Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis

Abstract

Target of rapamycin (TOR), a master sensor for growth factors and nutrition availability in eukaryotic species, is a specific target protein of rapamycin. Rapamycin inhibits TOR kinase activity viaFK506 binding protein 12 kDa (FKBP12) in all examined heterotrophic eukaryotic organisms. In Arabidopsis, several independent studies have shown that AtFKBP12 is non-functional under aerobic condition, but one study suggests that AtFKBP12 is functional during anaerobic growth. However, the functions of AtFKBP12 have never been examined in parallel under aerobic and anaerobic growth conditions so far. To this end, we cloned the FKBP12 gene of humans, yeast, and Arabidopsis, respectively. Transgenic plants were generated, and pharmacological examinations were performed in parallel with Arabidopsis under aerobic and anaerobic conditions. ScFKBP12 conferred plants with the strongest sensitivity to rapamycin, followed by HsFKBP12, whereas AtFKBP12 failed to generate rapamycin sensitivity under aerobic condition. Upon submergence, yeast and human FKBP12 can significantly block cotyledon greening while Arabidopsis FKBP12 only retards plant growth in the presence of rapamycin, suggesting that hypoxia stress could partially restore the functions of AtFKBP12 to bridge the interaction between rapamycin and TOR. To further determine if communication between TOR and auxin signaling exists in plants, yeast FKBP12 was introduced into DR5::GUS homozygous plants. The transgenic plants DR5/BP12 were then treated with rapamycin or KU63794 (a new inhibitor of TOR). GUS staining showed that the auxin content of root tips decreased compared to the control. DR5/BP12 plants lost sensitivity to auxin after treatment with rapamycin. Auxin-defective phenotypes, including short primary roots, fewer lateral roots, and loss of gravitropism, occurred in DR5/BP12 plants when seedlings were treated with rapamycin+KU63794. This indicated that the combination of rapamycin and KU63794 can significantly inhibit TOR and auxin signaling in DR5/BP12 plants. These studies demonstrate that TOR is essential for auxin signaling transduction in Arabidopsis.

Keywords: FKBP12; KU63794; auxin; rapamycin; target of rapamycin.

Figure 1

The expression pattern of AtFKBP12 in Arabidopsis and the identification of transgenic lines of AtFKBP12, HsFKBP12 and PAtFKBP12::GUS in Arabidopsis. (A) The GUS staining of PAtFKBP12::GUS and P35S::GUS transgenic lines. 5 DAG (days after germination) seedlings were stained. (a,b) bar = 0.5 cm; (c–f) bar = 2 mm; (g) bar = 0.2 mm. (B) qRT-PCR to detect expression pattern of AtFKBP12 in different Arabidopsis tissues. (C) qRT-PCR to detect expression level of AtFKBP12 or HsFKBP12 in transgenic lines. Asterisks denote Student's t-test significance compared with WT (^*P < 0.05;^**P < 0.01). (D) The protein expression level in three FKBP12 transgenic lines. AtFKBP12 and HsFKBP12 used anti-HA antibody; BP12-2 used anti-MYC antibody. (E) The multiple protein sequence alignment of ScFKBP12, HsFKBP12, and AtFKBP12. Significant mutations were marked with red box.

Figure 2

Rapamycin sensitivity tests of the transgenic plants with ScFKBP12, HsFKBP12, and AtFKBP12. (A) Dose-dependent effect of rapamycin on the growth and development of WT and three transgenic plants. The concentration of rapamycin ranged from 0 to 5 μM (10 DAG). Bar = 1 cm. (B,C) The root hair and leaf growth of WT and three transgenic plants on 0.5 MS medium supplied with DMSO or rapamycin (5 μM). (B) Bar = 1 mm; (C) Bar = 0.5 cm. (D,E) Quantitative analysis and comparison of the root length and the fresh weight (%) of WT and three transgenic plants after treatment with rapamycin. Error bars indicate ±SD for quadruplication.

