Primary Fatty Alcohols Are Major Components of Suberized Root Tissues of Arabidopsis in the Form of Alkyl Hydroxycinnamates^,

Fatty alcohols that are not covalently linked to the polymer suberin in Arabidopsis roots are soluble waxes in the form of alkyl hydroxycinnamates.

Abstract

Suberin is a complex hydrophobic polymer that acts as a barrier controlling water and solute fluxes and restricting pathogen infections. Suberin is deposited immediately outside of the plasmalemma in the cell wall of certain tissues such as endodermis of roots, aerial and underground periderms, and seed coats. Suberin consists of a variety of fatty acid derivatives polymerized with glycerol and phenolics. In this study, we show using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry techniques that most of the fatty alcohols not covalently linked to the suberin polymer are in the form of alkyl hydroxycinnamates (AHCs), with alkyl caffeates predominating. Such compounds are not restricted to the periderm of mature roots but also are present in the endodermis of younger roots, where they are not extracted by rapid dipping in chloroform. Analysis of several mutants affected in key enzymes involved in the biosynthesis and export of suberin monomers suggests that the formation of the suberin polymer and associated waxes involves common pathways and occurs concomitantly in Arabidopsis (Arabidopsis thaliana) roots. Although fatty alcohols represent only minor components of the suberin polymer in Arabidopsis roots, this study demonstrates that they constitute the major aliphatics of suberin-associated waxes in the form of AHCs. Therefore, our results indicate that esterified fatty alcohols, both soluble and polymerized forms, represent major constituents of Arabidopsis root suberized barriers, being as abundant as α,ω-dicarboxylic and unsubstituted fatty acids. In addition, our results show that suberized layers represent a major sink for acyl-lipid metabolism in Arabidopsis roots.

Suberin is a hydrophobic plant biopolymer that acts mainly as a protective barrier to control the movements of water, solutes, and gases and to limit pathogen infections (Ranathunge et al., 2011; Vishwanath et al., 2013; Andersen et al., 2015). Suberin is deposited on the inside face of the cell wall of various tissues, such as the endodermis and exodermis of primary roots, as well as in the periderm of mature roots that have undergone secondary growth. Underground storage organs, like potato (Solanum tuberosum) tubers, also possess a suberized periderm. In aerial parts, suberin is found in the bundle sheath cells of grass leaves and in the periderm of mature stems with secondary growth (e.g. tree bark). Additionally, it is deposited in abscission zones, as suberization is part of the wound-healing process of plants (Dean and Kolattukudy, 1976).

Suberin is a complex heteropolymer made up of aliphatics, phenolics, and glycerol (Bernards, 2002). Typical aliphatic monomers are ω-hydroxy acids and α,ω-dicarboxylic acids, but suberin also contains long-chain (C16 and C18) and very-long-chain (C20 or greater) unsubstituted fatty acids and primary fatty alcohols. The predominant phenolic components of suberin are hydroxycinnamic acids, especially ferulic acid. The outer bark of cork-oak tree (Quercus suber) and the periderm of potato tubers are suberin-rich tissues that have been used for decades as models to study the composition and structure of suberin. Recently, analyses by electrospray ionization-tandem mass spectrometry and high-resolution NMR of suberin oligomeric blocks obtained from partial depolymerization experiments have provided new insights about the macromolecular structure of this complex polymer (Graҫa and Santos, 2006; Graҫa et al., 2015). In both cork and potato skin suberin, glycerol-α,ω-diacid-glycerol diesters form the core structure of the aliphatic polyester, whereas glycerol-ω-hydroxy-acid-ferulic acid diesters are likely responsible for linking to the neighboring aromatics and carbohydrates of the cell wall. Whether suberin comprises both a polyaliphatic and a polyaromatic domain or should be restricted to the glyceroaliphatic polyester that colocalizes with a lignin-type polyphenolic domain remains a matter of debate (Bernards, 2002; Graҫa, 2015).

Solvent-extractable waxes are typically found in suberized tissues. These compounds are usually extracted by brief immersion in chloroform (Li et al., 2007; Molina et al., 2009; Kosma et al., 2012) or by extensive extraction in solvent (Schreiber et al., 2005; Yang and Bernards, 2006; Serra et al., 2010). A recent survey of the soluble waxes associated with the periderm or exodermis of various plants using chloroform dipping showed that suberin-associated waxes (or root waxes) are widespread among higher plants, but with highly variable composition and content (Kosma et al., 2015). Most species contained linear aliphatics in the form of fatty acids (free or esterified to glycerol at the sn-2 position), free fatty alcohols, alkanes (and their midchain oxygenated derivatives), and sterols, with total amounts varying from about 0.03 µg cm⁻² in pea (Pisum sativum) to 5.17 µg cm⁻² in Arabidopsis (Arabidopsis thaliana). Ten of the 11 species analyzed in this study contained alkyl hydroxycinnamates (AHCs) with contents ranging from 2 to 93 mol % of total waxes. Notably, four of 11 species were dominated by AHCs, including one member of the Solanaceae and three members of the Brassicaceae. In the periderm of freshly harvested potato tubers, the amount of suberin-associated waxes reaches 10 µg cm⁻², and the composition is dominated by saturated alkyl ferulates (i.e. ferulic acid linked by an ester bond to C16 to C32 fatty alcohols) and free primary alcohols, which represent 47% and 33% of the total, respectively, while alkanes (15% of the total) and free fatty acids (5% of the total) are minor components (Serra et al., 2009). Storing potato tubers for 3 to 4 weeks significantly increases the amount of soluble waxes (2 to 4 times) as well as the quantity of the polymerized aliphatics (45%–100%; Schreiber et al., 2005; Serra et al., 2010). Nevertheless, soluble waxes of potato tubers represent only 5% to 20% of the total periderm lipids, such that most of the aliphatics are polymerized.

In Arabidopsis, root waxes have been detected so far by chloroform dipping using roots of 6- to 8-week-old soil-grown plants with a fully developed periderm (Li et al., 2007; Molina et al., 2009; Kosma et al., 2012; Vishwanath et al., 2013) but not in roots of 3- to 4-week-old tissue culture-grown plants (Molina et al., 2009; Vishwanath et al., 2013). Arabidopsis root waxes are particularly enriched in AHCs, which can represent up to 90% of the total (Kosma et al., 2015). Also present in lesser proportions are free fatty acids, fatty alcohols, alkanes, monoacylgycerols, and sterols (Li et al., 2007; Molina et al., 2009; Kosma et al., 2012, 2015; Vishwanath et al., 2013). Despite varying proportions having been reported, all studies indicated that 18:0, 20:0, and 22:0 alkyl caffeates are the most abundant components of the suberin-associated waxes in Arabidopsis. The fatty alcohol:caffeoyl-coenzyme A transferase (FACT) responsible for the synthesis of alkyl caffeates in Arabidopsis root has been characterized, and null mutants of the FACT gene have a nearly complete lack of alkyl caffeate esters (Kosma et al., 2012).

Over the past 8 years, reverse genetics approaches in Arabidopsis have allowed for the identification of key enzymes involved in the synthesis of the major suberin aliphatic monomers, such that the suberin biosynthetic pathway is now well described (Franke et al., 2012; Vishwanath et al., 2015). Interestingly, several genes involved in the synthesis or export of compounds found in the suberin polymer also are important for the synthesis or export of root waxes, which indicates shared biosynthetic pathways. GPAT5 encodes a glycerol 3-phosphate acyltransferase producing sn-2 monoacylglycerols that are found in the suberin polymer as well as in the suberin-associated waxes (Li et al., 2007). In the same way, three fatty acyl reductases (FAR1, FAR4, and FAR5) catalyze the formation of the 18:0, 20:0, and 22:0 fatty alcohols found in both the suberin polyester and root waxes (Domergue et al., 2010; Kosma et al., 2012). Recently, three ABCG transporters (ABCG2, ABCG6, and ABCG20) were shown to be involved in the export of suberin monomers and waxes in Arabidopsis roots (Yadav et al., 2014).

In our previous study (Vishwanath et al., 2013), we showed that only 20% of the fatty alcohols present in Arabidopsis roots were covalently linked to the suberin polymer, while the remaining 80% were found in a soluble nonpolymeric fraction. Such a partition was obtained using roots from 4-week-old tissue culture-grown plants that did not contain any detectable suberin-associated wax components in the fraction obtained by rapid chloroform dipping of roots. When fully mature roots of plants grown in soil for 6 weeks were used, 19% of all fatty alcohols were found in the root waxes extracted by chloroform dipping, 24% of the total was recovered in the polymerized fraction, but the majority (i.e. more than 56% of all fatty alcohols) was recovered in the soluble fraction (Vishwanath et al., 2013). To further characterize these soluble fatty alcohols, we developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify AHCs in crude root total lipid extracts. Our analyses showed that most of the soluble fatty alcohols present in the nonpolymeric fraction are in the form of alkyl caffeates and alkyl coumarates. These results suggest that all the soluble fatty alcohols of roots from 4-week-old tissue culture-grown plants may represent suberin-associated root waxes that are not extracted by rapid dipping of roots in chloroform. In addition, our results show that soluble and polymerized fatty alcohols together represent a major part of Arabidopsis suberized layers and that suberin-associated lipids represent a major sink for acyl-lipid metabolism in Arabidopsis roots. Finally, measurements of AHCs and total fatty alcohol contents in the soluble fractions of several suberin mutants further confirmed that soluble and polymerized fatty alcohols are produced and exported by the same subset of proteins.

