Specific roles of alpha- and gamma-tocopherol in abiotic stress responses of transgenic tobacco

Abstract

Tocopherols are lipophilic antioxidants that are synthesized exclusively in photosynthetic organisms. In most higher plants, alpha- and gamma-tocopherol are predominant with their ratio being under spatial and temporal control. While alpha-tocopherol accumulates predominantly in photosynthetic tissue, seeds are rich in gamma-tocopherol. To date, little is known about the specific roles of alpha- and gamma-tocopherol in different plant tissues. To study the impact of tocopherol composition and content on stress tolerance, transgenic tobacco (Nicotiana tabacum) plants constitutively silenced for homogentisate phytyltransferase (HPT) and gamma-tocopherol methyltransferase (gamma-TMT) activity were created. Silencing of HPT lead to an up to 98% reduction of total tocopherol accumulation compared to wild type. Knockdown of gamma-TMT resulted in an up to 95% reduction of alpha-tocopherol in leaves of the transgenics, which was almost quantitatively compensated for by an increase in gamma-tocopherol. The response of HPT and gamma-TMT transgenics to salt and sorbitol stress and methyl viologen treatments in comparison to wild type was studied. Each stress condition imposes oxidative stress along with additional challenges like perturbing ion homeostasis, desiccation, or disturbing photochemistry, respectively. Decreased total tocopherol content increased the sensitivity of HPT:RNAi transgenics toward all tested stress conditions, whereas gamma-TMT-silenced plants showed an improved performance when challenged with sorbitol or methyl viologen. However, salt tolerance of gamma-TMT transgenics was strongly decreased. Membrane damage in gamma-TMT transgenic plants was reduced after sorbitol and methyl viologen-mediated stress, as evident by less lipid peroxidation and/or electrolyte leakage. Therefore, our results suggest specific roles for alpha- and gamma-tocopherol in vivo.

Figure 1.

The tocopherol, tocotrienol, and plastoquinone biosynthetic pathway in plants. This figure represents the enzymatic reactions and intermediates that are involved in the biosynthesis of tocopherol, tocotrienol, and plastoquinone. Arabidopsis mutants of the corresponding pathway genes are given in parentheses behind the enzymes. DMPBQ, 2,3-dimethyl-5-phytyl-1,4-benzoquinone; HPP, p-hydroxyphenylpyruvate; MSBQ, 2-methyl-6-solanesyl-1,4-benzoquinone; phytyl-DP, phytyl-diphosphate; solanesyl-DP, solanesyl diphosphate; HPPD, HPP dioxygenase; HST, homogentisate solanesyltransferase; MPBQMT (vte3), MPBQ/MSBQ methyltransferase; PK (vte5), phytol kinase; PPK, phytyl phosphate kinase.

Figure 2.

RNAi-mediated silencing of HPT results in tocopherol deficiency in transgenic tobacco plants. A, Representation of the T-DNA from the binary intron-spliced hairpin RNA (RNAi) expression construct used for transformation of tobacco plants. The 650-bp StHPT PCR fragment from the potato EST (accession no. BI919738) was cloned between the cauliflower mosaic virus 35S promoter and the ocs terminator of the Bin19-derived vector in sense and antisense orientation, separated by intron 1 of potato GA 20-oxidase (200 bp) using the introduced BamHI and SalI restriction sites. B, The total tocopherol content (α-, γ-, and δ-tocopherol) in fully expanded leaves of HPT:RNAi transformants and wild type was quantified fluorometrically by HPLC and normalized to fresh weight.

Figure 3.

RNAi-mediated silencing of γ-TMT lead to accumulation of γ-tocopherol and a deficiency in α-tocopherol in transgenic tobacco plants. A, Representation of the T-DNA from the binary intron-spliced hairpin RNA (RNAi) expression construct used for transformation of tobacco plants. The 625-bp Stγ-TMT PCR fragment from the potato EST (accession no. BQ116842) was cloned between the cauliflower mosaic virus 35S promoter and the ocs terminator of the Bin19-derived vector in sense and antisense orientation, separated by intron 1 of potato GA 20-oxidase (200 bp) using the introduced BamHI and SalI restriction sites. B to D, Contents of tocopherols in fully expanded leaves of γ-TMT:RNAi transformants and wild type were determined by HPLC. The transgenic plant lines are ordered from left to right with increasing α- to γ-tocopherol ratio. B, α-Tocopherol content. C, γ-Tocopherol content. D, Total tocopherol content (sum of α-, γ-, and δ-tocopherol). E, The ratio of α- to γ-tocopherol in wild-type and transgenic plants. The diagrams depict data of single measurements for individuals selected for further study in the T₀ generation screening. The data were later reproduced with all tested individuals of the lineage progenies.

Figure 4.

