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. 2021 May 14;17(5):e1009540. doi: 10.1371/journal.pgen.1009540

Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1

Zhenzhen Zhang 1,2, Ying Sun 3, Xue Jiang 1, Wenfei Wang 1,*, Zhi-Yong Wang 2,*
Editor: Li-Jia Qu4
PMCID: PMC8153450  PMID: 33989283

Abstract

Sugar, light, and hormones are major signals regulating plant growth and development, however, the interactions among these signals are not fully understood at the molecular level. Recent studies showed that sugar promotes hypocotyl elongation by activating the brassinosteroid (BR) signaling pathway after shifting Arabidopsis seedlings from light to extended darkness. Here, we show that sugar inhibits BR signaling in Arabidopsis seedlings grown under light. BR induction of hypocotyl elongation in seedlings grown under light is inhibited by increasing concentration of sucrose. The sugar inhibition of BR response is correlated with decreased effect of BR on the dephosphorylation of BZR1, the master transcription factor of the BR signaling pathway. This sugar effect is independent of the sugar sensors Hexokinase 1 (HXK1) and Target of Rapamycin (TOR), but requires the GSK3-like kinase Brassinosteroid-Insensitive 2 (BIN2), which is stabilized by sugar. Our study uncovers an inhibitory effect of sugar on BR signaling in plants grown under light, in contrast to its promotive effect in the dark. Such light-dependent sugar-BR crosstalk apparently contributes to optimal growth responses to photosynthate availability according to light-dark conditions.

Author summary

Genetic studies of the brassinosteroid (BR) deficient mutants revealed its essential role in seedling development in the dark, but subsequent studies showed no significant difference in BR level between seedlings grown under light and darkness. We recently observed that light does affect BR levels in Arabidopsis, but in a sugar dependent manner. In the dark, sugar increases BR level as well as BR sensitivity by stabilizing the steroid response factor BZR1 through the Target of Rapamycin (TOR) signaling pathway. However, the BR level is decreased by sugar under light and by darkness on sugar-free medium. These observations raised the question of how the combinations of light and sugar modulate BR signaling. We addressed this question using genetic physiological analyses and found interestingly that sugar inhibits brassinosteroid response in light-grown plants by stabilizing the glycogen synthase kinase 3 homolog BIN2 and attenuating the dephosphorylation of BZR1, but independently of TOR. Our results indicate that sugar acts through distinct pathways to promote and inhibit BR signaling in dark and light conditions. Our work illustrates an intricate three-way crosstalk whereby the combination of light and sugar signals modulate the brassinosteroid signaling pathway to optimize growth according to both environmental and metabolic conditions.

Introduction

Plant growth is highly sensitive to environmental light conditions, the levels of endogenous hormones, and the availability of photosynthates (sugars). Sugar not only provides essential material and energy for growth, but also functions as signaling molecules. The sugar signaling pathways mediate plant responses to starvation (low sugar) or excess of sugar, mostly through modulating hormonal pathways [1–5]. How light modulates the sugar-hormone interactions to optimize growth is not well understood.

Brassinosteroids (BR) are a major class of growth-promoting hormones that regulate a wide range of developmental and physiological processes, including photomorphogenesis. BR plays an essential role in plant developmental responses to darkness, so called skotomorphogenesis, as the BR-deficient mutants show strong de-etiolation or constitutive photomorphogenesis phenotypes in the dark [6]. While it was widely speculated that light would reduce BR levels to promote photomorphogenesis, experimental measurement showed surprisingly no significant difference in BR level between seedlings grown under light and those grown in the dark [7]. Further studies uncovered light-BR crosstalk through interactions between downstream components of the signaling pathways [8,9]. However, recent studies suggest that BR level and sensitivity are modulated by the combination of light and sugar conditions [2,10].

BR-responsive gene expression is mediated by the Brassinazole-Resistant 1 (BZR1) family transcription factors [11]. Both nuclear localization and DNA-binding activity of BZR1 are inhibited due to phosphorylation by the GSK3-like kinase Brassinosteroid-Insensitive 2 (BIN2) [12–16]. BR signaling through the BRI1 receptor kinase leads to inactivation and degradation of BIN2 [11,17,18], and dephosphorylation of BZR1 by protein phosphatase 2A (PP2A) [19]. Unphosphorylated BZR1 accumulates in the nucleus, where it recruits the TOPLESS family repressors to inhibit gene expression [20,21] and interacts with transcription factors of other hormonal and light signaling pathways to promote shoot cell elongation [8,22–24].

BZR1 protein level is regulated through several mechanisms. Sugar signaling through Target of Rapamycin (TOR) stabilizes BZR1. When seedlings are shifted from light to darkness and undergo starvation, BZR1 is degraded due to TOR inactivation [2,25]. The degradation of BZR1 and its homolog under starvation and stress conditions involves the autophagy pathway [2,26]. Phosphorylated BZR1 is degraded by the proteasome following PUB40-mediated ubiquitination in roots of Arabidopsis [27]. The dephosphorylated BES1, a homolog of BZR1, can be ubiquitinated and degraded by the SINAT E3 ligase in a light dependent manner [28]. In addition, BZR1 is also modified and stabilized by the small ubiquitin-like modifier (SUMO), and salt stress induces deSUMOylation of BZR1 to inhibit growth [29].

