The TOR Pathway Is Involved in Adventitious Root Formation in Arabidopsis and Potato

Abstract

In the agriculture industry, adventitious root formation is a core issue of plants asexual propagation. However, the underlying molecular mechanism of adventitious root formation is far beyond understanding. In present study we found that target of rapamycin (TOR) signaling plays a key role in adventitious root formation in potato and Arabidopsis. The core components of TOR complex including TOR, RAPTOR, and LST8 are highly conserved in potato, but the seedlings of potato are insensitive to rapamycin, implying FK506 Binding Protein 12 KD (FKBP12) lost the function to bridge the interaction of rapamycin and TOR in potato. To dissect TOR signaling in potato, the rapamycin hypersensitive potato plants (BP12-OE) were engineered by introducing yeast FKBP12 (ScFKBP12) into potato. We found that rapamycin can significantly attenuate the capability of adventitious root formation in BP12-OE potatoes. KU63794 (KU, an active-site TOR inhibitor) combined with rapamycin can more significantly suppress adventitious root formation of BP12-OE potato than the single treatments, such as KU63794 or rapamycin, indicating its synergistic inhibitory effects on potato adventitious root formation. Furthermore, RNA-seq data showed that many genes associated with auxin signaling pathway were altered when BP12-OE potato seedlings were treated with rapamycin + KU, suggesting that TOR may play a major role in adventitious root formation via auxin signaling. The auxin receptor mutant tir1 was sensitive to TOR inhibitors and the double and quadruple mutants including tir1afb2, tir1afb3, and tir1afb1afb2afb3 displayed more sensitive to asTORis than single mutant tir1. Consistently, overexpression of AtTIR1 in Arabidopsis and potato can partially overcome the inhibitory effect of asTORis and promote adventitious root formation under asTORis treatments. These observations suggest that TOR signaling regulates adventitious root formation by mediating auxin signaling in Arabidopsis and potato.

Keywords: AtTIR1; adventitious root formation; auxin; potato; target of rapamycin.

Figure 1

Conservative evolution of StTOR in potato. (A) The location of StTOR in chromosome and the difference of two different transcripts of StTOR in potato. The difference places of two transcripts were marked with ^*. (B,C) Similarity comparison of protein and phylogenetic tree between StTOR and its homologs from other species (St, Solanum tuberosum, At, Arabidopsis thaliana, Sc, Saccharomyces cerevisiae, Hs, Homo sapiens). N-term region includes HEAT repeats domain and FAT domain. (D) Comparison of kinase domain of StTOR and its homologs from other species. The conserved sites (catalytic base, catalytic loop stability, and activation loop) were marked with red box.

Figure 2

ScFKBP12 can bridge the interaction of StTOR and rapamycin in potato. (A) BP12-OE17 could respond to rapamycin but not WT, bar = 1 cm. (B,C) Rapamycin could effectively reduce the fresh weight and adventitious root (AR) numbers in BP12-OE lines. Asterisks denote Student's t-test significance compared with WT (^*P < 0.05; ^**P < 0.01). Each value represents the mean ± SD of 3 independent experiments.

Figure 3

The function of StTOR in regulation of potato seedlings growth. (A) The phenotype of potato explants growth under medium with different TOR inhibitors or DMSO, bar = 1 cm. (B) The leaf growth of potato explants, bar = 1 cm. (C,E) The root hair growth and the root hair length of BP12-OE lines under different treatment. Asterisks denote Student's t-test significance compared with WT (^**P < 0.01), bar = 1 mm. (D,F) The fresh weight and adventitious root (AR) numbers of BP12-OE lines. Asterisks denote Student's t-test significance compared with WT (^**P < 0.01). Each value represents the mean ± SD of 3 independent experiments.

Figure 4

StTOR functions in regulating adventitious root formation. (A) Adventitious root formation of WT and BP12-OE17 explants under different TOR inhibitors in potato; DMSO was used as control, bar = 1 cm. (B,C) Adventitious root (AR) numbers in different time of WT and BP12-OE lines under DMSO and RAP + KU. (D) The AtTOR expression level during adventitious root formation in Arabidopsis, bar = 0.2 mm. Each value represents the mean ± SD of 3 independent experiments, for (B,C) n = 20, DAC means days after cutting.

