Silencing of StKCS6 in potato periderm leads to reduced chain lengths of suberin and wax compounds and increased peridermal transpiration.

Very long chain aliphatic compounds occur in the suberin polymer and associated wax. Up to now only few genes involved in suberin biosynthesis have been identified. This is a report on the isolation of a potato (Solanum tuberosum) 3-ketoacyl-CoA synthase (KCS) gene and the study of its molecular and physiological relevance by means of a reverse genetic approach. This gene, called StKCS6, was stably silenced by RNA interference (RNAi) in potato. Analysis of the chemical composition of silenced potato tuber periderms indicated that StKCS6 down-regulation has a significant and fairly specific effect on the chain length distribution of very long-chain fatty acids (VLCFAs) and derivatives, occurring in the suberin polymer and peridermal wax. All compounds with chain lengths of C(28) and higher were significantly reduced in silenced periderms, whereas compounds with chain lengths of C(26) and lower accumulated. Thus, StKCS6 is preferentially involved in the formation of suberin and wax lipidic monomers with chain lengths of C(28) and higher. As a result, peridermal transpiration of the silenced lines was about 1.5-times higher than that of the wild type. Our results convincingly show that StKCS6 is involved in both suberin and wax biosynthesis and that a reduction of the monomeric carbon chain lengths leads to increased rates of peridermal transpiration.

Introduction

Protection against dehydration and pathogens is crucial for the survival of land plants. The epidermis of primary plant organs, for example, leaves and fruits, is covered by a thin impermeable membrane, the cuticle, which is composed of a lipidic cutin polymeric layer and of waxes, which are embedded in the cutin polymer and deposited on its outer surface. In plant organs in a secondary developmental state (shoots, roots, and tubers) the epidermis is replaced by a suberized periderm. The outer layer (phellem) of the periderm consists of tightly packed dead cells. Primary cell walls of the phellem are coated from the inside by suberin and embedded by waxes (Esau, 1965). Similar to cutin, suberin is a glycerol-based polyester of hydroxylated fatty acids (Moire ), but it also contains an aromatic domain attached to the aliphatic polymeric domain (Bernards and Razem, 2001). Furthermore, the aliphatic domain of suberin, compared with cutin, is composed of monomers with longer chain lengths (up to C32) and contains larger amounts of α,ω-diacids (Kolattukudy, 1980; Nawrath, 2002, Franke ). Soluble waxes, mostly VLCFA (C20–C34) and their derived compounds, are often extracted from suberin tissues. Peridermal wax composition has been reported for cork oak (Quercus suber) (Silva ) and potato (Schreiber ). Studies on cuticular and peridermal transpiration have shown the importance of waxes for establishing the water barrier of these membranes (Soliday ; Vogt ; Schreiber and Riederer, 1996; Schreiber ; Kerstiens, 2006). However, it is not yet clear exactly how waxes seal polymer membranes such as cuticles or periderms, rendering them essentially water impermeable (Kerstiens ; Lendzian, 2006). There is evidence that waterproofing abilities of lipids are related to their carbon chain length (Gibbs ; Patel ), but a simple relationship between chain lengths of cuticular or peridermal waxes and water permeability of cuticles or periderms have not yet been established.

VLCFA (fatty acids >C18) are direct components and important precursors for aliphatic suberin monomers and wax compounds. Biosynthesis of VLCFAs in plants is catalysed by endoplasmic reticulum (ER)-located fatty acid elongase (FAE) complexes that extend fatty acids from C16 and C18 to C20 and higher (Samuels ). FAE sequentially adds two-carbon moieties from malonyl-CoA to the fatty acid, through a condensing enzyme, and the resulting product is reduced in three subsequent steps catalysed by distinct enzymes (details reviewed in Shepherd and Griffiths, 2006; Samuels ). The initial condensation step is catalysed by a KCS. This reaction is the rate-limiting step in fatty acid elongation (Suneja ; Cassagne ) and the most sensitive to the chain length of the substrate (Todd ). KCSs are encoded by a multigene family with 21 members in Arabidopsis (Joubès ). Genetic approaches in planta showed the involvement of the KCS enzymes such as AtKCS6/AtCER6/CUT1 (Millar ) and AtKCS1 (Todd ) in the biosynthesis of the cuticular wax components of Arabidopsis and the role of LeCER6 in tomato fruit cuticular wax synthesis (Vogg ; Leide ). Arabidopsis kcs1 roots were also affected in α,ω-diacids, characteristic monomers of suberin, suggesting a role for this enzyme in suberin synthesis (Todd ). The kcs1 mutant, in addition, did not show a complete block in root VLCFAs and derivatives, pointing to a redundant role of other KCS enzymes. Consistent with this redundant function, evidence for a role of the Arabidopsis DAISY gene (AtKCS2) in root suberin biosynthesis has also been obtained recently (Franke ).

