Multiple Facets of Arabidopsis Seedling Development Require
Indole-3-Butyric Acid–Derived Auxin
Lucia C. Strader,aDorthea L. Wheeler,a,b,1Sarah E. Christensen,a,bJohn C. Berens,a,2
Jerry D. Cohen,cRebekah A. Rampey,band Bonnie Bartela,3
aDepartment of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005
bDepartment of Biology, Harding University, Searcy, Arkansas 72143
cDepartment of Horticultural Science and Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, Minnesota
Levels of auxin, which regulates both cell division and cell elongation in plant development, are controlled by synthesis,
inactivation, transport, and the use of storage forms. However, the specific contributions of various inputs to the active
auxin pool are not well understood. One auxin precursor is indole-3-butyric acid (IBA), which undergoes peroxisomal
b-oxidation to release free indole-3-acetic acid (IAA). We identified ENOYL-COA HYDRATASE2 (ECH2) as an enzyme
required for IBA response. Combining the ech2 mutant with previously identified iba response mutants resulted in enhanced
IBA resistance, diverse auxin-related developmental defects, decreased auxin-responsive reporter activity in both untreated
and auxin-treated seedlings, and decreased free IAA levels. The decreased auxin levels and responsiveness, along with the
associated developmental defects, uncover previously unappreciated roles for IBA-derived IAA during seedling develop-
ment, establish IBA as an important auxin precursor, and suggest that IBA-to-IAA conversion contributes to the positive
feedback that maintains root auxin levels.
Auxin is a key phytohormone that directs both cell division and
cell elongation, thus regulating critical aspects of plant growth
and development (reviewed in Perrot-Rechenmann, 2010). The
active auxin indole-3-acetic acid (IAA) is a potent growth regu-
lator, and its levels are modulated through synthesis, regulated
transport, and storage forms (reviewed in Woodward and Bartel,
2005b). However, the relative importance of various pathways
that contribute to the active auxin pool is not well understood.
is two carbons longer than the IAA side chain and is shortened to
IAA in numerous plants (Fawcett et al., 1960; reviewed in Epstein
and Ludwig-Mu ¨ller, 1993) in a peroxisome-dependent manner
revealed that the auxin activity of IBA requires conversion to IAA
through a multistep process similar to fatty acid b-oxidation,
which removes two-carbon units from fatty acyl-CoA molecules.
Proteins required for full IBA responsiveness include the ATP
binding cassette (ABC) transporter PEROXISOMAL ABC TRANS-
PORTER1 (PXA1), which may transport IBA into the peroxisome
for b-oxidation (Zolman et al., 2001), and proteins required for
2005), PEX5 (Zolman et al., 2000), PEX6 (Zolman and Bartel,
2004), and PEX7 (Woodward and Bartel, 2005a; Ramo ´n and
Bartel, 2010). In addition, several peroxisomal enzymes are re-
quired for IBA responsiveness, including a 3-ketoacyl-CoA thio-
lase (Zolman et al., 2000) and several acyl-CoA oxidase (ACX)
(Adham et al., 2005) enzymes, which also act in fatty acid
b-oxidation, and the apparent IBA b-oxidation enzymes INDOLE-
3-BUTYRIC ACID RESPONSE1 (IBR1) (Zolman et al., 2008), IBR3
(Zolman et al., 2007), and IBR10 (Zolman et al., 2008).
MutationsinIBR1,IBR3, or IBR10conferIBAresistancewithout
altering IAA response or causing dependence on exogenous
carbon sources for postgerminative growth (Zolman et al., 2000,
2007, 2008), consistent with the possibility that the encoded
peroxisomal enzymes act directly in IBA-to-IAA conversion. IBR1
et al., 2008), IBR3 resembles acyl-CoA dehydrogenases/
oxidases(Zolman etal.,2007),and IBR10 appears tobeanenoyl-
CoA hydratase (Zolman et al., 2008). A triple mutant defective in
these three IBR enzymes displays enhanced IBA response
defects (Zolman et al., 2008), converts IBA to IAA inefficiently,
and exhibits expansion defects in root hairs and cotyledons
suggestive of lowered IAA levels (Strader et al., 2010).
Here, we describe the identification of an IBA response mutant,
ech2-1, from a new screen using dark-grown seedlings. The
peroxisomally localized ENOYL-COA HYDRATASE2 (ECH2) en-
zyme is required for full response to applied IBA, and ech2
phenotypes are synergistic with previously identified ibr mutants.
Moreover, combining ech2 with other ibr mutations results in
multiple striking developmental defects, reduced responsiveness
to both IBA and IAA, and decreased IAA levels in root tips,
1Current address: University of Iowa Carver College of Medicine, Iowa
City, IA 52242.
2Current address: Baylor College of Medicine, Houston, TX 77030.
3Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Bonnie Bartel
WOnline version contains Web-only data.
The Plant Cell, Vol. 23: 984–999, March 2011, www.plantcell.org ã 2011 American Society of Plant Biologists
revealing important contributions of IBA to auxin levels in devel-
ECH2 Is Required for IBA Responsiveness
Endogenous auxin generally promotes cell expansion, but su-
praoptimal auxin levels can inhibit cell and consequently organ
expansion in both roots and hypocotyls. Classical auxin re-
sponse mutant screens are based on resistance to natural or
synthetic auxins in light-grown root elongation assays (reviewed
in Woodward and Bartel, 2005b). Because several mutants
exhibiting strongly auxin-resistant root elongation are not mark-
edly IBA resistant in dark-grown hypocotyl assays (Strader et al.,
2008a), we employed a hypocotyl resistance (HR) screen for
genes necessary for IBA response in dark-grown seedlings and
isolated mutants with long hypocotyls on an inhibitory IBA con-
centration. In dark-grown wild-type seedlings, hypocotyl elon-
gation is inhibited in the presence of the naturally occurring active
auxin IAA, the auxin precursor IBA, the synthetic auxin 2,4-D, and
the synthetic auxin precursor 2,4-dichlorophenoxybutyric acid
(2,4-DB; Figure 1A). Hypocotyl elongation of the auxin-resistant
mutant axr1-3 (Estelle and Somerville, 1987; Leyser et al., 1993)
was strongly resistant to IAA, 2,4-D, and 2,4-DB, but only slightly
HR7 isolate was strongly resistant to the auxin precursors IBA
and 2,4-DB but sensitive to the active auxins IAA and 2,4-D
et al., 2010) and 2,4-DB (Hayashi et al., 1998) require peroxi-
somal carboxyl side chain shortening to release active auxins,
the specific resistance of HR7 to IBA and 2,4-DB suggested a
We mapped the causative mutation in HR7 to a 592-gene
region on thelower arm of chromosome 1(Figure1B) and looked
for peroxisomally targeted enzymes in the mapping interval. One
such candidate was ECH2 (At1g76150), which encodes a per-
oxisomal enoyl-CoA hydratase implicated in the b-oxidation of
unsaturated fatty acids (Goepfert et al., 2006). We sequenced
ECH2 from HR7 genomic DNA and identified a G-to-A base
change at position 371 (using the A of the ATG as position 1) that
causes a Gly36-to-Glu missense mutation (Figure 1B) in a
residue conserved (Figure 1C) in many ECH2 homologs (Figure
1D). We named the mutation in HR7 ech2-1.
