The plant TOR kinase tunes autophagy and meristem activity for nutrient stress-induced developmental plasticity

Abstract

Plants, unlike animals, respond to environmental challenges with comprehensive developmental transitions that allow them to cope with these stresses. Here we discovered that antagonistic activation of the Target of Rapamycin (TOR) kinase in Arabidopsis thaliana roots and shoots is essential for the nutrient deprivation-induced increase in the root-to-shoot ratio to improve foraging for mineral ions. We demonstrate that sulfate limitation-induced downregulation of TOR in shoots activates autophagy, resulting in enhanced carbon allocation to the root. The allocation of carbon to the roots is facilitated by the specific upregulation of the sucrose-transporter genes SWEET11/12 in shoots. SWEET11/12 activation is indispensable for enabling sucrose to act as a carbon source for growth and as a signal for tuning root apical meristem activity via glucose-TOR signaling. The sugar-stimulated TOR activity in the root suppresses autophagy and maintains root apical meristem activity to support root growth to enhance mining for new sulfate resources in the soil. We provide direct evidence that the organ-specific regulation of autophagy is essential for the increased root-to-shoot ratio in response to sulfur limitation. These findings uncover how sulfur limitation controls the central sensor kinase TOR to enable nutrient recycling for stress-induced morphological adaptation of the plant body.

Figure 1

Sulfur deficiency leads to organ-specific regulation of TOR activity that is critical for the increased root-to-shoot ratio. A–D, Immunological detection of S6K phosphorylation status and S6K abundance (A) with specific antisera against S6K-p and S6K1/2 in the shoots and roots of wild-type plants hydroponically grown under optimal sulfate (500 µM MgSO₄, +S) or limited sulfate supply (1 µM MgSO₄, −S) for 5 weeks. Organ-specific regulation of TOR in response to sulfur deficiency, as determined by calculating the ratio of S6K-p and S6K (B, n=3, each replicate represents a pool of 3–9 plants). Data for TOR activity in wild-type shoots were obtained from Dong et al. (2017). Quantification of fresh weight of shoots (C) and roots (D) of plants grown under optimal or limited sulfur supply, demonstrating substantial correlation of TOR activity and growth in both organs. E and F, Growth phenotype (E) and root-to-shoot ratio (F) of wild-type plants and loss-of-function mutants of TORC1 subunits (rap78 and lst8-1-1, Kravchenko et al., 2015) grown under optimal (500 µM MgSO₄, +S) or limited sulfate supply (1 µM MgSO₄, −S) for 5 weeks (n=3, each replicate represents a pool of 3–9 plants. Scale bar, 3 cm. (b, c, d, f). Different letters indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak method based One-way ANOVA (P<0.05). G and H, Sulfur deficiency-induced increase in the root-to-shoot ratio of the wild type in the absence or presence of the TOR inhibitor AZD 8055 (AZD, 0.5 µM). Phenotypes (G) and quantification of organ growth and root-to-shoot ratio (H) of the TOR-inhibited plants and the nontreated wild type (control) under full sulfur supply (500 µM sulfate, +S, white) or low sulfur supply (1 µM sulfate, −S, yellow). (n=3 representing a pool of 20 seedlings). I and J, Sulfur deficiency-induced increase in the root-to-shoot ratio of the wild type and the β-estradiol inducible TOR-RNAi line tor-es1 in the presence of β-estradiol (1µM). Phenotypes (I) and quantification of organ growth and root-to-shoot ratio (J) of the TOR-inhibited plants and the nontreated wild type (Col-0) under full sulfur supply (500 µM sulfate, +S, white) or low sulfur supply (1 µM sulfate, −S, yellow). (n=3 representing a pool of 10 seedlings). TOR-inhibited seedlings (G–J) were unable to increase the root-to-shoot ratio in response to sulfur deficiency. Scale bar, 1 cm (G–J). Asterisks indicate groups that were different from the untreated wild type identified by pairwise multiple comparisons with a Holm-Sidak method based One-way ANOVA (P<0.05, n is as specified in panel captions [G–J]). Data are presented as mean ± SD in B, F, H, and J.

