Download The Nonspecific Lipid Transfer Protein AtLtpI-4 Is

The Nonspecific Lipid Transfer Protein AtLtpI-4 Is
Involved in Suberin Formation of Arabidopsis thaliana
Crown Galls1[OPEN]
Rosalia Deeken*, Stefanie Saupe, Joern Klinkenberg, Michael Riedel, Jana Leide, Rainer Hedrich, and
Thomas D. Mueller
Department of Molecular Plant Physiology and Biophysics (R.D., S.S., R.H., T.D.M.) and Department
of Ecophysiology and Vegetation Ecology (M.R., J.L.), Julius-von-Sachs-Institute, University of Wuerzburg,
D–97082 Wuerzburg, Germany; and Independent Junior Research Groups, Leibniz Institute of Plant
Biochemistry, D–06120 Halle, Germany (J.K.)
ORCID IDs: 0000-0003-3044-3171 (R.D.); 0000-0002-1390-1575 (M.R.); 0000-0003-1909-3350 (J.L.); 0000-0003-1862-7357 (T.D.M.).
Nonspecific lipid transfer proteins reversibly bind different types of lipid molecules in a hydrophobic cavity. They facilitate
phospholipid transfer between membranes in vitro, play a role in cuticle and possibly in suberin formation, and might be involved
in plant pathogen defense signaling. This study focuses on the role of the lipid transfer protein AtLTPI-4 in crown gall development.
Arabidopsis (Arabidopsis thaliana) crown gall tumors, which develop upon infection with the virulent Agrobacterium tumefaciens
strain C58, highly expressed AtLTPI-4. Crown galls of the atltpI-4 loss-of-function mutant were much smaller compared with those
of wild-type plants. The gene expression pattern and localization of the protein to the plasma membrane pointed to a function of
AtLTPI-4 in cell wall suberization. Since Arabidopsis crown galls are covered by a suberin-containing periderm instead of a cuticle,
we analyzed the suberin composition of crown galls and found a reduction in the amounts of long-chain fatty acids (C18:0) in the
atltpI-4 mutant. To demonstrate the impact of AtLtpI-4 on extracellular lipid composition, we expressed the protein in Arabidopsis
epidermis cells. This led to a significant increase in the very-long-chain fatty acids C24 and C26 in the cuticular wax fraction.
Homology modeling and lipid-protein-overlay assays showed that AtLtpI-4 protein can bind these very-long-chain fatty acids.
Thus, AtLtpI-4 protein may facilitate the transfer of long-chain as well as very-long-chain fatty acids into the apoplast, depending
on the cell type in which it is expressed. In crown galls, which endogenously express AtLtpI-4, it is involved in suberin formation.
Crown gall tumors develop on plants upon infection
with virulent Agrobacterium tumefaciens strains that
harbor a tumor-inducing plasmid. A segment of the
tumor-inducing plasmid, the T-DNA, becomes integrated into the host genome, and the expression of
T-DNA-encoded oncogenes causes proliferation of
the transformed plant cells (Chilton et al., 1977;
Thomashow et al., 1980; Klee et al., 1984; Lichtenstein
et al., 1984). The developing crown galls do not differentiate an epidermis and, therefore, are not covered by
a cuticle, which prevents water loss. Hypoxic conditions and the phytohormones abscisic acid and ethylene
1
This work was supported by the Deutsche Forschungsgemeinschaft (grant no. GRK1342 [TP A7]).
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Rosalia Deeken ([email protected]).
R.D. and T.D.M. conceived and designed the experiments; J.K.,
S.S., J.L., M.R., and T.D.M. performed the experiments; R.D., S.S.,
J.L., M.R., and T.D.M. analyzed the data; J.L., M.R., T.M., R.H., and
R.D. contributed materials/reagents/tools; T.D.M., R.H., and R.D.
wrote the article.
[OPEN]
Articles can be viewed without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.16.01486
induce drought stress-protective mechanisms, affecting
the lipid composition in Arabidopsis (Arabidopsis thaliana) crown galls. For example, two desaturases, the
stearoyl-acyl carrier protein desaturase SAD6 and the
fatty acid desaturase FAD3, are highly expressed and
enrich the unsaturated fatty acid pool, which helps in
maintaining membrane integrity (Klinkenberg et al.,
2014). Second, a suberin-containing periderm-like layer
covers the surface of crown galls to minimize water loss
and avoid pathogen invasion (Efetova et al., 2007).
In general, suberin lamellae are incorporated into the
apoplast to form a diffusion barrier in specialized cells
and tissues such as the periderm and endodermis of the
root (Nawrath et al., 2013). The formation of suberin can
be promoted by stimuli such as wounding, drought
stress, and pathogen invasion (Reinhardt and Rost, 1995;
North et al., 2004; Enstone and Peterson, 2005). Suberin
consists of a polymer matrix, which contains polyaromatics and polyaliphatics (Bernards, 2002; Graça and
Santos, 2007; Schreiber, 2010). These are embedded in a
glycerol-based polyester backbone and are impregnated
with suberin-associated waxes. The wax-like compounds
belong to different substance classes of aliphatic constituents such as fatty acids, alkanols, and alkanes with a
chain length ranging from C16 to C30 (Nawrath, 2002).
Components of the polyaromatic domain are ferulic acid
Plant PhysiologyÒ, November 2016, Vol. 172, pp. 1911–1927, www.plantphysiol.org Ó 2016 American Society of Plant Biologists. All Rights Reserved. 1911
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Deeken et al.
and coumaric acid (Franke et al., 2005; Mattinen et al.,
2009). The polyaliphatic fraction consists mainly of longchain alkanoic acids, primary alkanols, v-hydroxy alkanoic acids, 2-hydroxy alkanoic acids, a,v-alkanedioic
acids, and, to a lesser extent, very-long-chain primary
alkanols and alkanoic acids (Schreiber et al., 1999;
Kolattukudy, 2001; Franke et al., 2009).
The cuticle is synthesized specifically by epidermal
cells and covers all primary plant surfaces to prevent
water loss and pathogen invasion. It consists of the cutin
polymer and soluble cuticular lipids (Jeffree, 1996;
Kolattukudy, 2001; Kunst and Samuels, 2003; Schreiber,
2010; Yeats and Rose, 2013). The latter are deposited as
cuticular waxes within (intracuticular wax) as well as on
the outer part of (epicuticular wax) the cutin polymer. At
ambient temperatures, the epicuticular wax layer forms
a plant type-specific pattern of wax films or wax crystals.
The predominant compounds of the Arabidopsis cutin
polymer are saturated aliphatic oxygenated fatty acids
and unsaturated monomers with carbon chain lengths of
C16 and C18. The cuticular wax of leaves consists mainly
of aliphatic very-long-chain alkanes and primary alkanols, whereas alkanoic acids, alkyl esters, alkanones,
alkanals, and secondary alkanols are present only in
lower amounts (Rashotte et al., 2001; Li-Beisson et al.,
2013). The chain lengths of the aliphatic wax components
range from 20 to 34 carbon units.
Formation of the diffusion barriers requires the
translocation of lipid compounds across the plasma
membrane. Transport of aliphatic lipids across the
plasma membrane involves plasma membrane-localized
transporters of the G family of ATP-binding cassette
proteins (ABCG transporters). In Arabidopsis, the ABCG
transporter ABCG11 has been associated with both cutin
and suberin biosynthesis (Bird et al., 2007; Panikashvili
et al., 2007, 2010), while CER5/ABCG12 (Pighin et al.,
2004) and ABCG13 (Panikashvili et al., 2011) are required for the export of cuticular wax monomers
(Panikashvili and Aharoni, 2008; McFarlane et al., 2010).
For cuticular wax assembly, also nonspecific lipid
transfer proteins (nsLTPs) likely facilitate the transfer of
aliphatic compounds (Lee et al., 2009; Kim et al., 2012).
The Arabidopsis LTPG1 has been associated with
cuticular wax biosynthesis (DeBono et al., 2009).
LTPG proteins contain a sequence motif for a glycosylphosphatidylinositol (GPI) anchor that localizes the
proteins at the plasma membrane. GPI-anchored nsLTPs
also have been postulated to be involved in suberin
formation (Edstam et al., 2013). Most other nsLTPs are
synthesized as precursor proteins, with an N-terminal
signal peptide that targets them to the apoplastic space
(Thoma et al., 1994; Pagnussat et al., 2012).
The Arabidopsis genome comprises 49 nsLTP genes,
which encode 7- to 9-kD small, basic proteins and which
are grouped into 10 different classes based on amino acid
sequence identity (AtLtpI–AtLtpIX and AtLTPY; Boutrot
et al., 2008). Eighteen of the 49 genes are arranged in
tandem duplication repeats on the five Arabidopsis
chromosomes, suggesting functional redundancy. Type I
nsLTPs are characterized by a long tunnel-like cavity,
while type II proteins comprise two adjacent hydrophobic cavities. Biochemical and structural characterization has shown that nsLTPs nonspecifically bind
different types of lipid molecules. For instance, spinach
(Spinacia oleracea) nsLTP can transfer phospholipids between membranes (Kader et al., 1984). The crystal
structure of nsLTP1 from wheat (Triticum aestivum)
demonstrates the binding of two lyso-myristoylphosphatidylcholine molecules, which are inserted
head to tail in the two hydrophobic cavities of this type
II LTP (Charvolin et al., 1999). Three-dimensional (3D)
models of the maize (Zea mays) nsLTP suggest that fatty
acids such as hexadecanoic (C16) and octadecanoic (C18)
acid also serve as lipid-binding partners (Dong et al.,
1995; Han et al., 2001). The biological roles of nsLTPs
are considered to be very diverse. For example, they
have been shown to be involved in cuticle formation
and to contribute to plant defense responses against
fungal and bacterial pathogens (Molina and GarcíaOlmedo, 1997; Jung et al., 2009). The Arabidopsis
nsLTP-encoding genes DEFECTIVE IN INDUCED RESISTANCE1 (DIR1) and AZELAIC ACID INDUCED1
(AZI1) participate in long-distance signaling of bacterial
infections (Maldonado et al., 2002; Wang et al., 2004).
The aim of this study was to investigate the role of the
nsLTP AtLtpI-4 (formerly named LTP2; Arondel et al.,
2000) from Arabidopsis in grown gall physiology.
Crown gall development was induced upon infecting
Arabidopsis inflorescence stems with the A. tumefaciens
strain C58. Analysis of the spatiotemporal expression
pattern and transcript levels of the AtLtpI-4 gene gave
insights into putative AtLtpI-4 functions. The loss-offunction mutant atltpI-4 and ectopic overexpression of
AtLtpI-4 in epidermal cells revealed an impact of
AtLtpI-4 on extracellular lipid deposition. The functionality of AtLtpI-4 observed in planta was further
analyzed using a lipid-protein-overlay assay and 3D
modeling.
RESULTS
Arabidopsis Crown Galls Express AtltpI-4 and the Loss-ofFunction Mutant Shows Reduced Crown Gall Growth
According to microarray data, out of the 49 nsLTP
members only AtLtpI-4 is expressed in crown gall tumors
of Arabidopsis induced by the virulent A. tumefaciens
strain C58 (Deeken et al., 2006). This observation was
confirmed using Arabidopsis lines expressing the GUS
enzyme under the control of the AtLtpI-4 promoter.
Strong GUS expression was observed in cross sections of
3-week-old crown galls, induced on Arabidopsis inflorescence stems by A. tumefaciens strain C58 (Fig. 1A). The
Arabidopsis line GK-639E08, which harbors a T-DNA
insertion in the first exon of the AtltpI-4 gene locus
(Supplemental Fig. S1A), was then employed to determine the role of AtLtpI-4 in crown gall development.
Plants of line GK-639E08 did not express any AtLtpI-4
transcripts in leaves and flowers; therefore, the line is a
loss-of-function mutant (Supplemental Fig. S1, B and C).
1912
Plant Physiol. Vol. 172, 2016
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Crown Gall Suberin Formation Involves AtLtpI-4
Figure 1. Expression of AtLtpI-4 is essential for the
development of wild-type-like Arabidopsis crown
galls. A, Representative cross section of a crown
gall tumor (Tu) at an Arabidopsis inflorescence
stem (St) of the transgenic plant line AtLtpI-4pro:
GUS, expressing GUS under the control of the
AtLtpI-4 promoter. Blue indicates areas of GUS
activity. B, Representative images of crown galls
28 d after inoculation of the virulent A. tumefaciens strain C58 into inflorescence stems of Arabidopsis wild-type (Columbia-0 [Col-0]) and
atltpI-4 mutant plants. C, Weights of 28-d-old
crown galls from wild-type and atltpI-4 mutant
plants. Mean values 6 SD represent data from
three independent growth experiments, each
comprising at least 10 plants. ***, P , 0.01
according to Student’s t test. Modified from Saupe
(2014).
