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RESEARCH ARTICLE
The cyclophilin A DIAGEOTROPICA gene affects auxin transport
in both root and shoot to control lateral root formation
Maria G. Ivanchenko
1,
*, Jinsheng Zhu
2
, Bangjun Wang
2,8
, Eva Medvecka
3
, Yunlong Du
4,5
, Elisa Azzarello
6
,
Stefano Mancuso
6
, Molly Megraw
1
, Sergei Filichkin
1
, Joseph G. Dubrovsky
7
, Jir
ıFriml
3,4
and Markus Geisler
2
ABSTRACT
Cyclophilin A is a conserved peptidyl-prolyl cis-trans isomerase (PPIase)
best known as the cellular receptor of the immunosuppressant
cyclosporine A. Despite significant effort, evidence of developmental
functions of cyclophilin A in non-plant systems has remained obscure.
Mutations in a tomato (Solanum lycopersicum) cyclophilin A ortholog,
DIAGEOTROPICA (DGT), have been shown to abolish the
organogenesis of lateral roots; however, a mechanistic explanation of
the phenotype is lacking. Here, we show that the dgt mutant lacks auxin
maxima relevant to priming and specification of lateral root founder cells.
DGT is expressed in shoot and root, and localizes to both the nucleus
and cytoplasm during lateral root organogenesis. Mutation of ENTIRE/
IAA9, a member of the auxin-responsive Aux/IAA protein family of
transcriptional repressors, partially restores the inability of dgt to initiate
lateral root primordia but not the primordia outgrowth. By comparison,
grafting of a wild-type scion restores the process of lateral root
formation, consistent with participation of a mobile signal. Antibodies
do not detect movement of the DGT protein into the dgt rootstock;
however, experiments with radiolabeled auxin and an auxin-specific
microelectrode demonstrate abnormal auxin fluxes. Functional studies
of DGT in heterologous yeast and tobacco-leaf auxin-transport systems
demonstrate that DGT negatively regulates PIN-FORMED (PIN) auxin
efflux transporters by affecting their plasma membrane localization.
Studies in tomato support complex effects of the dgt mutation on PIN
expression level, expression domain and plasma membrane
localization. Our data demonstrate that DGT regulates auxin transport
in lateral root formation.
KEY WORDS: Auxin response, Auxin transport, Lateral root
initiation, Cyclophilin A, DIAGEOTROPICA
INTRODUCTION
The formation of root branches, known as ‘lateral roots’, continues
throughout the entire lifespan of a plant. In most eudicot plants such
as Arabidopsis and tomato, lateral root meristems form de novo from
cells in the pericycle cell layer of the parent root (for a recent review,
see Lavenus et al., 2013a). This process comprises several distinct
phases. First, some of the pericycle cells adjacent to a protoxylem
pole in the basal region of the root apical meristem (also referred to
as the transition zone) undergo pre-selection or ‘priming’(De Smet
et al., 2007; Moreno-Risueno et al., 2010). In the differentiation
zone of the root, selected pericycle cells become specified as lateral
root founder cells; these cells undergo asymmetric anticlinal
(perpendicular to the root surface) division, giving rise to a file of
short cells referred to as the stage I primordium (Malamy and
Benfey, 1997). Cells in the stage I primordium divide periclinally
(parallel to the root surface) to form a two-cell layered primordium
(stage II). Further development generates a dome-shaped advanced
primordium, then a recognizable meristem forms and the new lateral
root emerges through the overlying tissues of the parent root.
A number of studies have highlighted the organogenetic power of
the plant hormone auxin. Auxin biosynthesis, perception, signaling
and polar transport (PAT) are all required for normal lateral root
formation (reviewed by Benková et al., 2009; Lavenus et al., 2013b;
Overvoorde et al., 2010; Vanneste and Friml, 2009). PAT and auxin
responses are tightly interlinked and difficult to resolve in planta
(Vieten et al., 2005). Expression of the auxin-responsive reporter
gene DR5 in vascular cells in the Arabidopsis root apical meristem
associates with priming of adjacent pericycle cells (De Smet et al.,
2007; Moreno-Risueno et al., 2010), whereas DR5 expression in
pericycle cells in the root differentiation zone of Arabidopsis and
tomato marks their specification as lateral root founder cells
(Benková et al., 2003; Dubrovsky et al., 2008; Himanen et al.,
2002). Both types of DR5 expression patterns are abolished upon
application of the auxin efflux inhibitor 1-N-naphthylphthalamic
acid (NPA), indicating that these auxin responses depend on
PAT (De Smet et al., 2007: Himanen et al., 2002). In Arabidopsis,a
gain-of-function mutation solitary root (slr-1/iaa14) leads to
accumulation of a stabilized form of SLR/IAA14, a member of
the Aux/IAA protein family of transcriptional repressors, and
expression of mIAA14 in wild-type plants inhibits lateral root
formation (Fukaki et al., 2002). A close tomato SLR ortholog is
ENTIRE (E)/Sl-IAA9 (Wu et al., 2012). RNAi lines with decreased
Sl-IAA9 gene expression (Wang et al., 2005) and loss-of-function
e/iaa9 mutants (Zhang et al., 2007) show shoot morphological
defects but normal root development.
In Arabidopsis, PAT relies on two major families of membrane-
localized auxin efflux proteins, PIN and ABCB, and a family of
auxin influx proteins, AUX1/LAX (reviewed by Vanneste and
Friml, 2009). Dynamic recycling of PINs to and from the plasma
membrane is essential for PIN functionality (Geldner et al., 2001;
Grunewald and Friml, 2010; Kleine-Vehn et al., 2008). In the root,
auxin runs from the base towards the tip (acropetal stream) and from
the tip towards the base (basipetal stream). Using a self-referencing
IAA-specific microelectrode that permits noninvasive continuous
recordings of auxin flux rate along the root, it is possible to detect
Received 22 May 2014; Accepted 11 December 2014
1
Oregon State University, Department of Botany and Plant Pathology, 2082 Cordley
Hall, Corvallis, OR 97331, USA.
2
University of Fribourg, Department of Biology –
Plant Biology, CH-1700 Fribourg, Switzerland.
3
Institute of Science and Technology
Austria, Am Campus 1, Klosterneuburg A-3400, Austria.
4
VIB Department of Plant
Systems Biology and Department of Plant Biotechnology and Genetics, Ghent
University, Gent 9052, Belgium.
