1 short title: lipid intermediate toxicity in seedlings 2139 catabolism activity on seedlings....

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Short title: Lipid intermediate toxicity in seedlings 1

Author for contact: Bethany K. Zolman, University of Missouri – St. Louis 2

One University Blvd, R223 Research Building, St. Louis MO 63121 3

[email protected] 4

314-516-6205 5

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Ying Li, Yu Liu, and Bethany K. Zolman 7

University of Missouri – St. Louis 8

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Metabolic alterations in the enoyl-CoA hydratase 2 mutant disrupt peroxisomal pathways in 10

seedlings 11

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One sentence summary: Changes in enoyl-CoA hydratase2 activity result in metabolic 13

alterations that influence seedling development through a toxic effect. 14

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Author contributions: Ying Li and Bethany K. Zolman designed the project. Ying Li and Yu Liu 16

performed the experiments and analyzed the data. Ying Li, Yu Liu, and Bethany K. Zolman 17

wrote the article. Bethany K. Zolman agrees to serve as the author responsible for contact and 18

ensures communication ([email protected]). 19

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This research was supported by the National Institutes of Health (1R15GM116090-01) and the 21

University of Missouri Research Board. 22

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Plant Physiology Preview. Published on May 28, 2019, as DOI:10.1104/pp.19.00300

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Abstract 24

Mobilization of seed storage compounds, such as starch and oil, is required to provide energy 25

and metabolic building blocks during seedling development. Over 50% of fatty acids in 26

Arabidopsis thaliana seed oil have a cis-double bond on an even-numbered carbon. Degradation 27

of these substrates requires peroxisomal fatty acid β-oxidation plus additional enzyme activities. 28

Such auxiliary enzymes, including the enoyl-CoA hydratase ECH2, convert (R)-3-hydroxyacyl-29

CoA intermediates to the core β-oxidation substrate (S)-3-hydroxyacyl-CoA. ECH2 was 30

suggested to function in the peroxisomal conversion of indole-3-butyric acid (IBA) to indole-3-31

acetic acid (IAA), as ech2 seedlings have altered IBA responses. The underlying mechanism 32

connecting ECH2 activity and IBA metabolism is unclear. Here, we show that ech2 seedlings 33

have reduced root length, smaller cotyledons, and arrested pavement cell expansion. At the 34

cellular level, reduced oil body mobilization and enlarged peroxisomes suggest compromised -35

oxidation. ech2 seedlings accumulate 3-hydroxyoctenoate (C8:1-OH) and 3-hydroxyoctanoate 36

(C8:0-OH), putative hydrolysis products of catabolic intermediates for α-linolenic acid and 37

linoleic acid, respectively. Wild-type seedlings treated with 3-hydroxyoctanoate have ech2-like 38

growth defects and altered IBA responses. ech2 phenotypes are not rescued by sucrose or auxin 39

application. However, ech2 phenotypes are suppressed in combination with the core -oxidation 40

mutants mfp2 or ped1 and ech2 mfp2 seedlings accumulate less C8:1-OH and C8:0-OH than 41

ech2 mutants. These results indicate that ech2 phenotypes require efficient core β-oxidation. Our 42

findings suggest that low ECH2 activity causes metabolic alterations through a toxic effect of the 43

accumulating intermediates. These effects manifest in altered lipid metabolism and IBA 44

responses leading to disrupted seedling development. 45

46

47

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50



3

Introduction 51

The earliest stages of plant development, including germination and seedling establishment, are 52

critical transition points for dormant seeds to mature into flowering plants. Proper development 53

requires the close coordination and tight regulation of multiple physiological pathways. For 54

instance, mobilization of storage compounds, such as starch and oil, is required to provide both 55

energy and metabolic building blocks. Meanwhile, hormonal signals direct the growth and define 56

the morphology of developing plants in conjunction with environmental signals. 57

Triacylglycerol (TAG) is the major storage compound in oilseed plants. In Arabidopsis, 35-40% 58

of seed dry weight is comprised of oil (Li-Beisson et al. 2010), which is packed in oil bodies. Oil 59

bodies are synthesized from the endoplasmic reticulum in embryonic cells and accumulate in dry 60

seeds (Kim et al. 2013). Upon germination, storage lipids are quickly consumed to provide 61

energy and metabolic substrates for seedling development before photosynthesis initiates. The 62

consumption of oil bodies begins with the release of free fatty acids (FFAs) via TAG hydrolysis 63

by the lipase SDP1 (SUCROSE DEPENDENT1; (Eastmond 2006)). FFAs are used either in 64

membrane biogenesis or metabolized through β-oxidation in peroxisomes, which are organelles 65

housing oxidative reactions. 66

Many proteins are required for fatty acid catabolism (Figure 1). The import of fatty acyl-CoA 67

esters by the ATP-dependent transporter PXA1/CTS/PED3 (PEROXISOMAL ABC 68

TRANSPORTER/COMATOSE/PEROXISOME DEFECTIVE) is coupled with CoA-hydrolysis 69

(De Marcos Lousa et al. 2013; Footitt et al. 2002; Hayashi et al. 2002; Nyathi et al. 2010; 70

Zolman et al. 2001). Inside the organelle, fatty acids are re-esterified with CoA by the long-chain 71

acyl-CoA synthetases (LACS) LACS6 and LACS7 (Fulda et al. 2004). The activated acyl-CoA 72

is oxidized at its β-carbon by acyl-CoA oxidase (ACX1-ACX6), followed by sequential 73

hydration, dehydrogenation, and thiolytic cleavage steps catalyzed by a multifunctional protein 74

(MFP2 or AIM1) and 3-ketoacyl-CoA thiolase (KAT1, KAT2/PED1, and KAT5), respectively 75

(reviewed in (Kao et al. 2018)). These enzyme activities comprise the core β-oxidation cycle. 76

Overall, the acyl-CoA chain is shortened by two carbons during each round of β-oxidation. The 77

two carbons are released as acetyl-CoA, which can be condensed to 4-carbon compounds via the 78

peroxisomal glyoxylate cycle. These compounds can be used as carbon skeletons, as well as the 79

precursor of hexose and sucrose (with the integration of cytosolic and mitochondrial metabolism) 80



4

to support cell wall biosynthesis and fuel seedling growth (reviewed in (Böttcher and Pollmann 81



5

2009; Graham 2008)). Mutants defective in fatty acid β-oxidation enzymes result in 82

developmental abnormalities or growth retardation. Mutants with strong defects have arrested 83

seedling establishment, while partial loss-of-function lines have short roots and hypocotyls; these 84

phenotypes can be rescued by application of sucrose as an exogenous carbon source, which 85

precludes the need for β-oxidation metabolites (Hu et al. 2012; Hayashi et al. 1998). 86

Saturated fatty acids are catabolized through the core β-oxidation spiral after CoA attachment. 87

However, a large proportion of fatty acids in seed storage oils are unsaturated, which require 88

additional auxiliary peroxisomal activities for degradation. For example, Figure 1B shows the 89

complete degradation of petroselinic acid, a fatty acid with 18 carbons and one double bond in 90

the cis-configuration at an even-numbered (Δ6) carbon (C18:1Δ6-cis). Following core β-91

oxidation, degradation of unsaturated molecules occurs via one of two auxiliary pathways: the 92

reductase/isomerase pathway or the hydratase/epimerase pathway (reviewed in (Graham 2008)). 93

For the reductase/isomerase pathway, 2, 4-dienoyl-CoA reductase (DECR) activity has been 94

detected in Arabidopsis and a putative gene DECR/SDRB was identified (Reumann et al. 2007). 95

Three Arabidopsis genes were identified that encode Δ3, Δ2-enoyl-CoA isomerases (AtECI1-3), 96

whose activities were characterized when expressed heterologously in yeast (Goepfert et al. 97

2008). The alternative hydratase/epimerase pathway requires a 3-hydroxyacyl-CoA epimerase 98

and two stereo-specific enoyl-CoA hydratases. The 3-hydroxyacyl-CoA epimerase activity was 99

characterized initially in cucumber (Preisig-Muller et al. 1994) and may be completed by one of 100

the multifunctional enzymes in other plants (Graham 2008). MFP2 and ECH2 confer the stereo-101

specific enoyl-CoA hydratase 1 and 2 activity, respectively. Analysis of the carbon flux in the β-102

oxidation pathway of AtECH2RNAi lines suggested ECH2 converts (R)-3-hydroxyacyl-CoA 103

intermediates to the (S)-3-hydroxyacyl-CoA core β-oxidation intermediates (Goepfert et al. 104

2006). 105

Mutant analysis revealed that ECH2 activity mobilizing storage lipids is important for cell 106

expansion and proliferation (Katano et al. 2016; Strader et al. 2011). Interestingly, ech2-1 107

phenotypes also suggested the involvement of ECH2 in the metabolism of Indole-3-butyric acid 108

(IBA), a precursor and potential storage form of Indole acetic acid (IAA) (Strader et al. 2011). 109

IAA affects plant growth and development by directing cell division and elongation (Velasquez 110

et al. 2016; Majda and Robert 2018). The conversion of IBA to IAA is achieved through removal 111

of two carbons from the butanoate side chain, a β-oxidation-like pathway also occurring in 112



6

peroxisomes (Zolman et al. 2000; Strader et al. 2010). Enzymes acting in IBA to IAA conversion 113

were identified through forward genetic screening for mutants that are resistant to the inhibition 114

effect of IBA on root and/or hypocotyl elongation, known as IBA-response (ibr) mutants 115

(Strader et al. 2011; Zolman et al. 2000). Based on sequence homology to characterized 116

enzymatic domains, IBR3, IBR10, and IBR1 were predicted to act at successive steps of the IBA 117

β-oxidation pathway (Figure 1A; (Zolman et al. 2008; Zolman et al. 2007)). ECH2 has been 118

hypothesized to act at the enoyl-CoA hydratase step and/or the IAA-CoA hydrolase step, as 119