In the present work, a reverse genetic approach was used in potato to investigate the role of a KCS (referred to as StKCS6) in suberin biosynthesis. Potato is a very good model for studying suberin biosynthesis, structure and function since (i) the periderm can easily be isolated from the tuber, (ii) sufficient amounts are obtained for chemical analyses, and (iii) it is an excellent system for measuring peridermal transpiration (Vogt ; Schreiber ). After isolation of the coding sequence of StKCS6, the gene was stably down-regulated by RNAi-mediated silencing in potato plants cv. Desireé. The effects of StKCS6 deficiency on the chain length distribution of aliphatic suberin monomers and wax compounds was measured and related to peridermal transpiration.

Materials and methods

Plant material and growth conditions

Potato plants cv. Desirée were propagated in vitro and tubers were grown in the greenhouse. For in vitro propagation, stem cuttings were cultured in MS media (Duchefa) supplemented with 2% (w/v) sucrose and grown in growth cabinets under a light/dark photoperiod cycle of 16/8 h at 22 °C. In vitro plants were transferred to soil and grown for about 2 months in the greenhouse for tuber production. Tubers were harvested from 8-week-old plants and stored at room temperature before analysis.

Cloning of the full-length StKCS6 sequence

The full-length sequence of StKCS6 (Acc. No. ACF17125) was obtained based on the TC136686 expressed sequence tag (EST), by amplification of the 5′- cDNA missing region using the 5′- rapid amplification of cDNA ends (RACE) system (Invitrogen) according to the manufacturer's protocols. A full-length StKCS6 sequence was PCR-amplified from a cDNA tuber skin, using the gene-specific primers 5′-TGCCTTATCATCAGCACCTTTATGTGT-3′ and 5′-CCAACTTTTCCTTGTGGATCTTCTTGT-3′ and the Advantage Polymerase (Promega). The PCR products were cloned into pCR4-TOPO (Invitrogen) and sequenced using the BigDye Terminator 3.1 kit (Applied Biosystems). The StKCS6 genomic sequence (Acc. No. EU616538) was PCR amplified using potato genomic DNA as a template and primers corresponding to the most upstream and downstream known sequences of StKCS6, 5′-CTACAATCAACAATTCCCTCCTTT-3′ and 5′-TCCAGCTGTCTGATGATCCA-3′, respectively.

Plasmid construction

The silencing of StKCS6 in potato plants was carried out by means of a 290 bp fragment that encompasses the nucleotides from 1392-CDS to 190-3′UTR, therefore 100 bp were from the 3′-end CDS and 190 bp from the 3′-UTR. This fragment was specifically PCR amplified using the primers 5′-TTGGAAGTGTAACCGCACAA-3′ and 5′-TCCAGCTGTCTGATGATCCA-3′ bearing at their 5′-ends the attB1 and attB2 recombinant sequences, respectively, and potato tuber skin cDNA as a template, which was previously synthesized from total RNA using the SuperScript II RT (Invitrogen) and an oligo(dT)16 primer. The PCR product was cloned into the donor plasmid pDONR207 (Invitrogen) by BP clonase II recombination (Gateway Technology, Invitrogen). The binary destination vector (pBIN19RNAi) was obtained by subcloning the Gateway RNAi cassette from pH7GWIWG2(II) (www.psb.rug.ac.be/gateway/) (Karimi ) into the pBIN19 vector. For this purpose, the RNAi cassette was excised by a partial XbaI digestion and a HindIII digestion, and inserted into the XbaI-HindIII sites of the pBIN19 plasmid. This cassette included a chloramphenicol resistance marker (CmR) and two ccdB (in cursive) genes flanked by recombinant attR1 (in cursive) and attR2 (in cursive) sequences in inversed orientations, separated by an intron. The StKCS6-RNAi fragment from pDONR207 was transferred into the binary destination vector using LR clonase II (Invitrogen). The PCR insert and vector were incubated for 5 min at 65 °C before the clonase was added to improve cloning efficiency and incubated overnight at room temperature. Recombination replaced the ccdB genes by the StKCS6 fragment yielding a hairpin construct able to trigger StKCS6 mRNA degradation. Restriction enzyme digestion was used to verify the recombinant construct.

Plant transformation for RNAi-mediated silencing

Potato plants cv. Desirée were transformed as previously described by Banerjee . Potato leaves were infected with the Agrobacterium tumefaciens strain GV2260 transformed with the RNAi recombinant plasmid in accordance with Hofgen and Willmitzer (1988). Kanamycin-resistant plants were regenerated and grown until tuber development and analysed for StKCS6 mRNA accumulation in the tuber skin.

