Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway

Abstract

Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity.

Keywords: Kinematic analysis; PP2A catalytic subunit; phosphorylation of S6 kinase; rapamycin; real-time quantitative PCR; seed viability..

Fig. 1.

Enhanced growth of Arabidopsis Tap46 constitutive overexpression (OE) lines. (A) Immunoblotting with anti-HA antibodies to measure expression of HA:Tap46 fusion protein in WT (Col-0) and seven independent Tap46 OE lines. Thirty micrograms of total protein isolated from seedlings were subjected to immunoblotting. Anti-α-tubulin antibody was used as a loading control. (B) Immunoblotting with anti-Tap46 antibodies of protein extracts from WT and Tap46 OE (OE-5 and OE-10) seedlings. (C) Seedling growth of WT, OE-5, and OE-10 lines on MS medium under light and dark conditions 4 days after sowing. (D) Representative leaf series of WT, OE-5, and OE-10 plants grown for 3 weeks in soil. (E) WT, OE-5, and OE-10 plants grown for 3 weeks in soil. (F) WT and OE-10 plants grown for 4 weeks in soil. (G) Relative expression levels of Tap46 protein in WT, OE-5, and OE-10 seedlings based on quantification of the immunoblot band intensities in (B). Levels of endogenous and HA-tagged Tap46 were combined for the OE lines. (H) Average hypocotyl length of WT and Tap46 OE seedlings grown under dark conditions 4 days after sowing (n = 30). Asterisks denote the statistical significance of the differences between WT and Tap46 OE samples: *, P ≤ 0.05; **, P ≤ 0.01. (I) Average silique length of WT and Tap46 OE lines (n = 30). This figure is available in colour at JXB online.

Fig. 2.

Kinematic analysis of leaf growth. Seeds were imbibed at 4°C for 24 hours before sowing. First leaves were collected from WT, OE-5, and OE-10 plants at 4, 7, 11, 17, 21, and 25 DAC. (A) Average areas of first leaves. (B) Average areas of epidermal cells on the abaxial side of first leaves. (C) Calculated numbers of epidermal cells per leaf.

Fig. 3.

Analysis of seed enlargement in Tap46 overexpression lines. (A) Mature seeds of WT, OE-5, and OE-10 plants. Scale bars, 500 µm. (B) Average seed length (n = 500). (C) Average seed mass per 1000 seeds (n = 3). (D) Average seed number per silique (n = 30). (E) Tetrazolium staining to determine seed viability. WT, OE-5, and OE-10 seeds were incubated under accelerated ageing conditions for 0–48 hours. Viable seeds are stained red (and appear here as the darker seeds). (F) Germination rate (%) of WT, OE-5, and OE-10 seeds after accelerated ageing treatment for 0–48 hours (n = 100). This figure is available in colour at JXB online.

Fig. 4.

Enhanced growth of Arabidopsis DEX-inducible Tap46 overexpresion (DOE) lines. (A) WT seedling growth on MS medium containing ethanol [(–)DEX] or 10 μM DEX [(+)DEX] at 6 DAS. (B) Seedling growth of DOE-3 and DOE-11 lines on (–)DEX and (+)DEX medium 6 DAS. (C) Representative leaf series of soil-grown DOE-11 plants that were sprayed with ethanol or 30 µM DEX for 16 DAG. (D) Average area of the first leaves of soil-grown DOE-11 plants that were sprayed with ethanol or DEX for 4 days after germination. (E) Average area of the largest rosette leaves of soil-grown DOE-11 plants that were sprayed with ethanol or DEX for 16 days after germination. (F) Soil-grown DOE-11 seedlings sprayed with ethanol or DEX for three weeks. (G) Seedling growth 4 DAS on (–)DEX or (+)DEX medium under dark conditions. (H) Average hypocotyl length of dark-grown seedlings on (–)DEX and (+)DEX medium 4 DAS (n = 30). (I) Real-time quantitative RT-PCR analysis of Tap46 transcript levels. Transcript levels in (+)DEX samples are expressed relative to those in (–)DEX-24 hour samples. Values represent means ±SD of three replicates per experiment. (J) Immunoblotting to measure accumulation of HA:Tap46 fusion protein in DOE lines. Thirty micrograms of total protein isolated from different DOE lines (nos. 3, 4, and 11) with or without DEX treatment for 24 hours were subjected to immunoblotting with anti-HA antibodies (left). HA:Tap46 protein accumulation was detected by immunoblotting in DOE-11 seedlings after 6 and 24 hours of DEX treatment (right). Anti-α-tubulin antibody was used as a loading control. This figure is available in colour at JXB online.

