Download Kanr T-DNA Supplemental Figure 1. Transgenic complementation of

A
CAC1A-R
CAC1A-KO-5
At5g16390 (CAC1A)
JL-202
B
T-DNA Kanr
CAC1A-KO-5
C
CAC1A-X
RB
CAMBIA-1
35S
Bar
LB
Supplemental Figure 1. Transgenic complementation of the cac1a-1 mutant allele.
The figures show schematic representations of the CAC1A alleles that were tracked in the transgenic
complementation of the cac1a-1 mutation. Exons are represented by blue boxes, introns are
represented by pink boxes, UTRs are represented by white boxes, and intergenic chromosomal
regions are represented by gray lines. Locations of primer-sets used for allele-specific PCR assays
(see Supplemental Figure 2) are indicated with red arrows.
(A) Structure of the native CAC1A allele (At5g16390). The yellow-shaded box represents the
genomic region cloned into vector pCABMIA3300 for genetic complementation (see panel C).
(B) Structure of the cac1a-1 allele showing the position of the T-DNA insertion, which contains the
kanamycin resistance gene (Kanr).
(C) Structure of the ectopic CAC1A allele used to transgenically complement the mutant cac1a-1
allele. Black shaded elements represent genetic elements that were part of the T-DNA inserted at this
locus: 35S = CaMV 35S promoter; Bar = bar gene from Streptomyces hygroscopicus, conferring
resistance to glufosinate; LB = T-DNA left border; RB = T-DNA right border.
Supplemental Figure 2. Representative PCR genotyping results for transgenic
complementation of the cac1a-1 mutant allele.
Heterozygous cac1a-1 mutant plants were transformed with CAC1A genomic fragment
(see panel C of supplemental Figure 1). Twelve T2 plants derived from 3 independent
transgenic events, were genotyped individually to detect the presence of the endogenous
CAC1A (At5g16390) allele (using primers CAC1A-KO-5 and CAC1A-R), the cac1a-1
mutant allele (using primers CAC1A-KO-5 and JL-202), and CAC1A transgenic allele
(using primers CAC1A-X and CAMBIA-1). Position of primers are shown in
supplemental Figure 1.
Supplemental Figure 3. Generation of BCCP1- and BCCP2-specific antibodies.
(A) Alignment of BCCP1 and BCCP2 amino acid sequences generated by the
BESTFIT algorithm of GCG software package (Genetics Computer Group,
Madison, WI). Identical residues are black-shaded and conservative substitutions
are grey-shaded. The boxed sequences indicate the region with low amino acid
similarity between the two proteins. The BCCP1 and BCCP2 cDNAs coding for
this region were PCR amplified and cloned into an expression vector. Using the
expressed peptides, BCCP1- and BCCP2-specific antisera were generated.
Aliquots of protein extracts prepared from Arabidopsis flowers were subjected to
SDS-PAGE and western blot analyses. Identical blots were probed with
streptavidin and anti-BCCP1 antisera (B) or with streptavidin and anti-BCCP2
antisera (C).
Supplemental Figure 4. The CAC1A- and CAC1B-specific probes used
in RNA hybridization experiments. CAC1A and CAC1B sense RNAs
were transcribed from pBluescript SK (+/-) and pSPORT 1 clones,
respectively, and subjected to agarose electrophoresis. Following
northern blot transfer of the RNAs to nitrocellulose membrane, they were
subjected to hybridization with DIG-labeled antisense CAC1A RNA
(positions 451-1114 of the CAC1A cDNA), and DIG-labeled antisense
CAC1B RNA (positions 451-1217 of the CAC1B cDNA).