RNA isolation and mRNA expression analyses

Total RNA was isolated from potato tissues using the guanidine hydrochloride method (Logemann ). Real-time RT-PCR analysis was carried out as described previously by Soler . StKCS6 gene-specific forward and reverse primers, designed with Primer Express 2.0 (Applied Biosystems), were 5′-AACCGCACAATCAAGACACCA-3′ and 5′-TCTCTGGATGAACACTGGGT-3′, respectively. Real-time polymerase chain reactions were performed in an optical 96-well plate with an ABI PRISM 7300 Sequence Detector System (Applied Biosystems), using SYBR Green to monitor dscDNA synthesis. Reactions contained 1× Power SYBR Green Master Mix reagent (Applied Biosystems), 900 nM of gene-specific primer, and 5 μl of a 10-fold dilution of the previously synthesized cDNA in a final volume of 20 μl. The following standard thermal profile was used for all PCRs: 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A dissociation step was performed after amplification to confirm the presence of a single amplicon. To estimate variation in the technique, four technical replicates were carried out. Data were analysed with 7300 SDS 1.3.1 software (Applied Biosystems). To generate a baseline-subtracted plot of the logarithmic increase in fluorescence signal (ΔRn) versus cycle number, baseline data were collected between cycles 3 and 15. The amplification plot was analysed with an Rn threshold of 0.2 to obtain Ct (threshold cycle) values. The amplification efficiency for each gene was calculated based on five dilutions of template ranging from 1–10−3 and the equation E=10–1/slope. The relative mRNA abundance for StKCS6 was calculated as (1) (Pfaffl, 2001):

A mix with equal amounts of each sample was used as the control to standardize data, and adenine phosphoribosyl transferase (APRT) was used as the reference to normalize data. Forward and reverse primers for APRT (Nicot ) were 5′-GAACCGGAGCAGGTGAAGAA-3′ and 5′-GAAGCAATCCCAGCGATACG-3′, respectively. The absence of genomic DNA contamination was checked using non-Retrotranscriptase Controls (RT–) and the absence of environmental contamination using Non-Template Controls (NTC).

For the RT-PCR analyses with incremental cycle numbers, the procedure described by Soler was used. For each putative StKCS6-RNAi silenced line and for the wild type, first-stranded cDNA was synthesized from 0.5 μg of total RNA using the Superscript II (Invitrogen) in a 20 μl reaction and then 2.5-fold diluted. The total RNA used was previously treated with TURBO DNase (Ambion) to prevent genomic contamination and a further purification step was performed with the CleanUp protocol of RNeasy Plant Mini kit (Qiagen). For the PCR, 100 μl reactions were used with equal amounts of cDNA (1 μl of the diluted cDNA) as a template and gene-specific primers for the housekeeping StACTIN (TC119084) (5′-CCTTGTATGCTAGTGGTCG-3′ and 5′-GCTCATAGTCAAGAGCCAC-3′) and for the target StKCS6 (5′-TGGGTGGTGCTGCTATACTTT-3′ and 5′-TCCTTTCGCCTCG-ATGTAAC-3′). Aliquots of 10 μl were taken every three cycles from cycle 18 to 30 and every six from cycle 30 to 36 and analysed by agarose gel electrophoresis stained by ethidium bromide. To discard possible genomic DNA contaminations, the StACTIN and StKCS6 primers were designed complementary to two exons flanking an intron.

Isolation of periderm membranes

Freshly-harvested tubers 3–8 cm long were rinsed in tap water to remove adhering soil and stored for 21 d at room temperature in the dark, for periderm isolation as described by Vogt . Periderm discs (1 cm diameter) punched out from tubers using a cork borer were immersed in a mixture of 2% (v/v) cellulase (Celluclast; Novo Nordisc, Bagsvaerd, Denmark) and 2% (v/v) pectinase (Trenolin Super DF; Erbslöh, Geisenheim, Germany) dissolved in 10−2 M citric buffer (pH 3.0, adjusted with KOH). Sodium azide (1 mol m−3; Sigma) was added to prevent bacterial growth. During the incubation for 4 d, the solution was changed 2–3 times until it was clear. Isolated periderm membranes were washed twice in 2×10−2 M boric buffer pH 9.0 for 24 h and then carefully washed with deionized water. All the incubations were carried out at room temperature with shaking at 30 rpm. Isolated periderm discs were dried and stored at room temperature until used.

In a strict sense, potato periderm is composed of three distinct tissues: the suberized tissue (phellem), the cambial layer (phellogen), and the phelloderm. During this enzymatic isolation only the suberized phellem tissue is obtained, whereas phellogen and phelloderm are removed. However, the term periderm is used instead of phellem, in accordance with a number of different authors in the past (Vogt ; Stark ; Schreiber ).