RESULTS

Fatty Alcohols and Hydroxycinnamates Are Found Mainly in the Soluble Lipid Fraction of Arabidopsis Roots

We first carefully examined the distribution of fatty alcohols and hydroxycinnamates in three different fractions corresponding to surface lipids, soluble lipids, and polymerized lipids (Fig. 1A). Surface lipids were extracted by dipping the roots for 90 s in chloroform, and these corresponded to the so-called root waxes. Soluble lipids were then isolated by exhaustive solvent extractions using hot isopropanol and a series of various chloroform/methanol mixtures, and these comprised all membrane and storage lipids as well as all other soluble lipids. The remaining insoluble residue corresponded to cell wall material together with polymerized lipid phenolics (i.e. the suberin polymer). When the surface lipid fraction from roots of 4-week-old tissue culture-grown wild-type (Columbia-0 [Col-0]) seedlings was directly silylated and analyzed by gas chromatography-mass spectrometry (GC-MS), only traces of AHCs (mainly alkyl coumarates) were detected at the end of the gas chromatography (GC) run. However, when this fraction was first transmethylated, before silylation and analysis by GC-MS, the GC profile was much cleaner, and 18:0 to 22:0 fatty alcohols as well as coumaric, ferulic, and caffeic acids were readily detected (Fig. 1, B and C). Nevertheless, when the soluble and polymerized lipid fractions were similarly analyzed, their fatty alcohol and hydroxycinnamate contents were much higher than that of the root surface lipids, especially those of the soluble fraction (Fig. 1, B and C). In total, more than 76% of the fatty alcohols and hydroxycinnamates were found in the soluble lipid fraction, with about 17% in the polymerized lipid fraction and less than 7% in the surface lipid fraction. In addition, there were some differences in the distribution of the various hydroxycinnamates and fatty alcohol chain lengths. While the surface lipid and polymerized lipid fractions contained similar amounts of each fatty alcohol chain length, the soluble lipid fraction was enriched in very-long-chain fatty alcohols, suggestive of a different spatial distribution of specific chain lengths of fatty alcohols. As shown in Figure 1B, the soluble fraction contained 2 and 3 times more 22:0 than 20:0 and 18:0 fatty alcohols, respectively. Conversely, the polymerized lipid fraction did not contain any 24:0-OH but more than one-fourth of all 18:0-OH. This fraction also contained only 1% of all caffeate, less than 20% of all coumarate, and more than 31% of all ferulate, in agreement with ferulate being enriched in the suberin polymer. In contrast, the soluble fraction contained nearly all of the caffeate (96%) and most of the coumarate (78%). Since Arabidopsis root waxes were shown recently to be dominated by long-chain alkyl caffeates (Kosma et al., 2015), these partitions suggested that the fatty alcohols present in the soluble fraction of 4-week-old roots might be in the form of AHCs, although they were largely not extracted by rapid dipping in chloroform.

Figure 1.

Distribution of fatty alcohols and hydroxycinnamates between surface, soluble, and polymerized lipids from roots of 4-week-old tissue culture-grown wild-type seedlings. A, Protocol used to isolate surface lipids (root waxes), soluble lipids (mainly containing cellular membrane constituents), and polymerized lipids (corresponding to suberin polymer). B, Fatty alcohol contents of the different fractions. Acyl chains were released by acid-catalyzed transmethylation, and hydroxyl groups were silylated before separation by GC and detection by mass spectrometry. C, Coumarate, ferulate, and caffeate contents of the different fractions. Hydroxycinnamic acids were released by acid-catalyzed transmethylation, and hydroxyl groups were silylated before separation by GC and detection by mass spectrometry. Mean values are shown in nmol mg⁻¹ delipidated dry residue (DR) ± sd of five replicates.

Soluble Fatty Alcohols Are Present Mainly in the Form of AHCs

The soluble lipid fraction was a combination of all the solvent extractions that were used to leave behind a residue enriched in suberin polymer. This fraction, therefore, contained the vast majority of lipids present in the different root cell layers. Attempts to further fractionate this soluble lipid fraction using classical thin-layer chromatography to isolate free fatty alcohols and/or AHCs failed, most probably because of the complexity of this fraction. Therefore, we synthesized 18:0, 20:0, and 22:0-AHC standards and developed a targeted LC-MS/MS approach to quantify such compounds in the soluble fraction (see “Materials and Methods”). Separation was achieved using a reverse-phase column. Quantification was performed using positive multiple reaction monitoring (MRM) mode and calibration curves with 19:0-caffeate as an internal standard (Supplemental Fig. S1). The 10 AHCs that had been chemically synthesized showed the same tandem mass spectrometry fragmentation behavior in positive ionization mode. AHCs being esters of a fatty alcohol and a phenolic acid, they all dissociated into characteristic fragments corresponding to the phenolic acylium ion, the hydrated phenolic acylium ion, and the fatty alcohol-specific fragment.

LC-MS/MS analysis of the soluble fraction from roots of 4-week-old tissue culture-grown wild-type (Col-0) plants revealed the presence of high AHC amounts, dominated by alkyl caffeates that represented about 80% of the total. Alkyl ferulates were very minor components (less than 2%), while alkyl coumarates accounted for about 19% of the total AHCs (Table I). Alkyl ferulates and alkyl coumarates had very similar chain length distributions dominated by 22:0 species, which represented about 44% of the total, while 20:0 and 18:0 species accounted for about 34% and 20% of the total, respectively. Alkyl caffeates were even more enriched in 22:0 species (64% of alkyl caffeates), such that 22:0-caffeate alone represented more than half of all AHCs (Table I). In agreement with the presence of traces of 24:0-fatty alcohols in the soluble lipid fraction from roots (Vishwanath et al., 2013), 24:0-AHCs (about 2% of the total) also were detected (Table I).

Table I. Quantification of the soluble fatty alcohols present in the form of AHCs.

AHC and fatty alcohol contents of the soluble fraction from roots of 4-week-old tissue culture-grown wild-type (Col-0) plants were determined by LC-MS/MS and GC-MS as described in “Materials and Methods.” Data are expressed in nmol mg⁻¹ delipidated dry residue, and each value is the mean ± sd from four replicates.

Sample	Quantification Method	18:0-OH	20:0-OH	22:0-OH	24:0-OH
Alkyl ferulate	LC-MS/MS	0.03 ± 0.01	0.06 ± 0.01	0.07 ± 0.01	0.00 ± 0.00
Alkyl coumarate	LC-MS/MS	0.36 ± 0.10	0.56 ± 0.11	0.71 ± 0.10	0.03 ± 0.01
Alkyl caffeate	LC-MS/MS	1.13 ± 0.22	1.32 ± 0.21	4.57 ± 0.55	0.12 ± 0.02
Total fatty alcohols in the form of AHCs	LC-MS/MS	1.66 ± 0.22	2.15 ± 0.22	5.42 ± 0.64	0.16 ± 0.05
Soluble fatty alcohols	GC-MS	1.99 ± 0.37	2.90 ± 0.49	5.67 ± 0.12	0.16 ± 0.02
Soluble fatty alcohols in the form of AHCs (%)		81.1 ± 13.0	74.3 ± 13.0	90.5 ± 10.5	98.8 ± 1.7

Overall, AHCs with 22:0 alkyl chains represented about 58% of the total, while 20:0 species were slightly more abundant than 18:0 species (about 23% and 18% of the total, respectively). The relative quantities of the different fatty alcohol chain lengths present in the soluble fraction also were determined by GC-MS after transmethylation and silylation. The proportions of the different chain lengths obtained with such an analysis were in very good agreement with those found by LC-MS/MS (Table I). In addition, since GC-MS analysis allowed quantification of the total amount of esterified fatty alcohols present in the soluble fraction, it could be calculated from the LC-MS/MS analysis that more than 85% of these fatty alcohols were present in the form of AHCs (Table I). In terms of acyl chain length, 81% and 74% of the 18:0 and 20:0 fatty alcohols, respectively, were present in the form of AHCs, while the longer fatty alcohols (22:0 and 24:0) were found almost exclusively combined with phenolics (Table I).

Several Suberin Mutants Are Affected in Their Soluble AHC Content and Composition

The compositions of the AHCs found in the soluble fraction of 4-week-old in vitro-grown wild-type Col-0 roots (this study) and in the taproot waxes of mature roots (Kosma et al., 2015) were very similar. AHCs consist of fatty alcohols esterified with hydroxycinnamates, both of which also are found in the suberin polymer. To test whether the genes involved in the synthesis of AHCs in taproot waxes or in the synthesis of the suberin polymer also participate in the synthesis of the AHCs that we found in the soluble fraction, we analyzed by LC-MS/MS the soluble fraction of the roots from several mutants affected in suberin deposition (grown 4 weeks in vitro). For these analyses, the minor amounts of surface lipids were not extracted by rapid dipping in chloroform before delipidation, such that the resulting soluble lipid fraction contained both surface lipids and soluble lipids.