Because ech2-1 carried a mutation in a putative peroxisomal
enzyme, we compared the IBA responsiveness of ech2-1 in
hypocotyl and root elongation assays to mutants identified in
screens for IBA-resistant root elongation (see Supplemental
Table 1 online) (Zolman et al., 2000), including mutants defective
in peroxisomal enzymes IBR1 (Zolman et al., 2008), IBR3 (Zolman
et al., 2007), IBR10 (Zolman et al., 2008), ACX3 (Adham et al.,
2005), and PEROXISOME DEFECTIVE1 (PED1) (Lingard and
Bartel, 2009); a mutant defective in the peroxisomal transporter
PXA1 (Zolman et al.,2001); and mutants defective in peroxisome
biogenesis factors PEX4 (Zolman et al., 2005), PEX5 (Zolman
et al., 2000), PEX6 (Zolman and Bartel, 2004), and PEX7 (Ramo ´n
and Bartel, 2010). Like ech2-1, these mutants displayed IBA-
resistant hypocotyls: ped1-96, pxa1-1, pex4-1, pex6-1, and
pex7-2 displayed IBA resistance similar to ech2-1, whereas
ibr1-2, ibr3-1, ibr10-1, acx3-6, and pex5-1 displayed more
moderate resistance (Figure 2B). In addition, ech2-1 displayed
strongly IBA-resistant roots, similar to the ibr10-1, ped1-96,
pxa1-1, pex5-1, and pex6-1 mutants (Figures 2C and 2D).
Although ech2 RNA interference (RNAi) lines have normal 2,4-DB
responsiveness in spite of 5- to 10-fold reductions in ECH2
mRNA levels (Goepfert et al., 2006), we found that ech2-1 hypo-
cotyl (Figure 1A) and root (see Supplemental Figure 1A online)
elongation were 2,4-DB resistant. This 2,4-DB resistance indi-
cated that the ech2-1 defect was general to chain-elongated
more severely impaired ECH2 function than the previously char-
acterized ech2 RNAi lines (Goepfert et al., 2006).
Wild-type Arabidopsis seedlings metabolize fatty acids stored
in seeds by peroxisomal b-oxidation to fuel growth prior to
photosynthesis (Hayashi et al., 1998). Mutants defective in IBR1,
IBR3, or IBR10 display IBA resistance without dependence on
exogenous fixed carbon sources for postgerminative growth
(Zolman et al., 2000, 2007, 2008), consistent with the possibility
that these enzymes are required for IBA-to-IAA conversion but
not for fatty acid b-oxidation. Mutants defective in PXA1 or
PED1,however,display bothIBAresistance and dependence on
sucrose to fuel growth following germination (Hayashi et al.,
1998; Zolman et al., 2001), suggesting roles in both IBA and fatty
acid b-oxidation. Dark-grown ech2-1 hypocotyls, like those
of the wild type and the ibr mutants, elongated normally
with or without sucrose (Figures 2E and 2F), suggesting that
seed storage fatty acids are metabolized normally in ech2.
Together, the IBA resistance and sucrose independence of
ech2-1 suggest that ECH2 functions in IBA-to-IAA conversion
To test whether the ech2-1 lesion caused the observed IBA-
response defects, we transformed ech2-1 with constructs driv-
ing N-terminally tagged versions of an ECH2 cDNA from the
strong 35S promoter from cauliflower mosaic virus. ech2-1 was
rescued by both 35S:HA-ECH2 and 35S:YFP-ECH2 constructs
(Figures 3A and 3C), indicating that the IBA response defects
resulted from reduced ECH2 function and that the N-terminal
tags did not interfere with ECH2 function. Yellow fluorescent
protein (YFP)-ECH2 localization in the ech2 35S:YFP-ECH2 line
exhibited subcellular punctate fluorescence resembling the size
and shape of peroxisomes (Figure 3D) and colocalized with a
BODIPY probe (Figure 3E) that stains peroxisomes (Landrum
et al., 2010). This localization is consistent with previous reports
of peroxisomal localization of YFP-ECH2 in transiently trans-
fected onion epidermal cells (Goepfert et al., 2006; Reumann
et al., 2007) and identification of ECH2 in the proteome of
peroxisomes purified from Arabidopsis leaves (Reumann et al.,
2007) and cell cultures (Eubel et al., 2008).
Because ECH2 is an enoyl-CoA hydratase (Goepfert et al.,
2006) and IBR10 resembles enoyl-CoA hydratases (Zolman
et al., 2008), we examined whether ECH2 and IBR10 acted
IBR10 in both ech2-1 and ibr10-1. As previously demonstrated
(Zolman et al., 2008), overexpressing IBR10 complemented
ibr10-1 (Figure 3B). However, IBA responsiveness was not
Developmental Roles of IBA-Derived Auxin 985
restored in ibr10 accumulating hemagglutinin (HA)-ECH2 to
levels similar to the levels that complemented ech2-1 (Figure
3A; see Supplemental Figure 2 online), indicating that additional
ECH2 did not compensate for decreased IBR10 activity. Like-
wise, overexpressing IBR10 failed to restore IBA responsiveness
to the ech2-1 mutant (Figure 3B; see Supplemental Figure 2
online). These data suggest that ECH2 and IBR10, although
members of the same protein superfamily (Figure 1D), are
unlikely to act redundantly at the same step of IBA-to-IAA
conversion (Figure 2G).
ech2-1 Enhances Effects of Mutants with Reduced
We examined ech2-1 in combination with the previously char-
Figure 1. ECH2 Is Required for Hypocotyl IBA Response.
(A) Mean hypocotyl lengths (+SE; n $ 14) of dark-grown wild type (Wt), axr1-3, and isolate HR7 (ech2-1) on various natural and synthetic auxins.
(B) Recombination mapping with the indicated markers (see Supplemental Table 2 online) localized HR7 to a 2-Mb region on chromosome 1 (dark bar)
containing592 predicted genes between LW104 and LW109 with 1/384 and 8/462 flanking recombinants. Examination of the ECH2 (At1g76150) gene in
this region revealed a G-to-A mutation at position 371 in HR7 DNA that results in a Gly36-to-Glu substitution.
(C) The ech2-1 mutation disrupts a conserved Gly (asterisk). Sequences from predicted ECH2 homologs (accession numbers in [D]) and MFE2
homologs were aligned using the MegAlign program (DNAStar; full alignment and accession numbers are in Supplemental Figure 5 online).
(D) Phylogenetic tree of ECH2, IBR10, MFE2, MFP2, AIM1, and relatives. Protein portions corresponding to the hydratase domains were aligned using
ClustalW (alignment in Supplemental Figure 6 online and Supplemental Data Set 1 online), and the unrooted phylogram was generated using PAUP
4.05b (Swofford, 2001) by performing the bootstrap method with 500 replicates. Bootstrap values are shown at the nodes.
986 The Plant Cell
Figure 2. Hormone Responses of ech2 and Other Peroxisomal Mutants.
(A) Five-day-old wild-type (Wt) and ech2-1 seedlings following growth in the dark on medium supplemented with ethanol (Mock) or 20 mM IBA. Bar = 1 cm.
(B) Mean hypocotyl lengths (+SE; n $ 12) of 5-d-old dark-grown wild-type, ech2-1, ibr1-2, ibr3-1, ibr10-1, acx3-6, ped1-96, pxa1-1, pex4-1, pex5-1,
pex6-1, and pex7-2 grown on medium supplemented with ethanol (Mock) or IBA at concentrations indicated.
(C) Eight-day-old wild-type and ech2-1 seedlings following growth in the light on medium supplemented with ethanol (mock) or 10 mM IBA. Bar = 1 cm.
(D) Mean root lengths (+SE; n $ 10) of 8-d-old seedlings listed in (B) grown in the light on medium supplemented with ethanol (Mock) or IBA at
(E) Five-day-old wild-type and ech2-1 seedlings following growth in the dark in the presence (0.5%) and absence of an exogenous carbon source
(sucrose). Bar = 1 cm.
(F) Mean hypocotyl lengths (+SE; n $ 12) of 5-d-old seedlings listed in (B) grown as in (E).
(G) Schematic of a proposed IBA-to-IAA conversion pathway (Zolman et al., 2008) showing possible enzymatic activities for ECH2, IBR1, IBR3, and
IBR10, along with the PED1 thiolase, which may act in both IBA and fatty acid b-oxidation.