Measurement of peridermal permeance

Peridermal permeability was measured using a gravimetric method previously described by Schönherr and Lendzian (1981) and Schreiber . Isolated periderm membranes were soaked overnight in deionized water, laid flat on plastic sheets and left to dry at room temperature for 24 h. To avoid the effect of lenticels on water permeability measurements, small chambers (diameter 6 mm) and periderm fragments free of lenticels were used. Freshly-harvested and 21-d-stored periderm membranes, from two and three StKCS6-RNAi independent lines respectively, together with those from wild-type were mounted on water-filled transpiration chambers (300 μl) made of stainless steel with the physiological inner side of the isolated periderm facing the inner side of the chambers. Once mounted, the transpiration chambers were turned upside down to ensure direct contact between water and periderm. Chambers were kept in closed polyethylene boxes containing dry silica gel and stored at 25 °C, and the weight loss caused by evaporation of water across the periderm to the silica gel was determined at regular intervals using an analytical balance (Analytic AC210S, Sartorius). Transpiration kinetics was obtained by plotting the amounts of water (g), which had diffused across the periderm, against time (s). Permeances (P, m s−1) were calculated from the slopes (F, g s−1) of the linear regression lines and following the equation: P=F×(A×Δc)−1, where A (2.83×10−3 m2) corresponds to the exposed area and Δc (106 g m−3) represents the driving force, provided by the concentration of water in the chamber (Schreiber ).

Gas chromatography-mass spectrometry analysis

Wax and suberin extraction and gas chromatography–mass spectometry (GC–MS) and gas chromatography–flame ionization detector (GC–FID) analysis were carried out as previously described (Schreiber ). The total amount of periderm material used for each analysis ranged from 2 mg to 3 mg, corresponding to one isolated periderm disc. Three independent StKCS6 down-regulated lines were used for these studies. Wax was extracted from isolated periderms at room temperature for 18 h in a mixture (1:1; v/v) of chloroform and methanol. Chloroform/methanol extracts were used for wax analysis without further purification. For the depolymerization of suberin, wax-free periderms were dried for at least 24 h in a dessicator containing silica gel, and subsequently trans-esterified by incubation at 70 °C for 18 h with fresh methanol/boron trifluoride (∼10% BF3 in methanol; Fluka) carefully stored in the fridge and closed in a N2 gas atmosphere to prevent its oxidation and generation of artefacts (Kolattukudy and Agrawal, 1974; Zeier and Schreiber, 1998).

Monomers released after trans-esterification (suberin) and wax extracts obtained by chloroform/methanol extraction were identified as trimethylsilyl (TMS) derivatives by means of gas chromatography and a quadrupole mass selective detector HP 5971A (Hewlett-Packard) and quantified by GC-FID (HP 5890 Series II; Hewlett-Packard) using the HPChemStation software (Hewlett-Packard), by comparison with an internal standard (2 μg of tetracosane for wax and 10 μg dotriacontane for suberin). TMS derivatives were prepared after complete solvent evaporation (N2 gas) by adding 100 μl of chloroform, and derivatized with a 1:1 (v/v) mixture of 20 μl pyridine (GC-grade; Merck) and 20 μl of N,O-bis-trimethylsilyltrifluoroacetamide (BSTFA, Machery-Nagel) for 40 min at 70 °C.

Periderm microscopy

For light and fluorescence microscopy, small fragments of tuber periderm and isolated periderm discs were included in fresh potato blocks and cut using a rotation microtome (Anglia Scientific). Sections were mounted on slides in water and observed in brightfield and epifluorescence on a Leica DMR-XA optical microscope (Leica Microsystems, Wetzlar, Germany).

For scanning electron microscopy (SEM), small fragments of tuber periderm were fixed under vacuum with 4% formaldehyde in phosphate-buffered saline (pH 7.5) at room temperature for at least 48 h. Fragments were dehydrated with an increasing ethanol concentration series, exchanged through amyl-acetate, and critical-point dried. The pieces were mounted on copper stubs and coated with gold. Specimens were observed using a Zeiss DSM 960A SEM (Zeiss, Oberkochen, Germany). Digital images were collected and processed using the Quartz PCI 5.10 (Quartz Imaging Corporation).

For transmission electron microscopy (TEM), sections of periderm dissected into 1×1 mm2 were immediately placed in 100 mM sodium cacodylate buffer (pH 7.0) containing 2.5% (w/v) glutaraldehyde and 2% (w/v) paraformaldehyde for 2–4 h. Vacuum was applied until samples were submerged. Tissues were washed three times with 100 mM sodium cacodylate buffer (pH 7.0), and subsequently fixed overnight in 100 mM sodium cacodylate buffer (pH 7.0) containing 1% (w/v) osmium tetroxide. Samples were then washed with 100 mM sodium cacodylate buffer (pH 7.0), dehydrated in an acetone series (30–100% by 10% steps, 10 min each step), and infiltrated with Spurr's epoxy resin (1:2, 1:1, and 2:1 resin:acetone (v/v) and pure resin for 4 h, overnight, 3 h, and 5 h, respectively). Infiltrated tissues were placed in moulds, and incubated at 60 °C for 2 d. Embedded materials were thinly sectioned using an ultramicrotome RMC MT-XL (Tucson, USA). Sections were collected onto 200-mesh copper grids, stained with 2% (w/v) uranyl acetate for 15 min, and rinsed for 30 min, before being observed and photographed with the TEM ZEISS EM910 (Germany) at an accelerating voltage of 60 kV. Images were obtained on Kodak Electron Microscope film 4489 and scanned by HP 6100C (Hewlett-Packard).

