Article Roots Nurients Barberon 2016

Article
Adaptation of Root Function by Nutrient-Induced

Plasticity of Endodermal Differentiation
Graphical Abstract Authors
Marie Barberon,
Joop Engelbertus Martinus Vermeer,
Damien De Bellis, ..., Junpei Takano,
David Edward Salt, Niko Geldner
Correspondence
marie.barberon@unil.ch (M.B.),
niko.geldner@unil.ch (N.G.)
In Brief
The suberization layer of the root system
responds to a range of nutrient stresses,
and its deposition is regulated by stress
hormones, revealing a surprising level of
functional and anatomical plasticity in
adult roots.
Highlights
d The endodermis, a plant epithelium, later coats itself with
hydrophobic suberin
d Suberization can be enhanced or reversed, depending on

nutrient availabilities
d This stress-hormone-regulated plasticity allows adaptive

regulation of root function
Barberon et al., 2016, Cell 164, 447–459

January 28, 2016 ª2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2015.12.021
Article

Marie Barberon,1,* Joop Engelbertus Martinus Vermeer,1,5 Damien De Bellis,1,2 Peng Wang,3 Sadaf Naseer,1
Tonni Grube Andersen,1 Bruno Martin Humbel,2 Christiane Nawrath,1 Junpei Takano,4 David Edward Salt,3
and Niko Geldner1,*
1Department of Plant Molecular Biology, University of Lausanne, 1015 Lausanne, Switzerland
2Electron Microscopy Facility, University of Lausanne, 1015 Lausanne, Switzerland
3Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 3UU, UK
4Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
5Present address: Institute of Plant Biology, University of Zürich, 8008 Zürich, Switzerland
*Correspondence: marie.barberon@unil.ch (M.B.), niko.geldner@unil.ch (N.G.)

http://dx.doi.org/10.1016/j.cell.2015.12.021
SUMMARY root hair cells of the epidermis, repeated transport through

the cytoplasmic bridges of plasmodesmata, and export into
Plant roots forage the soil for minerals whose con- the xylem (Figure 1F). In contrast, the pathway with the longest
centrations can be orders of magnitude away from apoplastic component would be through the cell wall space of
those required for plant cell function. Selective epidermal and cortical cells, entering the symplast only by up-
uptake in multicellular organisms critically requires take at the outer plasma membrane of the endodermis (Fig-
epithelia with extracellular diffusion barriers. In ure 1F). An entirely apoplastic pathway is only possible at the
few sites where CSs have not yet formed or are broken, and it
plants, such a barrier is provided by the endodermis
is unclear whether this pathway can be of physiological rele-
and its Casparian strips—cell wall impregnations
vance (Clarkson, 1993; White, 2001). The coupled trans-cellular
analogous to animal tight and adherens junc- pathway would require repeated uptake and efflux of nutrients
tions. Interestingly, the endodermis undergoes sec- through the concerted action of influx and efflux transporters,
ondary differentiation, becoming coated with hy- a possibility that has been substantiated by a number of recent
drophobic suberin, presumably switching from an publications showing strictly polar distribution of numerous
actively absorbing to a protective epithelium. Here, transporters in root cells (reviewed in Barberon and Geldner,
we show that suberization responds to a wide range 2014).
of nutrient stresses, mediated by the stress hor- Recently, the discovery of sgn3 (schengen3), a mutant dis-
mones abscisic acid and ethylene. We reveal a strik- playing discontinuous CS, allowed to address the relevance of
ing ability of the root to not only regulate synthesis of these tight-junction-like structures in nutrient transport (Pfister
et al., 2014). The lack of an apoplastic barrier in sgn3 led to sur-
suberin, but also selectively degrade it in response to
prisingly mild phenotypes under non-challenging growth condi-
ethylene. Finally, we demonstrate that changes in tions but robustly displays a deficiency in the macronutrient
suberization constitute physiologically relevant, K (Pfister et al., 2014). Interestingly, the endodermis undergoes
adaptive responses, pointing to a pivotal role of the a second state of differentiation, which amounts to a direct
endodermal membrane in nutrient homeostasis. progression from a state of an actively absorbing epithelium
(state I) to a state that is thought to be exclusively protective in
INTRODUCTION nature (state II). State II endodermis is characterized by the for-
mation of the hydrophobic suberin polymer all around its surface
The Casparian strip (CS)-bearing root endodermis forms a para- (Meyer and Peterson, 2013). The ability of state II endodermis to
cellular transport barrier that defines two separate spaces within act as a protective barrier is highlighted by the fact that, in many
a root: the central nutrient-transporting stele, with its xylem plant species, environmental stresses such as drought can lead
network of interconnected hollow tubes, and the outer cortex, to a ‘‘cortical dieback,’’ leaving the endodermis as the outermost
whose intercellular (apoplastic) space is connected to the soil. protective layer of the root (Meyer and Peterson, 2013). In ani-
On their path from the root periphery to its center, mineral nutri- mals, specialized protective epithelia are often stratified (multi-
ents can be radially transported through three different path- layered) and arise from single-layered epithelia through complex
ways: symplastic; apoplastic; or coupled trans-cellular pathway processes of trans-differentiation and proliferation, contrasting
(Barberon and Geldner, 2014; Geldner, 2013; Robbins et al., the simple progression seen in the case of the root endodermis
2014; Andersen et al., 2015). The symplastic and apoplastic (Koster and Roop, 2004; Senoo et al., 2007). Endodermal suber-
pathways are best regarded as describing two extremes in the ization occurs in a ‘‘switch-like’’ manner, in which individual
degree to which a nutrient travels through either the intercon- endodermal cells rapidly suberize in an apparently random
nected cytosol of root cells or through the apoplastic space. fashion. This leads to a zone of ‘‘patchy’’ suberization that later
The longest symplastic pathway would be an uptake into the develops into a zone of continuous suberization (Kroemer,
Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc. 447
Figure 1. Suberin Interferes with Uptake
from the Apoplast
(A) Median and surface views of FY staining for
suberin. Arrows indicate suberin exclusion from
the CS; insets, zooms onto the CS region. The
scale bar represents 10 mm.
(B) FDA and PI penetration across root cell layers.
Picture corresponds to differentiated endodermis
prior to suberization, with individual channels and
overlay (merge). The scale bar represents 50 mm.
(C) Schematic of endodermal differentiation. Three
different zones are highlighted: undifferentiated,
non-suberized, and suberized zone. In the suber-
ized zone, we distinguish between stages of pat-
chy and continuous suberization.
(D) FDA penetration after 1 min in WT roots and in
ELTP::CDEF1 in similar positions. The scale bar
represents 25 mm.
(E) FDA penetration to the stele (IN) or blocking
before reaching the endodermis (OUT) in WT,
ELTP::CDEF1, casp1 casp3, esb1, and sgn3 mu-
tants roots. FDA penetration was evaluated after
1 min for all genotypes in the same part of the
corresponding root zones in WT plants. Data
correspond to frequency of FDA penetration per
genotype (percentage of roots for which FDA was
observed IN or OUT; n = 20).
(F) Models for radial transport of nutrients in
differentiated roots before and after suberization
(modified after Andersen et al., 2015).
co, cortex; en, endodermis; ep, epidermis. See
also Figure S1.
functional role of suberization. It has previ-

ously been associated with salt and
drought stress resistance (Baxter et al.,
2009; Höfer et al., 2008; Krishnamurthy
et al., 2009, 2011) and to be important for
growth under waterlogged conditions in
rice (Shiono et al., 2014). In Arabidopsis,
the enhanced suberin 1 (esb1) and myb36
mutants were found to display enhanced
suberization in early stages of endodermis
differentiation as a consequence of their
partial CS barrier defects, making their
associated mineral phenotypes difficult to
interpret (Baxter et al., 2009; Hosmani
et al., 2013; Kamiya et al., 2015). Here,
we visualize that suberin establishes a bar-
rier for uptake from the apoplast into the
endodermis. We find that endodermal su-
berization is highly plastic in response to
many nutritional stress conditions, which
1903; Figure 1C). Suberin is deposited as a secondary cell wall in exert their effect on suberin through the ethylene and abscisic
the form of lamellae between the plasma membrane and the pri- acid (ABA) hormonal pathways. Unexpectedly, ethylene applica-
mary cell wall, eventually coating the entire endodermal surface tion induces disappearance of suberin from state II endodermal
(Haas and Carothers, 1975; Robards et al., 1973). Recent data cells, suggesting that progression from an absorbing to a protec-
clearly indicate that these lamellae are not affecting the apoplastic tive epithelium can be reversed. We show that this surprising plas-
(paracellular) transport across the endodermis that is blocked by ticity is physiologically relevant as it can alleviate or enhance
the CS (Hosmani et al., 2013; Kamiya et al., 2015; Naseer et al., nutrient deficiency phenotypes of mutants and affect their ability
2012; Pfister et al., 2014). This begs the question as to the precise to tolerate elevated salinity.
448 Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc.
RESULTS FDA uptake even in the late parts of the root that are suberized
in WT (Figures 1D and 1E). Moreover, esb1 and casp1 casp3
CS and Suberin Lamellae Are Distinct Subcellular mutants—which both display enhanced and earlier suberization
Structures (Hosmani et al., 2013; Figure S1C)—displayed an earlier block in
In order to better delineate the precise cellular function of endo- FDA uptake (Figures 1E and S1D). Transport is not affected in
dermal suberization from that of CS, we investigated Fluorol the CS-defective mutant sgn3 that has no apparent defect
Yellow (FY)-stained WT roots using two-photon microscopy. in suberization (Figures 1E, S1C, and S1D). Taken together,
FY is a highly sensitive and reliable fluorescent stain specifically these results establish FDA uptake as a visual assay for suberin
labeling suberin in whole-mount roots (Lux et al., 2005; Naseer lamellae function in roots. We propose that suberin lamellae for-
et al., 2012). Our analysis revealed that suberin forms around mation regulates the apoplastic and trans-cellular transport
differentiated endodermal cells and is excluded from the CS route but leaves the symplastic route unaffected (Figure 1F).
(Figure 1A). This is consistent with earlier ultrastructural studies
(Haas and Carothers, 1975; Robards et al., 1973) and corrobo- The Plant’s Nutritional Status Regulates Endodermal
rates recent work that clearly separates formation of suberin Suberization
from the earlier formation of CS (Kamiya et al., 2015; Naseer In order to study the influence of nutritional stress on suberin
et al., 2012). Previously, a strong, suberin-deficient line was deposition along the root, we analyzed well-characterized
established by expressing a suberin-degrading enzyme specif- nutrient transporter mutants under conditions where they were
ically in the endodermis. This CASP1::CDEF1 (CASPARIAN STRIP indistinguishable from WT plants in terms of root length, thus
DOMAIN PROTEIN 1::CUTICLE DESTRUCTING FACTOR 1) line excluding secondary effects on suberization through altered
was shown not to affect uptake of an apoplastic tracer (propidium root development (Figure S2A). We analyzed 5-day-old seed-
iodide [PI]; Naseer et al., 2012). It was previously hypothesized that lings of the metal-deficient iron-regulated transporter 1 (irt1)
suberin lamellae could affect the direct uptake from the apoplast mutant (Henriques et al., 2002), the Fe- and Mn-deficient natu-
into endodermal cells (Barberon and Geldner, 2014; Geldner, ral-resistance-associated macrophage proteins (nramp1) mutant
2013; Kamiya et al., 2015; Ranathunge et al., 2011; Robbins (Cailliatte et al., 2010), the K-deficient stelar k+ outward rectifier
et al., 2014; Andersen et al., 2015). (skor) mutant (Gaymard et al., 1998), and the S-deficient sulfate
transporter (sultr1;1 sultr1;2) double mutant (Barberon et al.,
Endodermal Suberin Interferes with Uptake from the 2008). We observed a striking reduction in the amount of suber-
Apoplast ization in irt1 and nramp1 mutants, with a delayed and mainly
We therefore tested whether fluorescein diacetate (FDA), a dye discontinuous (patchy) suberization (Figure 2A). In contrast,
that only becomes fluorescent after its uptake into living cells, skor and sultr1;1 sultr1;2 mutants developed suberin lamellae
could be utilized as a tracer for the presence of functional earlier and rapidly went into a stage of continuous suberization
suberin lamellae in the endodermis. This dye has been used (Figure 2A). These variations are specific to suberin lamellae,
in previous studies to monitor the mobility through plasmodes- because the formation of a functional CS network, scored as
mata and in the phloem and xylem after several minutes or block in PI penetration, was similar in WT and in all investigated
hours of incubation (Botha et al., 2008; Melnyk et al., 2015; mutants (Figure 2B). Hence, different nutritional deficiencies can
Oparka et al., 1994). We observed that, in the non-suberized affect suberin lamellae development in opposite ways. We next
endodermis, carrying only CS, FDA could enter the endodermis addressed whether these effects are specific to transporter mu-
and even the pericycle after only 1 min of incubation (Figure 1B). tants or can also be induced by corresponding nutritional stress
Next, we monitored FDA uptake in the suberized develop- conditions. Again, plants were grown under moderate nutritional
mental zone over 16 min (Figure S1A; Movie S1) and observed stress where no root growth defects were apparent (Figures
that FDA did not enter the endodermis anymore after 1 min but S2B–S2D). FY staining revealed that suberization is delayed
only after about 8 min of staining (Figure S1A; Movie S1). This upon germination in Fe-, Mn-, and Zn-deficient conditions
suggested that suberization restricts fast FDA uptake into the (Figure 2C), whereas it is enhanced upon germination in K- and
endodermis. Its slower uptake into endodermal cells might be S-deficient conditions, matching the mutant analysis (Figures
occurring indirectly from the cortex through plasmodesmata, 2D and 2E).
known to be present in suberized endodermal cells (Haas Until now, only enhancement of suberization has been re-
and Carothers, 1975; Robards and Robb, 1974). Additionally, ported and this was mainly restricted to conditions of excess
the initially very thin suberin coating of endodermal cells might salt or drought stress (Baxter et al., 2009; Beisson et al., 2007;
not cause a qualitative block but only a delay in FDA uptake Krishnamurthy et al., 2009, 2011; Ranathunge et al., 2011). In
from the apoplast. Consistent with our observations, rice, for example, cultivars exposed to salt stress accumulate
carboxyl-FDA unloading from the phloem was shown to appear more suberin in their exodermis and endodermis (Krishnamurthy
in pericycle cells, but it was blocked at the level of the endo- et al., 2009, 2011), and we confirmed here that salt also leads to
dermis (Oparka et al., 1994). an increase in suberization in Arabidopsis roots (Figure 2F;
In order to demonstrate that suberin indeed delays uptake of Kosma et al., 2014). Importantly, this enhanced suberization oc-
FDA into the endodermis, we performed the same assay in curs at salt concentrations that do not interfere with overall root
a suberin-deficient ELTP::CDEF1 line (ENDODERMAL LIPID development (Figure S2E) and can be observed already after
TRANSFER PROTEIN::CDEF1, equivalent to CASP1::CDEF1 24 hr of treatment (Figure S2H). We conclude that suberin
described above; Figures S1B and S1C). We observed fast lamellae deposition is highly plastic and responds to a broad
Figure 2. Nutrient Status Affects Suberization
(A) FY staining of suberin in nutrient transporters mu-
tants. Pictures taken in similar parts of the root of
WT, irt1, nramp1, skor, and sultr1;1 sultr1;2 are shown
(upper panel). The scale bar represents 50 mm. Suberin
deposition was quantified along the root axis, using
three different zones (see Figure 1C): non-suberized;
patchy; and continuous; n = 10. Data are presented as
percentage of endodermal cells.
(B) Establishment of the apoplastic barrier, determined
as the number of endodermal cells after onset of elon-
gation at which PI is blocked by the endodermis. Data
are represented as mean.
(C–F) FY staining of suberin in WT seedlings germinated
in +Mx (+Fe, +Mn, and +Zn), Fe, Mn, and Zn (C),
in + or K (D), in + or S (E), and in presence of NaCl (F).
Pictures in similar parts of the root are shown (upper
panels; scale bars: 100 mm), and quantifications along
the root axis are presented (bottom panels); n R 6.
Error bars represent SD; different letters indicate sig-
nificant differences between genotypes or growth
conditions (p < 0.05). See also Figure S2.
range of nutritional stresses, a feature that had not been previ- Suberization Requires Endodermal ABA Signaling
ously appreciated. When we genetically manipulated ABA biosynthesis (aba-defi-
cient 2 [aba2]) or responses (aba-insensitive 3 [abi3], aba-insen-
Suberin Development Is Controlled by ABA sitive 5 [abi5], and aba-insensitive 4 [abi4]), we found a strong
In order to establish the signaling pathways mediating this delay in suberin deposition, which formed in a highly discontin-
nutrient-induced plasticity, we searched online databases for uous pattern (Figure S3G). Previously, ABA signaling specifically
conditions modulating expression of the main biosynthetic in meristematic cortical and endodermal cell layers was shown
genes driving endodermal suberization (Hruz et al., 2008). to be required for lateral root quiescence during salt stress
Consistent with many previous studies, we found a major effect (Duan et al., 2013). In order to suppress endodermal ABA
of ABA as an inducer of suberin-associated genes (Boher et al., signaling specifically in differentiating endodermal cells of pri-
2013; Cottle and Kolattukudy, 1982; Kosma et al., 2014; Soliday mary roots, we expressed abi1-1 (aba-insensitive 1-1) under
et al., 1978; Yadav et al., 2014). In order to establish whether the CASP1 and ELTP endodermis-specific promoters (Roppolo
ABA-mediated enhancement of suberization is an immediate, et al., 2011; Wyrsch et al., 2015; Figure S1E). abi1-1 is a domi-
short-term response of the endodermis, we sought to establish nant-negative allele of the PP2C-type protein phosphatase
a live marker for suberization. The glycerol-3-phosphate acyl- ABI1, suppressing ABA signaling (Leung et al., 1997). We first
transferase GPAT5 is one in a suite of suberin biosynthesis addressed whether endodermal expression of abi1-1 affects
enzymes that has been particularly well characterized (Beisson suberin development in non-stress conditions and observed a
et al., 2007), and GPAT5-driven GUS activity has been shown strong delay in suberin formation in both ELTP::abi1-1 and
to perfectly match the pattern of suberin deposition in Arabidop- CASP1::abi1-1 plants (Figures 3E and S3H). This indicates that
sis roots (Naseer et al., 2012). We therefore generated the tran- local, endodermal ABA signaling is a critical component for su-
scriptional reporter line GPAT5::mCITRINE-SYP122 (driving berization even under standard growth conditions. When treated
expression of a fluorescently tagged plasma membrane protein). with ABA, no enhanced suberization was observed, although
Using this line, we observed ABA effects on suberin biosynthesis some degree of ABA induction of the ELTP promoter—but not
in live roots. At ABA concentrations that did not affect root the CASP1 promoter—was found also in cortical tissues (Fig-
growth (1 mM; Figure S3A), we found that GPAT5 expression is ure S1F). Thus, effects of ABA on endodermal suberization are
induced in the endodermis after only 3 hr of ABA treatment, pro- a local and early response to this stress hormone.
gressing in a wave that rapidly extended toward the root tip
(Figures 3A and S3B; Movies S2 and S3). Developmental staging Ethylene Signaling Interferes with Suberin
after 20 hr of ABA treatment revealed an early and continuous Accumulation
onset of GPAT5 expression, in contrast to the initially patchy With ABA as a positive regulator of suberization, we wondered
pattern of GPAT5 expression observed in untreated roots which signal could account for decreases in suberin accumula-
(Figure 3B). Moreover, we found that, upon ABA treatment, tion, as observed in Fe, Zn, and Mn deficiencies. Fe deficiency
GPAT5 expression is also induced in cortical cells, initiating is associated with ethylene production, and application of the
as patches of expression before becoming continuous, some- ethylene precursor ACC (1-aminocyclopropane-1-carboxylic
times even extending into epidermal cells (Figure 3B). FY stain- acid, ethylene precursor) mimics morphological, physiological,
ing confirmed extension of suberization to the root tip and and molecular Fe deficiency responses, whereas ethylene inhib-
cortex (Figures 3C and S3C). An expansion of suberization itors abolish some of those responses (Garcı́a et al., 2010; Li and
into the cortex should profoundly impact the water and nutrient Li, 2004; Lingam et al., 2011; Lucena et al., 2006; Romera and
transport capacity of ABA-treated roots. We therefore sought Alcantara, 1994; Romera et al., 1999; Schmidt et al., 2000). We
independent confirmation for the ABA-induced presence of therefore investigated the role of ethylene in suberization. We es-
suberin in cortical cells through ultrastructural analysis. We tablished conditions of ACC treatment that only slightly affected
found that, in both untreated and ABA-treated plants, the root primary root development (Figure S4A) and found that ACC
endodermis developed suberin lamellae, with size increases application results in a strong reduction of suberin accumulation
upon ABA treatment (Figures 3D and S3D). In the cortex, by in newly formed parts of the root (Figure 4A). Yet, to our surprise,
contrast, suberin lamellae-like secondary cell walls were ACC treatment also caused disappearance of suberin staining in
observed exclusively after ABA treatment (Figures 3D and older parts of the root that had developed suberin lamellae prior
S3D). Presence of CS was not detected outside of the endo- to the treatment (Figures 4A and S4B). Results with 1 mM ACC
dermis, even below the hypocotyl root junction of ABA-treated were variable, reaching from an absence of detectable FY signal
plants (Figure S3E). Finally, direct chemical quantification of to a mere reduction, but became stronger and less variable at
suberin upon ABA treatment shows a strong increase in root higher doses of ACC (Figure 4A). We confirmed this effect of
suberin content (43% increase of total suberin monomer con- ACC by electron microscopy on root sections taken 2 mm below
tent when compared to non-treated seedlings; Figure S3F). the hypocotyl-root junction. After treatment with ACC for 24 hr,
Hence, our data establish that ABA induces a rapid suberiza- absence or only patches of suberin lamellae could be observed
tion response, making it a prime candidate for mediating the in many older endodermal cells, something only rarely observed
enhanced suberization we observe upon nutrient deficiencies. in wild-type plants (Figure 4B). In cells where suberin lamellae
Importantly, ABA does not only enhance suberization but also were still present, their thickness was significantly reduced
alters its developmental progression, leading to ectopic depo- (Figure S4C). We also observed a modest reduction, but not
sition in young root parts, as well as in the cortex. an absence of GPAT5::mCITRINE-SYP122 signal upon ACC
Figure 3. ABA Signaling Controls Suberization
(A) Time series of GPAT5::mCITRINE-SYP122 expression upon ABA treatment. Four-day-old plants were transferred on 0.53 MS containing 1 mM ABA. Images
correspond to Z projections extracted from Movie S3 at 0, 2, 3, and 13 hr after ABA treatment. Arrows indicate the onset of GPAT5 expression. The scale bars
represent 200 mm.
(B) GPAT5::mCITRINE-SYP122 expression (green) along the root developmental stages in seedlings either untreated or treated with 1 mM ABA for 20 hr prior to
observation. Z projections (upper panels) and cross section views are presented (bottom panels). Root cell layers are highlighted by PI (red). Asterisks show one
cortical cell per cross section view. The scale bars represent 50 mm.
(C) FY staining in seedlings either untreated or treated with 1 mM ABA for 20 hr. Pictures taken in older parts of the root are presented as LUT of Z projections. The
scale bars represent 50 mm.
(D) TEM showing cell wall (CW) and suberin lamellae (SL) in endodermis (end) and cortex (co) of roots either untreated or treated with 1 mM ABA for 20 hr prior to
observation. ep, epidermis; pe, pericycle. Sections were performed 2 mm below the hypocotyl-root junction. The scale bars represent 200 nm.
(legend continued on next page)
Figure 4. Ethylene Signaling Controls Su-
berization
(A) FY staining in WT seedlings grown 4 days and
transferred to 0.53 MS plates containing 1, 2, or
5 mM ACC for 20 hr. Pictures taken at different root
developmental stages (upper panels: late suberi-
zation; middle panels: early suberization), scale
bars: 50 mm, and quantification of suberization
along the root are presented (bottom panel). n R 8;
error bars: SD.
(B) TEM showing cell wall (CW) and suberin
lamellae (SL) in endodermis (en) of roots of WT
seedlings either untreated or treated with 1 mM
ACC for 24 hr prior to observation. Pictures are
shown (pe, pericycle; scale bars: 200 nm), and
quantification of the number of suberized endo-
dermal cells per root section presented as box
plot; n R 15; *** represent statistically significant
difference (p value < 10E 4). Note that the data for
the untreated condition are the same as the ones
presented in Figure S3D.
(C) FY staining for suberin in mutants affected in
ethylene signaling. Pictures taken in similar parts of
the root are shown (upper panels), and quantifi-
cation of suberization along the root is presented
(bottom panel). The scale bars represent 50 mm;
n = 10; error bars: SD.
(D) FY staining of ctr1 mutant either untreated or
treated with 1 mM ABA for 20 hr prior to observa-
tion. Pictures taken in similar parts of the root
are shown (upper panels), and quantifications of
suberization along the root are presented (bottom
panel). The scale bars represent 50 mm; n R 7;
error bars: SD. *** represents statistically signifi-
cant difference (p value < 10E 5).
(A and C) Different letters indicate significant
differences between genotypes and growth con-
ditions. See also Figure S4.
monomer content when compared to

the non-treated plants; Figure S4F). Our
observations indicate that ACC de-
creases suberin biosynthesis to a certain
extent but must also trigger active degra-
dation of pre-existing suberin lamellae.
Ethylene-signaling mutants corroborated
our results. We found that both etr1 and
ein3 (ethylene-resistant 1 and ethylene-
insensitive 3) mutants, defective in the
ethylene-signaling pathway, display a
slightly enhanced suberization that is not
treatment (Figure S4D). The effect of ACC was specific to suberin affected by ACC application (Figure 4C). Importantly, the ctr1
lamellae, because CS autofluorescence appears unaltered in the (constitutive triple response 1) mutant, exhibiting constitutive
endodermis of ACC-treated roots (Figure S4E). Chemical quan- ethylene responses, displayed a major delay or near absence
tification of suberin upon ACC treatment revealed a strong of suberization, comparable to the effect of ACC applications
decrease in root suberization (40% decrease of total suberin (Figure 4C). Interestingly, however, ABA treatment could partially
(E) FY staining in ELTP::abi1-1 lines either untreated (Unt) or treated with 1 mM ABA for 20 hr prior to observations. Pictures taken in similar parts of the root are
shown (left panel), scale bars: 50 mm, and quantification of suberization along the root presented (right panel); n = 10. Error bars represent SD; different letters
indicate significant differences between genotypes and growth conditions (p < 0.05).
See also Figure S3.
restore suberization in the ctr1 mutant (Figure 4D). Taken 1.5-fold reduction (Figures 6A and S6). This ionomic profile dis-
together, these observations support antagonism of ethylene- plays similarities with the analysis performed on the sgn3 mutant,
and ABA-signaling pathways in regulating suberization. where—despite a dramatic apoplastic bypass due to interrupted
CS—plants managed to maintain WT levels of most elements
ABA and Ethylene Signaling Mediate Nutrient- (Pfister et al., 2014). This reinforces the idea that plants have
Deficiency-Induced Plasticity of Endodermal homeostatic backup systems that are able to incompletely
Suberization compensate for defects in endodermal diffusion barriers.
Having identified ABA and ethylene as major regulators of endo- Despite the comparatively mild ionomic defects, rosette size
dermal suberization, we asked whether the nutrient-induced and weight as well as seed production were reduced in the
changes in suberization are mediated by these two antagonistic suberin-deficient genotypes (Figures 6B, S5B, and S5C). Yet,
hormones. We found that, in etr1 and ein3 mutants, suberization overall plant development and reproduction was not severely
is not affected in Fe, Mn, or Zn deficiency (Figure 5A). The reduc- impaired. Suberin-deficient plants develop chlorosis of the leaf
tion in suberin accumulation under metal-deficient conditions is margins in older leaves (Figure 6C), a characteristic K-deficiency
therefore largely dependent on an intact ethylene-signaling symptom (Marschner, 1995; Pfister et al., 2014). Together with
pathway. In addition, decrease of suberization in Fe deficiency our initial findings that a K channel mutant and K deficiency in-
can be blocked by the ethylene perception inhibitor AgNO3, as crease suberization, this suggests that enhanced suberization
well as the ethylene biosynthesis inhibitor AVG (Figure S4G). In is an adaptive response to K deficiency, assisting the plant in
order to ascertain that Fe conditions induce a reduction of maintaining K homeostasis under limiting conditions. Having
pre-existing suberin, we undertook transfer experiments and found that suberin-deficient genotypes also consistently dis-
found a decrease of suberization 48 hr after transfer in both the played increased Na accumulation (Figures 6A and S6) and
newly formed and older root parts that had pre-existing suberin. that salt treatment leads to increases in suberization, we tested
This was associated with a moderate reduction of GPAT5 the response of our suberin-deficient genotypes to salt stress.
expression, comparable to ACC treatments (Figures S2F and As would be predicted for a protective role of suberin in salt
S2G). As expected, suberin degradation upon transfer was stress, suberin-deficient genotypes grown on media containing
blocked in both etr1 and ein3 mutants. These observations sup- 100 mM NaCl showed increased reduction of root length and
port a model whereby ethylene mediates the nutrient-induced reduced seed production when compared to WT (Figures 6D,
reduction of suberization (Figure S4H). Moreover, we found S5B, and S5C). We wanted to extend this model to other nutri-
that suberization in S or K deficiency is not increased in ents and substantiate the idea that the striking increases and
ELTP::abi1-1 lines (Figure 5B), whereas it still occurs in etr1 decreases of suberization represent a physiologically relevant
and ein3 mutants (Figures S4I and S4J), showing that this response. We therefore combined our suberin-deficient line
nutrient effect on suberization is mediated by endodermal ABA with transporter mutants, arguing that nutrient transporter
signaling but is largely ethylene independent. Increased suberi- mutants leading to an increase of suberization should show
zation upon salt treatment was also shown to entirely depend an enhanced phenotypic severity in a suberin-deficient back-
on endodermal ABA signaling (Figure 5C). We conclude that ground. By contrast, mutants that cause a decrease of suber-
ABA and ethylene signaling mediate suberin plasticity in ization might be alleviated by a total absence of suberin. In
response to nutritional cues in opposite ways (Figure 5D). accordance with this, we found that sultr1;1 sultr1;2 mutants—
displaying increased suberization—showed increased pheno-
Suberization Is an Adaptive Response to Nutrient typic severity in the background of a suberin-deficient line,
Stresses consistent with a positive role of increased suberization under
We finally wanted to understand whether the plasticity of suber- S-deficient conditions (Figures 6E, S5D, and S5E). More tellingly,
ization in response to nutritional stress reflects a physiological however, we observed that irt1—the Fe-deficient mutant dis-
response by the plant that favorably modulates transport and playing decreases in suberin—actually benefits from a complete
homeostasis of limiting nutrients. To this purpose, we first stud- absence of suberin, especially in conditions of external Fe supply
ied the growth and ionomic phenotypes caused by an absence (Figures 6F, S5F, and S5G). This demonstrates that not only in-
of suberization in the suberin-deficient lines ELTP::CDEF1 and creases but also decreases in suberization are physiologically
CASP1::CDEF1. We ensured that these lines show an absence adaptive responses.
of suberin not only in 5-day-old seedlings on plates (Figures
S1B and S1C; Naseer et al., 2012) but also in the whole root sys- DISCUSSION
tem of adult plants grown 3 weeks in soil (Figure S5A). Elemental
profiling experiments using inductively coupled plasma-mass Multiple Inputs Positively and Negatively Regulate
spectrometry (ICP-MS) on rosette leaves in three independent Endodermal Suberization
laboratories with different growth conditions (short or long We have shown here that plant roots have a surprising capacity
days; soil or hydroponic) revealed that accumulation of most el- to adapt their level of suberization to a wide range of nutrient
ements remained surprisingly unaltered (Figures 6A and S6). stresses. We now show that not only excess of salt—or
Nevertheless, Li, Na, and As content was invariably found higher drought—but also deficiencies of required elements such as K,
in the suberin-deficient genotypes (ranging from 1.2- to 1.6-fold, Fe, or S can lead to changes in endodermal suberization. A num-
2.4- to 3.3-fold, and 1.3- to 2-fold increase, respectively) and K ber of earlier studies had established a connection between ABA
content was found invariably decreased, ranging from a 1.2- to and enhanced suberization in various organisms. Here, we now
Figure 5. Effects of Nutrients on Suberization Are Mediated through ABA and Ethylene Signaling
(A–C) FY staining for suberin in mutants affected in ethylene signaling germinated in +Mx (+Fe, +Mn, and +Zn), Fe, Mn, or Zn (A) and in ELTP:abi1-1
germinated in + or S and + or K (B) or with 0 or 500 mM NaCl (Unt, untreated; C). (A–C) Pictures taken from the same part of the root and quantifications of
suberization along the root are presented. The scale bars represent 50 mm; error bars: SD (n R 8, n R 8, and n R 5, respectively); different letters indicate
significant differences between growth conditions for a given genotype (A) or between genotypes and growth conditions (B and C).
(D) Model for the signaling controlling suberization in which ABA and ethylene are mediating the plasticity of suberization in response to nutritional cues in
opposite ways.
See also Figure S4.
Figure 6. Suberization Affects Plant Development and Homeostasis
(A) Overview of ionomic analysis performed in ELTP::CDEF1 and CASP1::CDEF1 leaves in three independent laboratories (Hokkaido, Lausanne, and Aberdeen)
using three growth systems—plates (Aberdeen Exp2), hydroponics (Hokkaido Exp1, 2, and 3), or soil (Lausanne Exp1 and 2 and Aberdeen Exp1)—and two day
length conditions—short days (Aberdeen Exp1 and Hokkaido Exp1, 2, and 3) or long days (Lausanne Exp1 and 2, Aberdeen Exp2). Elements were determined by
ICP-MS. Color code indicates significant changes of accumulation in ELTP::CDEF1 or CASP1::CDEF1 leaves compared to WT leaves after multiple or binary
comparisons, respectively (p < 0.01). For the numerical values, see Figure S6 source data. n.d., not determined.
(B and C) Phenotypes of 4-week-old ELTP::CDEF1 lines grown in soil. Different letters indicate significant change between genotypes. (B) Pictures (upper panels)
and fresh weight (bottom panel) are presented. Data are represented as mean ± SD; n = 12. (C) Occurrence of the K-deficiency-like phenotype determined as the
percentage of plants displaying at least one yellow leaf (as illustrated in the picture on the left side) versus percentage of plants displaying only green leaves. Bars
correspond to SD; data correspond to the mean of two independent experiments with a total n R 41.
(D) Response of ELTP::CDEF1 lines to salt treatments. Plants were germinated and grown in 0.53 MS plates containing 100 mM NaCl for 13 days. Data represent
primary root length; error bars: SD; different letters indicate significant differences between genotypes (p < 0.05).
(E) Suberin degradation exacerbates the sultr1;1 sultr1;2 phenotype. WT, sultr1;1 sultr1;2 mutants and four independents sultr1;1 sultr1;2/ELTP::CDEF1 lines
were grown in soil for 4 weeks. Arrows indicate yellow-brown leaves observed in sultr1;1 sultr1;2/ELTP::CDEF1 lines.
(F) Suberin degradation partially rescues irt1 phenotype of Fe metal deficiency. WT, irt1, and four independent irt1/ELTP::CDEF1 lines were grown 4 weeks in soil
watered with 0, 0.5, or 1 mM Fe-EDDHA.
See also Figures S5 and S6.
demonstrate that response to ABA is not only required for stress- cells. Yet, membrane surfaces of epidermis, cortex, and endo-
induced suberization but also for its establishment under non- dermis can only be considered as equivalent if one assumes
stress conditions. This response to ABA is rapid and requires that cytoplasmic mobility of nutrients is high and diffusion
local ABA perception in endodermal cells. The antagonistic ac- through plasmodesmata is unrestricted, both of which might
tion of ethylene was unexpected. It is especially striking that its not be the case for many nutrients. In cases where mobility in
application can lead to disappearance of preformed suberin the apoplast is high, nutrients could—through diffusion, mass
lamellae, which demonstrates an entirely unexpected flexibility flow, or ‘‘solvent drag’’—move toward the endodermis, leading
in the regulation of this secondary cell wall. Interestingly, a broad to higher local concentrations at the endodermal uptake surface.
array of enzymes that can potentially degrade suberin and/or In such cases, regulating endodermal suberization could have a
cutin are present in the Arabidopsis genome, making it easily comparatively strong impact on uptake rates. Finally, the impor-
conceivable that ethylene causes disappearance of suberin tance of endodermal suberization is also evident in the case of a
lamellae by upregulation of these genes (Yeats et al., 2014). coupled trans-cellular transport, as it would interrupt a ‘‘bucket
Indeed, we have demonstrated that simple endodermal overex- brigade’’ at its last crucial step. Our data strongly support the
pression of one of these family members, CDEF1, can abrogate idea that regulating suberization specifically affects two trans-
suberin accumulation in the endodermis (Naseer et al., 2012). A port pathways, the apoplastic and the coupled trans-cellular
reduction or disappearance of suberin in already suberized pathway, whereas it leaves the symplastic pathway unaltered.
endodermal cells can be seen as a reversal of endodermal differ-
entiation in which a protective epithelium might regain the possi- Does Suberization Quantitatively Affect Nutrient
bility to directly absorb nutrients again. It will be very important to Uptake?
establish through transcriptomic and molecular marker analysis The hydrophobicity of suberin, as evidenced in macroscopic,
whether reversal of endodermal suberization upon ethylene highly suberized structures like cork, often leads to the assump-
treatment is associated with a re-programming of those cells in tion that formation of suberin lamellae represents an effective,
order to regain capacity for nutrient absorption. The fact that a qualitative block for uptake of charged, possibly polar mole-
decrease of suberization under Fe deficiency improves plant cules. This assumption has been challenged in the case of water
growth certainly suggests that the newly non-suberized cells re- transport, where it was proposed that suberization causes a
gain some degree of activity. partial, rather quantitative resistance to water flow, which is
dependent on the degree of suberization (Schreiber, 2010).
A Framework for Interpreting the Consequences of Our findings of an extensive quantitative regulation of suberin
Endodermal Suberization in response to certain deficiencies would fit the idea of suberiza-
Our findings have several important implications for our under- tion introducing a resistance rather than a block. We find it plau-
standing of root function. It is well known that a specific nutrient sible that an often less than hundred-nanometer-thick suberin
deficiency, or knockout of a specific transporter, can have unex- lamellae around an endodermal cell might not provide a strictly
pected effects on the accumulation of unrelated elements. This qualitative block to the uptake of charged molecules. Better
is often explained in terms of cross-regulations of transporter ac- quantitative assays for probing suberin functionality will be
tivities, such as a change in membrane polarization or pH needed to address this question. A variant of the FDA assay
gradient that can disturb secondarily active transport processes, presented here could provide such a quantitative readout if
for example. Our findings now allow for an alternative, straight- bypass of FDA through plasmodesmata could be suppressed,
forward explanation of such pleiotropic effects. Changes in for example.
endodermal suberization might secondarily affect a number of
unrelated nutrients whose transport also depends on the endo-
Conclusions
dermal membrane surface. Previously, suberization of endo-
In summary, our findings reveal an unexpected plasticity of
dermal cells has often been confounded with CS formation, as
endodermal suberization in response to nutrient deficiency and
these were often assumed to be suberized structures (Rana-
stress hormone levels. The ability of roots to not only increase
thunge et al., 2011). This led to the assumption that suberization
but also to strongly decrease preformed suberin is bound to be
somehow affects both the paracellular barrier properties of the
of central importance for the adaptation of roots to different
endodermis as well as the direct uptake of nutrient into the endo-
soil environments and should be considered in studies of root
dermal cytoplasm. This work, together with previous works, now
developmental plasticity that often focus on the more easily
clearly delineates the function of CS from that of suberin lamellae
observable trait of root system architecture. It would be of great
(Hosmani et al., 2013; Kamiya et al., 2015; Naseer et al., 2012;
interest to study the degree to which variation of endodermal su-
Pfister et al., 2014). In current models, CS are involved in block-
berization exists within and across species and to investigate
ing transport in between endodermal cells, whereas suberin
whether it has been a target for natural selection.
lamellae exclusively suppresses uptake into the cytoplasm of
the endodermis. We have shown that suberization is largely
restricted to the endodermis, with some extension into the cor- EXPERIMENTAL PROCEDURES
tex under stress conditions. Regulating specifically uptake

Plant Material and Constructions
across the endodermal membrane only interferes with a minor Arabidopsis thaliana ecotype Columbia was used for most experiments. Gene
percentage of potential uptake surface because of the large numbers, mutants, and transgenic lines used and generated in this study are
area provided by the cortex and the root hair-bearing epidermal described in Supplemental Experimental Procedures.
Growth Conditions Andersen, T.G., Barberon, M., and Geldner, N. (2015). Suberization-the sec-
For most in vitro assays, plants were germinated on 0.53 MS (Murashige and ond life of an endodermal cell. Curr. Opin. Plant Biol. 28, 9–15.
Skoog) agar plates. Seeds were surface sterilized, sown on plates, incubated 2 Barberon, M., and Geldner, N. (2014). Radial transport of nutrients: the plant
to 3 days at 4 C, and grown vertically in growth chambers at 22 C, under root as a polarized epithelium. Plant Physiol. 166, 528–537.
continuous light (100 mE). All the microscopic analysis (FDA uptake, FY stain-
Barberon, M., Berthomieu, P., Clairotte, M., Shibagaki, N., Davidian, J.C., and
ing, PI uptake, CS autofluorescence, promoter analysis, and ultrastructure
Gosti, F. (2008). Unequal functional redundancy between the two Arabidopsis
analysis) were performed on 5-day-old seedlings. When seedlings were sub-
thaliana high-affinity sulphate transporters SULTR1;1 and SULTR1;2. New
jected to short-term treatment, e.g., transfer to ABA or ACC media, the transfer
Phytol. 180, 608–619.
was done in a way that seedlings were 5 days old at the point of analysis. De-
tails of media and growth conditions used in this study are described in Sup- Baxter, I., Hosmani, P.S., Rus, A., Lahner, B., Borevitz, J.O., Muthukumar, B.,
plemental Experimental Procedures. Mickelbart, M.V., Schreiber, L., Franke, R.B., and Salt, D.E. (2009). Root su-
berin forms an extracellular barrier that affects water relations and mineral
nutrition in Arabidopsis. PLoS Genet. 5, e1000492.
Fluorescence Microscopy
Confocal laser-scanning microscopy experiments were performed either on a Beisson, F., Li, Y., Bonaventure, G., Pollard, M., and Ohlrogge, J.B. (2007). The
Zeiss LSM 700, a Zeiss LSM 710, or a Zeiss LSM 710 NLO two-photon micro- acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and
scope. Excitation and detection parameters are presented in Supplemental root of Arabidopsis. Plant Cell 19, 351–368.
Experimental Procedures. Methods for visualizing CS autofluorescence, PI Boher, P., Serra, O., Soler, M., Molinas, M., and Figueras, M. (2013). The po-
penetration, and FY staining for suberin were previously described (Alassi- tato suberin feruloyl transferase FHT which accumulates in the phellogen is
mone et al., 2010; Naseer et al., 2012; Pfister et al., 2014) and are presented induced by wounding and regulated by abscisic and salicylic acids. J. Exp.
in detail in Supplemental Experimental Procedures. For visualization of FDA Bot. 64, 3225–3236.
transport, seedlings were incubated for 1 min in 0.53 MS FDA (5 mg.ml 1), Botha, C.E.J., Aoki, N., Scofield, G.N., Liu, L., Furbank, R.T., and White, R.G.
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Electron Microscopy Cailliatte, R., Schikora, A., Briat, J.F., Mari, S., and Curie, C. (2010). High-affin-
Micrographs were taken from ultrathin sections of 50 nm thick cut transversally ity manganese uptake by the metal transporter NRAMP1 is essential for Arabi-
at 2 mm from the hypocotyl-root junction. Details are presented in Supple- dopsis growth in low manganese conditions. Plant Cell 22, 904–917.
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Clarkson, D.T. (1993). Roots and the delivery of solutes to the xylem. Philos.
Trans. R. Soc. Lond. B Biol. Sci. 341, 5–17.
ICP-MS
Cottle, W., and Kolattukudy, P.E. (1982). Abscisic acid stimulation of suberiza-
Ionomic analyses were performed by ICP-MS from leave samples as previ-
tion : induction of enzymes and deposition of polymeric components and
ously described (Pfister et al., 2014). Details are presented in Supplemental
associated waxes in tissue cultures of potato tuber. Plant Physiol. 70,
Experimental Procedures.
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Duan, L., Dietrich, D., Ng, C.H., Chan, P.M.Y., Bhalerao, R., Bennett, M.J., and
SUPPLEMENTAL INFORMATION Dinneny, J.R. (2013). Endodermal ABA signaling promotes lateral root quies-
cence during salt stress in Arabidopsis seedlings. Plant Cell 25, 324–341.
Supplemental Information includes Supplemental Experimental Procedures,
Garcı́a, M.J., Lucena, C., Romera, F.J., Alcántara, E., and Pérez-Vicente, R.
six figures, and three movies and can be found with this article online at
(2010). Ethylene and nitric oxide involvement in the up-regulation of key genes
http://dx.doi.org/10.1016/j.cell.2015.12.021.
related to iron acquisition and homeostasis in Arabidopsis. J. Exp. Bot. 61,
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ACKNOWLEDGMENTS
Gaymard, F., Pilot, G., Lacombe, B., Bouchez, D., Bruneau, D., Boucherez, J.,
Michaux-Ferrière, N., Thibaud, J.-B., and Sentenac, H. (1998). Identification
We thank the Central Imaging Facility for expert technical support; John
and disruption of a plant shaker-like outward channel involved in K+ release
Danku, Kayo Konishi, and Tomoko Shimizu for ICP-MS analysis; and Lothar
into the xylem sap. Cell 94, 647–655.
Kalmbach and Sarra Ammar for technical assistance. Jean Daraspe of the
Electron Microscopy Facility of the University of Lausanne is thanked for tech- Geldner, N. (2013). The endodermis. Annu. Rev. Plant Biol. 64, 531–558.
nical expertise and advice. Lothar Kalmbach and Satoshi Fujita are thanked for Haas, D.L., and Carothers, Z.B. (1975). Some ultrastructural observations on
critical reading of the manuscript. We thank Rochus Franke for helpful discus- endodermal cell development in Zea mays roots. Am. J. Bot. 62, 336–348.
sions and Luis Lopez-Molina, Grégory Vert, Françoise Gosti, Catherine Curie, Henriques, R., Jásik, J., Klein, M., Martinoia, E., Feller, U., Schell, J., Pais,
Malcolm Bennett, and Isabelle Chérel for sharing published materials. This M.S., and Koncz, C. (2002). Knock-out of Arabidopsis metal transporter
work was funded by grants from the Swiss National Science Foundation gene IRT1 results in iron deficiency accompanied by cell differentiation de-
(SNSF) and the European Research Council (ERC) to N.G., a Human Frontiers fects. Plant Mol. Biol. 50, 587–597.
Science Program (HFSP) grant to J.T. and N.G., and the UK Biotechnology and
Höfer, R., Briesen, I., Beck, M., Pinot, F., Schreiber, L., and Franke, R. (2008).
Biological Sciences Research Council Grant (BB/L027739/1) to D.E.S. M.B.
The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid omega-hy-
was supported by a EMBO long-term postdoctoral fellowship and J.E.M.V.
droxylase involved in suberin monomer biosynthesis. J. Exp. Bot. 59, 2347–
and T.G.A. by Marie-Curie intra-European fellowships.
2360.
Hosmani, P.S., Kamiya, T., Danku, J., Naseer, S., Geldner, N., Guerinot, M.L.,
Received: August 2, 2015
and Salt, D.E. (2013). Dirigent domain-containing protein is part of the machin-
Revised: October 16, 2015
Accepted: November 26, 2015 ery required for formation of the lignin-based Casparian strip in the root. Proc.
Published: January 14, 2016 Natl. Acad. Sci. USA 110, 14498–14503.
Hruz, T., Laule, O., Szabo, G., Wessendorp, F., Bleuler, S., Oertle, L., Wid-
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Supplemental Figures
(legend on next page)
Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc. S1

Figure S1. Suberin Interferes with Uptake from the Apoplast, Related to Figure 1
(A) Time series of FDA uptake in WT seedlings along root cell layers in the suberized developmental zone. Incubation of FDA for 1 min reveals that FDA uptake is
blocked in suberized endodermis as shown in Figures 1D, 1E, and S1D. Longer incubation shows that FDA can still be transported in the suberized endodermis
after 8 to 10 min (less than 1 min in non suberized endodermis). Scale bars: 25 mm.
(B) FY staining in WT and ELTP::CDEF1 roots. Pictures from similar areas of the root are presented. Scale bars: 25 mm.
(C) Suberin deposition was quantified, after FY staining along the root, using three different zones (as described in Figure 1C): non-suberized zone (white), zone of
patchy suberization (gray) and zone of continuous suberization (yellow), n = 20, error bars: standard deviation. Different letters indicate significant differences
between genotypes (p < 0.05). Note that no quantification was performed for ELTP::CDEF1 line which displays no suberization.
(D) FDA penetration after 1 min in WT, ELTP::CDEF1 line and in casp1 casp3, esb1 and sgn3 mutants roots. Pictures are representative of FDA penetration in the
same root positions for the different genotypes, corresponding to suberized, non-suberized and undifferentiated in WT. Scale bars: 25 mm.
(E) Developmental series of CASP1::NLS3xmVENUS and ELTP::NLS3xmVENUS expressions (in Green) in suberized (left panels), non-suberized (middle panels)
and undifferentiated zones (right panels) from 5-day-old seedlings. *for the undifferentiated zone pictures were taken 3 to 5 cells prior to CS differentiation.
(F) CASP1::NLS3xmVENUS and ELTP::NLS3xmVENUS expression (in Green) in seedlings treated 20h with 1 mM ABA prior to observation. Pictures were taken in
the suberized zone. Arrows indicate ELTP::NLS3xmVENUS expression in cortical cells.
(A and D) ep: epidermis, co: cortex, en: endodermis. (E and F) Root cell layers are highlighted by PI (red). Scale bars: 50 mm.
S2 Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc.

Figure S2. Nutrient Status Affects Suberization, Related to Figure 2
(A) WT, irt1, nramp1, skor and sultr1;1 sultr1;2 mutants 5 days after germination in 0.5x MS.
(B) WT seedlings 5 days after germination in 0.5x MS (+Mx) or in 0.5X MS deficient in Fe (-Fe, 10 mM Fe-EDTA), Mn (-Mn, 0 mM) and Zn (-Zn, 0 mM).
(C) WT seedlings 5 days after germination in 0.5x MS based media at different KCl concentrations (0, 25 or 500 mM). -K and + K refer to 0 and 500 mM KCl
respectively.
(D) WT plants 5 days after germination in 0.5x Hoagland based media at different K2SO4 concentrations (0, 25, 250 or 500 mM). -S and +S refer to 0 and 500 mM
K2SO4 respectively.
(E) WT plants 5 days after germination in 0.5x MS with or without 500 mM NaCl.
(A–E) Pictures are presented (upper panels) and corresponding root lengths were determined (bottom panels). Data are represented as mean ± standard de-
viation, n R 15. ANOVA analysis did not indicate significant differences (p < 0.05) for any of the growth conditions.
(F and G) Seedlings were grown 3 days in 0.5x MS containing 50 mM Fe-EDTA and transferred 2 days to 0.5x MS containing 0 or 50 mM Fe-EDTA (- or +Fe
respectively).
(F) FY staining for suberin after transfer to + or -Fe. Pictures taken at different root developmental stages (upper panels: late suberization, middle panels: early
suberization), scale bars: 50 mm, and quantification of suberization along the root are presented (bottom panel), n = 6, error bars: standard deviation, ** and
* represent significant differences between + and -Fe for the zone of continuous suberization and the non-suberized zones (p < 0.005 and p < 0.05 respectively).

(G) GPAT5::mCITRINE-SYP122 expression after transfer to + or -Fe. Pictures are presented as Z projections taken in the zone of late suberization, scale
bars: 50 mm.
(H) FY staining for suberin in WT seedlings grown 4 days in 0.5x MS and transferred 1 day in 0.5x MS containing 0, 100 or 200 mM NaCl. Pictures (upper panels),
scale bars, 50 mm and quantification of suberization along the root are presented (bottom panel), n R 5, error bars, standard deviation, different letters indicate
significant differences between conditions (p < 0.05).

Figure S3. ABA Signaling Controls Suberization, Related to Figure 3
(A) WT seedlings grown 4 days in 0.5x MS and transferred to 0.5x MS containing 0 or 1 mM ABA for 1 day. Pictures are shown (upper panel) and the corresponding
root length measured (bottom panel). Data are represented as mean ± standard deviation, n R 15. No significant differences (p < 0.05) were observed after t test.
(B) Time series of GPAT5::mCITRINE-SYP122 expression after ABA treatment. 4-day-old seedlings were either untreated or treated with 1 mM ABA. Pictures
correspond to Z projections extracted from Movies S2 and S3 at 0, 2, 3 and 13 hr after treatment. Images for ABA treatment are the same as those presented in
Figure 3A. Arrows indicate the onset of GPAT5 expression. Scale bars: 200 mm.
(C) FY staining of suberin in 5-day-old seedlings untreated or treated with 1 mM ABA for 20h prior to observation. Pictures taken in young part of the root are
presented as Z projections. Scale bars, 50 mm.
(D) Quantification of suberin lamellae ultrastructure upon ABA treatment: occurrence (left panel) and width (right panel). The occurrence of suberized cells in
endodermis and cortex were quantified in 15 independent root sections for each condition performed 2 mm below the hypocotyl-root junction. Data are rep-
resented as box plot, different letters indicate significant differences between genotypes (p < 0.05). The suberin lamellae width was determined in 15 independent
root sections for each condition performed 2 mm below the hypocotyl-root junction. For each section the suberin lamellae width in 3 independent endodermal

cells were done in both the outer (toward the cortex) and the inner domains (toward the stele). Data are represented as mean ± standard error (n R 45), * and
*** significant difference compared to untreated condition with t test (p < 0.05 and p < 10E-05 respectively).
(E) Casparian strip autofluorescence after clearing in the older part of 5-day-old roots treated or not with 1 mM ABA for 20h prior to observation. Scale bars: 20 mm.
(F) Polyester composition in 5-day-old roots either untreated or treated with 1 mM ABA for 20h prior to measurement. Individual suberin monomer composition (left
panel) and the corresponding total amount of suberin monomers (right panel) are presented. Data are represented as mean ± standard deviation (n R 3 pools of
200-300 roots). FA, fatty acid; DCA, dicarboxylic fatty acid; uOH, u-hydroxy fatty acid. * and ** represent significant difference compared to the untreated
condition with t test (*p < 0.05; **p < 10E-05).
(G) FY staining for suberin in 5-day-old mutants affected in ABA biosynthesis and signaling.
(H) FY staining for suberin in 5-day-old CASP1::abi1-1 lines impaired in endodermal ABA signaling, either untreated (Unt) or treated with 1 mM ABA for 20h.
(G and H) Pictures taken in similar parts of the root are shown (left panels), scale bars: 50 mm and quantification of suberization along the root are presented (right
panels) n R 10. Error bars: standard deviation, different letters indicate significant differences between conditions (p < 0.05).

A B WT 4-day-old D GPAT5::mCITRINE-SYP122 E
untreated
untreated ACC 1 μM untreated
CS autofluorescence
Late suberization
Fluorol Yellow
ACC 1 μM
ACC 2 μM
untreated ACC 1 μM ACC 1 μM

C untreated
CS autofluorescence
Suberin lamellae width (nm) 70 ACC
ACC 5 μM
Early suberization
60 **
* *
50 **
**
40 *
Root length (cm)
a 30
1
b ab
0.8 b
20
0.6
0.4 10
0.2
0 Relative intensity Relative intensity
0
0 1 2 5 inner outer overall 0 250 0 250
ACC (μM)
F H WT etr1 ein3
200 800 untreated ACC
Total ion count
(AU/mg DW)
Ion count (AU/mg DW)
600
Transfer 48h +Fe

150 400 **
** *
200
100
0
Unt ACC
*
*
*
** ** *
50
*
* * * * * ** * **
0 *
16:0 18:2 18:1 20:0 22:0 24:0 16:0 18:2 18:1 18:0 16:0 18:2 18:1
Transfer 48h -Fe
FA DCA ωOH
G AgNO3 AVG
2 μM 5 μM 10 μM 2 μM 5 μM 10 μM
+Fe -Fe -Fe -Fe -Fe -Fe -Fe -Fe
Fluorol Yellow
% of endodermal cells
120
100 b
80 a ac a c c
120
60
100
bc b c bc 40
80 a a ac
d 20 ab’ c’
60 ad’ d’ b’ b’
0
40 +Fe -Fe +Fe -Fe +Fe -Fe
b’ b’c’d’
20 a’ b’c’ b’c’d’ a’c’ a’c’
0
a’d’ WT etr1 ein3
+Fe -Fe -Fe -Fe -Fe -Fe -Fe -Fe Zone of continuous suberization
2 μM 5 μM 10 μM 2 μM 5 μM 10 μM
Zone of patchy suberization
AgNO3 AVG
Non-suberized zone
Zone of continuous suberization Zone of patchy suberization Non-suberized zone
I J
120 120
100 100
80 a ab 80 a c
bc c bc b bd bc
c d
60 60
40 40
20 a’ a’ bc’ b’ a’ ac’ 20 a’ b’ bc’ c’ a’ bc’

0 0
+K -K +K -K +K -K +S -S +S -S +S -S
WT etr1 ein3 WT etr1 ein3
Zone of continuous suberization Non-suberized zone Zone of continuous suberization Non-suberized zone
Zone of patchy suberization Zone of patchy suberization
Figure S4. Ethylene Signaling Controls Suberization, Related to Figures 4 and 5

(A) WT seedlings were grown 4 days in 0.5x MS and transferred 1 day to 0.5x MS containing 1 to 5 mM ACC. Pictures are shown (upper panels) and the cor-
responding root lengths were measured (bottom panel). Data are represented as mean ± standard deviation, n R 10. Different letters indicate significant dif-
ferences between genotypes (p < 0.01).
(B) FY staining for suberin in 4-day-old WT seedlings (i.e. before ACC treatments). Picture corresponds to the older part of the root. Scale bar: 50 mm.
(C) Quantifications of suberin lamellae width after 1 day of ACC treatment determined in 15 independent root sections for each condition performed 2 mm below
the hypocotyl-root junction. Width was measured only in endodermal cells with remaining suberin after ACC treatment. For each section the suberin lamellae
width in 3 independent endodermal cells were measured in both the outer (toward the cortex) and the inner domain (toward the stele). Data are represented as
mean ± standard error (n R 45), *** significant difference compared to the untreated condition (p < 10E-07). Note that the data presented for the untreated
condition are the same as the ones presented in Figure S3D.

(D) GPAT5::mCITRINE-SYP122 expression in plants grown 4 days and untreated or treated with 1 mM ACC for 20 hr. Z projections at the onset of GPAT5
expression (bottom panels) and in the older root part (upper panels) are shown. Scale bars: 100 mm.
(E) Casparian strip autofluorescence after clearing in the older part of roots untreated or treated with 1 mM ABA for 20h prior to observation. Scale bars: 20 mm.
(F) Polyester composition in 5-day-old roots under control conditions or treated with 1 mM ACC for 44h prior to measurement. Individual suberin monomer
composition and the corresponding total amount of suberin monomers (inset) are presented. Data are represented as mean ± standard deviation (n R 3 pools of
200-300 roots). FA, fatty acid; DCA, dicarboxylic fatty acid; uOH, u-hydroxy fatty acid. * and ** represent significant difference compared to the CT condition (*p <
0.05; **p < 10E-05). Note that the data presented for the CT condition are the same as the ones presented in Figure S3F.
(G) FY staining for suberin in 5-day old seedlings germinated in 0.5x MS containing 10 or 50 mM Fe-EDTA (- or +Fe) and 2 to 10 mM AgNO3 or 2 to 10 mM AVG.
Pictures are presented (upper panels) and quantification of suberization along the root are presented (bottom panels) n R 5.
(H) FY staining for suberin in 5-day-old WT, etr1 and ein3 seedlings grown 3 days in 0.5x MS containing 50 mM Fe-EDTA and transferred for 2 days in 0.5x MS
containing 0 or 50 mM Fe-EDTA (- or + Fe). Pictures (upper panels: transfer to +Fe, middle panels: transfer to -Fe) and quantification of suberization along the root
are presented (bottom panels) n R 5.
(I and J) Quantification of suberization along the root in 5-day-old WT, etr1 and ein3 seedlings grown in I, 0.5x MS containing 0 or 500 mM KCl (- or + K) or in J, 0.5x
Hoagland containing 0 or 500 mM K2SO4 (- or +S).
(G and H) Scale bars, 50 mm. (G–I) Error bars, standard deviation, different letters indicate significant differences between conditions (p < 0.05).

A WT ELTP::CDEF1 WT ELTP::CDEF1
4000
Relative intensity
0
BrightField BrightField Fluorol Yellow Fluorol Yellow
B WT CASP1::CDEF1 C
untreated NaCl 100 mM untreated NaCl 100 mM
WT CASP1::CDEF1
100 200 300 400 500 600 700

Number of siliques per plant
*
*
*
*
NaCl : 0 100 0 100 mM

500 WT ELTP::CDEF1#37
D a ELTP::CDEF1#21 ELTP::CDEF1#6
E
400 ELTP::CDEF1#3 sultr1;1 sultr1;2
Weight (mg)
sultr1;1 sultr1;2 / ELTPCDEF1#1

300
100 a
sultr1;1 sultr1;2 / ELTPCDEF1#13
Sensitivity to S Deficiency
bc bc
bc sultr1;1 sultr1;2 / ELTPCDEF1#16
200 b
sultr1;1 sultr1;2 / ELTPCDEF1#2 80
100 d d d d d
60 b
0
Yellow Green 40 c
100 c
80
% of plants
20
60
40 0
20 WT
0 sultr1;1-sultr1;2
WT 1#21 F1#3 1#37 F1#6 ltr1;2 F1#1 1#13 1#16 F1#2 sultr1;1-sultr1;2/ELTP::CDEF1#1
EF DE EF DE -su DE EF EF DE
: : CD P::C ::CD P::C ltr1;1 P::C ::CD ::CD P::C sultr1;1-sultr1;2/ELTP::CDEF1#13
LTP ELT LTP ELT su / E LT LTP LTP /ELT
E E
1 ; 2 ; 2 /E ; 2 /E 1;2
ltr r1 r1 ltr
su ult -sult 1-su
;1- ;1-s ;
ltr1 1 1;1 ultr1
su sultr sultr s
F G
50
Sensitivity to Fe Deficiency
WT ELTP::CDEF1#37 a
500 ELTP::CDEF1#21 ELTP::CDEF1#6
irt1 40 c
a irt1 / ELTP::CDEF1#2 c c
400 a irt1/ELTP::CDEF1#8 c
Fresh weight (mg)
30
a
300 bc c
b b 20 b
b b b
b b b
200
bc 10
d c
100 d
e de
d c WT 0 irt1/ELTP::CDEF1#4
0 irt1 irt1/ELTP::CDEF1#5
0 0.5 1.5
Fe (μM) irt1/ELTP::CDEF1#1 irt1/ELTP::CDEF1#8
Figure S5. Suberization Affects Plant Development and Homeostasis, Related to Figure 6
(A) FY staining for suberin in 3-week-old WT and ELTP::CDEF1 plants grown in soil. The whole root system was observed with a fluorescent stereomicroscope.
Bright field (left panels) and fluorescence (right panels) are presented. Scale bars, 5 mm, arrows indicate the hypocotyl-root junction.
(B and C) Phenotype of CASP1::CDEF1 line upon salt stress. Prior to salt treatment (100 mM NaCl), plants were grown on plates 2.5 weeks and transferred to soil
for 10 more days.
(B) Pictures were taken after 3 weeks of salt treatment.
(C) Silique production was evaluated after 3 weeks of salt treatment by counting the number of siliques per plants. Data are represented as box plots, n R 10,
(* and *** represent significant difference compared to WT plants grown in the same condition, p < 0.05 and p < 1E-04 respectively).
(D) Phenotypic characterization of sultr1;1 sultr1;2/ELTP::CDEF1 lines grown 4 weeks in soil. Fresh weight of WT, 4 independent ELTP::CDEF1 lines, sultr1;1
sultr1;2 mutant and 4 independent sultr1;1 sultr1;2/ELTP::CDEF1 lines was determined (upper panel) as well as the percentage of plants displaying yellow leaves
(bottom panel). Note that the data for WT and ELTP::CDEF1 line are presented in Figures 6B and 6C.

(E) Sensitivity to sulfur deficiency was evaluated as the relative root length in sulfur deficient (–S) compared to the root length in sulfur sufficient (+S) of 9-day-old
plants.
(F) Fresh weight of WT, 3 independent ELTP::CDEF1 lines, irt1 mutant and 3 independent irt1/ELTP::CDEF1 lines grown 4 weeks in soil watered with 0, 0.5 or
1.5 mM Fe.
(G) Sensitivity to iron deficiency of irt1/ELTP::CDEF1 lines in vitro, evaluated as the relative root length in –Fe compared to the root length in +Fe of 9-day-old
plants.
(D–G) Data are represented as mean ± standard deviation (n = 12, n R 15, n R 9 and n R 10 respectively). Different letters indicate significant differences between
genotypes (p < 0.05).
Lausanne Exp1, soil, long days Lausanne Exp2, soil, long days
Element WT ELTP::CDEF1#21 ELTP::CDEF1#6 WT ELTP::CDEF1#21 ELTP::CDEF1#6
(ppm) Mean ±SD Mean ±SD pvalue Mean ±SD pvalue Mean ±SD Mean ±SD pvalue Mean ±SD pvalue
As 0.16 ± 0.00 0.77 ± 0.25 1.32E-02 0.78 ± 0.19 1.12E-02 0.11 ± 0.00 0.50 ± 0.08 2.10E-03 0.60 ± 0.13 4.85E-04
B 76.24 ± 2.42 95.93 ± 5.28 6.42E-03 89.83 ± 4.75 4.56E-02 61.02 ± 3.48 96.89 ± 28.75 1.36E-01 84.89 ± 15.95 4.23E-01
Ca 33373.86 ± 643.80 39936.13 ± 1634.57 7.34E-02 36844.55 ± 1938.60 4.55E-01 45971.62 ± 10290.96 41059.55 ± 1071.80 9.49E-01 41249.22 ± 1110.32 9.60E-01
Cd 0.49 ± 0.01 0.37 ± 0.05 3.56E-02 0.41 ± 0.05 2.19E-01 0.39 ± 0.05 0.37 ± 0.03 6.52E-01 0.34 ± 0.01 1.95E-01
Co 0.65 ± 0.03 1.32 ± 0.35 1.73E-02 1.11 ± 012 1.06E-01 0.72 ± 0.03 1.00 ± 0.06 3.12E-02 1.06 ± 0.15 1.02E-02
Cu 6.02 ± 0.22 6.79 ± 0.40 1.82E-01 6.93 ± 0.29 1.02E-01 5.06 ± 0.16 5.81 ± 0.07 1.74E-02 6.12 ± 0.29 1.90E-03
Fe 120.97 ± 6.12 143.86 ± 16.08 6.32E-02 121.91 ± 2.76 9.99E-01 115.86 ± 2.71 128.76 ± 9.12 1.53E-01 130.63 ± 3.93 8.90E-02
K 29853.84 ± 1897.13 17502.78 ± 747.57 1.60E-03 18371.82 ± 603.39 2.56E-03 32159.23 ± 1048.05 19270.02 ± 475.49 1.98E-04 19822.85 ± 882.64 2.71E-04
Li 3.76 ± 0.09 9.68 ± 1.17 1.28E-03 9.77 ± 1.21 1.16E-03 3.45 ± 0.28 7.48 ± 0.41 1.43E-03 8.15 ± 0.70 4.87E-04
Mg 8415.14 ± 168.91 11753.33 ± 113.33 1.54E-03 11185.05 ± 311.79 5.02E-03 8429.40 ± 70.07 11087.30 ± 328.53 1.09E-04 11085.95 ± 171.04 1.10E-04
Mn 169.05 ± 2.27 146.80 ± 15.73 9.77E-02 141.85 ± 4.33 4.16E-02 370.28 ± 32.07 108.72 ± 12.31 1.10E-06 116.28 ± 12.85 1.40E-06
Mo 11.83 ± 0.37 7.64 ± 0.37 8.35E-05 10.97 ± 0.99 3.19E-01 6.23 ± 0.38 5.67 ± 1.65 9.75E-01 6.85 ± 1.26 8.06E-01
Na 1209.20 ± 114.93 4672.91 ± 281.32 4.00E-04 4684.17 ± 814.20 3.91E-04 878.38 ± 47.09 3330.87 ± 365.09 7.61E-04 3668.55 ± 625.48 3.09E-04
Ni 1.35 ± 0.05 2.10 ± 0.22 3.28E-03 1.74 ± 0.05 8.97E-02 1.40 ± 0.04 1.72 ± 0.18 1.45E-02 1.83 ± 0.03 2.60E-03
P 8552.83 ± 219.38 8111.85 ± 50.66 2.84E-01 8954.85 ± 246.18 3.51E-01 6555.26 ± 418.09 11786.12 ± 910.09 2.83E-02 18238.92 ± 3062.61 1.57E-04
Rb 6.15 ± 0.09 3.75 ± 0.03 1.41E-04 4.24 ± 0.12 6.82E-04 6.36 ± 0.05 4.27 ± 0.20 2.36E-04 4.15 ± 0.15 1.58E-04
Se 5.12 ± 0.28 8.62 ± 0.59 5.67E-04 7.66 ± 0.59 4.66E-03 5.78 ± 0.59 8.02 ± 0.41 8.10E-04 8.45 ± 0.31 2.47E-04
SO 19375.23 ± 496.70 22089.28 ± 714.83 1.05E-01 21018.77 ± 1590.41 4.19E-01 17665.80 ± 296.87 20472.37 ± 418.15 4.06E-04 20379.80 ± 52.84 5.18E-04
Sr 87.78 ± 1.70 114.37 ± 3.91 3.35E-02 113.52 ± 8.18 3.91E-02 100.24 ± 4.56 118.49 ± 4.42 1.42E-02 112.61 ± 6.04 7.80E-02
Zn 88.18 ± 5.43 75.28 ± 3.66 1.29E-02 74.45 ± 2.00 9.05E-03 83.44 ± 2.49 73 28 ± 3.36 2.22E-02 72.26 ± 0.87 1.39E-02
Hokkaido Exp1, hydropony, short days
Aberdeen Exp1, soil, short days Element WT ELTP::CDEF1#8 ELTP::CDEF1#9

(μg.g -1 DW) Mean ±SD Mean ±SD pvalue Mean ±SD pvalue
Element WT CASP1::CDEF1#8
B 57.75 ± 1.80 73.92 ± 10.40 3.85E-02 83.11 ± 7.72 5.67E-03
(ppm) Mean ±SD Mean ±SD pvalue
Ca 32288.31 ± 2656.58 37866.92 ± 5061.47 1.53E-01 37197.57 ± 2802.29 2.61E-01
As 0.90 ± 0.48 1.78 ± 0.64 6.91E-05
Co 1.26 ± 0.21 1.10 ± 0.45 7.96E-01 1.26 ± 0.33 1.00E+00
B 74.21 ± 16.89 84.19 ± 24.12 1.67E-01
Cs 73.17 ± 11.62 98.16 ± 6.84 3.60E-02 104.20 ± 15.91 1.85E-02
Ca 42516.21 ± 4356.74 43615.50 ± 3846.30 4.70E-01
Cu 19.15 ± 3.02 20.22 ± 1.91 7.79E-01 24.18 ± 0.57 4.16E-02
Cd 0.51 ± 0.08 0.45 ± 0.06 7.19E-02
Fe 143.23 ± 30.42 157.19 ± 35.56 7.90E-01 149.66 ± 16.08 9.57E-01
Co 0.52 ± 0.04 0.51 ± 0.04 4.62E-01
K 43244.03 ± 4592.93 33899.02 ± 3523.68 4.04E-02 35103.41 ± 5213.48 9.48E-02
Cu 4.06 ± 0.58 4.15 ± 0.62 6.59E-01
Mg 7770.87 ± 137.74 11249.03 ± 433.60 4.00E-07 10393.93 ± 158.45 6.20E-06
Fe 124.83 ± 9.72 131.14 ± 17.13 1.78E-01
Mn 371.36 ± 52.02 363.15 ± 102.82 9.89E-01 413.20 ± 78.74 7.82E-01
K 26543.95 ± 5010.41 22179.06 ± 2176.09 7.42E-03
Mo 9.46 ± 3.79 10.63 ± 4.91 9.26E-01 13.37 ± 4.38 5.02E-01
Li 9.18 ± 1.43 11.04 ± 1.30 6.95E-04
Na 3919.58 ± 754.28 8774.82 ± 1151.21 8.44E-04 8022.72 ± 1556.31 3.97E-03
Mg 13416.91 ± 1667.30 15364.89 ± 1438.64 1.78E-03
P 6388.55 ± 616.55 6824.82 ± 2373.09 9.41E-01 6483.17 ± 2166.66 9.98E-01
Mn 53.81 ± 8.22 46.44 ± 3.22 5.65E-03
Sr 327.29 ± 23.55 341.78 ± 65.14 9.11E-01 366.36 ± 51.51 5.80E-01
Mo 7.21 ± 5.40 7.27 ± 3.58 9.71E-01
Zn 78.67 ± 13.99 65.14 ± 4.87 1.87E-01 75.50 ± 7.51 9.07E-01
Na 314.18 ± 53.18 739.16 ± 203.13 1.14E-10
Ni 1.19 ± 0.17 1.33 ± 0.12 1.64E-02 Hokkaido Exp2, hydropony, short days
P 6428.94 ± 493.26 6339.87 ± 309.88 5.76E-01
Element WT ELTP::CDEF1#8 ELTP::CDEF1#9
Rb 64.07 ± 10.95 55.55 ± 10.30 3.42E-02 (μg.g -1 DW) Mean ±SD Mean ±SD pvalue Mean ±SD pvalue
Se 133.29 ± 17.53 126.68 ± 12.72 2.59E-01 B 48.05 ± 2.20 63.40 ± 6.45 6.84E-04 64.55 ± 5.82 3.04E-04
SO 4787.76 ± 733.36 4898.35 ± 578.90 6.55E-01 Ca 43560.72 ± 876.06 44357.23 ± 3781.68 1.00E+00 36352.26 ± 10498.09 4.36E-01
Sr 11.30 ± 13.84 117.17 ± 11.56 2.21E-01 Co 1.29 ± 0.31 0.88 ± 0.23 8.33E-01 0.73 ± 0.33 5.84E-01
Zn 73.00 ± 4.66 70.93 ± 3.71 1.94E-01 Cu 8.81 ± 1.04 9.37 ± 0.95 1.00E+00 10.02 ± 2.85 1.00E+00
Aberdeen Exp2, plates, long days Fe 145.67 ± 92.17 104.30 ± 42.93 1.00E+00 525.21 ± 631.27 1.00E+00
K 38744.61 ± 998.95 30248.09 ± 2378.24 7.41E-01 30668.97 ± 4953.03 7.78E-01

Element WT CASP1::CDEF1#8
Mg 8908.99 ± 100.66 12411.47 ± 1304.86 2.27E-02 11810.14 ± 1463.66 7.70E-02
(ppm) Mean ±SD Mean ±SD pvalue
Mn 338.37 ± 8.15 308.20 ± 35.68 9.98E-01 267.77 ± 115.54 9.11E-01
As 0.41 ± 0.08 0.54 ± 0.05 3.95E-02
Mo 35.66 ± 2.46 26.78 ± 3.72 9.98E-01 25.78 ± 12.46 1.00E+00
B 344.72 ± 126.41 344.85 ± 34.59 9.98E-01
Na 2206 ± 605.81 5884.57± 1681.05 5.95E-04 7683.98 ± 1256.77 3.90E-06
Ca 15319.50 ± 2654.57 11833.70 ± 605.25 4.29E-02
P 8285.98 ± 678.06 8907.35 ± 452.90 9.95E-01 8011.22 ± 603.31 1.00E+00
Cd 1.17 ± 0.09 0.66 ± 0.15 1.02E-03
Zn 55.30 ± 4.00 45.76 ± 3.93 9.94E-01 43.30 ± 9.09 9.84E-01
Co 0.87 ± 0.17 0.69 ± 0.30 3.39E-01
Cu 12.21 ± 1.96 11.58 ± 1.76 6.47E-01 Hokkaido Exp3, hydropony, short days
Fe 49.57 ± 1.96 45.87 ± 2.46 5.71E-02 Element WT ELTP::CDEF1#8 ELTP::CDEF1#9
K 57847.88 ± 3828.34 39251.69 ± 2949.87 2.52E-04 (μg.g -1 DW) Mean ±SD Mean ±SD pvalue Mean ±SD pvalue
Li 0.16 ± 0.02 0.25 ± 0.01 1.36E-04 B 54.00 ± 4.46 78.46 ± 10.14 4.32E-02 60.32 ± 3.33 3.27E-01
Mg 6728.76 ± 1029.34 6783.60 ± 649.56 9.31E-01 Ca 17911.93 ± 1789.25 34511.49 ± 3610.25 2.84E-03 31101.58 ± 5275.64 4.07E-04
Mn 124.47 ± 13.32 63.32 ± 8.54 2.56E-04 Co 0.27 ± 0.05 0.43 ± 0.12 5.89E-01 0.34 ± 0.10 4.58E-01
Mo 1.33 ± 0.26 1.44 ± 0.30 5.81E-01 Cu 13.77 ± 2.00 25.08 ± 5.22 3.85E-02 17.31 ± 4.87 4.17E-01
Na 5142.13 ± 334.86 17022.15 ± 823.39 1.81E-07 Fe 175.48 ± 119.97 360.17 ± 253.07 8.97E-01 180.04 ± 117.48 9.99E-01
Ni 0.31 ± 0.04 0.39 ± 0.17 3.90E-01 K 40781.13 ± 4864.01 42342.40 ± 6857.75 3.14E-01 31013.00 ± 1492.69 2.19E-02
P 8193.43 ± 818.90 8234.09 ± 315.33 9.29E-01 Mg 8236.10 ± 545.83 12013.64 ± 1015.67 4.30E-02 10295.92 ± 1124.14 1.12E-02
Rb 8.17 ± 2.10 10.99 ± 1.17 5.75E-02 Mn 114.77 ± 22.99 204.52 ± 36.80 2.63E-01 189.10 ± 63.95 5.30E-02
Se 8395.48 ± 476.90 9104.79 ± 1163.73 3.02E-01 Mo 1.45 ± 0.43 5.48 ± 1.64 2.23E-02 5.60 ± 2.24 4.25E-03
SO 4.50 ± 0.61 4.92 ± 0.39 2.92E-01 Na 7564.50 ± 888.77 10221.94 ± 1134.97 4.49E-01 7941.22 ± 1193.08 8.48E-01
Sr 3.54 ± 0.60 3.93 ± 0.37 3.13E-01 P 6878.06 ± 1591.78 8454.47 ± 1263.98 9.99E-01 7366.78 ± 1026.55 8.29E-01
Zn 33.97 ± 5.19 183.30 ± 200.80 1.88E-01 Zn 48.09 ± 5.47 75.22 ± 8.88 8.02E-03 55.21 ± 6.56 2.90E-01
(legend on next page)
Figure S6. Ionomic Analysis, Related to Figure 6
Ionomic analysis performed in ELTP::CDEF1 and CASP1::CDEF1 lines in three independent laboratories (Hokkaido, Lausanne, Aberdeen), using 3 growth
systems – plates (Aberdeen Exp2), hydroponics (Hokkaido Exp1, 2 and 3) or soil (Lausanne Exp1 and 2 and Aberdeen Exp1) - and 2 day-length conditions: short
days (Aberdeen Exp1 and Hokkaido Exp1, 2 and 3) or long days (Lausanne Exp1 and 2, Aberdeen Exp2). Elements were determined by ICP-MS. Data are
represented as mean ± Standard deviation (SD). For Lausanne and Hokkaido experiments Analysis of variance (ANOVA) and Tukey’s test as post hoc analyses
were performed and for Aberdeen experiments t test were performed to determine the significant differences to WT (corresponding p values are presented). Note
that in the Hokkaido Exp1, data of ion content in WT leaves has been published before (Pfister et al., 2014).
Cell
Supplemental Information

Marie Barberon, Joop Engelbertus Martinus Vermeer, Damien De Bellis, Peng Wang,
Sadaf Naseer, Tonni Grube Andersen, Bruno Martin Humbel, Christiane Nawrath,
Junpei Takano, David Edward Salt, and Niko Geldner
SUPPLEMENTAL EXPERIMENT
Plant material and constructions

The following mutants and transgenic lines were used in this study: casp1-1 casp3-1 (Roppolo
et al., 2011); esb1-1 (Baxter et al., 2009); sgn3-3 (Pfister et al., 2014); irt1-2 (Henriques et al.,
2002); nramp1-1 (Cailliatte et al., 2010); skor-1 (Gaymard et al., 1998); sultr1;1 sultr1;2
(Barberon et al., 2008); aba2-1 (Leung et al., 1997); abi3-8 (Nambara et al., 2002); abi5-3
(Finkelstein and Lynch, 2000); abi4 (Finkelstein et al., 1998); etr1-1 (Chang et al., 1993),
ein3-1 (Chao et al., 1997), ctr1-1 (Kieber et al., 1993) and CASP1::CDEF1 (Naseer et al.,
2012).
Plasmids were constructed using Gateway technology (Lifesciences). To generate pEN4-
ELTPpro-1, 464bp upstream of ELTP was amplified and recombined into pDONRP4-P1. To
generate pEN-1-abi1-1-2, abi1-1 was amplified from pGreen229-UAS::abi1-1GR [Kindly
provided by Malcolm Bennett, Nottingham, UK]) and recombined into pDONR221.
ELTP::CDEF1, CASP1::abi1-1 and ELTP::abi1-1 were generated by recombining the
corresponding entry clones into pH7m24GW,3 (Plant Systems Biology, Ghent University,
Belgium). pEN1-CDEF1-2 and pEN4-CASP1pro-1 are described else where (Naseer et al.,
2012; Roppolo et al., 2011). GPAT5::mCITRINE-SYP122 was obtained by recombining the
two previously generated entry vectors for the GPAT5 promoter (Naseer et al., 2012) and the
mCITRINE-SYP122 fusion (Vermeer et al., 2014). CASP1::NLS3xmVENUS is described in
Vermeer et al., (2014). To generate ELTP::NLS3xmVENUS, the CASP1 promoter of
pGreen229-CASP1::NLS3xmVENUS, was replaced by the ELTP promoter using KpnI
digestion resulting in pGreen229-ELTP::NLS3xmVENUS. Plants were transformed using the
floral dip method (Clough and Bent, 1998).
The corresponding gene numbers are as follow: CASP1, At2g36100; CASP3, At2g27370;
ESB1, At2g28670; SGN3, At4g20140; IRT1, At4g19690; NRAMP1, At1g80830; SKOR,
At3g02850; SULTR1;1, At4g08620; SULTR1;2, At1g78000; ABA2, At1g52340; ABI3,
At3g24650; ABI5, At2g36270; ABI4, At2g40220; ETR1, At1g66340; EIN3, At3g20770;
CTR1, At5g03730; CDEF1, At4g30140; ELTP, At2g48140; ABI1, At4g26080; GPAT5,
At3g11430; SYP122, At3g52400.
Growth conditions
For in vitro assays, seeds were surface sterilized, sown on plates, incubated 2 to 3 days at 4°C
and grown vertically in growth chambers at 22 °C, under continuous light (100 µE). For metal
deficiency responses plants were germinated on Fe, Mn or Zn-free 0.5x MS agar plates,
pH5.7, sucrose 1%, supplemented with 0, 10 or 50 µM Fe-EDTA, 0, 50 µM MnSO4 (- or +
Mn) and 0 or 50 µM ZnSO4 (- or + Mn) respectively. +Mx refers to the corresponding 0.5x
MS media containing 50 µM Fe-EDTA, 50 µM MnSO4 and 50 µM ZnSO4. For suberin
analysis after germination -Fe condition corresponds to 10 µM Fe-EDTA in order to limit root
growth defects while for suberin analysis after transfer to + or -Fe and physiological
responses to Fe deficiency -Fe condition corresponds to 0 µM Fe-EDTA. For S deficiency
responses, plants were germinated on a S-free Hoagland modified media (Barberon et al.,
2008), pH 6.1, glucose 10 mM, 0.8% ultrapure agarose (Invitrogen), supplemented with 0, 25,
250 or 500 µM K2SO4. -S corresponds to 0 µM K2SO4 in both suberin analysis and
physiological responses to S starvation while +S corresponds to 500 and 250 µM K2SO4
respectively. For K deficiency plants were germinated on K-free 0.5x MS based media as
described in (Gruber et al., 2013) supplemented with 0, 25 or 500 µM KCl. In suberin
analysis, -K corresponds to 0 µM KCl while +K corresponds to 500 µM KCl. In soil, Fe
supply was performed with Fe-EDDHA solution (Sequestrene, http://www.fertiligene.com) at
the indicated concentration. For salt treatments in plates seeds were germinated on 0.5x MS
containing NaCl at the indicated concentration. For salt treatments on plates seeds were
germinated on 0.5x MS containing NaCl at the indicated concentration. For the
characterization of CASP1::CDEF1 growth in presence of NaCl plants were grown 2 weeks
on plates (Aberdeen Exp2 conditions, see below), transferred to soil in a green house
(Aberdeen summer long day conditions, 22.0°C), fertilized with 0.25 Hoagland, 1ml.l-1 Fe-
HBED for 10 days and then watered with the same solution containing 100 mM NaCl. For
ELTP::CDEF1, irt1/ELTP::CDEF1 and sultr1;1 sultr1;2/ELTP::CDEF1 phenotypic
characterizations in soil, plants were grown as described for the ionomic analysis performed
in Lausanne (Lausanne Exp1 and 2).
For ionomic analysis, Lausanne experiments 1 and 2 (Exp1 and 2) corresponds to plants
grown 4 weeks in soil at 14h/10h light/dark, 23°C/19°C, 100 µE light. Aberdeen experiment 1
(Exp1) corresponds to 5 weeks old plants grown on soil at 10h/24h light/dark, 22°C,
Humidity 35%, 100 µE light, fertilized with 0.25 Hoagland, 1 ml.l-1 Fe-HBED. Aberdeen
experiment 2 (Exp 2) corresponds to 2 week-old plants grown on MGRL, 1% sucrose, 1.2%
Type E agar plates (Fujiwara et al., 1992) at 16h/8h, 21°C/19°C, light/dark. For Hokkaido
experiments 1,2,3 (Exp1, 2 and 3), plants were grown hydroponically as described (Takano et
al., 2001), with slight modifications. Environmental parameters in a growth chamber are as
follows: 10h/14h light/dark cycle, 22°C under fluorescent lamps, 70% humidity. The seeds
were sown on rockwool and grown supplied with hydroponic media (Fujiwara et al., 1992)
supplemented with 50 µM Fe-EDTA. For Hokkaido Exp 1 plants were additionally supplied
with 3 µM CsCl and 10 µM SrCl2 after 14 days and further grown for 11 days. Please note
that the values obtained for WT plants have been previously published (Pfister et al., 2014).
11
Hokkaido Exp2 Exp3 boron was provided as B (stable isotope of B) and leaves were
collected from 46 and 26-day-old plants respectively.
Hormone and inhibitor treatments

3 or 4-day-old seedlings were transferred to 0.5x MS plates supplemented with hormones for
2 or 1 day respectively as indicated in figure legends. ABA was used as a 50 mM stock
solution in methanol, ACC as a 20 mM stock solution in water. Transfer and solvent controls
were done to ensure that neither affected suberization of roots under our growth conditions.
For inhibitor treatments AgNO3 was used as a 200 mM stock solution and AVG
(Aminoethoxyvinylglycine) as a 10 mM stock solution both dissolved in water.
Fluorescence microscopy
Confocal laser scanning microscopy experiments were performed either on a Zeiss LSM 700,
a Zeiss LSM 710 or a Zeiss LSM 710 NLO 2-Photon microscope. Excitation and detection
windows were set as follows: Zeiss LSM 700: FDA/mCITRINE/FY/CS 488 nm, SP 640; PI
555 nm, SP 640. ; Zeiss LSM 710: mCITRINE 488 nm, 495-604 nm; Zeiss LSM 710 NLO 2-
Photon: FY 960 nm, 500-550 nm (non-descanned).
Casparian strip autofluorescence after clearing was visualized as previously described
(Alassimone et al., 2010; Naseer et al., 2012). For visualization of the apoplastic barrier, 5-
day-old seedlings were incubated in the dark for 10 min in a fresh solution of 15 mM (10
mg/ml) Propidium Iodide (PI) and rinsed twice in water (Alassimone et al., 2010; Naseer et
al., 2012). For quantification, ‘‘onset of elongation’’ was defined as the point where the
length of an endodermal cell in a median optical section was more than three times its width.
From this point, cells in the file were counted until the endodermal cells blocked the PI
penetration to the stele. Experiments were repeated at least 3 times.
For most experiments suberin lamellae were observed in 5-day-old roots after Fluorol Yellow
(FY) staining as described in (Naseer et al., 2012; Pfister et al., 2014). Seedlings were
incubated in FY 088 (0.01%w/v, lactic acid) at 70°C for 30 min, rinsed with water and
counterstained with aniline blue (0.5% w/v, water) at RT for 30 min in darkness, washed, and
observed with an epifluorescence microscopes with GFP filter or with a LSM700 confocal or
a LSM710 NLO 2-photon microscope. Suberin patterns were observed and counted from the
hypocotyl junction to the onset of endodermal cell elongation. Three distinct patterns were
considered: continuous suberin lamellae, patchy suberin lamellae (corresponding to the area
where suberin lamellae establish) and non-suberized (corresponding to the young part of the
root) (Figure 1 C). Experiments were repeated at least 3 times. For analysis of suberization
from plants grown 3 weeks in soil, plants were carefully removed from the soil to minimize
mechanical damage of the root system, washed in water to remove most of the soil attached to
the root, sectioned at the level of the hypocotyl and the whole root system was stained as
describe above. Observations were done with a fluorescent stereomicroscope. FDA
accessibility to the endodermis (IN) or blocking in the cortex (OUT) was scored in 3 different
zones along the root corresponding in WT plants to the suberized zone, the non-suberized
zone and the undifferentiated zone, (see Figure 1 C for more details). Results are presented as
the frequency of FDA IN or OUT for each genotype, in 20 independent seedlings observed
one by one.
Electron microscopy
Plants were fixed in glutaraldehyde solution (EMS, Hatfield, PA) 2.5% in phosphate buffer
(PB 0.1 M [pH 7.4]) for 1h at RT and postfixed in a fresh mixture of glutaraldehyde 2.5% in
osmium tetroxide 1% (EMS) with 1.5% of potassium ferrocyanide (Sigma, St. Louis, MO) in
PB buffer for 1h at RT. The samples were then washed twice in distilled water and
dehydrated in acetone solution (Sigma, St Louis, MO, US) at graded concentrations (30%-40
min; 50% - 40 min; 70% - 40 min; 100% - 3x1h). This was followed by infiltration in Spurr
resin (EMS, Hatfield, PA, US) at graded concentrations (Spurr 33% in acetone-12h; Spurr
66% in acetone-12h; Spurr 100%-2x8h) and finally polymerized for 48h at 60°C in an oven.
Ultrathin sections of 50 nm thick were cut transversally at 2 mm below the hypocotyl-root
junction, using a Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria), picked up on
a copper slot grid 2x1mm (EMS, Hatfield, PA, US) coated with a polystyrene film (Sigma, St
Louis, MO, US). Sections were post-stained with uranyl acetate (Sigma, St Louis, MO, US)
4% in H2O for 10 min, rinsed several times with H2O followed by Reynolds lead citrate in
H2O (Sigma, St Louis, MO, US) for 10 min and rinsed several times with H2O.
Micrographs were taken with a transmission electron microscope FEI CM100 (FEI,
Eindhoven, The Netherlands) at an acceleration voltage of 80kV with a TVIPS TemCam-
F416 digital camera (TVIPS GmbH, Gauting, Germany). Measurements of the suberin
lamella thickness were done at an acceleration voltage of 80 kV and 20,000 magnification
(pixel size of 0.5101423 nm) with a TVIPS TemCam-F416 digital camera (TVIPS GmbH,
Gauting, Germany) using the software EM-MENU 4.0 (TVIPS GmbH, Gauting, Germany).
ICP-MS
For Lausanne and Aberdeen experiments, leaves were cleaned by rinsing with ultrapure water
and dried in an oven at 60ºC for 48 hours. The samples together with blank controls were
digested with 0.90 ml concentrated nitric acid (Baker Instra-Analyzed; Avantor Performance
Materials) and diluted to 10.0 ml with ultrapure water (18.2 MΩcm). The internal standard
Indium (In) was added to the acid prior to digestion for monitoring technical errors and
plasma stability in the ICP-MS instrument. After samples and controls were prepared,
elemental analysis was performed with an ICP-MS (NexION 300D; PerkinElmer) coupled to
Apex desolvation system and SC-4 DX autosampler (Elemental Scientific Inc., Omaha, NE,
USA), monitoring these elements: Li, B, Na, Mg, P, S, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se,
Rb, Sr, Mo and Cd. All samples were normalized to calculated weights, as determined with a
heuristic algorithm using the best-measured elements, the weights of the samples and the
solution concentrations (Lahner et al., 2003) are detailed at www.ionomicshub.org. For the
Hokkaido experiments (hydroponic conditions), the shoots of plants were harvested, dried in
an air incubator at 60°C for more than 60h, and the dry weights were measured. The tissues
were digested with 3 ml of 61% HNO3 (for boron determination; Wako Pure Chemicals,
Osaka, Japan) in a tube at 110°C in a DigiPREP apparatus (SCP Science, Quebec, Canada)
until complete dryness. The residues were dissolved in 10 ml of 2% HNO3 and analyzed for
elements by inductively coupled plasma mass spectrometry (ICP-MS) (ELAN, DRC-e;
Perkin–Elmer, Waltham, MA, USA).
Chemical analysis
For the analysis of aliphatic suberin components, a protocol described previously was
followed with slight modifications (Li-Beisson et al., 2013). Briefly, after the incubation in
hot isopropanol/0.01% butylated hydroxytoluene, roots were de-lipidated by a series of
solvent incubations (8-16h for each step with one additional solvent change) under constant
agitation. De-lipidated roots were dried under reduced pressure for 2-3 days resulting in 2-3
mg of residue. Aliphatic polyester components were trans-esterified to methanol by base
catalysis. Subsequently, monomers were acetylated, redisolved in chloroform and analyzed
with a gas chromatography system coupled to a mass spectrometer and a flame ionization
detector (Agilent 6890N GC Network systems) on a HP‐5MS column (J&W Scientific) using
the following temperature profile: 2 min at 90°C, temperature increase by 8°C/min to 200°C,
by 2°C/min to 250 °C and 15°C to 310°C that was held for 10 min. Amounts of suberin
monomers were calculated from 3-4 replicates per treatment.
Statistical analysis
All statistical analyses were done in the R environment (R Core Team, 2015). For multiple
comparisons between genotypes, one-way ANOVA was performed, and Tukey’s test
subsequently used as a multiple comparison procedure. Binary comparisons were performed
using Student t-test. When the data didn’t follow the linear model assumption Kruskal-Wallis
and nonparametric Tukey’s test were performed for multiple comparison and Wilcoxon-
Mann-Whitney test was used for binary comparison. For analysis of suberization along the
root only the zone of continuous suberization and the non-suberized zones were compared,
different letters (a,b,c or a’,b’,c’ respectively) indicating significant differences for a given
zone.
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Article

Adaptation of Root Function by Nutrient-Induced

d Suberization can be enhanced or reversed, depending on

d This stress-hormone-regulated plasticity allows adaptive

Barberon et al., 2016, Cell 164, 447–459

Adaptation of Root Function by Nutrient-Induced

*Correspondence: marie.barberon@unil.ch (M.B.), niko.geldner@unil.ch (N.G.)

SUMMARY root hair cells of the epidermis, repeated transport through

functional role of suberization. It has previ-

monomer content when compared to

tex under stress conditions. Regulating specifically uptake

(legend on next page)

Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc. S1

S2 Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc.

(legend continued on next page)

Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc. S3

S4 Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc.

Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc. S5

S6 Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc.

untreated ACC 1 μM ACC 1 μM

Transfer 48h +Fe

20 a’ a’ bc’ b’ a’ ac’ 20 a’ b’ bc’ c’ a’ bc’

Figure S4. Ethylene Signaling Controls Suberization, Related to Figures 4 and 5

(legend continued on next page)

Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc. S7

S8 Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc.

BrightField BrightField Fluorol Yellow Fluorol Yellow

100 200 300 400 500 600 700

NaCl : 0 100 0 100 mM

sultr1;1 sultr1;2 / ELTPCDEF1#1

Cell 164, 447–459, January 28, 2016 ª2016 Elsevier Inc. S9

Element WT ELTP::CDEF1#21 ELTP::CDEF1#6 WT ELTP::CDEF1#21 ELTP::CDEF1#6

Hokkaido Exp1, hydropony, short days

Aberdeen Exp1, soil, short days Element WT ELTP::CDEF1#8 ELTP::CDEF1#9

K 38744.61 ± 998.95 30248.09 ± 2378.24 7.41E-01 30668.97 ± 4953.03 7.78E-01

(legend on next page)

Adaptation of Root Function by Nutrient-Induced

Plant material and constructions

Hormone and inhibitor treatments

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