We recently observed that sugar decreases the BR level in Arabidopsis plants grown under light. However, after shifting light-grown Arabidopsis seedlings into darkness, the BR levels increased in seedlings grown on media containing sugar but decreased in those grown on sugar-free media, suggesting light-dependent effects of sugar on the BR pathway [2]. Therefore, we further tested how sugar affects BR responses under light conditions. We found that, in contrast to the positive effects of sugar on growth and BZR1 accumulation in the dark, high levels of sugar attenuated the BR promotion of hypocotyl elongation and the BR-induced BZR1 dephosphorylation in Arabidopsis grown under constant light. Further, sugar increased the level of BIN2. The inhibitory effects of sugar on hypocotyl elongation and BZR1 dephosphorylation are independent of the sugar sensors Hexokinase 1 (HXK1) and TOR. The results suggest that sugars act through distinct pathways to promote and inhibit BR signaling under different light-dark conditions. Such an intricate three-way crosstalk is likely important for optimizing growth according to both environmental condition and endogenous metabolic status.

Results

Sucrose inhibits BR-induced hypocotyl elongation in light

To test the effects of sugar on BR responses under light-grown conditions, we grew Arabidopsis seedlings on media containing various concentrations of sucrose with or without BR for five days under constant light. BR increased the hypocotyl lengths of seedlings grown on a sugar-free medium but had little effect on the seedlings grown on high concentrations (90 and 150 mM) of sucrose (Figs 1A and S1). These sucrose-dependent phenotypes were not caused by osmotic effects since the same concentrations of mannitol did not reduce BR’s promotion of hypocotyl elongation (Fig 1B). Compared to plants expressing wild type BZR1-CFP, transgenic plants expressing bzr1-1D-CFP, a hypermorphic mutant form that is more effectively dephosphorylated by PP2A [12,19], had slightly shorter hypocotyls without BR treatment and longer hypocotyls after growth on BR-containing medium (Fig 1A and 1C), consistent with previous observations [30]. Sucrose had much weaker inhibitory effects on BR responsive hypocotyl elongation of the bzr1-1D-CFP than BZR1-CFP seedlings (Fig 1A and 1C). Taken together, the results suggest that sugar inhibits BR-induced hypocotyl elongation by inactivating BZR1.

Fig 1. Sucrose inhibits the BR-induced hypocotyl elongation and dephosphorylation of BZR1 in light.

Fig 1

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(A-B) Hypocotyl length of transgenic Arabidopsis seedlings expressing BZR1-CFP (A and B) or bzr1-1D-CFP (C) grown on medium containing 100 nM epi-brassinolide (eBL) and various concentrations of sucrose (A and C) or mannitol (B). **** P<0.0001, *** P< 0.001, * P<0.05 (Student’s t test). Error bars indicate the standard error of the mean (SEM, three replicates). (D-F) Immunoblot analysis of BZR1-CFP protein in the same batches of seedlings as in penal A to C. Histone H3 was probed as a loading control. P-BZR1 indicates phosphorylated BZR1; BZR1 indicates dephosphorylated BZR1. S, sucrose; M, mannitol.

Sucrose attenuates BR-induced dephosphorylation of BZR1 in light

As BR promotes hypocotyl elongation through dephosphorylation of the BZR1 family transcription factors, we further examined whether sugar affects the phosphorylation status of BZR1. As shown in Fig 1D, exogenous BR treatment promoted dephosphorylation of BZR1 (as shown by the band shift in gel) in seedlings grown on mannitol or low concentrations of sugar (0 and 30 mM), but the effect on BZR1 dephosphorylation is decreased by high concentrations of sucrose (90 mM and 150 mM) (Figs 1D and 1E and S2). Furthermore, sucrose significantly decreased the BR effects on the expression of BZR1 target genes CPD, DWF4 and SAUR-AC (S3 Fig), consistent with the effects on BZR1 phosphorylation. The bzr1-1D protein was more dephosphorylated than BZR1 under all conditions, although high concentrations of sugar also increased the phosphorylation of bzr1-1D (Fig 1F). These results indicate that sucrose inhibits BR-induced BZR1 dephosphorylation and activation in light-grown seedlings.