Figure 5

The summary of basic information of the transcriptome data. (A) The number of clean bases in the treatments DMSO, rapamycin, KU, and rapamycin + KU. (B) The proportion of total clean reads in the four transcriptome libraries that mapped to the reference diploid potato genome. (C) The DEGs of all three transcriptome data from rapamycin, KU, and rapamycin + KU compared with the control DMSO. (D) Hierarchical clustering of the DEGs in the treatments of rapamycin, KU, and rapamycin + KU and the control DMSO. The blue bands indicate low gene expression quantity; the red bands indicate high gene expression quantity.

Figure 6

The mutations of auxin receptors were sensitive to asTORis in Arabidopsis. (A) Adventitious root formation of WT and auxin receptor mutant under different asTORis treatment; DMSO was used as control, bar = 1 cm. (B) Adventitious root (AR) numbers of WT and auxin receptor mutants under different asTORis treatment; DMSO was used as control. (C,D) Adventitious root numbers in different time of WT and auxin receptor mutants under DMSO and AZD treatment. Asterisks denote Student's t-test significance compared with DMSO (^*P < 0.05; ^**P < 0.01). Each value represents the mean ± SD of 3 independent experiments, for (C,D) n = 30.

Figure 7

The overexpression of AtTIR1 can enhanced adventitious root formation ability of BP12-2 under TOR inhibitors treatment in Arabidopsis. (A) Adventitious root formation of WT, BP12-2, and TIR1/BP12 OE-40 under different TOR inhibitors treatment, bar = 1 cm. (B,C) The fresh weight and adventitious root (AR) numbers of WT, BP12-2, and TIR1 /BP12 OE-40 under different TOR inhibitors treatment, DMSO was used as control. (D) Adventitious root numbers in different time of WT and BP12-2 and TIR1/BP12 OE-40 under R + K treatment. (E) The expression of auxin primary response genes under TOR inhibitors treatment in BP12-2 and TIR1/BP12-OE40. Four days old BP12-2 and TIR1/BP12-OE40 seedlings grew in 0.5 MS medium with low light; then the root of seedlings was removed and stems were transferred to the medium containing TOR inhibitors [RAP (5μM), KU (5μM), RAP (5μM) + KU (5μM); DMSO was used as control] under normally growth condition 48 h. Asterisks denote Student's t-test significance compared with BP12-2 (^*P < 0.05; ^**P < 0.01). Each value represents the mean ± SD of three independent experiments, for (D) n = 30.

Figure 8

TOR was involved in TIR1 stabilization in Arabidopsis. (A) The phenotype of TIR1-GUS /BP12 line in medium with rapamycin, bar = 1 cm. (B) The GUS staining of TIR1-GUS /BP12 line under different TOR inhibitors treatment; DMSO was used as control, bar = 1 cm. (C,D) The relative expression of GUS and the relative activity of GUS in TIR1-GUS /BP12 line under different TOR inhibitors treatment, DMSO was used as control. Asterisks denote Student's t-test significance compared with DMSO (^*P < 0.05; ^**P < 0.01). Each value represents the mean ± SD of three independent experiments.

Figure 9

The overexpression of AtTIR1 in potato can enhance the adventitious root formation of potato explant under different asTORis treatment. (A) Phylogenetic tree of auxin receptors in Arabidopsis and its homologs genes in potato. (B) The expression level of auxin receptor homologs genes in potato under TOR inhibitors treatment. Three weeks old BP12-OE17 seedlings grew in MS liquid medium and then were transfer to medium containing TOR inhibitors [RAP (10 μM), KU (10 μM), RAP (10 μM) + KU (10 μM); DMSO was used as control] under normally growth condition 48 h. (C) Adventitious root formation of WT and TIR1-OE4 under different asTORis treatment, bar = 1 cm. (D) Adventitious root (AR) numbers of WT and TIR1-OE4 under different asTORis treatment; DMSO was used as control. Asterisks denote Student's t-test significance compared with WT (^*P < 0.05). (E) Adventitious root numbers in different time of WT and TIR1-OE lines under AZD treatment. Asterisks denote Student's t-test significance compared with DMSO (^*P < 0.05). Each value represents the mean ± SD of three independent experiments, for (E) n = 20.