(H) ech2 enhances IBA resistance of ibr mutant hypocotyls. Mean hypocotyl lengths (+SE; n $ 12) of 7-d-old wild-type, ech2-1, ibr1-2, ech2-1 ibr1-2,
ibr3-1, ech2-1 ibr3-1, ibr10-1, ech2-1 ibr10-1, ibr1-2 ibr3-1 ibr10-1, and ech2-1 ibr1-2 ibr3-1 ibr10-1 pregerminated and then grown in the dark on
medium supplemented with ethanol (0 mM IBA) or 20 to 140 mM IBA.
(I) ech2 enhances IBA resistance of ibr mutant roots. Mean root lengths (+SE; n $ 12) of 10-d-old lines listed in (H) that were pregerminated and then
grown in the light on medium supplemented with ethanol (0 mM IBA) or 10 to 80 mM IBA.
Developmental Roles of IBA-Derived Auxin 987
ibr10-1 (Zolman et al., 2008), and ibr1-2 ibr3-1 ibr10-1 (Zolman
et al., 2008) mutants. ibr1-2 and ibr3-1 were mildly resistant to
low IBA concentrations in dark-grown hypocotyl elongation as-
says (Figure 2H) and light-grown root elongation assays (Figure
2I), whereas ech2-1 and ibr10-1 were more strongly resistant
(Figures 2H and 2I). ech2-1 resistance to the inhibitory effects of
IBA on dark-grown hypocotyl and light-grown root elongation
were slightly enhanced when combined with ibr1 or ibr3 and
greatly enhanced when combined with ibr10 (Figures 2H and 2I).
Similarly, ech2-1 2,4-DB resistance was enhanced when com-
bined with ibr10 (seeSupplemental Figure 1Aonline). Combining
ech2-1 with ibr mutants did not confer sucrose dependence to
dark-grown hypocotyls (see Supplemental Figure 1B online),
suggesting that the encoded enzymes do not function redun-
dantly in fatty acid b-oxidation. From here on, we will refer to
ech2-1 as ech2. The ech2 ibr1 ibr3 ibr10 quadruple mutant
displayed only slightly greater IBA resistance in hypocotyl elon-
gation than the ech2 ibr10 double mutant (Figure 2H). The strong
IBA resistance of ech2 ibr10 and ech2 ibr1 ibr3 ibr10 prompted
us to examine these mutants for the developmental conse-
quences of impeding IBA-to-IAA conversion.
Mutants Deficient in IBA-to-IAA Conversion Have Cell
Several lines of evidence suggest that IBA-derived IAA con-
tributes to root hair and cotyledon cell expansion. Mutants
defective in the ABCG36/PDR8/PEN3 and ABCG37/PDR9/
PIS1 ABC transporters, which probably function as IBA effluxers
(reviewed in Strader and Bartel, 2011), display lengthened root
hairs (Strader et al., 2008b; Strader and Bartel, 2009; Ru ˚z ˇic ˇka
et al., 2010), suggesting that increased IBA accumulation in root
hairs can increase auxin levels. Additionally, abcg36 mutants
display enlarged cotyledons, a second high-auxin phenotype
(Strader and Bartel, 2009). Both of these abcg36 developmen-
tal phenotypes are suppressed when combined with the ibr1,
ibr3, and ibr10 mutations, and the ibr1 ibr3 ibr10 triple mutant
displays short root hairs and small cotyledons (Strader et al.,
Like ibr1 ibr3 ibr10 (Strader et al., 2010), ech2 ibr1 ibr3 ibr10
seedlings exhibited smaller cotyledons than the wild type (Fig-
ure 4A). Moreover, the ech2 ibr1 ibr3 ibr10 quadruple mutant
exhibited defects in cotyledon vascular patterning (Figure 4B).
Because Arabidopsis cotyledons grow bycell expansion without
Figure 3. ech2 Is Rescued by ECH2 but Not IBR10.
(A) Overexpression of HA-tagged ECH2 restores IBA sensitivity to ech2-1
but not ibr10-1. Top: Immunoblot analysis of (left to right) 5-d-old light-
grown wild type (Wt), wild type carrying 35S:HA-ECH2, ech2-1, two
independent ech2-1 lines carrying 35S:HA-ECH2, ibr10-1, and ibr10-1
carrying 35S:HA-ECH2. Anti-HA and anti-HSC70 antibodies were used
to detect HA-ECH2 and HSC70 (loading control), respectively. Bottom:
Mean normalized root lengths (+SE; n $ 11) of 8-d-old light-grown lines
shown in the immunoblot above the graph.
(B) Overexpression of IBR10 restores IBA sensitivity to ibr10-1 but not
ech2-1. Mean normalized root lengths (+SE; n $ 9) of 8-d-old light-grown
wild type, two independent wild-type lines carrying 35S:IBR10, ech2-1,
four independent ech2-1 lines carrying 35S:IBR10, ibr10-1, and ibr10-1
(C) YFP-tagged ECH2 rescues ech2-1. Mean normalized root lengths
(+SE; n $ 11) of 8-d-old light-grown wild type, ech2-1, and ech2-1
(D) YFP-ECH2 localizes to punctate structures in Arabidopsis cells.
Confocal images of root epidermal cells from 4-d-old ech2-1 expressing
YFP-ECH2 counterstained with propidium iodide to visualize cell walls.
Bar = 20 mm.
(E) YFP-ECH2 localizes to peroxisomes in Arabidopsis cells. Confocal
images of root epidermal cells from 4-d-old wild type expressing a
peroxisomally targeted YFP derivative (YFP-PTS1; left panels) (px-yk;
Nelson et al., 2007) and ech2-1 expressing YFP-ECH2 (right panels). The
top panel of each pair shows the fusion protein fluorescence and the
bottom panel of each pair shows fluorescence from 8-(4-nitrophenyl)-
BODIPY, which allows visualization of peroxisomes (Landrum et al.,
2010). Bar = 10 mm.
988 The Plant Cell
cell division after germination (Mansfield and Briarty, 1996), this
small size likely results from decreased cell expansion. Indeed,
ech2 ibr1 ibr3 ibr10 cotyledon epidermal cells were smaller than
interdigitation (Figure 4C), suggesting a role for IBA-derived IAA
in shaping cotyledon pavement cells. The smaller size of ech2
ibr1 ibr3 ibr10 persisted in the first several true leaves, but older
plants eventually produced wild-type-sized rosette leaves (Fig-
ure 4D). The smaller early leaves of the quadruple mutant were
accompanied by slower development and a delay in the time to
flowering (Figure 4E), but ech2 ibr1 ibr3 ibr10 plants did not
display marked morphological defects at maturity (see Supple-
mental Figure 3 online).
Root hairs are long, tubular outgrowths from certain root
epidermal cell files that increase root surface area to aid in water
and nutrient uptake. Because root hair expansion is auxin
regulated, root hairs provide a sensitive single-cell model to
study auxin levels and response (reviewed in Grierson and
Schiefelbein, 2002). The ibr1 ibr3 ibr10 triple mutant exhibits
short root hairs (Figure 4E) (Strader et al., 2010) that can be
rescued by applying auxin (Strader et al., 2010). We found
and enhanced the short-root hair phenotype of ibr10 and
ibr1 ibr3 ibr10 (Figure 4F; see Supplemental Figure 4 online),
suggesting that ECH2 loss may decrease auxin levels in root
ech2 ibr10 Displays Reduced
To assess local effects of ech2 and ibr10 on auxin-responsive
transcription, we examined ech2, ibr10, and ech2 ibr10 carrying
DR5-GUS, a reporter driving b-glucuronidase (GUS) expression
from a synthetic auxin-responsive promoter (Ulmasov et al.,
1997). Auxinlevels (Petersson etal.,2009)andDR5-GUS activity
(Sabatini et al., 1999) are high in the root meristem. As previously
reported (Zolman et al., 2008), DR5-GUS activity was similar in
ibr10 and wild-type root tips (Figure 5A). Similarly, ech2 did not
display notably altered DR5-GUS activity (Figure 5A). However,
DR5-GUS activity in ech2 ibr10 was undetectable in roots
stained for the optimal time to visualize GUS activity in the wild
type and was only observed after longer staining periods (Figure
5A). The differences in DR5-GUS activity between the wild type
the small differences in 3-d-old seedlings were more apparent in
7-d-old seedlings (Figure 5A). The reduced DR5-GUS activity
suggested that active auxin levels were decreased in ech2 ibr10
Mutants Deficient in IBA-to-IAA Conversion Have
The decreased DR5-GUS activity and short root hairs suggested
that auxin levels were low in ech2 ibr10 roots. To determine if
ECH2 also impacted hypocotyl development, we examined two
auxin-regulated processes in ech2 mutants: apical hook curva-
Figure 4. ech2 Enhances ibr1 ibr3 ibr10 Cell Expansion Defects.