5
Key Laboratory of Agro-biodiversity and Pest
Management of Education Ministry of China, Yunnan Agricultural University,
Kunming 650201, China.
6
LINV–DIPSAA–Universitadi Firenze, Viale delle idee 30,
Sesto Fiorentino 50019, Italy.
7
Universidad Nacional Autonoma de Mexico, Instituto
de Biotecnologı
a, Departamento de Biologı
a Molecular de Plantas, Apartado
Postal 510-3, 62210 Cuernavaca, Morelos, Mexico.
8
School of Life Sciences,
Southwest University, Chongqing 400715, China.
*Author for correspondence (ivanchem@science.oregonstate.edu)
712
© 2015. Published by The Company of Biologists Ltd
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Development (2015) 142, 712-721 doi:10.1242/dev.113225
DEVELOPMENT
a distinct peak at the root transition zone (Mancuso et al., 2005;
Santelia et al., 2005), correlating with a PIN-dependent auxin
‘reflux loop’from peripheral towards central vascular cells (Blilou
et al., 2005). Less is known about how auxin transporters are
regulated by protein interactions that may influence their
conformation and thus affect trafficking, stability or activity.
Trafficking of ABCBs from the ER to the plasma membrane and
their functionality on the membrane is maintained by the peptidyl-
prolyl cis-trans isomerase (PPIase) FKBP42/TWD1, which does
not interact with PIN auxin transporters (Bouchard et al., 2006;
Wang et al., 2013; Wu et al., 2010).
Similar to FKBPs, cyclophilins (Cyps) display a PPIase activity
in vitro, suggesting they act in protein folding (Schiene-Fischer
and Yu, 2001). FKBPs and Cyps are commonly referred to as
immunophilins due to their high affinity for the immunosuppressive
drugs FK506 and cyclosporine A, respectively. Cyclophilin A
consists of only the core PPIase domain, localizes primarily in the
cytosol and nucleus, and is highly conserved from yeast to humans
[reviewed by Wang and Heitman (2005)]. In higherplants, cyclophilin
A has been linked to auxin-regulated development throughthe cloning
of the diageotropica (dgt) mutation in tomato (Oh et al., 2006). DGT
possessesPPIase activity and might affect plant development through
physiological refolding of target proteins (Oh et al., 2006). One of
the most remarkable phenotypes of dgt is the lack of lateral root
primordium organogenesis (Ivanchenko et al., 2006). In dgt,the
expression of members of the auxin-regulated Aux/IAA gene family
is abnormal to a different degree, depending on organ and
developmental stage (Balbi and Lomax, 2003; Mignolli et al., 2012;
Mito and Bennett, 1995; Nebenführ et al., 2000). Protoplasts from dgt
hypocotyls do not swell but instead decrease in volume when treated
with auxin or antibodies against AUXIN BINDING PROTEIN 1
(ABP1), further suggesting an abnormal auxin response (Christian
et al., 2003). Previous work has reported unchanged auxin transport
in dgt root and shoot, which has led to the hypothesis that DGT
regulates auxin perception or signaling but plays no role in PAT
(Daniel et al., 1989; Muday et al., 1995). Here, we show that DGT is
required for effective auxin transport in planta and in heterologous
auxin-transport systems that lack plant-specific components of auxin
perception and signaling. In contrast to TWD1 involved in regulation
of ABCB auxin transporters, DGT appears to regulate PIN
transporters.
RESULTS
The dgt mutant lacks auxin maxima related to pericycle cell
priming and founder cell specification
We analyzed auxin signals in vascular cells in the root apical
meristem related to pericycle cell priming using DR5:GUS and
IAA2:GUS reporters. DR5 was expressed in vascular cells in wild-
type root tips (Fig. 1A), similar to the pattern reported in
Arabidopsis (De Smet et al., 2007). Remarkably, vascular DR5
signals were completely absent in dgt, although expression in the
quiescent center region (QC) and the central root cap (columella)
was present and even appeared increased compared with wild type.
Treatment with the auxin transport inhibitor NPA abolished DR5
expression in the vascular cells of wild-type roots, causing them to
resemble untreated dgt roots. Upon a pulse treatment with IAA
(5 µM for 3 h), DR5 activity increased in the wild-type vascular
cells but no expression was induced in dgt. On comparing the effect
of a longer IAA treatment (5 µM for 40 h) with that of the synthetic
auxins NAA and 2,4-D, postulated to be inefficiently transported by
the auxin efflux and influx transporter, respectively (Marchant et al.,
1999), we found that IAA and NAA induced multiple lateral root
primordia in the wild-type root tip, whereas 2,4-D increased DR5
expression without primordium induction. None of these treatments
Fig. 1. The dgt mutant lacks auxin signals related
to pericycle cell priming and lateral root
organogenesis. (A) DR5:GUS auxin reporter
expression in wild-type and dgt root tips after
treatments as indicated. Note reporter expression in
vascular cells in the wild-type root apical meristem and
its absence in dgt (arrowhead). The dgt root exhibits
DR5 expression only in the QC region and central root
cap (arrow). Treatment with auxin transport inhibitor
NPA (10 μM for 20 h) abolishes DR5 expression in the
vascular region of wild type. Pulse treatment with IAA
(5 μM for 3 h) increases DR5 expression in stele of the
wild-type root tip but has no effect on dgt. Treatment
with IAA or NAA for 40 h induces lateral root primordia
in the wild-type root tip but not in dgt. 2,4-D increases
DR5 expression at the root tip in wild type but not in
dgt. (B) IAA2:GUS reporter expression in newly
emerged lateral roots induced through meristem
decapitation shows strong expression in stele of wild
type and more peripheral expression in dgt.
(C) Tomato root anatomy (one half of the root is
shown). (D) Wild-type lateral root primordia showing
DR5 expression at the primordia tips. (E) The
differentiation zone of wild-type and dgt roots after
treatment with 5 μM IAA for 3 h. Arrows in D,E denote
divided pericycle cells that do not show DR5
expression. c, cortex; cc, central cylinder; e,
endodermis; ep, epidermis; p, pericycle; x, xylem.
Scale bars: 100 μm in A,B; 30 μm in D,E.