No differences in AHC content and the composition of soluble lipids were observed in the asft mutant compared with the wild type (Fig. 2A), in agreement with this mutant having less ferulate in the suberin polymer but being unaffected in its taproot waxes (Molina et al., 2009). In contrast, the soluble fraction of the kcs2 kcs20 double mutant displayed a 50% decrease in the content of both 22:0 and 24:0 AHCs, which was coupled with a 2-fold increase in 18:0 and 20:0 AHCs. The far1 far4 far5* mutant (the asterisk indicates that FAR5 was down-regulated in the far1 far4 double knockout background using an artificial microRNA approach; Vishwanath et al., 2013) was strongly affected, as LC-MS/MS analyses showed a nearly complete lack of C20 or greater AHCs together with a 30% decrease in 18:0 AHC content (Fig. 2A). Such a strong effect is in agreement with our previous study (Vishwanath et al., 2013), which showed, according to GC-MS analyses, major reductions in the content of all fatty alcohols in the soluble fraction of roots from the far1 far4 far5* mutant lines. In the fact mutant, a drastic reduction (99%) in alkyl caffeates, a moderate decrease (52%) in alkyl ferulates, and a slight increase (20%) in C20 or greater alkyl coumarates were observed (Fig. 2A). Finally, LC-MS/MS analyses of the soluble fraction from the abcg2 abcg6 abcg20 mutant revealed that the alkyl caffeate and alkyl ferulate levels were close to zero, 20:0 and 22:0 alkyl coumarates were decreased, while 18:0 alkyl coumarate was increased slightly (Fig. 2A). Interestingly, for the far1 far4 far5*, fact, and abcg2 abcg6 abcg20 mutant lines, the variations in AHC content and composition that we measured in the soluble fractions of 4-week-old in vitro-grown roots were virtually identical to those reported in previous studies for the taproot waxes of 7-week-old soil-grown plants (Kosma et al., 2012; Vishwanath et al., 2013; Yadav et al., 2014).

Figure 2.

AHC contents and composition of the soluble fractions from roots of wild-type and suberin mutant lines grown for 4 weeks in tissue culture. A, AHC contents, sorted into individual compounds, of soluble fractions from roots of wild-type, asft, kcs2 kcs20, far1 far4 far5*, fact, and abcg2 abcg6 abcg20 mutant lines grown in tissue culture for 4 weeks. AHCs were separated and quantified by LC-MS/MS. B, Total fatty alcohol contents of the soluble fractions and fatty alcohol contents in the form of AHCs in the soluble fractions from roots of wild-type, asft, kcs2 kcs20, far1 far4 far5*, fact, and abcg2 abcg6 abcg20 mutant lines grown in tissue culture for 4 weeks. Fatty alcohols are sorted into individual chain lengths. Fatty alcohols present in the form of AHCs were calculated from A, whereas the total fatty alcohol contents of the soluble fractions were determined by GC (Supplemental Fig. S2). Mean values are shown in nmol mg⁻¹ delipidated dry residue (DR) ± sd of four replicates. Significant differences were assessed by Student’s t test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

The same soluble fractions and the corresponding dry residues containing the suberin polymer were transmethylated and silylated to analyze their acyl chain compositions by GC-MS (Supplemental Fig. S2). LC-MS/MS only allowed the quantification of the fatty alcohols present in the form of AHCs, while GC-MS analyses quantified the total fatty alcohol amounts of the soluble fraction. In the wild type and the asft and kcs2 kcs20 mutant lines, most of the fatty alcohols (about 80%) were in the form of AHCs (Fig. 2B). In contrast, only about 40% the remaining fatty alcohols present in the far1 far4 far5* mutant were in the form of AHCs. In the fact and abcg2 abcg6 abcg20 mutant lines, the proportion of fatty alcohols in the form of AHCs dropped below 20%, indicating that most of the fatty alcohols present in the soluble fraction from these lines were not combined with phenolics. Taproot waxes of the fact and abcg2 abcg6 abcg20 mutant lines, which were strongly affected in their AHC contents, presented higher levels of free fatty alcohols (Kosma et al., 2012; Yadav et al., 2014). Although the LC-MS/MS analyses that we performed did not allow the quantification of free fatty alcohols because of their very weak ionization, it is likely that there were also more free fatty alcohols in the soluble fractions of 4-week-old in vitro-grown roots from the fact and abcg2 abcg6 abcg20 mutant lines.

Suberin Lamellae Are Not Affected in far1 far4 far5* and fact Mutant Lines

In the fact mutant, the soluble fraction of 4-week-old in vitro-grown roots contained wild-type amounts of total fatty alcohols (Fig. 2B) but 70% less AHCs (Fig. 2A), while the suberin polymer composition and content were unaffected (Supplemental Fig. S2). In the far1 far4 far5* mutant, total amounts of fatty alcohols and AHCs were reduced by 80% and 85%, respectively (Fig. 2), while the suberin polymer was affected only slightly, with a 40% decrease in the level of total fatty alcohols, which are low-abundance components of the polymer (Supplemental Fig. S2). In order to test if these changes in AHCs and/or total fatty alcohol content affected the lamellar structure of the suberin layer, we performed transmission electron microscopy analysis of mature roots (Fig. 3). In the case of the fact mutant, the mature zone of roots (approximately 2 cm below the shoot-root junction) from plants grown in soil for 6 to 7 weeks was used, while in the case of the far1 far4 far5* mutant, plants were grown 4 weeks in tissue culture and mature roots about 5 mm below the rosette were used. At this developmental stage, the taproot of Arabidopsis seedlings had already undergone secondary growth, as indicated by the presence of xylem vessels surrounded by phloem and pericycle layers. A periderm often was visible, although the state of these outermost cell layers was variable, sometimes being present and well conserved and sometimes being in a state of dissolution or completely lost. Examination of intact periderm cells indicated that the suberin ultrastructure in the fact and far1 far4 far5* mutant lines was similar to that of wild-type roots (Fig. 3). The typical light and dark lamellation of the suberized cell wall layer was observed in all four lines, suggesting that major alterations in the soluble waxes associated with the suberin polymer did not cause abnormalities in the lamellar architecture of the suberin layer.

Figure 3.

Transmission electron microscopy of the suberin layer of peridermal cells from roots of wild-type and suberin mutant plants. Transverse sections were made in the mature zone of the roots (below the shoot-root junction), and periderm cells were used to acquire transmission electron microscopy images. Wild-type and far1 far4 far5* seedlings were grown for 4 weeks in tissue culture on vertically oriented plates, and sections about 5 mm below the rosette were made, whereas for the fact mutant, the mature zone of roots (approximately 2 cm below the shoot-root junction) from plants grown in soil for 6 to 7 weeks was used. Bars = 100 nm.

The Suberin Layer Represents a Major Sink for Fatty Acyl Chains in Roots

GC-MS analysis of the acyl chain compositions of both the soluble fraction and the polymerized lipid fraction containing the suberin polymer provided insights into fatty acyl chain partitioning in mature Arabidopsis roots (grown for 4 weeks in vitro). We first considered that all the 2-hydroxy fatty acids found in both the soluble and polymerized fractions were part of sphingolipids for the two following reasons. First, when analyzing separately the different solvent mixtures used to delipidate the roots, the first two steps (isopropanol and chloroform:methanol [1:1, v/v]) solubilized about 90% of all acyl chain types except 2-hydroxy fatty acids, of which more than 60% of the total was solubilized by the last more polar steps (i.e. chloroform:methanol [1:2, v/v] and methanol; Supplemental Fig. S3A). Second, when aqueous and saline (2 m NaCl) washes were used to delipidate the roots, as is typically done for seed coat analysis (Molina et al., 2006), more than 85% of the 2-hydroxy fatty acids were eliminated from the polymer fraction (Supplemental Fig. S3B), in agreement with these 2-hydroxy fatty acyl chains being components of the highly glycosylated sphingolipids that are not solubilized by classical chloroform-methanol mixtures (Buré et al., 2011). To evaluate the distribution of acyl chains in Arabidopsis roots, we then considered that all the unsubstituted fatty acids present in the soluble fraction were part of membrane glycerolipids, whereas all ω-hydroxy acids, α,ω-dicarboxylic acids, and fatty alcohols (polymerized and soluble), together with the fatty acids found in the polymeric fraction, were part of suberized layers. All fatty alcohols can be considered as suberin-specific acyl chains because 20% of them are part of the polymer (Vishwanath et al., 2013), while most of the soluble fatty alcohols are in the form of AHCs (Fig. 2B) and, therefore, probably associated with the polymer in the form of waxes. With such considerations, membrane lipids accounted for 64% of all root acyl chains, with glycerolipids and sphingolipids representing 55% and 9% of the total, respectively (Fig. 4A; Supplemental Fig. S4). The suberized layers contained around 36% of all the acyl chains present in Arabidopsis roots grown for 4 weeks in vitro, representing a major sink for root acyl-lipid metabolism.