Sorbitol-induced osmotic stress in wild-type tobacco and tobacco lines silenced for HPT:RNAi and γ-TMT:RNAi. Two-week-old seedlings of Samsun NN, wild type (A), γ-TMT:RNAi-55 (B), and HPT:RNAi-28 (C) tobacco seedlings were subjected to Murashige and Skoog medium containing different amounts of sorbitol (0, 200, 300, and 400 mm, from left to right). Pictures of five representative individuals were taken after 4 weeks of sorbitol stress. D, Tocopherol composition in leaves of the wild-type and transgenic tobacco plants shown in A to C after 4 weeks of sorbitol stress of the concentration indicated above the columns. The depicted results are from a single representative experiment with four replicate samples ± sd.

Figure 5.

Salt-induced stress in HPT:RNAi and γ-TMT:RNAi transgenic and wild-type tobacco plants. Samsun NN, wild type (A), γ-TMT:RNAi-55 (B), and HPT:RNAi-28 (C) tobacco seedlings were subjected to Murashige and Skoog medium containing different amounts of NaCl. The phenotype of five representative individuals after 4 weeks of salt stress is shown for every treatment, with 0, 200, 300, and 400 mm NaCl given in columns from left to right. D, Tocopherol composition in leaves of the wild-type and transgenic tobacco plants shown in A to C after 4 weeks of salt stress of the concentration indicated above the columns. The depicted results are from a single representative experiment with four replicate samples ± sd.

Figure 6.

Total tocopherol, ascorbate, dedydroascorbate, chlorophyll, and carotenoid contents in wild-type and transgenic tobacco leaves subjected to NaCl and sorbitol stress. Tobacco seedlings of wild-type Samsun NN (left bracket), γ-TMT:RNAi-55 (middle bracket), and HPT:RNAi-28 (right bracket) were subjected to oxidative and osmotic stress by supplementing 0 to 400 mm salt (A–C) or sorbitol (D–F) in 100 mm increments to the Murashige and Skoog culture medium. The numbers below the diagrams indicate the following conditions: 1, 0 mm; 2, 100 mm; 3, 200 mm; 4, 300 mm; 5, 400 mm of sodium chloride (A–C) or sorbitol (D–F). A and D, Total carotenoid (black bars) and total tocopherol contents (gray bars). B and E, Dehydroascorbate (black bars) and total ascorbate contents (gray bars). C and F, Chlorophyll contents (chlorophyll a, black bars; chlorophyll b, gray bars) were assessed in leaves after 4 weeks of stress. Total tocopherol was measured by HPLC (A and D), while chlorophyll (C and F), carotenoid (A and D), total ascorbate, and dehydroascorbate (DHA) were determined spectrophotometrically. The data shown are means from five replicate samples ± sd from one representative experiment. For ascorbate, significantly different values for identical treatments were classified into groups a and b, as indicated by the respective letter above the bar.

Figure 7.

Lipid peroxidation in leaves from HPT:RNAi and γ-TMT:RNAi and wild-type tobacco plants after 4 weeks of osmotic and salt stress. Wild-type Samsun NN (WT, left bracket, black bars), γ-TMT:RNAi-55 (middle bracket, gray bars), and HPT:RNAi-28 (right bracket, gray bars) tobacco plants were treated with varying amounts between 0 and 400 mm of sodium chloride (top section) or sorbitol (bottom section). The numbers below the diagrams indicate the following conditions: 1, 0 mm; 2, 100 mm; 3, 200 mm; 4, 300 mm; 5, 400 mm of NaCl (A) or sorbitol (B). Lipid peroxidation was assayed from leaf samples after 4 weeks of stress by determining the amount of MDA. Data shown is the mean and sd of five independent measurements.

Figure 8.

Ion leakage in wild-type and transgenic tobacco leaves after methyl viologen treatment. Fully expanded source leaves of 8-week-old tobacco plants of wild-type Samsun NN (left bracket, black bars), γ-TMT:RNAi-55 (middle bracket, gray bars), and HPT:RNAi-28 (right bracket, gray bars) were sprayed with 1 mL of the indicated methyl viologen solution on 2 d prior to the harvest of leaf disc samples. The numbers given behind the genotype acronym at the bottom indicate the following conditions: 0, 0 μm; 5, 5 μm; 20, 20 μm; 50, 50 μm methyl viologen. Ion leakage is given in percent of maximum leakage after boiling. The data shown are means from five replicate samples ± sd from one representative experiment.

Figure 9.

Carbohydrate and amino acid contents in leaves from HPT:RNAi and γ-TMT:RNAi and wild-type tobacco plants after 4 weeks of salt and sorbitol stress. Wild-type Samsun NN (WT, left bracket), γ-TMT:RNAi-55 (middle bracket), and HPT:RNAi-28 (right bracket) tobacco plants were treated with varying amounts between 0 and 300 mm of sodium chloride (left column) or sorbitol (right column). The numbers below the diagrams indicate the following conditions: 1, 0 mm; 2, 100 mm; 3, 200 mm; 4, 300 mm of either NaCl (left) or sorbitol (right). The contents of the soluble sugars Glc (A and G), Fru (B and H), and Suc (C and I), of starch (D and J), the compatible solute Pro (E and K), and total amino acids (F and L) were assayed from leaf samples after 4 weeks of stress. The depicted data are mean and sd of five independent measurements from one representative experiment.