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RESEARCH ARTICLE Development (2015) 142, 712-721 doi:10.1242/dev.113225
DEVELOPMENT
induced DR5 expression in the vascular cells of dgt or primordium
formation (Fig. 1A). Although dgt is unable to form lateral roots,
these can be induced in both wild type and dgt following meristem
decapitation. In induced lateral roots, which are thinner and allow
for more precise tissue assessment in tomato, IAA2 reporter
expression was strong in wild-type vascular cells, whereas in dgt
it was decreased in vascular and increased in peripheral tissues
(Fig. 1B). Furthermore, upon gravitropic stimulation wild-type roots
reoriented their growth direction and demonstrated asymmetric
IAA2:GUS reporter expression on the lower side of the root, whereas
dgt roots did not reorient, and did not show asymmetric IAA2:GUS
expression (supplementary material Fig. S1). Thus, dgt root tips
displayed spatial abnormalities in auxin reporter expression that
could be justifiably interpreted as resulting from a defect in PAT.
We next analyzed auxin signals in pericycle cells in the dgt root
differentiation zone. The tomato root anatomy is similar to that of
Arabidopsis except that the cortex has three cell layers instead of one
(Fig. 1C). In the differentiation zone of the wild-type root, DR5 was
expressed in lateral root primordia and increased at the primordia tips
(Fig. 1D). It appears that in tomato, some primordia are initiated by
relatively longer stretches of divided pericycle cells but those extra
cells do not show DR5 expression and do not participate in further
primordiumdevelopment (Fig. 1D, arrows). Because, intomato, DR5
expression was insufficiently strong at early stages of primordium
organogenesis, we analyzed rootsthat were pulse-treated with IAA for
3 h (F ig. 1E). In these roots, DR5 expression/signal was clearly seen in
founder cells and stage I primordia in wild type, apparently associated
with primordia centers and absent from short pericycle cells at the
primordia periphery. In dgt,noDR5 expression was observed in this
zone. Although some dgt roots exhibited short pericycle cells
apparently resulting from anticlinal pericycle cell division, these
cells did not show DR5 expression (Fig. 1E). We conclude that, in
tomato, DR5 marks auxin signals associated with primordium
initiation and growth; however, such signals are absent in dgt.
To confirm that DR5 expression in vascular cells at the wild-type
tomato root tip is indeed related to the process of pericycle cell
priming, as has been shown in Arabidopsis, we treated seedlings
with a 3 h pulse of IAA, transferred them onto fresh agar plates, and
marked the position of the root tips. As roots elongated below the
mark, the DR5-positive vascular tissues remained just above the
mark. Analysis of the root zone above the mark in a time course
revealed DR5 activity in pericycle cells 20 h after the treatment, and
a few hours later a primordium formed, then a lateral root emerged
(Fig. 2). However, no primordium organogenesis or lateral root
formation was observed in dgt, consistent with the lack of DR5
response in vascular cells of the root apical meristem (Fig. 2).
DGT tissue-specific expression and subcellular localization
DGT:GUS expression was observed in roots, cotyledons and leaves,
and is apparently associated with the vasculature in these tissues
(Fig. 3A-C). DGT activity was broader in cross-sections through the
middle part of the meristem (Fig. 3D), but was restricted to the central
cylinder and pericycle and endodermis near the transition and
elongation zone, where it was observed predominantly at the phloem
poles (Fig. 3E). At the beginning of the differentiation zone, some
DGT expression was also detected in early-stage lateral root
primordia (Fig. 3F). To observe the subcellular localization of
DGT in pericycle cells, we stably expressed a DGT:mCherry-DGT
fusion in Arabidopsis, which has thinner roots that are amenable to
confocal microscope imaging of inner tissues. Consistent with the
DGT:GUS expression in tomato, mCherry-DGT fluorescence was
observed in lateral root founder cells and lateral root primordia in
Arabidopsis roots (Fig. 3G). At the subcellular level, DGT
was observed in the nucleus and the cytoplasm in early-primordia
cells, consistent with the subcellular localization of yeast and
mammalian cyclophilin A (reviewed by Wang and Heitman, 2005).
Transformation the DGT:mCherry-DGT construct into dgt restored
the lateral root formation defect, demonstrating the functionality
of the mCherry-DGT fusion (supplementary material Fig. S2).
The DGT expression pattern supports participation in lateral root
primordium organogenesis and suggests a nuclear and a cytoplasmic
function of DGT.
DGT and E/Sl-IAA9 pathways overlap partially
Transformation of the Arabidopsis IAA14:mIAA14-GFP construct in
tomato resulted in a dramatic reduction in lateral root formation
(Fig. 4A) similar to that observed in Arabidopsis (Fukaki et al.,2002),
indicating conservation of the SLR-governed pathway in tomato. To
test whether DGT genetically interacts with the tomato SLR ortholog
E, we generated a dgt e double mutant. Lateral root formation was
partially restored in the dgt e background, although the appearance
of root branches was much delayed (Fig. 4B). Comparing the
primordium development, we found that wild-type and the esingle
mutant roots exhibited primordia of all stages, dgt roots rarely
exhibited primordium initiation, and dgt e roots predominantly
exhibited structures resembling stage I primordia (Fig. 4C). On
exposure to 5 µM IAA for 40 h, the wild type and the emutant roots
formed lateral root primordia close to the root apex as expected,
whereas dgt and the dgt e double mutant were equally insensitive to
the treatment (Fig. 4D). Thus, downregulation of Epartially restored
the primordium initiation in dgt but not the primordium outgrowth
and the ability to respond to exogenously applied auxin.
Grafting of a wild-type shoot partially restores the lateral root
defect in dgt
Because shoot-derived auxin is known to stimulate the outgrowth of
lateral root primordia in Arabidopsis (Bhalerao et al., 2002), and
application of the auxin transport inhibitor NPA was shown to
Fig. 2. Induction of lateral root organogenesis after treatment with 5 μM
IAA for 3 h. At ‘0’h post-treatment, DR5 expression is increased in developing
vascular cells in the wild-type root tip, but is not observed in dgt.At20h,
adjacent pericycle cells demonstrate DR5 expression and primordium
formation in wild type, and at 72 h a lateral root is formed; this process does not
take place in dgt roots. Panels (0-25 h) show images from cleared roots; panels
(72 h) show live roots on agar plates. e, endodermis; p, pericycle; v, vascular
cells; x, xylem. Scale bars: 50 μm (0, 20 and 25 h); 100 μm (72 h).
