Down-regulation of tomato PHYTOL KINASE strongly impairs tocopherol biosynthesis and affects prenyllipid metabolism in an organ-specific manner.

Tocopherol, a compound with vitamin E (VTE) activity, is a conserved constituent of the plastidial antioxidant network in photosynthetic organisms. The synthesis of tocopherol involves the condensation of an aromatic head group with an isoprenoid prenyl side chain. The latter, phytyl diphosphate, can be derived from chlorophyll phytol tail recycling, which depends on phytol kinase (VTE5) activity. How plants co-ordinate isoprenoid precursor distribution for supplying biosynthesis of tocopherol and other prenyllipids in different organs is poorly understood. Here, Solanum lycopersicum plants impaired in the expression of two VTE5-like genes identified by phylogenetic analyses, named SlVTE5 and SlFOLK, were characterized. Our data show that while SlFOLK does not affect tocopherol content, the production of this metabolite is >80% dependent on SlVTE5 in tomato, in both leaves and fruits. VTE5 deficiency greatly impacted lipid metabolism, including prenylquinones, carotenoids, and fatty acid phytyl esters. However, the prenyllipid profile greatly differed between source and sink organs, revealing organ-specific metabolic adjustments in tomato. Additionally, VTE5-deficient plants displayed starch accumulation and lower CO2 assimilation in leaves associated with mild yield penalty. Taken together, our results provide valuable insights into the distinct regulation of isoprenoid metabolism in leaves and fruits and also expose the interaction between lipid and carbon metabolism, which results in carbohydrate export blockage in the VTE5-deficient plants, affecting tomato fruit quality.

Introduction

Tocopherols are potent lipid-soluble antioxidants synthesized only by photosynthetic organisms and, together with tocotrienols, are collectively referred to as vitamin E (VTE) compounds (Kamal-Eldin and Appelqvist, 1996; DellaPenna and Pogson, 2006). Since plants are the major source of VTE required for human nutrition, understanding of the mechanisms underlying its synthesis and accumulation in crop species is of great interest (Grusak and DellaPenna, 1999; Fitzpatrick ). The antioxidant function of tocopherols relies on their ability to scavenge peroxyl radicals, limiting lipid oxidation of polyunsaturated fatty acids (PUFAs) (Serbinova ; Traber and Atkinson, 2008), and also singlet oxygen (1O2) (Di Mascio ; Kaiser ; Fukuzawa ). In plants, light-driven photosynthetic processes are the main contributors to reactive oxygen species (ROS) production in chloroplasts owing to electron transport chains and photosensitizing molecules such as chlorophyll (Chl) (Edreva, 2005; Demmig-Adams ). The delicate equilibrium between ROS production and their detoxification in chloroplast, which determines damage, protection, or signaling response, is controlled by a diversified ROS-scavenging system, including non-enzymatic antioxidant mechanisms (Edreva, 2005; Foyer and Noctor, 2005). Tocopherols, as part of the photoprotective machinery, are particularly involved in controlling the level of 1O2 in photosystem II (PSII), and the extent of lipid peroxidation in thylakoid membranes especially under stress conditions (Triantaphylidès and Havaux, 2009; Rastogi ; Miret and Munné-Bosch, 2015). Beyond photoprotective roles, tocopherol is also involved in seed longevity, seedling germination (Sattler ; Mène-Saffrané ), and photoassimilate export (Maeda , 2008; Asensi-Fabado ); although, for the latter, the precise underlying mechanism remains elusive (Maeda ).

Accumulation of tocopherol in plant tissues is a tightly controlled process, and several studies determined that tocopherol levels change significantly during plant growth and development, as well as in response to environmental stimuli including high light, low temperature, salt, and osmotic stress (Munné-Bosch, 2005; Maeda ; Abbasi ; Loyola ; Quadrana ; Eugeni-Piller ). Additionally, transgenic approaches have demonstrated that VTE content correlates with the expression of the biosynthesis- and recycling-related genes (reviewed by DellaPenna and Mène-Saffrané, 2011). Tocopherol synthesis occurs in plastids and requires two precursors, a prenyl side chain and a tyrosine catabolite-derived head group (Fig. 1). The prenyl moiety phytyl diphosphate and homogentisate derived from the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) and the shikimate pathway, respectively, are condensed by homogentisate phytyl transferase (VTE2), the only enzyme unique for tocopherol synthesis. From this precursor, the four naturally occurring tocopherol forms (α-, β-, γ-, and δ-tocopherol), which vary in the methylation pattern of the chromanol ring, are synthesized via the action of dimethyl-phytylquinol methyl transferase (VTE3), tocopherol cyclase (VTE1), and tocopherol γ-methyl transferase (VTE4). These enzymes are also responsible for the synthesis of the other tocochromanol compounds, which include not only tocotrienols but also plastochromanol (PC-8), a product of plastoquinone (PQ-9) cyclization (Zbierzak ).

Fig. 1.

Schematic view of tocopherol biosynthetic and related pathways. The genes are the following: 1-deoxy-d-xylulose-5-P synthase (DXS); geranylgeranyl diphosphate reductase (GGDR); 4-hydroxyphenylpyruvate dioxygenase (HPPD); homogentisate phytyl transferase (VTE2); 2,3-dimethyl-5-phytylquinol methyltransferase (VTE3); tocopherol cyclase (VTE1); γ-tocopherol-C-methyl transferase (VTE4); phytoene synthase (PSY); phytoene desaturase (PDS); chloroplast-specific β-lycopene cyclase (LCYβ); chromoplast-specific β-lycopene cyclase (CYCβ); chlorophyll synthase (CHLG); staygreen 1 (SGR1); pheophytinase (PPH); pheophorbide a oxygenase (PAO); phytol kinase (VTE5); farnesol kinase (FOLK); homogentisate solanesyl transferase (HST); solanesyl diphosphate synthase (SPS). Abbreviated intermediate metabolites are: glyceraldehyde 3-phosphate (GA3-P); 1-deoxy-d-xylulose-5-P (DXP); isopentenyl diphosphate (IDP); dimethylallyl diphosphate (DMADP); geranylgeranyl diphosphate (GGDP); hydroxyphenylpyruvate (HPP); homogentisate (HGA); chlorophyllide a (Chlide a); geranylgeranyl-chlorophyll a (Chl a GG); phytylated chlorophyll a (Chl a Phy); pheophytin a (Phein a); pheophorbide a (Pheide a); red chlorophyll catabolite (RCC); 2-methyl-6-geranylgeranylbenzoquinol (MPBQ); 2,3-dimethyl-6-geranylgeranylbenzoquinol (DMBQ); 2-methyl-6-solanyl-1,4-benzoquinol (MSBQ); plastoquinol-9 (PQH2-9); plastochromanol-8 (PC-8).

