Sucrose-activated TOR and phyA signaling alleviates shade-mediated inhibition of leaf development in Arabidopsis

Abstract

To over-top the neighbors, shade-intolerant plants trigger shade avoidance responses with increasing the elongation of stem-like organs at the expense of leaf development. Shade-induced elongation growth has been extensively studied, while the regulatory mechanisms underlying shade-suppressed leaf development remain poorly understood. Here we find sucrose can rescue shade-inhibited leaf development, which requires the functions of phytochrome A (phyA) and TARGET OF RAPAMYCIN (TOR) complex. We confirm an interaction between PHYA and LETHAL WITH SEC THIRTEEN 8 (LST8), a component of TOR complex. Shade and phyA affect TOR activity, in turn TOR affects the protein stability of PHYA. The sucrose–TOR mediated accumulation of PHYA requires CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)/SUPPRESSOR OF PHYA-105 (SPA) complex. Transcriptome analysis further reveals that sucrose and phyA regulate the expression of leaf development-related genes under shade, particularly those involved in cytokinin metabolism. Our work uncovers a critical role for TOR and PHYA in linking light signaling with cellular energy status, a mechanism that fine-tunes leaf development specifically under shade conditions.

Shade limits leaf growth. Here, Wang et al. show that sucrose alleviates shade-repressed leaf development by activating TOR and stabilizing PHYA, thereby regulating cytokinin and leaf development-related genes.

Introduction

Plants are photoautotrophic organisms and rely on sunlight to drive photosynthesis. When growing under potentially reduced light availability, as encountered in dense plant communities, they face a threat to growth and development. Plants detect the proximity of neighboring plants by perceiving changes in the ratio of red to far-red (R/FR) light and overall light quantity. Due to its profound ecological and economic significance, research on how plants perceive shading and trigger adaptive morphological and physiological responses has expanded dramatically. The key components of the shade signal transduction pathways linking photoreceptors to functional responses have been elucidated¹.

In Arabidopsis thaliana, shade light converts phytochrome B (phyB) to its inactive form. PhyB inactivation releases the repression of PHYTOCHROME-INTERACTING FACTORS (PIFs), allowing them to promote the transcription of elongation-related target genes^2–5. To prevent excessive stem elongation under shade, plants deploy multiple negative feedback mechanisms. Shade-accumulated phytochrome A (phyA) attenuates shade-induced elongation by suppressing the auxin and brassinosteroid signaling pathway^6,7, and elevating early-evening expression of core circadian-clock components⁸. Shade-induced atypical bHLH proteins LONG HYPOCOTYL IN FAR-RED 1 (HFR1) and PHYTOCHROME RAPIDLY REGULATED 1 (PAR1) /PAR2 inhibit PIFs activity by forming non–DNA-binding heterodimers^9,10, with KIDARI (KDR) modulating their repressive capacity^11,12. The bZIP transcription factor ELONGATION HYPOCOTYL5 (HY5) acts as a repressor of hypocotyl growth under longer-term shade conditions¹³. Meanwhile, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) re-accumulates in the nucleus to promote the stabilization of PIFs and to target HY5 and HFR1 for degradation, thereby promoting shade-avoidance response^14,15. These precise regulatory mechanisms ensure appropriate stem elongation, facilitating light foraging and enabling plants to outcompete their neighbors.

As a consequence, leaf primordium growth is rapidly arrested under canopy shade, tentatively ensuring that plant resources are redirected toward extension growth¹⁶. This growth arrest in the leaf depends on auxin-induced cytokinin breakdown¹⁷. PIF7 is reported to evict and substitute for ANGUSTIFOLIA3 (AN3, a transcriptional coactivator stimulating cell division during leaf development) in regulating the expression of genes associated with leaf cell proliferation¹⁸. Compared to the extensive research on shade-induced hypocotyl elongation, studies on shade-repressed leaf development are relatively scarce.

The TARGET OF RAPAMYCIN (TOR) kinase is a central growth regulator that integrates growth and development with carbon availability, nutrient status, and energy levels¹⁹. The TOR kinase is activated by light, sugars (such as sucrose), nitrogen availability, and other factors, promoting cell proliferation and growth²⁰. For instance, in de-etiolating Arabidopsis seedlings, photoreceptors, including phytochrome A, activate the auxin pathway to facilitate TOR-dependent phosphorylation of ribosomal protein S6 (RPS6), thereby mediating light-induced cotyledon opening²¹. Light sustains auxin biosynthesis required for TOR activation, and TOR is essential for light-dependent stem cell activation²². Glucose-activated TOR phosphorylates and activates the transcription factors E2Fa/E2Fb, promoting root growth and true leaf formation by enhancing cell division in the root meristem and shoot apex, respectively^22,23. These studies suggest the possibility that boosting TOR signaling might rescue leaf development suppressed by shade.

Our results reveal that exogenous sucrose alleviates shade-induced inhibition of leaf development through a mechanism dependent on both the TOR complex and phyA. We find that PHYA interacts with the TOR complex, suggesting a direct link between light and metabolic signaling pathways. Shade and phyA influence TOR activity, while TOR in turn regulates the stability of PHYA. Sucrose promotes PHYA protein accumulation in a manner that requires both the TOR and COP1/SPA complexes. We also identify the downstream genes of sucrose and phyA related to leaf development. Together, our findings suggest that TOR and phyA function as central integrators of light and carbon signaling and act as key regulators of leaf development under shade.

Results

TOR and phyA are involved in sucrose-rescued leaf development under canopy shade

To establish comparative light environments, we used three-color LEDs to control spectral composition and irradiance, generating simulated white light (WL, Red: 20 µmol·m⁻²·s⁻¹, Blue: 20 µmol·m⁻²·s⁻¹, Far Red: 5 µmol·m⁻²·s⁻¹; R/FR = 4) and simulated shade (SH, Red: 10 µmol·m⁻²·s⁻¹, Blue: 5 µmol·m⁻²·s⁻¹, Far Red: 60 µmol·m⁻²·s⁻¹; R/FR = 0.17) (Supplementary Fig. 1a). Upon exposure to SH, Col-0 seedlings exhibited increased hypocotyl elongation as well as a reduction in true leaf size. However, sucrose application significantly increased the area of the first leaf, as well as hypocotyl length under shade compared to equimolar sorbitol (Fig. 1a, Supplementary Fig. 1b). The mechanism of sucrose-induced hypocotyl elongation has been studies in several literatures^24–26. To focus on the effects on leaf primordium growth and mitotic activity in the shoot apex, we examined seedlings expressing the transcriptional mitotic reporter pCYCB1;1::GUS, as cell division is essential for leaf organogenesis. Consistent with the observed seedling growth responses, both leaf primordium size and pCYCB1;1::GUS expression indicated that sucrose reactivated shoot meristem activity under shade (Fig. 1b). This effect was abolished by the TOR inhibitor Torin 2 (Fig. 1b). We also take advantage of a mutant line with estradiol-inducible RNA-mediated inhibition of TOR expression (tor-es, Supplementary Fig. 1c) to confirm that TOR is required for sucrose-mediated promotion of leaf development under both WL (Fig. 1c) and SH (Fig. 1d).

Fig. 1. TOR and phyA are involved in sucrose-rescued leaf development under canopy shade.

a The first true leaf area of Col-0 under simulated white light (WL) and shade (SH) treatment (scale bar, 2 mm). b The leaf primordium area and GUS staining intensity of pCYCB1;1::GUS seedlings under WL and SH (scale bar, 0.1 mm). c, d The first true leaf area of Col-0, tor-es and Col-0 treated with 5 μM Torin 2 under WL (c) or SH (d) treatment (scale bar, 2 mm). e, f The first true leaf area of Col-0, phyA-211, phyB-9, hy5 and pif7-1 under WL (e) or SH (f) treatment (scale bar, 2 mm). g The leaf primordium area of Col-0, tor-es, and phyA-211 under SH treatment (scale bar, 0.1 mm). In a–g, data are presented as mean values ± SD. Numbers above the bars indicate n, the number of independent seedlings analyzed. The letters above the scatter indicate significant differences (P < 0.05) determined by two-way ANOVA, followed by Tukey’s multiple comparison test. Sor: sorbitol (30 mM; black dots); Suc: sucrose (30 mM; red dots); Suc + Torin 2: sucrose (30 mM) plus Torin 2 (5 μM; blue dots). For exact P-values, see Source Data.