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RESEARCH ARTICLE Development (2015) 142, 712-721 doi:10.1242/dev.113225
DEVELOPMENT
inhibit lateral root formation (Reed et al., 1998), we tested whether
grafting of a wild-type scion would improve the lateral root
formation in the dgt rootstock. Seedlings were grafted at the middle
of the hypocotyl as soon as they germinated. At this stage, neither
wild type nor dgt had root branches, and the primary wild-type root
had on average seven primordial, whereas primordia were very rare
in dgt. Older soil-grown dgt plants also had root systems much
smaller than those in wild type. Grafted seedlings were analyzed for
root development at 12 days post-grafting (Fig. 5). As expected, root
development was minimal in self-grafted dgt seedlings compared
with self-grafted wild-type plants (Fig. 5A). The grafting of a dgt
scion onto a wild-type rootstock did not affect the root development.
However, when a wild-type scion was grafted onto a dgt rootstock,
the development of the dgt rootstock was significantly improved,
confirming an earlier report (Zobel, 1973). Histological markers in
the differentiation zone were improved, including asymmetric
pericycle cell division, early-stage primordium formation and DR5
expression in primordia (Fig. 5B). At the root tip, DR5 expression
was missing in vascular cells of self-grafted dgt plants, and was
restored in some of the plants with a dgt rootstock grafted on a wild-
type shoot (Fig. 5C). Furthermore, root growth in a dgt1-1 (AC
background) rootstock was restored upon grafting of an ethylene
overproducing Epinastic (Epi) mutant scion (VFN8 background)
and emutant scion (AC background) but not dgt-dp (Chatham
background), indicating that the effect was a property of the DGT
protein and not the genetic background used for grafting
(supplementary material Fig. S3). Thus, grafting improved the
auxin responses and lateral root formation in the dgt rootstock,
consistent with participation of a mobile signal. We therefore tested
whether DGT could move from the shoot into the root in grafted
plants. An Arabidopsis cyclophilin A antibody (Lippuner et al.,
1994) detects DGT in wild-type tissues but not in dgt tissues in
western blots (Oh et al., 2006). Using this antibody, we could not
detect any DGT signal in dgt rootstocks grafted on wild-type scions
(n=5 plants) (supplementary material Fig. S4), ruling out the
possibility that DGT movement restored the lateral root formation in
grafted dgt rootstocks.
Measurements of auxin transport detect abnormal PAT
fluxes in dgt
To investigate defects in PAT in dgt, transport of radiolabelled IAA
was assayed. Root IAA transport from the root-shoot junction to the
root tip (root-ward) was increased in dgt (Fig. 6A), whereas
transport from the root tip toward its base (shoot-ward) was
decreased (Fig. 6B). By comparison, movement of benzoic acid
(BA), assayed as a diffusion control, was unchanged between dgt
and wild type (Fig. 6A,B). Using an IAA-specific microelectrode,
we then analyzed the IAA influx velocity along the root tip. In wild
type, the transition between the meristem and elongation zone was at
0.85±0.06 mm from the root apex; in dgt it was at 0.58±0.04 mm
(Fig. 6C). An IAA influx peak averaging 188 fmoles cm
−2
s
−1
was
recorded in this zone in wild type that was dramatically reduced to
106 fmoles cm
−2
s
−1
in presence of NPA, as expected (Fig. 6C,D).
In dgt, the IAA influx peak averaged only 98 fmoles cm
−2
s
−1
,
comparable to that in NPA-treated wild-type roots, and even more
strikingly was completely unaffected by the presence of NPA
(Fig. 6C,D). Thus, the dgt root tip seems to be inefficient in
generating an IAA reflux loop at the transition zone and supplying
auxin into vascular cells involved in lateral root formation.
Modulating the DGT level results in changes in cellular IAA
efflux, and subcellular localization and functionality of PIN
auxin transporters
Protoplasts prepared from dgt leaves had an increased IAA efflux
compared with wild type, indicating that DGT is a negative PAT
regulator at the cellular level (Fig. 7A). To separate the effect of
DGT on PAT from that on auxin signaling, we then used a yeast
(Saccharomyces cerevisiae) auxin-transport system. HA-DGT had
no effect on its own, but reduced Arabidopsis PIN2-driven and
synergistic ABCB1/PIN1-mediated IAA efflux, apparently acting
Fig. 3. DGT expression pattern and subcellular
localization. (A-F) Expression of DGT:GUS
reporter in tomato. (A) Five-day-old seedling,
(B) cotyledon, (C) leaf-1 primordium, (D-F)
cross-sections through the middle of the meristem
(D), transition zone (E) and the beginning of the
differentiation zone (F) of an 8-day-old seedling.
Asterisks indicate the pericycle cell layer and an
arrow indicates an early primordium. pp, phloem
pole; xp, xylem pole. (G) Expression of DGT:
mCherry-DGT construct in Arabidopsis.p,
pericycle; x, xylem. Scale bars: 5 mm in A; 300 μm
in B,C; 50 μm in D-F; 20 μminG.
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RESEARCH ARTICLE Development (2015) 142, 712-721 doi:10.1242/dev.113225
DEVELOPMENT
in line with the above-described function as a negative regulator of
auxin efflux (Fig. 7B). HA-DGT had no significant effect on
ABCB1 alone (Fig. 7B), indicating that DGT might act preferably as
a negative regulator of PIN transporters. Because PIN1 is not
functional in S. cerevisiae without ABCB1 (Blakeslee et al., 2007;
Kim et al., 2010), we re-tested the effect of DGT on PIN1 in a
tobacco (Nicotiana benthamiana) leaf transport system (Henrichs
et al., 2012). Analogous to the yeast system, a mCherry-DGT fusion
had a negative effect on PIN1-driven IAA efflux but no significant
effect on ABCB1-driven IAA efflux, demonstrating a preferential
regulation of PIN transporters (Fig. 7C).