(A) Seven-day-old light-grown ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings
display decreased cotyledon size compared with wild-type (Wt) seed-
lings. Bar = 1 cm.
(B) Eight-day-old light-grown ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings
display aberrant vascular patterning. Bar = 1 mm.
(C) Five-day-old (top panels) and 7-d-old (bottom panels) ech2-1 ibr1-2
ibr3-1 ibr10-1 seedlings display small cotyledon epidermal cells. Con-
focal images of propidium iodide–stained cells are shown. Bar = 50 mm.
(D) Soil-grown ech2-1 ibr1-2 ibr3-1 ibr10-1 plants are smaller than the
wild type at 21 d (top panel) but begin to recover by 26 d (bottom panel).
Two wild-type (left) and quadruple mutant (right) plants are shown. Bar =
(E) Soil-grown ech2-1 ibr1-2 ibr3-1 ibr10-1 plants flower later than the
wild type. Profile of plants shown in (D). Bar = 1 cm.
(F) ech2 mutants display short root hairs. Mean root hair lengths (+SE;
n = 500) of 5-d-old wild-type, ech2-1, ibr10-1, ech2-1 ibr10-1, ibr1-2
ibr3-1 ibr10-1, and ech2-1 ibr1-2 ibr3-1 ibr10-1 light-grown seedlings.
Inset: Root hairs from 5-d-old wild-type and ech2-1 ibr1-2 ibr3-1 ibr10-1
light-grown seedlings. Bar = 500 mm.
Developmental Roles of IBA-Derived Auxin 989
exhibit closed cotyledons and form an apical hook to protect the
(Goeschl et al., 1966; Guzma ´n and Ecker, 1990). Auxin response
is necessary for proper apical hook formation and maintenance,
and auxin-resistant and auxin-deficient mutants often are defec-
tive in apical hook curvature (Tian and Reed, 1999; Harper et al.,
2000; Friml et al., 2002; Stepanova et al., 2008; Vandenbussche
et al., 2010; Zˇa ´dnı ´kova ´ et al., 2010).
We found that apical hook curvature was slightly decreased in
dark-grown ech2 and ibr10 single mutants and that ech2 ibr10,
hook curvature in both 3-d-old and 4-d-old seedlings, with ech2
ibr1 ibr3 ibr10 exhibiting the greatest curvature defect (Figures
5B and 5D). The ech2 ibr1 ibr3 ibr10 mutants were defective in
both apical hook formation, reaching a maximal angle of only
1308(versus 1808 in the wild type), and apical hook maintenance,
beginning to open when wild-type hooks were still largely closed
(Figure 5D). Auxin promotes both hook formation and hook
maintenance, and as previously observed (Friml et al., 2002;
Vandenbussche et al., 2010; Zˇa ´dnı ´kova ´ et al., 2010), DR5-GUS
activity was concentrated in the inner side of the apical hook in
the wild type (Figure 5C). ech2 and ibr10 displayed decreased
DR5-GUS activity in the inner face of the apical hook, and DR5-
GUS activity in ech2 ibr10 was barely detectable in this region
(Figure 5C), suggesting that the apical hook curvature defects in
ech2 mutants were caused by decreased auxin response in this
Growth of seedlings at high temperature results in increased
auxin levels and hypocotyl elongation (Gray et al., 1998). We
found that ech2 and ech2 ibr1 ibr3 ibr10 displayed shorter
hypocotyls than the wild type when grown at elevated temper-
atures (Figure 5E). Because the increased IAA at high temper-
ature is thought to result from increased IAA synthesis (Gray
et al., 1998), the high-temperature short-hypocotyl phenotype of
ech2 is consistent with the possibility that ECH2 promotes IAA
synthesis from IBA.
Figure 5. ech2 ibr10 Displays Decreased Auxin Reporter Activity, Apical
Hook Formation, and High-Temperature Hypocotyl Elongation.
(A) ech2 ibr10 displays decreased DR5-GUS activity in root tips. Three-,
five-, and seven-day-old light-grown wild-type (Wt), ech2-1, ibr10-1, and
ech2-1 ibr10-1 seedlings carrying the DR5-GUS construct (Ulmasov
et al., 1997; Zolman et al., 2008) were stained for GUS activity for 1 or 3 h.
Bar = 100 mm.
(B) ech2 mutants display decreased apical hook formation. Three-
day-old (top panel) or 4-d-old (bottom panel) wild-type, ech2-1, ibr10-1,
ech2-1 ibr10-1, ibr1-2 ibr3-1 ibr10-1, and ech2-1 ibr1-2 ibr3-1 ibr10-1
dark-grown seedlings are shown. Bar = 0.5 cm.
(C) ech2 ibr10 displays decreased DR5-GUS reporter activity in apical
hooks. Three-day-old dark-grown wild-type, ech2-1, ibr10-1, and ech2-1
ibr10-1 seedlings carrying the DR5-GUS construct were stained for
GUS activity for 4 h. Bar = 100 mm.
(D) ech2 ibr1 ibr3 ibr10 displays decreased apical hook formation and
maintenance. Mean apical hook angles (6SE; n $ 20) of wild-type and
ech2-1 ibr1-2 ibr3-1 ibr10-1 dark-grown seedlings are shown.
(E) ech2 mutants display decreased high temperature–induced hypo-
cotyl elongation. Mean hypocotyl lengths (+SE; n = 16) of wild-type,
ech2-1, ibr1-2 ibr3-1 ibr10-1, and ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings
grown for 8 d at 22 or 288C under yellow-filtered light.
990 The Plant Cell
Mutants Deficient in IBA-to-IAA Conversion Have
Decreased Lateral Root Production and Smaller
Auxin is a critical regulator of both lateral root initiation (reviewed
in Casimiro et al., 2003) and lateral root emergence (Swarup
et al., 2008). We examined lateral root initiation in the ech2 and
ibr10 lines carrying the DR5-GUS reporter, which facilitates
detection of lateral root primordia (LRP). In the absence of
treatment, we did not detect any lateral roots or LRP in 8-d-old
ech2 ibr10 seedlings (Figures 6A and 6B). Intriguingly, the LRP
(Figure 6A), suggesting that auxin levels in these LRPs were
sufficient to generate the lateral root but insufficient to highly
activate the DR5-GUS reporter. Wild-type seedlings responded
to IBA treatment with increased lateral root production (Figures
6A and 6B). Consistent with roles for ECH2 and IBR10 in IBA-
to-IAA conversion, ech2, ibr10, and ech2 ibr10 were less re-
sponsive to IBA promotion of lateral root initiation (Figures 6A
and 6B). Treatment withthe synthetic auxin 1-naphthaleneacetic
acid (NAA) increased lateral root production in all the tested lines
(Figures 6A and 6B), indicating that the lack of LRPs in the ech2
but by decreased active auxin in these tissues. Interestingly, the
lateral roots and LRPs produced by ech2 ibr10 following NAA
treatment displayed reduced DR5-GUS activity (Figure 6A),
suggesting that even with NAA treatment, auxin levels in these
LRP were sufficient to generate the lateral root but insufficient to
highly activate the DR5-GUS reporter.