In addition to the de novo synthesis, the phytyl diphosphate precursor may also originate from Chl turnover or degradation, by the release of a phytol moiety from the tetrapyrrole ring. Intriguingly, the precise identity of the tocopherol biosynthesis-related phytol hydrolase remains to be determined. In Arabidopsis thaliana, the absence of the known dephytylating enzymes, pheophytinase (PPH) and chlorophyllase (CLH), in triple mutants does not alter seed tocopherol content, whereas seed-specific PPH-overexpressing transgenic lines exhibit modestly increased tocopherol levels (Zhang ). The hydrolyzed phytol is sequentially phosphorylated by two enzymes, phytol kinase (VTE5) and phytyl-phosphate kinase (VTE6) (Ischebeck ; Valentin ; vom Dorp ). VTE5 has been characterized in Arabidopsis where its mutant allele, vte5, causes a substantial reduction of the tocopherol content in seeds and to a lesser extent in leaves (Valentin ). Furthermore, based on sequence similarity, a locus encoding a putative VTE5 paralog was identified in Arabidopsis, which further was characterized as a farnesol kinase (FOLK) (Fitzpatrick ). However, its involvement in tocopherol biosynthesis was not addressed. So far, VTE5 has only been characterized in Arabidopsis, and its contribution to tocopherol content is largely unknown in other species and organs, such as in edible fleshy fruits. Moreover, the impact of VTE5 deficiency on plant metabolism remains unexplored. Solanum lycopersicum is an interesting model species for studying tocopherol metabolism. Besides being an important food crop worldwide, the fruits are a significant source of VTE for the human diet (Chun ). Additionally, tomato ripening, which encompasses the conversion of chloroplasts into chromoplasts, couples Chl degradation and an active MEP pathway (Seymour ), both sources of the prenyl precursor for tocopherol biosynthesis (Almeida ). A previous study on the regulation of tocopherol biosynthesis in this species demonstrated a strong correlation between VTE5 mRNA levels and the contents of Chl and tocopherol in tomato leaves and fruits, suggesting the contribution of phytol recycling to tocopherol biosynthesis (Quadrana ). Moreover, expression analysis of senescence-related tomato mutants suggested that maintenance of the de novo phytyl diphosphate synthesis might, at later ripening stages, compensate for the lack of Chl-derived phytol for tocopherol production in fruits (Almeida ).

To better understand the extent of the contribution of the VTE5-dependent phytol pathway to tocopherol biosynthesis in source and sink organs, we functionally characterized VTE5-like genes in tomato. Tocopherol content was dramatically compromised in both leaves and fruits of SlVTE5-knockdown plants. In contrast, analyses of the folk mutant genotype ruled out SlFOLK as a major contributor to phytol kinase activity required for tocopherol biosynthesis. Additionally, VTE5 deficiency differentially impacts fatty acid phytyl ester and prenyllipid metabolism in fruits and leaves, and also has consequences in photosynthesis and sugar partitioning.

Materials and methods

Plant material, growth conditions, and sampling

Seeds of tomato (Solanum lycopersicum, cv. Micro-Tom) were obtained from the Laboratory of Hormonal Control of Plant Development (www.esalq.usp.br/tomato). The folk-1 tomato mutant was isolated from an ethyl methanesulfonate (EMS)-mutagenized Micro-Tom collection from INRA, France. Plants were grown in a greenhouse under automatic irrigation at an average temperature of 25 °C, 11.5h/13h (winter/summer) photoperiod, and 250–350 μmol m−2 s−1 of incident photoirradiance. Source (the first fully expanded leaf) and sink (the first apical leaf not fully expanded) leaves were sampled. Fruits were harvested at mature green, 1 d after breaker (B+1), 3 d after breaker (B+3), and ripe (B+6) stages at 35, 38, 40, and 43 d after anthesis, respectively. Samples were frozen in liquid N2 and stored at –80 °C. All biochemical analyses were performed in the T1 generation. For photosynthesis and yield evaluation, an independent experiment in the T2 generation was performed. Destructive harvest took place at a point where the largest possible numbers of fruits were ripe without visible over-ripening (15 weeks old) (Vicente ). At harvest time, aerial biomass was weighed and all the fruits were counted and weighed.

Phylogenetic analysis

For phylogenetic analysis, Blastp searches were performed using the protein sequences of A. thaliana VTE5 (At5g04490) and FOLK (At5g58560) as queries against the tomato genome (http://solgenomics.net). Homologous sequences from other plant species were retrieved by Blastp from the Phytozome database (http://phytozome.jgi.doe.gov/pz/portal.html). Nicotiana benthamiana sequences were obtained from the Sol Genomics Network database (http://solgenomics.net). The sequences were aligned using the MUSCLE package available in the MEGA 5.0 software with default parameters (Tamura ), and Neighbor–Joining phylogenies with 5000 bootstrap replications were created with the distances calculated according to the best model indicated by MEGA 5.0.

Generation of SlVTE5-RNAi transgenic lines

Transgenic plants expressing a SlVTE5-specific intron-spliced hairpin sequence under the control of the Cauliflower mosaic virus 35S promoter were obtained for RNAi-mediated silencing of the Solyc03g071720 locus. A 237bp fragment of SlVTE5 was amplified by PCR using the primers RNAi-VTE5-F and RNAi-VTE5-R listed in Supplementary Table S1 available at JXB online. PCR products were cloned into pENTR/d-TOPO vector (Invitrogen) via directional cloning, and then recombined into the binary vector pK7GWIWG2 (Karimi ) to generate pK7GWIWG2(I)-SlVTE5. Agrobacterium-mediated transformation (strain EHA105) of S. lycopersicum was performed according to Pino . The presence of the transgene in T0, T1, and T2 kanamycin-resistant plants was detected by PCR in genomic DNA using 35S-right and RNAi-VTE5-R primers (see Supplementary Table S1).

Identification of the folk-1 tomato mutant by TILLING

Mutations in SlFOLK were identified by screening an EMS-mutagenized tomato population (Just ) essentially as described in Okabe . TILLING (Targeting Induced Local Lesions In Genomes) unlabeled external primers and internal primers 5′ labeled with IRDye 700 and IRDye 800 dye are listed in Supplementary Table S1 at JXB online. Induced point mutations were identified using the mismatch-specific endonuclease ENDO 1. Digested DNA fragments were separated on a Li-Cor DNA analyzer (LI-Cor, Lincoln, NE, USA). The mutation analysis was performed using PARSESNP (Taylor and Greene, 2003) and SIFT (Ng and Henikoff, 2003) software. Homozygous mutant plants were identified by sequencing of the tilled M3 family. Phenotypic characterization was performed in M4 plants homozygous for the folk-1 allele using the corresponding segregating individuals homozygous for the FOLK wild-type allele as control genotype.

qPCR analysis

RNA extraction, cDNA synthesis, and real-time quantitative PCR (qPCR) assays were performed as described by Quadrana . Primer sequences are listed in Supplementary Table S1 at JXB online. qPCRs were performed in a 7500 real-time PCR system (Applied Biosystems) using 2× SYBR Green Master Mix reagent (Applied Biosystems). Expression values were normalized against the geometric mean of two reference genes, CAC and EXPRESSED, according to Quadrana . A permutation test lacking sample distribution assumptions (Pfaffl ) was applied to detect statistical differences (P<0.05) in expression ratios using the algorithms in the fgStatistics software package (Di Rienzo, 2009).