Figure 4.

Acyl chain distribution in roots and stems, in root suberized layers and stem cuticle, and global acyl chain composition of suberized layers from roots of wild-type plants. A, Total acyl chain distribution in roots of wild-type plants grown for 4 weeks in tissue culture and in stems of wild-type plants grown for 6 to 7 weeks in soil. For roots, the soluble fraction derived from the delipidation procedure and the remaining dry residue containing the suberin polymer were each transmethylated, derivatized, and their acyl chain composition was determined by GC-MS. Membrane glycerolipids accounted for all unsubstituted and very-long-chain fatty acids present in the soluble fraction, while sphingolipids accounted for all 2-hydroxy fatty acids found in both the soluble and polymerized fractions. Suberized layers accounted for all acyl chains, except 2-hydroxy fatty acids, found in the polymerized fraction and for all suberin-type acyl chains (ω-hydroxy acids, α,ω-dicarboxylic acids, and fatty alcohols) found in the soluble fraction. For stems, waxes were first extracted, derivatized, and quantified by GC-MS. Dewaxed stems were then transmethylated, derivatized, and their acyl chain composition was determined by GC-MS. Membrane glycerolipids and sphingolipids accounted, respectively, for all unsubstituted fatty acids and all 2-hydroxy fatty acids present in the dewaxed stems. Cuticle accounted for all acyl chains present in waxes as well as all cutin-type acyl chains present in the dewaxed stems. B, Total acyl chain distribution in suberized layers of roots of wild-type plants grown for 4 weeks in tissue culture and in the cuticle of stems of wild-type plants grown for 7 weeks in soil. Suberin polymer accounted for all acyl chains except 2-hydroxy fatty acids found in the polymerized fraction, while suberin waxes accounted for all suberin-type acyl chains (ω-hydroxy acids, α,ω-dicarboxylic acids, and fatty alcohols) found in the soluble fraction. For stems, waxes accounted for all acyl chains present in chloroform extracts, while cutin accounted for all cutin-type acyl chains present in the dewaxed stems (for details, see Supplemental Fig. S5). C, Global acyl chain composition of the suberized layers from roots of wild-type plants grown for 4 weeks in tissue culture. Fatty acids accounted for unsubstituted fatty acids present in the polymerized fraction, while ω-hydroxy acids, α,ω-dicarboxylic acids, and fatty alcohols accounted for compounds founds in both the soluble and polymerized fractions. Mean values are shown in μg mg⁻¹ delipidated dry residue (DR) ± sd of four replicates.

For comparison, we evaluated the acyl chain distribution in stems of 7-week-old plants (Supplemental Fig. S5). Interestingly, we found very similar proportions, since glycerolipids and sphingolipids accounted for 59% and 7% of the total, respectively, whereas about 33% of all stem acyl chains were part of the cuticle layer (Fig. 4A). Nevertheless, the distribution within the cuticle and suberin layers clearly differed, since the cuticle of stems was principally made of soluble waxes (the cutin polymer accounting for only 7% of the total), whereas the suberized layers were principally made of the suberin polymer, as suberin-like acyl chains found in the soluble fraction accounted for only 24% of the total (Fig. 4B). These acyl chain distributions not only highlight the importance of suberized layers in the global lipid metabolism of Arabidopsis roots but also allowed new considerations about its global composition. As shown in Figure 4C, if all polymerized and soluble suberin-like acyl chains (fatty alcohols, but also ω-hydroxy and α,ω-dicarboxylic acids) are considered as components of the root suberin barriers, the composition of suberized layers is still dominated by ω-hydroxy acids, which represent 32% of all acyl chains. Nevertheless, fatty alcohols then also become major constituents, being as abundant as α,ω-dicarboxylic acids (each class representing about 25% of the total) and slightly more abundant than unsubstituted fatty acids (19% of the total; Fig. 4C).

Suberin Deposition (Both Polymer and Associated Waxes) Starts within the First Week of Growth and Represents up to 55% of All Root Acyl Chains in Plants Grown in Soil for 8 Weeks

To understand the kinetics of Arabidopsis root suberization, we then performed a developmental study by analyzing the fatty acyl chain compositions of the root soluble and polymerized fractions in plants grown in vitro over a 4-week period (Supplemental Fig. S6). In 1-week-old roots, glycerolipids contained most of the fatty acyl chains (about 67 mol %), with suberized layers (suberin polymer and associated waxes) already accounting for about one-fourth of all fatty acyl chains (24 mol %; Fig. 5A). In the following weeks, the relative fatty acyl chain content of glycerolipids decreased to around 53 mol %, while that of suberin and associated waxes increased to reach 41 mol % at week 4. In contrast, the fatty acyl chain level in sphingolipids was stable, accounting for 5 to 9 mol % over the 4-week period (Fig. 5A). The acyl chain composition of suberized layers (suberin polymer and associated waxes) was nearly similar at all four time points, except that α,ω-dicarboxylic acids decreased slightly during the 4-week period (from 30 to 25 mol %), while ω-hydroxy acids increased continuously (from 25 to 36 mol %; Fig. 5B). When considering the partition of the different types of acyl chains between the polymer and soluble fractions over the 4 weeks, the distribution was very similar at all stages (Fig. 5C). Altogether, these data suggest that the deposition of suberin in roots grown in vitro occurs mainly within the first 3 weeks and that the composition of the suberized layers as well as the partition of acyl chains between the soluble and polymerized fractions remain about the same within the 4-week period.

Figure 5.

Fatty acyl chain distribution in roots of wild-type seedlings grown over a 4-week period in tissue culture. A, Total acyl chain distribution in roots of wild-type plants grown for 1, 2, 3, or 4 weeks in tissue culture. The amounts of the acyl chains present in membrane glycerolipids, sphingolipids, and suberized layers were determined as in Figure 4A. B, Fatty acyl chain composition of the suberized layers from roots of wild-type plants grown for 1, 2, 3, or 4 weeks in tissue culture. The amounts of the different acyl chain types were determined as in Figure 4C. C, Amounts (in percentage of total) of the different types of acyl chains found in the suberin polymer from roots of wild-type plants grown for 1, 2, 3, or 4 weeks in tissue culture. Mean values are shown in mol % ± sd of four replicates.

Using the promoter:GUS lines that we generated to study the spatiotemporal gene expression pattern of FAR1 (Domergue et al., 2010; Vishwanath et al., 2013), we found that, in 1-week-old roots, FAR1 expression first appeared after the elongation zone of the root tip and was confined to the endodermis all along the root, including in the oldest part situated just below the hypocotyl (Fig. 6). To confirm that aliphatic suberin deposition occurred only in the endodermis of 1-week-old roots, we performed histochemical staining using the lipophilic dye Sudan III (Brundrett et al., 1991) and the berberine-Aniline Blue fluorescent procedure (Brundrett et al., 1988). Both stains confirmed that suberin deposition was restricted to the endodermal layer, even in the most mature part of the root below the hypocotyl, where no periderm-like staining was observed (Fig. 6). Furthermore, quantitative PCR analyses using in vitro-grown roots indicated that the expression of several genes involved in suberin biosynthesis, including FACT, FAR1, and FAR4, which are responsible for the synthesis of most AHCs, was at about the same levels over the 4-week period of growth (Supplemental Fig. S7A). Altogether, these results suggest that the suberin acyl monomers detected after 1 week of in vitro growth represent suberized endodermis and that this tissue probably also contains root waxes in the form of AHCs.

Figure 6.

Expression pattern of FAR1 and histochemical staining of suberized endodermis in 1-week-old roots grown in tissue culture. Primary root tissues were collected from 1-week-old seedlings grown on vertical plates in tissue culture. A to C, Expression patterns of FAR1 were detected using the transgenic promoter:GUS line as reported by Domergue et al. (2010). D to G, Histochemical staining using the lipophilic suberin dye Sudan III (D and E) or the berberine-Aniline Blue fluorescent procedure (F and G) was performed on full-length roots, and images taken just below the hypocotyl (D and F) and around the middle of the root (E and G) are presented. Bars = 100 µm.

We then performed similar analyses using soil-grown plants in order to evaluate how the suberization of Arabidopsis roots occurs under more normal growing conditions. Plants were grown during an 8-week period, and all roots were carefully separated from the soil every 2 weeks before analyzing the fatty acyl chain compositions of both the soluble and polymerized fractions by GC-MS (Supplemental Fig. S8). In 2-week-old roots, glycerolipids and suberized layers (suberin polymer and associated waxes) contained similar amounts of fatty acyl chains (44 mol%), while sphingolipids accounted for about 11 mol% (Fig. 7A). During the next 6 weeks, the fatty acyl chain contents of sphingolipids remained about the same, whereas that of glycerolipids increased slightly between weeks 2 and 4 before decreasing during the next 4 weeks. Conversely, the amounts of fatty acyl chains found in suberized layers first decreased but then increased constantly to represent as much as 55 mol % at week 8 (Fig. 7A). The partition of all the different types of acyl chains between the polymer and soluble fractions was not significantly different over the 8-week period (Fig. 7B). About 95% of all α,ω-dicarboxylic acids, 87% of all ω-hydroxy acids, and 17% of all fatty alcohols and unsubstituted fatty acids were systematically found in the polymer (Fig. 7B). The fatty acyl chain composition of the suberized layers (suberin polymer and associated waxes) also remained similar between weeks 2 and 8, with ω-hydroxy acids being systematically the major type of acyl chain (around 35% of the total), while α,ω-dicarboxylic acids, fatty alcohols, and fatty acids accounted, each, for 20% to 24% of the total (Fig. 7C; Supplemental Fig. S8).