To further test whether sucrose decreases the sensitivity to BR, we treated seedlings grown on media containing 30 mM and 90 mM sucrose with BR for different times. BR caused more rapid dephosphorylation of BZR1 protein in seedlings grown under the low sucrose condition (30 mM) than the high sucrose (90 mM) condition (Fig 2). The total BZR1 protein level is lower on 30 mM sucrose media than on 90 mM sucrose (1:1.8) before BL treatment but reached similar levels after 60 min BL treatments (Fig 2A), suggesting that the difference in phosphorylation is not due to a difference in BZR1 protein level. Base on three biological replicate results, the 15-minute BR treatments of seedlings grown in the low-sugar condition caused similar BZR1 dephosphorylation (ratio of dephosphorylated BZR1 to phospho-BZR1, BZR1/P-BZR1) to that caused by 60-minute BR treatments of seedlings grown in the high sugar condition (Fig 2B).

Fig 2. High level of sucrose inhibits BR-induced dephosphorylation of BZR1.

Fig 2

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(A) Immunoblot analysis of BZR1-CFP in seedlings grown in 30 mM or 90 mM sucrose medium after treatment with 1 μM brassinolide (BL) for the indicated time. Numbers below the image show the relative level of total BZR1 protein. (B) Quantification of the ratio between dephosphorylated and phosphorylated BZR1 (P-BZR1) using results shown in (A) and two additional biological repeats. (C) Immunoblot of BZR1-CFP in seedlings grown for five days on media containing 30 mM or 90 mM sucrose and different concentrations of BL after two-day germination in 30 mM sucrose medium. The relative levels of total BZR1 protein are shown below the image. (D) Quantification of the BZR1/P-BZR1 ratio using results shown in (C) and two additional biological replicate samples. Error bars indicate the SEM.

We further analyzed BZR1 in seedlings grown on media containing low (30 mM) or high (90 mM) concentrations of sucrose and various concentrations of brassinolide (BL). The results show that a higher BL concentration is required in high sugar condition than in low sugar condition to induce a similar increase of the BZR1/P-BZR1 ratio (Fig 2C and 2D). In the presence of BL (10 nM or higher), BZR1 was more dephosphorylated under low-sugar conditions than high-sugar conditions while the total BZR1 protein level remained similar. Collectively, these results demonstrate that high concentrations of sucrose inhibit BR signaling upstream of BZR1, specifically by inhibiting BR-induced BZR1 dephosphorylation, in light-grown seedlings.

Sucrose suppresses BR signaling upstream of BIN2

Phosphorylation of BZR1 at Ser173 causes its binding and inhibition by the 14-3-3 proteins [16]. It has been reported that sugar increases the amount of many proteins that bind to the 14-3-3 proteins [31,32]. It was proposed that sugar may promote the binding of the 14-3-3 proteins to their target proteins and protect them from degradation by proteolysis [32]. Therefore, sugar may inhibit dephosphorylation or degradation of phospho-BZR1 by enhancing its binding to the 14-3-3 proteins. To test this hypothesis, we analyzed sugar effects on hypocotyl elongation and BZR1 dephosphorylation in transgenic plants that express the BZR1S173A–CFP protein, which contains the S173A mutation that abolishes 14-3-3 binding [16]. The hypocotyl elongation induced by BR was inhibited by sucrose in the BZR1S173A–CFP plants, and the BR-induced dephosphorylation of BZR1S173A was also inhibited by sucrose, similar to wild-type BZR1 (S4 Fig). Thus, these results indicate that sucrose inhibits BR-induced dephosphorylation of BZR1 dephosphorylation and hypocotyl elongation independent of the 14-3-3 proteins.

BR induces BZR1 dephosphorylation by inactivating BIN2. We thus tested whether BIN2 mediates the phosphorylation of BZR1 on high sugar medium. It is reported that BIN2 interacts directly with BZR1 through a 12-amino acid BIN2-docking motif (DM) near the BZR1 C terminus. Deletion of this motif (bzr1-ΔDM) abolishes the interaction of BZR1 with BIN2 and prevents BIN2 phosphorylation of BZR1 in vivo [33]. Sugar was unable to cause phosphorylation of the bzr1-ΔDM protein (Fig 3A and 3B), suggesting that BIN2 mediates the sugar-promoted phosphorylation of BZR1. Further, the bin2-triple mutant with loss-of-function of BIN2 and its close homologs, bin2bil1bil2, showed reduced sensitivity to sugar (Fig 3C and 3D). In the presence of sugar, BR induced more dramatic hypocotyl elongation and BZR1 dephosphorylation in the bin2bil1bil2 seedlings than wild type (Figs 3C and 3D and S5). These results suggest that sugar inhibition of BR responsiveness is dependent on BIN2 family proteins. Interestingly, we found that sugar increased the BIN2 level in the absence and presence of exogenous BR (Fig 3E). Together these results support a scenario that sugar causes accumulation of BIN2 protein to increase BZR1 phosphorylation and reduce BR responsive growth.

Fig 3. Sucrose inhibition of BR signaling depends on the regulation of BIN2.