ECH2 also contains a hot dog domain associated with potential thioesterase activity (Strader et al. 120

2011). Additionally, the PED1 thiolase was predicted to have overlapping functions on IBA to 121

IAA conversion based on the IBA-resistant phenotype of a ped1 mutant (Lingard and Bartel 122

2009). Genetic evidence indicates that IBA-derived IAA is important in germination, root 123

branching, and cell expansion for both roots and leaves (Spiess et al. 2014; Strader et al. 2011; 124

Zolman et al. 2008). 125

Here, we have investigated the role of ECH2 in fatty acid metabolism and IBA to IAA 126

conversion further to better understand how peroxisomal processes are orchestrated to regulate 127

the early developmental stages of plants. Metabolic profiling of ech2 reveals an over-128

accumulation of (R)-3-hydroxy-5-octenoic acid, a putative hydrolysis product of the presumed 129

ECH2 substrate for α-linolenic acid (ALA) catabolism. Epistasis analysis of mutants disrupting 130

ECH2 and other lipid catabolism enzymes has revealed a link between β-oxidation efficiency, 131

over accumulation of pathway intermediates, and altered growth phenotypes and IBA responses. 132

133

134



7

Results 135

ech2 has altered seedling development, but normal adult growth 136

Arabidopsis development requires the concerted effort of fatty acid metabolism and hormone 137

signaling. We examined the effects of disrupting IBA to IAA conversion and fatty acid 138

catabolism activity on seedlings. Mutants defective in each pathway were examined for 139

cotyledon growth and primary root elongation. The tested IBA-response (ibr) mutants included 140

ibr1, ibr3, ibr10, and ech2. For fatty acid catabolism, acx3, mfp2, aim1, and ped1 were used. 141

Each line was grown on sucrose-containing media for 7 days. Only ech2 showed obviously 142

reduced cotyledon size and primary root length (Figure 2 and Supplemental Figure S1). 143

Moreover, ech2 cotyledons had reduced pigmentation compared with wild-type (Wt) seedlings 144

(Figure 2A and Supplemental Figure S1). However, true leaves and the overall size of ech2 145

rosettes were comparable to that of wild type (Figure 2C and 2D). These results suggest that 146

decreased ECH2 activity disrupts normal cotyledon development, but this defect has a reduced 147

effect during adult development. The altered seedling development of ech2 was rescued in 148

transgenic plants overexpressing ECH2 (Supplemental Figure S2A and S2B). 149

Similar seed weight and germination rate between wild type and ech2 (Supplemental Figure S3) 150

suggest embryonic defects are unlikely the cause of retarded ech2 development. Thus, we 151

hypothesize the seedling growth phenotype can be attributed to compromised post-germination 152

development. The normal growth of true leaves in ech2 suggests that the heterotrophic nature of 153

early seedling development may be a key to cause the growth defect. 154

155

ech2 cotyledons have reduced pavement cell area and complexity 156

To characterize the defects of ech2 in cotyledon development, pavement cells were imaged at 157

days 5 and 12. Notably, ech2 pavement cells not only had reductions in cell area, but also in the 158

levels of interlocking with adjacent cells. Wild-type pavement cells are shaped like a jigsaw 159

puzzle of interlocked pieces with lobes (convex) and indentations (concave). ech2 pavement 160

cells were shaped more like irregular rectangles, with few lobes and indentations (Figure 2E). 161

Circularity was used as an index for complexity of pavement cells based on the independence of 162

cell area (Zhang et al. 2011). A higher circularity indicates less complexity. In both wild-type 163



8

and ech2 cotyledon pavement cells, increased cell area, with more lobes and indentations, was 164

noted at day 12 compared to day 5 (Figure 2F). However, the circularity of ech2 cotyledon 165

pavement cells was consistently higher than that of wild type, indicating ech2 has reduced 166

pavement cell complexity throughout cotyledon development (Figure 2F). Notably, pavement 167

cells in the ech2 true leaves were comparable to that of wild type in area and lobe number, 168

consistent with normal growth of ech2 beginning with the first true leaves (Figure 2F). 169

Transgenic complementation lines also showed restoration of cell patterning (Supplemental 170

Figure S2C), consistent with the restoration of cotyledon size noted above. 171

Lobe and indentation formation involves two antagonistic Rho GTPase signaling pathways 172

(Feiguelman et al. 2018). ROP2 and ROP4 activate the effector RIC4 to promote cortical 173

diffusion of F-actin and outgrowth of lobes. ROP6 suppresses ROP2, activating the effector 174

RIC1 to promote microtubule organization and indentation formation (Fu et al. 2005). ech2 175

pavement cell morphology resembled that of a mutant disrupted in both ROP2 and ROP4 (Xu et 176

al. 2010). To test if the Rho GTPase signaling pathway is compromised in ech2, expression 177

levels of ROP2, ROP4, and ROP6 along with the RIC4 and RIC1 effectors were examined in 178

cotyledons and true leaves. Consistently, ROP2 and ROP4 were downregulated in ech2 seedlings 179

at day 3 when cotyledons emerged (~2 fold lower than Wt), but not at day 25, when true leaves 180



9

were well developed (Supplemental Figure S4A and S4B). RIC4 expression levels paralleled 181

those of ROP2 and ROP4 at day 3. These results suggest that the reduced complexity of ech2 182

cotyledon pavement cells might be related to compromised Rho GTPase signaling. 183

Despite ech2-1 having defects in IBA responses, auxin application did not rescue the complexity 184

or the area of the ech2 cotyledon pavement cells (Supplemental Figure S5). Although a negative 185

result in this experiment is not definitive of a direct contribution, our results suggested 186

contributors other than reduced IBA-derived IAA levels in ech2 disrupt cotyledon development 187

and led us to explore other metabolic pathways in more detail. 188

189

ech2 shows reduced fatty acid catabolism 190

β-oxidation mutants typically have improved growth in the presence of sucrose (Germain et al. 191

2001; Pinfield-Wells et al. 2005; Rylott et al. 2006; Thazar-Poulot et al. 2015). Exogenous 192

sucrose supplants the need for lipid metabolism and promotes growth and development. We 193

examined ech2 sucrose dependence as a first step in characterizing its -oxidation activity. We 194

compared seedlings grown on medium with or without sucrose under light and dark conditions. 195

In the light, ech2 had improved cotyledon and primary root development when sucrose was 196

added (Supplemental Figure S6), suggesting the beneficial effect of sucrose as energy source and 197

carbon supply on ech2 seedling establishment. While not completely restored, growth was more 198

similar to wild type. On the contrary, under darkness, ech2 showed similar hypocotyl elongation 199

regardless of the presence of sucrose (Supplemental Figure S7). These results indicate that 200

sucrose affects seedling development of ech2 only when light is present, unlike the well-201

characterized -oxidation mutants mfp2 and ped1 (Supplemental Figure S7; (Lingard and Bartel 202

2009; Rylott et al. 2006)). 203

We next visualized potential disruptions of fatty acid metabolism in ech2 by examining oil 204

bodies and peroxisomes with confocal microscopy. Many fatty acid β-oxidation mutants with 205

impaired lipid mobilization retain oil bodies and have enlarged peroxisomes, including mfp2 and 206

ped1 (Rinaldi et al. 2016). We visualized and counted oil bodies in wild-type and ech2 cotyledon 207

pavement cells. Oil bodies in wild-type cotyledon pavement cells were abundant on day 3, but 208

significantly fewer on days 4 and 5 (Figure 3A). On day 3, the total oil body area, as indicated by 209

oil body fluorescence, was more in ech2 than that in wild type. The number of oil bodies in ech2 210



10

was comparable with wild-type samples (Figure 3B), suggesting that oil bodies in ech2 were 211

either larger or aggregated. At days 4 and 5, however, ech2 not only had increased oil body area, 212

but also an increased number relative to wild type, although the trend of mobilization over time 213

remained consistent in ech2 samples. Peroxisomes also were imaged in wild-type and ech2 214

seedlings expressing a peroxisomally-targeted GFP (Woodward and Bartel 2005). The number of 215

peroxisomes in cotyledon pavement cells decreased on day 5 compared with day 4 in both wild 216

type and ech2. However, more peroxisomes were present in ech2 and the overall organelle size 217

was larger than in wild type (Figure 3C and 3D). These results suggest retarded lipid 218

mobilization when ECH2 function is disrupted. 219

To characterize the effect of losing ECH2 enoyl-CoA hydratase activity on acyl-CoA oxidase 220

products in core -oxidation reactions, electrospray ionization tandem mass spectrometry (ESI-221

MS/MS) was used. CoA conjugated intermediates were quantified from β-oxidation reactions 222

using extracts from 3-d-old seedlings with an acyl-CoA (C18:1, n-9) substrate. Supplemental 223

Figure S8 shows the relative abundance of C18:1 (n-9) substrate and the resulting 2-trans-enoyl-224

CoA, 3-hydroxyacyl-CoA, and 3-ketoacyl-CoA intermediates. Accumulation of 2-trans-enoyl-225

CoA, the potential ECH2 substrate, was significantly higher in ech2 than in wild type (~1.4 fold). 226



11

In contrast, accumulation of 3-hydroxyacyl-CoA, the presumed ECH2 product, was significantly 227

lower in ech2 than in wild type (0.55 fold). Increased upstream substrates coupled with reduced 228

products indicate that -oxidation was reduced in ech2. While these results suggest that the 229

mutation compromises ECH2 enoyl-CoA hydratase activity on acyl-CoA oxidase products, a 230

large amount of substrate is processed properly. Further, the changes are not to the degree of 231

defects seen in mfp2 mutant lines (Rylott et al. 2006) and suggest a minor role for ECH2 in the 232

core -oxidation pathway. 233

234

Efficient core β-oxidation and TAG hydrolysis are required for ech2 phenotypes 235