To identify potential regulators involved in this process, we examined sucrose responses in several shade-signaling mutants, including phyA-211, phyB-9, hy5, and pif7-1. Under WL, all tested mutants showed sucrose-induced enlargement of the first true leaf (Fig. 1e). However, under shade, phyA-211 was completely insensitive to sucrose in terms of both first leaf area and leaf primordium size (Fig. 1f, g), implying a shade-specific regulatory effect in leaves. Notably, phyA-211 still responded to sucrose with hypocotyl elongation (Supplementary Fig. 1d, e). By contrast, phyB-9 and pif7-1 showed larger first leaf areas with sucrose treatment, while the sucrose-induced hypocotyl elongation under shade was abolished in these mutants (Fig. 1f, Supplementary Fig. 1d, e), suggesting that the regulatory roles of these factors in sucrose responses are tissue-dependent.

To further confirm the role of phyA on leaf development, we assessed sucrose responses in two additional phyA mutants (SALK_014575 and SALK_020360) (Supplementary Fig. 1f), which also exhibited insensitivity to sucrose in the first leaf area, leaf primordium size, and pCYCB1;1::GUS expression under shade (Supplementary Fig. 1g–i). Moreover, the sucrose insensitivity of phyA-211 was rescued by 35S::PHYA-YFP complementation under shade (Supplementary Fig. 1j, k).

Collectively, these results support the roles of TOR and phyA in mediating sucrose-induced leaf development under shade.

PHYA interacts with TOR complex and they mutually regulate each other

In Arabidopsis, the functional TOR complex consists of TOR, RAPTOR, and LST8, which collectively regulate plant growth and development. RAPTOR binds to the HEAT repeat domain of AtTOR, maintaining its activation status²⁷, while LST8 interacts directly with the kinase domain of AtTOR, modulating its kinase activity toward specific substrates²⁸. We examined the effects of sucrose on leaf development in several TOR complex mutants, including raptor1-1 (RAPTOR1B: SALK_078159), raptor1-2 (RAPTOR1B: SALK_006431), and lst8-1 (LST8-1: SALK_208632) (Supplementary Fig. 2a), all of which showed reduced sucrose responsiveness in terms of leaf area expansion under both WL (Fig. 2a) and SH (Fig. 2b), consistent with the phenotype observed in the tor-es mutant (Fig. 1c, d).

Fig. 2. PHYA interacts with TOR complex and they mutually regulate each other.

a, b The first true leaf area of Col-0, raptor1-1, raptor1-2, lst8-1, and 35S::LST8-1-FLAG under WL (a) or SH (b) treatment (scale bar, 2 mm). Data are presented as mean values ± SD. Numbers above the bars indicate n, the number of independent seedlings analyzed. The letters above the scatter indicate significant differences (P < 0.05) determined by two-way ANOVA, followed by Tukey’s multiple comparison test. c Interactions between LST8-1 and PHYA detected via a semi-in vitro GST pull-down assay. d Interactions between PHYA and LST8-1 were detected via a luciferase complementary imaging (LCI) assay. e The complex of LST8-1, TOR and PHYA was identified by co-immunoprecipitation in Arabidopsis. f The protein levels of phosphorylated S6K (pS6K) and total S6K in Col-0 with sorbitol or sucrose treatment under WL and SH. g The protein levels of phosphorylated S6K (pS6K) and total S6K in Col-0 and phyA-211 with sorbitol or sucrose treatment under SH. h The phosphorylation of phyA in the in vitro kinase experiment was detected by the phos-tag assay. The arrow indicates the phosphorylated band. In c, and e–h, each experiment was independently repeated three times with similar results, and representative data are shown. For exact P-values, see Source Data.

To explore the genetic relationship between TOR and phyA, we assessed the leaf area phenotypes of phyA-211 and the 35S::PHYA-YFP/phyA-211 transgenic line upon treatment with Torin 2 (Supplementary Fig. 2b, c). Under shade, the attenuated inhibitory effect of Torin 2 in phyA-211 is likely due to the already severely reduced leaf size in this mutant, limiting the extent of further suppression. In 35S::PHYA-YFP/phyA-211 plants, Torin 2 treatment abolished sucrose sensitivity even in the presence of PHYA overexpression (Supplementary Fig. 2c). Considering that phyA-211 mutants were sucrose-insensitive, this finding indicates that neither PHYA overexpression nor elevated TOR activity alone is sufficient to restore the sucrose responsiveness. Consequently, the sucrose-dependent rescue of leaf development in shade requires the combined presence of functional phyA and active TOR signaling.

Large-scale phosphoproteomics and interactomics studies have been conducted to map TOR signaling events in plants²⁹. Notably, PHYA was shown to be a potential interactor of LST8 (Supplementary Fig. 2d). We examined the protein-protein interaction between PHYA and LST8 using multiple approaches. In a half in vitro pull-down assay, GST-LST8-1 purified from E. coli pulled down PHYA from shade-treated Col-0 seedlings (Fig. 2c). In a luciferase complementation imaging (LCI) assay, nLUC-tagged LST8-1 interacted with cLUC-tagged PHYA when transiently expressed in Nicotiana benthamiana leaf cells (Fig. 2d). In CoIP experiments using a 35S::LST8-1-FLAG transgenic line, LST8-1-FLAG co-immunoprecipitated with TOR and PHYA, as detected by native TOR and PHYA antibodies (Fig. 2e). These results support that PHYA interacts with TOR complex.

We next investigated whether phyA and TOR mutually influence each other on biochemical characteristics. Using the phosphorylation of T449 in the TOR substrate protein ribosomal S6 kinase 1 (S6K1) as a conserved indicator of endogenous TOR kinase activity³⁰, we assessed TOR activity using phospho-S6K and total-S6K antibodies. TOR activity was suppressed under shade compared to WL, while sucrose efficiently reactivated TOR kinase under both conditions (Fig. 2f, Supplementary Fig. 2e). Additionally, sucrose-induced TOR activation under shade was reduced in the phyA-211 mutant (Fig. 2g, Supplementary Fig. 2f), consistent with previous published data that phyA positively regulates the activation of TOR under far-red light conditions²¹. These findings indicate that light, phyA, and sucrose signals tightly regulate TOR kinase activity.

To investigate how the sucrose–TOR signaling pathway regulates phyA, we hypothesized that sucrose-activated TOR might directly phosphorylate phyA, given the kinase activity of the TOR complex. However, phos-tag gel electrophoresis failed to resolve mobility shifts between phosphorylated and non-phosphorylated forms of endogenous phyA following sucrose treatment. To further investigate potential phosphorylation events in vivo, we conducted LC-MS/MS analysis to identify phosphorylation sites on phyA. The phyA protein was immunoprecipitated from 35S::PHYA-YFP/phyA-211 seedlings treated with shade and 30 mM sucrose for 6 h. We made multiple attempts but were unsuccessful, with only one exception where three potential sites (S13, S500, and T597) were observed, though this result could not be replicated. Therefore, we sought to test whether TOR can directly phosphorylate phyA using an in vitro kinase assay. We used recombinant phyA protein purified from insect cells as the substrate and endogenous TOR complex immunoprecipitated from the 35S::LST8-1-FLAG transgenic line as the kinase source. Following Phos-tag analysis, we observed an increased intensity of the upper PHYA band corresponding to its phosphorylated form, which decreased upon Torin 2 treatment (Fig. 2h). Phosphopeptide enrichment and LC-MS/MS analysis of the in vitro reaction identified multiple phosphorylated residues (Supplementary Fig. 2g); however, the relevance of these sites in vivo and their direct regulation by TOR remain to be clarified.

Sucrose–TOR signaling increases the protein level of PHYA under shade

To further investigate the effects of sucrose and TOR activity on phyA, we examined endogenous PHYA protein levels. Five-day WL-grown seedlings were treated with sucrose, sorbitol, or sucrose combined with Torin 2. Half of the seedlings remained under WL, and the other half were transferred to shade for 6 h (Supplementary Fig. 3a). Consistent with a previous report⁶, PHYA was accumulated under shade, as detected using a native PHYA antibody (Fig. 3a, Supplementary Fig. 3a, b). Sucrose significantly enhanced PHYA accumulation, whereas Torin 2 reversed this effect (Fig. 3a, Supplementary Fig. 3a, b). Then we extended the duration of sucrose treatment. WL-grown seedlings were pretreated with sucrose, sorbitol, or sucrose with Torin 2 for 6 h under WL, then transferred to shade and collected at 0, 2, and 8 h. Sucrose markedly increased PHYA protein levels under shade, slightly increased under WL, and this effect was effectively blocked by Torin 2 (Supplementary Fig. 3a, c). Given the low PHYA protein levels and the slight effect of sucrose on PHYA protein levels under WL, we mainly focused on shade conditions in the following experiments.