Figure 2

Sulfur deficiency results in organ-specific autophagy induction. A, Immunological detection of GFP-release (Free GFP) from the autophagy marker GFP-ATG8a) in shoots and roots of GFP-ATG8a marker lines grown under optimal sulfate (500 µM MgSO₄, +S) or limited sulfate supply (1 µM MgSO₄, −S). B, Sulfur deprivation significantly increases the ratio of free GFP to GFP-ATG8a in the shoots due to autophagy induction (quantification of immunological detection, n=3, P<0.05). C and D, Autophagy induction in the shoots and roots of sulfur-deprived wild type (n=3, each replicate represents a pool of 3–9 plants, mean±s.e.m., t test, *P<0.05). (D) Total levels of unmodified ATG8a protein (ATG8a) and lipidated ATG8a (ATG8a-PE) were quantified after immunological detection (C) with a specific antiserum under optimal sulfate (+S) or limited sulfate supply (−S) in shoots and roots. E, Sulfur deprivation specifically induces the transcription of diverse ATG8 gene family members in wild-type shoots. Data were extracted from two published microarray data sets (Dong et al., 2017; Forieri et al., 2017). The shoots and roots used for these microarrays were harvested from the same individuals to allow direct comparison. Plants were grown hydroponically for 2 weeks under short-day conditions with full sulfur supply (500 µM sulfate) and were then transferred to low sulfur supply (1 µM sulfate) or full sulfur supply for 5 weeks. The heat map depicts significant changes in steady-state transcript levels induced upon sulfur deficiency as x-fold of control treatment (transcript –S/transcript +S, brown = upregulation in response to sulfur deficiency, turquoise = downregulation in response to sulfur deficiency). White indicates no significant change in transcript level in response to –S in this organ (n=3, P<0.05). F, Verification of transcriptional induction of ATG8a specifically in wild-type shoots upon sulfur-deprivation by qRT-PCR with specific primers as described in the Materials and Methods section. Data are presented as mean ± s.e.m. Asterisks indicate statistically significant differences between +S and –S conditions and were determined by Student’s t test (n=4, P<0.05).

Figure 3

Organ-specific autophagy induction contributes to the increased root-to-shoot ratio under sulfur deficiency. A and B, Growth phenotype (A) and quantification of the root-to-shoot ratio (B) of 7-week-old wild-type and autophagy-deficient mutants that were grown under optimal sulfate (500 µM MgSO₄, +S) or limited sulfate supply (1 µM MgSO₄, −S) for 5 weeks. Scale bar, 2 cm. Data are presented as mean ± SD (n=3 or 4, each replicate represents a pool of 3–9 plants). Different letters indicate statistically significant differences determined by One-way ANOVA (P<0.05). C, Time-resolved analysis of the sulfur deprivation-induced increase in the root-to-shoot ratio in the wild type (black) and the atg5-1 mutant (orange). Plants were grown hydroponically under optimal sulfur supply (500 µM sulfate) for the first 2 weeks and then transferred to limited sulfur supply (1 µM sulfate) for the indicated time points (n=3 or 4, each replicate represents a pool of 3–9 plants). Data are presented as mean ± s.e.m. Asterisks indicate statistically significant differences between wild type grown under +S conditions and other conditions, as determined by Student’s t test.

Figure 4

Leaf-specific autophagy induction contributes to sulfur deficiency-induced carbon allocation from the shoot to the root. A and B, PCA of metabolomes extracted from shoots (A) and roots (B) of wild-type and three autophagy-deficient mutants that were grown under optimal (500 µM MgSO₄, +S) or limited sulfate supply (1 µM MgSO₄, −S) for 5 weeks. The colored zone displays the 95% confidence interval for the respective genotype (n = 3). C and D, Volcano plot of sulfur-deficiency-induced metabolite changes in the shoot (C) and root (D) of the wild type and the group of autophagy-deficient mutants (atg5-1, atg5-3, atg2-1). Statistically significant metabolite changes between both groups are labeled in red (>2-fold, P < 0.1). E, Sulfur deficiency-induced fold-change of sucrose levels in the shoots and roots of the wild-type and autophagy-deficient mutants (n=3 or 4, mean±s.e.m., t test, *P<0.05). F, Carbon deposition in the shoots and roots of wild-type and atg5-1 plants from the third to the fifth week of sulfur depletion. The sum of newly fixed carbon (root+shoot) in the respective genotype was set to 1 in each condition. This allows for direct comparison of relative carbon deposition in the shoot and the root of both genotypes under both sulfur regimes. Different letters indicate statistically significant differences determined by one-way ANOVA (n=4, mean±s.e.m., P<0.05). G and H, Growth phenotype (G) and root length (H) of wild type and atg5-1 (red) grown for 2 weeks on AT medium supplemented with 500 µM sulfate (+S), 0 µM sulfate (−S), or 0 µM sulfate + 1% sucrose (−S, +Suc) or 0.1 mM proline (−S, +Pro) for 2 weeks. White arrows indicate the root tip. Scale bar = 1 cm. AZD8055 (1 µM) was applied to inhibit TOR under –S+Suc (+AZD). Different letters indicate statistically significant differences determined by one-way ANOVA (n = 10, P<0.05). I, Root meristem activity of wild-type seedlings grown under +S or –S conditions, as determined by Edu staining. Sucrose was applied to seedlings grown on –S medium 24 h prior to the detection of root meristem activity. Scale bar = 25 µm.