To test if ECH2 loss affects mutants defective in other peroxisomal pathways, higher-order 236

mutants were analyzed. Previous work demonstrated that ibr1 ibr3 ibr10 ech2 displayed similar 237

small cotyledons to ech2 (Strader et al. 2011). The same response was observed in other ibr 238

mutant combinations containing ech2 (Supplemental Figure S9A). However, ech2 mfp2 did not 239

have a size reduction and showed comparable cotyledon area to wild-type and the mfp2 parent 240

lines (Figure 4). Notably, the small cotyledons of ibr1 ibr10 ech2 also were rescued by 241

incorporation of mfp2 (Supplemental Figure S9B). These results demonstrate that loss of MFP2 242

can suppress the defects associated with ECH2 dysfunction regardless of IBA to IAA conversion 243

levels. Pavement cells in ech2 mfp2 cotyledons were of similar area and complexity to wild type 244

and mfp2 (Figure 4B-4D), suggesting that mfp2 suppresses the ech2 defects in cotyledon 245

development by promoting cotyledon cell expansion. 246

Previous studies suggested ech2 had reduced levels of IBA-derived IAA, as it produced fewer 247

lateral roots and developed shorter root hairs than wild type (Strader et al. 2011). This phenotype 248

also was suppressed by combination with mfp2, as ech2 mfp2 produced more lateral roots and 249

developed longer root hairs than ech2 (Figure 5). Similarly, IBA responses were tested based on 250

the resistance of dark-grown seedlings to the inhibitory effects of IBA on hypocotyl elongation. 251

Figure 5E and 5F show that ech2 mfp2 showed IBA sensitivity in hypocotyl growth compared to 252

ech2, further supporting a suppressive effect of mfp2 on ech2 phenotypes. These results suggest 253

that mfp2 suppressed the IBA to IAA conversion defects associated with losing ECH2 activity. 254



12

Phenotypic assays also were done on combination lines incorporating other core -oxidation 255

mutants. Incorporation of ped1 with ech2 showed identical responses as mfp2 combination 256

(Figure 4A and Supplemental Figure S9B), supporting the above model that the direct disruption 257

of fatty acid -oxidation rescues ech2 mutant phenotypes. Similarly, analysis of ech2 sdp1, 258

which also disrupts TAG lipase, showed that sdp1 suppresses ech2 phenotypes (Supplemental 259

Figure S10A and S10B). However, ech2 acx3 and ech2 aim1combination mutants showed 260

morphology similar to ech2 (Supplemental Figure S10C). mfp2 and ped1 have stronger 261

reductions in fatty acid metabolism than acx3 and aim1 (Supplemental Figure S7, (Lingard and 262

Bartel 2009; Rylott et al. 2006)). As demonstrated previously, phenotypes in acx mutant lines 263

and aim1 can be masked by redundancy, expression domains, and substrate specificity (Adham 264

et al. 2005; Rylott et al. 2003; Wiszniewski et al. 2009; Khan et al. 2012). For these reasons, we 265

hypothesize that lines combining ech2 and these mutations may be less likely to show altered 266

phenotypes due to enzyme activity remaining at these steps. Taken together, these results suggest 267

that active TAG hydrolysis and efficient fatty acid core -oxidation are required for ech2-linked 268

phenotypes. 269



13

270

ech2 over-accumulates short-chain 3-hydroxy fatty acids 271

The rescue of ech2 phenotypic defects by addition of mutations disrupting TAG hydrolysis or 272

core fatty acid β-oxidation suggests the accumulation of intermediates may be causing the defect 273

in ech2 cell expansion and resulting growth deficiencies. Based on the involvement of ECH2 274

activity in the catabolism of fatty acids with an even-numbered cis-double bond, potential 275

candidates include (R)-3-hydroxy fatty acids. 276

To detect such compounds, we performed a direct scan of negatively charged lipid metabolites 277

by ESI-MS. A unique peak (m/z = -157.2) was identified in ech2 seedlings (Figure 6). The m/z 278

matches the [M-H]ˉ ion of hydroxyoctenoic acid (C8:1 hydroxy fatty acid). To confirm the 279

identity of the compound, GC-MS was used to analyze the butyldimethylsilyl- derivatized lipid 280

mixture and the fragment fingerprint of each unique compound in ech2 samples was analyzed. 281

As expected, a unique peak was identified in the ech2 sample (Supplemental Figure S11). 282

Analysis of the fragment fingerprint suggests the peak represents an 8-carbon fatty acid, with an 283

ω-3 double bond and a 3-hydroxy group (Supplemental Figure S11). Another peak of m/z = -284

159.3, which matches the [M-H]ˉ ion of hydroxyoctanoic acid (C8:0 hydroxy fatty acid), was 285

identified at a lower abundance (Figure 6A). These short-chain 3-hydroxy fatty acids are likely 286

the hydrolysis products of the corresponding β-oxidation acyl-CoA intermediates of α-linolenic 287



14

acid (ALA, C18:3) and linoleic acid (LA, C18:2), both of which have a double bond at the 288

twelfth carbon requiring ECH2 for efficient degradation. (Figure 6B). Short-chain 3-hydroxy 289

fatty acids were not detectable in ech2 rosette leaves (Supplemental Figure S12) and 290

complementation lines had normal metabolism (Supplemental Figure S2F), supporting our 291

earlier finding that loss of ECH2 led to seedling-specific alterations. 292

293

The peak area in the ESI-MS scan was used to quantify the amount of these accumulated 294

compounds. While almost absent in wild type and mfp2, up to 20% of total FFA pool was 295

comprised of 3-hydroxyoctenoic acid and 3-hydroxyoctanoic acid in ech2 (Figure 6C). 296

Interestingly, the ech2 mfp2 double mutant accumulated much less 3-hydroxy fatty acids 297

compared with ech2 seedlings. As discussed before, MFP2 is one of the two primary enzymes 298

that catalyze the core β-oxidation steps upstream of ECH2 for degradation of fatty acids with 299

even-numbered cis-double bond (Figure 1); its absence would severely limit the amount and rate 300

of substrates supplied to ECH2. These results suggest the supply of substrate is kinetically 301



15

important to the over accumulation of the hydroxyoctenoic and hydroxyoctanoic acid products in 302

ech2 mutants. 303

304

Wild-type seedlings treated with 3-hydroxyoctanoic acid show ech2-like responses in many 305

different aspects 306

The fatty acid metabolic analysis on ech2 and ech2mfp2 suggests a connection between short-307

chain 3-hydroxy fatty acid accumulation and ech2 phenotypes. To test if there is a direct 308

connection, wild-type seedlings treated with 3-hydroxyoctanoic acid were examined for seedling 309

morphology, cotyledon pavement cell expansion, peroxisome morphology, and IBA response. 310

Wild-type seedlings treated with 50 µM 3-hydroxyoctanoic acid had comparable root length and 311

cotyledon area to mock-treated ech2 seedlings (Figure 7A to 7D). 2-hydroxyoctanoic acid was 312

applied to test if the position of hydroxyl group affects the inhibitory effectiveness. A similar 313

strong toxic effect was observed (Figure 7A). However, compared with 3-hydroxyoctanoic acid, 314

2-hydroxyoctanoic acid displayed a stronger inhibition effect on root elongation, but less 315

inhibition on cotyledon expansion (Figure 7B to 7D). These results demonstrate that 3-316

hydroxyoctanoic acid treatment can cause ech2-like morphology alterations in wild-type 317

seedlings, and fatty acids with hydroxy group at different positions have distinct effects on root 318

and cotyledon development. 319

To further examine the effect of treatments on pavement cell patterning, pavement cells were 320

imaged (Figure 7E). Notably, pavement cells of wild-type seedlings treated with 3-321

hydroxyoctanoic acid showed reductions in both cell area and complexity, with similar levels to 322

that of mock-treated ech2. (Figure 7F). The same reductions were not detected in wild-type 323

seedlings treated with 2-hydroxyoctanoic acid, even at applied concentrations sufficient to cause 324

significantly reduced root elongation (Figure 7B). These results indicate that treatment of 3-325

hydroxyoctanoic acid, but not 2-hydroxyoctanoic acid, can cause ech2-like defects in pavement 326

cell development in wild type seedlings. 327

To examine the effect of treatments on peroxisome morphology, wild-type and ech2 seedlings 328

expressing a peroxisomally-targeted GFP were treated similarly. Peroxisomes in cotyledon 329

pavement cells were imaged (Supplemental Figure S13A). As shown above, ech2 seedlings had 330

an increased peroxisome size and number compared to that of wild-type mock-treated seedlings 331



16

(Supplemental Figure S13B and S13C). Significant gains in peroxisome size and number were 332

observed in 3-hydroxyoctanoic acid-treated wild-type seedlings (Supplemental Figure S13B and 333

S13C). 2-hydroxyoctanoic acid-treated wild-type seedlings had mildly increased peroxisome size 334

but comparable peroxisome number to that of mock-treated wild-type seedlings (Supplemental 335

Figure S13B and S13C). These results suggest that 3-hydroxyoctanoic acid treatment can cause 336

ech2-like defects in peroxisome morphology. 337

To test if the hydroxy fatty acid treatment can induce an altered IBA response in wild-type 338

seedlings, root elongation and lateral root density were examined in wild-type seedlings grown in 339

media with both hydroxyoctanoic acid and IBA. Wild-type seedlings showed increasing 340

inhibition of root elongation with increasing IBA concentrations, while ech2 showed IBA-341

resistant responses (Figure 8A and 8B). Intriguingly, wild-type seedlings treated with both 3-342

hydroxyoctanoic acid and IBA had reduced inhibition of root elongation compared to IBA 343

treatment alone, indicating an ech2-like altered IBA response caused by the fatty acid treatment 344