TargetP searches (at http://www.cbs.dtu.dk/services/TargetP/) did
not reveal any canonical subcellular localization signals in DGT, with
the exception of a potential palmitoylation signal at the C terminus;
such signals are important for protein targeting to the plasma
membrane and/or interactions with membrane proteins. To explore
how DGT could functionally affect auxin transporters, we analyzed
the colocalization of DGT with Arabidopsis PIN1 and ABCB1 upon
co-expression in N. benthamiana leaves. When expressed alone,
DGT localized predominantly in the nucleus, in addition to signals in
the cytoplasm and the cell periphery as expected; PIN1 and ABCB1
localized predominantly to the plasma membrane, consistent with
previous results (Henrichs et al., 2012) (Fig. 7D). When DGT was co-
expressed with ABCB1, the localization of ABCB1 did not change
significantly, but most of the DGT signal disappeared from the
nucleus and appeared on the cell periphery (Fig. 7E), suggesting that
ABCB1 may directly or indirectly affect localization and putative
nuclear and cytoplasmic function of DGT. When DGT was co-
expressed with PIN1, the localization of both proteins was modified:
Fig. 4. Partial overlap between DGT and ENTIRE (E)/Sl-IAA9 pathways.
(A) Inhibition of lateral root formation in tomato upon expression of Arabidopsis
IAA14:mIAA14-GFP construct.(B) Comparison of wild-type and mutant
phenotypes. Lateral root formation is partially restored in dgt e mutant
compared with dgt. (C) Quantification of primordia stages: ∼3-cm-long root
apices were excised from each genotype and primordia quantified after root
clearing. The percentages of stage I primordia are indicated. Only two
primordia, both from stage I, were found in 20 dgt roots. Data are mean±s.e.m.
(D) Inability of dgt and dgt e mutants to form primordia upon treatment with IAA
(5 µM for 40 h). Morphogenesis of primordia in the transition zone in wild type
and eis marked with asterisks. The larger cell size in dgt and dgt e indicates
that the IAA treatment did not suppress cell elongation in these genotypes.
Scale bar: 30 μminD.
Fig. 5. Grafting of a wild-type shoot partially restores development in the
dgt rootstock. (A) Root system phenotypes in grafted tomato seedlings.
Arrowheads indicate the sites of the graft junctions. The tissue identity is
indicated. (B) Improvement of primordium organogenesis in dgt roots upon
grafting of a wild-type scion. (C) DR5 expression in root tips of grafted plants.
n(dgt/dgt)=10 roots from eight plants, n(dgt/wt)=11 roots from three plants,
n(wt/dgt)=14 roots from five plants, n(wt/wt)=11 roots from three plants. The
number of roots with DR5 expression in vascular tissues is indicated. Scale
bars: 10 mm in A; 30 μminB;50μminC.
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RESEARCH ARTICLE Development (2015) 142, 712-721 doi:10.1242/dev.113225
DEVELOPMENT
a significant proportion of PIN1 shifted from the plasma membrane
to the nuclear periphery, whereas DGT increased on the cell
periphery (Fig. 7F). The PIN1 internalization following DGT co-
expression explains the negative effect of DGT on PIN-driven auxin
efflux, and the lack of DGT effect on ABCB1 localization is in line
with unchanged ABCB1-driven auxin efflux (Fig. 7B,C). Together,
the data supported a function of DGT in PAT that was independent of
auxin signaling, and identified distinct interactions of DGT with
different types of auxin transporters.
DGT affects PIN expression and localization to the plasma
membrane at the root tip
We analyzed the expression of PIN mRNAs in the apical 1 cm
region of the root. We found no significant change in tomato PIN1a,
blevels, whereas the expression of PIN2 was reduced (Fig. 8A). We
also analyzed the PIN protein behavior using Arabidopsis PIN1 and
PIN2 antibodies. Tomato PIN1 and PIN2 showed a typical polar
Fig. 6. dgt roots show defects in polar auxin transport. (A,B) Acropetal (A)
and basipetal (B) IAA and benzoic acid (BA) transport in wild-type (wt) and dgt
roots. IAA and BA radioactivity sections 5-10 mm (5), 10-15 mm (10) and
15-20 mm (15) from the source; data are mean±s.e.m. (n=4). (C) Images of a
wild-type and a dgt root; arrowheads indicate the length of the meristem.
(D) IAA influx profiles and peak influx rates (inset) along wild-type (wt) and
dgt root tips in the absence and presence of NPA. Data are mean±s.e.m.
(n=12). Asterisk indicates statistically significant differences between
genotypes; # indicates statistically significant differences between treatments
(P≤0.01, t-test). Scale bar: 50 μm.
Fig. 7. Effect of DGT expression level on PIN- and ABCB-mediated auxin
efflux. (A) dgtleaf protoplasts exhibit increased IAA export (mean±s.e.m.;
n=4). (B) Co-expression of DGT blocks Arabidopsis PIN2 and synergistic
PIN1/ABCB1 IAA export in yeast (mean±s.e.m.; n=4). PIN1 is inactive in the
absence of ABCB1 in the yeast S. cerevisiae. (C) Co-transfection of N.
benthamiana leaves with mCherry-DGT significantly reduces Arabidopsis
PIN1 but not ABCB1 IAA export activity. IAA efflux was calculated relative to
the initial export, where vector control was set to 100% (mean±s.e.m.; n=4).
(D) Single transfection results in nuclear and weak cytoplasmic and PM
labeling (arrow) for mCherry-DGT and PM locations for ABCB1-YFP and
PIN1-YFP, respectively. (E,F) Co-transfection of mCherry-DGT does not alter
ABCB1-YFP location (E) but shifts PIN1-YFP to the nuclear periphery
(F, arrows). Both ABCB1 (E) and PIN1 co-expression (F) enhance mCherry-
DGT presence at the cell periphery. Typical results from four independent
experiments are shown in each case. (A-C) Significant differences
(unpaired t-test with Welch’s correction, P<0.05) are indicated by asterisks.
Scale bars: 20 μm.
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DEVELOPMENT
localization known from Arabidopsis, with PIN1 localizing on the
lower/rootward face of cells in central tissues, and PIN2 on the
upper/shootward face of cells in more peripheral tissues (Fig. 8B,C).