Transcription of the AtLtpI-5 gene (formerly called LTP1;
Arondel et al., 2000), which is highly homologous to
AtLtpI-4 potentially due to gene duplication (Boutrot
et al., 2008), was approximately 1.5- and 2-fold increased
in atltpI-4 mutant leaves and flowers, respectively.
Inoculation of the A. tumefaciens strain C58 into inflorescence stems of the atltpI-4 mutant induced the development of crown galls that were very small compared
with those of wild-type plants (Fig. 1B). The amount of
crown gall material harvested from the atltpI-4 mutant
plant was about four times smaller than that of the wild
type (Fig. 1C). Transcription of AtLtpI-5 was induced
380-fold in the mutant crown galls, which is much
stronger than in leaves and flowers of the mutant
(Supplemental Fig. S1D). The expression of two other
type I nsLTP genes, AtLtpI-11 (2-fold) and AtLtpI-12 (1.7fold), was increased only marginally, whereas transcript
levels of AtLtpI-6 and LTPG2 remained unchanged in the
atltpI-4 mutant crown galls. Consistently, the latter two
are not members of the nsLTP gene family according to
the classification by Boutrot et al. (2008). Furthermore,
AtLtpI-7 was not expressed in either of the crown gall
genotypes. These data suggest that the expression of
AtLtpI-4 is essential for normal crown gall development and that its function cannot be replaced by other
LTPs.
The AtLTPI-4 Gene Is Expressed in Cells Undergoing Cell
Wall Modifications
To gain insights into the function of AtLtpI-4 in
crown gall development, the spatiotemporal expression
pattern of the gene was monitored in three GUSexpressing Arabidopsis lines. GUS activity was observed in the papillae of the stigma of Arabidopsis
flowers at all developmental stages (Fig. 2, A–D). Mature sepals, petals, and anthers, as well as the sepal and
petal abscission zones of mature siliques, showed blue
staining only at late developmental stages of fruit ripening (Fig. 2, C–F). Blue spots with a ring-like structure
were observed on rosette leaves, indicating AtLtpI-4
promoter activity in basal cells of the trichome at a
certain developmental stage (Fig. 2, G and H). In addition, GUS expression was found in cross sections of
the hypocotyl stele from seedlings, in the youngest cell
layers of the xylem, and in the root periderm from
plants in the secondary growth phase (Fig. 2, I and J).
Blue staining also was detected in cells adjacent to the
sclerenchyma in cross sections from the inflorescence
stem (Fig. 2K).
The GUS staining pattern suggests that AtLtpI-4 is
expressed in cells undergoing cell wall modifications.
Consequently, stimuli that induce such modifications
and are associated with crown gall development were
tested with respect to the activation of AtLtpI-4 gene
expression. Wounding and long-term treatment of root
cultures with the stress phytohormone abscisic acid
(ABA) are known to induce the suberization of cell
walls. AtLtpI-4 transcription started to increase as
early as 45 min after wounding of Arabidopsis leaves
(Supplemental Fig. S2A). ABA treatment of Arabidopsis root cultures also induced AtLtpI-4 transcription
from 12 h on (Supplemental Fig. S2B). In contrast,
inoculation of Arabidopsis roots with the virulent
A. tumefaciens strain C58 did not alter AtLtpI-4
Plant Physiol. Vol. 172, 2016
1913
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Deeken et al.
Figure 2. Tissue-specific expression pattern of the
Arabidopsis AtLtpI-4 gene in plant lines expressing GUS under the control of the AtLtpI-4 promoter (AtLtpI-4pro:GUS). A to F, Different
developmental stages of flowers and siliques:
flower bud (A), young flower (B), mature flower
(C), developing silique (D), mature silique (E), and
petal and sepal abscission zone of a mature silique
(F). G and H, Fully expended rosette leaf (G) and
enlargement of a leaf section shown in G (H). I to
K, Cross sections of hypocotyl (I), main root in the
secondary growth phase (J), and segment of the
inflorescence stem with a vascular bundle and
sclerenchyma during secondary growth (K). Blue
areas indicate AtLtpI-4 promoter-driven GUS activity, and pink areas indicate safranin-stained
secondary cell walls. Arrows point to blue-stained
cells. Images from one representative GUSexpressing line out of three are shown. Ca, Cambium; Co, cortex; En, endodermis; Ph, phloem; St,
stele; Xy, xylem. Modified from Saupe (2014).
transcription within 48 h compared with the untreated
control (Supplemental Fig. S2C). A. tumefaciens attachment to the surface of intact roots was observed by
transmission microscopy (data not shown). These
findings indicate that signals that induce cell wall
modifications rather than pathogen defense positively
regulate AtLtpI-4 gene expression.
The AtLTPI-4 Protein Localizes to the Plasma Membrane
The involvement of AtLtpI-4 in cell wall modifications requires localization of the protein in the cell wall
or plasma membrane. Hence, subcellular localization of
the AtLtpI-4 protein was studied using fluorescent reporter genes. Its C-terminal end was fused with the
red-fluorescing protein mCherry (AtLTPI-4:mCherry).
The coding sequence of Arabidopsis AtLtpI-5 also
was fused to mCherry (AtLtpI-5:mCherry). This construct served as a cell wall marker, because the
AtLtpI-5 protein was shown to be located in the cell
wall (Thoma et al., 1994). The mVenus-labeled anion
channel SLAH3 (SLAH3:mVenus) was used as a
plasma membrane marker (Geiger et al., 2011). All
three constructs were transformed into the disarmed
A. tumefaciens strain GV3101 and transiently expressed
in Nicotiana benthamiana leaves. Leaves that emitted
fluorescence associated with the marker proteins
were analyzed by confocal laser scanning microscopy. Coexpression of the fusion proteins AtLtpI-5:
mCherry and SLAH3:mVenus in epidermal tobacco
(Nicotiana tabacum) cells demonstrated that AtLtpI-5and SLAH3-derived fluorescence partially overlap at
the boundary of epidermal cells (Supplemental Fig.
S3, A–D). A similar superposition was observed with
AtLtpI-4:mCherry and SLAH3:mVenus coexpression
(Supplemental Fig. S3, E–H). These findings suggest
that the AtLtpI-4 protein, like AtLtpI-5, resides in the
cell wall. Since the resolution of the confocal laser
scanning microscope is not sufficient to unambiguously
localize the fluorescence signal to the plasma membrane or cell wall, the N. benthamiana leaf areas with
labeled epidermis cells were plasmolyzed. The red
fluorescence associated with AtLtpI-5 was now observed in the cell wall (Fig. 3, A–C). Thus, upon
plasmolysis, AtLtpI-5:mCherry no longer colocalized
with the plasma membrane marker SLAH3:mVenus.
In contrast to AtLtpI-5:mCherry, AtLtpI-4:mCherry
clearly colocalized with SLAH3:mVenus in the
plasma membrane, as indicated by the overlapping
yellow and red fluorescence signals, which were particularly visible in Hechtian strands (Fig. 3, D–F, arrows). Thus, the cell biological approach indicates that
the AtLtpI-4 protein is associated with the plasma
membrane.
1914
Plant Physiol. Vol. 172, 2016
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Crown Gall Suberin Formation Involves AtLtpI-4
Figure 3. Plasmolysis assay with transiently transformed tobacco leaves
visualizes differences in the subcellular localization of the AtLtpI-4 and
AtLtpI-5 proteins. A to C, Coexpression of the two fusion proteins AtLtpI5 with mCherry (AtLtpI-5:mCherry) and SLAH3 with mVenus (SLAH3:
mVenus) in leaf epidermal cells. AtLtpI-5:mCherry serves as a cell wall
marker, and SLAH3:mVenus indicates plasma membrane localization.
A to C, The red color of AtLtpI-5:mCherry marks the cell wall (A), and
the yellow color of SLAH3:mVenus marks the plasma membrane (B);
the overlay of red and yellow colors shows the difference in the localization of AtLtpI-5 and SLAH3 (C). D to F, Epidermis cells coexpressing
AtLtpI-4 fused to mCherry (AtLtpI-4:mCherry) and SLAH3:mVenus. The
red color of AtLtpI-4:mCherry (D) and the yellow color of SLAH3:
mVenus (E) are both in the plasma membrane; the overlay of the two
colors in the plasma membrane is shown in F. For plasmolysis, leaf discs
were incubated with 1 M KNO3 for 8 min and inspected with a confocal
laser-scanning microscope. White arrows indicate cell wall areas, yellow arrows point to the plasma membrane, and gray arrows mark
Hechtian strands connecting the cytoplasm of plasmolyzed cells.
Modified from Saupe (2014).
The Composition of Crown Gall Suberin Is Altered in
atltpI-4 Mutants
The pattern of AtLtpI-4 gene expression and the association of the protein with the plasma membrane
point toward a role for AtLtpI-4 in suberin formation.
Hence, the suberin load and the composition of enzymatically hydrolyzed and washed crown gall material
from the atltpI–4 mutant and the wild type were analyzed using gas chromatography-flame ionization detection and gas chromatography-mass spectroscopy.
Although tumor growth was highly reduced in the
atltpI-4 mutant plants, crown galls of the atltpI-4 mutant
and the wild type contained similar amounts of total
suberin when normalized to mg dry weight of the isolated tumor material (Supplemental Fig. S4). The fraction of solvent-soluble wax represented approximately
one-third of the total suberin. In the suberin-associated
wax portion, the amount of the unsaturated alkanoic
acid C18:1 was reduced in the small crown galls of the
atltpI-4 mutant compared with the wild type (Fig. 4A).
All other compounds with significantly different
amounts were increased, such as nonacosanoic acid
(C 29), triacontanol (C 30), alkyl esters (C 40–C 43), and
sitostenone. In the insoluble suberin polyester fraction,
the quantity of octadecanoic acid (C18) was lower in
atltpI-4 mutant crown galls, while the amounts of some
compounds of the 2-hydroxy alkanoic acid class (C18,
C22, and C24) were increased (Fig. 4B). Thus, in suberized crown galls, the loss of AtLtpI-4 expression caused
a decrease in C18 alkanoic acids.
To compare the wax composition of the crown gall
with that of the inflorescence stem, stem segments were
harvested above the crown galls of 12-week-old atltpI-4
mutant and wild-type plants. The total amount of
solvent-soluble wax was similar in inflorescence stems
of the mutant and the wild type. The atltpI-4 mutant
contained 0.25 6 0.02 mg mg21 dry weight and the wild
type contained 0.19 6 0.07 mg mg21 dry weight cuticular waxes. Similarly, no difference was found in the
monomer composition of the cuticular wax between the
two genotypes (Supplemental Fig. S5). Inflorescence
stem and crown gall waxes shared alkanoic acids (C20–
C28), primary alkanols (C20–C30), and alkanes (C27–C31)
of the same carbon chain length as well as C29 alkanone.
The content of these aliphatic compounds was higher in
crown galls than in stems. In addition, the spectrum
particularly of alkanoic acids is larger in crown gall
waxes toward compounds with longer (greater than 28)
carbon chain lengths.
Ectopic Expression of AtLTPI-4 in the Epidermis Alters
Cuticular Wax Composition
AtLtpI-4 was then ectopically expressed in Arabidopsis epidermal cells, a cell type in which the gene is
not endogenously expressed but that produces the cuticle. The ectopic epidermal expression of AtLtpI-4,
therefore, enabled us to study the impact of AtLtpI-4
on the composition of waxes in a different environment. The CER5/ABCG12 promoter, which is active
exclusively in epidermal cells of aboveground tissues
(Pighin et al., 2004), was used to drive the expression of
AtLtpI-4. Three independently transformed plant lines
(ABCG12pro:AtLtpI-4 lines 1, 4, and 11) with different
transcript levels of AtLtpI-4 in leaves were chosen for
analysis. The highest transcript levels were found in
line ABCG12pro:AtLtpI-4 #11, which was 5-fold higher
than the levels in wild-type leaves (Supplemental Fig.
S2D).
The composition of cuticular waxes of adult leaves
was analyzed using gas chromatography-flame ionization detection and gas chromatography-mass spectroscopy. Transgenic and wild-type plants did not
show a significant difference in the total cuticular wax
quantity (Kruskal-Wallis test: H [3, n = 11] = 6.24, P =
0.10). The total cuticular wax amounts were 1.03 6 0.03
mg cm22 for the wild type, 0.89 6 0.14 mg cm22 for
ABCG12pro:AtLtpI-4 line 1, 0.93 6 0.12 mg cm22 for line
4, and 0.60 6 0.23 mg cm22 for line 11. However, considerable differences were detected between line 11,
with the highest AtLtpI-4 expression levels, and the
wild type in the amounts of single aliphatic components (Fig. 5). The absolute amounts of alkanoic acids
Plant Physiol. Vol. 172, 2016
1915
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Deeken et al.
