Fig 2: GSK3s function redundantly in etiolated seedling growth. (A) Amino acid sequence alignment of BIN2, BIL1, and BIL2 performed using ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 21 May 2021)) and displayed using Expasy Boxshade (https://embnet.vital-it.ch/software/BOX_form.html (accessed on 21 May 2021). Highly conserved amino acids are indicated by black shading, whereas gray indicates less highly conserved residues. Additional N-terminal amino acids and different C-terminal tails are boxed in red and blue, respectively. Hypocotyl length of bin2-3bil1bil2 (B) and bin2-1 (C) mutants treated with or without 30 mM sucrose. Error bars for both (B) and (C) represent the standard deviations (n > 18). Inset in (C) is the image of 5-day-old dark-grown seedlings of the homozygous bin2-1(-/-) mutant. Scale bars represent 3 mm. Data were statistically evaluated using Student’s t-test, and the significance of differences (*, p < 0.05; n.s., not significant) is indicated.

Fig 3: Roles of S6K2 in skotomorphogenesis. (A) Interaction between S6K2 and BIN2. The interaction was determined using a split-luciferase complementation assay in Nicotiana benthamiana. The full-length coding sequences of S6K2 and BIN2 were ligated into the pCAMBIA-35S-NLuc and pCAMBIA-35S-CLuc vectors, respectively. The empty vectors were used as negative controls. (B) In vitro phosphorylation sites of BIN2 catalyzed by S6K2 was identified by in vitro kinase assay followed by mass spectrometric analysis [31], of which S203 was found to be an in vivo phosphorylation site based on proteomics analyses [32] (http://epsd.biocuckoo.cn/ accessed on 21 May 2021). (C) Hypocotyl length of wild-type (WT) etiolated seedlings treated with different concentrations of S6K inhibitor (LY2584702 tosylate). (D) Growth inhibition of Ws-2 and bin2-3bil1bil2 mutant induced by S6K inhibitor. Growth inhibition (%) = (1 − Lt / Ln). Lt and Ln represent the hypocotyl length of etiolated seedlings grown in medium with or without the S6K inhibitor, respectively. Error bars represent the standard deviations (SD) (n > 20). Data were statistically evaluated using Student’s t-test, and the significance of differences (*, p < 0.05; n.s., not significant) is indicated.

Fig 4: Sucrose influences the mRNA levels of GSK3s and the protein abundance of BIN2. Expression patterns of the BIN2 (A), BIL1 (B), and BIL2 (C) genes in wild-type (WT) etiolated seedlings treated with 30 mM sucrose or 1 μM AZD. (D) Comparison of the mRNA levels of BIN2, BIL1, and BIL2 in untreated WT etiolated seedlings. Values are presented as the means ± SD of three independent sets of experiments. (E) Immunoblot analysis of BIN2 protein in WT etiolated seedlings grown in the dark on medium supplemented with 30 mM sucrose or 1 μM AZD. Data were statistically evaluated using Student’s t-test, and the significance of differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001) is indicated.

Fig 5: A model for sucrose-promoted skotomorphogenesis in the dark. The model illustrates the mechanisms whereby exogenously supplied sugar enhances brassinosteroid (BR) biosynthesis [29], the interaction between BRI1 and BAK1 [42], and the activation of the TOR-S6K2 pathway, resulting in the inhibition of GSK3 kinases and the enhancement of BIN2 degradation. Inactivation of BIN2 and its homologs leads to the accumulation of BZR1/2 for transcriptional regulation (including the upregulated expression of CESA genes [48]) and the enhancement of CESA complex activity [50] for cellulose synthesis, which may synergistically promote skotomorphogenesis in the dark.