Supporting Information: Figure Legends
Figure S1. Expression pattern of CBL2 (a-d) and CBL3 (e-h) during Arabidopsis
thaliana development. Promoter:GUS analyses were performed as described in
supporting information materials and methods. After staining, tissues were bleached
with 70 % Ethanol for 24 h at 37 °C. Pictures were recorded with a stereomicroscope.
a&e: 7 day old seedlings, b&f: 14 day old seedlings, c&g: leaves of 5 week old plants,
d&h: flowers of 5 week old plants.
Figure S2. Molecular characterization of cbl2-1 and cbl3 T-DNA insertion lines and
generation of the cbl2/3rf double mutant. (a) Schematic illustration of the T-DNA
insertion in the CBL2 gene. The location of the insertion in the cbl2-1 allele is
depicted by a triangle. Boxes illustrate exons, lines illustrate introns. Arrows indicate
the position of the primers used for PCR and RT-PCR experiments. (b) Transcript
analysis of cbl2-1 mutant by RT-PCR using 40 cycles. (c) Schematic illustration of the
T-DNA insertion in the cbl3 allele. Boxes illustrate exons, lines illustrate introns. The
triangle marks the position of the T-DNA insertion. Arrows indicate the position of the
genomic primers used for PCR and RT-PCR experiments. Bar = 100 bp. (d)
Transcript analysis of cbl3 mutant by RT-PCR using 40 cycles. (e) PCR analysis of
homozygous cbl2/3rf plants. Primer combination for CBL2 WT: CBL2 F + CBL2 R,
primer combination for CBL2 T-DNA: CBL2 F + LB_GABI, primer combination for
CBL3 WT: CBL3 F + CBL3 R, primer combination for CBL3 T-DNA: CBL3 R +
LB_GABI. K=water control (f) Transcript analysis of homozygous cbl2/3rf plants by
RT-PCR using 25 cycles. Primer combinations are as depicted for transcript analysis
(a) and (c). (g) Transcript analysis of cbl2/3rf mutant by quantitative RT-PCR
performed on three batches of individual plants from 7 day old seedlings per line.
ACTIN2 was uses as reference. Indicated transcription values are relative values with
transcription of CBL2 and CBL3 in the wild type sample set to 1 (N=3, error
bars=SEM). (h) Growth comparison of 6 week old Col-0, cbl2-1, cbl3 and cbl2/3rf
plants grown in soil in a 16h light / 8h dark cycle.
Figure S3. Transcript analyses of the cbl2-2 single mutant and generation of the
cbl2/3lf double mutant. (a) Schematic illustration of the T-DNA insertion position in
cbl2-2 allele. Boxes illustrate exons, lines illustrate introns, thin boxes represent
untranslated exons in the 5’-UTR. Bar=100 bp. The triangle marks the position of the
T-DNA insertion. Arrows indicate the position of the primers used for PCR. CBL2 F2
primer was used as forward primer together with CBL2 R to identify homozygous
plants. (b) Transcript analysis of homozygous cbl2-2 mutants using 40 PCR cycles.
Primers CBL2 F + CBL2 R were used for transcript analysis. (c) Transcript analysis
of cbl2/3lf mutant by RT-PCR using 40 PCR cycles. Primer combinations for RT-PCR
were the same as for single mutants. K=water control (d) Growth comparison of 6
week old Col-0, cbl2-2, cbl3 and cbl2/3lf plants grown 6 weeks in soil in a 16h light /
8h dark cycle.
Figure S4. Complementation of cbl2/3rf plants with either CBL2 or CBL3 restores
wild type seed size and seed weight. (a) Growth phenotype of Col-0, cbl2/3rf,
CBL2/cbl2/3rf and CBL3/cbl2/3rf mutants after 6 weeks grown in soil in a 16h light /
8h dark cycle. (b) Quantification of seed square by transmitted light scanning. Values
are mean ± SD (N≥250). (c) Determination of thousand seed weight. Batches of 1000
seeds from individual plants were measured. Values are mean ± SD (N=5).
Figure S5. cbl2/3lf mutant seeds exhibit reduced seed weight and size, while vhaa2/vha-a3 seeds are not affected. (a) Quantification of seed size by transmitted light
scanning. Values are mean ± SD (N≥250) (b) Determination of hundred seed weight.
Batches of 100 seeds from individual plants were measured. Values are mean ± SD
(N=5). (c) Measurement of total fatty acid content in single seeds. Values are mean ±
SD (N≥5, *** P<0.0001 compared to WT) (d) Total fatty acid composition of wild type,
cbl2/3rf and cbl2/3lf double mutants.
Figure S6. Comparison of cbl2/3rf and vha-a2/vha-a3 mutants seed ionic profiles
using ion chromatography. (a) comparison of cation profiles (b) comparison of anion
profiles. Seeds from 5 individual plants for each line were analyzed. Bars = SEM.
Figure S7. (a) Representative pictures of Col-0, cbl2/3rf and cbl2/3lf seedlings 7
days post germination. (b) Schema of seed distribution in the petri dish in this
experiment.
Figure S8. Complete loss of CBL2 and CBL3 function in cbl2/3lf mutants induces an
aberrant embryo development. (a) Rate of normal and misshaped embryos in Col-0,
cbl2/3rf and cbl2/3lf mutants. In total 100 seeds from different plants were analyzed.
(b-d) Normal developed embryos of cbl2/3lf mutants. (b) globular stage, (c) walking
stick stage (d) mature embryo without seed coat. Bars=100 µm.
Figure S9. Expression pattern of CBL2 and CBL3 in Arabidopsis thaliana seeds
during different developmental stages. Seeds were prepared from siliques at different
developmental stages. Promoter:GUS assay was performed as described in
materials and methods. After staining, seeds were incubated for 4 h with
ethanol:acetat 1:1 and bleached over night in a modified Hoyer´s medium. Pictures
were obtained with a stereomicroscope with DIC module at the same magnification.
(a-d) Expression of CBL2 at (a) globular, (b) heart, (c) linear cotyledon and (d)
mature stage. (e-h) Expression of CBL3 at (e) globular, (f) heart, (g) linear cotyledon
and (h) mature stage. Bars=100 µm.
Table S1. Primers used in this study. Restriction sites are underlined.
Method S1.