RNAi-mediated tocopherol deficiency impairs photoassimilate export in transgenic potato plants

Abstract

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and the photosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have been proposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherol cyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacks a phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in this study, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a protein with high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescent protein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption of TC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence of tocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to be a consequence of vascular-specific callose deposition observed in source leaves. CO2 assimilation rates and photosynthetic gene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting a carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity. This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreased tocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from source leaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherol reduction.

Figure 1.

TC activity of recombinant StSXD1 protein in E. coli and intracellular targeting of StSXD1 in leaves. A, The premature (columns 3 and 6) and mature part (columns 2 and 5) of StSXD1 cDNA was expressed in E. coli cells and used for TC assay with DMGQ and DMPQ as substrates. The products, γ-tocotrienol and γ-tocopherol, were quantified by fluorescence HPLC. The values represent the mean of three independent experiments and sd. E. coli cells transformed with an empty vector pQE11 served as control (columns 1 and 4) and accumulated less than 2 ng γ-tocotrienol or 1 ng γ-tocopherol/mg of protein. B–J, GFP fusion proteins of full-length StSXD1 (StSXD1:GFP) and N-terminal TP (StSXD1TP:GFP) as well as GFP protein alone were transiently expressed in tobacco epidermal cells by microprojectile bombardment and analyzed by confocal microscopy after 20 h. Green color indicates GFP fluorescence and red color reveals chlorophyll (Chl) fluorescence. B, Intracellular localization of StSXD1:GFP in terminal trichome cell. C, Chl fluorescence indicates chloroplast distribution. D, Merged image. E, Intracellular localization of StSXD1TP:GFP in stomata cell. F, Chl fluorescence of chloroplasts. G, Merged image. H, Intracellular localization of free GFP in stomata cell. I, Chl fluorescence of chloroplasts. J, Merged image. Bars represent 10 μm.

Figure 2.

RNAi-mediated silencing of StSXD1 leads to suppression of TC activity and results in tocopherol deficiency as well as DMPQ accumulation in transgenic potato plants. A, Schematic structure of the binary intron-spliced hairpin RNA (RNAi) expression construct used for transformation of potato plants. StSXD1 fragments (765 bp, nts 503–1,268 of StSXD1 cDNA; accession no. AY536918) in sense and antisense orientation separated by intron 1 of potato GA20 oxidase (200 bp) were placed between the cauliflower mosaic virus 35S promoter and the ocs terminator of the Bin19-derived vector using the indicated restriction sites. B, TC activity. C, Tocopherol contents (α- and γ-tocopherol). D, DMPQ levels in source leaves of StSXD1-silenced transgenic lines (StSXD1-RNAi-18, -14, -21, -27, and -22) compared to wild type. TC activity was determined using DMGQ as substrate and tocopherols, as well as the prenyl quinone precursor DMPQ, were quantified by fluorescence HPLC. Samples were taken from source leaves 5 weeks after transfer form tissue culture and data represent the means ± se of 5 individual plants.

Figure 3.

Carbohydrate steady-state levels and spatial distribution of starch accumulation in leaves of tocopherol-deficient potato plants. A, Soluble sugar and starch contents in three different leaf stages (11th, 8th, and 3rd leaf from the top) of 59-d-old plants at the end of the dark period. Two samples were taken from each leaf and averaged; values are given as means ± se of 5 independent plants, and soluble sugars represent the sum of Glc, Fru, and Suc. B, Qualitative determination of starch accumulation in leaves of StSXD1-RNAi-22 and wild type after an extended dark period (16 h) by iodine (KI) staining. Bars represent 2 cm.

Figure 4.

Amino acid concentrations in source leaves of StSXD1-silenced potato plants. Leaf samples were taken at the end of the light period from lower source leaves (leaf 11 from the top) of 59-d-old plants. Total amino acid levels represent the sum of single amino acid concentrations determined by HPLC. Values are given as means (n = 4) ± se.

Figure 5.

Growth response, leaf morphology, and tuber yield of tocopherol-deficient potato plants. A, Visible phenotype of transgenic lines and wild type after 9 weeks in the greenhouse. Bar represents 50 cm. B, Phenoytpic alterations of lower source leaves from StSXD1-RNAi-22 and -27 compared to wild type. Arrowheads indicate necrotic spots at the leaf margins. C, Tuber yield of StSXD1-RNAi and wild-type plants grown for 13 weeks in the greenhouse. Data represent means ± se of 5 plants.

Figure 6.

Callose concentrations and distribution in source leaves of tocopherol-deficient potato plants. A, Callose contents in lower source leaves (leaf 11 from the top) of 59-d-old potato plants at the end of the photoperiod. Data represent the means (n = 5) ± se and are given as β-1,3-glucan pachyman equivalents. B, Immunofluorescence analysis of vascular-specific callose accumulation in StSXD1-RNAi-22 compared to wild type. Samples were taken from lower source leaves of 59-d-old potato plants and callose was detected in longitudinal section using a monoclonal anti-β-1,3-glucan antibody and Alexa Fluor 488 antimouse IgG as fluorescence marker. Callose-derived fluorescence signals in vein class III-associated cells were detected by confocal laser scanning microscopy and superimposed with bright field images. Bars represent 50 μm.

Figure 7.

Photosynthetic capacity in tocopherol-deficient potato lines. A, Starch accumulation in three different leaf stages (11th, 8th, and 5th leaf from the top) of wild type and StSXD1-RNAi-22 plants selected for CO₂ gas-exchange measurements. B, Light response curves of StSXD1-RNAi-21, -27, and -22 plants in comparison to wild type. Photosynthetic rates were determined at 400 μm m⁻¹ CO₂ concentration using a LICOR LI6400 instrument. Plants were analyzed 8 weeks after transfer to the greenhouse and data represent means ± se of 4 plants.

Figure 8.

Gene expression analysis of StSXD1-silenced plants. Total RNA was isolated from lower and upper source leaves (11th and 8th leaves from the top of 59-d-old StSXD1-RNAi lines and control plants (wild type), and transcript levels were analyzed by northern blotting. Thirty micrograms of RNA were loaded per lane and hybridized with cDNA probes of photosynthetic (rbcS, cab), defense-related (Pin2), as well as JA (AOC) and Pro (P5CS) biosynthetic genes. Additional probing with an 18S cDNA served as loading control.