(Figure 8B). Similarly, the effective concentration of IBA in lateral root stimulation was reduced 345

by concurrent application of the fatty acid substrate (Figure 8C). On the contrary, wild-type 346

seedlings treated with 2-hydroxyoctanoic acid showed comparable root elongation and root 347

branching to mock-treated wild-type seedlings under each IBA concentration (Figure 8B and 8C). 348

Again, these results suggest that treatment of 3-hydroxyoctanoic acid, but not 2-hydroxyoctanoic 349

acid, on wild-type seedlings can cause an altered IBA response similar to ech2 seedlings. 350



17

351



18

ech2 responds differently to treatments with unsaturated fatty acid depending on bond position 352

Considering the function of ECH2 in the degradation of unsaturated fatty acids on even-353

numbered carbon (Figure 1 and Goepfert et al. 2006), seedlings with low ECH2 activity are 354

hypothesized to accumulate (R)-3-hydroxyacyl-CoA and/or associated hydrolyzed intermediates 355

when treated with unsaturated fatty acids on even-numbered carbon, but not with unsaturated 356

fatty acids on odd-numbered carbon. This differential metabolic accumulation may lead to 357

different physiological responses. To test this hypothesis, wild-type and ech2 seedlings were 358

treated with ∆7-cis nonadecenoic acid (C19:1 ∆7-cis) and ∆10-cis nonadecenoic acid (C19:1 359

∆10-cis). Root elongation of seedlings were tested. In both wild type and ech2, significant 360

reductions of seedling root elongation were observed when treated with either substrate (Figure 361

9). At this concentration, the reductions of root elongation in wild-type seedlings treated with 362

either fatty acid were comparable. However, the inhibition of root elongation in ech2 seedlings 363

treated with ∆10-cis nonadecenoic acid was stronger compared to ech2 seedlings treated with 364

∆7-cis nonadecenoic acid (Figure 9). These results show that wild-type seedlings are equally 365

inhibited by ∆10-cis nonadecenoic acid and ∆7-cis nonadecenoic acid treatments; ech2 seedlings 366

are more sensitive to ∆10-cis nonadecenoic acid than ∆7-cis nonadecenoic acid treatment. 367

368

369

370



19

Discussion 371



20

Peroxisomes are important organelles that harbor lipophilic pathways required for plant growth, 372

development, and responses to changing environmental conditions. Fatty acid catabolism and 373

IBA to IAA conversion are peroxisomal pathways that occur via similar β-oxidation mechanisms. 374

Mutants exclusively defective in IBA to IAA conversion were defined by their aberrant 375

responses to IBA; exogenous sucrose is not required for their seedling establishment, ruling out 376

alterations in fatty acid metabolism. However, other mutants have disruptions in both IBA-377

response pathways and fatty acid metabolism. These mutations typically fall into two classes: 378

mutations disrupting overall peroxisome import or activity and those in enzymes with a primary 379

function in fatty acid β-oxidation that also impact IBA metabolism (Hu et al. 2012). Here, we 380

focused on ech2, which has defects in both peroxisomal pathways while also having unique early 381

growth defect. 382

383

ech2 has unique growth and metabolic phenotypes 384

The ech2-1 mutant, referred to here as ech2, was identified in a screen for mutants with altered 385

IBA response (Strader et al., 2011). The study demonstrated the requirement of IBA-derived 386

IAA in Arabidopsis seedling development by characterizing phenotypes of the quadruple mutant 387

ibr1ibr3ibr10ech2. The strong IBA resistance displayed in this line suggested reductions in IBA 388

to IAA conversion (Strader et al., 2011). Moreover, the altered IBA response of ech2 was 389

rescued in transgenic lines carrying 35S:HA-ECH2. Here, we further characterized the ech2 390

mutant in seedling morphology, and found that ech2 alone has reduced cotyledon expansion and 391

short root length as seen in ibr1ibr3ibr10ech2 (Figure 2 and Strader et al., 2010). A similar 392

reduced cotyledon expansion phenotype was observed in ech2-2 (Mana Katano, 2016). To 393

confirm these phenotypes result from the loss of ECH2 function, a 35S:ECH2 construct was 394

transformed into the ech2 background. Homozygous ech2(ECH2) transgenic lines had normal 395

growth responses in phenotypic assays, including both the seedling development and metabolic 396

phenotypes (Supplemental Figure S2). Notably, studies of an ECH2 RNAi line showed a wild-397

type physical appearance and normal responses to the IBA analog 2,4-DB (Goepfert et al., 2006). 398

These discrepancies suggest that ECH2 activity is more reduced in the ech2 mutant than in the 399

ECH2 RNAi line (Goepfert et al., 2006 and Strader et., 2010). Therefore, we focused on 400



21

expanding studies of the role of ECH2 using the ech2-1 mutant and an ech2(ECH2) 401

complementation line. 402

ech2 phenotypes are related to alterations reported in two peroxisomal pathways: fatty acid -403

oxidation and IBA to IAA conversion. ech2 cotyledons have retarded oil body mobilization and 404

enlarged peroxisomes (Figure 3), similar to other mutants with impaired fatty acid metabolism 405

(Rinaldi et al. 2016). The root growth defect of ech2 is alleviated on media with sucrose 406

(Supplemental Figure S6), similar to fatty acid catabolism mutants including sdp1 and mfp2 407

(Supplemental Figure S14; (Thazar-Poulot et al. 2015)). ech2 also is resistant to exogenous IBA 408

application (Figure 5) and has small cotyledons and short root hairs (Figures 2 and 5), similar to 409

the triple IBA-response mutant ibr1 ibr3 ibr10 (Strader et al. 2010). These mutant phenotypes 410

suggest the ECH2 enzyme affects both fatty acid catabolism and IBA to IAA conversion. 411

However, the compromised fatty acid metabolism of ech2 is distinct from that of mfp2 and ped1. 412

ech2 peroxisomes are larger than in wild type (Figure 3), but smaller than in mfp2 and ped1 413

(Rinaldi et al. 2016). Also, the increased peroxisome numbers in ech2 (Figure 3) have not been 414

reported in either mfp2 or ped1. Compared with mfp2 and ped1, reductions in hypocotyl 415

elongation of dark-grown ech2 are minor and not rescued by sucrose (Supplemental Figure S7). 416

These distinctions suggest ech2 disrupts fatty acid metabolism to only a mild degree. Further, 417

auxin did not restore root hair elongation to the level of wild type (Supplemental Figure S15). 418

Both ech2 and wild-type root hairs elongate with auxin treatment. Unlike ibr1 ibr3 ibr10, which 419

has short root hairs that recover to lengths similar to wild type following auxin application 420

(Strader et al. 2010), the differential between wild type and ech2 remains consistent plus or 421

minus auxin, suggesting the additional auxin does not overcome or is not the primary cause of 422

inhibited root hair elongation. Finally, the ech2 small cotyledon phenotype is not rescued by 423

exogenous sugar or auxin (Supplemental Figure S5 and S6). These observations led us to 424

consider potential underlying mechanisms other than the reduced production of energy/short-425

chain carbons or IBA-derived auxin. 426

Alternatively, ech2 responses could be due to decreased production of a particular product or 427

indirect effects resulting from reduced intermediates required for a secondary pathway or 428

accumulation of substrates or intermediates with potentially toxic consequences. We investigated 429

the hypothesis that ech2 phenotypes result from the toxic accumulation of molecules that 430



22

normally require ECH2 activity to be effectively metabolized. A previous report described a 431

feeding experiment in which ECH2 RNAi lines expressing a polyhydroxyalkanoate (PHA) 432

synthase were incubated with even cis-unsaturated fatty acids. Accumulation of PHA9:0 433

produced from corresponding 3-hydroxyacyl-CoAs (C9:0-OH) was observed, suggesting ECH2 434

is required to metabolize (R)-3-hydroxyacyl-CoAs (Goepfert et al. 2006). Arabidopsis seeds 435

accumulate large amounts of polyunsaturated fatty acids, primarily LA and ALA, which contain 436

even cis-double bonds at the Δ12 carbon (O'Neill et al. 2003). ECH2 RNAi lines also over-437

accumulated PHA8:0 and PHA8:1, corresponding to the metabolic intermediates of endogenous 438

LA and ALA (Goepfert et al. 2006). Although these results are consistent with our toxic 439

intermediate hypothesis, intermediate accumulation levels under normal background may not 440

result from the accumulation of PHA. Additionally, the accumulation of a novel product can 441

have broader impacts to the metabolic and/or signaling network in cells and the consequences of 442

such accumulation need to be examined in the context of plant morphology and development. 443

To explore ech2 metabolic changes, we profiled the fatty acid spectrum in wild type and mutants. 444

We detected 3-hydroxyoctanoic acid (C8:0-OH) and 3-hydroxyoctenoic acid (C8:1-OH) 445

accumulation in ech2 (Figure 6). C8:1-OH levels are ~7 fold higher than C8:0-OH (Figure 6C). 446

This pattern is opposite to the ALA and LA distribution in Arabidopsis seeds, which typically 447

have 21% ALA and 33% LA (O'Neill et al. 2003). It is possible that ALA is metabolized to a 448

higher degree than LA due to unknown enzyme specificities, C8:0-OH-CoA could be further 449

metabolized by alternative pathways, or acyl-CoA thioesterases prefer C8:1-OH-CoA for 450

hydrolysis. 451

In addition to the accumulation of these intermediates directly related to ECH2 loss, we also 452

found an accumulation of acetate (Figure 6C). The lack of other FFAs (Supplemental Table S1) 453

suggests the stall happens specifically, while the overall functionality of β-oxidation is less 454

affected. Our in vitro β-oxidation assay show ech2 also has reduced enoyl-CoA hydratase 1 455

activity, but in a minor way compared with mfp2 (Supplemental Figure S8; (Rylott et al. 2006)). 456

