Supplementary Materials for
Characterization of Arabidopsis Mutants Insensitive to High Sugar Concentrations
Methods
Isolation of sucrose-insensitive mutants and analysis of sensitivity to sugars. To isolate sin
mutants, seeds were sown on 1/2 MS solid media containing 438 mM sucrose (15%) and
incubated at 22 ± 1°C under the same photoperiodic conditions as described previously. After
2 weeks, seedlings that had formed a relatively normal shoot with an expanded cotyledon, true
leaves, and elongated roots were transplanted to soil and grown to maturity. Seeds were
harvested from these putative mutants and rescreened to distinguish between wild type plants
that showed somaclonal variation and true mutants.
Chlorophyll and anthocyanin assays. Chlorophyll and anthocyanin level were measured
using the procedures described previously by Laby (Laby et al., 2000).
Analysis of mannose-sensitivity for sin and other mutants. To assess other type of sugar
sensitivity, sin and other sugar-insensitive (aba2, aba4, gin2, and sis1) mutant seeds were
sterilized and sown on 1/2 MS media plates containing 1.7 mM mannose and grown under
approximately 80–100 μEm-2sec-1 fluorescent light with a long day photoperiod of 16 h of light
and 8 h of dark. Typically, 100-150 seeds were scored per assay.
Figure legends
Fig. S1 The sin mutant seedlings were insensitive to a high concentration of exogenous sucrose.
(A) The sin25 mutant was isolated from screening based on seedling establishment at a high
sucrose concentration. All sin mutants showed a high germination rate (B) and normal
development (C) in media containing a very high concentration (430 mM) of sucrose. Seed
germination and shoot growth were assessed on three replica plates; error bars indicate standard
deviations.
Fig. S2 The sin mutant seedlings were insensitive to a high concentration of exogenous sucrose
and glucose like other sugar-insensitive mutants (aba2, aba4, gin2, and sis1), but not mannose.
Seeds of wild-type and sugar mutants were sown on 1/2 MS medium supplemented with 300
mM sucrose, glucose, sorbitol and 1.7 mM mannose, and incubated at 22 °C under continuous
light. Seed germination (A to D) and shoot development (E to H) were measured at 14 days
after incubation, respectively. Germination was defined as the emergence of the radicle from
the seed coat. Data represent the means of five independent assays ± SD.
Fig. S3 Sensitivity of wild type and sugar mutants to a very high concentration of sucrose (430
mM). Seeds of wild-type and sugar mutants were sown on 1/2 MS medium supplemented with
430 mM sucrose and incubated at 22°C under continuous light. Seed germination (A) and leaf
development (B) were measured at 3 days and 10 days after incubation, respectively.
Germination was defined as the emergence of the radicle from the seed coat. Data represent
the means of five independent assays ± SD.
Fig. S4 Accumulation of chlorophyll and anthocyanin in wild-type and sin mutant seedlings.
(A) For chlorophyll determination, seedlings were grown under continuous light for 2 weeks
on 1/2 MS medium containing either 30 or 150 mM sucrose. (B) To determine anthocyanin
content in the mutants, seedlings were grown under continuous light for 2 weeks on 1/2 MS
medium supplemented with 30 mM sucrose and then for an additional week on medium
containing either 30 or 150 mM sucrose. Anthocyanin levels were scaled relative to values
obtained for Col-0 seedlings grown on 150 mM sucrose. Results are presented as the means of
three independent assays. Error bars indicate standard deviations. FW, fresh weight.
Fig. S5 Phenotypes of sin and other sugar-insensitive mutants on high concentration of NaCl.
Seeds of Col-0, sin25 and other sugar-insensitive (aba2, aba4, gin2, and sis1) mutant lines
were placed on MS medium containing high concentrations of NaCl (200 mM) and allowed to
germinate and grow. Germination yield (A) and leaf size (B) of the wild type and mutants were
analyzed from 2-week-old seedlings grown on the indicated salt medium after germination.
Error bars indicate standard deviations calculated from three independent experiments.
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Fig. S5