Fig. 3. Sucrose–TOR signaling increases the protein level of PHYA under shade.

a, b The protein levels of PHYA in Col-0 (a) or 35S::PHYA-YFP/phyA-211 (b) with sorbitol, sucrose or sucrose plus Torin 2 treatment under WL and SH. c The protein levels of PHYA in Col-0, tor-es, raptor1-2, and lst8-1 with sorbitol or sucrose treatment under SH. d Subcellular fluorescence of PHYA-YFP in response to sorbitol, sucrose, or sucrose plus Torin 2 treatment under SH. Fluorescent signals at the top of the hypocotyl were observed using confocal microscopy (scale bar, 100 μm for top images and 10 μm for bottom images). The average fluorescence intensity was quantified using ImageJ. Data are presented as mean values ± SD. Numbers above the bars indicate n, the number of independent seedlings analyzed. The letters above the scatter indicate significant differences (P < 0.05) determined by one-way ANOVA, followed by Tukey’s multiple comparison test. The experiment was independently repeated three times with similar results, and representative data are shown. e The protein levels of PHYA in Col-0 with sorbitol, sucrose, or sucrose plus Torin 2 treatment, analyzed via cell fractionation assay. Numbers under the bands denote ImageJ-quantified PHYA band intensities after normalization to total protein in the sorbitol group. The experiment was independently repeated three times with similar results, and representative data are shown. f The protein levels of PHYA in Col-0, atg5, and atg7 with sorbitol or sucrose treatment under SH. g The effects of the autophagy inhibitors E64d and 3-MA on PHYA protein levels in Col-0 with sorbitol or sucrose treatment under SH. In (a–c, f, g), western blot band intensities were quantified using ImageJ and normalized to β-tubulin protein levels. Data are presented as mean values ± SD (n = 3 independent biological replicates). The letters above the scatter indicate significant differences (P < 0.05) determined by one-way ANOVA (a, b) or two-way ANOVA (c, f, g), followed by Tukey’s multiple comparison test. For exact P-values, see Source Data.

We performed the same treatments using 35S::PHYA-YFP/phyA-211 transgenic lines and detected PHYA-YFP protein levels. We got similar results that sucrose increased PHYA-YFP protein accumulation under shade, while Torin 2 blocked this effect (Fig. 3b, Supplementary Fig. 3d, e). PHYA transcript levels remain stable following sucrose treatment (Supplementary Fig. 3f), indicating that sucrose enhances PHYA protein accumulation under shade at the post-transcriptional level.

We also checked the accumulation of PHYA by application of sucrose in TOR complex mutants, including tor-es, raptor1-2 and lst8-1, under shade conditions. These mutants exhibited diminished sucrose responsiveness of PHYA accumulation (Fig. 3c, Supplementary Fig. 3g), confirming the requirement of the TOR complex for sucrose-induced accumulation of PHYA. Thus, our phenotypical (Supplementary Fig. 2b, c) and biochemical (Figs. 2f–h, 3a–c) data suggested a coordinated and mutually reinforcing relationship between phyA and TOR.

Then we observed the subcellular localization of PHYA-YFP. Sucrose treatment enhanced nuclear PHYA-YFP accumulation, as evidenced by increased fluorescence intensity (Fig. 3d). To further validate this observation, we performed a subcellular fractionation experiment using the Col-0 and 35S::PHYA-YFP/phyA-211 seedlings. Consistent with the imaging results, sucrose promoted nuclear accumulation of PHYA under shade (Fig. 3e, Supplementary Fig. 3h).

Autophagy functions in the degradation and recycling of cellular components, particularly under stresses involving nutrient deficiency³¹. In contrast, under favorable conditions such as sugar supplementation, activated TOR inhibits autophagy initiation, which may contribute to increased protein stability^19,24. To determine whether autophagy is involved in sucrose-mediated regulation of PHYA degradation, we examined PHYA protein levels in atg5 and atg7 mutants (defective autophagy)³², as well as in Col-0 seedlings treated with the autophagy inhibitor 3-methyladenine (3-MA) or the protease/autophagy inhibitor E64d³³. If sucrose stabilized PHYA primarily by inhibiting autophagy, an increase in PHYA levels without sucrose treatment, and no further increase in PHYA levels with sucrose application would be expected in autophagy-deficient mutants or autophagy inhibitor-treated plants, as the autophagy-mediated degradation pathway is already disrupted. In fact, despite an increase in basal PHYA levels, sucrose still promoted PHYA accumulation in both the autophagy mutants and inhibitor-treated Col-0 seedlings (Fig. 3f, g, Supplementary Fig. 3i, j). These results suggest that autophagy contributes to PHYA basal level, but the sucrose-induced PHYA stabilization likely involves additional regulatory pathways.

The effect of sucrose on PHYA accumulation requires COP1 and SPAs

As a light-labile phytochrome, PHYA accumulates to a high level when light is scarce (Very Low Fluence Response or VLFR) or when the R/FR is very low (FR-High Irradiance Response, FR-HIR)^34,35. Upon exposure to white light, PHYA is rapidly degraded through the 26S proteasome pathway, mediated by CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)/SUPPRESSOR OF PHYA-105 (SPA) complex^36,37. To examine the effect of sucrose on PHYA degradation, we compared the degradation rate of PHYA in Col-0 seedlings treated with sorbitol, sucrose, or the proteasome inhibitor MG132 upon transfer from SH to WL. To specifically monitor protein degradation rather than de novo synthesis, cycloheximide (CHX) was included to block new protein synthesis. Similar to MG132 treatment, sucrose blocked the disappearance of PHYA, suggesting that sucrose interfered with PHYA proteasomal degradation (Fig. 4a, Supplementary Fig. 4a).

Fig. 4. The effect of sucrose on PHYA accumulation requires COP1 and SPAs.

a The protein levels of PHYA in Col-0 during the transition from SH to WL with 100 μM CHX plus 30 mM sorbitol, 30 mM sucrose, or 50 μM MG132 treatment. b, c The protein levels of PHYA in cop1-4 (b) or spa1spa2spa3spa4 (c) during the transition from SH to WL with 100 μM CHX plus 30 mM sorbitol or 30 mM sucrose treatment. In a–c, western blot band intensities were quantified using ImageJ and normalized to β-tubulin levels. Data are presented as mean values ± SD (n = 3 independent biological replicates). The asterisks represent statistical significance relative to the sorbitol treatment group (CHX plus sorbitol), using a two-sided Student’s t-test, * P < 0.05, ** P < 0.01, ‘n.s.’ indicating no significant difference. d The protein levels of PHYA in Col-0, cop1-4 and spa1spa2spa3spa4 with sorbitol, sucrose or sucrose plus Torin 2 treatment under SH. Data are presented as mean values ± SD (n = 3 independent biological replicates). The letters above the scatter indicate significant differences (P < 0.05) determined by one-way ANOVA, followed by Tukey’s multiple comparison test. e The interaction between COP1 and PHYA in 35S::YFP-COP1/cop1-5 seedlings with sorbitol, or sucrose, or sucrose plus Torin 2 treatment under SH, detected via co-immunoprecipitation. Numbers beneath the bands denote ImageJ-quantified ratios of PHYA (IP) to the corresponding COP1 (IP) signals, normalized to the value of the sorbitol control. The experiment was independently repeated three times with similar results, and representative data are shown. f The first true leaf area of Col-0, phyA-211, cop1-4, cop1-4phyA-211, spa1spa2spa3spa4, and spa1spa2spa3spa4phyA-211 under SH treatment (scale bar, 2 mm). Data are presented as mean values ± SD. Numbers above the bars indicate n, the number of independent seedlings analyzed. The letters above the scatter indicate significant differences (P < 0.05) determined by two-way ANOVA, followed by Tukey’s multiple comparison test. For exact P-values, see Source Data.

