Tocopherol deficiency reduces sucrose export from salt-stressed potato leaves independently of oxidative stress and symplastic obstruction by callose

Abstract

Tocopherol cyclase, encoded by the gene SUCROSE EXPORT DEFECTIVE1, catalyses the second step in the synthesis of the antioxidant tocopherol. Depletion of SXD1 activity in maize and potato leaves leads to tocopherol deficiency and a 'sugar export block' phenotype that comprises massive starch accumulation and obstruction of plasmodesmata in paraveinal tissue by callose. We grew two transgenic StSXD1:RNAi potato lines with severe tocopherol deficiency under moderate light conditions and subjected them to salt stress. After three weeks of salt exposure, we observed a strongly reduced sugar exudation rate and a lack of starch mobilization in leaves of salt-stressed transgenic plants, but not in wild-type plants. However, callose accumulation in the vasculature declined upon salt stress in all genotypes, indicating that callose plugging of plasmodesmata was not the sole cause of the sugar export block phenotype in tocopherol-deficient leaves. Based on comprehensive gene expression analyses, we propose that enhanced responsiveness of SnRK1 target genes in mesophyll cells and altered redox regulation of phloem loading by SUT1 contribute to the attenuation of sucrose export from salt-stressed SXD:RNAi source leaves. Furthermore, we could not find any indication that elevated oxidative stress may have served as a trigger for the salt-induced carbohydrate phenotype of SXD1:RNAi transgenic plants. In leaves of the SXD1:RNAi plants, sodium accumulation was diminished, while proline accumulation and pools of soluble antioxidants were increased. As supported by phytohormone contents, these differences seem to increase longevity and prevent senescence of SXD:RNAi leaves under salt stress.

Keywords: Oxidative stress; SnRK1 signalling; potato; salt stress; starch accumulation; sucrose export defective; tocopherol; tuber yield..

Fig. 1.

Total tocopherol content of StSXD1-silenced potato leaves. Total toopherol levels were calculated on a fresh weight (FW) basis, for StSXD1:RNAi-21 and -22 potato lines and the wild type (WT), at top (leaf 3, left panel), middle (leaf 8, middle panel), and bottom (leaf 11, right panel) whorl positions. Samples were collected 19 days after the onset of treatments, consisting of irrigation either with 150mM NaCl (salt stress, black bars) or water (control, white bars). Data represent the mean ± SE of four individual plants.

Fig. 2.

Carbohydrate levels in source leaves of tocopherol-deficient potato plants upon salt treatment. Data represent sucrose, total soluble sugars (glucose, fructose, and sucrose) expressed as hexose equivalents and starch contents (expressed as glucose equivalents) of middle (leaf 8, left panel) and bottom (leaf 11, right panel) leaves of StSXD1:RNAi-21 and -22 potato lines after 19 days’ treatment (salt stress, black bars; control, white bars). at the end of the light period. Samples were collected 12h after the onset of light. Data represent the mean ± SE of four individual plants. Data were analysed by t-test; significant differences between the transgenic lines and the wild type (WT) within a treatment are indicated by a black asterisk (control treatment) or a white asterisk (stress treatment), while diamonds indicate significant differences between control and salt stress within a genotype (P < 0.05).

Fig. 3.

Diurnal carbohydrate turnover in source leaves of StSXD1-silenced potato plants challenged with salt stress. Sucrose, total soluble sugars (sum of glucose, fructose, and sucrose) expressed as hexose equivalents and starch contents (expressed as glucose equivalents) were measured in middle leaves (leaf 8) at the end of the light (white bars) and dark (black bars) period. Light samples were collected as stated in the legend to Fig. 2; dark samples were collected immediately before the end of the subsequent dark period. Left panels, control plants; right panels, salt-treated plants; WT, wild type. Data represent the mean ± SE of four individual plants. Data were analysed by t-test and significant differences between light and dark period within a genotype are indicated by an asterisk (P < 0.05).

Fig. 4.

Callose content and tissue distribution in source leaves of StSXD1-silenced potato plants 19 days after the onset of salt treatment. (A) Callose content in middle (leaf 8, left panel) and bottom (leaf 11, right panel) leaves of control (white bars) and salt-treated (black bars) plants. Data are given as β-1,3-glucan pachyman equivalents and represent the mean ± SE of four individual plants. Data were analysed by t-test; significant differences between the transgenic lines and the wild type (WT) within a treatment are indicated by a black asterisk (control treatment) or a white asterisk (stress treatment), while diamonds indicate significant differences between control and salt stress within a genotype (P < 0.05). (B) Fluorescence microscope images of potato middle leaves stained with aniline blue, showing class II and III veins and surrounding mesophyll in the wild type (WT) (left images), SXD:RNAi-21 line (middle images), and SXD:RNAi-22 line (right images). Upper images, control treatment; lower images, salt-stress treatment. Callose was observed in both tocopherol-deficient potato lines as bright spots along the phloem, mainly in control conditions. Arrowheads indicating callose appositions have been included in some images for the sake of clarity. Images are representative of four individual plants per genotype and treatment. The bar in the bottom right image represents 100 μm for all panels except for the top left image, which bears its own reference bar of 100 µm.

