Supporting Information
The following supplemental materials are available.
Table S1. Primer sequences used in this study.
Figure S1. Phylogenetic analysis of UVI4 and CCS52homologous genes in Solanaceae species. The full-length amino acid sequences of UVI4 and CSS52 were isolated through BLAST analysis in the website (https://solgenomics.net/) and analyzed in the software MEGA X to determine their relationships. The bootstrap values are shown at the nodes. (A) Phylogenetic relationship among UVI4 homologous genes. (B) Phylogenetic relationship among CCS52 homologous genes.
Figure S2. Amino acid sequence alignment of tomato CCS52 homologs. Tomato CCS52 homologous genes were isolated from the websitewww.solgenomics.net. Alignment of amino acid sequences was performed using online multiple sequence alignment program Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).
Figure S3. Subcellular protein localization. Fully expanded leaves of N. benthamiana were infiltrated with A. tumefaciens strain GV3101 containing different constructs with 35S promoter-driven GFP or SlCCS52genes fused with GFP.
Figure S4. Gene expression of APC/C core genes in hypocotyls at different growth stages after seed germinated. Hypocotyls of tomato cultivar ‘Heinz1706’ grown on agar with MS salts were harvested at day 1, 3, 5 and 7 (1d, 3d, 5d and 7d) after seeds germinated. qRT-PCR analysis of SlAPC2 and SlAPC11 in hypocotyls using SlCBL1 as a reference gene. The transcript abundance of each gene at day 1 was used for all comparisons to calculate the relative expression. Data shown are means ± SE calculated from three independent experiments. Letters on the top of each column indicate statistically significant differences (p < 0.05) determined by least significance difference test (LSD) in DPS software.
Figure S5. White light represses hypocotyl elongation through the suppression of endoreduplication. Tomato seedlings of cultivar ‘Heinz 1706’ were grown on MS medium in the dark (A-D) and white light (E-H) conditions. (A, E) Morphology of tomato seedlings. (B, F) Hypocotyl length. (C, G) Diameter in the middle of hypocotyl. Data represents means ± SE (n > 15). Different letters indicate statistical significance (p < 0.05) determined by LSD test in DPS Software. (D, H) Nuclear DNA distribution in hypocotyls. Data are presented by means ± SD. EI values are on the top of columns.
Figure S6. Gene expression ofSlUVI4 and SlCCS52s in hypocotyls at different growth stages in the dark and white light condition. Transcript abundance ofSlUVI4 (A), SlCCS52B (B), SlCCS52A1 (C), andSlCCS52A2 (D) in hypocotyls was analyzed by qRT-PCR. SlCBL1 transcript abundance was used for data normalization. Data shown are means ± SE calculated from three independent experiments.
Figure S7. Heat promotes the endoreduplication to improve hypocotyl elongation of different tomato cultivars. Two tomato cultivars ‘Alisa Craig’ (A-C) and ‘Moneymaker’ (D-F) were grown on NMS and MS agar plates at 25°C and 33°C for days. (A, D) Hypocotyl length. (B, E) Diameter in the middle of hypocotyl. Data represents means ± SE (n > 15). Different letters indicate statistical significance (p < 0.05) determined by LSD test in Data Processing System (DPS) Software. (C, F) Nuclear DNA ploidy distribution in hypocotyls. Data represents means ± SD. EI values are on the top of columns.
Figure S8. Exogenous application of plant hormones affects hypocotyl elongation. Tomato seedlings of cultivar ‘Heinz 1706’ were grown on MS medium supplemented with different concentration of plant hormones IAA (A, B), BR (C, D), GA3 (E, F) and ACC (G, H). (A, C, E, G) Hypocotyl length. (B, D, F, H) Diameter in the middle of hypocotyls. Data represents means ± SE (n > 15). Different letters indicate statistical significance (p < 0.05) determined by LSD test in DPS Software.
Figure S9. Deletion mutations ofSlUVI4 generated using Crispr-Cas9 technology. (A) Target sites of the guide RNAs in Crispr-Cas9 gene editing system. (B) DNA sequence information of SlUVI4 cr-1 and cr-2 mutations. Nucleotides in blue are target sequences. The red hidden lines indicate missing nucleotides. Black lines indicate the protospacer-adjacent motif (PAM).
Figure S10. Overexpression ofSlCCS52A2 inhibits tomato hypocotyl elongation. (A) Morphology of 7-day seedlings grown on MS medium. (B) Hypocotyl length. (C) Diameter in the middle of hypocotyl. Data represents means ± SE (n > 15). Different letters indicate statistical significance (p< 0.05) determined by LSD test in DPS Software. (D) Nuclear DNA ploidy distribution in hypocotyls. Data are presented by means ± SD. EI values are on the top of columns.
Figure S11. A possible model forSlUVI4 and SlCCS52B coordinated hypocotyl elongation under normal and stress conditions. Under normal growth condition, light represses SlUVI4 andSlCCS52B coordinated endoreduplication to inhibit hypocotyl elongation. MS salts and sugar promote SlUVI4 and SlCCS52Bcoordinated endoreduplication to enhance hypocotyl elongation. Under stress condition, salt triggers ethylene production which repressesSlUVI4 and SlCCS52B coordinated endoreduplication to inhibit hypocotyl elongation, while ethylene itself promotes endoreduplication via unknown cell cycle machinery. Heat-stimulated auxin production and auxin-mediated ethylene production reorchestrateSlUVI4 and SlCCS52B coordinated endoreduplication to promote hypocotyl elongation.