ASR1 mediates glucose-hormone cross talk by affecting sugar trafficking in tobacco plants

Abstract

Asr (for ABA, stress, ripening) genes are exclusively found in the genomes of higher plants, and the encoded proteins have been found localized both to the nucleus and cytoplasm. However, before the mechanisms underlying the activity of ASR proteins can be determined, the role of these proteins in planta should be deciphered. Results from this study suggest that ASR is positioned within the signaling cascade of interactions among glucose, abscisic acid, and gibberellins. Tobacco (Nicotiana tabacum) transgenic lines with reduced levels of ASR protein showed impaired glucose metabolism and altered abscisic acid and gibberellin levels. These changes were associated with dwarfism, reduced carbon dioxide assimilation, and accelerated leaf senescence as a consequence of a fine regulation exerted by ASR to the glucose metabolism. This regulation resulted in an impact on glucose signaling mediated by Hexokinase1 and Snf1-related kinase, which would subsequently have been responsible for photosynthesis, leaf senescence, and hormone level alterations. It thus can be postulated that ASR is not only involved in the control of hexose uptake in heterotrophic organs, as we have previously reported, but also in the control of carbon fixation by the leaves mediated by a similar mechanism.

Figure 1.

Asr1 expression in tobacco transgenic lines. Western- (A) and northern-blot (B) analyses of three overexpressing (S) and three silenced (AS) transgenic lines (T0). Schemes of the constructs used for plant transformation are shown in the top side of each figure. Bars = 100 bp. C, qRT-PCR expression analyses in source leaves from T4 homozygous lines (left, antisense plants; right, overexpressing plants). Asterisks indicate statistically significant differences by the permutation test (P < 0.05). n = 3 to 5. WT, Wild type.

Figure 2.

Phenotypic characterization of the Asr1 transgenic lines. A, Visual phenotype of 6-week-old plants of three independent T0 silenced transgenic lines (left). Expanded seventh leaves from a wild-type (WT) plant and a T4 homozygous silenced plant (AS 2 4; right). Fresh matter accumulated in leaves (B), stems (C), and roots (D) of independent T0 transgenic lines. Phenotypic characterization of T4 homozygous lines: plant height (E), internode 4 to 8 length (F), flowering time measured as the number of total leaves of the plant at the time of the setting of flower buds (G), and matter accumulation in leaves, stems, and roots (H). Data represent the means ± se of measurements from five to eight plants per line. Asterisks indicate statistically significant differences by Student’s t test (P < 0.05). AS, Silenced plants; S, overexpressing plants. [See online article for color version of this figure.]

Figure 3.

Effect of altered Asr1 expression on photosynthetic parameters of T4 plants. A, CO₂ assimilation rate or A; B, stomatal conductance or G; and C, transpiration rate or E, measured at the indicated photon flux density (PFD) in fully expanded leaves of T4 plants at vegetative stage. Light-gray squares, AS 2 2; light-gray rhombuses, AS 2 4; black triangles, wild type; dark-gray circles, S 22 2; dark-gray crosses, S 22 3. The data represent the mean ± se of measurements from four to six plants per line. Asterisks indicate statistically significant differences by Student’s t test (P < 0.05).

Figure 4.

Leaf carbohydrate contents in Asr1 transgenic lines. A, Carbohydrate contents in source leaves of T0 plants at a light time point (1 pm) and a dark time point (5 am). B, Diurnal changes in carbohydrate contents in leaves from T4 homozygous plants. At each time point, samples were taken from mature source leaves and data represent the means ± se of measurements from four to six plants per line. L, Light period; D, dark period; FW, fresh weight. Light-gray squares, AS 2 2; light-gray rhombuses, AS 2 4; black triangles, wild type (WT); dark-gray circles, S 22 2; dark-gray crosses, S 22 3. Asterisks indicate statistically significant differences relative to wild-type controls by Student’s t test (P < 0.05).

Figure 5.

Glc assimilation in Asr1 transgenic leaves (T0). Leaf discs were cut from 6-week-old plants at the onset of the light period and incubated with [U-¹⁴C]Glc. Soluble and insoluble fractions were measured in a scintillation counter. Soluble fractions were subsequently separated by ion-exchange chromatography. Total [U-¹⁴C]Glc uptake and soluble, insoluble, neutral, acid, and basic fractions are shown on the left. Total uptake rate, absolute soluble, insoluble, neutral, acid, and basic metabolites are shown on the right. Sugars from neutral fractions were separated on TLC plates, and Glc-to-Suc ratios are presented at bottom left. Data represent the mean ± se of measurements from three plants per line. Asterisks indicate statistically significant differences by Student’s t test (P < 0.05). WT, Wild type.

Figure 6.

ABA and GA metabolism in Asr1 tobacco plants and GA₃ sensitivity of Asr1-silenced plants. ABA (A) and GA (B) levels were measured by LC-MS/MS in source leaves from 6-week-old plants. ABA and GA data are expressed in ng g dry weight⁻¹ and pg g dry weight⁻¹, respectively, and represent the means ± se of measurements from five independent plants per line. Asterisks indicate statistically significant differences compared with wild-type (WT) controls by Student’s t test (P < 0.05). Species in brackets were not detected. GA₃₄ was convincingly detected, but levels were below the reliable level of quantification. Reconstruction of GA pathway was done based by Yamaguchi (2008) and MacMillan (1997). Continuous and dashed lines indicate single and multiple reactions, respectively. 13ox, 13-Oxidase; 20ox, 20-oxidase; 3ox, 3-oxidase; 2ox, 2-oxidase. Exogenous GA₃ (10 μm) was applied to young seedling as described in “Materials and Methods.” Pictures of GA₃-treated (right) and untreated (left) silenced transgenics (T4) and control plants (C). Plant height along growth (D). Gray squares, AS 2 2; gray triangles, AS 2 4; black triangles, wild type; continuous lines, untreated plants; dashed lines, treated plants. [See online article for color version of this figure.]

Figure 7.

Expression analyses of sugar sensors and transporters in Asr1 tobacco lines. mRNA levels of sugar transporters Ht1 (A), Sut2 (B), Vgt (C), and Tmt3 (D) and sugar sensors Hexokinase1 (E) and SnRk1.1 (F) were quantified by qRT-PCR in leaves of T4 lines relative to wild-type (WT) plants. Asterisks indicate statistically significant differences by the permutation test (P < 0.05). n = 3 to 5. Ratio = (E_target)^{ΔCp target(MEAN control – MEAN sample)} ((E_target)^{ΔCp target(MEAN control – MEAN sample)})^–1 (Pfaffl 2001).