The decreased enoyl-CoA hydratase 1 activity in ech2 may be an indirect inhibitory effect of 457

accumulated abnormal FFAs, since previous reports suggest ECH2 only possesses enoyl-CoA 458

hydratase 2 activity (Goepfert et al., 2006). The functional core β-oxidation precludes the 459

complete disruption of fatty acid catabolism that might result from loss of ECH2. 460



23

461

Efficient substrate supply is required for the accumulation of fatty acid intermediates and 462

resulting phenotypes in ech2-containing mutants 463

We did epistatic analysis to investigate the genetic interactions between ech2 and other mutants 464

defective in peroxisomal enzymes. Our results showed that ibr higher-order mutants containing 465

ech2 displayed similar growth phenotypes to ech2 (Supplemental Figure S9A), consistent with 466

previous results describing ibr1 ibr3 ibr10 ech2 (Strader et al. 2011). However, mutants 467

disrupting enzymes upstream of ECH2 in fatty acid metabolic pathways suppress the ech2 468

growth phenotypes, including mfp2, ped1 and sdp1. The fatty acid catabolism defects determine 469

the severity of the growth defects in ech2-containing mutant combinations. In the catabolism of 470

even cis-unsaturated fatty acids, SDP1, MFP2, and PED1 catalyze the steps preceding ECH2 471

activity to generate (R)-3-hydroxyacyl-CoAs (Figure 1). ECH2 disruption would disrupt the 472

substrate-product balance, resulting in accumulation of (R)-3-hydroxyacyl-CoAs. If this step is 473

stalled for a prolonged period, these intermediates could build up or be degraded to recycle CoA 474

and produce free (R)-3-hydroxy fatty acids. Concurrent reductions of an upstream enzyme and 475

ECH2 reduce the supply of these substrates and minimize accumulation of (R)-3-hydroxy fatty 476

acids, thus restoring the metabolic balance and reducing the phenotypic severity. Free fatty acid 477

profiles of wild type and mutant lines support this hypothesis. In ech2 mfp2, accumulation of 478

short-chain hydroxy fatty acids and acetate were suppressed compared with ech2 (Figure 6 and 479

Supplemental Table S1). 480

The ech2 FFA profile also suggests the importance of the hydratase/epimerase pathway to 481

degrade fatty acids with an even-numbered cis-double bond. Previous results demonstrated the 482

alterative reductase/isomerase pathway (Figure 1) contributes to FFA degradation (Allenbach 483

and Poirier 2000). However, the dramatic 3-hydroxy-5-octenoic acid accumulation in ech2 (~20% 484

of total FFA pool; Figure 6C) leads to questions about activity of the reductase/isomerase 485

pathway. The production of the ECH2 substrate requires one round of core β-oxidation of the 2-486

trans-4-cis-dienoyl-CoA intermediate and hydration by MFP2; these steps are not reversible 487

(Figure 1; (Graham 2008), and therefore accumulating intermediates cannot be degraded by the 488

reductase/isomerase pathway. Auxiliary enzymes, including 3-hydroxyacyl-CoA epimerase, 2, 4-489

dienoyl-CoA reductase (DECR), and Δ3, Δ2-enoyl-CoA isomerases (AtECI), are not well 490



24

studied and future mutant analysis and metabolic profiling are necessary to understand the 491

activation of and biological importance for the reductase/isomerase pathway. 492

493

Accumulation of fatty acid intermediates affects auxin metabolism 494

The IBA-response mutant ibr1 ibr3 ibr10 ech2 has an ~20% reduction in IAA levels, indicating 495

IBA conversion is a major contributor to the auxin pool (Strader et al. 2011). One of the most 496

surprising observations in this study is the restoration of IBA sensitivity when ech2 was crossed 497

with fatty acid catabolism mutants. Previous studies hypothesized ECH2 as a shared enoyl-CoA 498

hydratase acting in both fatty acid catabolism and IBA to IAA conversion (Goepfert et al. 2006; 499

Strader et al. 2011). If the overlap between these two peroxisomal pathways happens at the 500

catalytic level, we would hypothesize combination mutants to have unchanged or enhanced IBA 501

resistance, as the additional fatty acid catabolism mutant would also negatively affect IBA to 502

IAA conversion. This opposite observation led us to hypothesize other effects resulting from loss 503

of ECH2 that affect these pathways (Figure 5). 504

We also uncovered an association between short-chain 3-hydroxy fatty acids and IBA resistance 505

in ech2-containing mutants. Consistently, treatment of 3-hydroxyoctanoic acid on wild-type 506

seedlings caused an altered IBA response similar to that of ech2 seedlings (Figure 8). The 507

accumulation in the mutant or feeding in the wild-type seedlings could be due to direct 508

disruption of peroxisomal IBA pathways. The unexpected over-accumulation of free acetate in 509

ech2 (Figure 6C) led to a second potential hypothesis. Unlike long-chain and very long-chain 510

acyl-CoAs, acetyl-CoA is the β-oxidation end product. Increased conversion of acetyl CoA to 511

acetate is not likely the result of a direct feedback effect of elevated intermediates. Instead, it 512

may reflect the increased need for local or global CoA recycling as a result of stalled (R)-3-513

hydroxy acyl-CoA in ech2. A similar effect could affect the co-localized IBA(-CoA) to IAA(-514

CoA) pathway, decreasing its efficiency and causing the IBA resistance phenotype. CoA 515

limitations have been suggested previously to affect IBA metabolism in mutants disrupting the 516

ACX proteins required for -oxidation as well (Adham et al. 2005). It is necessary to study 517

these genetic, metabolic, and regulatory interactions systematically, to understand the full picture 518

of peroxisome function in the context of auxin homeostasis and its influence on early plant 519

development. 520



25

521

Position of hydroxyl group and double bond affect the inhibitory specificity and potency of FFAs 522

3-hydroxyoctanoic acid treated wild-type seedlings phenocopied ech2 (Figure 7-8, and 523

Supplemental Figure 14). Treatment with 2-hydroxyoctanoic acid resulted in similar root defects 524

but milder cotyledon defects than ech2 (Figure 7). The different structures of the two 525

hydroxyoctanoic acids may make them effective against different targets, as 2-hydroxyoctanoate 526

inhibits medium chain acyl-CoA synthetase in mammals (Kasuya et al. 1996), while we 527

hypothesize 3-hydroxyoctanoate would competitively inhibit/saturate enoyl-CoA hydratases. It is 528

plausible the competitive inhibition can happen on acetyl-CoA synthetases in Arabidopsis, the 529

closest homologues of mammalian medium chain acyl-CoA synthetase, by both 2- and 3-530

hydroxyoctanoate, and other FFAs at higher concentrations. The additional inhibitory effect of 3-531

hydroxyoctanoic acid may contribute more to the cotyledon defect with the overlapping effects 532

on peroxisomal β-oxidation pathways. 533

Application of FFAs has general adverse effects on plant growth (Fan et al. 2013; Li et al. 2011). 534

To distinguish the general inhibition effect from defects specific to the loss of ECH2, it is 535

beneficial to study the positional effect of double bond on the specificity and potency of the 536

applied FFA to wild type and ech2. We compared the inhibitory effect of two nonadecenoic 537

acids (C19:1, cis-Δ7, and cis-Δ10), and found they differentially inhibited the growth of wild 538

type and ech2 (Figure 9). Notably, wild type showed no significant difference under the effect of 539

either C19:1, displaying only the general FFA toxicity effect. In contrast, ech2 was further 540

inhibited by C19:1-cis-Δ10 than by C19:1-cis-Δ7. This experiment further strengthens the 541

importance of ECH2 to the proper degradation of even cis-double bond FFAs. 542

543

Perspectives 544

We have investigated the metabolic alterations underlying the ech2 phenotypes. Loss of ECH2 545

led to aberrant accumulation of intermediates from fatty acid catabolism pathways, resulting in 546

seedling growth defects. A metabolic shift following the simultaneous loss of MFP2 reduced the 547

growth defects of ech2. We also have gained insights into potential interactions between 548

peroxisomal pathways. ech2 accumulates FFA intermediates that are not present under normal 549



26

conditions, resulting in exaggerated phenotypic responses. From this information, we can make 550

valuable inferences and form new hypotheses about the roles of FFAs in metabolic crosstalk and 551

potential signaling between peroxisomal pathways. 552

Our results and current hypotheses are illustrated in Figure 10. 3-hydroxy fatty acids accumulate 553

when ECH2 levels are low, as exemplified in this work by the ech2 mutant. In turn, this 554

metabolic defect reduces IBA to IAA conversion and impacts epidermal cell expansion, possibly 555

through the ROP pathway. At the cellular level, these metabolic changes lead to reduced oil body 556

mobilization and increases in peroxisome number and size. Simultaneous reduction of core -557

oxidation pathways, as exemplified by the double mutants in our studies, reduce the need for 558

ECH2 activity because a decreased amount of substrate is available for metabolism. Future 559

studies, including the direct profiling of IBA to IAA conversion intermediates and cell-specific 560

testing of upstream effectors and downstream signaling components of the ROP pathway may 561

reveal additional components of and connections between these metabolic and signaling 562

pathways. 563

Better understanding the mechanism underlying FFA lipotoxicity, especially of hydroxy fatty 564

acids, also has great benefits for industrial purposes. Hydroxy fatty acids are frequently used as 565

additives or raw materials for chemical feedstocks, lubricants, inks, and plastics (Lee et al. 2015). 566

Metabolic engineering approaches have been used to produce hydroxy fatty acids in Arabidopsis 567

seeds. However, transgenic plants successfully producing such compounds have low seed oil and 568

poor seedling establishment, similar to ech2 (Lunn et al. 2018a; Lunn et al. 2018b). Further 569

studies of the potential downstream processes of FFA signaling as well as transcriptome and 570

proteome changes in ech2 and FFA treated plants will greatly improve our understandings of 571

peroxisomal lipid metabolism and regulation in seeds and developing plants. 572

573

574



27

Methods 575

Plant materials 576

Arabidopsis thaliana mutants are in the Colombia (Col-0) background. ech2-1 has a G-A 577

mutation in At1g76150 (Gly36-to-Glu substitution) and was originally isolated as IBA resistant 578

in hypocotyl growth (Strader et al. 2011). ibr1-2, ibr3-1, and ped1-96 are EMS-based point 579

mutations that have been described previously (Lingard and Bartel 2009; Zolman et al. 2008; 580