Figure 5

Antagonistic regulation of SWEET11 and SWEET12 in shoots and roots is crucial for sulfur-starvation-induced shoot-to-root sucrose transport. A, Venn diagram comparing sulfur deficiency-induced (orange) and sulfur deficiency-repressed (blue) transcripts in leaves and roots of wild type. Five hundred twenty-two transcripts were identified, which are antagonistically regulated in both organs in response to sulfur deprivation (fold change>1.25 and P<0.05). Data were extracted from a microarray experiment that was performed by our group using precisely the same sulfur supply and developmental stage of plants described here (Forieri et al., 2016; Dong et al., 2017). B Functional category analysis of these antagonistically regulated genes in both organs using DAVID software (P<0.05, FDR<0.25). C, Organ-specific analysis of the relative transcript levels of SWEET sucrose transporters in response to sulfur deficiency. Significantly upregulated (red) or downregulated (blue) genes are indicated by the color code in the heat map (n=3, P<0.05). D–E, Growth phenotype (D) and the root-to-shoot ratio (E) of wild-type and sweet11 sweet12 double mutants grown under optimal (500 µM MgSO₄, +S, white) or limited sulfate supply (1 µM MgSO₄, −S, yellow) for 5 weeks. Data are presented as mean ± SD. Different letters indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak method based one-way ANOVA (n=3, each replicate represents a pool of 3–9 plants, P<0.05). Scale bar, 3 cm.

Figure 6

Sucrose transport by SWEET11 and SWEET12 is crucial for the maintenance of root TOR-activity in sulfur-deprived plants. A, Immunological detection of S6K phosphorylation status and S6K abundance with specific antisera against S6K-p and S6K1/2 in the roots of wild type and sweet11 sweet12 grown under optimal sulfate (500 µM MgSO₄, +S) or limited sulfate supply (1 µM MgSO₄, −S) for 5 weeks. B, Regulation of TOR in response to sulfur deficiency in wild type and sweet11 sweet12, as determined by calculating the ratio of S6K-p and S6K. Data are presented as mean ± SD. Different letters indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak method based one-way ANOVA (n =3 or 4, each replicate represents a pool of 3–6 plants, P<0.05). C and D, Growth phenotype (C) and root length (D) of wild-type and sweet11 sweet12 plant seedlings for 1 week on AT medium supplemented with 500 µM sulfate (+S), 0 µM sulfate (−S), 1% sucrose (+Suc), and 1 µM AZD 8055 (AZD). Data are presented as mean ± s.e.m. Different letters indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak method based one-way ANOVA (n = 20–30, each replicate represents a pool of 3–9 plants, P<0.05). Scale bar, 1 cm.

Figure 7

Model for the role of the organ-specific regulation of TOR and autophagy in modulating the root-to-shoot ratio in response to external sulfur supply. A, Under optimal external sulfur supply, glucose (Glc) levels are high in the shoot and root of the wild type and trigger the activation of TOR kinase via the established glucose-TOR signal relay. TOR represses autophagy and activates the shoot apical meristem (SAM) and root apical meristem (RAM). B, External sulfur limitation causes downregulation of the glucose-TOR signal relay, as observed in the shoots of sir1-1 (Dong et al., 2017), but at the same time triggers the transcriptional reprogramming of sucrose transporters (e.g. SWEET12, SWEET11, and SUC1) in the wild type. This reprogramming causes the efficient loading of sucrose (Suc) into the phloem (blue pipe) for transport to the root. The induction of autophagy in the shoot contributes to the remobilization of carbon under sulfur limitation. In the root, the sucrose is converted to Glc to maintain TOR activity (gray dashed arrow). This TOR activity inhibits autophagy in the root and activates the apical root meristem. The antagonistic activation of apical meristems in the root and shoot by TOR is the key for developmental plasticity in response to sulfur limitation and, in combination with enhanced carbon/Suc supply from the shoot, causes an increase in the root-to-shoot ratio. In the root, Suc has dual functions: It serves as the signal regulating TOR activity, and it provides the energy and carbon needed for root growth.