When we quantified lateral root emergence in response to
auxins, we were surprised to find that ech2 ibr10 and ech2 ibr1
in response to IBA but also in response to IAA and NAA (Figure
6B), suggesting that ECH2 and IBR10, and by implication IBA-
but also to active auxins. We quantified LRPs in untreated roots
and found that 8-d-old ech2 ibr1 ibr3 ibr10 seedlings had no
deficiency at 8 d, ech2 ibr1 ibr3 ibr10 did display emerged lateral
roots after 12 (Figure 6D) and 21 d (Figure 6E). However,
emerged lateral roots in ech2 ibr1 ibr3 ibr10 did not reach wild-
type numbers even after 21 d, resulting in a reduced root system
in the quadruple mutant (Figures 6D and 6E).
Because ECH2 and IBR10 were necessary for full response
not only to IBA, but also to active auxins in 4-d-old lateral root
formation assays(Figures 6A and 6B),weexamined DR5-GUS in
ech2 ibr10 following a shorter exposure to active auxins to
determine ifECH2 and IBR10 alsocontribute to morerapidauxin
responses. We found that ech2 ibr10 displayed dramatically
decreased DR5-GUS activity in response to short (2-h) expo-
in whole seedlings (Figure 7B). Furthermore, we found that ech2
ibr1 ibr3 ibr10 displayed mild resistance to IAA in root elongation
assays (Figure 7C), confirming that ECH2 and IBR10 are needed
for full response to active auxins.
The root apical meristem is a region near the root tip that
undergoes indeterminate growth, generating new root tissue
through balanced cell division and differentiation. Establishment
of an auxin maximum in the root tip is necessary to maintain the
quiescent center, a small group of cells within the meristem that
undergo only occasional cell divisions, and an auxin gradient is
necessary to maintain root meristematic activity (reviewed in
Iyer-Pascuzzi and Benfey, 2009). Consistent with reduced DR5-
measured as the distance between the quiescent center and the
with the wild type in the ech2 ibr1 ibr3 ibr10 mutant (Figures 8A
and 8C). Additionally, root width, measured in the cell elongation
zone, was reduced by ;20% in ech2 ibr1 ibr3 ibr10 (Figure 8D).
In spite of the reduced size of the root apical meristem in the
meristem organization (Figure 8B).
The ech2 ibr1 ibr3 ibr10 Mutant Has Reduced Auxin Levels
Although ibr1 ibr3 ibr10 seedling phenotypes are suggestive of
low auxin levels, overall IAA levels are unchanged in ibr1 ibr3
ibr10 seedlings (Strader et al., 2010), indicating that any such
reductions are minor and/or local. Because the reduced DR5-
GUS activity in untreated ech2ibr10 seedlings (Figures 5A and7)
along with the various ech2 ibr10 and ech2 ibr1 ibr3 ibr10
developmental phenotypes (Figures 4 to 6 and 8) suggested that
auxin levels were further reduced, we used gas chromatography–
mass spectrometry to measure free IAA levels in the root tips of
ech2 ibr1 ibr3 ibr10 and the wild type. We found a 20% reduction
of free IAA in the quadruple mutant compared with the wild type
(Figure 8E). This reduction indicates that IBA-to-IAA conversion
normally provides a significant input into the free IAA pool in
Peroxisomal Enzymes Implicated in IBA-to-IAA Conversion
Screens for IBA resistance have revealed an IBA-to-IAA conver-
sion pathway involving peroxisomal enzymes resembling fatty
acid b-oxidation enzymes, suggesting that IBA undergoes
b-oxidation to free IAA and acetyl-CoA (Figure 2G). IBR3 resem-
bles acyl-CoA dehydrogenases/oxidases and may oxidize IBA-
CoA to the a,b-unsaturated thioester (Zolman et al., 2007). Both
IBR10 (Zolman et al., 2008) and ECH2 appear to be enoyl-CoA
hydratases and may produce an IBA hydroxylacyl-CoA thioester
intermediate. IBR1, a 3-hydroxylacyl-CoA-dehydrogenase-like
protein, may perform the 3-hydroxyacyl-CoA dehydrogenase
step in IBA-to-IAA conversion (Zolman et al., 2008). Removal of
acetyl-CoA to give IAA-CoA may be catalyzed by the thiolase
PED1 (Zolman et al., 2000), which acts in the analogous step
in fatty acid b-oxidation (Hayashi et al., 1998). No genes anno-
tated as encoding acyl-CoA synthetases or thioesterases have
emerged from IBA response screens; however, either ECH2 or
IBR10 may perform the thioesterase step, as discussed below.
alleles have been isolated in IBA response screens, and some of
these mutations result in premature stop codons. Moreover,
T-DNA insertion alleles disrupting IBR1 and IBR3 have been
Developmental Roles of IBA-Derived Auxin991
Figure 6. ech2 ibr10 and ech2 ibr1 ibr3 ibr10 Are Defective in Lateral Root Production.
(A) ech2 ibr10 displays decreased DR5-GUS activity in lateral roots. Four-day-old light-grown wild-type (Wt), ech2-1, ibr10-1, and ech2-1 ibr10-1
seedlings carrying DR5-GUS were transferred to medium supplemented with ethanol (mock) or the indicated auxin and grown for an additional 4 d
under yellow-filtered light at 228C prior to staining for GUS activity for 1 h. Lateral roots and LRPs are highlighted with arrowheads. Bar = 200 mm.
(B) ech2 mutants produce fewer lateral roots than the wild type. Emerged lateral roots of wild-type, ech2-1, ibr10-1, ech2-1 ibr10-1, ibr1-2 ibr3-1
ibr10-1, and ech2-1 ibr1-2 ibr3-1 ibr10-1 were counted 4 d after transfer of 4-d-old seedlings to medium supplemented with either ethanol (mock) or the
indicated auxins (mean + SE, n $ 12).
(C) ech2 ibr1 ibr3 ibr10 displays fewer LRPs. Eight-day-old wild-type and ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings were cleared, and the number and
stage of LRPs recorded (+SE, n = 18). Stage A spans of the first anticlinal division of a pericycle cell to an LRP with three cell layers. Stage B includes
unemerged lateral roots with more than three cell layers. Stage C includes emerged lateral roots shorter than 0.5 mm. Stage D consists of emerged
lateral roots longer than 0.5 mm.
(D) Twelve-day-old wild-type, ech2-1, ibr10-1, ech2-1 ibr10-1, ibr1-2 ibr3-1 ibr10-1, and ech2-1 ibr1-2 ibr3-1 ibr10-1 light-grown seedlings. Bar = 1 cm.
(E) Twenty-one-day-old wild-type and ech2-1 ibr1-2 ibr3-1 ibr10-1 light-grown plants. Bar = 1 cm.
992 The Plant Cell
characterized (Zolman et al., 2007, 2008; Wiszniewski et al.,
2009). By contrast, no T-DNA insertions have been reported in
IBR10 or ECH2, and only single alleles have emerged from IBA
response screens: ibr10-1, which results in a 26–amino acid in-
frame deletion (Zolman et al., 2008), and ech2-1, a missense
allele (Figure 1), consistent with the possibility that null alleles of
ibr10 and ech2 may confer more severe defects than the alleles
that have been recovered to date. Unlike the 2,4-DB resistance
of ech2-1 (Figure 1A; see Supplemental Figure 1 online), ech2
RNAi lines retain normal 2,4-DB responsiveness (Goepfert et al.,
2006), suggesting that lines eliminating ECH2 function were not
recovered in this study. Regardless of whether ibr10-1 or ech2-1
is a null allele, the observation that each confers greater IBA
resistance than likely ibr1 or ibr3 null alleles suggests that the
former mutations more effectively reduce IBA-to-IAA conversion
than the latter.