Zolman et al. 2007; Zolman et al. 2000). ibr10-1 contains a large in frame deletion in At4g14430 581

(Zolman et al. 2008). Insertion mutants include acx3-6 (SALK_044956, (Adham et al. 2005)), 582

mfp2-2 (SALK_098016C, (Rylott et al. 2006)), aim1-1 (SALK_023469, (Richmond and 583

Bleecker 1999)) and sdp1 (SALK_102887, (Eastmond 2006; Thazar-Poulot et al. 2015)). All 584

mutants were crossed with ech2-1 to generate higher-order mutants for assays. A p35S: PTS2-585

GFP (Woodward and Bartel 2005) wild-type line was crossed into mutant lines to examine 586

peroxisome morphology. 587

588

Plant growth and phenotypic assays 589

Seeds were surface-sterilized and imbibed at 4°C in darkness for 3-4 days, and then plated on 590

plant nutrient (PN) medium (Haughn and Somerville 1986) solidified with 0.6% (w/v) agar. 0.5% 591



28

(w/v) sucrose was added (PNS) when indicated. Plates were incubated at 22°C in continuous 592

light. Seedlings were used for phenotypic assays, transferred to soil and grown at 18-22°C in 593

long-day conditions (16 hours light, 8 hours darkness). 594

To measure cotyledon area, seedlings were grown on PNS under white light for 7 d. Cotyledons 595

were imaged by confocal microscopy and the area was measured using NIH Image software 596

(https://imagej.nih.gov/ij/). To examine root elongation, seedlings were grown for 7-9 d and 597

removed from the agar to measure the primary root length. 598

To examine the effect of sucrose on seedling establishment, seeds were plated on PN or PNS 599

medium for 7 d, and then the cotyledon area and root length were measured. For dark grown 600

seedlings, seeds were plated on PN or on PNS medium for 1 d under white light, and then 601

transferred to dark for an additional 5 days growth. Hypocotyl lengths were measured. 602

To examine the effect of auxin on cotyledon pavement cells expansion, surface sterilized seeds 603

were germinated in liquid half strength MS (Toshio and Folke 1962) with mock or indicated 604

concentration of Naphthalene-1-acetic acid (NAA; GoldBio, St. Louis, MO) (Xu et al. 2010). 605

Cotyledons were detached and pavement cells were imaged at 3d. 606

To measure root hair length, seedlings were grown at 22°C in continuous light for 7 days on PNS 607

or PNS with indicated concentration of auxin. When auxin was added, plates were incubated 608

under yellow filters to protect indolic compound from breakdown (Stasinopoulos and Hangarter 609

1990). Seedlings were removed and immersed in 5% glycerol (v/v). Root hairs were imaged 610

using an EVOS Floid cell Imaging Station and measured using NIH Image software. 611

To examine IBA response, surface sterilized seeds were plated on PNS or PNS with indicated 612

concentrations of IBA (GoldBio, St. Louis, MO) for 1 d under light with yellow filters, and then 613

transferred to dark for an additional 5 days growth. Hypocotyl lengths were measured. For root 614

elongation assays, surface sterilized seeds were plated on media for 7 days under light with 615

yellow filters. And then root lengths were measured. 616

Statistical analysis was completed for each assay as indicated using GraphPad Prism. 617

618

Vector Construction and Plant Transformation 619



29

For complementation of the ech2 mutant, ECH2 and HA tag coding sequence were amplified 620

from the pEarleyGate 201 plasmid containing a N-terminal HA-fusion ECH2 (Strader 2011). 621

PCR products were digested and inserted into 35SFlast plant expression vector (Ge X., Dietrich 622

C., Matsuno M., Li G., Berg H., Xia Y. (2005)), replacing the Flag/Strep tag within the multiple 623

cloning sites BamHI and SalI. 624

35S-HA-ECH2 was introduced into Agrobacterium tumefaciens GV3101 (Koncz et al. 1992) and 625

transformed into ech2 using the floral dip method (Clough and bent 1998). Transformants were 626

selected on media supplemented with 25 µg/ml Kanamycin and verified with genomic PCR. 627

628

Confocal Microscopy and Organelle Quantification 629

Seedlings were submerged in propidium iodide (10 μg/mL; Molecular Probes) for at least 10 min 630

to label cell walls. Adaxial cotyledon pavement cells were imaged using a Zeiss LSM 700 laser 631

scanning confocal microscope with a 20X or 63X lens. Area and circularity [4(area/perimeter2)] 632

of each cell were determined using NIH Image software. Number of lobes was automatically 633

counted by Lobefinder software (Wu et al. 2016) and the PaCeQuant plugin in NIH Image 634

software (Moller et al. 2017). Lobes were manually counted for confirmation. 635

For Nile red Staining, seedlings were submerged in 5 μg/ml Nile red for at least 10 min. 636

Cotyledon oil bodies were imaged under confocal microscope through a 63X oil immersion lens. 637

The same lens was used to detect GFP signals to image the peroxisomes in seedlings with p35S: 638

PTS2-GFP background. Samples was excited with a 488-nm laser line and fluorescence 639

emission was collected for best signals of indicated fluorescent probes. 640

For each 202.5 x 202.5 μm confocal image, fluorescence area and organelle number was 641

measured using NIH Image software. Images were converted to 8-bit binary images. The 642

Threshold tool was used to designate objects (oil bodies or peroxisomes) with manual settings. 643

Fluorescence area and objects number were measured by the Analyze Particles function. 644

645

Fatty acid β-oxidation assays 646

After surface sterilization, seeds were plated on PNS medium covered with filter paper. β-647

oxidation assays were performed according to (Rylott et al. 2006). Briefly, total proteins were 648



30

extracted and desalted by disposable PD MidiTrap G-10 columns (GE Healthcare) using the 649

centrifugation protocol. Each assay was initiated by adding acyl-CoA substrate to the reaction 650

mix containing desalted protein. The relative abundance of acyl-CoA, enoyl-CoA, hydroxyacyl-651

CoA, ketoacyl-CoA in the β-oxidation series were determined by an ESI-MS/MS based method 652

adapted from (Rylott et al. 2006). 653

654

RNA Extraction and Expression Analysis 655

Seedlings or rosette leaves were collected at the indicated time and homogenized in liquid 656

nitrogen. RNA was extracted using the IBI Mini Total RNA Kit (Plant; MidSci). Expression 657

levels of genes of interest were determined and normalized to that of Ubiquitin10 (At4G05320) 658

with the BioRad CFX96 Real Time PCR System using Bullseye EvaGreen qPCR Mix (MidSci). 659

No reverse transcriptase and no cDNA reactions were used as negative controls. Primers for each 660

gene are listed in Supplemental Table S2. Experiments were performed with both biological and 661

technical triplicates. 662

663

Feeding experiments 664

Surface-sterilized seeds were placed at 4ْC for 72 h, and then transferred to liquid PN containing 665

0.5% sucrose, 2% pluronic-127 (Millipore Sigma), and fatty acids at the indicated concentration. 666

Constant light and agitation (70 rpm) were applied. 2-hydroxyoctanoic acid was purchased from 667

Millipore Sigma; 3-hydroxyoctanoic acid was purchased from Matreya LLC (www. 668

Matreya.com). ∆7-cis nonadecenoic acid and ∆10-cis nonadecenoic acid were purchased from 669

NU-CHEK-PREP, INC (www.nu-chekprep.com). 670

671

Lipid extraction 672

Total lipid extraction was performed according to method of (Liu and Wang 2016). Briefly, fresh 673

tissues (3-d old seedlings or 21-d rosette leaves) were incubated in preheated (75 °C) isopropanol 674

for 15 min to inactivate phospholipase D activity (Liu et al. 2015). Up to five extractions using 675

lipid extraction solution (Chloroform: methanol = 2:1 (v/v)) were performed. All the extracts 676



31

were collected and lipids phases were washed with 1 M KCl and water sequentially. Lipids were 677

dried under nitrogen evaporator, and then diluted with methanol for downstream analysis. 678

679

Analysis of free fatty acids by ESI-MS and GC-MS 680

FFAs in extracted lipid samples were analyzed directly by ESI-MS. The scanning method was 681

adapted from (Li et al. 2011) with slight modification. Briefly, lipid samples were scanned in 682

negative ion mode (-4500kV, -Q1) between 50 and 400 Da on API 4000 mass spectrometry. 683

Heptadecanoic acid (17:0) was used as internal standard. Acquired data were subjected to 684

background subtraction and normalization to internal standard and dry weight of plant material. 685

Five biological repeats were used for each genotype and treatment. 686

Agilent 5975C GC/MSD system was used to characterize novel fatty acid intermediates. Lipid 687

samples were hydrolyzed by 0.5 M HCl in acetonitrile/water (9:1) at 100 °C for 1 h, followed by 688

chloroform extraction. The organic phase was pooled and dried under nitrogen gas and the 689

derivatization was performed by adding 50 µl MTBSTFA (with 1% TBDMCS) and 50 µl N,N-690

dimethylformamide and incubating at 70°C for 1 h. Samples were separated by J&W DB-5ms 691

capillary column (Agilent, 122-5562G), from 80 °C to 310 °C with a ramping rate at 20 °C/min. 692