Figure 4. Suberin monomer compounds of crown galls from Arabidopsis wild-type (Col-0) and atltpI-4 mutant plants. Absolute
amounts of solvent-soluble (wax; A) and solvent-insoluble (polyesters; B) esterified suberin monomers are given per mg dry
weight (DW) from crown galls induced by the virulent A. tumefaciens strain C58. Mean values 6 SD of five samples of pooled
crown galls are shown. *, P # 0.05 (Mann-Whitney U test).
with carbon chain lengths of C26 or less were increased
significantly, whereas those with carbon chain lengths
greater than C26 were reduced. The amounts of C28, C32,
C33, and C35 alkanes, C26 and C28 primary alkanols, and
C50 and C52 alkyl esters also were reduced significantly.
The wax compositions of lines 1 and 4 with lower
AtLtpI-4 expression levels showed similar, although
less pronounced, differences. In summary, upon ectopic expression in epidermal cells, AtLtpI-4 only
marginally affected the total amount of the wax load
(line 11) but caused a clear shift in chain length distribution within very-long-chain aliphatic lipids (VLCALs),
with significant increases only in C24 and C26 alkanoic
acids in the cuticular wax fraction.
Epidermal Expression of AtLtpI-4 Alters Plant Morphology
and Properties of the Cuticle
In accordance with the requirements of a proper cuticle composition for normal plant development, ectopic epidermal expression of AtLtpI-4 caused several
morphological changes in the ABCG12pro:AtLtpI-4
transgenics: (1) the last internode of the main inflorescence
stem was extremely short (Fig. 6A); (2) the first flowers
of the stunted inflorescence stems were malformed,
with small brownish sepals and an unusually large
number of trichomes on the surface (Fig. 6, B and C); (3)
flowers from stunted inflorescences did not give rise to
siliques; (4) lesion formation was frequently found at
the edges of rosette leaves (Fig. 6, D and E); and (5)
leaves generally harbored a large number of single- or
double-branched trichomes (Table I), with the numbers
of branching points correlating with AtLtpI-4 transcript
levels in the transgenic lines.
Further inspection of the ABCG12pro:AtLtpI-4 plant
lines revealed differences in the physical properties of
their leaf surface. The contact angles of water droplets
were significantly smaller in line 11 compared with the
wild-type Wassilewskija (Ws-2), suggesting a less hydrophobic surface (Fig. 6F). Furthermore, analysis of
the permeability of the cuticle for solvents confirmed
that the cuticle of ABCG12pro:AtLtpI-4 leaves is more
permeable (Fig. 6G). Incubation of leaves in ethanol
caused a more efficient release of chlorophyll from
ABCG12pro:AtLtpI-4 plants compared with the wild
type. The quantitative data of the chlorophyll-leaching
and contact-angle assay again reflected well the
1916
Plant Physiol. Vol. 172, 2016
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Crown Gall Suberin Formation Involves AtLtpI-4
Figure 5. Cuticular wax composition of rosette leaves of 12-week-old Arabidopsis plants with ectopic expression of AtLtpI-4 in
the epidermis. Absolute amounts of wax compounds are given per leaf surface area of wild-type plants and three different lines
expressing AtLtpI-4 under the control of the ABCG12 promoter (ABCG12pro:LTP#1, ABCG12pro:LTP#4, and ABCG12pro:
LTP#11). Mean values and 25th and 75th percentiles of two independent experiments for line CER5pro:LTP#11 and three of the
other genotypes are shown. *, P , 0.05 according to Kruskal-Wallis ANOVA (posthoc test for multiple comparisons). Ca,
Campesterol; b-S, b-sitosterol; Ch, cholesterol; St, sterol (not further identified); N.i., not identified.
different levels of AtLtpI-4 transcripts determined in the
three independent transgenic lines.
The impact of epidermal AtLtpI-4 expression on the
ultrastructure of epicuticular waxes of ABCG12pro:
AtLtpI-4 plants was examined by scanning electron
microscopy of the leaf and stem surfaces. Scanning
electron micrographs from wild-type Arabidopsis
plants and the three ABCG12pro:AtLtpI-4 lines depicted
differences in the epicuticular wax crystal structure.
Whereas the leaf surface of the wild type was smooth,
with very few wax crystals (Fig. 7A), the surface of
ABCG12pro:AtLtpI-4 plants contained unevenly distributed plate-like wax crystals (Fig. 7B). Tube-shaped
wax crystals covered the inflorescence stem surface of
wild-type and ABCG12pro:AtLtpI-4 plants, the density
of which was lower for the transgenics (Fig. 7, C and D).
Furthermore, the surface of the trichomes of ABCG12pro:AtLtpI-4 plants was rather smooth and did not
contain the papillae-like structures seen on wild-type
trichomes (Fig. 7, E and F). Taken together, the ectopic epidermal expression of AtLtpI-4 influences the
VLCAL composition in cuticular waxes, and this has
an impact on the wax surface structure and plant
development.
The AtLtpI-4 Protein Binds Very-Long-Chain Aliphatic
Fatty Acids in Vitro
Our chemical cuticular wax analyses suggested that
AtLtpI-4 binds C24 and C26 very-long-chain fatty acids
in addition to C18 long-chain fatty acids, which already
had been demonstrated by others (Han et al., 2001).
Therefore, we modeled potential lipid interactions of
AtLtpI-4 in silico employing 3D homology modeling
and experimentally analyzed its lipid-binding properties using an in vitro lipid-protein-overlay assay. Several structures of nsLTP protein-lipid complexes exist in
the protein structure database (Protein Data Bank
[PDB]), which can be used as templates for homology
modeling of AtLtpI-4 lipid-protein complexes: (1)
nsLTP from maize in complex with a C18 fatty acid (PDB
entry 1FK4); (2) the same protein with 12-hydroxy
octadec-9-enoic acid (hydroxy C18:1) bound (PDB entry
1FK7; Han et al., 2001); and (3) nsLTP1 from rice
(Oryza sativa; PDB entry 1UVB; Cheng et al., 2004),
whose structure was determined in complex with
two hexadec-9-enoic acid (C16:1) molecules. On the
basis of these templates, we obtained a 3D homology
model of AtLtpI-4, which exhibits the typical foura-helix-bundle architecture (Fig. 8A, green ribbons)
Plant Physiol. Vol. 172, 2016
1917
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Deeken et al.
Figure 6. Effects of ectopic AtLtpI-4 expression in
Arabidopsis epidermis cells on plant morphology
and leaf surface properties. A to E, Morphological
phenotypes of the wild type and a representative
plant of the ABC12pro::AtLtpI-4 transgenic lines.
A, Retarded elongation growth of the primary inflorescence of the transgenic plant (arrow) but not
the wild-type plant. B, Enlargement of the boxed
primary inflorescence shown in A. C, Enlargement
of a malformed flower at the end of the primary
inflorescence of the ABCG12pro:AtLtpI-4 plant
shown in B. D and E, Plant rosettes (D) and single
rosette leaves (E), of which only the transgenic
ones display lesions at the leaf edges (arrows). F
and G, Contact angles (F) of water droplets
pipetted onto the rosette leaf surface (mean
values 6 SD of six droplets on three leaves of three
plants each) and chlorophyll-leaching assay (G)
with rosette leaves soaked in 80% ethanol of wildtype plants and the three ABC12pro:AtLtpI-4 lines
(mean values 6 SD of four leaves of three plants
each). FW, Fresh weight. ***, P , 0.001 according
to Student’s t test. Modified from Saupe (2014).
and comprises a hydrophobic cavity in the protein core
(Fig. 8B). Fatty acid substrates can be accommodated,
with the carboxylate head group of the fatty acid potentially forming hydrogen bonds with the hydroxyl
group of Tyr-81 (Y81), the main chain carbonyl of
Arg-45 (R45), and the side chain amino group of Lys-36
(K36; Fig. 8A). To gain insight into how very-long-chain
fatty acids might bind to AtLtpI-4, we took advantage
of the ligand interaction data found in the templates
listed above. The two structures from the maize LTP,
each with octadecanoic acid (C18) bound, show that the
fatty acid is inserted in the cavity with the aliphatic
chain bending over near the end between the carbon
atoms at positions 14 and 15 (Supplemental Fig. S6A).
These two carbon atoms of the C18 fatty acid thereby
form a sharp turn. The torsion angles within this segment allow the last three carbon atoms of the aliphatic
chain to point back upward to the tunnel entry, where
the carboxylic acid group is located. In the structure of
rice nsLTP1, the protein harbors two hexadecenoic
acids (C16:1) side by side and arranged head to tail in
the hydrophobic cavity, with their polar head groups
being located close to the two entries of the cavity
(Supplemental Fig. S6B). Since the helix packing of all
the above-mentioned nsLTPs does not differ, this suggests that the hydrophobic cavity of LTPI-type members
likely can accommodate two aliphatic chains side by
side or one very-long-chain fatty acid, if the aliphatic
chain is folded in a hook-like conformation. Therefore, the
information about the localization of the C18 and the
two molecules of C16:1 fatty acids within the nsLTPs
(Supplemental Fig. S6C) was combined to provide a
structural model of how a very-long-chain fatty acid,
such as hexacosanoic acid (C26), could be accommodated in the hydrophobic cavity of the AtLtpI-4
protein (Supplemental Fig. S6D).
In this model, the very-long-chain fatty acids C22 to
C28 would bend at carbon atoms 14 and 15 (Supplemental
Fig. S6D), such that the downstream carbon chains point
upward toward the carboxylate head group at the upper
cavity entry (Fig. 8, A and B). The resulting torsion angle
at the carbon atom positions 13 to 16 is 22°, whereas for an
extended aliphatic carbon chain, it is between 80° and
160°. According to this theoretical model, fatty acids with
more than 28 carbon atoms would not fit into the hydrophobic cavity of AtLtpI-4 due to the interference of the
last carbon atom with the carboxylate head group and the
amino acid residues that seal off the cavity entry: Ile (I79),
Pro (P80), and Tyr (Y81; Fig. 8A). Taken together, the
AtLtpI-4 homology models suggest that very-long-chain
fatty acids with chain lengths between 22 and 28 carbon
atoms also can be accommodated in the hydrophobic
cavity of the Arabidopsis AtLtpI-4 protein.
The very-long-chain fatty acid-LTP interaction
hypothesis of the in silico model was then tested by a
lipid-protein-overlay assay. To do so, His/V5-tagged
AtLtpI-4 protein was heterologously expressed in
Escherichia coli and tobacco Bright Yellow-2 (BY-2) callus cells (Supplemental Fig. S7). The overlay assays
revealed that the His/V5-tagged AtLtpI-4 proteins
1918
Plant Physiol. Vol. 172, 2016
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Crown Gall Suberin Formation Involves AtLtpI-4
Table I. Number of trichomes and trichome branches on the leaf
surface of Arabidopsis wild-type and ABCG12pro:AtLtpI-4 plants
Trichomes were counted for 12 leaves (three leaves of four plants
each). The absolute number of trichomes and trichome branch points
on the upper leaf surface of Arabidopsis wild-type plants and the
transgenic lines 1, 4, and 11 were counted using a Leica MZ6 microscope. The percentages of zero, one, and two trichome branch
points were calculated from the total number of trichomes. Modified
from Saupe (2014).
Arabidopsis Genotype
Wild type
ABCG12pro:AtLtpI-4#1
ABCG12pro:AtLtpI-4#4
ABCG12pro:AtLtpI-4#11
No. of Trichome
Branch Points
0
1
2
–
–
1%
3%
29%
44%
41%
54%
71%
56%
58%
43%
Total No. of
Trichomes
508
495
508
479
from E. coli and BY-2 cells indeed bound very-longchain fatty acids with a carbon chain length of C22 to
C26 (Fig. 8, C and D). The AtLtpI-4 protein produced in
E. coli also bound C27 and, to a lesser extent, C28 fatty
acids (Fig. 8C). The in vitro assay demonstrates that the
AtLtpI-4 protein is able to bind fatty acids with more
than 22 and less than 28 carbon atoms. Thus, AtLtpI-4
can bind the C24 and C26 fatty acids, which were increased in the cuticular wax fraction of the transgenic
plant lines with epidermal expression of AtLtpI-4.
Arabidopsis crown galls, which contain high levels of
ABA and are covered by cell layers with suberized
walls, also express high levels of the AtLtpI-4 gene
(Efetova et al., 2007). Arabidopsis epidermis cells,
however, do not express the AtLtpI-4 gene endogenously; therefore, AtLtpI-4 does not play a role in the
cuticle formation of Arabidopsis. Overall, the AtLtpI-4
gene expression pattern points to the involvement of
AtLtpI-4 in processes that require cell wall modifications, such as suberization.