Supplemental Table I. Characterization of T-DNA tagged alleles of BCCP subunit genes
Gene
Name
Gene locus
Allele
CAC1A
At5g16390
cac1a-1
1st intron, 4nt
deletion
SALK_120571
At5g15530
Characterization
T-DNA border
5'
3'
Embryo lethal
LB
LB
-181, 1nt
deletion
No phenotype, normal
level BCCP1
LB
LB
SALK_023225
-288 to -234,
54nt deletion
No phenotype, normal
level BCCP1
LB
RB
SALK_050082
1st exon
Possible T-DNA
translocation a
Not
found
LB
SALK_138637
Not found
T-DNA not found
Not
found
Not
found
GT_3_38620
Not found
T-DNA not found
Not
found
Not
found
SALK_025081
N/A
NOT
CHARACTERIZED
N/A
N/A
GABI_217E08
N/A
NOT
CHARACTERIZED
N/A
N/A
cac1b-1
(SALK_056228)
3rd intron
No phenotype
Not
found
LB
cac1b-2
(SALK_070569)
1st exon and
intron junction,
64nt deletion
No phenotype
LB
RB
(BCCP1)
CAC1B
Insert Position
(BCCP2)
a
see text for more details.
Supplemental Table II. PCR primers used to characterize CAC1A antisense plants and TDNA tagged alleles for htACCase subunit genes
Primer
Sequence
Purpose
JL-202
5'-CATTTTATAATAACGCTGCGGACATCTAC-3'
T-DNA left border for Wisconsin lines
XR-2
5'-TGGGAAAACCTGGCGTTACCCAACTAAT-3'
T-DNA right border for Wisconsin lines
LB
5'-CGTTCTTTAATAGTGGACTCTTGTTCCAA-3'
T-DNA left border for SALK lines
RB
5'-GCAATAATGGTTTCTGACGTATGTGCTTA-3'
T-DNA right border for SALK lines
inv6-2
5'-GCTAAGCACATACGTCAGAAACCATTATT-3'
transposon left border primer for GT lines
spm31
5'-GCTTGTTGAACCGACACTTTTAACATAAG-3'
transposon right border primer for GT lines
CAC1A-1
5'-GTTGAGAAAAATCAGTTTGCCTCTCTTTT-3'
CAC1A gene 5' primer
CAC1A-KO-5
5'-TACACGATTCCTTCCTCGATTAAGATAAG-3'
CAC1A gene 5' primer
CAC1A-X
5'-TGTTAGGTTTGTTAGTTTGGTGAGGAAGA-3'
CAC1A gene 5' primer
CAC1A-L
5'-CTTTCCGTTATTCTCCGATTACATCTACC-3'
CAC1A gene 5' primer
CAC1A-2
5'-CAAAAAGATTCATGTATCCTCAACATCCT-3'
CAC1A gene 3' primer
CAC1A-3
5'-CTTCAATGAGTTTTTCTCCTTTCCTTCAG-3'
CAC1A gene 3' primer
CAC1A-R
5'-TATTGTAGTCGAAGTGGAACTGATTCTCG-3'
CAC1A gene 3' primer
CAC1B-5
5'-GACTAATGGTGGGTATATGAACGGAAAAG-3'
CAC1B gene 5' primer used for cac1b-1
CAC1B-3
5'-CATTACAGGAGGCATTGAGTGATAAACTG-3'
CAC1B gene 3' primer used for cac1b-1
CAC1B-a
5'-GCGGTTTGGTGAAGTTAGTTAAAAGAGTT-3'
CAC1B gene 5' primer used for cac1b-2
CAC1B-b
5'-GCAGGAGAAGAACAGAACAGAGAATTATG-3'
CAC1B gene 3' primer used for cac1b-2
pC-F
5'-CATCTTATGCCCAGCAAATGGC-3'
CAC1A cDNA 5' primer
pC-R
5'-CAGTGACAACGAAGGGGAAACG-3'
CAC1A cDNA 3' primer
CAMBIA-1
5'-TAACAATTTCACACAGGAAACAGCTATGA-3'
pCAMBIA3300 vector primer
A-F
5'-CCTCAACCTCAAGCTCCT-3'
CAC1A cDNA 5' primer
A-R
5'-AACAGTAGGAAGTGACGATT-3'
CAC1A cDNA 3' primer
B-F
5'-CAGCAAGCTGTACCACCA-3'
CAC1B cDNA 5' primer
B-R
5'-GAGTGGAGGATGAGACGA-3'
CAC1B cDNA 3' primer