Notably, PIN1 signals were essentially missing in stele tissues at the
dgt root tip (Fig. 8B). We could not assess the PIN1 plasma
membrane localization in those cell files due to the low expression
level. In the cell files where PIN1 was normally present its plasma
membrane localization appeared normal (Fig. 8B, inset). By
contrast, PIN2 plasma membrane localization was modified
showing a notably broader localization domain with ‘fuzzy’
appearance in the wild type when compared with a narrow more-
compact signal in dgt (Fig. 8C, inset). The PIN2 signal distribution
along the membrane (measured in pixels) was similar in wild type
and dgt (17.2±2.7 vs. 16.2±3.3, respectively) but the distribution of
the signal across the plasma membrane reached 5.0±1.4 in wild type
and only 2.4±0.9 in dgt (P=6.17519E-36; mean±s.d.; n=266 cells
from 13 roots in wild type and 191 cells from eight roots in dgt). In
the wild type, the mean PIN2 signal intensity at the plasma
membrane was 60.64±0.9043 and inside the cell it was 51.12±
0.8276 (ratio inside/PM: 0.8446), whereas in the dgt roots the PIN2
signal at the membrane was 54.55±1.099, and inside the cell it was
42.22±0.9123 (ratio inside/PM: 0.7663). Thus, the PIN2 signal in
dgt roots was overall lower but more sharply defined at the plasma
membrane, and the proportion of PIN2 allocated to the plasma
membrane was significantly higher (P<0.05).
In an attempt to analyze the subcellular trafficking of PIN2, we
also tried treatments with the trafficking inhibitor BFA that in
Arabidopsis interferes with the constitutive endocytic recycling of
PIN proteins to the plasma membrane and leads to PIN
internalization (Geldner et al., 2001; Kleine-Vehn et al., 2008).
The BFA treatment in tomato roots was ineffective as we did not see
the typical ‘BFA compartments’with internalized PIN proteins, as
observed in Arabidopsis, presumably due to different arrangements
of BFA-sensitive and -insensitive ARF GEFs in tomato when
compared with Arabidopsis. Altogether, the results show that the
dgt mutation affects the PIN expression domain and expression
level, as well as the plasma membrane localization of PIN proteins.
DISCUSSION
Our results show that DGT is required for the generation of PAT-
driven auxin maxima that are essential for lateral root formation.
Two earlier works reported unchanged PAT in dgt (Daniel et al.,
1989; Muday et al., 1995). However, it is important to note that both
studies detected increased transport of radiolabelled IAA in dgt
hypocotyls (Daniel et al., 1989, Fig. 2) and from the root base
towards the root tip (Muday et al., 1995, Fig. 8) but interpreted this
as ‘normal’. We observed increased root transport from the root-
shoot junction to the root tip and decreased transport from the root
tip toward the root base, demonstrating clearly abnormal PAT fluxes
and explaining our earlier findings of increased auxin level and
abnormal distribution along the dgt root tip (Ivanchenko et al.,
2006). Although more auxin moves from the aerial parts of dgt into
the root, it is abnormally distributed and no response maxima occur
in stele tissues related to lateral root initiation. Low auxin supply
into the stele of dgt is evident from low expression of auxin-
responsive DR5,IAA2 and PIN1 signals, and inability of the
e/sl-iaa9 mutation to restore the outgrowth of lateral root primordia.
The increased PAT in the dgt shoot might result from increased
cellular efflux, occur in response to PAT deficiencies in the root, or
be related to a putative yet unknown function of DGT in leaves,
whereas the decreased basipetal PAT at the root tip correlated with
decreased expression of PIN2.
The root tip is the most dynamic root region with respect to PAT.
In the tip, auxin is moved down the vascular tissues mainly by PIN1,
and redirected at the transition zone from peripheral into vascular
tissues in a ‘reflux loop’by PIN2, PIN3 and PIN7, providing stable
auxin circulation through the meristem (Blilou et al., 2005). The
IAA influx peak recorded at the dgt transition zone with an
IAA-specific microelectrode was reduced to 50% and was
insensitive to NPA, a potent auxin-efflux inhibitor, consistent
with inefficient IAA supply into vascular cells. This defect was
much greater than those reported in loss-of-function Arabidopsis
twd1 mutant (Bouchard et al., 2006; Wang et al., 2013) and pin2
mutant in blue light conditions (Wan et al., 2012), the peaks of
which average at ∼80% of wild type. The more severe dgt
phenotype argues that multiple transporters, PIN1, PIN2, and
potentially also PIN3 and PIN7, might be regulated by DGT.
We also observed that grafting of a wild-type scion partially
rescued the auxin response in the root tip vasculature of the dgt
rootstock, leading to primordium initiation, and antibodies did not
detect DGT movement into the rootstock. We therefore hypothesize
that the rescue was achieved through improving auxin transport
from the wild-type scion. A recent study in Arabidopsis has shown
Fig. 8. PIN expression and plasma membrane localization in dgt root tips.
(A) Relative expression of tomato PIN1a,PIN1b and PIN2 mRNA in root tips of
wild-type and dgt seedlings (combined values from three experiments
performed in duplicate). Data are mean±s.e.m., P<0.01. (B) Immunodetection of
tomato PIN1 in root tips of wild type and dgt (arrowheads). (C) Immunodetection
of tomato PIN2 in root tips of wild type and dgt (arrowheads). PIN2 signal is
more condensed in dgt compared withwild type. PIN signals are present in stele
of wild-type roots (asterisks) but absent in dgt. Representative images are
from four independent experiments with total of 60 roots per group. Scale bars:
50 μminB;75μminC.
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RESEARCH ARTICLE Development (2015) 142, 712-721 doi:10.1242/dev.113225
DEVELOPMENT
that radiolabelled auxin moves down the vasculature from the shoot
through plasmodesmal connections in the phloem and accumulates
at the root tip, but that the signal is barely perceptible in NPA-treated
plants, demonstrating a strong dependence on PAT (Bishopp et al.,
2011). Thus, the simplest explanation of our grafting results is that
in the grafted wild-type scion, auxin is more successfully channeled
into the vasculature, allowing for movement into the vasculature of
the dgt rootstock. Auxin transport from developing true leaves has
been reported to stimulate the emergence of lateral root primordia
(Bhalerao et al., 2002), whereas basipetal PAT from the root tip has
been proposed to stimulate the primordium initiation (Casimiro
et al., 2001). Our results show that, at least in tomato, the shoot is
important for root primordium initiation.