The tiny crown galls of the loss-of-function mutant
displayed an altered composition of aliphatic suberin
monomers. Surprisingly, the extremely high expression
levels of the close homolog AtLtpI-5 in the tiny crown
galls of the mutant could not functionally replace the
loss of atltpI-4 function. However, the strong induction
of AtLtpI-5 gene expression may cause an increase in the
aliphatic lipids, with elevated amounts in mutant
crown galls. This feed-forward response to the loss of
atltpI-4 function could be an attempt of the mutant to
balance the disordered suberin lipid composition in
crown galls. The formation of suberin requires aliphatic constituents (alkanoic acids, primary alkanols,
a,v-alkanedioic acids, v-hydroxy alkanoic acids, and
DISCUSSION
AtLtpI-4 Is Involved in Suberin Formation during Crown
Gall Development
In Arabidopsis crown galls, expression of the type I
nsLTP AtLtpI-4 is highly up-regulated and the Arabidopsis atltpI-4 knockout mutant develops only very
small crown galls, suggesting an essential role of this
nsLTP in normal crown gall development. Plant nsLTP
genes are encoded by multigene families and are
expressed under abiotic (Yubero-Serrano et al., 2003) as
well as biotic (Blilou et al., 2000; Gomès et al., 2003)
stress conditions. The proteins exert different physiological functions and are thought to be involved in the
secretion of extracellular lipophilic material. The Arabidopsis nsLTPs DIR1 (Maldonado et al., 2002) and
AZI1 (Wang et al., 2004) have been shown to play a role
in pathogen defense. We found that the involvement of
AtLtpI-4 in pathogen signaling is rather unlikely, because A. tumefaciens infection of Arabidopsis roots did
not induce the expression of the AtLtpI-4 gene. Instead,
the gene was expressed in the papillae of the pistil, leaf
abscission zones of siliques, and the root periderm,
tissue types known to contain suberized cell walls.
Wounding and long-term treatment of roots with the
stress hormone ABA, stimuli usually causing suberization (Bleecker and Patterson, 1997; Roberts et al.,
2002; Enstone and Peterson, 2005; Schreiber et al., 2005),
also induced the expression of AtLtpI-4. Furthermore,
Figure 7. Scanning electron micrographs of air-dried leaf and inflorescence stem surfaces from Arabidopsis wild-type and transgenic
ABC12pro:AtLtpI-4 plants. A and B, Adaxial surfaces of rosette leaves of
wild-type (A) and ABC12pro:AtLtpI-4 (B) plants. C and D, Inflorescence
stem surfaces of wild-type (C) and ABC12pro:AtLtpI-4 (D) plants. E and F,
Trichomes of the adaxial rosette leaf surfaces of wild-type (E) and
ABC12pro:AtLtpI-4 (F) plants. One representative image is shown for one
out of three plants from three independent lines each. Modified from
Saupe (2014).
Plant Physiol. Vol. 172, 2016
1919
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Deeken et al.
Figure 8. 3D homology model and in vitro binding properties of Arabidopsis AtLtpI-4. A, Ribbon plot of the 3D homology model
of AtLtpI-4 showing the four-helical-bundle motif (green). Amino acid residues lining the hydrophobic cavity and interacting with
the aliphatic lipid molecules are shown as sticks and are labeled. The four fatty acid molecules with differing carbon atom chain
lengths from 22 to 28 are displayed in different shades of blue. B, Sectional model of the AtLtpI-4 homology model highlighting
the large cleft building the hydrophobic cavity for the aliphatic fatty acid molecule C26. C and D, Lipid-protein-overlay assays,
with dots of equimolar amounts of saturated and unsaturated fatty acids of different carbon chain lengths used for immunodetection of the hexa-His-tagged AtLtpI-4 protein expressed in E. coli cells (C) and in tobacco BY-2 cell suspension (D). One
representative overlay assay is shown; the assays were done in triplicate. Modified from Saupe (2014).
2-hydroxy alkanoic acids) with a chain length ranging
from 16 to 30 carbon atoms (Schreiber et al., 1999;
Kolattukudy, 2001; Franke and Schreiber, 2007). The
lipid monomers or oligomers/polymers have to be
transferred across the plasma membrane into the apoplastic space for suberin assembly in plant cell walls. The
involved transport mechanisms need to be located at the
plasma membrane and/or the cell wall. The nsLTPs are
located in the cell wall (Thoma et al., 1994; Maldonado
et al., 2002), can be attached to the plasma membrane by
a GPI anchor (DeBono et al., 2009; Lee et al., 2009), or can
bind to proteinaceous receptor molecules in the plasma
membrane (Wang et al., 2009). For instance, the tobacco
nsLTP1 binds jasmonic acid, and this complex exhibits a
strong binding affinity to elicitin receptors in the plasma
membrane (Blein et al., 2002; Buhot et al., 2004). AtLtpI-4
also might be attached to the plasma membrane via a
receptor, because we observed the protein at the plasma
membrane, although it lacks a GPI anchor.
It has been postulated by their abundant expression
in epidermal cells and altered lipid profiles of LTPdeficient mutants that plant nsLTPs are involved in the
export of lipids to the plant surface (Sterk et al., 1991;
Thoma et al., 1994; Pyee and Kolattukudy, 1995; Kader,
1996; DeBono et al., 2009). The double null mutant of
the two GPI-anchored nsLTPs ltpg1/ltpg2 showed a
1920
Plant Physiol. Vol. 172, 2016
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Crown Gall Suberin Formation Involves AtLtpI-4
decrease in C29 alkane in stem waxes, a characteristic
compound of cuticular waxes of Arabidopsis inflorescence stems and siliques (DeBono et al., 2009). In contrast,
the cuticular wax of the atltpI-4 mutant stems showed no
alteration in the content of C29 alkane or of any other wax
compound compared with the wild type. This finding
reflects that AtLtpI-4 is endogenously not expressed in
epidermal cells and, therefore, is not involved in cuticle
formation on inflorescence stems. In crown galls, the loss
of atltpI-4 function caused a reduction in C18 long-chain
fatty acids. From our 3D model (Supplemental Fig. S6A)
and structure analyses by Han et al. (2001), we assume
that AtLtpI-4 can bind C18 fatty acids. Therefore, it seems
likely that AtltpI-4, as was postulated for the GPIanchored nsLTPs by Edstam et al. (2013), is involved in
suberin formation. Taking the above-mentioned findings together, we conclude that AtLtpI-4 is required for
periderm formation during crown gall development.
The periderm-like layer is essential for normal crown
gall growth, because it protects the crown gall from
desiccation and pathogen invasion (Efetova et al., 2007).
AtLtpI-4 Affects Extracellular Lipid Composition
To verify the idea that AtLtpI-4 affects extracellular
lipid composition, we at first tried to overexpress AtLtpI4 in Arabidopsis under the control of the cauliflower
mosaic virus 35S promoter. However, no transformants
could be obtained in three independent attempts to
generate stable transgenic lines. We then expressed the
AtLtpI-4 gene specifically in Arabidopsis epidermal cells
instead, a cell type that endogenously does not express
AtLtpI-4. The advantage of this approach is that one can
analyze the impact of AtLtpI-4 overexpression on extracellular lipid composition, because epidermis cells
exclusively produce the plant cuticle with cuticular
waxes as the outermost layer. Such plants with ectopic
overexpression of AtLtpI-4 in the epidermis showed an
increase in the amounts of two very-long-chain fatty
acids (C24 and C26), while homologs of carbon chain
lengths greater than C26 were decreased. Similarly, in
other aliphatic lipids, such as alkanes, primary alkanols,
and alkyl esters, homologs with chain lengths of C26 or
greater were decreased. Thus, the increase in the amount
of two fatty acids, which serve as precursors for the biosynthesis of VLCALs of the same and of longer chain
lengths, was accompanied by a decrease in VLCALs
downstream of the biosynthetic pathway of cuticular
waxes (Samuels et al., 2008). Principally, the shift in
composition and chain length distribution observed may
result from direct or indirect influences of AtLtpI-4 on
different steps in biosynthetic and transport processes.
Taking the results from lipid-protein-overlay assays and
3D homology modeling into account (see below), we
conclude that AtLtpI-4 preferentially binds and transports C24 and C26 fatty acids and, hence, removes these
substrates from downstream lipid biosynthesis.
The consequence of a shift from VLCALs with longer
lengths toward VLCALs with shorter carbon chain
lengths and more polar groups is an altered ultrastructure formed by epicuticular waxes. This most
likely results in an altered surface hydrophobicity of the
transgenic Arabidopsis leaves. It is known that the
composition of aliphatic lipids determines the microstructure of wax crystals and that this has an impact on
the water-repelling property of the leaf surface (Jeffree
et al., 1975; Barthlott and Neinhuis, 1997). The surface of
the rosette leaves of wild-type Arabidopsis plants is
smooth and contains almost no wax crystals. In contrast, the leaf surfaces of the Arabidopsis mutants
knobhead and bicentifolia (bcf1) are covered with platelike epicuticular wax crystals as a result of an altered
cuticular wax composition (Jenks et al., 1996). This
change in cuticular wax chemistry and ultrastructure
increases the surface permeability and loss of water,
even though the total wax load is not markedly changed
(Jenks et al., 2002; Xiao et al., 2004; Li et al., 2007). Other
than that, morphological abnormalities such as malformed glaucous leaves, organ fusions, or reduced sterile
flowers also occur when the quality or quantity of the
cuticular wax is affected. This is the case in the mutants
bcf1 and cuticular defect and organ fusion1 (Jenks et al., 1996;
Ukitsu et al., 2007). Similarly, an impaired cuticle formation causes dwarfism and abnormal leaf development,
with a reduced number and altered morphology of trichomes due to the loss of function of the extracellular a/b-hydrolase bodyguard or the suppression of
b-KETOACYL REDUCTASE expression (Kurdyukov
et al., 2006; Beaudoin et al., 2009). The transgenic plant
lines with gain of AtLtpI-4 function in the epidermis
displayed almost all of the above-described morphological abnormalities, such as reduced trichome branching
and leaf bending, but organ fusions were not observed.
The above-mentioned chemical and physical phenotypes
of the transgenic plants suggest that AtLtpI-4 affects the
composition of extracellularly deposited lipids.
To transfer C24 and C26 fatty acids into the apoplastic
space, the AtLtpI-4 protein needs to bind very-longchain fatty acids. To demonstrate this, we performed
3D homology modeling and lipid-protein-overlay
assays. According to the 3D model, the AtLtpI-4 protein can bind very-long-chain fatty acids as well as the
long-chain fatty acid C18. Analyses of the tertiary
structures of nsLTPs suggested a flexible hydrophobic
cavity with openings at both ends (Dong et al., 1995;
Lerche et al., 1997). The structures of maize nsLTP,
which were obtained from cocrystallization with model
substrates, reveal that aliphatic lipids of a chain length
shorter than 20 carbon units, such as decanoic acid (C10;
PDB entry 1FK0), tetradecanoic acid (C14; PDB entry
1FK1), hexadec-9-enoic acid (C16:1; PDB entry 1FK3),
and octadecanoic acid (C18; PDB entry 1FK4), can be
accommodated in this cavity (Han et al., 2001). However, these structures show that residual cavities in the
hydrophobic pocket, resulting from the short aliphatic
chain, were filled with water molecules. The presence of
water molecules possibly destabilizes and weakens
lipid binding, as these polar solvent molecules disrupt
or attenuate the hydrophobic interactions between the
Plant Physiol. Vol. 172, 2016
1921
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Deeken et al.
lipids and the protein. Hence, shorter fatty acids possibly might bind with lower affinities, whereas longer
fatty acids bind tighter, as they completely fill up the
hydrophobic chamber and thereby expel all solvent
molecules from the pocket. This hypothesis is consistent
with data indicating lower affinities for shorter aliphatic chains (Gomar et al., 1996) and structure analyses that show that LTP proteins also can accommodate
quite bulky cargo, such as two fatty acids inside rice or
wheat LTP1 (Charvolin et al., 1999; Cheng et al., 2004)
or a prostaglandin B2 inside wheat nsLTP (TassinMoindrot et al., 2000). Thus, LTP proteins seem to
have a highly adaptable cleft (Lerche et al., 1997) that
allows them to bind a wide range of hydrophobic
molecules, with binding strength being mainly scaled
by the number of interactions and restrained by geometric boundaries of the cleft volume (Zachowski et al.,
1998). Noteworthy, another more quantitative computational study, which analyzed the binding characteristics of rice nsLTP2 using molecular docking to embed
various fatty acids and subsequently calculating the
interaction energy for the lipid, also suggests that fatty
acids with longer aliphatic chains can bind to LTP
proteins with higher affinities (Tousheh et al., 2013).