Figure 7.

Fatty acyl chain distribution in roots of wild-type plants grown over an 8-week period in soil. A, Total acyl chain distribution in roots of wild-type plants grown for 2, 4, 6, or 8 weeks in soil. The amounts of the acyl chains present in membrane glycerolipids, sphingolipids, and suberized layers were determined as in Figure 4A. B, Amounts (in percentage of total) of the different types of acyl chains found in the suberin polymer from roots of wild-type plants grown for 2, 4, 6, or 8 weeks in soil. C, Fatty acyl chain composition of the suberized layers from roots of wild-type plants grown for 2, 4, 6, or 8 weeks in soil. The amounts of the different acyl chain types making the suberized layers were determined as in Figure 4C. Mean values are shown in mol % ± sd of four replicates.

We also analyzed using LC-MS/MS the AHC content of the soluble fractions from the roots of soil-grown plants and found striking differences between the different developmental stages (Fig. 8A). In 2-week-old roots, more than 69% of all AHCs were alkyl coumarates, with alkyl caffeates and alkyl ferulates accounting for 28% and 3% of the total AHC content, respectively. In addition, 18:0 and 20:0 alkyl chains largely dominated at 2 weeks, representing 58% and 26% of the total, respectively, while 22:0 alkyl chains accounted for only 16% of the total and 24:0 alkyl chains were below the detection level. In the following weeks, the amounts of alkyl ferulates decreased, and 18:0 alkyl coumarate decreased steadily, whereas the levels of 20:0 alkyl coumarate remained unchanged. Inversely, the levels of 22:0 and 24:0 alkyl coumarates and 18:0 alkyl caffeate steadily increased slightly. The most significant changes were the sharp increases in 20:0 alkyl caffeate between weeks 2 and 6 and in 22:0 and 24:0 alkyl caffeates between weeks 2 and 8 (Fig. 8A). These variations perfectly reflected those observed when analyzing the same soluble fractions by GC-MS (Supplemental Fig. S8). As shown in Figure 8B, the ferulate and coumarate contents decreased with time, while the level of caffeate increased. Also, whereas 18:0 fatty alcohols dominated at week 2 (representing 45% of all fatty alcohols), 22:0 was the major fatty alcohol chain length at week 8 (representing 62% of all fatty alcohols). With such variations, the AHC composition of 8-week-old roots was dominated by alkyl caffeates, which represented 75% of all AHCs, and by 22:0 alkyl chain lengths, which accounted for 56% of the total (Fig. 8A). Finally, when comparing the levels of fatty alcohols present in the form of AHCs (measured by LC-MS/MS) with the total amount of fatty alcohols (measured by GC-MS), only 70% of all fatty alcohols of the soluble fraction were in the form of AHCs at week 2, whereas nearly all of them were esterified with hydroxycinnamic acids at weeks 4 to 8 (Fig. 8C).

Figure 8.

AHC, hydroxycinnamate, and fatty alcohol contents of the soluble fractions from roots using wild-type plants grown over an 8-week period in soil. A, AHC contents, sorted into individual compounds, of the soluble fractions from roots of wild-type plants grown for 2, 4, 6, or 8 weeks in soil. AHCs were separated and quantified by LC-MS/MS. B, Hydroxycinnamate and fatty alcohol contents and composition of the soluble fractions from roots of wild-type plants grown for 2, 4, 6, or 8 weeks in soil. Acyl chains and hydroxycinnamic acids were released by acid-catalyzed transmethylation, and hydroxyl groups were silylated before separation by GC and detection by mass spectrometry. C, Total fatty alcohol contents and fatty alcohols in the form of AHCs in the soluble fractions from roots of wild-type plants grown for 2, 4, 6, or 8 weeks in soil. Fatty alcohols present in the form of AHCs were calculated from A, whereas total fatty alcohol contents were calculated from B. Mean values are shown in nmol mg⁻¹ (A and C) or μg mg⁻¹ (B) delipidated dry residue (DR) ± sd of four replicates. Significant differences were assessed by Student’s t test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

DISCUSSION

In Arabidopsis, suberin-associated root waxes have only been extracted so far by rapid dipping in chloroform using 6- to 8-week-old taproots with a fully developed periderm (Li et al., 2007; Molina et al., 2009; Kosma et al., 2012, 2015; Vishwanath et al., 2013). Our results here suggest that such compounds are present much earlier in the development of roots and that the rapid dipping in chloroform procedure extracts only a portion of all suberin-associated waxes present in Arabidopsis roots. The LC-MS/MS targeted approach that we developed showed that most of the fatty alcohols not covalently linked to the suberin polymer are in the form of AHCs. Whereas the presence of alkyl ferulate esters in tree barks and potato periderm has been known for decades (Brooker, 1959; Adamovics et al., 1977; Bernards and Lewis, 1992), alkyl caffeates predominate in Arabidopsis suberin-associated root waxes. We propose that the soluble fatty alcohols of Arabidopsis roots should be considered as part of the suberized layers in the form of associated waxes. With such considerations, fatty alcohols, both soluble and polymerized, then represent a major constituent of suberized layers in Arabidopsis roots, being, for example, as abundant as α,ω-dicarboxylic and unsubstituted fatty acids. In addition, our results suggest that suberized layers represent a major sink for the global metabolism of acyl chains in Arabidopsis roots.

Soluble Fatty Alcohols Not Extracted by Rapid Dipping in Chloroform Are Predominantly in the Form of Alkyl Caffeates and Represent Suberin-Associated Waxes

In this study, we used a targeted LC-MS/MS method to show that 75% to 90% of the soluble fatty alcohols that are not extracted by rapid dipping in chloroform are in the form of AHCs in roots of 4-week-old tissue culture-grown Arabidopsis seedlings (Table I; Fig. 2B). Concerning the phenolic part, caffeic acid was most abundant, being present in about 80% of all AHCs, while for the fatty alcohol part, the saturated C22 chain length dominated, being present in about 55% of all AHCs. As such, 22:0 alkyl caffeate was the most abundant molecular species, representing around 55% of the total (Table I; Fig. 2A). In mature roots grown for 8 weeks in soil, alkyl caffeates accounted for up to 75% of the total AHC content, with 22:0 alkyl caffeate alone representing 56% of all AHC molecular species (Fig. 8A). Interestingly, the soluble fractions of roots from plants grown for 4 weeks in tissue culture or for 8 weeks in soil had nearly identical AHC compositions, while the total content was about 35% greater in soil-grown plants. Although we observed important variations in AHC composition according to the developmental stage of the roots grown in soil (Fig. 8A), 70% to 100% of all the fatty alcohols from the soluble lipid fraction were systematically detected in the form of AHCs (Fig. 8C). Since this fraction combined the different solvent extractions conducted over several days at room temperature to separate the suberin polymer from all soluble lipids, it might have been subjected to breakdown and hydrolysis, such that the amount of free fatty alcohols (as well as those of ω-hydroxy and α,ω-dicarboxylic acids) may be overestimated. Recently, Kosma et al. (2015) reported that up to 90 mol % of all compounds identified in Arabidopsis root waxes from 6- to 7-week-old soil-grown plants were AHCs, with alkyl caffeates accounting for 90% of the total AHC content and 22:0 alkyl caffeate being the most abundant molecular species (49% of all AHCs). These values support the idea that most wild-type Arabidopsis root waxes are indeed AHCs, the other components (free fatty acids, alkanes, monoacylgycerols, as well as free fatty alcohols) being nearly negligible. Altogether, these analyses indicate that the vast majority of the fatty alcohols that are not covalently linked to the suberin polymer are present in the form of AHCs in Arabidopsis roots whatever the developmental stage.