In contrast to the Arabidopsis immunophilin TWD1, which has
been demonstrated to act as a positive regulator of ABCB-driven
auxin efflux (Bouchard et al., 2006; Wang et al., 2013; Wu et al.,
2010), our functional studies implicate DGT as a negative regulator
of auxin efflux that preferentially affects PIN transporters at the
cellular level. First, protoplasts from dgt leaves displayed an
increased IAA efflux, whereas overexpression of DGT in tobacco
leaves reduced the PIN-mediated IAA efflux. Second, in a yeast-
based auxin-transport system, which lacks plant-specific auxin
responses, DGT co-expression still reduced the PIN-mediated IAA
efflux, providing strong evidence that DGT affects PIN
functionality independently of auxin signaling. As to how DGT
could affect auxin transporters at the protein level, DGT reduced the
PIN1 plasma membrane localization simultaneously with reducing
the PIN1-mediated auxin efflux upon co-expression in tobacco
leaves. This result agrees with previous observations in Arabidopsis
showing that increasing PIN levels at the plasma membrane leads to
elevated auxin efflux (Paciorek et al., 2005; Robert et al., 2010).
Furthermore, the proportion of PIN2 on the plasma membrane
versus inside the cell was also increased at the dgt root tip and
displayed a sharper signal when compared with wild type. Together,
the data demonstrate that DGT is implicated in a mechanism related
to membrane localization of PINs. However, both PIN1 and
ABCB1 were also able to modify the subcellular localization of
DGT upon co-expression in tobacco leaves, suggesting complex
inter-relationships among all three types of proteins. Whether
DGT interacts with PINs (and ABCBs) directly or via other proteins
or molecules remains to be determined. Given that the gene
transcription of DGT is downregulated by auxin at the root tip
(Ivanchenko et al., 2013), that PIN expression is upregulated in
Arabidopsis (Vieten et al., 2005), and PIN and ABCB activities
interact synergistically (Blakeslee et al., 2007), one can envision an
extremely complex functional feedback between DGT level, auxin
level, functionality and membrane localization of PINs, and
maintenance of PAT fluxes at the plant level. This complexity is
evident in the observed complex effect of the dgt mutation on the
expression levels of the PIN genes and their expression domain, and
on PIN protein localization to the plasma membrane at root tip.
It has been shown that mutations in the rice cyclophilin gene
OsCYP2 cause a similar inability to form lateral roots (Kang et al.,
2013; Zheng et al., 2013) and mutations in the DGT ortholog in the
moss Physcomitrella patens cause auxin-resistant phenotypes (Lavy
et al., 2012), demonstrating a conservation of DGT-like function in
auxin-regulated development. Several aspects of cyclophilin A
function have also emerged in non-plant systems, suggesting it to be
a multifunctional protein. Cyclophilin A has been linked to
regulation of protein activity (Brazin et al., 2002; Colgan et al.,
2004), protein interactions (Zander et al., 2003; Sorin and Kalpana,
2006) and protein trafficking (Ansari et al., 2002; Galigniana et al.,
2004). Another suggested function of yeast and mammalian
cyclophilin A is regulation of gene expression at the level of
chromatin folding (Arévalo-Rodríguez et al., 2000; Pijnappel et al.,
2001; Arévalo-Rodríguez and Heitman, 2005; Lu et al., 2006). Our
findings demonstrate a role of a plant cyclophilin A in polar auxin
transport. Some phenotypes of the dgt mutant cannot be directly
explained by a defect in auxin transport, e.g. the inability of dgt to
respond to exogenously applied auxin with increased DR5 auxin
reporter expression at the root tip, and the partial restoration of
lateral root formation by a loss of ENTIRE (IAA9). Therefore, in
addition to affecting auxin-regulated gene expression via regulating
PAT, DGT could also have a direct effect on auxin signaling, or even
act more broadly on gene expression, possibilities suggested by the
nuclear expression of DGT. In addition, the dgt root tip shows an
increased level of hydrogen peroxide (H
2
O
2
) (Ivanchenko et al.,
2013), and this oxidative environment could contribute to
decreasing the auxin sensitivity of dgt due to auxin oxidation, to
which grafted plants might be less susceptible because of the direct
auxin delivery into vascular tissues.
MATERIALS AND METHODS
Plant material and growth conditions
Wild-type tomato (Solanum lycopersicum), dgt1-1 and entire (e)mutants
in the Ailsa Craig (AC) background, and Arabidopsis seedlings in the
Columbia 0 (Col ) background were used unless otherwise stated. The
dgt1-1 and dgt-dp tomato mutant alleles (Oh et al., 2006), the emutant
(Zhang et al., 2007), Epi mutant (Fujino et al., 1988), transgenic tomato
DR5:GUS line (Dubrovsky et al., 2008), tomato IAA2:GUS line
(Dubrovsky et al., 2011) and IAA14:mIAA14-GFP construct (Fukaki
et al., 2002) have been reported. Tomato and Arabidopsis seedlings were
grown in 0.2× MS agar medium with vitamins (PhytoTechnology). Indole-
3-acetic acid (IAA) (Sigma), N-naphthalene-acetic acid (NAA) (Sigma),
2,4-dichloro phenoxyacetic acid (2,4-D) (Sigma) and NPA (Chem
Service) were used at concentrations and exposure times as indicated.
For grafting, tomato seedlings were germinated in vermiculite moistened
with 0.2× MS liquid medium and grafted as described in Arabidopsis
(Turnbull et al., 2002).
Cloning procedures and plant transformation
For the DGT:GUS construct, the 5′flanking region of the DGT gene from
−1389 to +36 bp was cloned between the KpnI and BamHI sites in a
pCAMBIA1300 vector (http://www.cambia.org), and the GUS-coding
sequence between the BamHI and SalI sites. A TGA stop codon was
introduced at the end of GUS. For the DGT:mCherry-DGT construct, the 5′
region of the DGT gene was cloned between the HindIII and SalI sites of
pCAMBIA1300, and an mCherry-DGT in-frame fusion was introduced
between the SalI and BamHI sites. For expression in N. benthamiana leaves,
the mCherry-DGT fusion was amplified by PCR and cloned under 35S
constitutive promoter between BamHI and SpeI sites in pCB302-3. For yeast
expression, HA-DGT fusion was generated by PCR and cloned between
BamHI and SalI sites in pRS314CUP. Arabidopsis transformation was
performed by the floral dip method, and tomato transformation as described
previously (Ivanchenko et al., 2006).
Histological analyses and microscopy
GUS staining was performed as described previously (Ivanchenko et al.,
2006) and westernblot as described previously (Oh etal., 2006). For meristem
and lateral root primordium analyses, roots were cleared as described
previously (Malamy and Benfey, 1997), and mounted in saturated chloral
hydrate solution in 10% glycerol. For tissue sectioning, roots stained for GUS
were imbedded in Technovit 7100 (Electron Microscopy Sciences). Root
samples were analyzed under a Zeiss Axiovert microscope with differential
interference contact (DIC) optics. Confocal microscopy in Arabidopsis roots
and N. benthamiana leaves was performed using an inverted Zeiss LSM 510
Meta (Carl Zeiss) microscope with ×63 (NA 1.2, C-Apochromat) objective
with water immersion.