According to our 3D model, carbon chains with more
than 28 carbons likely do not fit into the hydrophobic
cavity of the AtLtpI-4 protein due to the limited cavity
size. The lipid-protein-overlay assay was consistent
with this structure hypothesis and showed that the
AtLtpI-4 protein binds very-long-chain fatty acids
ranging from C22 or greater to C28 or less. The differences in binding C27 and C28 very-long-chain fatty acids
to the AtLtpI-4 protein derived from E. coli or tobacco
cells are likely due to the fact that the protein obtained
from E. coli was, first, purified and, second, applied at
higher concentration than the protein from BY-2 cells.
The latter was used as cell lysate, because of too-low
protein expression levels in plant cells. Furthermore,
our lipid-protein-overlay assay did not show binding of
the AtLtpI-4 protein to C16 to C20 long-chain fatty acids.
We explain this finding with the above-mentioned hypothesis that shorter fatty acids do not completely occupy the cavity and, thus, do not provide a sufficiently
high affinity to retain the AtLtpI-4 protein on the blot
surface during stringent washing of the membrane.
Taken together, in planta (epidermal expression), in silico
(3D models), and in vitro (lipid-protein-overlay) data
suggest that AtLtpI-4 is able to bind, in addition to longchain, also very-long-chain fatty acids.
CONCLUSION
This study provides evidence that the nsLTP AtLtpI-4 is
essential for crown gall growth. In crown galls, the
AtLtpI-4 protein, which is attached to the plasma membrane, functions in suberin assembly by facilitating the
extracellular delivery of long-chain fatty acids. However,
when AtLtpI-4 is ectopically overexpressed in epidermal
cells, it can also increase the amount of very-long-chain
fatty acids extracellularly in cuticular wax. Thus, the
AtLtpI-4 protein can transfer long-chain as well as verylong-chain fatty acids into the extracellular space and
functions as an nsLTP. Although AtLtpI-4 exhibits a
broad lipid-binding specificity, the type of cargo might be
limited by the substrate availability of the cell type in
which the nsLTP is expressed, thereby explaining the
differences observed in the range of transported fatty
acids in crown galls and epidermal cells. However, as the
expression of AtLtpI-4 in Arabidopsis is endogenously
limited to cell types that require suberization, like the
periderm of wounded tissues or the crown gall, its native
cargo consists of very-long-chain aliphatic compounds.
MATERIALS AND METHODS
Plant Material, Treatment, and Growth Conditions
The Arabidopsis (Arabidopsis thaliana) ecotypes used in this study were Ws-2
and Col-0 and the T-DNA-tagged line GK-639E08 (Nottingham Arabidopsis
Stock Centre; accession no. N736747). The homozygosity of the T-DNA-tagged
line was tested as described at http://wmd3.weigelworld.org using the
primers AtLtpI-4-LP, AtLtpI-4-RP, and BP (Supplemental Table S1A). Plants
were cultivated in a growth chamber (Percival Scientific; AR-60L S; www.
percival-scientific.com) on sterilized soil under a day/night cycle with 12 h of
light (80 mmol m22 s21) at 22°C/12 h of dark at 16°C and 60% relative humidity.
Arabidopsis root cultures were cultivated according to a protocol from Efetova
et al. (2007). Three-week-old root cultures were treated with 100 mM ABA or the
solvent methanol as a control. Cultivation of the T-DNA-transformed tobacco
(Nicotiana tabacum) cell culture line BY-2 was carried out as described by Nagata
et al. (1992) in the presence of 50 mg mL21 kanamycin. For wounding, Arabidopsis leaves were pricked 10 times with a set of 20 needles.
Preparation of Plasmid Constructs
The AtLtpI-4pro:GUS construct was generated by amplifying a 1,874-bp
genomic DNA fragment upstream of the AtLtpI-4 translational start site
(Arabidopsis DNA from ecotype Ws-2) and fused upstream to the GUS coding
sequence (CDS). The Gateway TA-TOPO cloning strategy was applied
according to the manufacturer’s instructions (Invitrogen) using the entry vector
pCR8/GW/TOPO and the binary destination vector pMDC164 (Curtis and
Grossniklaus, 2003). Primers used for PCR amplification were AtLtpI-4-Pro
sense and AtLtpI-4-Pro antisense (Supplemental Table S1A). Cloning of the
fusion protein constructs with the reporter genes mCherry and mVenus was
performed by applying the uracil excision-based cloning (USER) strategy, as
described by Nour-Eldin et al. (2006). The AtLtpI-5 and AtLtpI-4 coding sequences
were PCR amplified with the polymerase Phusion Cx (Finnzymes) and the primer
pairs UAtLtpI-5/UAtLtpI-4:mCherry sense and UAtLtpI-4 antisense and
UAtLtpI-5 antisense (Supplemental Table S1A). The resulting PCR products were
inserted at the 59 end of the reporter genes into a modified pSAT plasmid (Tzfira
et al., 2005), containing a UBIQUITIN10 promoter, followed by a uracil excisionbased cloning cassette (Nour-Eldin et al., 2006) and the mCherry coding sequence.
With the resulting plasmids and the primer pair UAtLtpI-4/AtLtpI-5:mCherry
sense and UAtLtpI-4/AtLtpI-5:mCherry antisense (Supplemental Table S1A), the
fragments AtLtpI-4:mCherry and AtLtpI-5:mCherry were PCR amplified and
transferred into the binary vector pCambia3300 (Cambia).
The ABCG12pro:AtLtpI-4 construct also was generated via the uracil excisionbased cloning strategy. A 2,618-bp fragment upstream of the ABCG12 start codon,
the putative ABCG12 promoter, and the AtLtpI-4 coding sequence were PCR
amplified and fused using the primer pairs ABCG12-Pro sense/ABCG12-Pro
antisense and AtLtpI-4-CDS sense/USERAtLtpI-4-CDS antisense (Supplemental
Table S1A). The overlapping PCR, which combines the AtLtpI-4 coding sequence with the new promoter, was performed using the primers USERABCG12Pro sense and USERAtLtpI-4-CDS antisense (Supplemental Table S1A). The
ABCG12pro:AtLtpI-4 construct was finally cloned into the binary vector pCambia3300 (Cambia).
To express the AtLtpI-4 protein in BY-2 cells, the AtLtpI-4 coding sequence
including the sequence encoding for the signal peptide was fused to a nucleotide
1922
Plant Physiol. Vol. 172, 2016
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Crown Gall Suberin Formation Involves AtLtpI-4
sequence encoding six His residues and a V5 epitope (AtLtpI-4:6xHis/V5) by
applying the directional TOPO cloning strategy according to the manufacturer’s
protocol (Invitrogen, Life Technologies). For PCR amplification, the primer pair
AtLtpI-4TOPO sense and AtLtpI-4TOPO antisense (Supplemental Table S1A)
was used, and for cloning the vector, the primer pair pcDNA3.1-D-V5/His-TOPO
was used. To obtain the final expression vector, the uracil excision-based cloning
strategy together with the primers AtLtpI-4USER sense and AtLtpI-4USER antisense (Supplemental Table S1A) was applied to transfer the AtLtpI-4:6xHis/V5
gene fusion into the binary vector pCambia2300 (Cambia).
For expression of the AtLtpI-4 protein in Escherichia coli cells, the nucleotide
sequence of the V5 epitope was fused to the 39 end of the AtLtpI-4 coding sequence lacking a stop codon and the export signal peptide (75 bp downstream of
the start codon). This AtLtpI-4:V5/HIS-DNA construct was inserted into the pET28b expression vector (Novagen, Merck) and cloned in cells of the E. coli strain
GenX [genotype BL21(DE3); www.genlantis.com] as follows. For PCR amplification, primers were used that contained NcoI and SalI restriction sites (AtLtpI-4NcoI sense and V5-SalI antisense; Supplemental Table S1A). The PCR product was
digested with the restriction enzymes NcoI and SalI (Fermentas, Thermo Fisher
Scientific), and the resulting fragment was inserted into the pET-28b vector linearized with NcoI and SalI using T4 ligase (Fermentas, Thermo Fisher Scientific).
All constructs were verified by sequencing (GATC; www.gatc-biotech.com).
X-100, 0.5 mM potassium ferricyanide, and 0.5 mM potassium ferrocyanide) for
20 min at 37°C. Chlorophyll from stained tissues was removed by incubation in
70% (v/v) ethanol at room temperature overnight. To visualize lignified cell
walls, cross sections were counterstained with 0.01% (w/v) safranin O (Alfa
Aesar) in 70% (v/v) ethanol for 5 s and then washed with distilled water for
20 min. For the preparation of GUS-stained semithin (15 mm) cross sections, the
dehydrated samples were embedded in 2-hydroxyethyl methacrylate according to the manufacturer’s protocol (Agar Scientific Electron Technology) and
sliced with a Leica RM 2165 microtome (Leica Microsystems).
Agrobacterium tumefaciens Strains, Plant Transformation
Procedures, and Crown Gall Harvest
Microscopic Analysis
The virulent nopaline-catabolizing A. tumefaciens strain C58 (no. 584; MaxPlanck-Institute for Plant Breeding Research) and the disarmed strain GV3101
(pMP90; Koncz and Schell, 1986) were used. Binary vectors were transformed
into agrobacteria using the Electroporator 2510 (Eppendorf). The growth of
agrobacteria and the infiltration of leaves with agrobacteria were carried out as
described by Efetova et al. (2007). Three-week-old Arabidopsis root cultures
(250 mL) were inoculated with a 150-mL suspension of virulent A. tumefaciens
strain C58 prepared according to Gohlke et al. (2013).
For the transient transformation of Nicotiana benthamiana, the disarmed strain
GV3101 was mixed with strain 19K to prevent gene silencing and infiltrated
into leaves according to the protocol of Latz et al. (2007). The fluorescence of
AtLtpI-4:mCherry, AtLtpI-5:mCherry, and SLAH3:mVenus was inspected 72 h
after infiltration. For leaf infiltration, a, optical density at 600 nm = 0.5 (Romeis
et al., 2001) was used.
Stably transformed plant lines were generated using floral dipping and the
disarmed A. tumefaciens strain GV3101 (Clough and Bent, 1998). Transgenic
seeds were selected on one-half-strength Murashige and Skoog medium
(Duchefa) supplemented with 1% (w/v) Suc and an antibiotic or herbicide,
depending on the resistance genes. Resistant progeny were screened for homozygosis by PCR using the cloning primers. Three independent lines of the
homozygous T3 generation were used in the experiments.
A stably transformed BY-2 cell suspension culture was generated by cocultivation with A. tumefaciens strain GV3101 harboring a binary vector. The
BY-2 cell suspension was divided under sterile conditions in 5-mL aliquots,
transferred to six-well plates, and mixed with 20 to 30 mL of A. tumefaciens
suspension, prepared after Gohlke et al. (2013). Two days after cocultivation at
21°C in the dark, 1 mL of the culture was transferred to 49 mL of growth medium (Nagata et al., 1992) with 200 mg L21 timentin/ticarcillin (Duchefa) and
cultivated for 10 d in the dark at 21°C. Thereafter, 5 mL of A. tumefaciens-free
transformed BY-2 cells was suspended in 45 mL of growth medium with
kanamycin (25 mg mL21). Ten days later, 5 mL of the transformed BY-2 cell
culture was diluted in 45 mL of growth medium with kanamycin (50 mg mL21)
and from then on subcultured in the same manner twice per week.
For induction of crown gall development, the virulent A. tumefaciens strain
C58 was inoculated at the base of young primary inflorescence stems (3–5 cm
long) of 8-week-old Arabidopsis plants, as described by Gohlke et al. (2013).
A. tumefaciens suspensions were injected four times using a syringe with a
needle attached. This procedure was repeated turning the stem by 90°. Three
weeks after inoculation, tumor material was carefully separated from the inflorescence stems using a scalpel and a binocular microscope (Leica MZ6; Leica
Microsystems) to prevent contamination with stem material.
GUS Staining Assay
GUS staining was performed by incubating plant tissue in GUS staining
solution (150 mM sodium phosphate buffer, pH 7, 10 mM EDTA, 0.5 mg mL21
5-bromo-4-chloro-3-indolyl b-D-glucuronic acid [Duchefa], 0.1% [v/v] Triton
Quantitative Real-Time PCR
Poly(A) mRNA was isolated from Arabidopsis organs and tissues using
Dynabeads Oligo (dT) according to the manufacturer’s protocol (Invitrogen, Life
Technologies). For the synthesis of cDNA, 10 mL of poly(A) mRNA was reverse
transcribed using Moloney murine leukemia virus reverse transcriptase as
described by the supplier (Promega). A 20-fold dilution of the cDNA reaction
was used for PCR amplification with the ABsolute QPCR SYBR Green Capillary
Mix (Abgene) in a Realplex Mastercycler (Eppendorf). Transcript numbers were
calculated according to the protocol of Szyroki et al. (2001). Primers used for
real-time PCR are listed in Supplemental Table S1B.
