The Arabidopsis TOR kinase links plant growth, yield, stress resistance and mRNA translation

Abstract

Plants, unlike animals, have plastic organ growth that is largely dependent on environmental information. However, so far, little is known about how this information is perceived and transduced into coherent growth and developmental decisions. Here, we report that the growth of Arabidopsis is positively correlated with the level of expression of the TARGET OF RAPAMYCIN (TOR) kinase. Diminished or augmented expression of the AtTOR gene results in a dose-dependent decrease or increase, respectively, in organ and cell size, seed production and resistance to osmotic stress. Strong downregulation of AtTOR expression by inducible RNA interference also leads to a post-germinative halt in growth and development, which phenocopies the action of the plant hormone abscisic acid, to an early senescence and to a reduction in the amount of translated messenger RNA. Thus, we propose that the AtTOR kinase is one of the contributors to the link between environmental cues and growth processes in plants.

Figure 1

The AtTOR gene regulates plant growth. (A,D) Expression of the AtTOR gene in the leaves (A) and roots (D) of the control line (Col0 wild type) and in RNAi-silenced (35-7 and 65-1) and RNAi-overexpressing lines (Gabi mutants G166 and G548; Salk mutants S7817 and S7846). Plants were grown in vitro for 15 days and relative expression levels were determined by real-time quantitative PCR (RT–PCR) with the control line as a reference (100 arbitrary units) in each experiment. The mean values±s.d. of three independent experiments are shown. (B) Col0 WT plants, an RNAi-silenced line (35-7) and the G548 AtTOR-overexpressing line grown in the greenhouse for 36 days. (C,F) Leaf (C) and root growth (F) curves in hydroponic cultures of control Col0 (cross), G548 (diamonds), G166 (circles), 35-7 (squares) and 65-1 (triangles) lines. The mean values±s.d. of three independent experiments are shown and correspond to the weight of individual plants. (E) Length of the root system in control, AtTOR-overexpressing and AtTOR-silenced lines. Plants were grown in hydroponic conditions for 49 days. ND, not determined; RNAi, RNA interference.

Figure 2

AtTOR expression is correlated with the size of rosette leaves and epidermal cells. The size of rosette leaves and adaxial epidermal cells was determined for wild type (Col0), the AtTOR-silenced 65-1 and 35-7 lines, and for the G548- and G166-overexpressing lines. Leaf size (right axis) was measured on leaves 5 and 6 and averaged (n=4). Epidermal cell size (left axis) was measured on the adaxial side of leaf 5 (n=3 × 4). See supplementary Fig S2 and supplementary information online for details.

Figure 3

AtTOR expression is correlated with sensitivity to osmotic stress. (A) Col8 wild type and S7817 seedlings were grown for 2 weeks on growth media without nitrogen (left) or on media supplemented with 100 mM nitrate (right). (B) Col8 wild type and AtTOR-overexpressing line S7817 were grown in vitro for 15 days with different nitrogen supplements (0, 35 or 100 mM nitrate). The length of the primary root was then measured at different time points. Values represent means±s.d. of ten individual plants. The differences in root growth are only statistically significant for 100 mM nitrate. (C) Col0 wild type, AtTOR-silenced (35-71 and 36-6) and AtTOR-overexpressing (G166 and G548) lines were grown in vitro for 15 days with 35 mM KCl, and the length of the primary root was determined at the end of the culture. Values represent means±s.d. of ten individual plants. (D) Seeds of Col0 wild type and of the AtTor-silenced 35-7 and 65-1 lines were sown on growth medium containing 5 mM mannitol. Representative 35-7 and 65-1 seedlings are shown.

Figure 4

Conditional silencing of AtTOR expression by ethanol-inducible RNA interference phenocopies in plants the action of rapamycin in animal and yeast cells. (A) After 6 weeks of culture without ethanol, Arabidopsis plants (Landsberg erecta) carrying either an alcA:RNAi-TOR construct (control line, RNAi4), a 35S:ALCR alcA:RNAi-TOR construct (allowing for ethanol-inducible silencing of AtTOR, RNAi6) or a 35S:ALCR alcA:GUS construct (GUS) were grown in the greenhouse for 10 days with or without 5% (v/v) ethanol (EtOH). (B) Landsberg erecta wild type (Le) and the progeny of two independent lines carrying a 35S:ALCR alcA:RNAi-TOR construct were sown in vitro with (10 or 50 mM) or without ethanol. Representative seedlings are shown. (C) Comparison of carbohydrate contents (log scale) in the control (Le), GUS, RNAi4 and two independent AtTOR-silenced lines as well as in the homozygous progeny of the RNAi6 line grown with ethanol. The RNAi6 line was grown for 10 days with or without ethanol (n=8), all other lines were grown for 27 days with ethanol (n=4). The mean values±s.d. are shown. (D) Close-up of an RNAi6-3 plantlet grown as in (B) with 50 mM ethanol. FW, fresh weight; RNAi, RNA interference.

Figure 5

Variations in AtTOR expression affect the messenger RNA translation machinery. (A) Absorbance profile at 254 nm of ribosomes purified by ultracentrifugation on a sucrose density gradient. The ribosomal pellet fraction was prepared (I) from shoots for the ethanol-inducible RNAi6 line, and (II) from plantlets grown in vitro in liquid medium for the partly silenced 35-7 line. Experiments were repeated at least three times and a typical experiment is shown for each line. Black lines correspond to control wild-type plants (Col0 and Landsberg erecta for, respectively, the 35-7 and RNAi6 lines). The positions of the 60S ribosomal subunits, monosomes (80S) and polysomes are indicated. (B) Increased and decreased expression of the Arabidopsis EBP1 gene in, respectively, AtTOR-overexpressing (G548 and G166) and AtTOR-silenced (35-7 and ethanol-induced RNAi6-3) lines. RNA for relative quantification by real-time RT–PCR was obtained from leaves. The EBP1 relative expression levels were determined as in Fig 1 with the control lines as reference (Col0 for G548, G166 and 35-7; ethanol-treated Landsberg erecta for RNAi6-3) in each experiment. The mean values±s.d. of three independent experiments are shown. EBP, human ErbB-3 epidermal growth factor receptor binding protein; OD, optical density; RT–PCR, reverse transcriptase–PCR.