All the microscopic analyses were performed by the Microscopy Service of the University of Girona.

Sequence alignment and phylogenetic analysis

Amino acid sequence alignments were performed using the ClustalW program at the European Bioinformatics Institute (EBI, http://www.ebi.ac.uk/Tools/clustalw/). The alignment was edited using the BioEdit Sequence Alignment Editor 7.0.1. (Hall, 1999). KCS transmembrane domains were identified according to Qin and conserved amino acidic residues in the catalytic triad (Cys, His, and Asn) according to Blacklock and Jaworski (2006).

Accession numbers

The StKCS6 genomic sequence (Acc. no. EU616538) and the StKCS6 coding sequence (Acc. no. ACF17125) were isolated. The StKCS6 coding sequence was compared to the partial sequence of tomato LeCER6 (TC125065), the Arabidopsis AtKCS6/AtCER6/AtCUT1 (Acc. no. NP_177020; At1g68530), and AtKCS5/AtCER60 (Acc. no. NP_173916; At1g25450), the cotton GhCER6 (Acc. no. ABA01490), and the barley HvCUT1 (Acc. no. ABG35744). Moreover AtKCS1 (Acc. no. NM_099994; At1g01120), AtKCS20 (Acc. no. NM_123743; At5g43760), and AtKCS2 (Acc. no. NM_100303; At1g04220) are also cited in the paper.

Results

StKCS6 isolation and expression pattern

An EST putatively encoding a KCS condensing enzyme was isolated from potato periderm (tuber skin) by suppression subtractive hybridization (SSH) using the tuber parenchyma as a control or driver tissue. A partial sequence for this potato EST was identified in the TIGR database (TC136686; http://www.tigr.org) and used to obtain the corresponding full-length coding sequence by 5′-RACE. The generated full-length sequence, which also matches at the 5′-end with TC150110, contains an open reading frame of 1491 bp and codes for a putative protein of 496 residues with a predicted molecular mass of 55.92 KDa. Amino acid sequence analysis showed high similarity to members of the KCS family: 99% similarity to the partial sequence of tomato LeCER6, 94% similarity to Arabidopsis AtKCS6/AtCER6/AtCUT1 and AtKCS5/AtCER60, 93% similarity to cotton GhCER6, and 88% similarity to barley HvCUT1. Based on the strong homology to LeCER6, this potato KCS is referred to as StKCS6. However, since the potato genome has not yet been sequenced and considering that homology to both AtKCS6/AtCER6/AtCUT1 and AtKCS5/AtCER60 is similar, it cannot unambiguously be assumed that StKCS6 is the potato orthologue of AtKCS6. The amino acid alignment for StKCS6 and the highly homologous KCS proteins is shown in Supplementary Fig. S1 at JXB online. Examination of the aligned sequences revealed highly conserved amino acid sequences in the neighbourhood of the catalytic triad (Cys, His, and Asn) (Blacklock and Jaworski, 2006) and the two predicted N-terminal transmembrane spanning domains (see Supplementary Figs S1, I, II at JXB online) (Qin ).

To examine the StKCS6 expression profile, StKCS6 transcript levels in different organs of potato were analysed by real-time PCR (Fig. 1A). StKCS6 expression was highest in the tuber periderm. Lower but still significant expression levels could be detected in the leaf, stem, and root, but not in the parenchyma of tubers.

Fig. 1.

StKCS6 transcript profile in potato organs and tuber tissues and its down-regulation in StKCS6-silenced lines. (A) Relative StKCS6 transcript accumulation (log-transformed) in potato stem (S), leaf (L), root (R), tuber-parenchyma (T-PAR), and tuber periderm (T-PER) was determined by real-time RT-PCR analysis. Values were normalized using the housekeeping reference gene adenine phosphoribosyl transferase. The data represent the mean ±sd of three replicates. (B) RT-PCR analysis with PCR incremental cycle numbers of the StKCS6 gene, to verify silencing of the StKCS6 transcript. Samples correspond to potato tuber periderms of independent StKCS6-RNAi transgenic lines. PCR products were analysed at the cycle numbers indicated in the top. Equal amount of cDNA was used as template for each sample as showed by the PCR amplification of StACTIN. Note that lines 5, 9, and 34 showed the highest StKCS6 down-regulation whereas line 23 was partially silenced and line 37 was non-silenced, showing an accumulation of the StKCS6 transcript comparable to that of the wild type.