Fig. 5.

Changes in sucrose exudation rate of source leaves upon salt treatment. Samples were taken from middle leaves (leaf 9) and were collected 19 days after the onset of treatments (salt stress, black bars; control, white bars). Data represent the mean ± SE of four individual plants. Data were analysed by t-test; significant differences between the transgenic lines and the wild type within a treatment are indicated by a black asterisk (control treatment) or a white asterisk (stress treatment), while diamonds indicate significant differences between control and salt stress within a genotype (P < 0.05). WT, wild tye.

Fig. 6.

Effects of salt treatment on sodium and calcium content in source leaves. Sodium (top panels) and calcium contents (bottom panels) are depicted on a dry weight (DW) basis. Left panels represent middle leaves (leaf 8); right panels represent bottom leaves (leaf 11). Samples were collected 19 days after the onset of treatments (salt stress, black bars; control, white bars) and data represent the mean ± SE of four individual plants. Data were analysed by t-test; significant differences between the transgenic lines and the wild type (WT) within a treatment are indicated by a black asterisk (control treatment) or a white asterisk (stress treatment), while diamonds indicate significant differences between control and salt stress within a genotype (P < 0.05).

Fig. 7.

Effects of salt treatment on the pool size of foliar soluble antioxidants in source leaves of StSXD1-silenced potato plants. Total glutathione (top panels) and total ascorbate content (bottom panels) are depicted. Left panels represent middle leaves (leaf 8); right panels represent bottom leaves (leaf 11). Samples were collected 19 days after the onset of treatments (salt stress, black bars; control, white bars) and data represent the mean ± SE of four individual plants. Data were analysed by t-test; significant differences between the transgenic lines and the wild type (WT) within a treatment are indicated by a black asterisk (control treatment) or a white asterisk (stress treatment), while diamonds indicate significant differences between control and salt stress within a genotype (P < 0.05).

Fig. 8.

Content of free amino acids in source leaves of tocopherol-deficient plants. (A) Amino acid contents in middle leaves (leaf 8). (B) Amino acid contents in bottom leaves (leaf 11). Total amino acid levels represent the sum of single free amino acid contents determined by HPLC. Samples were collected 19 days after the onset of treatments (salt stress, black bars; control, white bars) and data represent the mean ± SE of four individual plants. Data were analysed by t-test; significant differences between the transgenic lines and the wild type within a treatment are indicated by a black asterisk (control treatment) or a white asterisk (stress treatment), while diamonds indicate significant differences between control and salt stress within a genotype (P < 0.05).

Fig. 9.

Relative transcript accumulation of genes involved in sucrose export and SnRK1 target genes in source leaves of StSXD1-silenced potato plants. The transcript levels of the genes indicated were measured in middle leaves (leaf 8) of tocopherol-deficient and wild-type (WT) potato plants after 19 days of treatment (salt stress, black bars; control, white bars) with ubiquitin as the reference gene. Data represent the mean ± SE of 4–5 individual plants. Left column (responding SnRK1 target genes): ASN1 (asparagine synthetase), UDPglcE (UDP-glucose epimerase), and SuSy2 (sucrose synthase). Middle column (genes involved in sucrose export): SUT1 (H⁺-sucrose transporter), Snakin1 (SN1), and PVP1 (vacuolar H⁺-pyrophosphatase). right column (non-responding SnRK1 target genes): XTH5 (xyloglucan endotransglucosylase-hydrolase), TPS11 (trehalose 6-phosphate synthase), and SnRKα (α subunit of SnRK1, sugar non-fermenting related kinase1). Data were analysed by t-test; significant differences between the transgenic lines and the wild type within a treatment are indicated by a black asterisk (control treatment) or a white asterisk (stress treatment), while diamonds indicate significant differences between control and salt stress within a genotype (P < 0.05).

Fig. 10.

Phytohormone contents in source leaves of tocopherol-deficient potato plants exposed to 19 days’ salt treatment. Leaf content of ABA, ACC, SA, and of the cytokinins IPA and ZR are shown from top to bottom. Left panels represent middle leaves (leaf 8); right panels represent bottom leaves (leaf 11). Black bars, salt-stress treatment; white bars, control treatment. Data represent the mean ± SE of four individual plants. Data were analysed by t-test; significant differences between the transgenic lines and the wild type within a treatment are indicated by a black asterisk (control treatment) or a white asterisk (stress treatment), while diamonds indicate significant differences between control and salt stress within a genotype (P < 0.05).