To further investigate the effect of sucrose on COP1/SPA-mediated PHYA degradation, we assessed PHYA degradation in the cop1-4 and the spa1spa2spa3spa4 quadruple mutants after shifting seedlings from SH to WL, and found that sucrose no longer slowed PHYA disappearance in these backgrounds (Fig. 4b, c, Supplementary Fig. 4b, c). We next examined PHYA protein levels in the cop1-4 and spa1spa2spa3spa4 mutants following 6 h of sucrose treatment under shade. In both mutants, the sucrose-induced increase in PHYA protein levels was impaired (Fig. 4d, Supplementary Fig. 4d). These results indicated that sucrose-mediated accumulation of PHYA required COP1 and SPAs.

In co-immunoprecipitation (CoIP) experiments using 35S::YFP-COP1/cop1-5 transgenic line, sucrose application resulted in less amounts of PHYA immunoprecipitated with YFP-COP1, and Torin 2 blocked this effect (Fig. 4e, Supplementary Fig. 4e). These results suggested that sucrose disrupted the interaction between PHYA and COP1/SPA complex, leading to PHYA accumulation.

Genetically we detected the sucrose effects in cop1-4 and spa1spa2spa3spa4 mutants, and we also generated cop1-4phyA-211 and spa1spa2spa3spa4phyA-211 by crossing (Fig. 4f). Under shade, although the true leaf area and leaf primordia area were smaller in the cop1-4 and spa1spa2spa3spa4 mutants compared to Col-0 under shade, as we expected, their response to sucrose was enhanced, with sucrose-induced expansion being greater in cop1-4 ([Suc - Sor]/Sor: 1.22 on leaf, 2.35 on primordia) and spa1spa2spa3spa4 ([Suc - Sor]/Sor: 1.70 on leaf, 3.28 on primordia) than in Col-0 ([Suc - Sor]/Sor: 0.60 on leaf, 1.17 on primordia) (Fig. 4f, Supplementary Fig. 4f). Moreover, the sucrose response on leaf area in the cop1-4phyA-211 double mutant ([Suc - Sor]/Sor: 0.47) was weaker than that in the cop1-4 single mutant ([Suc - Sor]/Sor: 1.22). The spa1spa2spa3spa4phyA-211 quintuple mutant ([Suc - Sor]/Sor: 0.24) exhibited a significantly weaker response than the spa1spa2spa3spa4 quadruple mutant ([Suc - Sor]/Sor: 1.70) (Fig. 4f). These results suggest that COP1 and SPAs were required for the sucrose- and phyA- regulated leaf development.

Sucrose and phyA activate the expression of genes related to leaf development

To further investigate how sucrose and sucrose-accumulated PHYA affect the leaf development, we conducted RNA-sequencing using above-hypocotyl portions (including the cotyledons, first pair of true leaves, and petioles) to identify sucrose- and phyA- regulated genes under shade. Based on the Integrative Genomics Viewer (IGV) visualization of the PHYA locus and Gene Ontology (GO) analysis of differentially expressed genes in phyA-211 compared to Col-0, we verified the genotype of phyA-211 used in this study (Supplementary Fig. 5a, b). In Col-0, we identified 714 sucrose-induced genes and 857 sucrose-repressed genes (Fig. 5a, b, Supplementary Fig. 5c, d and Supplementary Data 1). However, in phyA-211, only 623 sucrose-induced genes were detected (Fig. 5a, b, Supplementary Fig. 5c, d, Supplementary Data 2), indicating that phyA is required for the full induction of a subset of sucrose-responsive genes.

Fig. 5. Sucrose and phyA activate the expression of genes related to leaf development.

a, b Volcano plot showing the differentially expressed genes (DEGs) between sorbitol and sucrose treatment in Col-0 (a) and phyA-211 (b). Differential expression analysis was performed using DESeq2 with two-sided statistical testing. P-values were adjusted for multiple comparisons using the Benjamini–Hochberg method. Genes with fold change (FC) > 2.0 or < 0.5 and adjusted P < 0.05 were considered significantly differentially expressed (red and blue, respectively). c Correlation analysis of EODFR-regulated genes (GSE17845, FC > 2.0, or FC < 0.5, adjusted P < 0.05) with sucrose-responsive genes under SH (our data). P values were adjusted for multiple comparisons using the Benjamini–Hochberg method. d The heatmap showing the expression levels of sucrose-induced genes in Col-0 and phyA-211 (n = 3 independent biological replicates). Based on expression patterns, sucrose-induced genes were categorized into three clusters (I1-I3). The boxplots display the normalized expression levels within each cluster (left figure). The boxplot boundaries reflect the interquartile range, the center line is the median, and the whiskers represent 1.5 × the interquartile range from the lower and upper quartiles. The violin plots show the log2 fold change (sucrose/sorbitol) of induced genes in Col-0 and phyA-211 (right figure), with the median indicated. The asterisks represent statistical significance, using a two-sided Student’s t-test, *** P < 0.001, ‘n.s.’ indicating no significant difference. e Gene Ontology (GO) analysis of sucrose-induced genes under SH. Bubble size represents the number of genes, and the colors indicate the P-value, Fisher’s one-tailed test (P < 0.05). f Expression levels of CIA1, OLI2, SWL1, and PFL2 in Col-0, tor-es and phyA-211 with sorbitol or sucrose treatment under SH. Gene expression levels were determined by RT-qPCR and normalized to the reference gene TUB4 (AT5G44340). Data are presented as mean values ± SD (n = 3 independent biological replicates). The letters above the scatter indicate significant differences (P < 0.05) determined by two-way ANOVA, followed by Tukey’s multiple comparison test. For exact P-values, see Source Data.

To determine the correlation between sucrose and the shade avoidance pathway on leaf development, we compared a list of previously published end-of-day far-red (EODFR)-responsive genes³⁸ (GSE17845, RNAs were prepared from leaf blades and petioles; EODFR-responsive genes in blades were defined as those exhibiting at least a two-fold difference, adjusted P < 0.05), against our sucrose treatment. The majority of EODFR-responsive genes were located in quadrants II and IV, indicating that sucrose inversely regulated these genes (Fig. 5c). This pattern aligns with the observed repression of shade responses by sucrose in leaves.

To further dissect the differential gene expression patterns between Col-0 and phyA-211, we categorized sucrose-induced genes into three clusters using mfuzz clustering: I1 (sucrose induction reduced in phyA-211), I2 (sucrose induction comparable between Col-0 and phyA-211), and I3 (sucrose induction increased in phyA-211) (Fig. 5d, Supplementary Data 3). Similarly, we divided sucrose-repressed genes into three clusters: R1 (sucrose repression comparable between Col-0 and phyA-211), and R2 + R3 (sucrose repression reduced in phyA-211) (Supplementary Fig. 5e, Supplementary Data 4). GO analysis revealed that genes in I1, which exhibited diminished sucrose induction in phyA-211, were enriched in terms related to “response to cytokinin”, “cytokinin-activated signaling pathway”, “trans-zeatin biosynthetic process”, and “cell wall biogenesis” (Fig. 5e, Supplementary Data 5). These processes may underlie the reduced sucrose response on leaf area observed in phyA-211. Additionally, genes in R2 and R3, which were repressed by sucrose in Col-0 but showed diminished repression in phyA-211, were significantly associated with “leaf senescence” (Supplementary Fig. 5f, Supplementary Data 6).

The genes in I1 and R2 + R3 clusters represent phyA-regulated sucrose-responsive genes. To determine whether these genes were also regulated by TOR, we analyzed their expression patterns in response to glucose in the tor-es mutant using the publicly available dataset GSE104146 (15 mM glucose-treated Col-0 and tor-es for 2 h). The fold changes of these genes were reduced in tor-es, mirroring their expression pattern in phyA-211 (Supplementary Fig. 5g). To verify this, we selected shade-repressed genes CIA1 (CHLOROPLAST IMPORT APPARATUS 1)³⁹, OLI2 (OLIGOCELLULA 2)⁴⁰, SWL1 (SNOW-WHITE LEAF1)⁴¹ and PFL2 (POINTED FIRST LEAF 2)⁴², which have been reported to positively regulate leaf development, and performed RT-qPCR to confirm their expression patterns (Supplementary Fig. 5h). Consistent with analysis in Supplementary Fig. 5g, the sucrose-induction of these genes was attenuated in phyA-211, tor-es and Torin 2-treated seedlings under shade (Fig. 5f, Supplementary Fig. 5i), indicating that TOR and phyA function cooperatively to regulate the expression of genes which are critical for leaf development under shade.