Figure 3

KU sensitivity tests of WT, DR5::GUS and DR5/BP12-OE transgenic lines and the identification of transgenic lines of DR5/BP12-OE transgenic lines. (A) The leaf formation of DR5::GUS and DR5/BP12-OE11 after treatment with different concentration of KU (10 DAG). Bar = 0.5 cm. (B) Dose-dependent inhibitory effect of KU on WT, DR5:: GUS and DR5/BP12-OE plants growth. The KU concentration ranged from 0 to 20 μM (10 DAG). Bar = 1 cm. (C–E) The quantitative analysis and comparison of fresh weight (%), root length (%) and lateral root initiation index (%) (LR initiation index) of DR5::GUS and DR5/BP12-OE plants after treating with KU at different concentration. Error bars indicate ±SD for quadruplication. (F,G) Identification of transgenic lines of DR5/BP12-OE lines by semi-qPCR, western blot (F, note: Since some results of the western blot located between DR:GUS and T3-5 are not associated with this manuscript, part of the membrane has been cut out), and qRT-PCR (G).

Figure 4

The inhibition of Arabidopsis by combined rapamycin with KU. (A) The growth of whole plant of DR5:: GUS and DR5/BP12-OE11 after treated with rapamycin and KU (10 DAG). Rapamycin concentration ranged from 0 to 5 μM, whereas KU was used at a final concentration of 5 μM. Bar = 1 cm. (B) The inhibitory effect of KU or rapamycin plus KU on root hair development and leaf formation of DR5:: GUS and DR5/BP12-OE11. Bar = 1 mm at the left and 0.5 cm in the right. (C,D) The quantitative analysis and comparison of root length (%) and fresh weight (%) of WT, DR5::GUS and DR5/BP12-OE plants after treatment with rapamycin or/and KU. Error bars indicate ±SD for quadruplication.

Figure 5

The inhibition of DR5:: GUS and DR5/BP12-OE11 by rapamycin or/and KU affect auxin levels. (A) The phenotype of DR5::GUS and DR5/BP12-OE11 treated with rapamycin or/and KU. DMSO was used as control (12 DAG). Bar = 1 cm. (B) The changed gravitropism of plant root. Plants grew for 12 din 0.5 MS medium with different TOR inhibitors or DMSO. Bar = 1 cm. (C) Rapamycin or/and KU affect auxin distribution in root tip, DR5:: GUS reporter was used as a marker of auxin distribution. Plants grew for 7 d in 0.5MS medium with different TOR inhibitors or DMSO. Bar = 0.1 mm. (D) Detection expression level of auxin synthesis-related genes by qRT-PCR. DR5/BP12-OE11 grew 12 days in 0.5 MS medium with different TOR inhibitors [RAP (5 μM), KU (5 μM), RAP (5 μM) +KU (5 μM); DMSO was used as control]. Each value represents the mean± SD of 3 independent experiments. Asterisks denote Student's t-test significance compared with control (^*P < 0.05;^**P < 0.01).

Figure 6

TOR was required for exogenous IAA response in Arabidopsis. (A) The phenotype of DR5/BP12-OE11 treated with IAA and rapamycin (8 DAG). Bar = 1 cm. (B) The root hair elongation of DR5:: GUS and DR5/BP12-OE11 with exogenous IAA and rapamycin. Bar = 1 mm. Error bars indicate ±SD for quadruplication. (C,D) The quantitative analysis and comparison of LR initiation index (%) and root hair length (%) of WT, DR5::GUS and DR5/BP12-OE plants after treatment with IAA and rapamycin. Error bars indicate ±SD for quadruplication. Asterisks denote Student's t-test significance compared with DR5:: GUS (^**P < 0.01).

Figure 7

The expression level of primaryauxin response gene family AUX/IAAs, SAURs, and GH3s were affected by TOR specific inhibitors. DR5/BP12-OE11 grew in 0.5 MS medium containing TOR inhibitors [RAP (5 μM), KU (5 μM), RAP (5 μM) + KU (5 μM); DMSO was used as control)] for 12 days. Each value represents the mean± SD of 3 independent experiments. Asterisks denote Student's t-test significance compared with control (^*P < 0.05; ^**P < 0.01).