Fig 3

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(A) Schematic presentation of BZR1-CFP and bzr1-ΔDM-GFP. (B) Immunoblot analysis of BZR1 protein in BZR1-CFP/Col-0 and bzr1-ΔDM-GFP /Col-0 seedlings grown on medium containing 100 nM eBL and different concentrations of sucrose (mM S). Histone H3 was probed as a loading control in the Immunoblot analysis. (C-D) Phenotypes and hypocotyl length of wild type (WS) and bin2bil1bil2 (bin2-triple) mutant seedlings grown on media containing no sucrose (-S) or 90 mM sucrose (+S) and 0 (-BL) or 10 nM BL (+BL). Bar = 5 mm. **** P<0.0001, *** P< 0.001, ** P<0.01 (Student’s t test). Error bars indicate the SEM (three replicates). (E) Anti-BIN2 immunoblot analysis of BIN2 proteins in seedlings grown on media containing 0 (-S) or 90 mM (+S) sucrose and 0 (-) or 10 nM (+) BL as indicated. The immunoblot was probed with an anti-Actin antibody as a loading control.

Sucrose restrains BR signaling in light through unknown sugar signaling pathway

To determine which sugar-signaling pathway mediates the suppression on BR signaling in light, we carried out genetic tests of known sugar-signaling mutants. TOR and HXK1 are known to mediate plant growth response to moderate and high concentrations of sugars, respectively [25,34]. We thus performed sugar and BR treatments in the HXK1-deficient mutant gin2-1 [25] and the estradiol-inducible TOR silencing line tor-es [34]. As HXK1 is the sensor of glucose, we grew seedlings with glucose and sucrose separately to distinguish their effect on BR signaling. The gin2-1 mutant and wild type plants showed similar sugar inhibition of BR-promoted hypocotyl elongation (Fig 4A and 4B), unlike that bzr1-1D mutant, which is less inhibited by sugar compared to wild type (Fig 4A and 4B). In addition, the effects of BR and sugar on the BZR1 phosphorylation level were similar in the gin2-1 background compared to wild type, with significant amounts of phosphorylated BZR1 remaining in the presence of both BR and high sugar (Fig 4C). The results suggest that HXK1 is not required for sugar inhibition of BR signaling.

Fig 4. Sucrose inhibits BR signaling independent of HXK1 and TOR.

Fig 4

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(A) Hypocotyl lengths of seedlings grown on medium containing 0 mM or 90 mM of mannitol (90M), glucose (90G), or sucrose (90S) and no BL (-BL) or 100 nM BL (+BL). (B) Quantitation of BR sensitivity. Relative hypocotyl elongation is calculated as the ratio between the increase of length caused by BR and the length without BR treatment. Error bars indicate the SEM (five independent experiments, n≥ 20). (C) Immunoblot analysis of BZR1 protein in BZR1-CFP/Col-0 and BZR1-CFP/gin2-1, grown under conditions as shown in (A). (D) Hypocotyl lengths of tor-es seedlings grown on media containing 0 mM or 90 mM (90S) sucrose, 0 (-BL) or 100 nM BL (+BL), and 0 or 1 μM estradiol (+Estradiol). (E) Quantitation of BR sensitivity based on data in (D) using the method described for (B). Error bars indicate the SEM (three independent experiments, n≥ 20). (F) Images of seedlings and immunoblot analysis of BZR1 protein in seedlings described in (D).

Consistent with TOR promoting plant growth, when TOR is inactivated by estradiol-inducible RNAi suppression in the tor-es seedlings, seedlings are smaller compared to tor-es untreated with estradiol (Fig 4D). However, these tor-es plants showed similar sugar inhibition of BR signaling as wild type, based on their hypocotyl elongation and BZR1 dephosphorylation status in response to sugar and BR treatments (Fig 4D–4F). These results suggest that TOR is not involved in sugar suppression of BR signaling in light-grown plants.

Discussion

Plant growth is highly sensitive to environmental signals and endogenous nutrient availability. Hormones, as internal growth regulators, are highly modulated by both environmental conditions and sugar availability to optimize growth and survival. Low sugar levels tend to be limiting for growth when plants are shaded or left in extended darkness, whereas surplus of photosynthate can also be inhibitory to growth. How plants deal with different sugar statuses under different environmental conditions is a key question relevant to crop yield.

We previously showed that sugar depletion in the dark causes BZR1 degradation and growth arrest; under such conditions, exogenous supply of sugar promotes BZR1 accumulation, thus enhancing BR promotion of shoot organ elongation [2,10]. Such sugar promotion of BR signaling in the dark is consistent with the need for maximum shoot elongation under shaded conditions while sugar is available, but arrest of such elongation when the sugar level is low. By contrast, we show in this study that for plants grown under full light, hypocotyl/shoot elongation is not a priority and a surplus of sugar is inhibitory to BR promotion of hypocotyl elongation. Such opposite effects of sugar on BR-dependent hypocotyl elongation are mediated by distinct mechanisms. In the dark, sugar increases BZR1 accumulation through TOR signaling. Under light, sugar increases BZR1 phosphorylation by increasing the level of BIN2 (Fig 5). It’s worth noting that sugar also increases BR hormone accumulation in the dark but decreases BR level under light [2]. Thus, sugar has consistent effects on BR level and BR sensitivity, but these sugar effects are switched under dark and light conditions.