If ibr1-2 and ibr3-1 are null alleles defective in dedicated
IBA-to-IAA conversion enzymes, then why is IBA resistance
enhanced when these alleles are combined with the ech2-1
missense allele? It is likely that alternate enzymes can catalyze
the same IBA-to-IAA conversion steps as IBR3 and IBR1. For
(Zolman et al., 2007), but also by ACX3 (Adham et al., 2005), a
peroxisomal protein that also b-oxidizes fatty acyl-CoA esters
(Froman et al., 2000). Because the ibr3 acx3 double mutant
displays enhanced IBA resistance compared with either parent
(Zolman et al., 2007),both IBR3 and ACX3 may oxidize IBA-CoA.
Similarly, mutants in the multifunctional enzyme ABNORMAL
INFLORESCENCE MERISTEM1 (AIM1), required for fatty
acid b-oxidation (Richmond and Bleecker, 1999), also are IBA
(Zolman et al., 2000) and 2,4-DB (Richmond and Bleecker, 1999)
resistant. AIM1 can carry out the hydration and dehydrogenation
steps of fatty acid b-oxidation (Richmond and Bleecker, 1999),
whereas the combined activities of IBR1 (Zolman et al., 2008),
IBR10 (Zolman et al., 2008), and/or ECH2 may perform the
hydration and dehydrogenation steps of IBA b-oxidation. Be-
cause aim1 is IBA resistant (Zolman et al., 2000), it is possible
that AIM1 may provide some IBA b-oxidation activity in addition
to that provided by IBR1, IBR10, and ECH2. Interestingly, the
enoyl-CoA domains of AIM1, IBR10, and ECH2 are dissimilar in
primary sequence (Figure 1D).
ECH2 is conserved throughout the plant kingdom and does
is more similar (;51% identical) to the enoyl-CoA hydratase
domain from metazoan MULTIFUNCTIONAL ENZYME (MFE2)
than it is the enoyl-CoA hydratase domains from IBR10 (11%
identical) or plant multifunctional enzymes such as AIM1 (;8%
identical) (Figure 1D). Unlike IBR10 and the plant multifunctional
enzymes, members of the ECH2 family and the metazoan MFE2
family contain hot dog domains (see Supplemental Figure 5
online), a motif associated with both enoyl-CoA hydratases and
CoA hydratase activity when heterologously expressed in Sac-
charomyces cerevisiae (Goepfert et al., 2006). That both ECH2
and IBR10 appear to encode enoyl-CoA hydratases (Figure 1D)
and that mutation of either leads to strong IBA resistance (Fig-
ure 2) present a paradox: Are both of these enzymes catalyzing
the enoyl-CoA hydratase step in IBA-to-IAA conversion? Our
Figure 7. ech2 ibr10 Displays Decreased Auxin Reporter Activity in
Response to Active Auxins.
(A) Seven-day-old light-grown wild-type (Wt) and ech2-1 ibr10-1 seed-
lings carrying DR5-GUS were transferred to medium supplemented with
ethanol (mock) or the indicated auxin and were incubated for 2 h at 228C
prior to staining for GUS activity for 1.5 h. Bar = 100 mm.
(B) Mean GUS activity (6SE; n = 16) of 7-d-old light-grown wild-type and
ech2-1 ibr10-1 seedlings carrying DR5-GUS treated for 2 h with ethanol
(mock treatment; 0 mM auxin) or the indicated auxin.
(C) Mean root lengths (6SE; n = 13) of 8-d-old light-grown wild-type and
ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings grown on medium supplemented
with ethanol (mock treatment; 0nM IAA) or various concentrations of IAA.
Developmental Roles of IBA-Derived Auxin 993
demonstration that overexpression of one cannot compensate
for loss of the other (Figure 3) suggests that ECH2 and IBR10
catalyze different reactions. Given the mechanistic and se-
quence similarity between hydrolases and hydratases (Pidugu
hydroxylacyl-CoA thioester intermediate and the other hydro-
lyzes IAA-CoA to release free IAA. Resolution of this question
awaits the availability of postulated substrates needed for the
biochemical characterization of these enzymes.
IBA-Derived IAA Plays Critical Roles in
defective in IBA efflux exhibit expanded root hairs and cotyle-
dons, whereas mutants defective in IBA-to-IAA conversion dis-
play smaller root hairs and cotyledons (Strader and Bartel, 2008;
Strader et al., 2010). IBA-derived IAA also plays a role in stamen
filament elongation and lateral root production; pxa1 mutants
display delayed filament elongation that is rescued by NAA
application (Footitt et al., 2007) and decreased lateral rooting
that is restored by IAA application (Zolman et al., 2001). The
(Woodward and Bartel, 2005a), a phenotype often associated
with low auxin levels in embryogenesis (reviewed in Mo ¨ller and
for IBA-derived IAA are supported and extended by the wide-
ranging auxin-related developmental defects in ech2 ibr1 ibr3
ibr10 seedlings, which included not only defects in lateral root
production and cotyledon and root hair cell expansion, but also
reduced cotyledon vasculature, delayed development, reduced
apical hook curvature, reduced temperature-induced hypocotyl
lengthening, reduced root width, smaller root meristems, and
shorter roots. Together, the diverse low-auxin phenotypes dis-
played by ech2 ibr1 ibr3 ibr10 seedlings, our demonstration that
and the high-auxin phenotypes displayed by mutants with de-
an IAA precursor in Arabidopsis and that IBA-derived IAA con-
tributes todevelopmentalprocessesinmultipleseedling tissues.
Previously characterized IAA biosynthetic mutants with low
auxin levels include higher-order mutants in the YUCCA and
(TAA1) families. Combinations of yuc1, yuc2, yuc4, and yuc6
exhibit various developmental defects, including altered plant
stature, floral organ patterning, and vascular development
(Cheng et al., 2006). Additionally, the yuc1 yuc4 yuc10 yuc11
quadruple mutant lacks a hypocotyl and a root meristem, indi-
cating that YUCCA-synthesized IAA is critical during embryo-
genesis (Cheng et al., 2007). Mutants of TAA1 were isolated for
altered shade avoidance (Tao et al., 2008), reduced ethylene
responses (Stepanova et al., 2008), and reduced auxin transport
inhibitor responses (Yamada et al., 2009). Like the yucca family,
combining taa1 with tar2 or tar1 and tar2 alters plant stature,
gravity response, vascular development, floral organ patterning,
and embryo development (Stepanova et al., 2008). IBA-derived
primarily during seedling development; we find no evidence in
Figure 8. ech2 ibr1 ibr3 ibr10 Displays Meristem Defects and Decreased
(A) Confocal images of propidium iodide–stained root tips from 7-d-old
heads delineate the top and bottom of the root meristem. Bar = 100 mm.
(B) Confocal images of propidium iodide–stained root tips from 7-d-old
light-grown wild-type and ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings. Bar =
(C) Mean meristem lengths (+SE; n $ 47) of 8-d-old light-grown wild-type
and ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings.
(D) Mean root widths (+SE; n $ 47) in the elongation zone of 8-d-old light-
grown wild-type and ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings.
(E) Mean IAA levels (+SE; n $ 3) in 5-mm root tips from light-grown wild-
type and ech2-1 ibr1-2 ibr3-1 ibr10-1 seedlings.
(F) A model for the effects of IBA-to-IAA conversion on the auxin pool.
IBA is converted to IAA in a process similar to peroxisomal fatty acid
b-oxidation (solid black arrow). IAA also can be converted to IBA (dashed
gray arrow; reviewed in Ludwig-Mu ¨ller, 2000). Reduced DR5-GUS ac-
tivity and decreased lateral root formation in ech2 ibr10 mutants treated
with active auxins suggests that IBA-to-IAA conversion is necessary
for full response to active auxins, either because auxin stimulates IBA-
to-IAA conversion (positive feedback loops; dotted gray arrows) or be-
cause additional auxin is required to overcomethe auxin deficit thatresults
from blocking IBA-to-IAA conversion. Ultimately, increased auxin levels
promote auxin signaling (dashed black arrow).
