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Figure S1. Genotyping, transcript and protein levels in the ca double mutants. (a)
Homozygous ca2 mutant plants were emasculated and pollinated with pollen from homozygous
cal2 or cal1 mutant plants. T3 plants were grown and leaves were used to extract genomic DNA
and subjected to gPCR using specific primers for both CA2 and CAL2 or CAL1 genes and T-DNA
border primers (LB). Double homozygous plants were identified for ca2cal2 and ca2cal1 double
mutants. (b) Transcript levels of WT and double mutant ca2cal1 plants were assessed by RT-PCR
at 40 cycles using specific primers using ACTINE2 as a housekeeping gene for normalization.
SYBR safe agarose gels were used to assess the presence or not of the entire CA2 and CAL1
transcripts. The ca2cal1 mutant is a double knockout. Different panels of the figures (a) and (b)
come from different gels with molecular markers (M). Sizes are indicated on the right side. (c)
Due to the T-DNA insertion at CAL2 locus causes a knockdown mutation, protein levels were
determined by 2-D gels and Western blot analysis using a CA specific antibody (which recognizes
all 5 CAs, Perales et al., 2005) in a non-lethal cal1cal2 double mutant compared to WT.
Supercomplex I+III2 and complex I lines, and 20-30 kDa zone are shown. CAL1 protein (black
arrow) is not detected and CAL2 protein (black arrow) level is 40% of the WT. The white arrow
indicates all three CA proteins. No detectable CA2 protein in the ca2 mutant was reported
(Perales et al., 2005).