MSD scan range was set to 50-550 Da. Unique peaks in ech2 samples but not Wt were 693

individually checked. Each significant unique ion was mapped to potential fragments from 694

pseudo-molecular ions due to the instability of the theoretical molecular ion of the double 695

derivatized 3-hydroxyoctenoic acid. The retention time was also considered. An alternative 696

derivatization method using methylation followed by silylation was used to further confirm the 697

results. 698

699

Accession Numbers 700

Sequence data from this article can be found in the EMBL/GenBank database or the Arabidopsis 701

Genome Initiative database under the following accession numbers: ECH2, AT1G76150; IBR1, 702

AT4G05530; IBR3, AT3G06810; IBR10, AT4G14430; MFP2, AT3G06860; AIM1, AT4G29010; 703

ACX3, AT1G06290; PED1, AT2G33150; SDP1, AT5G04040; ROP2, At1g20090; ROP4, 704

At1g75840; ROP6, At1g10840; RIC1, At2g33460; RIC4, At5g16490. 705



32

706

Supplemental Data 707

Supplemental Figure 1. Only ech2 shows reduced cotyledon size and primary root length. 708

Supplemental Figure 2. ech2 is rescued by ECH2. 709

Supplemental Figure 3. Dry seed weight and germination rate of ech2 seeds are comparable to 710

those of Wt. 711

Supplemental Figure 4. ech2 cotyledons have reduced ROP expression levels. 712

Supplemental Figure 5. Auxin application is not able to improve pavement cell expansion in 713

ech2 cotyledons. 714

Supplemental Figure 6. ech2 displays better seedling growth on sucrose-containing medium than 715

on medium with no sucrose. 716

Supplemental Figure 7. ech2 shows similar elongation reductions in dark-grown hypocotyl 717

regardless of the absence or presence of sucrose. 718

Supplemental Figure 8. Enoyl-CoA hydratase activity on acyl-CoA oxidase product is disrupted 719

in ech2 seedlings. 720

Supplemental Figure 9. Either mfp2 or ped1 can suppress the defects of ech2 in cotyledon 721

development regardless of the conversion levels of IBA to IAA. 722

Supplemental Figure 10. Efficient TAG hydrolysis and fatty acid core β-oxidation are required 723

for ech2 phenotypes. 724

Supplemental Figure 11. GC-MS is used to confirm the identification of the unique peak in ech2 725

samples. 726

Supplemental Figure 12. C8:1 hydroxy fatty acid (m/z=-157) and C8:0 hydroxy fatty acid (m/z=-727

159) are not accumulated in rosette leaves. 728

Supplemental Figure 13. 3-hydroxyoctanoic acid treatment affects peroxisome size and number. 729

Supplemental Figure 14. Sucrose application improves seedling establishment of light-grown 730

mfp2 and ech2 mfp2. 731



33

Supplemental Figure 15. Application of auxin does not restore root hair elongation defects in 732

ech2. 733

Supplemental Table 1. FFA content of 3-d-old Wt, ech2, mfp2, and ech2 mfp2 seedlings. 734

Supplemental Table 2. Primers used for qPCR. 735

736

Acknowledgments 737

We thank the Arabidopsis Biological Resource Center at Ohio State University for seed stocks 738

and the Salk Institute Genomic Analysis Laboratory for generating the sequence-indexed 739

Arabidopsis T-DNA insertion mutants. We appreciate the gifts of ech2-1 seeds and p35S: HA-740

ECH2 plasmid from Lucia Strader, and ped1-96 and p35S: PTS2-GFP wild-type seeds from 741

Bonnie Bartel. We gratefully acknowledge members of the Zolman lab for assistance generating 742

plant lines and Vanessica Jawahir and Lon Chubiz for critical comments on the manuscript. 743

744

Figure legends 745

Figure 1. Peroxisomal β-oxidation of fatty acids and indole-butyric acid (IBA). 746

(A) Fatty acids (green) and IBA (purple) are processed and transported to peroxisomes for 747

catabolism through β-oxidation. Required enzyme activities are indicated in the center of the 748

mechanism; known or predicted enzymes acting in each pathway are indicated outside the 749

arrows. The enzyme converting IBA to IBA-CoA is unknown and it is not clear if IBA is 750

transported into peroxisome in the form of IBA or IBA-CoA. 751

(B) The complete degradation of C18:1Δ6-cis is shown as an example of pathways involved in 752

catabolism of fatty acids with a cis-double bond on an even-numbered carbon. Auxiliary 753

enzymes are in red. The first double bond of unsaturated fatty acids is always more than two 754

carbons away from the CoA attachment site. Thus, at least one round of core β-oxidation is 755

required to function upstream of auxiliary pathways in the metabolism of unsaturated fatty acids. 756

Figure 2. ech2 has altered seedling development, but normal adult growth. 757

(A) Left, cotyledons of 7-d-old wild-type (Wt) and ech2 seedlings. Right, images of whole 758

seedlings. Bar = 1 cm. 759



34

(B) Mean cotyledon area and root length (±SD; n≥10) of 7-d-old Wt, ibr1-2, ibr3-1, ibr10-1, 760

ech2-1, acx3-6, mfp2-2, and ped1-96 seedlings. Bar = 1 cm. *** p < 0.005 in Students’ T-test 761

between Wt and mutants. 762

(C) Morphology of 25-d-old Wt and ech2 plants. Bar = 1 cm. 763

(D) Leaf spread of 25-d-old Wt (top) and ech2 (bottom) plants in order of emergence. White 764

arrows designate the cotyledons. Bar = 1 cm. 765

(E) Representative confocal microscopy images of propidium iodide-stained Wt and ech2 766

cotyledon pavement cells at day 5 (top) and day 12 (middle), and the first true leaf pavement 767

cells at day 12 (bottom). Bar = 50 μm. 768

(F) The area, lobe number, and circularity of pavement cells. Circularity = 4π(area)/(perimeter)²; 769

the value of circularity is between 0 and 1. A value of 1 indicates a perfect circle; 0 indicates a 770

shape with infinite complexity. 771

Data are shown ±SD; n>30. *** p < 0.005 in Students’ T-test between Wt and ech2. NS, not 772

significant. 773

Figure 3. ech2 has reduced oil body mobilization and enlarged peroxisomes. 774

(A) Confocal microcopy of cotyledon pavement cells in 3, 4, and 5-d-old Wt and ech2 seedlings. 775

Nile red was used to stain oil bodies (red). 776

(B) Nile-red puncta number and sum of nile-red puncta areas per 202.5 x 202.5 μm image (±SD; 777

n= 10). 778

(C) Confocal microcopy of peroxisomes in cotyledon pavement cells of 4 and 5-d-old Wt and 779

ech2 seedlings expressing peroxisomally targeted GFP (PTS2-GFP). Propidium iodide was used 780

to stain pavement cell membranes (magenta). 781

(D) PTS2-GFP puncta areas from 202.5 x 202.5 μm images shown in (C) were plotted (±SD; 782

n>100). 783

*** p < 0.005 in Students’ T-test. The number below the x-axis indicates how many puncta were 784

measured for Wt and ech2 on each day. 785



35

Results shown are representative of four independent experiments. For all panels, scale bar = 50 786

μm. 787

Figure 4. Either mfp2 or ped1 can suppress the deficiency of ech2 in cell expansion. 788

(A) Cotyledons of Wt, ech2, mfp2, ech2 mfp2, ped1, and ech2 ped1 seedlings five and seven 789

days after sowing. Bar = 1 cm. 790

(B) Confocal microscopy of propidium iodide-stained cotyledon pavement cells at day 5. Bar = 791

50 μm. 792

(C-D) The area (C) and interdigitation (D) of cotyledon pavement cell in 5-d-old seedlings (±SD; 793

n≥30). * p < 0.05; *** p < 0.005 in Students’ T-test between Wt and mutants. Interdigitation, an 794

index of interlocking levels between adjacent cells, was calculated by dividing lobe number by 795

cell area. 796

Figure 5. mfp2 suppresses the deficiency of ech2 in IBA to IAA conversion. 797

(A) 10-d-old Wt, ech2, mfp2, and ech2 mfp2 seedlings grown on medium with no hormone. Bar 798

= 1 cm. 799

(B) Lateral root densities were calculated by dividing lateral root number by primary root length 800

(SD±; n≥10). Statistical significance was determined by ANOVA and Tukey's multiple 801

comparisons test (P < 0.05). Letters in common among the groups indicate no significant 802

difference. 803

(C) Representative roots images of 7-d-old seedlings. Bar = 0.25 cm. 804

(D) Root hair length of 7-d-old seedlings was measured using ImageJ. Data are shown as a 805

histogram of the percent of root hairs of each length range ±SD; n≥10. 806

(E) Wt, ech2, mfp2, and ech2 mfp2 seedlings grown one day in light, followed by 5 days in the 807

dark on medium without hormone or with 20 μM IBA. Bar = 1 cm. 808

(F) Hypocotyl length of seedlings grown one day in light, followed by 5 days in the dark on 809

media with 0, 10, 15, or 20 μM IBA. Data shown are ±SD; n≥12. 810

Figure 6. ech2 over-accumulates short-chain 3-hydroxy fatty acids. 811



36

(A) ESI-MS spectra of lipid extracts of 3-d-old Wt, ech2, mfp2, and ech2 mfp2 seedlings. 812

(B) Putative β-oxidation of α-linolenic acid after the attachment of CoA. C8:1 hydroxy fatty 813

acid-CoA is labeled as the substrate of ECH2. α-linolenic acid also has cis double bonds at odd-814

numbered carbons (Δ9 and Δ15), and therefore complete degradation requires additional 815

auxiliary enzyme activities including the Δ3,5, Δ2,4-dienoyl-CoA isomerase (DCI/Isomerase; 816

(Graham 2008)). Linoleic acid-CoA should be catabolized similarly in spite of the absent double 817

bond at Δ15. 818

(C) Acetate, C8:1 hydroxy fatty acid (C8:1-OH), and C8:0 hydroxy fatty acid (C8:0-OH) content 819

of 3-d-old Wt, ech2, mfp2, and ech2 mfp2 seedlings (±SD; n=5). * p < 0.05; ** p < 0.01; *** p < 820