Sucrose and phyA activate cytokinin to regulate leaf development

Cytokinin-related terms (“response to cytokinin”, “cytokinin-activated signaling pathway”, and “trans-zeatin biosynthetic process”) were enriched in the phyA-regulated sucrose-responsive gene cluster (Fig. 5e). Previous studies have shown that canopy shade rapidly and transiently arrests leaf primordium growth by promoting cytokinin breakdown¹⁷. Our RT-qPCR analysis revealed that the shade conditions used in this study downregulated the expression of cytokinin biosynthesis genes (CYP735A1, IPT3, and LOG3) and cytokinin signaling pathway genes (ARR4 and ARR6), while upregulating the expression of cytokinin oxidase gene (CKX6) (Supplementary Fig. 6a).

We further investigated the effect of sucrose on the expression of these cytokinin-related genes. As we expected, sucrose exhibited a counter-regulatory effect against shade, and phyA-211 was insensitive to sucrose-induced changes in the expression of these genes (Fig. 6a), indicating that sucrose-accumulated phyA might activate cytokinin to modulate leaf development.

Fig. 6. Sucrose and phyA activate cytokinin to regulate leaf development.

a Expression levels of CYP735A1, IPT3, LOG3, ARR4, ARR6, and CKX6 in Col-0 and phyA-211 with sorbitol or sucrose treatment under SH. The expression levels of genes were normalized to TUB4 (AT5G44340). Data are presented as mean values ± SD (n = 3 independent biological replicates). The letters above the scatter indicate significant differences (P < 0.05) determined by two-way ANOVA, followed by Tukey’s multiple comparison test. b The leaf primordium area of Col-0, ckx6, phyA-211, and phyA-211ckx6 under SH treatment (scale bar, 0.1 mm). c The leaf primordium area of Col-0 and phyA-211 treated with 6-BA under SH treatment (scale bar, 0.1 mm). In (b, c), data are presented as mean values ± SD. Numbers above the bars indicate n, the number of independent seedlings analyzed. The letters above the scatter indicate significant differences (P < 0.05) determined by two-way ANOVA, followed by Tukey’s multiple comparison test. For exact P-values, see Source Data. d Proposed model illustrating how exogenous sucrose regulates true leaf development under canopy shade through the TOR and phyA signaling networks. Under shade, TOR activity is reduced, and phyA accumulates in the nucleus, where it is targeted for degradation by COP1/SPA. Exogenous sucrose activates TOR, which interacts with phyA, disrupting its interaction with COP1/SPA and enhancing phyA stability. Accumulated phyA regulates the transcription of downstream genes, including cytokinin biosynthesis and metabolism genes, thereby increasing active cytokinin levels, enhancing cytokinin signaling, and ultimately promoting true leaf development.

Then, we examined the sucrose response in the ckx6 mutant. Due to elevated cytokinin levels, ckx6 exhibited enlarged leaf primordia and reduced sensitivity to sucrose treatment (Fig. 6b). Genetically, the phyA-211ckx6 double mutant partially rescued the small leaf primordium phenotype of the phyA-211 single mutant, indicating that phyA regulates leaf primordium size, at least in part, through CKX6 (Fig. 6b). Moreover, exogenous cytokinin treatment (6-benzylaminopurine, 6-BA) partially restored the sucrose response in phyA-211 leaf primordia (Fig. 6c), implying that both cytokinin levels and signaling are impaired in phyA-211 during sucrose response. These results suggest that phyA regulates leaf development by modulating cytokinin pathways, encompassing both metabolism/biosynthesis and signaling.

Discussion

Plants have evolved sophisticated mechanisms to perceive and respond to the presence of neighbors. phyA is a negative regulator for shade-induced hypocotyl elongation⁶. Here, we found that when sucrose was exogenously supplied to shade-grown seedlings, TOR and phyA functioned as negative regulators of shade-repressed leaf development, at least partially by modulating cytokinin signal (Fig. 6d).

In this study, we investigated the role of phyA in leaf development under our shade conditions. Previous studies have demonstrated that phyB inactivation suppresses true-leaf development under EODFR, a condition effective in inducing a robust SAS while avoiding phyA activation^18,43. phyA and phyB serve as dominant photoreceptors under distinct shade intensities. Specifically, it has been established that phyB suppresses shade response under moderate shade (R/FR > 0.3), whereas phyA mediates the inhibitory effects under deeper shade with lower R/FR⁴⁴.

Exogenous sucrose application promotes growth in both hypocotyls and leaves under our shade conditions. We demonstrate a tissue-specific division of labor: sucrose-induced leaf development under shade is mediated predominantly by phyA, not phyB, while sucrose-induced hypocotyl elongation requires phyB and PIFs (Fig. 1f, Supplementary Fig. 1e). Thus, the regulatory mechanisms for sucrose responses differ between these tissues. Sucrose-induced hypocotyl elongation has been more extensively studied, for example, under far-red (FR) light in the presence of sucrose; overexpression of phyB interferes with phyA-mediated inhibition of hypocotyl elongation⁴⁵. Exogenous sugar has been shown to upregulate PIF gene expression to promote hypocotyl elongation in the dark²⁵. Although PIF7 has been reported to regulate leaf development under shade¹⁸, our findings suggest that in leaves, the positive regulatory role of phyA surpasses the repression of PIF7. Notably, although phyA, phyB, and PIFs are expressed in both hypocotyls and leaves⁴⁶, their functional dominance appears to differ between these tissues. Such tissue-specific functional dominance may reflect spatial differences in downstream signaling, including the cell type-specific expression, subcellular localization, or post-translational modification of co-regulators or interacting proteins, as well as chromatin accessibility or epigenetic states at their target loci.

As shown in Figs. 3a–c, 4e, sucrose disrupts the interaction between PHYA and COP1/SPA complex, thereby stabilizing PHYA in a manner dependent on TOR complex components. Given that TOR is an evolutionarily conserved Ser/Thr kinase that primarily functions through phosphorylation of downstream targets such as EIN2, E2Fa, and FIE^23,30,47, the most parsimonious model is that TOR directly phosphorylates PHYA, thereby reducing its degradation by the COP1/SPA complex. Our in vitro kinase assays (Fig. 2h) are consistent with this possibility, although the relevant phosphorylation sites and their functional significance in vivo remain to be determined. Alternatively, TOR may influence PHYA stability indirectly by modulating upstream kinases such as PPKs, which phosphorylate hinge-region residues (S590, T593, and S602) to promote COP1-dependent degradation^48–50, by regulating COP1 itself, whose activity is attenuated by phosphorylation at Ser20⁵¹, or via deubiquitinating enzymes, including UBP12/13, which interact with RAPTOR1B and stabilize COP1^29,52. TOR also controls multiple steps of protein synthesis via phosphorylation of translation factors^21,53, raising the possibility that it may affect PHYA accumulation at the translational level. Further studies are needed to distinguish among these mechanisms and clarify how TOR regulates PHYA stability.

TOR functions as a central integrator of nutrient, energy, and stress signaling networks. Here, we provide evidence for a regulatory relationship between TOR and PHYA that contributes to coordinating light signaling and nutrient availability. Under shade, phyA accumulates, while the activity of TOR is repressed (Figs. 2f, g, 3a, b). In contrast, under white light, phyA degrades, and TOR activity is enhanced (Figs. 2f, g, 3a, b). Light conditions likely prevent them from reaching high activity simultaneously, ensuring appropriate growth. However, exogenous sucrose supplementation may release this constraint, allowing both TOR and phyA to be activated and to cooperate in promoting true leaf development. In basic research, artificially creating a “coexistence” or “co-activation” state is a common strategy to transiently disrupt endogenous negative feedback or mutual repression, allowing exploration of regulatory limits and hidden cooperative interactions. However, plants have been reported to accumulate higher endogenous sugar levels under far-red–enriched conditions⁵⁴ or in phyB mutants^55,56. Under such physiological contexts, simultaneous activation of phyA and TOR may not be implausible. Our findings suggest that elevated endogenous sugar levels may activate TOR, which could promote PHYA stability and collectively alleviate the suppression of leaf development under shade conditions.