Fig 5. A model of sugar suppressing BR signaling to modify plant growth in light.

Fig 5

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In light, BR signaling inhibits BIN2 activity, which induces accumulation of active BZR1 to promote plant growth, whereas sugar induces the phosphorylation of BZR1 to suppress BR signaling probably through increasing BIN2 activity by unknown sugar signaling pathway. The black bars and arrows indicate previously reported mechanisms; the blue arrow indicates findings made in this study.

Genetic evidence suggests that distinct sugar signaling pathways are involved in the regulation of BZR1 in dark and light. While TOR mediates sugar-dependent stabilization of BZR1 in the dark [2] and BIN2 has been reported to be direct target of TOR-S6K signaling [3], inducible silencing of TOR made no obvious difference in the sugar effect on BZR1 phosphorylation or hypocotyl elongation responses to BR. TOR is inactivated when the sugar level is low, to trigger starvation response and growth arrest [34]. HXK1 is activated by high levels of glucose and mediate glucose inhibition of seedling growth and cotyledon greening [25]. There is also evidence showing that HXK1 mediates glucose positive regulation of BR signaling in promoting lateral root development [4]. It’s somewhat surprising that HXK1 is also not required for sugar inhibition of BR-induced hypocotyl elongation and BZR1 dephosphorylation (Fig 4A–4C).

Alternative sugar signaling pathways may mediate inhibition of BR signaling. The SnRK1 pathway is inhibited by sugar but activated by sugar/energy deficiency, to deal with nutrient deficiency stress conditions [35]. SnRK1 is not known to be involved in responses to high sugar levels.

Recent studies show that glucose signaling is also mediated by Regulator of G-protein Signaling 1 (RGS1), a seven transmembrane guanosine-triphosphatase-activating protein that keeps GPA1 (Gα of G protein) in its inactive state [36–38]. Glucose-induced endocytosis of RGS1 releases its inhibition of G-protein self-activation which is triggered by phosphorylation at its C-terminal region by either the WNK kinases [38,39] or several LRR-RLKs [40–42]. Brassinosteroid insensitive 1 Like 3 (BRL3) and BRI1-associated Kinase 1 (BAK1) phosphorylates RGS1 promoting its endocytosis [41,42]. However, early seedling development and root growth of rgs mutants showed similar response to BL as wild type [41]. Recently, it was reported that glucose at low concentration increases the interaction between BRI1 and BAK1 in a manner dependent on BR biosynthesis [43], consistent with sugar increasing BR level in dark-treated seedlings [2]. Interestingly, high concentrations of glucose caused endocytosis of BRI1 and BAK1. Whether these cell surface receptors contribute to the sugar inhibition of BR signaling remains to be tested [43].

O-GlcNAc and O-fucose modifications have recently emerged as major sugar sensing mechanisms that impact on several hormonal pathways in plants [44,45]. Little is known about the interactions between these O-glycosylation pathways and BR signaling.

Our study indicates that light modulates sugar-BR crosstalk, however, the underlying mechanism remains unclear. Recently, several reports showed that light signaling inhibits BR signaling through photoreceptors regulating the activity of BZR1/BZR2 (BES1). Upon light activation, Phytochrome B, cryptochromes, and UVR8 bind with the dephosphorylated BZR1/BES1 and inhibit their DNA-binding activity [9,46–49]. CRY1 also interacts with BIN2 and enhances the interaction of BIN2 with BZR1 [47]. It would be very interesting to test in future studies whether such direct interactions with phototransduction components rewire the crosstalk between sugar and BR pathways. The interactions between light and sugar pathways in modulating BR responses are important aspects of plant growth regulation that require further molecular investigation.

Materials and methods

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia-0 (Col-0), bzr1-1D [12], bzr1-1D-CFP/Col-0, BZR1-CFP/Col-0 [30], BZR1S173A-CFP/Col-0, Ler, gin2-1 [25], bzr1-ΔDM-GFP [33], BZR1-CFP/tor-es [2] and tor-es [50] were all grown in a greenhouse with a 16-hr light/8-hr dark cycle at 22–24°C for general growth and seed harvesting. All the plants were in Col-0 ecotype background except that gin2-1 is in Landsberg erecta ecotype and bin2,bil1,bil2 is in Wassilewskija (Ws).

Sugar and BR Treatments and hypocotyl elongation assays

Seeds sterilized by 75% (v/v) ethanol were grown on solid 1/2 MS medium (pH 5.7) with 0.4% phytagel (Sigma) and 1% sucrose. After three days of incubation at 4°C and two days of germination in light at 22–24°C, seeds were transferred into 1/2 MS medium containing different concentrations of sugar and BR and were grown in continuous light for another 5 or 6 days as indicated. TOR silencing was induced in tor-es by adding 1 μM β-estradiol (Sigma, E8875) in the medium. Pictures were taken and the hypocotyl lengths were measured with the Image J. Raw data is shown in S1 Data. Seedlings were harvested for Western Blot.