Down-regulation of StKCS6 in potato plants

Down-regulation of StKCS6 in potato was performed by RNAi-mediated silencing. To prevent silencing of non-target genes, a 290 bp sequence was used for RNAi, which corresponds to the last 100 bp of the coding region and 190 bp of the non-conserved 3′-untranslated region (UTR) of StKCS6. This fragment was inserted twice into the pBIN19RNAi vector to generate an inverted repeat construct that targets the StKCS6 mRNA. Transgenic plants, derived from independent transformation events, were grown in the greenhouse until tuber harvesting. Lines effectively silenced were identified by Northern blot analysis of RNA isolated from the tuber periderm (data not shown). Three lines (5, 9, and 34) were selected among those showing the highest degree of down-regulation and subsequently confirmed by RT-PCR analysis, using a non-silenced line (37), a partially silenced line (23), and wild-type potatoes as reference (Fig. 1B). These selected lines were propagated for additional tuber production, which were used in further analyses. Potato plants deficient for StKCS6 developed normally either in vitro or in pots. No changes were observed in the vegetative above and below-ground organs in comparison with wild-type plants. Tubers were harvested from 8-week-old plants and analysed after 21 d storage at room temperature (21-d-stored). Tubers of StKCS6-silenced potato plants showed no visual differences in comparison with the wild type; neither during tuber development, at harvest nor during storage at room temperature for a period up to 2 months. Freshly-harvested (0-d-stored) tubers were also analysed for comparison.

StKCS6 deficiency affects the chain length lipid profile

The effect of StKCS6 down-regulation on the chemical composition of the potato tuber periderm (suberin polymer and solvent-extractable waxes) was analysed in the three independent silenced lines (above mentioned) by GC-MS and quantified by GC-FID. Enzymatically isolated periderm membranes were used for the analysis. The total amount of aliphatic and aromatic suberin monomers quantified in freshly-harvested and 21-d-stored periderms was not significantly different between StKCS6-silenced lines and the wild type, except for the suberin aromatics of 21-d-stored periderms which were slightly increased in the StKCS6-silenced periderms (Fig. 2). The total amount of wax compounds in freshly-harvested periderms was also not significantly different between StKCS6-silenced lines and wild-type, but a significant decrease in these compounds was observed in 21-d-stored StKCS6-silenced periderms. Total amounts of suberin fractions increased with storage time, both in wild-type and silenced tubers (Fig. 2). This trend was also observed for the wax amounts in wild-type tubers, but not in StKCS6-silenced tubers, where wax amounts did not significantly increase after 21 d of storage compared to freshly-harvested tubers (Fig. 2).

Fig. 2.

Total amount of suberin and wax compounds per surface area (μg cm−2). The soluble fraction (wax) was obtained by treating the enzymatically isolated periderms with a mixture of chloroform:methanol. The suberin fraction was obtained after trans-esterification of wax-free periderms and the released monomers were classified as suberin aromatic or aliphatic compounds. Note that as a whole, periderms from 21-d-stored tubers showed an increased load of suberin and wax. Data represent the mean ±sd of the periderms from StKCS6-RNAi (suberin n=8: line 5 n=3, line 9 n=2, line 34 n=3; wax n=7: line 5 n=3, line 9 n=2, line 34 n=2) and wild-type (WT) (suberin n=5; wax n=7) tubers.

Analysis of chemical composition of the aliphatic suberin and wax fractions from 21-d-stored tubers showed that StKCS6 down-regulation results in a significant reduction of the lipid chain lengths (Fig. 3). Both, aliphatic suberin monomers (Fig. 3A) and wax constituents (Fig. 3B) displayed a reduction in compounds with chain length C28 and higher and a concomitant accumulation of those with chain length C26 and lower. For C25 alkane, the large variation observed in wild-type tubers hampers appreciating the effect of StKCS6 silencing, but the accumulation of this compound in StKCS6-silenced periderm was clearly observed in freshly-harvested periderm (see Supplementary Fig. S2 at JXB online) and in 60-d-stored tubers (not shown). Plotting the chain length distribution of all added compounds of the same chain length (Fig. 4) also plainly shows the impact of StKCS6 down-regulation on the shortening of chain length in the suberin and wax lipid profiles. Similar effects of StKCS6 silencing on wax and suberin chemical composition were observed in periderms isolated from freshly-harvested tubers (see Supplementary Fig. S2 and Fig. S3 at JXB online).

Fig. 3.

Chemical composition of suberin and wax fractions of StKCS6-RNAi and wild-type periderms from 21-d-stored tubers. (A) Absolute amounts (μg cm−2) of suberin monomers released after trans-esterification of wax-free periderms. (B) Absolute amounts (μg cm−2) of wax compounds obtained by treating the enzymatically isolated periderms with a mixture of chloroform:methanol. StKCS6-RNAi periderms show a decrease in C28 and longer VLCFA and derivatives of all substance classes, both in suberin and wax. Data represent the mean ±sd of the periderms from StKCS6-RNAi (suberin n=8: line 5 n=3, line 9 n=2, line 34 n=3; wax n=7: line 5 n=3, line 9 n=2, line 34 n=2) and wild-type (suberin n = 5; wax n=7) tubers.