Previous studies have shown that glucose promotes leaf development by activating TOR signaling, which phosphorylates E2Fa/b to induce cell cycle genes and enhance shoot apical meristem activity²². Here, we demonstrate that TOR promotes leaf development by stabilizing PHYA. Based on a published chromatin immunoprecipitation and RNA sequencing of phyA⁵⁷, we found the leaf development-related genes CIA1 and OLI2 as potential direct targets of PHYA. Combined with our RT-qPCR data (Fig. 5f, Supplementary Fig. 5i), the reduced expression of these genes might be responsible for the leaf phenotype of phyA-211. In addition to regulating these genes, phyA also modulates cytokinin levels by influencing the expression of several cytokinin biosynthesis and metabolism-related genes. Cytokinin is a positive regulator of cell proliferation in the shoot apical meristem (SAM)⁵⁸. During the proliferative phase, it can increase leaf cell number and contribute to leaf size control⁵⁹. Previously, we demonstrated that phyA negatively regulates the auxin signaling pathway by stabilizing AUX/IAA proteins⁶. Auxin in leaf primordia induces the expression of CKX6¹⁷. Whether directly or indirectly, phyA appears to regulate the expression of CKX6 and cytokinin biosynthesis genes, potentially elevating cytokinin levels and thereby influencing leaf primordia development. In addition to cytokinin levels, our RNA-seq analyses (Fig. 5e) and hyposensitivity of phyA-211 to 6-BA (Fig. 6c) suggest impaired CK signaling may underlie its diminished response to sucrose.

Our study provides insights into how plants dynamically adjust their growth strategies in response to changes in light conditions and nutrient availability. We show that this adaptive regulation is mediated by the TOR and phyA, which modulate the expression of leaf development-related genes, thereby enabling precise, individualized control of leaf growth. The exogenous application of nutrients, such as carbon or nitrogen fertilizers, may alleviate the arrest in leaf development, once TOR is activated.

Methods

Plant materials and growth conditions

The model used in this study is Arabidopsis thaliana, with all lines in the Columbia-0 (Col-0) background. The mutants analyzed included phyA-211⁶⁰, phyA-S1 (SALK_014575), phyA-S2 (SALK_020360), phyB-9^BC, pif7-1, hy5, atg5-1 (SAIL_129_B07), atg7-3 (SAIL_11_H07), cop1-4, spa1spa2spa3spa4, spa1spa2spa3spa4phyA-211, estradiol-inducible tor RNAi line (tor-es), raptor1-1 (SALK_078159), raptor1-2 (SALK_006431), lst8-1 (SALK_208632), and ckx6 (SALK_070071). Transgenic Arabidopsis plants included pCYCB1;1::GUS, 35S::PHYA-YFP/phyA-211, 35S::YFP-COP1/cop1-5 and 35S::LST8-1-FLAG. The pCYCB1;1::GUS/phyA-S1 and pCYCB1;1::GUS/phyA-S2 line were generated by crossing pCYCB1;1::GUS line with phyA-S1 and phyA-S2 respectively. The mutants cop1-4phyA-211 double mutant was generated by crossing cop1-4 with phyA-211. The phyA-211ckx6 double mutant was generated by crossing phyA-211 with ckx6.

To generate LST8-1 overexpression (LST8-FLAG) transgenic lines in the Col-0 background, the full-length LST8-1 coding sequence was amplified by PCR and cloned into the pCambia1300 vector to construct pCambia1300-LST8-1-FLAG. These constructs were transferred into Agrobacterium tumefaciens GV3101 and subsequently used to transform Col-0 plants via the floral dip method. Transgenic lines were selected on half-strength Murashige and Skoog (1/2 MS) nutrient medium (Duchefa Biochemistry, Haarlem, Netherlands) containing 50 mg/mL hygromycin.

For shade phenotypic analysis, Arabidopsis seeds were sterilized with 75% ethanol at room temperature for 15 min, then sown on 1/2 MS medium containing 1% agar but no sugar. The seeds were stratified at 4 °C for 3 days to promote uniform germination. After seed stratification, the plates were incubated in growth chamber (JIUPO, BPC500-2H, Fujian Jiupo Biotechnology Co., Ltd) under continuous simulated white light (LED light, JIUPO, JIUPO-5050TLED-300-S, Red: 20 µmol·m⁻²·s⁻¹, Blue: 20 µmol·m⁻²·s⁻¹, Far Red: 5 µmol·m⁻²·s⁻¹) at 22 °C for 3 days, and then transferred to either simulated canopy shade (LED light, JIUPO, JIUPO-5050TLED-300-S, Red: 10 µmol·m⁻²·s⁻¹, Blue: 5 µmol·m⁻²·s⁻¹, Far Red: 60 µmol·m⁻²·s⁻¹) or maintained under simulated white light. Leaf primordium area was measured after 1 day, hypocotyl length was measured after 3 days, and the first true leaf area was measured after 10 days.

First true leaf, leaf primordium area and hypocotyl length analysis and quantification

To analyze the effect of sucrose on true leaf and hypocotyl growth phenotypes, the seedlings were germinated on solid 1/2 MS medium and grown under continuous simulated white light (WL) without sucrose for 3 days, then transferred to 1/2 MS medium containing 30 mM sucrose or 30 mM sorbitol (as an osmotic control) and placed in shade (SH) or maintained under WL for 10 days (true leaf area) or for 3 days (hypocotyl length). A 30 mM sucrose solution is approximately equivalent to a 1% (w/v) concentration, which is commonly used for normal cultivation of Arabidopsis as well as for studies on sugar signaling⁶¹. Hypocotyl, cotyledons and first true leaves were scanned with an HP Scanjet8270 scanner. The hypocotyl length and first true leaf area were measured using NIH ImageJ software.

To analyze the leaf primordium area, seedlings were grown under continuous WL for 3 days, transferred to 1/2 MS medium containing 30 mM sorbitol or 30 mM sucrose, and then placed in SH or maintained under WL for 1 day. At least 30 seedlings were used for leaf primordium observations, which were conducted using a Motic BA310 microscope, and the area was measured using NIH ImageJ software. For the 6-BA rescue experiment, an additional 6-BA or DMSO was added to the medium containing sorbitol or sucrose.

GUS staining and quantification

The transgenic pCYCB1;1::GUS reporter line was used in this study. The seedlings were germinated on solid 1/2 MS medium and grown under continuous WL without sucrose for 3 days, transferred to 1/2 MS liquid medium containing 30 mM sorbitol, 30 mM sucrose, or 30 mM sucrose plus 5 μM Torin 2, and then placed in SH or maintained under WL for 1 day. For each treatment, over 30 seedlings were incubated in 2 mL of GUS staining solution (10 mM phosphate buffer pH7.4, 0.5 mM K₃[Fe(CN)₆], 0.5 mM K₄[Fe(CN)₆], 10 mM EDTA, 0.01% Triton X-100, 1 mg/mL X-Gluc [5-bromo-4-chloro-3-indolyl-b-D-glucuronide]) at 37 °C for 12 h. After staining, seedlings were destained in 75% ethanol at 37 °C for 24 h. The GUS staining area in the leaf primordium was visualized using a Motic BA310 microscope and quantified with the NIH ImageJ software.

Immunoblot assays

To test the effect of sucrose on PHYA protein level, seeds were germinated in 12-well plates (15–20 seeds per well) containing 1 mL of 1/2 MS liquid medium without sugar and grown under continuous WL for 5 days. Seedlings were then transferred to 1/2 MS medium containing 30 mM sorbitol, 30 mM sucrose, or 30 mM sucrose plus 5 μM Torin 2. The plates were then transferred to SH or maintained under WL for the indicated time before harvest. Total proteins were extracted with 2 × extraction buffer (100 mM Tris-HCl [pH 7.5], 300 mM NaCl, 2 mM EDTA, 10% [v/v] glycerol, 1% [v/v] TritonX-100, and protease inhibitor cocktail) and centrifuged at 12,000 × g for 10 min. The supernatant was mixed with 2 × SDS loading buffer (100 mM Tris-HCl [pH 6.8], 4% [w/v] SDS, 0.2% [w/v] BPB, 20% [v/v] glycerin, 2% [v/v] β-mercaptoethanol) and heated at 95 °C for 10 min. The proteins were separated by SDS/PAGE gels and transferred onto a polyvinylidene fluoride (PVDF) membrane. Anti-PHYA or anti-GFP antibody were used to detected PHYA protein levels, and anti-tubulin antibody was used to detect β-tubulin as a loading control. The western blot band intensities were quantitatively analyzed using NIH ImageJ software.