Primer design and Real-time quantitative PCR

Total RNA of above different sugar and BR treated seedlings was isolated by Spectrum Plant Total RNA Kit (Sigma-Aldrich, Shanghai, China). The primer sequences of BR responsive genes were listed in S1 Table. The qRT-PCR reactions were performed on QuantStudio 6 Flex Real-Time PCR System with TaKaRa Real-time qPCR Master Mix Kit. For each condition, the qRT-PCR experiments were performed with biological triplicates. Raw data is shown in S1 Data.

Protein extraction and immunoblot analysis

For protein extraction, plants were frozen in liquid nitrogen, ground, weighed, and added into corresponding 2× SDS buffer (0.125 mM Tris-HCl [pH 6.8], 4% SDS, 20% Glycerol and 2% β-mercapto-ethanol). Samples were heated for 10 min at 95°C, centrifuged at 10000g for 10 min, separated on a 7.5% (detecting BZR1 protein) or 15% (detecting Histone 3H protein) acrylamide gel and then blotted on PVDF membranes (Millipore, IPVH0010) in 192 mM glycine and 25 mM Tris-HCl with a Trans-blot Turbo blotting system (Bio-Rad) for 13 min. Membranes were blocked for 1 hour at room temperature in a blotting buffer (140 mM NaCl, 10 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, 0.5% skim milk, and 0.1% Tween20, pH 7.4). The gel blots were incubated with the primary antibodies (anti-GFP, Transgene, HT801, at 1:1000; anti-BZR1 and anti-BIN2 (home-made) at 1 μg/ml; anti-Actin (Sigma, A2228) at 1:5000 dilutions; anti-Histone H3 (Sangon BBI antibody, AB51007) at 1:2000 dilutions). The secondary antibodies were used at 1:5000 dilutions for 1 hour.

Supporting information

S1 Fig. Sucrose inhibits the BR-induced hypocotyl elongation.

(A) Phenotypes of BZR1-CFP and bzr1-1D-CFP grown on media containing no sugar (-S), 30 to 150 mM sucrose (S) or mannitol (M), as in Fig 1. Bar = 5 mm. (B) Phenotypes of Col-0 grown on medium containing 0 (-BL) or 10 nM brassinolide (+BL) and 0 (-S) or 90 mM sucrose (+S) for six days. (C) Hypocotyl length of seedlings shown in panel B. Error bars indicate the standard error of the mean (SEM, three independent experiments, n≥ 20). **** P<0.0001 (Student’s t test).

(TIF)

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S2 Fig. Sucrose inhibits the dephosphorylation of BZR1 protein induced by BR.

Immunoblot analysis of BZR1 protein with BZR1 antibody in Col-0 grown on medium containing 0 (-BL) or 10 nM brassinolide (+BL) and 0 (-S) or 90 mM sucrose (+S) for six days.

(TIF)

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S3 Fig. Sucrose inhibits BR responses of BZR1 target genes.

(A-C) Relative expression of CPD, DWF4 or SAUR-AC analyzed by qRT-PCR in Col-0 seedlings grown on medium containing 0 (-BL) or 10 nM brassinolide (+BL) and 0 (-S) or 90 mM (+S) sucrose for six days. Error bars indicate the SEM (three independent experiments). ** P< 0.01, * P<0.05 (Student’s t test).

(TIF)

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S4 Fig. Sucrose inhibits BR signaling independent of 14-3-3 protein.

(A) Hypocotyl length of BZR1S173A-CFP/Col-0 grown on medium containing 100 nM eBL and indicated concentrations of sucrose. (B) Immunoblot analysis of BZR1S173A-CFP protein in seedlings in (A). Histone H3 was probed as a loading control in the immunoblot analysis.

(TIF)

Click here for additional data file. (196.2KB, tif)
S5 Fig. Loss of BIN2 function enhances BR-induced BZR1 dephosphorylation in the presence of sugar.

Immunoblot analysis of BZR1 protein in wild type and the bin2-triple mutant grown on media containing 90 mM sucrose and 0 (-BL) or 10 nM BL (+BL) for six days. The ratio of dephosphorylated BZR1 to phosphorylated BZR1 (BZR1/P-BZR1) was measured.

(TIF)

Click here for additional data file. (156.7KB, tif)
S1 Table. List of primers used in this study.

(XLSX)

Click here for additional data file. (11.2KB, xlsx)
S1 Data. Raw data for all quantitative assays shown in the manuscript.