The AHC and free fatty alcohol contents reported here for the soluble lipid fraction (according to LC-MS/MS analyses) compared with previous studies of root waxes (according to GC-MS analyses) are not only a match for wild-type plants but also for the suberin mutants that we studied. Vishwanath et al. (2013) reported that the AHC content of the root waxes from the far1 far4 far5* triple mutant was decreased by 90% with a nearly complete lack of C20 and C22 molecular species. In our study, we found that the AHC content of the soluble lipid fraction from the far1 far4 far5* triple mutant was decreased by 85%, with C20 or greater AHCs being barely detected (Fig. 2A). Similarly, in the study from Yadav et al. (2014), the root waxes of the abcg2 abcg6 abcg20 triple mutant were nearly devoid of alkyl caffeates and alkyl ferulates, while the 18:0 alkyl coumarate and free fatty alcohol contents were increased. In our study, we found that the alkyl caffeate and alkyl ferulate levels were close to zero, while the 18:0 alkyl coumarate and free fatty alcohol contents were increased in the abcg2 abcg6 abcg20 triple mutant (Fig. 2). Finally, in the study from Kosma et al. (2012), the root waxes of the fact mutant lines were characterized by an absence of alkyl caffeates and by an increase in the levels of C20 or greater alkyl coumarate and free fatty alcohols. In this study, the soluble fraction of the fact mutant did not contain any alkyl caffeate, while free fatty alcohols, especially 20:0 and 22:0 fatty alcohols, were increased (Fig. 2). Altogether, these data show that the mutations in far1 far4 far5, fact, and abcg2 abcg6 abcg20 had about the same impact on the root waxes extracted by chloroform dipping of mature plants (grown 6 to 7 weeks in soil) as on the soluble fractions from plants grown 4 weeks in vitro. This perfect concordance further supports the idea that the AHCs that we identified in the soluble fraction represent suberin-associated waxes.

The major role of suberin in roots is to form a hydrophobic barrier controlling the movement of water, nutrients, and ions between the soil and the central cylinder and protecting the plant from soil pathogens (Vishwanath et al., 2015). It was shown recently that a fine, hormone-dependent tuning of the suberization process in the endodermal layer allows the plant to adapt to the surrounding nutrient availability (Barberon et al., 2016). Although the far1 far4 far5* and fact mutants were strongly affected in their AHC and/or fatty alcohol contents (Fig. 2B), we could never identify a clear physiological or developmental phenotype using simple biological assays. These mutants showed neither any increased sensitivity to salt or osmotic stress when grown in tissue culture nor any developmental delay or defect when grown on soil under different watering regimes. AHCs also were shown to display antibacterial activities toward the gram-negative bacterium Pseudomonas fluorescens (Baranowski and Nagel, 1982), so that such compounds may be important in protecting the plant root system from soil-borne plant pathogens. Therefore, studying the role of AHCs and free fatty alcohols in the context of biotic interactions represents a promising direction for future research.

Most Suberin-Associated Root Waxes Are Not Extracted by Rapid Dipping in Chloroform

When we tried to extract root waxes by chloroform dipping using roots from 4-week-old tissue culture-grown wild-type seedlings, GC-MS analysis revealed only very low amounts of alkyl coumarates and traces of alkyl ferulates. In addition, the vast majority of fatty alcohols (more than 76% of the total) were recovered in the soluble fraction and characterized by LC-MS/MS as AHCs. These results imply that most of the suberin-associated root waxes are not extracted by rapid dipping in chloroform. Although the exact localization of root waxes remains to be determined, it is most probable that they colocalize with the suberin polymer. Along the length of the root, deposition of the suberin lamellae is observed 7 to 8 mm from the root cap junction during state II endodermal differentiation (Martinka et al., 2012; Andersen et al., 2015), implying that the biosynthesis of the different monomers required to form the suberin barrier occurs in the very early stages of root development. Confocal microscopy imaging of transgenic Arabidopsis plants expressing the yellow fluorescent protein under the control of the FACT promoter revealed transcriptional activity in the endodermis of young roots (Kosma et al., 2012), suggesting that AHC biosynthesis is not restricted to the periderm but also occurs in the endodermis. The promoter:GUS lines that we generated to study the spatiotemporal expression of FAR1, FAR4, and FAR5 showed that all three FAR genes were expressed in the endodermis as well as in the periderm (Domergue et al., 2010; Vishwanath et al., 2013).

Histochemical staining of suberized layers together with the expression pattern of FAR1 (Fig. 6) strongly suggest that only the endodermis is suberized after 1 week of in vitro growth. Although we cannot completely rule out epidermal cell suberization, Arabidopsis root epidermal cell walls are not expected to be heavily suberized, since they are actively involved in solute acquisition from the soil, especially in early developmental stages (Nawrath et al., 2013). Therefore, it is highly probable that most, if not all, of the suberin acyl monomers that we detected after 1 week of in vitro growth represent suberized endodermis. Since the partition of the different types of acyl chains indicated that about 80% of all fatty alcohols were found in the soluble fraction after 1 week, these results further indicate that root waxes are produced in the endodermis of 1-week-old roots. In addition, the fact that FACT, FAR1, and FAR4, which are responsible for the synthesis of most AHCs, were expressed at similar levels over the 4-week period of growth (Supplemental Fig. S7A) further supports the presence of roots waxes in the endodermis at the very early stages of root development. In agreement with this hypothesis, the presence of compounds typical of waxes (fatty alcohols and alkanes) has been described in the endodermis of soybean (Glycine max) roots, which can be separated easily from the epidermis (Thomas et al., 2007).

Root waxes associated with the suberin layer of the endodermis probably would not be extracted by dipping the root for 90 s in chloroform because of their internal location, below the root epidermis and cortex layers. Similarly, the periderm being composed of several cell layers, it is also possible that the rapid dipping procedure does not extract the soluble waxes associated with suberin of the innermost cell layers of the periderm. Finally, root waxes are probably deposited within the polymer between the plasmalemma and the cell wall, and this internal cell wall location, in contrast to that of aerial cuticular waxes, also could prevent their complete extraction by the rapid chloroform dipping procedure. In agreement, Li et al. (2007) reported that a 10-s dip in chloroform was sufficient to recover 98% (w/w) of total surface waxes from Arabidopsis stems, while such a procedure extracted only 66% (w/w) of the root waxes. From our LC-MS/MS analyses, the total amount of AHCs present in the soluble lipid fraction from roots of 6-week-old soil-grown plants can be estimated as 1,017 ± 123 µg g⁻¹ fresh weight. Using rapid dipping in chloroform, Kosma et al. (2015) evaluated the root wax AHC amounts of Arabidopsis grown for 6 weeks in soil to be 388 ± 59 µg g⁻¹ fresh weight. Although these values were obtained from plants grown in different conditions, they suggest that the rapid dipping in chloroform procedure extracted at most 40% of all root waxes. In our previous study, we similarly found that only 25% of the soluble fatty alcohols present in the roots from 7-week-old wild-type plants grown in soil were extracted by rapid dipping in chloroform (Vishwanath et al., 2013). Therefore, all these data support the view that an important proportion of suberin-associated root waxes are not extracted using the rapid dipping in chloroform procedure, probably because of the internal location of suberized layers and possibly because of a tight association of root waxes within the suberin polymer.

The Suberin Polymer and Associated Waxes Are Synthesized Concomitantly

Our data showed that the acyl chain composition of root suberin was rather similar at different developmental stages. All the analyses we performed indicated that ω-hydroxy acids were slightly more abundant than α,ω-dicarboxylic acids, fatty alcohols (soluble and polymerized), and unsubstituted fatty acids, which were all about equally abundant (Figs. 4C, 5B, and 7C). Similarly, the partition of the different types of acyl chains between the soluble and polymerized fractions was very similar at the different developmental stages. While 80% to 95% of all ω-hydroxy and α,ω-dicarboxylic acids were found in the polymeric fraction, the soluble fraction systematically contained about 80% of all fatty alcohols (Figs. 5C and 7B). These results suggest that all the aliphatic monomers making the suberin layer (i.e. the polymer and associated root waxes) are synthesized concomitantly and that the endodermis and periderm likely have rather similar suberin compositions. The parallel formation of suberin polymer and root waxes is further supported by the involvement of a common set of proteins required for the synthesis of both components of suberized layers. As shown in this study, the condensing enzymes KCS2 and KCS20 are necessary for the formation of the very-long-chain aliphatics present in the suberin polymer as well as in associated waxes (Fig. 2A; Supplemental Fig. S2; Franke et al., 2009; Lee et al., 2009). Similarly, the fatty acyl reductases FAR1, FAR4, and FAR5 are collectively responsible for the biosynthesis of the fatty alcohols found as AHCs in root waxes and polymerized into the suberin polyester (Fig. 2A; Supplemental Fig. S2; Vishwanath et al., 2013). Finally, our results also confirmed that three ATP-binding cassette transporters, ABCG2, ABCG6, and ABCG20, could be involved in the export of the monomers making up the suberin polymer as well as the soluble waxes (Fig. 2A; Supplemental Fig. S2; Yadav et al., 2014). It should be pointed out that Yadav et al. (2014) reported that decreased expression of FAR1 and FAR4 may be at the origin of the reduced fatty alcohol content observed in the root waxes of abcg2 abcg6 abcg20. Using 4-week-old in vitro-grown roots, we also found using quantitative PCR analysis that the expression levels of FAR1 and FAR4 were decreased in abcg2 abcg6 abcg20 roots compared with wild-type roots, while that of FAR5 remained unaffected (Supplemental Fig. S7B). Interestingly, the expression of FACT was more than 85% reduced in abcg2 abcg6 abcg20 (Supplemental Fig. S7B), suggesting that a feedback regulation of AHC biosynthesis may occur in this mutant and be responsible for reduced AHC content. Nevertheless, the fact that the same proteins produce or export aliphatics present in both the suberin polymer and its associated waxes strongly supports that the synthesis of the two components of suberized root barriers is coordinated during root development.