To evaluate the effects of different light conditions on TOR activity, Col-0 seedlings were germinated on solid 1/2 MS medium and grown under continuous WL without sugar for 3 days, then transferred to shade or maintained in WL for 10 days, followed by treatment with 30 mM sorbitol or 30 mM sucrose in 1/2 MS liquid medium for 2 h.

To assess the extent of sucrose-induced TOR activation in Col-0 and phyA-211 under shade, seeds were grown in 12-well plates (15–20 seeds per well) in 1/2 MS liquid medium under continuous WL for 3 days without sugar, then treated with 30 mM sorbitol, 30 mM sucrose or 30 mM sucrose plus 5 μM Torin 2 in 1/2 MS liquid medium and simultaneously exposed to SH for 6 h. Endogenous TOR activity was assessed using anti-pS6K and anti-S6K antibodies.

Confocal microscopy and quantification of the microscopic data

For Confocal imaging, 35S::PHYA-YFP/phyA-211 seedlings were germinated and grown on solid 1/2 MS medium under continuous WL for three days, and then transferred to 1/2 MS liquid medium containing 30 mM sorbitol, 30 mM sucrose, or 30 mM sucrose plus 5 μM Torin 2, while simultaneously exposed to SH for 6 h. The top of the hypocotyl from each sample was scanned only once using a Leica TCS SP8 confocal microscope to minimize the influence of excitation laser light on the PHYA-YFP subcellular localization. Fluorescence intensity was measured and analyzed using NIH ImageJ software. First, a threshold value was set to determine the sum of fluorescence intensity (Integrated Density, IntDen) within a defined area. Then, IntDen was divided by the area to obtain the mean fluorescence intensity.

Cell fractionation

Cytoplasmic and nuclear protein isolation was performed as previously described⁶². Col-0 and 35S::PHYA-YFP/phyA-211 seedlings were germinated and grown on solid 1/2 MS medium under continuous WL for five days, and then treated with 30 mM sorbitol, 30 mM sucrose, or 30 mM sucrose plus 5 μM Torin 2 in 1/2 MS liquid medium and simultaneously exposed to SH for 6 h. The harvested samples were ground in liquid nitrogen, homogenized in lysis buffer (20 mM Tris-HCl [pH 7.5], 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl₂, 250 mM sucrose, 5 mM DTT, 25% [v/v] glycerol, and protease inhibitor cocktail) and filtered through Miracloth (Calbiochem). A total of 5% of the extract was retained as the total protein. The remaining extract was centrifuged at 1500 × g for 10 min, and the supernatant was collected as the cytoplasmic fraction. The pellet was washed five times with resuspension buffer (NRBT:20 mM Tris-HCl [pH 7.5], 2.5 mM MgCl₂, 0.2% [v/v] Triton X-100, 25% [v/v] glycerol, and protease inhibitor cocktail). After the final wash, the pellet was resuspended in NRB2 (20 mM Tris-HCl [pH 7.5], 250 mM sucrose, 10 mM MgCl₂, 0.5% [v/v] Triton X-100, 5 mM β-mercaptoethanol and protease inhibitor cocktail) and gently layered over an equal volume of NRB3 buffer (20 mM Tris-HCl [pH 7.5], 1.7 M sucrose, 10 mM MgCl₂, 0.5% [v/v] Triton X-100, 5 mM β-mercaptoethanol and protease inhibitor cocktail). After centrifugation at 16,000 × g for 1 h at 4 °C, the pellet was resuspended in lysis buffer as the nuclear fraction.

CHX degradation assay

Col-0, cop1-4 and spa1spa2spa3spa4 seedlings were grown in 12-well plates (15–20 seeds per well) in 1/2 MS liquid medium under continuous WL for 5 days, then placed in SH for 24 h, transferred to 1/2 MS liquid medium containing 100 μM CHX plus 30 mM sorbitol, 100 μM CHX plus 30 mM sucrose, or 100 μM CHX plus 50 μM MG132 (for Col-0), followed by transfer back to WL. Samples were collected at 0, 1, 2, 4, 8, and 24 h. The collected samples were ground in liquid nitrogen, extracted with 2 × extraction buffer, and separated via SDS/PAGE, following the protocol described above.

Luciferase complementation imaging (LCI) assay

The coding sequences of the LST8-1 and PHYA genes were cloned into either the N-terminal half (nLUC) or C-terminal half (cLUC) of the LCI vector to obtain LST8-1-nLUC and cLUC-PHYA. The resulting constructs were transformed into the Agrobacterium strain GV3101. Agrobacterium strains harboring different constructs were infiltrated into N. benthamiana leaves. After infiltration, the plants were kept in darkness for 24 h, followed by a 16 h light/8 h dark photoperiod for 48 h. Luciferase activity was observed using a CCD imaging apparatus.

Semi-in vivo pull-down assay

The full-length coding sequence of LST8-1 was cloned into pGEX4T-1. The recombinant plasmids were transformed into E. coli BL21 (DE3). To induce protein expression, transformed bacteria were incubation in 300 mL of LB medium containing 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37 °C for 5 h. The cells were harvested by centrifugation at 4000 × g, resuspended in 1 × PBS, and subsequently used for GST and GST-LST8-1 purification. For GST pull-down assays, five-day WL-grown Col-0 seedlings were treated with 30 mM sucrose and simultaneously exposed to SH for 6 h. The total plant protein was extracted using an extraction buffer (100 mM Tris-HCl [pH 7.5], 300 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail). After centrifugation at 16,000 × g for 10 min, the supernatant was incubated with pretreated GST-LST8-1 beads or GST beads (as a negative control) for 2 h. The beads were collected, washed five times with a buffer containing 1 × PBS, 0.1% Triton X-100 and protease inhibitor cocktail and resuspended in the 2 × SDS loading buffer. The immunoprecipitates and input samples were analyzed using SDS/PAGE followed by immunoblotting with anti-GST and anti-PHYA antibodies.

Co-immunoprecipitation (CoIP) assay

To detect the interaction between LST8-1 and PHYA, five-day WL-grown 35S::LST8-1-FLAG/Col-0 overexpression plants (germinated and grown on solid 1/2 MS medium) were treated with 30 mM sucrose and simultaneously exposed to SH for 6 h. The total plant protein was extracted with an extraction buffer (100 mM Tris-HCl [pH 7.5], 300 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail). The crude protein extract was filtered through Miracloth and centrifuged at 16,000 × g for 10 min. This fraction was used as the input control. For immunoprecipitation (IP), the protein extract was incubated with anti-FLAG magnetic beads or control beads at 4 °C for 2 h. The beads were collected, washed three times with the extraction buffer, and resuspended in the 2 × SDS loading buffer. Immunoprecipitates and input samples were analyzed using SDS/PAGE, followed by immunoblotting with anti-FLAG, anti-TOR and anti-PHYA antibodies.

To detect the effect of sucrose on the interaction between COP1 and PHYA, total protein was extracted from five-day WL-grown 35S::YFP-COP1/cop1-5 overexpression plants using the extraction buffer. Five-day WL-grown seedlings were treated with 30 mM sorbitol, 30 mM sucrose, or 30 mM sucrose plus 5 μM Torin 2, each supplemented with 50 μM MG132 in 1/2 MS liquid medium and simultaneously exposed to SH for 6 h. Anti-GFP and anti-PHYA antibodies were used in immunoblot analysis.