(XLSX)

Click here for additional data file. (215.5KB, xlsx)

Acknowledgments

We thank Dr. Jen Sheen for the mutant gin2-1 and tor-es. We thank Marica Margis-Pinheiro and Tina Tingting Wang for editing the manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by grants from National Institute of Health (NIH, R01GM066258 to Z-Y.W., https://www.nigms.nih.gov), the National Natural Science Foundation of China grant (NO. 31700254, http://www.nsfc.gov.cn)and FAFU-International Collaborative Program (KXb16005A, https://www.fafu.edu.cn) to W.W., the Postdoctoral Innovative Talent Support Program (BX201700052, http://www.chinapostdoctor.org.cn/index.html) and the China Postdoctoral Science Foundation (2018M642551, http://jj.chinapostdoctor.org.cn/website/index.html) to Z.Z. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Gregory P Copenhaver, Li-Jia Qu

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

3 Nov 2020

Dear Dr Wang,

Thank you very much for submitting your Research Article entitled 'Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1.' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by two independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review again a much-revised version. 

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Li-Jia Qu

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The present manuscript describes that sugar inhibits brassinosteroid (BR) signaling pathway in the light, and BR induction of hypocotyl elongation in seedlings grown under light is inhibited by increasing concentration of sucrose. Through phenotypic analysis and immunoblot analysis, the authors demonstrate that sucrose inhibits the BR-induced hypocotyl elongation and dephosphorylation of BZR1 in light. Furthermore, the authors find that high sucrose inhibits BR-induced dephosphorylation of BZR1. Moreover, the authors present that sucrose inhibition of BR signaling in light dependents on the regulation of BIN2. This work illustrates an intricate three-way crosstalk whereby the combination of light and sugar signals modulate the BR signaling pathway to optimize growth according to both environmental and metabolic conditions, which is of novelty and interests to researchers in the fields of light, phytohormone and sugar signaling. However, the authors need to address the comments and concerns I raised below.

Major points:

1. The authors used BZR1-CFP and bzr1-1D-CFP to analyze the hypocotyl lengths (Figure 1), but did not include wild type or bzr1 /mutant or BZR1-RNAi plants. These data are not sufficient to support the authors’ conclusion that sucrose inhibits the BR-induced hypocotyl elongation and dephosphorylation of BZR1. The authors should perform these analyses again with at least wild type plants.

2. As shown in n Figures 2 A and C, as sucrose concentration increases, the protein levels of P-BZR1 and BZR1 increase accordingly, and P-BZR1 and BZR1 proteins are not at the similar levels at the very beginning. Therefore, it’s not appropriate to conclude that high sucrose inhibits BR-induced dephosphorylation of BZR1. Please clarify this complication.

3. The authors stated that high concentration of sucrose inhibits BR-induced dephosphorylation of BZR1. Unfortunately, the authors did not detect the protein levels of P-BZR1 and BZR1 in bin2-triple mutant, which is important to support their conclusion.

4. The authors conclude that sucrose inhibits BR signaling independent on 14-3-3 proteins, HXK1 and TOR. But the authors did not explain how high concentration of sucrose may inhibit BR signaling. Please discuss the possible molecular mechanisms. Moreover, the results showing that sucrose inhibition of BR signaling is independent of HXK1 and TOR should be included in the supplemental materials.

Minor points:

1. In Figures 1F and 3B, the loading control protein histone H3 is not at the same level.

2. In Figure S1B, “Mock” and “eBL” are not located at the central of the line. “Histone H3” does not show the band correctly. The similar problems are found in Figures 1, 2, 3, and 4.

3. The data shown in Figures 3C and 3D should be analyzed by student’s t test.

4. Given the authors’ conclusion that sucrose inhibits BR signaling independent of HXK1 and TOR, it appears that the trend of BZR1 phosphorylation in BZR1-CFP/Col-0 and the BZR1-CFP/gin2-1 seedlings shown in Figure 4C is not consistent with this conclusion. Please clarify.

Reviewer #2: Brassinosteroids (BRs) are a family of plant steroid hormones that play crucial roles throughout plant growth and development. Through BRI receptor, BR signal inhibits BIN2 kinase and results in dephosphorylation and activation of the master transcription factors BZR1 and BES1 to regulate thousands of BR target genes. In dark, BR promotes hypocotyl growth, while under light, BR inhibit hypocotyl elongation. It was shown that sugar-TOR signaling promote hypocotyl growth by stabilizing BZR1 protein when light grown seedlings shift to darkness (ref1). However, the mechanism of how BR inhibit seedling hypocotyl elongation is still unclear. The authors reported an interesting finding that under light high sucrose inhibits BR signaling by stabilizing BIN2 kinase and therefore increasing BZR1 phosphorylation and degradation/inactivation. This paper connects BR signaling with nutrient status and provides interesting observation that BIN2 and BZR1 protein levels correlate with sugar inhibition

Specific concerns

1. As hypocotyl length is one measurement of BR/BZR1/BES1 activity, it would be nice to have seedling pics of those various sugar & BL treatments, at least for Fig 1, 2,3. Reference 1 is a good example for combing phenotypes and quantitative measurements.

2. do authors also have other evidence for BZR1 activity, such as target gene expression change by Q-RT-PCR and binding of BZR1 to its target genes by ChIP-PCR? Reference 2 provides RT and ChIP-PCR target genes. The reason is that WB exposure varies from gel to gel, which makes it hard to compare between samples. For example, in fig 4 D and F, the measurements of hypocotyl length are not consistent with the BZR1 band intensity between samples treated w/ and w/o Estradiol.