The absence of significant variation in the relative proportions of the different aliphatics making up suberin suggests in addition that the composition of suberin in the endodermis and periderm is similar. In the early stages of root development (i.e. after 1 week of in vitro growth), suberin comes exclusively from the endodermis (Fig. 6), whereas in later stages (i.e. after 8 weeks of growth in soil), the periderm, which contains several layers of suberized cells, should account for most of the root suberin. However, the overall compositions of suberin reported throughout this study are relatively similar. A noticeable exception might be the total content of ω-hydroxy fatty acids, which, in addition to being slightly higher than that of the other types of acyl chains, increased with age as the periderm developed in vitro (Fig. 5B). Such a situation was already observed in maize (Zea mays; Zeier et al., 1999) as well as in Arabidopsis, where suberin analyses along the length of the roots showed that the mature part with a fully developed periderm contains more ω-hydroxy fatty acids than younger parts (Höfer et al., 2008). Therefore, it could be that periderm suberin is richer in ω-hydroxy fatty acids than endodermal suberin.

Transmission electron microscopy imaging of intact periderm cells indicated that the lamellar architecture of the suberin layer was not affected in the roots from the fact and far1 far4 far5* mutant lines (Fig. 3). Since the soluble fraction of the far1 far4 far5* mutant contained 80% to 85% less AHCs and total fatty alcohols than that of wild-type plants (Fig. 2), our data suggest that the root wax components (i.e. AHCs) are not necessary to generate the typical light and dark lamellation of suberized layers. Similarly, since the soluble fraction of the fact mutant was characterized by large changes in its composition, specifically a 70% decrease in AHCs and concomitantly higher levels of free fatty alcohols (Fig. 2; Kosma et al., 2012), our data indicate that suberin ultrastructure is not affected by drastic changes in the composition of root waxes. In contrast, the kcs2 kcs20 double mutant was reported to present an altered lamellation of the suberin in the endodermis (Lee et al., 2009), while the suberin appearance of the abcg2 abcg6 abcg20 triple mutant was affected in both the endodermis and the mature root periderm (Yadav et al., 2014). These two mutant lines also have major alterations in their suberin polyester composition, and changes in the ratio of the different monomers comprising the polymer likely impact the lamellar structure of suberin. Also, the suberin-associated wax composition and/or content also are affected in the kcs2 kcs20 and abcg2 abcg6 abcg20 mutants (Fig. 2; Yadav et al., 2014), so that alterations in both polymerized and soluble monomers could modify the lamellation of suberized layers. Whichever the case, these data contradict the idea that the light bands of the suberin lamellae are made primarily of waxes (Soliday et al., 1979).

Suberized Layers Represent a Major Sink for Acyl Lipid Metabolism in Arabidopsis Roots

Analyzing the acyl chain composition of both the soluble and polymerized fractions allowed us to get insights about the partitioning of all acyl chains between the three major acyl pools of roots: sphingolipids, glycerolipids, and suberized layers. We considered that all the 2-hydroxy acids (those found in the soluble fraction as well as those recovered residually in the polymerized fraction) represent the acyl chains found in sphingolipids. Although a small proportion of the nonsubstituted fatty acids from the soluble fraction might be wax components, we considered that all these acyl chains reflect the glycerolipids that form cellular membranes. Finally, all fatty alcohols, ω-hydroxy acids, and α,ω-dicarboxylic acids (both soluble and polymerized) as well as all nonsubstituted fatty acids of the polymeric fraction were considered to belong to the suberized layers. With such considerations, we found that, although the majority of the acyl chains from roots of plants grown in vitro for 4 weeks were membrane components in the form of glycerolipids and sphingolipids, suberized layers nevertheless accounted for about 36% of all acyl chains (Fig. 4A). In the case of plants grown in soil for 8 weeks, the proportion of acyl chains found in root suberin reached 55% of the total (Fig. 7A). At that late developmental stage, although suberin deposition is restricted to two cell types, the endodermis and periderm, it accounted for more acyl chains than membranes, which are present in every living cell. Interestingly, the amounts of acyl chains present in glycerolipids dropped at the latter stages of root development from plants grown in vitro and in soil (Figs. 5A and 7A). Whether these decreases are related to the secondary growth of roots, which is characterized by the development of additional vascular tissues (i.e. xylem), remains to be determined. Nevertheless, our results indicate that a very large proportion (40%–55%) of the fatty acyl chains produced in roots is dedicated to the biosynthesis of suberized layers, demonstrating that suberin represents a major sink for acyl lipid metabolism in Arabidopsis roots. Similarly, Suh et al. (2005) reported that about 60% of the acyl chains of epidermal cells from (middle) Arabidopsis stems were found in surface lipids, with the epidermal wax load being about 8 times that of the cutin polyester load. Our analyses confirm that the cuticular barrier of stems is made primarily of soluble waxes (Fig. 4B) and report that the majority of the acyl chains constituting the suberized layers of Arabidopsis roots are, in contrast, part of the polymer (Fig. 4B), as in the periderm of potato tubers (Schreiber et al., 2005; Serra et al., 2010).

CONCLUSION

In this study, we showed that most of the fatty alcohols not covalently linked to the suberin polymer are present in the form of AHCs, with alkyl caffeates predominating, and represent suberin-associated waxes that are not extracted by rapid dipping in chloroform. Therefore, fatty alcohols, both soluble and polymerized, represent a major constituent of Arabidopsis suberized layers, being in total as abundant as α,ω-dicarboxylic and unsubstituted fatty acids. In addition, our results suggest that suberized layers represent, in Arabidopsis roots, a major sink for the biosynthesis of aliphatic acyl chains.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 was used in all experiments. For in vitro growth, seeds were sterilized, plated on Murashige and Skoog medium supplemented with 0.7% agar and 2.5 mm MES-KOH, pH 5.7, and stratified in the dark for 3 to 4 d at 4°C. Plates were then transferred to a controlled-environment growth chamber under long-day conditions (16 h of light and 8 h of darkness) at a temperature of 22°C, and seedlings were grown for up to 4 weeks. For soil-grown plants, seeds were sterilized, stratified in the dark for 3 d at 4°C, and then sown onto prewetted peat moss-vermiculite-perlite growing medium (Promix BX; Premier Horticulture Ltée). The seeds were germinated and grown at 22°C in a controlled-environment growth chamber with ambient humidity under long-day conditions. Seedlings were transferred to individual pots after 10 d and continued growing under the same conditions for up to 8 weeks of total growth. Seed stocks of far1-2 far4-1 far5* (Vishwanath et al., 2013), asft-1 (Molina et al., 2009), kcs2/daisy1 kcs20 (Lee et al., 2009), fact1 (Kosma et al., 2012), and abcg2-1 abcg6-1 abcg20-1 (Yadav et al., 2014) were used in this study.

Lipid Extractions

For the analysis of soluble and polymerized lipids from roots, freshly collected roots were immediately immersed in 4 mL of hot isopropanol. After incubation for 30 min at 85°C and then cooling, the isopropanol phase was collected. Roots were further extracted with 4 mL of chloroform:methanol (2:1, v/v) for 24 h at room temperature on a tube rotator, and the solvent phase was collected again. The delipidation of roots was completed by performing the same procedure with 4 mL of chloroform:methanol (1:1, v/v), 4 mL of chloroform:methanol (1:2, v/v), and 4 mL of 100% methanol. All the collected solvent phases were combined, evaporated under a gentle stream of nitrogen gas, and resuspended in chloroform:methanol (1:4, v/v). This fraction corresponded to the soluble lipids. The resulting solvent-extracted roots were dried in a fume hood at room temperature for 2 d and then in a desiccator for another 2 d, and this corresponded to the polymerized lipid fraction. When root waxes were extracted, freshly collected roots were first dipped in chloroform for 1.5 min before immersion in hot isopropanol. For the more extensive delipidation procedure, a series of supplemental extractions in water (24 h), 2 m NaCl (8 h), water (24 h), and 100% methanol (48 h), each at room temperature on a tube rotator, was added following the classical delipidation procedure.

Lipid Analyses by GC

For lipid analyses by GC, one-fifth of the soluble lipid fraction and 5 to 25 mg of solvent-extracted dried residues were used. Samples were depolymerized by transmethylation at 85°C for 3 h in closed glass tubes containing 1 mL of 5% (v/v) sulfuric acid in methanol and 5 μg each of heptadecanoic acid (17:0), pentadecanol (15:0-OH), and ω-hydroxy-pentadecanoic acid (ω-OH-15:0) as internal standards. After cooling, 2.2 mL of methyl-tert-butyl ether (MTBE) and 1 mL of NaCl (2.5%, w/v) were added successively. Tubes were mixed vigorously before centrifugation at 800g for 5 min at room temperature to promote phase separation. The upper MTBE phase was transferred to a fresh glass tube and washed with 1 mL of Tris-NaCl solution (100 mm Tris base, pH 8, in 0.09% NaCl). After mixing and centrifugation, the upper MTBE phase was again transferred to a fresh glass tube, avoiding any aqueous phase, and evaporated under a gentle stream of nitrogen. For silylation, 150 µL of 99% N,O-bis(trimethylsilyl)-trifluoroacetamide + 1% chlorotrimethylsilane was added and incubated at 110°C for 15 min. After cooling, the N,O-bis(trimethylsilyl)-trifluoroacetamide-chlorotrimethylsilane was evaporated under a gentle stream of nitrogen, and samples were resuspended in heptane:toluene (1:1, v/v). GC analyses was performed on an Agilent 6850 gas chromatograph equipped with an Agilent 5975 mass spectrometer as described previously (Domergue et al., 2010). For the analysis of root waxes, the chloroform extract was evaporated under a gentle stream of nitrogen, and samples were either silylated directly and analyzed by GC or treated as above to determine their total fatty alcohol and phenolic contents.