Leaf gas exchange and fluorescence measurements

Gas exchange and Chl fluorescence parameters were evaluated in 5-week-old plants using a portable open gas-exchange system incorporating infra-red CO2 and water vapor analyzers (LI-6400XT system; Li-Cor) equipped with an integrated modulated Chl fluorometer (LI-6400-40; Li-Cor). Reference [CO2] was held at 400 µmol mol−1 and the temperature at 25 °C for all measurements. Air humidity inside the leaf chamber was controlled to the externally measured greenhouse relative humidity (50–60%). Carbon assimilation rate (A), leaf stomatal conductance (g ), leaf dark respiration (R d), and fluorescence parameters were measured at a photosynthetic photon flux density (PPFD) of 600 µmol m−2 s−1 in the first fully expanded leaf between 10:00h and 14:00h. The parameters derived from Chl fluorescence, including light-adapted PSII maximum quantum efficiency (F′ v/F′ m), proportion of open PSII centers (photochemical quenching, qP), and PSII operating efficiency (ΦPSII), were calculated according to Genty .

Tocopherol, free phytol, and fatty acid phytyl ester quantification

Tocopherols were extracted and measured by HPLC as previously described (Yang ). For determination of fatty acid phytyl esters (FAPEs) and free phytol, total lipids were extracted with chloroform according to Lippold . Non-polar lipids were purified using chromatography on silica columns (Kieselgel 60; Merck). FAPEs were measured by direct infusion nanospray quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS; Agilent 6530 Accurate Mass Q-TOF) using methanol:chloroform:300mM ammonium acetate [665:300:35 (v/v/v); Welti ] as the solvent system. FAPEs were detected in the positive ion mode by neutral loss scanning for m/z 278.2974, a fragment characteristic for the phytol moiety. For phytol measurements, the non-polar lipid fraction was silylated and then phytol was quantified by GC-MS as previously described (Lippold ).

Prenylquinone and carotenoid profile

Prenylquinone and related compounds (α-tocopherolquinone, PQ-9, plastoquinol-9, hydroxy-plastoquinone, PC-8, hydroxy-plastochromanol, and ubiquinone-10) were analyzed by a targeted analysis of the lipidomic profile obtained by ultra-HPLC coupled with atmospheric pressure chemical ionization-quadrupole time-of-flight mass spectrometry (UHPLC-APCI-QTOF-MS) as described in Martinis with the following modifications. Briefly, 15mg of lyophilized tissue were exactly weighed and resuspended in 500 μl of tetrahydrofurane:methanol:water 42.5:42.5:15 (v/v/v). The mixture was homogenized using glass beads (1mm in diameter) for 3min at 30 Hz in a tissue lyser. After two rounds of centrifugation (3min, 14 000 g, and 4 °C), supernatants were transferred to vials. Prenyllipids were separated on a reverse-phase Acquity BEH C18 column (50×2.1mm, 1.7 μm) under the following conditions: solvent A=water; solvent B=methanol; 80–100% B in 3min, 100% B for 2min, re-equilibration at 90% B for 0.5min. The flow rate was 0.8ml min–1 and the injection volume was 2.5 μl. PQ-9 and PC-8 were quantified based on calibration curves obtained from standard compounds. Data were processed using MassLynx version 4.1 (Waters).

Carotenoids were extracted and detected as described in Almeida using an Agilent 1200 Series HPLC system coupled with a diode array detector on a reverse phase column [Zorbax Eclipse Plus C18 (150 mm×4.6mm, 5 μm), Agilent Technologies]. Compounds were identified at 440nm by their order of elution and absorption spectra (Gupta ), and co-migration with authentic standards (all-trans-lycopene, all-trans-β-carotene, lutein, violaxanthin, neoxanthin, and zeaxanthin). Relative quantification was performed based on chromatografic peak area normalized against sample dry weight.

Chlorophyll and chlorophyll catabolites

Chl and green catabolites (chlorophyllide, pheophorbide, and pheophytin) were extracted from 10mg of lyophilized tissue during 17h at –20 °C in 90% (v/v) acetone, 10% (v/v) 0.2M TRIS-HCl, pH 8.0, pre-cooled to –20 °C (5ml g−1 initial fresh weight). After centrifuging twice (2min, 16 000 g, 4 °C), supernatants were analyzed by HPLC as described (Langmeier ). Pigments were identified by their absorption spectra at 665nm. For quantification, peak areas were analyzed and referred to calibration curves built from known quantities of standard pigments (Schelbert ).

Quantification of soluble sugars and starch

A 10mg aliquot of the lyophilized samples was extracted five times with 1.5ml of 80% ethanol at 80 °C according to De Souza . Combined supernatants were dried under vacuum and re-suspended in 1ml of ultrapure water. To remove pigments, an extraction with 0.5ml of chloroform was performed. Alcohol-soluble sugar quantification was done by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD) (Dionex-ICS3000, Dionex). Sugar separation was carried out on a CarboPac PA1 column using isocratic elution of 150mM NaOH with a flow rate of 1ml min−1. The calibration curves were prepared using standard solutions of glucose, fructose, and sucrose with a concentration range from 50 μM to 200 μM.

For starch quantification, the dried insoluble material obtained after ethanol extraction was treated with α-amylase (120U ml−1, Megazyme) from Bacillus licheniformis and amyloglucosidase (30U ml−1, Megazyme) from Aspergillus niger according to Amaral . The glucose content obtained after starch hydrolysis was determined from extract aliquots of 20 μl and 50 μl for leaves and fruits, respectively, after an incubation with glucose oxidase/peroxidase and d-4-aminoantipirine (GOD/POD). Absorbance of quinoneimine dye, which is directly proportional to glucose concentration, was measured spectrophotometrically using an ELISA-type microplate reader at 490nm. A standard curve was prepared using high purity glucose solution (Sigma) ranging from 2.5 µg ml−1 to 12.5 µg ml−1.

Trolox equivalent antioxidant capacity (TEAC) assay

The antioxidant capacity of non-polar extracts was assayed as previously described (Re ), with minor modifications. The pre-formed radical 2,2′-azin-obis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+) was produced by oxidation of 7mM ABTS with potassium persulfate (2.45mM final concentration) dissolved in ultrapure water. The mixture was incubated in the dark at room temperature for 12–16h before use. The ABTS·+ solution was diluted with ethanol and adjusted to 0.70±0.02 absorbance units at 734nm. A 50 µl aliquot of diluted extract or Trolox standard was mixed with 150 µl of diluted ABTS·+ solution, and the absorbance was read at 734nm after 10min at 30 ºC. The ABTS·+ antioxidant capacity was reported as µmol of TEAC per gram of sample on a dry weight basis by comparison with a Trolox standard curve (0.015–0.50mM). Analyses were run in triplicate at two dilutions for a total of six assays per sample.

Transmission electron microscopy

Leaf segments were fixed at 4 °C in Karnovsky’s solution [2.5% glutaraldehyde, 2% (v/v) paraformaldehyde in 0.1M sodium phosphate buffer pH 7.2] for 24h. After washing in buffer, the samples were post-fixed in buffered 1% (w/v) osmium tetroxide, washed, dehydrated in a graded series of acetone, and embedded in Spurr resin. The resin was polymerized at 60 ºC. Ultrathin sections were stained with saturated uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), and observed using a Zeiss EM 900 transmission electron microscope.

Data analyses

Statistical analyses were performed using R statistical software (www.r-project.org). To determine significant differences between the transgenic lines and the control, data were analyzed by t-test or ANOVA followed by a Dunnett’s multiple comparison test with the level of significance set to 0.05.

Results

Tomato tocopherol contents are highly dependent on SlVTE5 but not on SlFOLK

By using the A. thaliana VTE5 protein sequence (At5g04490; Valentin ) as query, a survey for homologous sequences in the S. lycopersicum genome was performed in the Solanaceae Genomics Network (http://solgenomics.net/). Two loci were identified, Solyc03g071720 and Solyc09g018510. In order to establish the orthology relationships, a phylogenetic analysis was performed with VTE5 homologous protein sequences of 14 flowering species with completely sequenced genomes. The tree revealed two clades whose topology coincided with the established phylogenetic relationships between the analyzed species. One clade contains the Arabidopsis VTE5 protein sequence (Valentin ) that clustered together with Solyc03g071720. The other clade groups At5g58560, an earlier proposed VTE5 paralog that was further identified as a farnesol kinase (FOLK; Fitzpatrick ), together with Solyc09g018510. This analysis displayed VTE5 and FOLK proteins as sister clades, and the respective genes were named SlVTE5 and SlFOLK (see Supplementary Fig. S1 at JXB online). Both genes showed similar expression patterns, with the highest mRNA levels found in green tomato tissues (Supplementary Fig. S2).

In order to obtain experimental evidence regarding SlVTE5 function in tocopherol biosynthesis, transgenic SlVTE5-knockdown plants were generated by RNAi-mediated silencing. Out of eight primary transformants that showed reduced levels of SlVTE5 mRNA (see Supplementary Fig. S3 at JXB online), three lines with a reduction of >80% were selected for further analyses; SlVTE5-RNAi#1, SlVTE5-RNAi#7, and SlVTE5-RNAi#11 (Fig. 2A). Under normal growth conditions, these transgenic lines exhibited no evident morphological alterations and an apparently unaltered pattern of fruit degreening (Supplementary Fig. S3B).

Fig. 2.

Down-regulation of SlVTE5 expression and tocopherol content in SlVTE5-RNAi transgenic lines. (A) Relative expression of the SlVTE5 gene in the wild-type (WT) and SlVTE5-RNAi lines (#1, #7 and #11). Data are means ±SEM of five biological replicates. The asterisks denote statistically significant differences (permutation test, P<0.05). (B) Total tocopherol was measured in leaves, mature green, and ripe fruits of SlVTE5-RNAi lines. Data represent the mean ±SD of five biological replicates. The asterisks denote significant differences between the WT and the transgenic lines (ANOVA/Dunnett’s test, P<0.05).

HPLC analysis revealed that down-regulation of SlVTE5 resulted in a dramatic reduction (80–90%) of total tocopherol contents both in leaves and in fruits (Fig. 2B). Overall, no differences in tocopherol composition were observed (Supplementary Table S2 at JXB online) except for line #7 at the mature green stage. Notably, we detected only traces of tocotrienols in SlVTE5-RNAi lines (data not shown). These results ruled out the possibility that depletion of tocopherols could be compensated by tocotrienol production in these plants.

The VTE5 deficiency in transgenic lines would be expected to increase free phytol content (Valentin ). We therefore assayed the amount of this metabolite by GC-MS. While in mature leaves and ripe fruits of wild-type tomato plants the amount of free phytol ranged from 100 nmol g−1 DW to 190 nmol g−1 DW, in the counterparts from SlVTE5-RNAi lines this prenyl alcohol accumulated four to five times more (Fig. 3). Interestingly, the molar amount of free phytol that accumulated in leaves was of the same order of magnitude as the reduction observed in total tocopherol contents. Strikingly, the increase in the amounts of free phytol in transgenic ripe fruits was 10 times higher than the decrease in tocopherol (Supplementary Table S3 at JXB online), suggesting differential regulation in the response of phytol metabolism perturbations in source and sink tomato organs.

Fig. 3.

Free phytol content in leaves and ripe fruits of the SlVTE5-RNAi transgenic lines. Data represent the mean ±SD of at least three biological replicates. The asterisks denote significant differences between the wild-type (WT) and the transgenic lines (ANOVA/Dunnett’s test, P<0.05).

Due to sequence similarity between the tomato phosphatidate cytidylyltransferases proteins SlVTE5 and SlFOLK, and the lack of a complete functional characterization of the latter, the putative impact of SlFOLK on tocopherol metabolism was also explored. In this case, a TILLING-based molecular screening was applied to identify a loss-of-function mutation in SlFOLK using an EMS-mutagenized tomato collection. Among the identified mutants, one, named folk-1, displayed a G to A substitution disrupting the 3′ splicing site of intron 4. Sequence analyses of folk-1 cDNA from homozygous mutant plants revealed that this lesion led to the use of a cryptic splicing site in intron 4, producing an mRNA that lacks exon 4 and contains a fragment of intron 4 (see Supplementary Fig. S4A at JXB online). This abnormally spliced transcript of FOLK, which is the only isoform detected in the mutant, contains an in-frame premature stop codon that presumably leads to a truncated protein (Fig. 4A; Supplementary Fig. S4A, B). mRNA harboring premature termination codons can be recognized by the RNA surveillance machinery as aberrant; these transcripts may be targeted by the nonsense-mediated decay pathway, being rapidly degraded (Filichkin ). Expression analysis by qPCR showed that the amount of the abnormal mRNA in folk-1 corresponded to only 10% of the fully spliced transcript found in control plants (Supplementary Fig. S4C). Tocopherol levels and composition in plants homozygous for the folk-1 allele were much the same as those in control plants, suggesting a small, if any, contribution of SlFOLK to tocopherol biosynthesis in both leaves and fruits (Fig. 4B; Supplementary Table S2). Having demonstrated the major role of VTE5 in tomato tocopherol metabolism, we further performed a comprehensive phenotypic characterization of SlVTE5-RNAi lines.

Fig. 4.

Total tocopherol content in the folk-1 mutant. (A) Diagram showing the SlFOLK gene and fully spliced mRNA found in the wild-type (WT) and abnormally spliced mRNA found in the folk-1 mutant. Boxes and solid lines represent exons and introns, respectively. The premature stop codon is indicated by a black hexagon. (B) Total tocopherol was measured in leaves, mature green, and ripe fruits of M4 plants homozygous for the folk-1 allele. The corresponding segregating individuals homozygous for the FOLK WT allele were used as control. Data represent the mean ±SD of five biological replicates. No significant differences were observed (Student’s t-test, P>0.05).

Down-regulation of SlVTE5 boosted phytyl ester synthesis in leaves

Free phytol can be esterified directly with fatty acids derived from activated acyl groups. FAPEs increase during stress-associated Chl degradation (e.g. nitrogen deprivation) and senescence (Gaude ; Lippold ). To address the question of whether the increased phytol levels affect FAPE contents in the SlVTE5-RNAi lines, the level of these compounds was measured by direct infusion Q-TOF MS/MS. Notably, SlVTE5 knockdown resulted in a dramatic increase of FAPE content up to 10-fold in leaves. In contrast, fruits from SlVTE5-RNAi lines exhibited levels of FAPE identical to those of wild-type plants (Fig. 5). In addition to the total amount, FAPE composition was also highly affected in transgenic leaves (Fig. 6A). With the exception of palmitic (16:0) and linolenic (18:3) acids, analysis of the lipid profiles showed that the contribution of the different acyl chains was not proportional to the increment in total FAPE content observed in SlVTE5-RNAi lines. In particular, FAPEs containing oleic acid (18:1), hexadecatrienoic acid (16:3), and medium-chain fatty acids (10:0, 12:0, 14:0) exhibited a reduction in their relative content, while FAPEs containing stearic acid (18:0) and linoleic acid (18:2) became the predominant forms, increasing at least 2-fold in transgenic leaves compared with the wild-type. In SlVTE5-RNAi fruits, the FAPE composition at mature green and ripe stages remained almost unchanged (Fig. 6B, C).

Fig. 5.

Total fatty acid phytyl ester (FAPE) content in SlVTE5-RNAi transgenic lines. Data represent the mean ±SD of at least three biological replicates. The asterisks denote significant differences between the wild-type (WT) and the transgenic lines (ANOVA/Dunnett’s test, P<0.05).

Fig. 6.

Molecular species composition of fatty acid phytyl esters (FAPEs) in SlVTE5-RNAi transgenic lines. FAPEs were measured in leaves (A), mature green (B), and ripe fruits (C). Data represent the mean ±SD of at least three biological replicates.

Chlorophyll content is not affected in SlVTE5-RNAi lines

To examine whether SlVTE5 knockdown affects Chl metabolism, we determined Chl a, Chl b, and pheophytin a (Phein a) levels in leaves and fruits at three different ripening stages by HPLC. The contents of these compounds were largely unaltered in both tested organs, suggesting that accumulation of phytol did not significantly affect Chl and Phein a levels in tomato (Fig. 7).

Fig. 7.

Chlorophyll (Chl) and pheophytin a (Phein a) content in SlVTE5-RNAi transgenic lines. (A, C) Quantification of Chl and Phein a in leaves. (B, D) Quantification of Chl and Phein a in fruits at mature green (MG), breaker+1 (B+1), and breaker+3 (B+3) stage. Data represent the mean ±SD of at least three biological replicates. No significant differences were observed (ANOVA/Dunnett’s test, P>0.05).

SlVTE5 knockdown alters prenyllipid metabolism in fruits

In addition to tocopherols, the plastidial antioxidant network includes a variety of prenyllipids derived from the MEP isoprenoid pathway with strong antioxidant properties, such as carotenoids, prenylquinones (PQ-9), and other tocochromanols (e.g. PC-8) (Nowicka ). To investigate whether tocopherol deficiency in SlVTE5-RNAi lines is compensated by any other non-enzymatic antioxidant mechanism, we performed a comprehensive profiling of prenyllipids (Table 1). For determination of prenylquinone and their derivative compounds, a targeted analysis of lipidomic profile obtained by the UHPLC-QTOF MS method was performed, whereas carotenoids were quantified by HPLC. In leaves and ripe fruits of the transgenic plants, depletion of tocopherol was accompanied by a decrease of α-tocopherolquinone, an oxidized intermediate of the tocopherol redox cycle; yet, the level of this metabolite remained unchanged in mature green fruits. Remarkably, the levels of the photosynthetic electron carrier phylloquinone (vitamin K), another product of phytyl diphosphate-dependent biosynthesis, did not change between SlVTE5-RNAi lines and the wild-type control.

Table 1.

Changes in prenyllipid contents in leaves and fruits of SlVTE5-RNAi transgenic lines compared with the wild-type

Data were normalized to sample DW and expressed relative to the wild-type (WT) in each tissue.

Prenyllipids (relative amounts)	WT	SlVTE5-RNAi
		#1	#7	#11
Leaf
α-TQ	1.00±0.18	0.23±0.03	0.22±0.08	0.24±0.01
PC-8	1.00±0.30	0.52±0.19	0.54±0.19	0.36±0.07
PQ-9	1.00±0.18	0.83±0.15	0.87±0.20	0.63±0.12
PQH2-9	1.00±0.66	1.27±0.59	1.02±0.43	0.86±0.37
PC-OH	1.00±0.21	0.65±0.18	0.72±0.24	0.51±0.11
PQ-OH	1.00±0.20	1.01±0.26	1.04±0.27	0.70±0.19
UQ-10	1.00±0.24	0.83±0.23	0.81±0.12	0.81±0.10
Phylloquinone	1.00±0.16	0.87±0.12	0.84±0.12	0.81±0.12
β-Carotene	1.00±0.07	0.89±0.04	0.91±0.02	0.91±0.07
Lutein	1.00±0.07	0.97±0.06	0.96±0.03	0.95±0.03
Violaxanthin/neoxanthin	1.00±0.06	0.97±0.06	1.00±0.02	0.99±0.06
Mature green fruit
α-TQ	1.00±0.15	1.11±0.48	1.20±0.47	1.13±0.10
PC-8	1.00±0.15	1.89±0.26	1.78±0.05	1.88±0.19
PQ-9	1.00±0.15	2.11±0.46	2.22±0.42	2.22±0.26
PQH2-9	1.00±0.30	0.77±0.23	1.24±0.36	1.16±0.36
PC-OH	1.00±0.42	0.89±0.34	1.10±0.30	1.02±0.29
PQ-OH	1.00±0.26	1.31±0.49	1.74±0.47	1.52±0.32
UQ-10	1.00±0.13	2.13±0.24	1.76±0.28	1.93±0.34
Phylloquinone	1.00±0.27	1.23±0.18	1.25±0.32	1.32±0.16
β-Carotene	1.00±0.22	0.96±0.16	0.92±0.15	1.00±0.16
Lutein	1.00±0.22	0.98±0.10	1.09±0.11	1.31±0.15
Violaxanthin/neoxanthin	1.00±0.34	0.85±0.16	0.85±0.07	1.28±0.20
Ripe fruit
α-TQ	1.00±0.25	0.54±0.04	0.64±0.17	0.64±0.15
PC-8	1.00±0.15	2.29±0.34	1.67±0.20	2.07±0.50
PQ-9	1.00±0.17	2.40±0.54	2.24±0.20	1.76±0.26
PQH2-9	1.00±0.29	1.55±0.39	1.77±0.55	1.92±1.06
PC-OH	1.00±0.27	1.26±0.51	1.35±0.46	0.99±0.42
PQ-OH	1.00±0.07	2.27±1.03	2.24±0.80	2.10±0.94
UQ-10	1.00±0.14	2.40±0.38	1.96±0.53	2.13±0.24
Phylloquinone	1.00±0.18	1.11±0.18	1.32±0.50	1.41±0.50
Phytoene	1.00±0.09	0.68±0.11	0.72±0.19	0.62±0.10
Phytofluene	1.00±0.14	0.72±0.13	0.66±0.14	0.64±0.08
Neurosporene	1.00±0.22	0.73±0.24	0.97±0.18	0.60±0.20
ζ-Carotene	1.00±0.20	0.44±0.17	0.33±0.19	0.39±0.18
Lycopene	1.00±0.06	0.78±0.14	0.70±0.08	0.74±0.13
β-Carotene	1.00±0.06	0.87±0.20	0.86±0.16	0.90±0.14
Lutein	1.00±0.13	1.07±0.13	1.02±0.09	1.19±0.25

Values are represented as means ±SD. Terms in bold indicate a statistically significant difference by ANOVA/Dunnett’s test (P<0.05).

α-TQ, α-tocopherolquinone; PQ-9, plastoquinone-9; PQH2-9, plastoquinol-9; PQ-OH, hydroxy-plastoquinone; PC-8, plastochromanol-8; PC-OH, hydroxy-plastochromanol; UQ-10, ubiquinone-10.

The presence of the reduced and oxidized forms of PQ-9 in wild-type tomato leaves has already been reported (Jones ), and our data showed that PC-8 is also detected in S. lycopersicum, in both leaves and fruits (see Supplementary Fig. S5 at JXB online). PC-8 was less abundant than tocopherols in wild-type tomato leaves, as described for other species (Kruk ). In fruits, however, the amount of these tocochromanols was similar (Fig. 2; Supplementary Fig. S5).

The comparison of prenyllipid profiles between wild-type and SlVTE5-knockdown leaves revealed that PQ-9 forms (PQ-9, PQH2-9, and PQ-OH) remained unchanged. In contrast, a reduction to 50% of PC-8 content was observed in SlVTE5-RNAi lines, which was accompanied by lower levels of its oxidation product, PC-OH (Table 1). In sharp contrast, the prenylquinone pool of transgenic fruits was significantly increased. PQ-9 levels were >2-fold higher in the SlVTE5-RNAi lines, although the levels of the reduced form PQH2-9 were unchanged. Intriguingly, the levels of the mitochondrial prenylquinone UQ-10 were also ~2-fold increased in SlVTE5-RNAi fruits. In addition, PC-8 accumulated up to 2-fold in both mature green and ripe fruits of the transgenic lines (Table 1). These results suggest that the pool of PQ-9 and PC-8 contributes to fulfilling an antioxidant function in the transgenic fruits.

Carotenoid contents in leaves and mature green fruits remained largely unaltered in the SlVTE5-RNAi lines (Table 1). Although no differences in visual appearance were identified (see Supplementary Fig. S3B at JXB online), at the ripe stage, silenced fruits exhibited 30% less lycopene than those of wild-type plants, which was also accompanied by an equivalent reduction in levels of lycopene precursors (phytoene, phytofluene, and ζ-carotene).

Considering the differences observed in prenyllipid profiles, the total antioxidant capacity of SlVTE5-RNAi and wild-type tomato plants was evaluated in non-polar extracts by the TEAC assay. Transgenic ripe fruits showed a reduction in TEAC values in lines #1 and #11 (see Supplementary Fig. S6 at JXB online). These results emphasize the role of tocopherol and/or carotenoids in antioxidant protection, since the increase in prenyllipid contents exhibited in the fruits of the SlVTE5-RNAi lines did not compensate the TEAC values up to the values observed in wild-type plants.

VTE5 deficiency affects the expression of tocopherol metabolism-related genes

The biochemical profile described above showed that SlVTE5 knockdown results in an adjustment in prenyllipid and FAPE metabolism in an organ-specific manner. In order to understand whether these changes could be associated with differential gene expression regulation, mRNA levels of genes encoding proteins involved in MEP, shikimate, tocochromanol, carotenoid, and Chl metabolism (Quadrana ; Lira ; Almeida ), as well as in prenylquinone and FAPE synthesis were measured by qPCR (Fig. 1). Genes that showed significantly different mRNA levels in at least two transgenic SlVTE5-RNAi lines and, when applicable, the third followed the same trend are shown in Fig. 8. The complete set of data is shown in Supplementary Table S4 at JXB online.

Fig. 8.

Changes in gene expression levels of some key isoprenoid metabolism-related genes resulting from SlVTE5 down-regulation in both leaves and fruits.The amount of mRNA of the enzyme-encoding genes shown in Fig. 1 was quantified. Expression data are means ±SEM of three biological replicates of log2-fold changes compared with the corresponding organ of the wild-type control. Only genes that showed significantly different mRNA levels in SlVTE5-knockdown lines are shown (permutation test, P<0.05). For simplicity, solely data from SlVTE5-RNAi#7 are represented. The complete data set is available in Supplementary Table S4 at JXB online.

Genes of tocochromanol biosynthesis did not exhibit a consistent expression tendency in leaves of SlVTE5-RNAi lines. In fruit, the elevated expression of HPPD(1) and HPPD(2) is consistent with the increased prenylquinone content in silenced plants. Moreover, in transgenic ripe fruit, VTE3(1) up-regulation also coincided with the higher content of PC-8 and PQ-9. Intriguingly, the expression of the gene encoding the solanesyl diphosphate synthase (SPS), which catalyzes the production of the PQ-9 or PC-8 prenyl side chain, was decreased in transgenic mature green fruits, suggesting a negative feedback regulatory mechanism.

In leaves, an apparent reduction in the Chl degradation pathway was observed in SlVTE5-RNAi lines, as indicated by a down-regulation of STAY-GREEN1 (SGR1) and PHEOPHORBIDE A OXYGENASE (PAO) expression. Although Chl contents remained invariant, the observed transcriptional down-regulation may reflect a response to phytol accumulation. This scenario contrasts with that observed in fruits where SGR1 was up-regulated in transgenic plants compared with the wild-type. Additionally, ripe fruit of SlVTE5-RNAi lines showed higher mRNA levels of PHEOPHYTINASE (PPH).

Regarding carotenoid biosynthesis, only certain genes showed significant changes in their mRNA levels in leaves and mature green fruits of the SlVTE5-RNAi lines, although the biochemical profiles of these compounds remained unaltered when compared with the wild-type. In contrast, in ripe fruits, the increased levels of CHROMOPLAST-SPECIFIC β-LYCOPENE CYCLASE (CYC-β) transcripts could account for the reduction in lycopene and its immediate precursors verified in the transgenic lines.

Finally, the PALE YELLOW PETAL 1 (PYP1) gene, the ortholog of Arabidopsis PHYTYL ESTER SYNTHASE1 (PES1) (Ariizumi ), showed increased levels of transcripts in the leaves of the SlVTE5-RNAi lines, in agreement with the higher FAPE contents observed in these organs. Thus, the analysis of transcriptional profiles reinforces the specific regulation occurring in leaves and fruits.

Measurements of carbohydrate metabolism, photosynthesis, and yield parameters suggest carbon export impairment in SlVTE5-knockdown plants

To evaluate whether the observed tocopherol deficiency in SlVTE5-RNAi lines affects carbon fixation and partitioning, the starch and soluble sugar content, photosynthetic performance, and yield parameters were assessed. Leaves of 5-week-old transgenic plants showed up to a 4.5-fold increase in starch content accompanied by a 20% decrease in sucrose levels compared with the wild-type control in the middle of the light period (Fig. 9). Concomitantly, the carbon assimilation rates were reduced in SlVTE5-RNAi lines, while the efficiency of PSII activity (ΦPSII) displayed moderate reduction (Fig. 10). The chloroplast ultrastructure was mostly preserved in SlVTE5-knockdown plants (see Supplementary Fig. S7 at JXB online).

Fig. 9.

Starch and soluble sugar levels in source leaves in SlVTE5-RNAi transgenic lines. The first fully expanded leaves were harvested from 5-week-old plants in the middle of the light cycle. Starch is given in µg glucose equivalents. Data are means ±SD of five biological replicates. The asterisks denote significant differences between the wild-type (WT) and the transgenic lines (ANOVA/Dunnett’s test, P<0.05).

Gas-exchange and PSII efficiency parameters in SlVTE5-RNAi transgenic lines. (A) The response of carbon assimilation (A) to light intensity. (B) Leaf stomatal conductance (g ). (C) Leaf dark respiration (R d). (D) Light-adapted PSII maximum quantum efficiency (F′ v/F′ m). (E) PSII operating efficiency (ΦPSII). Data correspond to measurements in the first fully expanded leaf of 5-week-old plants and represent the means ±SD of five biological replicates. The asterisks denote significant differences between the wild-type (WT) and the transgenic lines (ANOVA/Dunnett’s test, P<0.05).

In agreement with carotenoid profiling and the gene expression pattern, transgenic plants displayed a delay in fruit development and ripening as indicated by the frequency of red and green fruits, as well as the red fruit yield compared with the control genotype at harvest time (Table 2). Moreover, they displayed a yield penalty evidenced by a modest reduction in the harvest index. These results suggest that efficiency in photosynthate partitioning is compromised by the tocopherol deficiency in transgenic lines.

Table 2.

Yield-associated traits of SlVTE5-RNAi transgenic lines

Trait	WT	SlVTE5-RNAi
		#1	#7	#11
Number of total fruits	36.2±5.8	37.4±4.7	40.8±4.3	35.8±1.5
Frequency red fruits (%)	63.9±6.9	49.6±7.8	54.0±11.5	53.3±8.8
Frequency green fruits (%)	37.3±6.9	50.4±7.8	46.0±11.5	46.7±8.8
Vegetative plant weight (g FW)	30.0±8.3	34.3±8.0	39.4±6.9	34.0±7.3
Total yield per plant (g FW)	114.4±17.2	115.9±11.3	125.9±28.8	101.6±16.9
Harvest index	0.79±0.02	0.77±0.04	0.76±0.02	0.75±0.03
Red yield/aerial biomass ratio	0.57±0.08	0.47±0.11	0.50±0.09	0.52±0.15

Vegetative plant weight was determined by weighing only the vegetative tissue (after harvesting the fruits) without the root. Harvest index was calculated as the ratio between total fresh yield per plant (red and green fruit mass) and aerial biomass (total yield+vegetative plant weight).

Values indicate the mean ±SD of phenotypic values (n=5) determined for 15-week-old plants. Statistically significant differences between the wild-type (WT) control and transgenic lines are indicated in bold (Student’s t-test, P<0.05).

Discussion

Several reports have dissected tocopherol biosynthesis, identified quantitative trait loci (QTLs) that determine VTE accumulation, and characterized regulatory mechanisms that control the tocopherol biosynthetic pathway (Almeida ; DellaPenna and Mène-Saffrané, 2011; Martinis et al., 2013, 2014; Quadrana , 2014; Zhang ; Zhang ). One well-defined metabolic constraint is the availability of phytyl diphosphate precursor for tocopherol biosynthesis, which can be derived from de novo biosynthesis via the MEP pathway and from Chl phytol tail recycling (Ischebeck ; Quadrana ; Zhang ). The phytol hydrolysis of Chl is the primary source of prenyl chain for tocopherol biosynthesis in Arabidopsis seeds (Valentin ). However, functional analysis of the phytol salvage pathway has been limited to this species. In this study, we investigated the contribution of VTE5-mediated phytyl diphosphate synthesis for tocopherol production and its impact on plant physiology in tomato. The comprehensive metabolite profiling, expression analyses, and evaluation of photosynthetic parameters performed in tomato SlVTE5-knockdown plants allowed us to gain insights into the interactions between phytol recycling, and lipid and carbon metabolism, exposing distinct metabolic adjustment in source and sink organs.

Even with an active de novo synthesis of phytyl diphosphate, down-regulation of SlVTE5 dramatically reduces tocopherol content in leaves and fruits in comparison with the wild-type genotype. These data demonstrated that in tomato, tocopherol production is mostly dependent on the Chl-linked pathway for phytyl diphosphate synthesis in both vegetative and reproductive organs. Furthermore, these findings suggest that SlVTE5 is the main—if not the sole—contributor to VTE5 activity. The analysis of the tomato folk-1 mutant suggested that SlFOLK is not involved in reactivation of free phytol.

Coincident with SlVTE5 down-regulation, free phytol, the substrate of the phytol kinase reaction, accumulates in leaves of transgenic lines. Interestingly, the amount of free phytol accumulated corresponds to the decrease in tocopherol content, suggesting that a large proportion of phytol diphosphate derived from the phytol phosphorylation pathway is used for tocopherol biosynthesis in tomato. Furthermore, FAPEs are strongly increased in the mature leaves of SlVTE5-RNAi lines, although the Chl levels remain unchanged. FAPEs are plastogobule compounds that represent a class of stress-induced lipids in higher plants, which may act as plastidial transient sinks for the deposition of fatty acids and phytol (Gaude ). Since the phytol moiety of FAPEs is mostly derived from Chl degradation (Lippold ), our findings raise an intriguing issue concerning the origin of the phytol that sustains FAPE synthesis in SlVTE5-knockdown plants. One possible explanation may be that perturbations in phytol metabolism caused by the lack of VTE5 activity promote an increase in Chl turnover in transgenic lines. In leaves, the increased steady-state phytol levels were channeled to FAPE synthesis, while in fruits phytol remained as the free form. Alternatively, the origin of FAPE-associated phytol might be explained by the impairment in catabolism, which in plants involves the production of phytenoyl-CoA in chloroplasts that is further degraded by α-oxidation in peroxisomes and mitochondria, similar to that described in animals (Araújo ). Notably, levels of phylloquinone, another phytyl diphosphate chain-containing molecule, were unchanged in both leaves and fruits, indicating that the VTE5-dependent phytol pathway does not affect vitamin K synthesis in tomato. This result coincides with the previous observation of feeding experiments of Arabidopsis seedlings with radiolabeled phytol (Ischebeck ).

Even under permissive growing conditions, VTE5-tocopherol deficiency not only impacts the lipid profile but also carbon metabolism and photosynthesis, having consequences on the tomato plant physiology. SlVTE5-RNAi lines showed higher starch accumulation in mature leaves that correlated with a lower CO2 assimilation rate and the reduction in PSII operating efficiency. This scenario suggests carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity (Adams ; Asensi-Fabado ). Moreover, a subtle reduction in the number of harvestable fruits and the harvest index was observed. These results could be indicative of sugar export blockage from leaves towards sink organs in SlVTE5-knockdown tomato plants. Photoassimilate export impairment mediated by tocopherol deficiency has been reported in the literature, such as in potato VTE1-RNAi lines (Hofius ; Asensi-Fabado ) and vte2 A. thaliana during low-temperature adaptation (Maeda , 2008), where carbon accumulation was verified in source leaves at the end of the light period. How tocopherol influences photoassimilate partitioning has not been precisely addressed yet. However, Song provided robust genetic evidence that alterations in extra-plastidic lipid metabolism are upstream of the defect in photoassimilate export in VTE-deficient plants, which is mediated by fatty acid desaturases (FADs). In particular, it was reported that VTE depletion led to increased linoleic acid (18:2) content and a reduced level of linolenic acid (18:3). Consistent with this, one of the acyl groups that mostly contribute to the increase in the total FAPEs in the chloroplasts of the SlVTE5-RNAi lines was 18:2. Additionally, our lipidomic data revealed that the plastidial digalactosyldiacylglycerol (DGDG), which mainly consists of pairs including 18:3 species, was reduced in leaves of SlVTE5-RNAi lines compared with the wild-type (see Supplementary Table S5 at JXB online), resembling the lipid alterations previously reported (Maeda , 2008). It has been proposed that changes in membrane lipid composition as a result of tocopherol deficiency might affect the properties of the secretory membrane systems (Maeda et al., 2008, 2014). Since tomato has been described as an apoplastic phloem loader (Muller ), we could speculate that the alteration of endomembrane vesicle formation affects the sucrose efflux mediated by SWEET proteins, which have been described as key players in phloem transport (Chen ; McCurdy and Hueros, 2014). Alternatively, the carbon export impairment observed in SlVTE5-knockdown tomato plants could be the result of the interaction between lipid and sugar metabolism by an as yet unidentified mechanism probably involving sugar sensing proteins, as proposed by Asensi-Fabado .

VTE5 deficiency triggered different metabolic responses in fruits compared with those described for leaves, reflecting the intrinsic physiological differences between organs and their corresponding plastids. First, fruits of SlVTE5-knockdown plants accumulated phytol in the free form rather than channeled into synthesis of FAPEs. This might be explained by the lowered availability of acyl donors inherent to fruit-specific lipid metabolism (Domínguez ). Secondly, the observation that the non-tocopherol prenylquinone pool, including PQ-9 and PC-8, is increased in fruits of SlVTE5-knockdown plants suggests that a regulatory compensation mechanism between the tocopherol and prenylquinone pathways exists in this organ. The ability of plastoquinol (PQH2-9), ubiquinol (UQH2-10), the reduced forms of PQ-9 and UQ-10, respectively, and PC-8 to scavenge ROS and inhibit lipid peroxidation has been demonstrated before (Kruk and Trebst, 2008; Nowicka ; Rastogi ). Moreover, PQ-9 and PC-8 have already been associated with inhibition of lipid peroxidation and 1O2 scavenging in VTE-deficient Arabidopsis vte2 mutants (Mène-Saffrané ). Likewise, in tomato leaves, the reduction of PQ-9 content by the virus-induced gene silencing approach resulted in increased tocopherol and UQ-10 levels (Jones ). Interestingly, the favorable biochemical environment for the accumulation of PQ-9 and PC-8 is supported not only by reduced flux through the tocopherol competing pathway but also by the HPPD and VTE3 up-regulation in this organ. Finally, SlVTE5-RNAi ripe fruits exhibited perturbations in the carotenoid pathway. The reduced amount of lycopene and its biosynthetic precursors can be explained by the higher transcript levels of SGR1 found at the mature green and ripe stages. Besides having an important role in the regulation of plant Chl degradation and senescence (Hörtensteiner, 2009), SGR1 also regulates lycopene and β-carotene biosynthesis by direct interaction with PSY(1), thereby inhibiting its activity (Luo ). Simultaneously, the failure of co-ordinate transcriptional repression of the CYC-β gene in SlVTE5-knockdown ripe fruits could also account for the reduced abundance of acyclic carotenoids. The amounts of β-carotene and lutein in transgenic fruits were similar to those in the wild-type at the expense of the preceding carotenoids. Additionally, it has been demonstrated that the level of PPH transcripts decreases during tomato fruit ripening (Lira ); however, SlVTE5-RNAi ripe fruits displayed higher levels of PPH transcripts than the wild-type. Thus, the comprehensive analysis of the biochemical and transcriptional data together with the results of the yield experiment indicates that the ripe profiled transgenic fruits are indeed less ripe than wild-type fruits. The reduction in carbohydrate export described above might be in part responsible for the delay in fruit development and ripening, resulting in a reduced proportion of mature fruits at harvest time in SlVTE5-knockdown plants.

The results presented here clearly show that in tomato VTE biosynthesis is largely dependent on the salvage pathway for phytyl diphosphate synthesis rather than the de novo synthesis from the MEP pathway in both leaves and fruits. VTE5 deficiency affected lipid metabolism, evidenced by the abundance and composition of FAPEs in leaves and prenylquinones in fruits. Together, these results exposed the complexities of the metabolic regulation that emerge from the isoprenoid pathway network, which involves a tight control between precursor supply and utilization highly dependent on the plastid type. Moreover, our data highlighted the cross-talk between lipid and carbon metabolism mediated by tocopherol that resulted in the impairment of carbon export in SlVTE5-knockdown tomato plants compromising fruit development and ripening.

Supplementary data

Supplementary data are available at JXB online.

Table S1. Primers used for each experiment.

Table S2. Tocopherol content and composition of SlVTE5-RNAi transgenic lines and the folk-1 mutant.

Table S3. Moles of prenyllipids found in SlVTE5-RNAi transgenic lines.

Table S4. Transcriptional profile of genes encoding isoprenoid metabolism-related enzymes.

Table S5. Changes in fatty acid-derived lipids in leaves of SlVTE5-RNAi transgenic lines compared with the wild-type.

Fig. S1. Phylogenetic analysis of VTE5 and FOLK proteins.

Fig. S2. Expression of SlVTE5 and SlFOLK.

Fig. S3. Fruit phenotype of SlVTE5-RNAi transgenic lines.

Fig. S4. Analysis of the folk-1 mutation.

Fig. S5. Plastoquinone (PQ-9) and plastochromanol (PC-8) levels in SlVTE5-RNAi transgenic lines.

Fig. S6. Trolox equivalent antioxidant capacity (TEAC) in leaves and fruits of SlVTE5-RNAi transgenic lines.

Fig. S7. Chloroplast ultrastructure resulting from SlVTE5 down-regulation.