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RESEARCH ARTICLE Development (2015) 142, 712-721 doi:10.1242/dev.113225
DEVELOPMENT
Auxin transport assays
Root acropetal (root-shoot junction to root tip) and basipetal (root tip to root
base) PAT measurements were performed as described previously (Lewis
and Muday, 2009). Continuous recordings of IAA fluxes at the root apex
with a self-referencing IAA-specific microelectrode were performed as
described previously (Mancuso et al., 2005). For NPA response, plants were
treated with or without 5 μM NPA for 2 h. Yeast IAA transport was
performed as described previously (Kim et al., 2010). Relative export from
yeast is calculated from retained radioactivity as follows: (radioactivity in
the yeast at time t=10 min)−(radioactivity in the yeast at time t=0)×(100%)/
(radioactivity in the yeast at t=0 min); mean values from four independent
experiments are presented. IAA export from N. benthamiana leaf tissue was
analyzed as described previously (Mravec et al., 2009; Henrichs et al.,
2012). Tomato protoplast assays were conducted as for tobacco, except that
enzyme digestion was performed overnight at room temperature. Relative
export from protoplasts is calculated from exported radioactivity as follows:
(radioactivity in the protoplasts at time t=xmin)−(radioactivity in the
protoplasts at time t=0)×(100%)/(radioactivity in the protoplasts at t=0 min);
mean values from four independent experiments are presented.
Quantification of PIN mRNA expression
RT-qPCR was performed as described previously (Ivanchenko et al., 2013).
Primers for tomato PIN1a (Bayer et al., 2009), PIN1b (Acc. HQ127074) and
PIN2 (Acc. HQ127077) were designed to include part of the 3′UTRs:
PIN1a F 5′-AGCACAGGGGTCATATTTGG, R 5′-TCCCAACAATTG-
ACCATTCA; PIN1b F 5′-TCCTGACATTCTTAGCACAGC, R 5′-TTTA-
TCTCCATGCCAATTGCT; PIN2 F 5′-CAGGACCAGCTGTTATTGCT,
R5′-CCAAGTCTACACACCAAGAAGC.
Analyses of PIN expression at the root tip
Roots from 8-day-old tomato seedlings were probed with Arabidopsis anti-
PIN1 or -PIN2 primary antibody (1:1000) and Cyanine Dye3 (Cy3)-
conjugated anti-rabbit secondary antibody (1:600) (Sigma) following a
whole-mount procedure as described for Arabidopsis (Sauer et al., 2006).
Images were acquired using a Zeiss LSM 700 upright confocal microscope.
To quantify PIN2 distribution in root epidermal cells of wild type and dgt,
measurements were performed with ImageJ2x software and analyzed with
GraphPad Prism6 software. Obtained data were tested by Mann–Whitney
test to assess significance. The PIN2 levels inside the cell were measured as
the mean gray value of pixel intensity using the ‘poligon’option, and the
PIN2 levels at the plasma membrane as the mean gray value of pixel
intensity using the ‘segmented lines’option with ‘line width’set to three
pixels. For each cell, the distribution of the PIN2 signal across the membrane
was measured in pixels as the length of the area possessing PIN2 signal
(‘thickness’of the PM signal). The distribution of the PIN2 signal along the
same plasma membrane (length of the PM domain with signal) was
measured to normalize for differences in cell size.
Acknowledgements
We thank S. Napsucialy-Mendivil, L. Charrier (Department of Biology, University of
Fribourg, Switzerland) and Kathy Cook (Microtechniques Laboratory, Oregon State
University, USA) for technical assistance; Hideriro Fukaki (Department of Biology,
Kobe University, Japan) for the IAA14:mIAA14-GFP construct; Chris Kuhlemeyer
(Institute of Plant Sciences, University of Bern, Switzerland) for the tomato PIN1a
sequence; Charles Gasser (University of California Davis, USA) for cyclophilin A
antibody; M. J. Ek Ramos and T. P. Devarenne (Department of Biochemistry and
Biophysics, Texas A&M University, USA) for advice in tomato protoplast
preparation; and The Tomato Genetics Resource Center at University of California,
Davis (http://tgrc.ucdavis.edu/) for seeds of the entire (e)andEpinastic (Epi)tomato
mutants.
Competing interests
The authors declare no competing or financial interests.
Author contributions
M.G.I., M.G. and J.F. designed experiments. M.G.I. generated expression constructs,
prepared transgenic Arabidopsis and tomato lines, analyzed reporter expression,
generated and analyzed dgt e mutants, and performed grafting experiments. J.Z. and
B.W. performed PAT measurements in tomato, N. benthamiana and S. cerevisiae
auxin transport assays, and confocal microscope imaging of protein localization. E.M.
analyzed PIN1and PIN2 expression intomato root tips. Y.D. analyzed PIN expression
in response to BFA treatment. E.A. and S.M. performed measurements of IAA fluxes
with an IAA-specific microelectrode. M.M. performed statistical analysis of PIN
expression in BFA-treated roots. S.F. performed RT-qPCRof PIN mRNA expression.
J.G.D. performed confocal microscope analyses of DGT:mCherry expression in
Arabidopsis roots. M.G.I. wrote the paper with inputs from M.G. and J.F. All authors
participated with data analysis and interpretation.
Funding
Financial support was provided by the USDA National Research Initiative
Competitive Grants Program [2007-35304-17728 to M.G.I.], by the Oregon State
University General Research Fund (to M.G.I.), by PRIN 2010-11 ‘PRO-ROOT’(to
S.M.), by Mexican CONACyT [127957, J.G.D.], by PAPIIT-DGAPA, by Universidad
Nacional AutonomadeMe
xico [IN204312 to J.G.D.], by the Odysseus program of
Research Foundation-Flanders (J.F.), by the Pool de Recherche of the University of
Fribourg (M.G.), by the Novartis Foundation (M.G.) and by Swiss NationalFunds (M.G.).
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.113225/-/DC1
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RESEARCH ARTICLE Development (2015) 142, 712-721 doi:10.1242/dev.113225
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