For the subcellular localization of fluorescing fusion proteins, confocal laser
scanning microscopy (TCS SP5 2; Leica) was used. The excitation wavelength
was set to 514 nm for mVenus and 561 nm for mCherry, and the emitted fluorescence was detected at 530 to 555 nm and 580 to 615 nm, respectively. The
fluorescence of mCherry was excited using a DPSS561 laser, and mVenus was
excited using an argon laser. Chlorophyll autofluorescence was detected at 650 to
700 nm. For all observations, a 253 objective (HC PL FLUOTAR 25 3 0.95,
water) was used. Overlay images (mCherry/mVenus) were created using the
software LAS AF (Leica Application Suite Advanced Fluorescence 2.4.1; Leica).
Plasmolysis was induced by incubating leaf sections in 1 M KNO3 for 10 min at
room temperature.
The Arabidopsis surface structure was analyzed using scanning electron
microscopy. Plant samples were fixed on a sample holder with conductive
carbon cement (Agar Scientific) and air dried for 4 d at room temperature. The
sample surface was then sputter coated three times for 50 s with gold-platinum at
25 mA (SCD 005; BAL-TEC). After surface coating, the samples were transferred
to the sample stage, observed, and digitally recorded with a field emission
scanning electron microscope (JSM-7500F; JEOL) employing an accelerating
voltage of 5 kV.
GUS-stained tissue and plant sections were inspected and documented with a
digital microscope (VHX-100; Keyence).
Protein Modeling
3D models of Arabidopsis AtLtpI-4 were generated by homology modeling
using the crystal structure(s) of nsLTP1 of maize (Zea mays; PDB entries 1FK0–
1FK7; Han et al., 2001) and rice (Oryza sativa; PDB entries 1UVA, 1UVB, and
1UVC; Cheng et al., 2004) as templates, which were determined as complexes
with different fatty acid molecules bound. First, an amino acid sequence
alignment was performed using the sequences of different Arabidopsis nsLTPs
and the sequences of maize and rice nsLTP1 using the software ClustalW and
the BLOSUM62 weight matrix. According to this alignment, AtLtpI-4 of Arabidopsis shares between 37% and 57% identity and about 57% to 69% similarity
at the amino acid sequence level with the sequences of the structure templates.
To obtain the homology model, amino acids differing between the target sequence of Arabidopsis AtLtpI-4 and the template nsLTP1 of maize (PDB entry
1FK0) were replaced using the tool ProteinDesign within the software package
QUANTA2008 (Accelrys). Deletions (loop between helices 1 and 2) and insertions (loop between helices 3 and 4) were modeled manually, assuming backbone conformations within the allowed regions of the Ramachandran plot. Van
der Waals overlaps between modeled side chains were eliminated by short
energy minimization (200 steps of an adopted-basis Newton-Raphson algorithm) and in vacuo molecular dynamics simulations (total 1 3 1,011 s, 1 3
1,015 s steps) using the CHARMM27 all-hydrogen force field and employing
only geometrical energy terms with a van der Waals energy cutoff of 11 Å. To
minimize drift from the original coordinates, the energy minimization/
molecular dynamics were done stepwise, first, with all backbone atoms kept
fixed and the amino acid side chains restrained by a harmonic potential of
Plant Physiol. Vol. 172, 2016
1923
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Deeken et al.
25 kcal mol21 Å22. The harmonic potential on the side chain atom position was
subsequently lowered to 10.5 and then to 0 kcal mol21 Å22. Finally, a short
energy minimization was performed with all atoms unrestrained.
Potential fatty acids fitting into the hydrophobic cavity formed by the four
helices were identified from analysis of the different template structures 1FK1-7
and 1UVB/1UVC (PDB entries) and the lipid molecules cocrystallized with
these nsLTPs (Han et al., 2001; Cheng et al., 2004). The structure 1UVB showed
that the hydrophobic cavity could accommodate two C16 fatty acid moieties in a
head-to-tail orientation (Cheng et al., 2004). Together with the template 1FK4,
which harbors a C18 fatty acid that adopts a hook-like conformation between
carbon atoms 14 and 18, longer aliphatic chains can be accommodated by
forming a sharp turn between carbon atoms 14 and 15 (Han et al., 2001). By
adopting this conformation, unsaturated fatty acids with a carbon chain length
between 22 and 26 were fitted into the cavity. These 3D models were again
energy minimized as described above by keeping the protein atoms restrained
by a strong positional harmonic restraint (50 kcal mol21 Å22) and applying no
restraints to the fatty acid molecule docked into the cavity. Inspection of the
carbon chain length showed a maximum length of 28 when slight conformational changes were applied to Tyr-81. The latter amino acid forms a cap to the
hydrophobic cavity, and its side chain conformation might be fixed through
hydrogen bond formation with the carboxylate group of the fatty acid moiety.
Protein Preparation
For heterologous expression of the AtLtpI-4 protein in bacteria, the plasmid
AtLtpI-4:V5/His was transferred into chemically competent cells of the E. coli
strain GenX [genotype BL21(DE3); www.genlantis.com] by heat shock (45 s at
42°C). Other E. coli strains also were tested for suitability to produce AtLtpI-4
protein, such as SoluBL21 (Amsbio) and BL21(DE3) Rosetta. However, expression in Gen-X produced the largest amount of soluble AtLtpI-4 protein.
After the induction of protein synthesis with 1 mM isopropyl b-D-thiogalactoside (Fermentas, Thermo Fisher Scientific), protein expression proceeded at
28°C overnight. Bacteria were harvested by centrifugation at 6,000g (5810R;
Eppendorf) for 15 min and resuspended in ice-cold binding buffer (50 mM
Na2HPO4, 300 mM NaCl, and 30 mM imidazole, pH 8). After cell lysis by sonication (20-s pulses and 20 s of resting for 10 min on ice [Bandelin Sonopuls
HD3200, 150W; amplitude, 60%]), cell debris were removed by centrifugation
at 10,000g for 30 min at 4°C and the clear supernatant was transferred to an
Ni2+-chelating Sepharose fast-flow column (Qiagen). The column material
was washed with binding buffer containing 0.1% (v/v) Triton X-100 to remove
impurities nonspecifically bound to the column. The protein then was eluted
using binding buffer supplemented with 500 mM imidazole. Protein-containing
fractions were dialyzed overnight at 4°C against 2 L of dialysis buffer (150 mM
NaCl and 50 mM HEPES, pH 8). As a second purification step, a preparative gel
filtration was applied using 20 mM Tris-HCl, pH 8, and 150 mM NaCl as a
running buffer and employing an XK16/60 Superdex 75 prep grade (GE
Healthcare). Fractions were analyzed by SDS-PAGE under nonreducing conditions, and fractions containing AtLtpI-4 protein of sufficient purity were
pooled, flash frozen, and stored at 280°C until further use.
For protein extraction from BY-2 suspension cell cultures, cells were homogenized in buffer (250 mM Suc, 2 mM EDTA, 2 mM dithiothreitol, 2 mM
phenylmethylsulfonyl fluoride, and 5% [w/v] polyvinylpyrrolodine in 30 mM
Tris-HCl, pH 7.5) prior to protein extraction. The homogenate was centrifuged
at 10,000g for 30 min (5810R; Eppendorf) at 4°C. To remove insoluble impurities, the supernatant was filtered using a mesh (pore size 22–25 mm; Miracloth;
Merck) and stored at 4°C until further use. Interaction analyses were performed
using the crude protein extract and detecting lipid binding via the V5 affinity
tag sequence and an anti-V5 antibody.
Western-Blot Analysis
For western-blot analyses, Tris-Tricine SDS-PAGE was performed as described by Schägger (2006) using a 4% (v/v) stacking gel, a 10% (v/v) spacer gel,
and a 16% (v/v) running gel for protein separation. After Tris-Tricine SDSPAGE, proteins were transferred onto nitrocellulose membranes (pore size,
0.2 mm; Whatman Protran; Sigma-Aldrich) using anode buffer I (300 mM TrisHCl, pH 10.4, and 20% [v/v] methanol), anode buffer II (25 mM Tris-HCl, pH
10.4, and 20% [v/v] methanol), and cathode buffer (25 mM Tris-HCl/40 mM
aminocaproic acid, pH 9.4, and 20% [v/v] methanol) at 50 mA and 20 V within
1 h. Protein detection was performed after washing twice with Tris-buffered
saline (TBS; 137 mM NaCl and 10 mM Tris-HCl, pH 8), subsequent blocking in
buffer (137 mM NaCl, 10 mM Tris-HCl, and 5% [w/v] albumin [AppliChem], pH
8), and washing twice with TBS supplemented with 0.05% (v/v) Tween 20.
Membranes were then incubated with a 1:10,000 dilution of the monoclonal
goat anti-His antibody (Invitrogen, Life Technologies) for 1 h at room
temperature. After removal of the primary antibody and washing with TBS
supplemented with 0.05% (v/v) Tween 20, the secondary antibody, a peroxidaseconjugated goat anti-mouse IgG (Invitrogen, Life Technologies), was added, and
the membranes were incubated in this solution overnight at 4°C. Chemiluminescence detection was performed according to the manufacturer’s instructions
(Pierce) and documented on x-ray films (Kodak-Biomax Light).
Protein-Lipid-Overlay Assay
Lipid-protein-overlay assays were carried out as described by Dowler et al.
(2002). Fatty acids and their derivatives dissolved in a 1:1 mixture of methanol:
chloroform were spotted (5 mg each; Invitrogen, Life Technologies and
A. Hansjakob, University of Wuerzburg) on nitrocellulose membranes of 0.2
mm pore size (Whatman Protran; Sigma-Aldrich). Protein binding and detection were performed according to western-blot analysis.
Chlorophyll-Leaching Assay
Rosette leaves of 8-week-old nonflowering Arabidopsis plants were used for
the chlorophyll-leaching assay and analyzed according to the protocol of Xia
et al. (2009). Chlorophyll absorbance of the solutions was measured at 664 and
647 nm using a spectral photometer (ATI UNICAM UV/Vis spectrophotometer
UV 4-500; Unicam). The chlorophyll concentration was calculated according to
the equation described by Xia et al. (2009): mmol chlorophyll g21 fresh weight =
(7.93 3 A664 + 19.53 3 A647) g21 fresh weight.
Contact-Angle Measurement
Contact-angle measurements were performed by pipetting 1 mL of distilled
water onto the surface of 8-week-old adult Arabidopsis rosette leaves and
employing the contact-angle system (OCA 15) together with the software SCA20
(Dataphysics Instruments). The means of contact angle values were calculated
from six droplets per leaf, three leaves per plant, and three plants per plant line.
Analysis of Cuticular Wax from Leaves
For the extraction of cuticular waxes, a fully expanded leaf of a 12-week-old
Arabidopsis plant was harvested from 10 different plants. Leaf surfaces were
cleaned by carefully rinsing with deionized water and dried with paper tissue.
Wax extraction was performed by immersion of these 10 leaves in 15 mL of
chloroform (99.9% or greater [v/v]; Roth) for 30 s to get one pooled sample. Three
pooled samples were prepared for each plant line. N-Tetracosane (C24; SigmaAldrich) was added as an internal standard to all samples, and the solvent was
evaporated under a gentle stream of N2. Chemical analyses were performed as
described by Ringelmann et al. (2009) using a 7890A gas chromatograph
(Agilent Technologies) and the column type 30 m DB-1HT (0.32 mm i.d., degrees of freedom = 0.1 mm; Agilent Technologies) for quantification. Accordingly, single compounds (where applicable as trimethylsilyl derivatives) were
identified by comparison of their mass spectra with those of published data,
commercially available databases, as well as authentic standards.
Analysis of Crown Gall Suberin, Suberin-Associated
Waxes, and Stem Cuticular Waxes
For suberin analysis, the crown gall tumor material was pooled. To collect the
same amount of crown gall material, three to four times more atltpI-4 mutant plants
were required than of the wild type. Stem material was collected separately from
sections above the crown galls. The crown gall and stem material was enzymatically digested with pectinase (Trenolin Super DF; Erbslöh) and cellulase (Celluclast; Novo Nordisk) in 20 mM citrate buffer, pH 3, supplemented with 1 mM
sodium azide according to Schönherr and Riederer (1986), and washed
exhaustingly with deionized water. The air-dried material was weighed, and the
soluble wax-like compounds were extracted using chloroform (Roth) at 40°C for
1 h. The insoluble crown gall material was transesterified with methanolic boron
trifluoride (1.3 M boron trifluoride in methanol; Fluka) at 70°C overnight to release
ester-bound suberin compounds. Depolymerized methyl ester monomers were
extracted with chloroform from an aqueous two-phase system (Leide et al., 2012).