Fig. 4.

Chain length profile of aliphatic suberin (A) and wax (B) constituents in StKCS6-RNAi and wild-type periderms from 21-d-stored tubers. StKCS6-deficient periderm shows a decrease in carbon chain length C28 and longer in both suberin and wax fraction (t test; *, P < 0.05; **, P < 0.01). Data represent the mean ±sd of the periderms from StKCS6-RNAi (suberin n=8: line 5 n=3, line 9 n=2, line 34 n=3; wax n=7: line 5 n=3, line 9 n=2, line 34 n=2) and wild-type (suberin n=5; wax n=7) tubers.

StKCS6 deficiency impairs the water barrier function

Periderms isolated from 21-d-stored tubers showed transpiration values significantly lower than those of periderms from freshly-harvested tubers (Fig. 5). As regards StKCS6-silenced lines, peridermal transpiration was higher than wild-type either at 21 d storage as at 0 d storage (Fig. 5). The transpiration increase was statistically significant for the two independent silenced lines analysed (5 and 34) at 21 d storage but only for line 5 at 0 d storage.

Fig. 5.

Periderm water permeances of StKCS6-RNAi and wild-type tubers from freshly-harvested and 21-d-stored tubers. After 21 d storage, periderms improve the water barrier properties showing a decreased water permeance (P; m s−1). At this stage, the StKCS6-silenced periderms have a 1.54-fold increase on water permeance when compared to wild-type (t test; **, P < 0.01). Data represent the mean and error bars the 95% confidence intervals for the periderms from wild-type freshly-harvested (n=11) and 21-d-stored (n=14) and for the StKCS6-RNAi freshly-harvested (line 5 n=7; line 34 n=11) and 21-d-stored (line 5 n=9; line 34 n=11) tubers.

Effect of StKCS6 down-regulation on suberin fine structure

Periderm samples obtained from 21-d-stored tubers were examined using light and electron microscopy in order to test whether StKCS6 silencing had an effect on the periderm fine structure or ultrastructure. No differences could be observed using light microscopy, SEM or TEM (Fig. 6), being the ultrastructural lamellation of the suberin polymer similar in StKCS6-silenced lines and the wild type.

Fig. 6.

Ultrastructure of StKCS6-silenced periderms. Transmission electron micrographs showing a detailed view of the cork cell walls ultrastructure of wild-type (A) and transgenic StKCS6-RNAi (B) periderms. The polysaccharide primary wall (PW) and tertiary wall (TW) as well as the suberized secondary wall (SW) formed by the typical suberin lamella show a normal development in the StKCS6-RNAi periderm.

Discussion

Results presented here convincingly show that potato StKCS6, which shares a high homology with LeCER6, AtCER6/CUT1/KCS6, AtCER60/KCS5, and GhCER6, is required for the biosynthesis of very long chain suberin and wax compounds in the potato tuber periderm. These results demonstrate that suberin monomers and wax compounds share common fatty acid precursors, as previously suggested by others (Li ). The ability of KCS6-like genes to elongate cuticular wax compounds in Arabidopsis (Jenks ; Millar ; Fiebig ; Hooker ) and tomato (Vogg ; Leide ) is well established. This is consistent with the expression of StKCS6 in aerial organs (Fig. 1A). Regarding the suberized tissues, although it was shown that AtKSC5/CER60 (Trenkamp ) and AtKCS6/CER6/CUT1 (Jenks ; Millar ) can use fatty acids longer than C24 as substrates, both genes are weakly or not expressed in roots (Joubès ). Therefore, they do not seem to contribute to root suberin biosynthesis in Arabidopsis and consistent with it, the chain length in Arabidopsis root periderms rarely exceeds C24. This is very different from the potato tuber periderm, where chain lengths of C30 and higher occur (Figs 3, 4). Thus, StKCS6 would be highly relevant for suberin and wax monomer biosynthesis in potato periderm (Figs 3, 4). It can reasonably be assumed that the biosynthesis of aliphatic compounds with chain length C28 and higher depend on the activity of StKCS6, as periderms isolated from silenced tubers show a significant decrease of this compound. This fact, together with the concomitant accumulation of compounds with chain lengths of C26 and lower, strongly suggests that StKCS6 preferentially elongates C26 substrates. However, it cannot be excluded that StKCS6 can also act on substrates other than C26, as KCS enzymes were reported to catalyse multiple sequential elongation reactions (Samuels ), being the putative orthologues of StKCS6 shown to act on a range of substrates. In planta studies suggested that Arabidopsis AtKCS6 acts on C24 and C26 fatty acids (Jenks ; Millar ) and that the tomato LeCER6 acts on fatty acids beyond C28 (Leide ). In vitro studies in yeast demonstrated that AtKCS5/AtCER60 produces C26, C28, and C30 fatty acids (Trenkamp ), while GhCER6 produces C26 fatty acids (Qin ). In general, activated fatty acids are considered to be the substrates for KCSs. However, it is still possible that fatty acids derivatives are also elongated by these enzymes, as speculated by different authors (Höfer ; Pollard ). The accumulation of compounds with chain lengths of C26 and lower in StKCS6-silenced tubers indicates that KCS enzymes other than StKCS6 do also contribute to the biosynthesis of VLCFAs in the potato periderm. These might correspond to homologues of the Arabidopsis AtKCS1, AtKCS20, and AtKCS2 genes that expressed in yeast produce fatty acids in a size range (Trenkamp ) that can be used as potential substrates by StKCS6. AtKCS1 (Todd ) and AtKCS2 (daisy) (Franke ) mutants actually showed a deficiency in root VLCFAs, while KCS20 is expressed in Arabidopsis roots and was also highlighted as a candidate for suberin biosynthesis in the cork bark of cork oak (Soler ). Since a non-conserved fragment was used for StKCS6 silencing, it can convincingly be assumed that StKCS6 deficiency is responsible of the observed alterations in periderm composition of StKCS6-RNAi plants. This is supported by the fact that identical effects on chemical composition are observed in the three independent StKCS6-silenced lines (line 5, 9, 34). On the other hand, the presence of C28 compounds and longer in suberin and waxes suggest that either the silencing was incomplete and/or there is redundancy in the activity of KCS enzymes in potato tuber periderm. The former is consistent with the detection of StKCS6 cDNA in different down-regulated lines (Fig. 1B). The latter agrees with substrate overlapping shown by several Arabidopsis KCSs in yeast (Trenkamp ) and with the expression of potato KCS enzymes other than StKCS6 in potato tuber (see Supplementary Table S1 at JXB online).

