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.