Before gas chromatographic analysis, solvent-soluble and transmethylated
1924
Plant Physiol. Vol. 172, 2016
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Crown Gall Suberin Formation Involves AtLtpI-4
components from crown galls and stems were transformed into the corresponding trimethylsilyl derivatives using N,O-bis-trimethylsilyl-trifluoroacetamide
(Macherey-Nagel) dissolved in pyridine (Merck). The qualitative and quantitative composition was determined by referring to n-dotriacontane (C32; SigmaAldrich) as an internal standard as described by Leide et al. (2007).
Accession Numbers
Sequence data from this article are available from the GenBank/EMBL data
libraries under the following accession numbers: ACTIN2/8 (AT3G18780/
AT1G49240), ABCG12 (At1g51500), AtLtpI-4 (At2g38540), AtLtpI-5 (At2g38530),
AtLtpI-6 (At3G08770), AtLtpI-7 (At3G51590), AtLtpI-11 (At5G59310), AtLtpI-12
(At5G59320), LTPG2 (AT3G43720), and SLAH3 (At5g24030). The T-DNA-tagged
Arabidopsis line of the AtLtpI-4 gene locus was obtained from the Nottingham
Arabidopsis Stock Centre with accession number N736747.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Gene structure of the atltpI-4 mutant locus and
expression pattern of LTP genes in Arabidopsis.
Supplemental Figure S2. Relative gene expression levels of AtLtpI-4 in
Arabidopsis wild-type plants (ecotype Ws-2) under different physiological conditions and upon ectopic expression in epidermal cells.
Supplemental Figure S3. Subcellular localization of Arabidopsis AtLtpI-4
and AtLtpI-5 proteins in tobacco epidermal cells.
Supplemental Figure S4. Total amount of solvent-soluble and insoluble
esterified suberin compounds in crown galls of Arabidopsis wild-type
(Col-0) and mutant (atltpI-4) plants.
Supplemental Figure S5. Cuticular wax monomer composition of the inflorescence stems above the crown gall from 12-week-old Arabidopsis
wild-type (Col-0) and mutant (atltpI-4) plants.
Supplemental Figure S6. Template structures for modeling of the AtLtpI4-fatty acid complexes.
Supplemental Figure S7. Heterologous expression of the AtLtpI-4 protein.
Supplemental Table S1. Primer pairs used in PCR for plasmid constructs
and primer pairs used for quantitative real-time PCR.
ACKNOWLEDGMENTS
We thank GABI-Kat (Max-Planck-Institute of Plant Breeding) and the
European Arabidopsis Stock Centre (University of Nottingham) for the
sequence-indexed Arabidopsis T-DNA insertion mutant; D. Geiger (University
of Wuerzburg) for providing the uracil excision-based cloning vectors used in
this study; and T.A. Cuin (University of Wuerzburg) for critical reading of the
article.
Received September 26, 2016; accepted September 27, 2016; published September 29, 2016.
LITERATURE CITED
Arondel V XII, Vergnolle C II, Cantrel C, Kader J (2000) Lipid transfer
proteins are encoded by a small multigene family in Arabidopsis thaliana.
Plant Sci 157: 1–12
Barthlott W, Neinhuis C (1997) Purity of the sacred lotus, or escape from
contamination in biological surfaces. Planta 202: 1–8
Beaudoin F, Wu X, Li F, Haslam RP, Markham JE, Zheng H, Napier JA,
Kunst L (2009) Functional characterization of the Arabidopsis b-ketoacylcoenzyme A reductase candidates of the fatty acid elongase. Plant Physiol
150: 1174–1191
Bernards MA (2002) Demystifying suberin. Can J Bot 80: 227–240
Bird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu X,
Yephremov A, Samuels L (2007) Characterization of Arabidopsis
ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is
required for cuticular lipid secretion. Plant J 52: 485–498
Bleecker AB, Patterson SE (1997) Last exit: senescence, abscission, and
meristem arrest in Arabidopsis. Plant Cell 9: 1169–1179
Blein JP, Coutos-Thévenot P, Marion D, Ponchet M (2002) From elicitins
to lipid-transfer proteins: a new insight in cell signalling involved in
plant defence mechanisms. Trends Plant Sci 7: 293–296
Blilou I, Ocampo JA, García-Garrido JM (2000) Induction of Ltp (lipid
transfer protein) and Pal (phenylalanine ammonia-lyase) gene expression in rice roots colonized by the arbuscular mycorrhizal fungus Glomus mosseae. J Exp Bot 51: 1969–1977
Boutrot F, Chantret N, Gautier MF (2008) Genome-wide analysis of the rice
and Arabidopsis non-specific lipid transfer protein (nsLtp) gene families
and identification of wheat nsLtp genes by EST data mining. BMC Genomics 9: 86
Buhot N, Gomès E, Milat ML, Ponchet M, Marion D, Lequeu J, Delrot S,
Coutos-Thévenot P, Blein JP (2004) Modulation of the biological activity of a tobacco LTP1 by lipid complexation. Mol Biol Cell 15: 5047–
5052
Charvolin D, Douliez JP, Marion D, Cohen-Addad C, Pebay-Peyroula E
(1999) The crystal structure of a wheat nonspecific lipid transfer protein
(ns-LTP1) complexed with two molecules of phospholipid at 2.1 A resolution. Eur J Biochem 264: 562–568
Cheng HC, Cheng PT, Peng P, Lyu PC, Sun YJ (2004) Lipid binding in rice
nonspecific lipid transfer protein-1 complexes from Oryza sativa. Protein
Sci 13: 2304–2315
Chilton MD, Drummond MH, Merio DJ, Sciaky D, Montoya AL, Gordon
MP, Nester EW (1977) Stable incorporation of plasmid DNA into
higher plant cells: the molecular basis of crown gall tumorigenesis. Cell
11: 263–271
Clough SJ, Bent AF (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J
16: 735–743
Curtis MD, Grossniklaus U (2003) A Gateway cloning vector set for highthroughput functional analysis of genes in planta. Plant Physiol 133:
462–469
DeBono A, Yeats TH, Rose JKC, Bird D, Jetter R, Kunst L, Samuels L
(2009) Arabidopsis LTPG is a glycosylphosphatidylinositol-anchored
lipid transfer protein required for export of lipids to the plant surface.
Plant Cell 21: 1230–1238
Deeken R, Engelmann JC, Efetova M, Czirjak T, Müller T, Kaiser WM,
Tietz O, Krischke M, Mueller MJ, Palme K, et al (2006) An integrated
view of gene expression and solute profiles of Arabidopsis tumors: a
genome-wide approach. Plant Cell 18: 3617–3634
Dong HS, Lee JY, Hwang KY, Kim KK, Suh WS (1995) High-resolution
crystal structure of the non-specific lipid-transfer protein from maize
seedlings. Structure 2: 189–199
Dowler S, Kular G, Alessi DR (2002) Protein lipid overlay assay. Sci STKE
2002: pl6
Edstam MM, Blomqvist K, Eklöf A, Wennergren U, Edqvist J (2013) Coexpression patterns indicate that GPI-anchored non-specific lipid
transfer proteins are involved in accumulation of cuticular wax, suberin
and sporopollenin. Plant Mol Biol 83: 625–649
Efetova M, Zeier J, Riederer M, Lee CW, Stingl N, Mueller M, Hartung
W, Hedrich R, Deeken R (2007) A central role of abscisic acid in drought
stress protection of Agrobacterium-induced tumors on Arabidopsis.
Plant Physiol 145: 853–862
Enstone DE, Peterson CA (2005) Suberin lamella development in maize
seedlings roots grown in aerated and stagnant conditions. Plant Cell
Environ 25: 444–455
Franke R, Briesen I, Wojciechowski T, Faust A, Yephremov A, Nawrath
C, Schreiber L (2005) Apoplastic polyesters in Arabidopsis surface tissues: a typical suberin and a particular cutin. Phytochemistry 66: 2643–
2658
Franke R, Höfer R, Briesen I, Emsermann M, Efremova N, Yephremov A,
Schreiber L (2009) The DAISY gene from Arabidopsis encodes a fatty acid
elongase condensing enzyme involved in the biosynthesis of aliphatic
suberin in roots and the chalaza-micropyle region of seeds. Plant J 57:
80–95
Franke R, Schreiber L (2007) Suberin: a biopolyester forming apoplastic
plant interfaces. Curr Opin Plant Biol 10: 252–259
Geiger D, Maierhofer T, Al-Rasheid KA, Scherzer S, Mumm P, Liese A,
Ache P, Wellmann C, Marten I, Grill E, et al (2011) Stomatal closure by
fast abscisic acid signaling is mediated by the guard cell anion channel
SLAH3 and the receptor RCAR1. Sci Signal 4: ra32
Plant Physiol. Vol. 172, 2016
1925
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Deeken et al.
Gohlke J, Scholz CJ, Kneitz S, Weber D, Fuchs J, Hedrich R, Deeken R
(2013) DNA methylation mediated control of gene expression is critical
for development of crown gall tumors. PLoS Genet 9: e1003267
Gomar J, Petit MC, Sodano P, Sy D, Marion D, Kader JC, Vovelle F, Ptak
M (1996) Solution structure and lipid binding of a nonspecific lipid
transfer protein extracted from maize seeds. Protein Sci 5: 565–577
Gomès E, Sagot E, Gaillard C, Laquitaine L, Poinssot B, Sanejouand YH,
Delrot S, Coutos-Thévenot P (2003) Nonspecific lipid-transfer protein
genes expression in grape (Vitis sp.) cells in response to fungal elicitor
treatments. Mol Plant Microbe Interact 16: 456–464
Graça J, Santos S (2007) Suberin: a biopolyester of plants’ skin. Macromol
Biosci 7: 128–135
Han GW, Lee JY, Song HK, Chang C, Min K, Moon J, Shin DH, Kopka
ML, Sawaya MR, Yuan HS, et al (2001) Structural basis of non-specific
lipid binding in maize lipid-transfer protein complexes revealed by
high-resolution x-ray crystallography. J Mol Biol 308: 263–278
Jeffree CE (1996) Structure and ontogeny of plant cuticles. In G Kerstiens,
ed, Plant Cuticles: An Integrated Functional Approach. Bios Scientific
Publishers, Oxford, pp 33–82
Jeffree CE, Baker EA, Holloway PJ (1975) Ultrastructure and recrystallization of plant epicuticular waxes. New Physiol 75: 539–549
Jenks MA, Eigenbrode SD, Lemieux D (2002) Cuticular waxes of Arabidopsis. The Arabidopsis Book doi/http://dx.doi.org/10.1199/tab.0016
Jenks MA, Rashotte AM, Tuttle HA, Feldmann KA (1996) Mutants in
Arabidopsis thaliana altered in epicuticular wax and leaf morphology.