In vitro TOR protein kinase assay

The AtphyA (containing PΦB) protein was expressed and purified from High Five™ insect cell line (BTI-TN-5B1-4; derived from Trichoplusia ni ovarian cells; Thermo Fisher Scientific) as previously described⁶³. The kinase assay is modified from what has been described previously^23,30. TOR complex was immunoprecipitated from 35S::LST8-1-FLAG seedlings. In brief, total protein was lysed by immunoprecipitation buffer (40 mM HEPES, pH 7.4, 2 mM EDTA, 10 mM pyrophosphate, 10 mM glycerol phosphate, 0.3% CHAPS and 1 × cocktail inhibitors) from 7-day-old seedlings grown in 1/2 MS solid medium with 15 mM sucrose. Prior to harvest, seedlings were transferred to 1/2 MS liquid medium containing 30 mM sucrose and incubated under WL for 6 h. Protein extracts were incubated with anti-FLAG magnetic beads or control beads at 4 °C for 2 h. The immunoprecipitated TOR complex was washed twice with low-salt wash buffer (40 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 mM pyrophosphate, 10 mM glycerol phosphate and 0.3% CHAPS) and once with kinase wash buffer (25 mM HEPES, pH 7.4 and 20 mM KCl). In-tube kinase assay was performed in 30 μL kinase reaction buffer (25 mM HEPES, pH 7.4, 50 mM KCl, 10 mM MgCl₂, 10 μM ATP and 1 μg AtphyA protein) with anti-FLAG magnetic beads (with or without 5 μM Torin 2) or control beads at 25 °C for 30 min. The reaction was stopped by adding 5 × SDS sample buffer and heating at 95 °C for 5 min. Proteins were separated by phos-tag SDS/PAGE gel and regular SDS/PAGE gel.

Phosphopeptide enrichment and LC–MS/MS analysis

For in vivo experiments, 35S::PHYA-YFP/phyA-211 seedlings grown under continuous WL for 5 days were treated with 30 mM sucrose and shade for 6 h. Total proteins were extracted using the coIP lysis buffer described above, supplemented with phosphatase inhibitors (PhosSTOP, Roche, 4906845001). PHYA-YFP proteins were immunoprecipitated using anti-GFP beads. The bead-bound proteins were incubated with 2 × SDS sample buffer, heated at 95 °C for 10 min, and subjected to enzymatic digestion followed by phosphopeptide enrichment. A single sample was analyzed without biological or technical replicates, as this initial exploratory analysis was designed to identify potential phosphorylation sites.

Peptide digestion was performed using the FASP protocol with Microcon PL-10 filters. After three rounds of buffer exchange with 8 M urea in 100 mM Tris-HCl (pH 8.0), proteins were reduced with 10 mM DTT at 37 °C for 30 min and alkylated with 30 mM iodoacetamide at 25 °C for 45 min in the dark. Samples were then exchanged into digestion buffer (30 mM Tris-HCl, pH 8.0) and digested with trypsin (enzyme-to-protein ratio 1:50) at 37 °C for 12 h. The filtrates were collected, combined after washing the filter twice with 15% ACN, and vacuum-dried.

Phosphopeptides were enriched using home-made TiO₂ microcolumns (peptide: TiO₂ = 1:10). Peptides were dissolved in loading buffer (1 M glycolic acid, 80% ACN, 5% TFA), loaded twice, washed with loading buffer followed by washing buffer (80% ACN, 1% TFA), and sequentially eluted with 2 M ammonium hydroxide and 1 M ammonium hydroxide in 30% ACN. Eluates were vacuum-dried.

LC–MS analysis was performed using an EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a home-made C18 analytical column (75 μm inner diameter × 25 cm, 1.9 μm) using a one-column configuration. The mobile phases consisted of solution A (0.1% formic acid) and solution B (0.1% formic acid in 80% ACN). Peptides were eluted at a flow rate of 200 nL min⁻¹ using a gradient of 5–8% B (2 min), 8–44% B (38 min), 44–70% B (8 min), 70–100% B (2 min), followed by 100% B (10 min). FAIMS was enabled with compensation voltages of −40 and −60 V. MS1 spectra were acquired at a resolution of 60,000, and precursor ions with charge states of 2–7 were selected for MS2 analysis. A dynamic exclusion of 45 s was applied with a cycle time of 1 s. MS2 spectra were acquired with HCD fragmentation (isolation window 1.6 m/z; resolution 15,000; normalized collision energy 30%; maximum injection time 30 ms).

Raw data were processed against the Arabidopsis TAIR database (32,790 entries) using Proteome Discoverer (version 2.4, Thermo Fisher Scientific) with the Mascot search engine (version 2.7.0, Matrix Science). The precursor and fragment mass tolerances were set to 10 ppm and 0.05 Da, respectively. Up to two missed cleavages were allowed. Carbamidomethylation of cysteine was set as a fixed modification, while oxidation of methionine and protein N-terminal acetylation were set as variable modifications. Phosphorylation of serine, threonine, and tyrosine residues was included as a variable modification for phosphopeptide analysis.

For in vitro experiments, kinase reaction products were mixed with 5 × SDS sample buffer, heated at 95 °C for 5 min, and supplemented with phosphatase inhibitors (PhosSTOP, Roche, 4906845001). A single sample was then subjected to enzymatic digestion, phosphopeptide enrichment, and database searching as described above. LC–MS analysis was performed using a nanoElute system (Bruker Daltonics) coupled to a timsTOF HT mass spectrometer (Bruker Daltonics) operating in PASEF mode. Peptides were separated on a C18 analytical column (75 μm inner diameter × 25 cm, 1.9 μm) at a flow rate of 300 nL min⁻¹. Data were acquired in DDA-PASEF mode over an m/z range of 100–1700, with ion mobility separation (1/K₀ 0.75–1.50 V·s·cm⁻²). MS/MS spectra were acquired with collision energies of 20–59 eV, and dynamic exclusion was enabled.

RNA sequencing

The Col-0 and phyA-211 seedlings grown under WL for 10 days were treated with 30 mM sorbitol or 30 mM sucrose in 1/2 MS liquid medium and simultaneously exposed to SH for 6 h. The above-hypocotyl portions, including the cotyledons, first pair of true leaves, and petioles, were excised, rapidly frozen in liquid nitrogen, and sent to Majorbio Company (http://www.majorbio.com) for standard RNA-seq library preparation and construction. Three independent replicates were prepared for each genotype of plants with sorbitol and sucrose treatment. Total RNA was extracted from the tissue samples using the TRIzol^® Reagent (Invitrogen) according to the manufacturer’s instructions. RNA-seq libraries were prepared using the TruSeq^TM RNA sample preparation kit from Illumina (San Diego, CA, USA) and sequenced on an Illumina HiSeq X Ten sequencer, with 150 bp paired-end reads. Differential expression analysis was performed using DESeq2. P-values were adjusted for multiple testing using the Benjamini–Hochberg method, and genes with an adjusted P-value < 0.05 and |log₂ fold change| > 1 were considered differentially expressed. The transcriptome data were divided into clusters using the mfuzz algorithm. The heatmap was plotted by https://www.bioinformatics.com.cn. Gene Ontology enrichment analysis was performed by the website Plant Regulomics (http://bioinfo.cemps.ac.cn/plant-regulomics/).

The RNA-seq data generated in this study were deposited in the NCBI GEO database (https://www.ncbi.nlm.nih.gov/gds) (accession number GSE291348).

Quantitative reverse-transcription PCR

Total RNA was extracted from Arabidopsis seedlings subjected to specific treatments using a TRIzol^® kit (Promega). Two micrograms of total RNA were reverse transcribed using All-in-One First-Strand Synthesis MasterMix (ShareBio) according to the manufacturer’s instructions. The cDNA was then subjected to real-time qPCR using a Real-Time System CFX96™ C1000 Thermal Cycler (Bio-Rad). The gene-specific primers used for qPCR are listed in Supplementary Data 7.

Quantification and statistical analysis

In this study, data are presented as mean ± SD, and the number (n) of samples or replicates is indicated in the figures or figure legends. Statistical significance was examined by two-sided Student’s t-test for pairwise comparisons and one-way or two-way analysis of variance (ANOVA) with Tukey’s test between multiple groups comparison indicated with asterisk or different letters (P < 0.05).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

J.W., F.L., and L.L. designed the experiments. J.W., F.L., and C.L. performed the experiments. L.L wrote the paper.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

The raw and processed RNA-seq data generated in this study were deposited in the NCBI GEO database under accession code GSE291348. Previously published RNA-seq datasets used in this study are available in the NCBI GEO database under accession numbers GSE17845 and GSE104146. Phosphoproteomic data were deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD075951. Source data are provided with this paper (10.6084/m9.figshare.30879302).

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-026-71841-x.