d. After 7d in growth chamber with continuous low intensity light or darkness, the plates were scanned using an EPSON scanner (Epson Perfection 4870 PHOTO). Hypocotyl lengths of dark-grown seedlings and roots of light-grown seedlings were measured using ImageJ (imagej.nih.gov/ij/). To study IAA, IBA, 2,4-D, 2,4-DB and OPDA responses, seeds were sown on plates containing 0.5% (w/v) sucrose and various concentrations of IAA (BioWORLD), IBA (Sigma-Aldrich), 2,4-D (Sigma-Aldrich), 2,4-DB (Sigma-Aldrich), OPDA (Cayman Chemical), or MeJA (Sigma-Aldrich), as described in figure legends. The plates were then stored at 4 °C for 2d and placed in growth chamber with continuous low-intensity light. Root length was quantified at 7d. All the data are representative of at least three independent experiments. For each experiment, n=50.
To detect photorespiratory phenotypes, 2w seedlings were transferred from plates to soil and placed in a growth cabinet with controlled environment at light intensity of 115 µmol photons m -2 s -1 , 20 ˚C, 16/8 photoperiod, and CO 2 concentration of 80 µL L -1 CO 2 (low CO 2 concentration) or 400 µL L -1 CO 2 (ambient air). After two weeks, plants were photographed with a COOLPIX 8800 VR camera (Nikon).

Visualization of oil bodies
www.plantphysiol.org on August 18, 2017 -Published by Downloaded from Copyright © 2014 American Society of Plant Biologists. All rights reserved. 0 Etiolated seedlings grown on 0.8% agar (w/v) for 5 or 7d were stained for 5 min with 1 µg mL -1 aqueous solution of Nile Red (Molecular Probes) as previously described (Greenspan et al., 1985;Linka et al., 2008). Images were recorded using an Olympus FluoView 1000 Spectralbased Laser Scanning Confocal Microscope system (excitation wavelength 450-500 nm; emission wavelength, > 528 nm), using the 60x objective. All the data are representative of at least three independent experiments. For each experiment, n=12.

Fatty acid analysis
Amount of fatty acids in seeds and seedlings were analyzed as fatty acid methyl esters (FAMEs) according to a published protocol (Li et al., 2006). Briefly, 50 seedlings or 100 seeds were placed in a tube with a screw cap. Added to the tube were 2 mL of 5% (v/v) H 2 SO 4 in methanol, 300 µL of Toluene, and 10-60 µg of Tri15:0TAG (amount used depends on the tissue) as internal standard. The tubes were caped and vortexed to ensure submergence of the sample, and later heated for 90 min at 85 °C. The resulting FAMEs were extracted using hexane and 0.9% NaCl, dried down under nitrogen gas, and resuspended in appropriate volume of hexane. FAMEs were analyzed by gas chromatography (GC) with a flame ionization detector (FID). Samples were separated on a DB-23 capillary column (30 m x 0.25 mm ID, 0.25 µm film thickness; J&W Scientific, Folsom, CA), and helium was used as carrier gas at a constant flow of 1.5 mL/min. For GC, oven temperature was maintained at 140 °C for 3 min, followed by a 5 °C per min increase until the oven reached 230°C, and a final 3 min at 230°C. Injector and detector were maintained at 250 °C throughout the analysis.

Statistical analysis
To reveal statistical difference between the mutants and the wild type, all data generated from the assays were subjected to an unpaired Student's t test. We considered differences between mutants and wild type plants with p<0.01 as significant.

SUPPLEMENTAL DATA
The following materials are available in the online version of this article: Supplemental Table S1. Lines without T-DNA insertion and lines for which homozygotes could not be identified. 1 Supplemental Table S2. Identification of peroxisomal proteins analyzed in this study from previous proteomic studies. Table S3. Measurements for the sucrose dependence assays. Table S4. Log2 expression values downloaded from the BAR expression browser for heatmap generation. Table S5. Primers used for genotyping and RT-PCR analysis.

Supplemental
Supplemental Figure S1. Schematics of peroxisomal gens analyzed in this study.  Percentage of 7d light-grown seedlings displaying resistance to 2,4-DB after growing on 1/2 LS medium supplemented with 0.5% Suc and 0.4, 0.8, or 1 µM of 2,4-DB is presented. Resistance was defined as having root length with no statistically significant differences from that on medium without 2,4-DB. Data represent means ± SE of three independent experiments. For each experiment, n≥30. Section and hashtag signs indicate mutants in Col-3 and Ws-4 backgrounds respectively. Asterisks indicate statistically significant changes from the wild type  or Ws-4). Student's t test, * p<0.01, ** p<0.001.  Percentage of radicle emergence from seeds on plain agar (0.8% agar) in dark without or with 1h light pre-treatment is presented. Data represent means ± SE of three independent experiments.

Supplemental
For each experiment, n = 50. Section and hashtag signs indicate mutants in Col-3 and Ws-4 backgrounds respectively. Asterisks indicate changes significantly different from that in the wild type (Col-0, Col-3, or Ws-4). Student's t test, * p<0.01, ** p<0.001. Percentage of radicle emergence from seeds grown on plain agar supplemented with 0, 2 or 5 µM of ABA for 5 and 10d is presented. Data represent relative means (treated vs. untreated) ± SE of three independent experiments. For each experiment, n = 100. Section and hashtag signs indicate mutants in Col-3 and Ws-4 backgrounds respectively. Asterisks indicate changes significantly different from that in the wild type (Col-0, Col-3 or Ws-4). Student's t test, *p<0.001.    Percentage of 7d light-grown seedlings displaying resistance to 2,4-DB after growing on 1/2 LS medium supplemented with 0.5% Suc and 0.4, 0.8, or 1 µM of 2,4-DB is presented. Resistance was defined as having root length with no statistically significant differences from that on medium without 2,4-DB. Data represent means ± se of three independent experiments. For each experiment, n≥30. Section and hashtag signs indicate mutants in Col-3 and Ws-4 backgrounds respectively. Asterisks indicate statistically significant changes from the wild type (Col-0, Col-3, or Ws-4). Student's t test, * p<0.01, ** p<0.001.    Percentage of radicle emergence from seeds on plain agar (0.8% agar) in dark without or with 1h light pretreatment is presented. Data represent means ± se of three independent experiments. For each experiment, n = 50. Section and hashtag signs indicate mutants in Col-3 and Ws-4 backgrounds respectively. Asterisks indicate changes significantly different from that in the wild type (Col-0, Col-3, or Ws-4). Student's t test, * p<0.01, ** p<0.001).