0.005 in Student’ T-test between Wt and mutants. 821

Figure 7. Wild-type seedlings treated with 3-hydroxyoctanoic acid displayed defects on root and 822

cotyledon development similar to ech2. 823

(A) 5-d-old Wt seedlings treated with different concentrations of either 3-hydroxyoctanoic acid 824

or 2-hydroxyoctanoic acid, and ech2 seedlings treated with mock and . Bar = 1 cm. 825

(B) Root length of seedlings shown in (A) ±SD; n>10. 826

(C-D) Cotyledon images (C) and cotyledon area (D) of 5-d-old Wt seedlings treated with mock, 827

0.05 mM 3-hydroxyoctanoic acid, 0.05 mM 2-hydroxyoctanoic acid, and ech2 seedlings treated 828

with mock. 829

Cotyledon area was measured with ImageJ and averages are shown ±SD; n>10. Bar = 1 cm. 830

(E) Representative confocal microscopy images of propidium iodide-stained cotyledon pavement 831

cells of 6-d-old Wt and ech2 seedlings treated with mock, Wt seedlings treated with 0.05 mM 832

C8:0 (3-OH), and Wt seedlings treated with 0.05 mM C8:0 (2-OH). Bar = 50 μm. 833

(F) The area (top), lobe number (middle), and circularity (bottom) of pavement cells. 834

Data are shown ±SD; n>30. Statistical significance was determined by ANOVA and Tukey's 835

multiple comparisons test (P < 0.05). Letters in common among the groups indicates no 836

significant difference. 837



37

Figure 8. Wild-type seedlings treated with 3-hydroxyoctanoic acid have ech2-like altered IBA 838

response. 839

(A) Representative images of 6-d-old Wt and ech2 seedlings grown in media with different 840

combinations of 3-hydroxyoctanoic acid (C8:0 3-OH), 2-hydroxyoctanoic acid (C8:0 2-OH), and 841

IBA. Scale bar = 1 cm. 842

(B) Root length of seedlings shown in (A). 843

(C) Lateral root density of seedlings shown in (A). 844

Data are shown ±SD; n>10. Statistical significance was determined by ANOVA and Tukey's 845

multiple comparisons test (P < 0.05). Letters in common among the groups indicates no 846

significant difference. 847

Figure 9. Δ7-cis nonadecenoic acid treatment and Δ10-cis nonadecenoic acid treatment inhibit 848

root elongation of ech2 seedlings to different levels. 849

(A) 5-d-old Wt (top) and ech2 (bottom) seedlings treated with mock, 5 mM Δ7-cis nonadecenoic 850

acid (C19:1 (Δ7-cis)), or 5 mM Δ10-cis nonadecenoic acid (C19:1 (Δ10-cis)). Scale bar = 1 cm. 851

(B) Root length of seedlings shown in (A) ±SE; n>10. 852

Statistical significance was determined by ANOVA and Tukey's multiple comparisons test (P < 853

0.05). Letters in common among the groups indicates no significant difference. 854

Figure 10. Schematic model of how low ECH2 activity alters metabolic pathways that influence 855

fatty acid catabolism and IBA to IAA conversion during seedling development in Arabidopsis 856

thaliana. 857

(A) ECH2 functions in the hydratase pathway for the degradation of unsaturated fatty acids with 858

a cis-double bond on an even-numbered carbon. 859

(B) Low ECH2 activity, indicated in this study by an ech2 mutant, results in accumulation of 860

short-chain 3-hydroxyacyl acids and acetate, which suppress IBA to IAA conversion and 861

cotyledon cell expansion via Rho GTPase signaling and other undetermined pathways. 862

(C) ech2 mfp2 displays phenotypes similar to mfp2. Low ECH2 activity has less impact on a cell 863

when core β-oxidation pathway activity also is reduced. In the ech2 background, MFP2 864



38

dysfunction results in the reduced production of acetyl-CoA, ECH2 substrates, and associated 865

metabolites, releasing the suppression on cell expansion and IBA to IAA conversion. Exogenous 866

sucrose still is required for seedling establishment in mfp2 and ech2 mfp2 due to the reduced 867

energy production when β-oxidation pathways are not active. 868

For all panels, bar = 1 cm. 869



39

870



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https://www.ncbi.nlm.nih.gov/pubmed?term=Adham AR%2C Zolman BK%2C Millius A%2C Bartel B %282005%29 Mutations in Arabidopsis acyl%2DCoA oxidase genes reveal distinct and overlapping roles in β%2Doxidation%2E Plant J&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Spiess GM%2C Hausman A%2C Yu P%2C Cohen JD%2C Rampey RA%2C Zolman BK %282014%29 Auxin Input Pathway Disruptions Are Mitigated by Changes in Auxin Biosynthetic Gene Expression in Arabidopsis%2E Plant Physiol 165 %283%29%3A1092%2D1104%2E doi%3A10%2E1104%2Fpp&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Stasinopoulos TC%2C Hangarter RP %281990%29 Preventing Photochemistry in Culture Media by Long%2DPass Light Filters Alters Growth of Cultured Tissues%2E Plant Physiology 93 %284%29%3A1365%2D1369%2E doi%3A10%2E1104%2Fpp&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Strader LC%2C Culler AH%2C Cohen JD%2C Bartel B %282010%29 Conversion of endogenous Indole%2D3%2DButyric Acid to Indole%2D3%2DAcetic Acid drives cell expansion in Arabidopsis seedlings%2E Plant Physiol 153 %284%29%3A1577%2D1586%2E doi%3A10%2E1104%2Fpp&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Strader LC%2C Wheeler DL%2C Christensen SE%2C Berens JC%2C Cohen JD%2C Rampey RA%2C Bartel B %282011%29 Multiple Facets of Arabidopsis Seedling Development Require Indole%2D3%2DButyric Acid Derived Auxin%2E Plant Cell 23 %283%29%3A984%2D999%2E doi%3A10%2E1105%2Ftpc&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Thazar%2DPoulot N%2C Miquel M%2C Fobis%2DLoisy I%2C Gaude T %282015%29 Peroxisome extensions deliver the Arabidopsis SDP1 lipase to oil bodies%2E Proceedings of the National Academy of Sciences 112 %2813%29%3A4158%2D4163%2E doi%3A10%2E1073%2Fpnas&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Toshio M%2C Folke S %281962%29 A revised medium for rapid growth and bio assays with tobacco tissue cultures%2E %2E Physiol Plant&dopt=abstract

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https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Toshio&as_ylo=1962&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Velasquez SM%2C Barbez E%2C Kleine%2DVehn J%2C Estevez JM %282016%29 Auxin and Cellular Elongation%2E Plant Physiol 170 %283%29%3A1206%2D1215%2E doi%3A10%2E1104%2Fpp&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Wiszniewski A%2C Zhou W%2C Smith S%2C Bussell J %282009%29 Identification of two Arabidopsis genes encoding a peroxisomal oxidoreductase%2Dlike protein and an acyl%2DCoA synthetase%2Dlike protein that are required for responses to pro%2Dauxins%2E Plant Mol Biol&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Woodward AW%2C Bartel B %282005%29 The Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 is necessary for peroxisome function and dependent on PEX5%2E Mol Biol Cell 16 %282%29%3A573%2D583%2E doi%3A10%2E1091%2Fmbc%2Ee&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Wu TC%2C Belteton SA%2C Pack J%2C Szymanski DB%2C Umulis DM %282016%29 LobeFinder%3A A Convex Hull%2DBased Method for Quantitative Boundary Analyses of Lobed Plant Cells%2E Plant Physiol 171 %284%29%3A2331%2D2342%2E doi%3A10%2E1104%2Fpp&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Xu T%2C Wen M%2C Nagawa S%2C Fu Y%2C Chen J%2DG%2C Wu M%2DJ%2C Perrot%2DRechenmann C%2C Friml J�%2C Jones AM%2C Yang Z %282010%29 Cell Surface%2D and Rho GTPase%2DBased Auxin Signaling Controls Cellular Interdigitation in Arabidopsis%2E Cell&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Zhang C%2C Halsey LE%2C Szymanski DB %282011%29 The development and geometry of shape change in Arabidopsis thalianacotyledon pavement cells%2E BMC Plant Biology 11 %281%29%3A27%2E doi&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Zolman BK%2C Martinez N%2C Millius A%2C Adham AR%2C Bartel B %282008%29 Identification and characterization of Arabidopsis Indole%2D3%2DButyric Acid response mutants defective in novel peroxisomal enzymes%2E Genetics 180 %281%29%3A237%2D251%2E doi%3A10%2E1534%2Fgenetics&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Zolman BK%2C Nyberg M%2C Bartel B %282007%29 IBR3%2C a novel peroxisomal acyl%2DCoA dehydrogenase%2Dlike protein required for indole%2D3%2Dbutyric acid response%2E Plant Mol Biol&dopt=abstract




https://www.ncbi.nlm.nih.gov/pubmed?term=Zolman BK%2C Silva ID%2C Bartel B %282001%29 The Arabidopsis pxa1 Mutant Is Defective in an ATP%2DBinding Cassette Transporter%2DLike Protein Required for Peroxisomal Fatty Acid %2DOxidation%2E Plant Physiol 127 %283%29%3A1266%2D1278%2E doi%3A10%2E1104%2Fpp&dopt=abstract





classes. Genetics 156 (3):1323-1337Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title


https://www.ncbi.nlm.nih.gov/pubmed?term=Zolman BK%2C Yoder A%2C Bartel B %282000%29 Genetic analysis of Indole%2D3%2Dbutyric Acid responses in Arabidopsis thaliana reveals four mutant classes%2E Genetics&dopt=abstract





1 short title: lipid intermediate toxicity in seedlings 2139 catabolism activity on seedlings....

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