Synthesis and Purification of AHC Standards

The synthesis and purification of nonadecyl (19:0) caffeate was done as described by Razeq et al. (2014). The 18:0, 20:0, and 22:0 alkyl ferulates, alkyl p-coumarates, and alkyl caffeates (the nine naturally occurring compounds) were synthesized according to the procedure reported previously by Nishimura et al. (2009) with minor modifications. Briefly, a chemoselective Mitsunobu esterification reaction was employed to couple the phenolic acids with the fatty alcohols in the presence of diisopropyl azodicarboxylate and triphenylphosphine. A typical procedure involved the very slow dropwise addition of a solution of diisopropyl azodicarboxylate (4.37 mL, 22.2 mmol) in dry tetrahydrofuran (20 mL) into a stirring solution of p-coumaric acid (3.64 g, 22.2 mmol), 1-octadecanol (3.0 g, 11.1 mmol), and triphenylphosphine (5.82 g, 22.2 mmol) in dry tetrahydrofuran (80 mL) at room temperature under N₂ atmosphere. The complete reaction mixture was stirred for an additional 1 h at room temperature, during which it was judged to be complete by thin-layer chromatography. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL) to give the crude product, which was then purified by flash chromatography on a silica gel (60 Å, 230–500 mesh) using a gradient (5%–15%) of ethyl acetate in hexane. The desired product was obtained as a pure crystalline white solid (3.6 g, 78%). The structure as well as the purity of the product were confirmed by melting point, chromatographic retardation factor, infrared spectroscopy, ¹H- and ¹³C-NMR, along with GC-MS analyses of the silylated product. The same procedure was followed to prepare the other eight compounds, and varying yields (67%–89%) were obtained similar to that reported before (Nishimura et al., 2009).

Lipid Analyses by LC-MS/MS

For lipid analyses by LC-MS/MS, the soluble lipid fraction was first diluted to 80 mg dry residue mL⁻¹ in chloroform:methanol (1:4, v/v). Two further dilutions, 1:3 and 1:15, were made for alkyl ferulate and alkyl coumarate/alkyl caffeate quantification, respectively. Each dilution contained 2 nmol µL⁻¹ 19:0-caffeate as an internal standard. LC-MS/MS analyses were performed using the liquid chromatography system Ultimate 3000 (Dionex) coupled to the mass spectrometer model QTRAP 5500 (ABSciex). Sample (5 μL) separations were carried out at room temperature on a reverse-phase Jupiter C4 50- × 1-mm column, with 300-Å pore size and 5-μm particles (Phenomenex). Eluent A was water with 0.1% (v/v) formic acid, and eluent B was acetonitrile with 0.1% (v/v) formic acid. The gradient elution program was as follows: 0 to 2 min, 60% B; 2 to 6 min, 60% to 75% B; and 6 to 26 min, 75% to 85% B. The flow rate was set at 100 μL min⁻¹. Tandem mass spectrometry (MRM mode) analyses were achieved in positive mode. Nitrogen was used for the curtain gas (set to 25), gas 1 (set to 25), and gas 2 (set to 10). Needle voltage was at +5,500 V without needle heating, the declustering potential was set at +200 V, and the entrance potential was set at 10 V. The collision gas was also nitrogen, collision energy was set at +25 eV, and the dwell time was set at 1.5 ms. To define the MRM transitions, the Q1 mass analyzer selected the protonated molecular ion [M+H]⁺ as precursor ion while the Q3 mass analyzer selected the phenolic acylium ion [R–CH=CH–C=O+H]⁺ (where R represents the phenolic ring) for alkyl caffeates and alkyl coumarates or its hydrated form [R–CH=CH–C=O+H₂O+H]⁺ for alkyl ferulates as daughter ions. The areas of the different AHC peaks were determined using MultiQuant software (version 2.1; ABSciex). Quantification was based on the signal from the internal standard 19:0-caffeate and calibration curves obtained using the nine synthesized 18:0, 20:0, and 22:0 AHCs.

Transmission Electron Microscopy

For transmission electron microscopy analysis, 4-week-old in vitro-grown Arabidopsis seedlings were gently removed from Murashige and Skoog medium. About 1-mm-long root sections were cut at the base of the rosette and fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer (pH 7.2). After a 10-min vacuum step at 200 mbar, the fixed samples were rinsed three times in 0.1 m phosphate buffer (pH 7.2), postfixed in 1% OsO₄ in 0.1 m phosphate buffer (pH 7.2) for 2 h at 4°C, rinsed three times in 0.1 m phosphate buffer (pH 7.2), stained with tannic acid (1% in water) for 30 min at room temperature, rinsed three times in water, dehydrated through an ethanol series (30%, 50%, 70%, 85%, 95%, and 100%), and impregnated in increasing concentrations of EPON 812 resin (Electron Microscopy Sciences) in propylene oxide over a period of 3 d before being polymerized at 60°C for 24 h. Sections 60 nm thick were imaged at 120 kV using a Tecnai Spirit FEI transmission electron microscope equipped with an Eagle 4K CCD.

Light Microscopic and Histochemical Techniques

One-week-old seedlings grown in vitro on vertical plates were used for all microscopic analyses. The expression pattern of FAR1 was detected in transgenic promoter:GUS lines as reported by Domergue et al. (2010). For Sudan III staining, seedlings were removed gently from the Murashige and Skoog medium, then full-length roots were cut at the base of the rosette and fixed with 2.5% formaldehde/glutaraldehyde in 0.1 m sodium calcodylate buffer, pH 7.4 (Electron Microscopy Sciences) using a gentle vacuum for 10 min, three times, with a 5-min rest in between. Fixed samples were then stained with 0.4% Sudan III in 96% ethanol for 30 s at 70°C in a dry bath and then thoroughly washed with distilled water. Stained samples were mounted in 30% glycerol and imaged under bright-field light on a BZ-X700 microscope (Keyence). For the berberine-Aniline Blue fluorescent procedure, whole seedlings were first cleared in 90% lactic acid saturated with chloral hydrate for 6 h at 37°C. Following several washes with distilled water, roots were incubated with 0.1% (w/v) berberine hemisulfate overnight at room temperature. Whole seedlings were then washed thoroughly with distilled water and counterstained with 0.5% (w/v) Aniline Blue for 60 min. Following several washes with distilled water, stained samples were mounted in water and imaged under UV light on a Carl Zeiss AxioZoom V16 microscope.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: FAR1, At5g22500; FAR4, At3g44540; FAR5, At3g44550; ASFT, At3g23840; KCS2/DAISY, At1g04220; KCS20, At5g43760; FACT, At5g41040; ABCG2, At2g37360; ABCG6, At5g13580; ABCG20, At3g53510; GPAT5, At3g11430; CYP86B1/RALPH, At5g23190; and CYP86A1/HORST, At5g58860.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. LC-MS/MS quantification of AHCs.

• Supplemental Figure S2. Acyl chain contents of the polymerized and soluble fractions from roots of wild-type and suberin mutant lines grown for 4 weeks in tissue culture.

• Supplemental Figure S3. Acyl chain compositions of the different solvent mixtures used to delipidate the roots and of the polymerized fraction after classical or extensive delipidation.

• Supplemental Figure S4. Acyl chain contents of the polymerized and soluble fractions of roots from wild-type plants grown for 4 weeks in tissue culture.

• Supplemental Figure S5. Acyl chain distribution in stem waxes of 7-week-old plants.

• Supplemental Figure S6. Acyl chain contents of the polymerized and soluble fractions of roots from wild-type plants grown from 1 to 4 weeks in tissue culture.

• Supplemental Figure S7. Relative transcript levels of suberin genes in roots from wild-type plants grown from 1 to 4 weeks in tissue culture and in 4-week-old in vitro-grown roots from wild-type and abcg2 abcg6 abcg20 plants.

• Supplemental Figure S8. Acyl chain contents of the polymerized and soluble fractions of roots from wild-type plants grown over an 8-week period in soil.

Glossary

AHC

alkyl hydroxycinnamate

LC-MS/MS

liquid chromatography-tandem mass spectrometry

Col-0

Columbia-0

GC-MS

gas chromatography-mass spectrometry

GC

gas chromatography

MRM

multiple reaction monitoring

MTBE

methyl-tert-butyl ether