Effects of StKCS6 deficiency allows to infer a direct relationship between chain length extension and water permeability in potato periderm

Periderm maturation is a process that takes place within the first 2–3 weeks after harvesting. During this time the tuber periderm (potato skin) acquires the complete lipid coverage (Schreiber ), becomes resistant to skinning (Lulai and Freeman, 2001) and attains the full water barrier properties (Lendzian, 2006). Our data (Fig. 2; see Supplementary Fig. S2 and Fig. S3 at JXB online) agree with previous results in potato cv. Desireé, since they show that the total lipid load increases during the storage period, even the lipid profile remains basically unchanged (Schreiber ). In addition, our results show that StKCS6 is relevant for the whole periderm maturation process as changes induced by StKCS6 deficiency were similar in the periderm of freshly-harvested tubers and 21-d-stored tubers.

StKCS6 silencing had no effect on the fine structure or ultrastructure of suberin and the typical lamellation was not altered (Fig. 6). This could be explained by the observation that the predominant monomer C18:1 α-ω diacid was not affected by gene silencing. By contrast, chemical treatments with inhibitors of fatty acid elongases of wound periderm of potato tubers (Soliday ) and of fibres of the green-lint mutant of cotton (Schmutz ) resulted in a greater chain length reduction of VLCFAs and derivatives, in a stronger decrease of the suberin predominant α,ω-diacid monomer, and showed pronounced effects on suberin lamellation. Moreover, in potato wound periderm the treatment with elongase inhibitors results in severe inhibition of the development of diffusion resistance of the tissue to water vapour (Soliday ). A weak but significant increase in peridermal transpiration by about 1.5-fold in 21-day-stored tubers of StKCS6-silenced lines was observed here (Fig. 5). This effect was less pronounced in periderms isolated from 0-d-stored tubers. Both the significant reduction in average chain length in periderm of freshly- harvested and 21-d-stored tubers of StKCS6-silenced lines and the reduction in total wax coverage in the periderm of stored tubers of StKCS6-silenced lines should be responsible for these increased rates of peridermal transpiration (Fig. 5). The reduction in total wax coverage in the periderm of stored tubers of StKCS6-silenced lines could also be explained by the chain length shortening itself, as shorter chain length means lower molecular weight. Results presented here demonstrate that potato periderm represents an excellent model system for studies aimed to analyse the relationship between transport properties of suberized barriers and their qualitative (chain length of wax compounds and suberin monomers; substance classes) and quantitative (total amounts of wax compounds and suberin monomers) chemical composition.

Supplementary data

Supplementary material may be found at JXB online.

Amino acid alignment of StKCS6 with the most similar KCS proteins of different species.

Wax compounds in the periderm of wild-type and StKCS6-RNAi lines, from freshly-harvested and 21-d-stored tubers.

Aliphatic suberin monomeric composition of wild-type and StKCS6-RNAi periderms from freshly-harvested and 21-d-stored tubers.

Potato ESTs corresponding to KCSs identified by in silico analysis.