Fig. 5.

Modulation of metabolic enzyme activities in DEX-inducible Tap46 overexpression and Tap46 RNAi (RNAi-16) plants. Enzyme activities were measured using protein fractions prepared from WT, DOE-11, and RNAi-16 seedlings that were grown in soil for 2 weeks and sprayed with ethanol [(–)DEX] or 30 μM DEX [(+)DEX] for 3 or 5 days. Data points represent mean ± SD of three experiments. Asterisks denote statistical significance of the differences between (–)DEX and (+)DEX samples on each day: *, P ≤ 0.05; **, P ≤ 0.01. (A) NR activities. (B) NiR activities. (C) GS activities.

Fig. 6.

Determination of the relationship between Tap46 and the TOR signalling pathway. (A) Phosphorylation of S6K1 and its mutant in DOE-11 seedlings upon DEX treatment. Flag-tagged WT S6K1 or mutant S6K1 [S6K1(m); T449A, non-phosphorylated form] were transiently expressed in protoplasts. Western blot analyses were performed with anti-Flag antibodies to detect total S6K1 protein and with anti-S6K1-P(T449) antibodies to detect S6K1 phosphorylated at T449. (B) Phosphorylation of S6K1 and its mutant in DEX-inducible Tap46 RNAi-16 seedlings upon DEX treatment. (C) Co-immunoprecipitation. Protein extracts were prepared from N. benthamiana leaves that expressed HA:Tap46 with S6K1:Myc or S6K2:Myc fusion proteins. After immunoprecipitation (IP) with anti-Myc antibodies, co-immunoprecipitated HA:Tap46 was detected by immunoblotting with anti-HA antibodies. To check IP efficiency, the precipitated fractions were also immunoblotted with anti-Myc antibodies (input). (D) Co-immunoprecipitation of HA:Tap46 with S6K1:Myc, S6K1(A):Myc, or S6K1(D):Myc fusion proteins. S6K1(T449A) is a non-phosphorylated form and S6K1(T449D) is a phospho-mimetic form. (E) Visualization of interactions of Tap46 with S6K1 and S6K2 using bimolecular fluorescence complementation (BiFC). YFP^N:Tap46 was expressed together with YFP^C:S6K1 or YFP^C:S6K2 fusion proteins in N. benthamiana leaves by agroinfiltration for confocal laser scanning microscopy. (F) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in TOR RNAi lines. Thirty micrograms of total protein isolated from Arabidopsis estradiol-inducible TOR RNAi seedlings after 3 and 7 days of ethanol (–EST) or 10 µM estradiol (+EST) treatment were subjected to immunoblotting with anti-Tap46 and anti-PP2Ac antibodies. Coomassie-stained Rubisco large subunit (rbcL) is shown as a loading control. (G) Immunoblot analysis to detect cellular Tap46 and PP2Ac protein levels in WT and Arabidopsis FKP12 overexpression lines upon rapamycin treatment. Seedlings were treated with 1 or 10 µM rapamycin for 24 hours prior to immunoblotting. This figure is available in colour at JXB online.

Fig. 7.

Real-time quantitative RT-PCR analysis of gene expression in DOE-11 seedlings. DOE-11 seedlings grown on MS medium for one week were treated with ethanol [(–)DEX] or 10 µM DEX [(+)DEX] for 3 days for real-time quantitative RT-PCR. Data points represent mean ± SD of three experiments. Asterisks denote statistical significance of the differences between the (–)DEX and (+)DEX samples: *, P ≤ 0.05; **, P ≤ 0.01. (A) Nitrogen metabolism-related genes. (B) Ribosomal protein genes. (C) Lignin biosynthesis genes. (D) Cell wall biosynthesis genes. (E) Cytochrome 450 genes. (F) Autophagy genes. (G) Lipid degradation-related genes.