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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. 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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.