Plant Physiol 110: 377–385
Jung HW, Tschaplinski TJ, Wang L, Glazebrook J, Greenberg JT (2009)
Priming in systemic plant immunity. Science 324: 89–91
Kader JC (1996) Lipid-transfer proteins in plants. Annu Rev Plant Physiol
Plant Mol Biol 47: 627–654
Kader JC, Julienne M, Vergnolle C (1984) Purification and characterization
of a spinach-leaf protein capable of transferring phospholipids from
liposomes to mitochondria or chloroplasts. Eur J Biochem 139: 411–416
Kim H, Lee SB, Kim HJ, Min MK, Hwang I, Suh MC (2012) Characterization of
glycosylphosphatidylinositol-anchored lipid transfer protein 2 (LTPG2) and
overlapping function between LTPG/LTPG1 and LTPG2 in cuticular wax export or accumulation in Arabidopsis thaliana. Plant Cell Physiol 53: 1391–1403
Klee H, Montoya A, Horodyski F, Lichtenstein C, Garfinkel D, Fuller S,
Flores C, Peschon J, Nester E, Gordon M (1984) Nucleotide sequence of
the tms genes of the pTiA6NC octopine Ti plasmid: two gene products
involved in plant tumorigenesis. Proc Natl Acad Sci USA 81: 1728–1732
Klinkenberg J, Faist H, Saupe S, Lambertz S, Krischke M, Stingl N, Fekete A,
Mueller MJ, Feussner I, Hedrich R, et al (2014) Two fatty acid desaturases,
STEAROYL-ACYL CARRIER PROTEIN D9-DESATURASE6 and FATTY
ACID DESATURASE3, are involved in drought and hypoxia stress signaling
in Arabidopsis crown galls. Plant Physiol 164: 570–583
Kolattukudy PE (2001) Polyesters in higher plants. Adv Biochem Eng Biotechnol 71: 1–49
Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the
tissue specific expression of chimaeric genes carried by a novel type of
Agrobacterium binary vector. Mol Gen Genet 204: 383–396
Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular
wax. Prog Lipid Res 42: 51–80
Kurdyukov S, Faust A, Nawrath C, Bär S, Voisin D, Efremova N, Franke
R, Schreiber L, Saedler H, Métraux JP, et al (2006) The epidermisspecific extracellular BODYGUARD controls cuticle development and
morphogenesis in Arabidopsis. Plant Cell 18: 321–339
Latz A, Ivashikina N, Fischer S, Ache P, Sano T, Becker D, Deeken R,
Hedrich R (2007) In planta AKT2 subunits constitute a pH- and Ca2+sensitive inward rectifying K+ channel. Planta 225: 1179–1191
Lee SB, Go YS, Bae HJ, Park JH, Cho SH, Cho HJ, Lee DS, Park OK,
Hwang I, Suh MC (2009) Disruption of glycosylphosphatidylinositolanchored lipid transfer protein gene altered cuticular lipid composition,
increased plastoglobules, and enhanced susceptibility to infection by the
fungal pathogen Alternaria brassicicola. Plant Physiol 150: 42–54
Leide J, Hildebrandt U, Hartung W, Riederer M, Vogg G (2012) Abscisic
acid mediates the formation of a suberized stem scar tissue in tomato
fruits. New Phytol 194: 402–415
Leide J, Hildebrandt U, Reussing K, Riederer M, Vogg G (2007) The developmental pattern of tomato fruit wax accumulation and its impact on
cuticular transpiration barrier properties: effects of a deficiency in a
b-ketoacyl-coenzyme A synthase (LeCER6). Plant Physiol 144: 1667–
1679
Lerche MH, Kragelund BB, Bech LM, Poulsen FM (1997) Barley lipidtransfer protein complexed with palmitoyl CoA: the structure reveals
a hydrophobic binding site that can expand to fit both large and small
lipid-like ligands. Structure 5: 291–306
Li Y, Beisson F, Koo AJK, Molina I, Pollard M, Ohlrogge J (2007) Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc Natl Acad Sci USA
104: 18339–18344
Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates
PD, Baud S, Bird D, DeBono A, Durrett TP, et al (2013) Acyl-lipid
metabolism. The Arabidopsis Book 11: e0161, doi/10.1199/tab.0161
Lichtenstein C, Klee H, Montoya A, Garfinkel D, Fuller S, Flores C,
Nester E, Gordon M (1984) Nucleotide sequence and transcript mapping of the tmr gene of the pTiA6NC octopine Ti-plasmid: a bacterial
gene involved in plant tumorigenesis. J Mol Appl Genet 2: 354–362
Maldonado AM, Doerner P, Dixon RA, Lamb CJ, Cameron RK (2002) A
putative lipid transfer protein involved in systemic resistance signalling
in Arabidopsis. Nature 419: 399–403
Mattinen ML, Filpponen I, Järvinen R, Li B, Kallio H, Lehtinen P,
Argyropoulos D (2009) Structure of the polyphenolic component of
suberin isolated from potato (Solanum tuberosum var. Nikola). J Agric
Food Chem 57: 9747–9753
McFarlane HE, Shin JJ, Bird DA, Samuels AL (2010) Arabidopsis ABCG
transporters, which are required for export of diverse cuticular lipids,
dimerize in different combinations. Plant Cell 22: 3066–3075
Molina A, García-Olmedo F (1997) Enhanced tolerance to bacterial pathogens
caused by the transgenic expression of barley lipid transfer protein LTP2.
Plant J 12: 669–675
Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the
“HeLa” cell in the cell biology of higher plants. Int Rev Cytol 132: 1–30
Nawrath C (2002) The biopolymers cutin and suberin. The Arabidopsis
Book 1: e0021, doi/10.1199/tab.0021
Nawrath C, Schreiber L, Franke RB, Geldner N, Reina-Pinto JJ, Kunst L
(2013) Apoplastic diffusion barriers in Arabidopsis. The Arabidopsis
Book 11: e0167, doi/10.1199/tab.0167
North GB, Martre P, Nobel PS (2004) Aquaporins account for variations in
hydraulic conductance for metabolically active root regions of Agave
deserti in wet, dry and rewetted soil. Plant Cell Environ 27: 219–228
Nour-Eldin HH, Hansen BG, Nørholm MH, Jensen JK, Halkier BA (2006)
Advancing uracil-excision based cloning towards an ideal technique for
cloning PCR fragments. Nucleic Acids Res 34: e122
Pagnussat L, Burbach C, Baluška F, de la Canal L (2012) An extracellular
lipid transfer protein is relocalized intracellularly during seed germination. J Exp Bot 63: 6555–6563
Panikashvili D, Aharoni A (2008) ABC-type transporters and cuticle assembly: linking function to polarity in epidermis cells. Plant Signal Behav 3: 806–809
Panikashvili D, Savaldi-Goldstein S, Mandel T, Yifhar T, Franke RB,
Höfer R, Schreiber L, Chory J, Aharoni A (2007) The Arabidopsis
DESPERADO/AtWBC11 transporter is required for cutin and wax secretion. Plant Physiol 145: 1345–1360
Panikashvili D, Shi JX, Bocobza S, Franke RB, Schreiber L, Aharoni A
(2010) The Arabidopsis DSO/ABCG11 transporter affects cutin metabolism in reproductive organs and suberin in roots. Mol Plant 3: 563–575
Panikashvili D, Shi JX, Schreiber L, Aharoni A (2011) The Arabidopsis
ABCG13 transporter is required for flower cuticle secretion and patterning of the petal epidermis. New Phytol 190: 113–124
Pighin JA, Zheng H, Balakshin LJ, Goodman IP, Western TL, Jetter R,
Kunst L, Samuels AL (2004) Plant cuticular lipid export requires an
ABC transporter. Science 306: 702–704
Pyee J, Kolattukudy PE (1995) The gene for the major cuticular waxassociated protein and three homologous genes from broccoli (Brassica
oleracea) and their expression patterns. Plant J 7: 49–59
Rashotte AM, Jenks MA, Feldmann KA (2001) Cuticular waxes on eceriferum mutants of Arabidopsis thaliana. Phytochemistry 57: 115–123
Reinhardt DH, Rost TL (1995) Salinity accelerates endodermal development and induces an exodermis in cotton seedling roots. Environ Exp
Bot 35: 563–574
Ringelmann A, Riedel M, Riederer M, Hildebrandt U (2009) Two sides of
a leaf blade: Blumeria graminis needs chemical cues in cuticular waxes of
Lolium perenne for germination and differentiation. Planta 230: 95–105
Roberts JA, Elliott KA, Gonzalez-Carranza ZH (2002) Abscission, dehiscence,
and other cell separation processes. Annu Rev Plant Biol 53: 131–158
1926
Plant Physiol. Vol. 172, 2016
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Crown Gall Suberin Formation Involves AtLtpI-4
Romeis T, Ludwig AA, Martin R, Jones JD (2001) Calcium-dependent protein
kinases play an essential role in a plant defence response. EMBO J 20: 5556–5567
Samuels L, Kunst L, Jetter R (2008) Sealing plant surfaces: cuticular wax
formation by epidermal cells. Annu Rev Plant Biol 59: 683–707
Saupe S (2014) Funktion des Lipidtransferproteins 2 (LTP2) und dessen
Rolle bei der Bildung von durch Agrobacterium tumefaciens induzierten
Wurzelhalsgallen an Arabidopsis thaliana. PhD thesis. Julius-MaximiliansUniversity of Wuerzburg, Wuerzburg, Germany, https://opus.bibliothek.
uni-wuerzburg.de/frontdoor/index/index/docId/10544
Schägger H (2006) Tricine-SDS-PAGE. Nat Protoc 1: 16–22
Schönherr J, Riederer M (1986) Plant cuticles sorb lipophilic compounds
during enzymatic isolation. Plant Cell Environ 9: 459–466
Schreiber L (2010) Transport barriers made of cutin, suberin and associated
waxes. Trends Plant Sci 15: 546–553
Schreiber L, Franke R, Hartmann K (2005) Wax and suberin development
of native and wound periderm of potato (Solanum tuberosum L.) and its
relation to peridermal transpiration. Planta 220: 520–530
Schreiber L, Hartmann K, Skabs M, Zeier J (1999) Apoplastic barriers in
roots: chemical composition of endodermal and hypodermal cell walls. J
Exp Bot 50: 1267–1280
Sterk P, Booij H, Schellekens GA, Van Kammen A, De Vries SC (1991)
Cell-specific expression of the carrot EP2 lipid transfer protein gene.
Plant Cell 3: 907–921
Szyroki A, Ivashikina N, Dietrich P, Roelfsema MR, Ache P, Reintanz B,
Deeken R, Godde M, Felle H, Steinmeyer R, et al (2001) KAT1 is not
essential for stomatal opening. Proc Natl Acad Sci USA 98: 2917–2921
Tassin-Moindrot S, Caille A, Douliez JP, Marion D, Vovelle F (2000) The wide
binding properties of a wheat nonspecific lipid transfer protein: solution
structure of a complex with prostaglandin B2. Eur J Biochem 267: 1117–1124
Thoma S, Hecht U, Kippers A, Botella J, De Vries S, Somerville C (1994)
Tissue-specific expression of a gene encoding a cell wall-localized lipid
transfer protein from Arabidopsis. Plant Physiol 105: 35–45
Thomashow MF, Nutter R, Montoya AL, Gordon MP, Nester EW (1980)
Integration and organization of Ti plasmid sequences in crown gall tumors. Cell 19: 729–739
Tousheh M, Miroliaei M, Asghar Rastegari A, Ghaedi K, Esmaeili A,
Matkowski A (2013) Computational evaluation on the binding affinity
of non-specific lipid-transfer protein-2 with fatty acids. Comput Biol
Med 43: 1732–1738
Tzfira T, Tian GW, Lacroix B, Vyas S, Li J, Leitner-Dagan Y, Krichevsky
A, Taylor T, Vainstein A, Citovsky V (2005) pSAT vectors: a modular
series of plasmids for autofluorescent protein tagging and expression of
multiple genes in plants. Plant Mol Biol 57: 503–516
Ukitsu H, Kuromori T, Toyooka K, Goto Y, Matsuoka K, Sakuradani E,
Shimizu S, Kamiya A, Imura Y, Yuguchi M, et al (2007) Cytological
and biochemical analysis of COF1, an Arabidopsis mutant of an ABC
transporter gene. Plant Cell Physiol 48: 1524–1533
Wang SY, Wu JH, Ng TB, Ye XY, Rao PF (2004) A non-specific lipid
transfer protein with antifungal and antibacterial activities from the
mung bean. Peptides 25: 1235–1242
Wang X, Wang H, Li Y, Cao K, Ge X (2009) A rice lipid transfer protein
binds to plasma membrane proteinaceous sites. Mol Biol Rep 36:
745–750
Xia Y, Gao QM, Yu K, Lapchyk L, Navarre D, Hildebrand D, Kachroo A,
Kachroo P (2009) An intact cuticle in distal tissues is essential for the
induction of systemic acquired resistance in plants. Cell Host Microbe 5:
151–165
Xiao F, Goodwin SM, Xiao Y, Sun Z, Baker D, Tang X, Jenks MA, Zhou
JM (2004) Arabidopsis CYP86A2 represses Pseudomonas syringae type
III genes and is required for cuticle development. EMBO J 23: 2903–
2913
Yeats TH, Rose JKC (2013) The formation and function of plant cuticles.
Plant Physiol 163: 5–20
Yubero-Serrano EM, Moyano E, Medina-Escobar N, Muñoz-Blanco J,
Caballero JL (2003) Identification of a strawberry gene encoding a nonspecific lipid transfer protein that responds to ABA, wounding and cold
stress. J Exp Bot 54: 1865–1877
Zachowski A, Guerbette F, Grosbois M, Jolliot-Croquin A, Kader JC
(1998) Characterisation of acyl binding by a plant lipid-transfer protein.
Eur J Biochem 257: 443–448
Plant Physiol. Vol. 172, 2016
1927
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
CORRECTION
Vol. 172: 1911–1927, 2016
Deeken R., Saupe S., Klinkenberg J., Riedel M., Leide J., Hedrich R., and Mueller T.D. The
Nonspecific Lipid Transfer Protein AtLtpI-4 Is Involved in Suberin Formation of Arabidopsis
thaliana Crown Galls.
In the Accession Numbers listed for this article, the AGI codes for the AtLtpI-4 and AtLtpI-5
genes were mistakenly reversed. The correct annotation should read as follows: AtLtpI-4
(At2g38530) and AtLtpI-5 (At2g38540).
www.plantphysiol.org/cgi/doi/10.1104/pp.17.00171
1936
Plant PhysiologyÒ, March 2017, Vol. 173, p. 1936, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved.