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

Abstract

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.

Keywords: Carotenoids; Solanum lycopersicum; chlorophyll; phytol; phytol kinase; prenyllipids; tocopherol; tomato; vitamin E..

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).

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).

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).

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).

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.

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).

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.

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).

Fig. 10.

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 _s). (C) Leaf dark respiration (R _d). (D) Light-adapted PSII maximum quantum efficiency (F′ _v/F′ _m). (E) PSII operating efficiency (Φ_PSII). 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).