994 The Plant Cell
ech2 ibr1 ibr3 ibr10 of the altered plant stature, floral organ
patterning, gravity response, or embryo development found in
higher-order yucca and taa1 family mutants, suggesting that the
ECH2 pathway is distinct from the YUCCA and TAA1 pathways.
However, because ech2 ibr1 ibr3 ibr10 mutants still respond to
very high IBA levels (Figure 2), we cannot discount the possibility
that residual IBA-to-IAA conversion occurs in the quadruple
mutant and that phenotypes similar to those found in higher-
order yucca and taa1 family mutants will be uncovered with a
more complete block in IBA-to-IAA conversion. Indeed, a recent
report of developmental alterations in mature plants resulting
from increased IBA glucosylation suggests that IBA contribu-
tions are not limited to the seedling stage (Tognetti et al., 2010).
Is IBA an Auxin Storage Form or Biosynthetic Intermediate?
Previous data showing that IBA can be synthesized from IAA
(reviewed in Ludwig-Mu ¨ller, 2000), together with demonstrations
in IAA-to-IBA conversion, and the observation that IBA is present
at even lower levels than IAA in Arabidopsis seedlings (Ludwig-
is difficult to reconcile with the important roles of IBA-derived
IAA in seedling development revealed here. It is tempting to
speculate that IBA may function as an intermediate in a de novo
IAA synthesis pathway in which IBA-to-IAA b-oxidation enzymes
catalyze the final steps. Plants synthesize IAA using several
pathways, none of which have been demonstrated at a level of
identified (reviewed in Strader and Bartel, 2008). Some pathways,
such as the Trp-independent pathway, are particularly ill defined,
with no identified intermediates between indole and IAA. Whether
IBA serves as an IAA biosynthetic precursor, storage form, or
both, the deep conservation of ECH2 and IBR10 homologs in the
ancient input to the free IAA pool.
Positive Feedback in Auxin Homeostasis
Positive feedback loops reinforcing auxin maxima are recurring
themes in plant development, controlling root meristem mainte-
nance, lateral root formation, vascular strand formation, and
apical dominance maintenance (reviewed in Petra ´s ˇek and Friml,
2009). Auxin canalization models to explain these maxima gen-
erally rely on the positive reinforcement provided by regulating
polar auxin transport; however, temporal and spatial regulation
of auxin synthesis also contributes to these maxima (reviewed in
Zhao, 2010). Combining ech2 with ibr10 resulted in decreased
activity of the DR5-GUS auxin-responsive reporter not only
following IBA treatment (Figure 6A) but also in untreated seed-
lings (Figures 5 to 7), consistent with the reduced IAA levels we
GUS activity also was reduced in ech2 ibr10 seedlings following
short- and long-term treatment with active auxins (Figures 6A
and 7). Our observation that ech2 ibr10 mutants have reduced
response not only to IBA, but also to IAA, suggests that auxin
response, even to exogenous auxin, is dampened when endog-
enous IAA levels are reduced. Indeed, when release of IAA from
amino acid conjugates is impaired, Arabidopsis seedlings show
reduced IAA levels and less responsiveness to exogenous IAA
(Rampey et al., 2004), consistent with the reduced auxin respon-
siveness that we find when IBA-to-IAA conversion is reduced.
The molecular mechanisms underlying these reinforcing effects
remain to be elucidated. Perhaps the IAA response machinery
does not operate efficiently below some threshold IAA level,
perhaps IAA response is impeded by the IBA that remains when
IBA-to-IAA conversion is impaired, or perhaps IAA promotes
(Figure 8F). Given the importance of IBA-derived IAA in seedling
development uncovered here, fully understanding the various
inputs to the active auxin pool will require integration of IBA
contributions into models of IAA homeostasis.
Growth Conditions and Phenotypic Assays
Arabidopsis thaliana mutants were in the Colombia (Col-0) background,
which was used as the wild type. Surface-sterilized seeds (Last and Fink,
1988)wereplatedonplant nutrient(PN) medium(Haughnand Somerville,
1986) supplemented with 0.5% (w/v) sucrose (PNS) solidified with 0.6%
(w/v) agar and grown at 228C under continuous illumination, unless
To examine auxin-responsive elongation, seeds were plated on PNS
supplemented with auxin and grown for 8 d under yellow-2208 (3-mm
indolic compound breakdown (Stasinopoulos and Hangarter, 1990) to
monitor light-grown root and hypocotyl elongation or for 1 d under yellow
long-pass filters followed by 4 d in the dark to monitor dark-grown
hypocotyl elongation. To promote uniform germination on high IBA con-
centrations (Figures 2H and 2I), surface-sterilized seeds were imbibed in
concentrations and incubating for 8 d under yellow-filtered light (root
in darkness (hypocotyl elongation).
To examine sucrose dependence, seeds were plated on unsupple-
mented PN or on PNS and grown for 1 d under white light before growth
for 4 d in the dark.
To examine cotyledon size, seedlings were grown for 7 d under white
light on PNS before cotyledons were removed, mounted, and imaged.
Cotyledon blade area was measured using NIH Image software.
To examine cotyledon vasculature, seedlings were grown for 8 d under
white light on PNS, cleared with an ethanol series followed by 1 week in a
chloral hydrate solution (80 g chloral hydrate, 20 mL glycerol, and 10 mL
To examine apical hooks, seeds were plated on PNS and grown for 1 d
under white light before additional growth in the dark. Apical hooks were
imaged, and apical hook angles were measured using NIH Image soft-
To examine lateral roots, seedlings were grown for 4 d on PNS under
yellow filters before transfer to PNS supplemented with the indicated
auxin concentrations and grown for an additional 4 d. Emerged lateral
roots were counted using a dissecting microscope.
To quantify LRPs, seedlings were grown for 8 d under white light on
PNS, cleared by incubating for 1 weekin chloral hydrate solution, mounted
in 50% glycerol, and examined using a Zeiss Axioplan 2 microscope.
Lateralroots and LRPswere counted andclassified into fourstages.Stage
A includes LRP with three or fewer cell layers, corresponding to previously
Developmental Roles of IBA-Derived Auxin 995
described Stages I to III (Malamy and Benfey, 1997), and Stage B includes
unemerged LRPswith four ormorecelllayers, correspondingto previously
described Stages IV to VIb (Malamy and Benfey, 1997). Stages C and D
include emerged lateral roots less than and greater than 0.5 mm, respec-
To examine root hair lengths, vertically grown 5-d-old seedlings grown
under white light were imaged using a dissecting microscope, and root
hairs lengths from the 4-mm root sections adjacent to the root-shoot
junction were measured using NIH Image software.
To quantify meristem size and root width, 8-d-old seedlings grown
under white light were fixed in ethanol:acetic acid (3:1) and mounted in
chloral hydrate solution. Root tips were imaged using a Zeiss Axioplan 2
microscope, and the distance between the quiescent center and the first
elongating cell was measured using NIH Image software.
HR7 (in the Col-0 background) was outcrossed to Landsberg erecta for
recombination mapping. DNA was isolated for mapping using markers
(see Supplemental Table 2 online) from F2 individuals displaying long
hypocotyls after 1 d in light and 4 d in darkness on 30 mM IBA. The ECH2
gene within the HR7 mapping interval was PCR amplified and sequenced
from HR7 genomic DNA.
PCR analysis (see Supplemental Table 3 online) of segregating F2
plants was used to identify higher-order mutants.