3. BIN2 has been reported to be a direct target of TOR-S6K signaling (ref 3). When the authors claimed that the sugar inhibition is independent of TOR, the possibility was not ruled out that high sugar might reduce TOR activity, which releases BIN2 inhibition and results in decreased BZR1 activity.

Minor issues:

1. Fig 3E, the label needs to correspond to samples.

2. In some figures, such as Fig1D and E, western exposure time varies a lot that makes the similar samples look so different (for example, Suc 0, eBL Vs Mannitol 0, eBL)

Reference

1. Zhang Z, Zhu J Y, Roh J, et al. TOR signaling promotes accumulation of BZR1 to balance growth with carbon availability in Arabidopsis[J]. Current Biology, 2016, 26(14): 1854-1860.

2. Oh E, Zhu J Y, Wang Z Y. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses[J]. Nature cell biology, 2012, 14(8): 802-809.

3. Xiong F, Zhang R, Meng Z, et al. Brassinosteriod Insensitive 2 (BIN2) acts as a downstream effector of the Target of Rapamycin (TOR) signaling pathway to regulate photoautotrophic growth in Arabidopsis[J]. New Phytologist, 2017, 213(1): 233-249.

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: No

Decision Letter 1

Li-Jia Qu

6 Apr 2021

Dear Dr Wang,

We are pleased to inform you that your manuscript entitled "Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1." has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Yours sincerely,

Li-Jia Qu

Section Editor: Plant Genetics

PLOS Genetics

Gregory Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have addressed almost all of my comments and concerns, and I am satisfied with the revised manuscript.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

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Reviewer #1: No

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Acceptance letter

Li-Jia Qu

12 May 2021

PGENETICS-D-20-01404R1

Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1.

Dear Dr Wang,

We are pleased to inform you that your manuscript entitled "Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1." has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Sucrose inhibits the BR-induced hypocotyl elongation.

    (A) Phenotypes of BZR1-CFP and bzr1-1D-CFP grown on media containing no sugar (-S), 30 to 150 mM sucrose (S) or mannitol (M), as in Fig 1. Bar = 5 mm. (B) Phenotypes of Col-0 grown on medium containing 0 (-BL) or 10 nM brassinolide (+BL) and 0 (-S) or 90 mM sucrose (+S) for six days. (C) Hypocotyl length of seedlings shown in panel B. Error bars indicate the standard error of the mean (SEM, three independent experiments, n≥ 20). **** P<0.0001 (Student’s t test).

    (TIF)

    Click here for additional data file. (800.3KB, tif)
    S2 Fig. Sucrose inhibits the dephosphorylation of BZR1 protein induced by BR.

    Immunoblot analysis of BZR1 protein with BZR1 antibody in Col-0 grown on medium containing 0 (-BL) or 10 nM brassinolide (+BL) and 0 (-S) or 90 mM sucrose (+S) for six days.

    (TIF)

    Click here for additional data file. (146.1KB, tif)
    S3 Fig. Sucrose inhibits BR responses of BZR1 target genes.

    (A-C) Relative expression of CPD, DWF4 or SAUR-AC analyzed by qRT-PCR in Col-0 seedlings grown on medium containing 0 (-BL) or 10 nM brassinolide (+BL) and 0 (-S) or 90 mM (+S) sucrose for six days. Error bars indicate the SEM (three independent experiments). ** P< 0.01, * P<0.05 (Student’s t test).

    (TIF)

    Click here for additional data file. (205KB, tif)
    S4 Fig. Sucrose inhibits BR signaling independent of 14-3-3 protein.

    (A) Hypocotyl length of BZR1S173A-CFP/Col-0 grown on medium containing 100 nM eBL and indicated concentrations of sucrose. (B) Immunoblot analysis of BZR1S173A-CFP protein in seedlings in (A). Histone H3 was probed as a loading control in the immunoblot analysis.

    (TIF)

    Click here for additional data file. (196.2KB, tif)
    S5 Fig. Loss of BIN2 function enhances BR-induced BZR1 dephosphorylation in the presence of sugar.

    Immunoblot analysis of BZR1 protein in wild type and the bin2-triple mutant grown on media containing 90 mM sucrose and 0 (-BL) or 10 nM BL (+BL) for six days. The ratio of dephosphorylated BZR1 to phosphorylated BZR1 (BZR1/P-BZR1) was measured.

    (TIF)

    Click here for additional data file. (156.7KB, tif)
    S1 Table. List of primers used in this study.

    (XLSX)

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    S1 Data. Raw data for all quantitative assays shown in the manuscript.

    (XLSX)

    Click here for additional data file. (215.5KB, xlsx)
    Attachment

    Submitted filename: Response to Reviewer comments-zw.docx

    Click here for additional data file. (34.1KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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