Vector Construction and Plant Transformation
ECH2 was amplified from the U21373 cDNA obtained from the ABRC
(Ohio State University) using Pfx Platinum Taq (Invitrogen) with 59-CAC-
CATGGCGACTAGCGATTCTGAATTCAATTC-39 and 59-TCATTACGCC-
AAGCAAACATCAAGAG-39. The resulting PCR product was captured
into the pENTR/D-TOPO vector (Invitrogen). The pENTR-ECH2 entry
clone was cut with PvuII to linearize the vector to prevent transformation
of Escherichia coli by the entry vector. ECH2 was recombined into the
LR Clonase (Invitrogen) to form pEarleyGate 104-ECH2 and pEarleyGate
201-ECH2, respectively, which express N-terminal YFP and HA fusions
with ECH2 driven by the cauliflower mosaic virus 35S promoter. Recom-
binant plasmids were transformed into Agrobacterium tumefaciens strain
GV3101 (Koncz and Schell, 1986), which was used to transform plants
using the floral dip method (Clough and Bent, 1998). Transformants were
selected on PNS plates supplemented with 7.5 mg/mL Basta (phos-
phinothricin), and lines homozygous for the transgene were selected in
subsequent generations. To monitor HA-ECH2 levels, protein from 12
5-d-old seedlings was analyzed by immunoblotting as previously de-
scribed (Strader et al., 2009), except that a 1:500 dilution of rat anti-HA
antibody (3F10; Roche) was used.
Protein sequences corresponding to the hydratase domains of ECH2,
IBR10, and relatives were aligned with Lasergene MegAlign (DNASTAR)
using the ClustalW default settings with the Gonnet series protein weight
matrix, then manually adjusted to optimize alignments. An unrooted
phylogram was generated using PAUP 4.05b (Swofford, 2001) by per-
forming the bootstrap method with 500 replicates with distance as the
optimality criterion and all characters weighted equally.
To histochemically examine GUS activity, seedlings carrying DR5-GUS
(Ulmasov et al., 1997) were treated as indicated, fixed in 90% acetone for
20 min at 2208C, rinsed twice in GUS buffer (0.1 M NaPO4, pH 7.0, 0.5
mM K3[Fe(CN6)], 0.5 mM K4[Fe(CN6)], 10 mM EDTA, and 0.01% Triton
X-100), and then incubated in GUS buffer supplemented with 0.5 mg/mL
5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid at378C for theindicated
mounted, and imaged using a Zeiss Axioplan 2 microscope.
For 4-methylumbelliferyl-b-D-glucuronide hydrate assays, 16 replicates
of three 7-d-old seedlings were treated as indicated prior to GUS activity
being monitored as previously described (Strader and Bartel, 2009).
Seedlings were counterstained in aqueous propidium iodide (10 mg/mL;
Molecular Probes) to label cell walls or stained with 5 mM 8-(4-nitro-
phenyl)-BODIPY (diluted in water from a 5 mM stock in 90% acetone;
Toronto Research Chemicals N503045) to label peroxisomes prior to
mounting in water for imaging. Propidium iodide–stained YFP-ECH2
seedlings were imaged through a 363 oil immersion lens on a Zeiss LSM
510 laser scanning confocal microscope equipped with a Meta detector.
Samples were excited with the 488-nm laser line from an argon laser, and
long-pass filter and false-colored yellow. Pixels resulting from fluores-
cence >545 nm were false-colored red.
Root apical meristem structure was imaged using propidium iodide–
stained seedlings examined through a 310 lens or a 340 oil immersion
lens on a Zeiss LSM 710 confocal laser scanning microscope. Samples
were excited with the 543-nm laser, and resultant fluorescence between
566 and 719 nm was collected.
Seedlings stained with the peroxisome-associated 8-(4-nitrophenyl)-
BODIPY fluorophore (Landrum et al., 2010) were imaged through a 3100
oil immersion lens on a Zeiss LSM 710 confocal laser scanning micro-
scope. To detect BODIPY and YFP, samples were excited with the 488-
and 514-nm lasers, and the resultant fluorescence between 493 and 516
nm, and 550 and 592 nm was collected, respectively. Images were
converted and channels merged using NIH Image software.
Quantification of IAA
For IAA analysis, 150 mL of homogenization buffer (65% isopropanol and
35% 0.2 M imidazole buffer, pH 7.0) containing 10 ng [13C6]IAA internal
standard (99 atom%; Cambridge Isotope Laboratories; Cohen et al.,
1986) were added to each tissue sample. Samples were homogenized
with two 3-mm tungsten carbide beads (Qiagen) in a Mixer-Mill (Qiagen)
for 5 min at 25 Hz, then incubated on ice for 1 h to allow the internal
standard to equilibrate with the endogenous IAA in the extract. After
equilibration, debris was pelleted by centrifugation for 5 min at 10,000g,
and 100 mL of supernatant was placed into a deep 96-well plate
(Continental Lab Products). IAA extraction and solid phase purification
was performed as described (Barkawi et al., 2008, 2010). The eluate, in
600 mL methanol, was methylated with 900 mL ethereal diazomethane
(Cohen, 1984) in 1.5-mL screw-capped glass vials. Samples were then
dried under N2in a 558C sand bath and resuspended in 30 mL ethyl
acetate before being injected into an Agilent 6890 GC/5973 MS (Agilent
Technologies) run in EI mode at 70 eV and equipped with a fused silica
capillary column (HP-5MS, 30 m 3 0.25-mm ID, 0.25-mm film). The
injector temperature was 2808C, and the GC oven temperature was
programmed to ramp from 70 to 2808C at 128C/min. Helium was used as
the carrier gas at a flow rate of 1 mL/min. Samples were analyzed in the
selective ion monitoring mode. IAA levels were calculated by monitoring
ions at mass-to-charge ratios (m/z) of 130 and 189 for endogenous IAA
using standard isotope dilution equations (Cohen et al., 1986).
996 The Plant Cell
Sequence data from this article can be found in the Arabidopsis Ge-
nome Initiative or GenBank/EMBL databases under accession number
At1g76150 (ECH2). Additional accession numbers are provided in Sup-
plemental Table 1 online (loci corresponding to mutants used) and in
SupplementalFigures5and 6online (sequences used for alignments and
The following materials are available in the online version of this article.
Supplemental Figure 1. ech2 ibr Mutants Are 2,4-DB Resistant (but
Not Sucrose Dependent).
Supplemental Figure 2. ech2 Rescue Experiments in Hypocotyl
Supplemental Figure 3. ech2 ibr Mutants Lack Morphological
Defects as Adult Plants.
Supplemental Figure 4. ech2 ibr Mutants Have Short Root Hairs.
Supplemental Figure 5. Alignment of ECH2 and MFE2 Family
Supplemental Figure 6. Alignment of ECH2, IBR10, MFE2, MFP2,
and AIM1 Family Members.
Supplemental Table 1. Mutant Alleles Used in This Study.
Supplemental Table 2. New Markers Used in HR7 Mapping.
Supplemental Table 3. PCR Analysis of Mutant Genotypes.
Supplemental Data Set 1. Text File of the Sequences and Alignment
Used for the Phylogenetic Analysis in Figure 1.
We thank Xing Liu (University of Minnesota) for assistance with mass
spectrometry, the ABRC for providing the ECH2 cDNA and YFP-PTS1
(px-yk), and Wendell Fleming and Jerrad Stoddard for critical comments
on the manuscript. This research was supported by the National
Science Foundation (MCB-0745122 to B.B. and MCB-0725149 and
IOS-0923960 to J.D.C.), the Robert A. Welch Foundation (C-1309 to
B.B.), a Howard Hughes Medical Institute Professors Grant (to B.B.), the
National Institutes of Health (1K99-GM089987 to L.C.S.), and the
Gordon and Margaret Bailey Endowment for Environmental Horticulture
(to J.D.C.). Confocal microscopy was performed on equipment obtained
through a Shared Instrumentation Grant from the National Institutes of
Received January 7, 2011; revised January 7, 2011; accepted March 5,
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