Figure S1. Genomic PCR of in vitro potato plants transformed with StPTB1prom
(top) and StPTB6prom (bottom) constructs using nptII-specific primers. Thirty
transgenic events from each construct able to root on kanamycin media were
screened using PCR to identify stably transformed lines. Positives were further
propagated and screened using X-gluc staining. (-) represents a negative
control, using non-transgenic WT genomic DNA as template; (+) is a positive
control using the original proStPTB1:GUS binary vector as template.
Line 12
Construct
Total #
lines
# with X-gluc
staining
Used for
analysis
StPTB1prom
22
3
3
StPTB6prom
15
7
3
Line 22
Line 26
Line 2
Line 16
Line 22
Figure S2. Screen of StPTB1prom (left panels) and StPTB6prom (right panels) transgenic lines
using 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) staining of whole in vitro plants. Lines
positive for PCR were screened by staining with X-gluc for 16 hr to determine relative
consistency and intensity of GUS staining across lines of the same construct. Plants were grown
two weeks in vitro prior to staining and were evaluated in triplicate. The six representative lines
chosen for further analyses are shown here.
Table S1. Promoter structure comparison of PTB genes from Arabidopsis (AtPTB3), potato (StPTB1 and -6), tobacco
(Tobacco1 and -2) and tomato (Tomato1 and -2). Promoter structures were constructed by aligning full-length mRNA
sequence with genomic sequence spanning approximately 1.8 kb upstream of the transcriptional start site (Upstream
region) to the start codon of the coding sequence of each gene (5´ UTR). All genes analyzed here exhibited intronic
sequence (Intron) within their 5´ UTRs. mRNA sequences of potato genes were generated using conventional cloning
methods, whereas sequences from other species are derived from the TAIR database (AtPTB3; accession number
AT1G43190) or the Sol Genomics Network (Tomato1 and -2; locus names Solyc12g019750 and Solyc10g080720,
respectively, Tobacco1 and -2; locus names Niben.v0.3.Scf24895145 and Niben.v0.3.Scf25190834, respectively).
Values are shown in bp.
Gene
Upstream region
5´ UTR
Intron
5´ UTR
AtPTB3
1,876
122
309
8
StPTB1
1,817
40
656
6
Tomato1
1,890
46
645
6
Tobacco1
1,832
78
650
7
StPTB6
1,868
64
1,654
7
Tomato2
1,894
36
1,571
7
Tobacco2
1,885
34
1,614
7
Table S2. Identification of conserved cis-regulatory elements within potato (StPTB1 and 6) and tomato (Tomato 1 and 2) PTB gene
promoters using Plant-CARE. Elements identified in each potato promoter were mapped and compared to their tomato ortholog to
determine genetic conservation of each element. Conservation was scored by a tomato element being within 200 bp of a potato
element of the same type. Percent conservation was calculated by the number of conserved elements and total elements within each
potato promoter. The lengths of the upstream sequence analyzed were 2,519 nt for StPTB1; 2,587 nt for Tomato1 (PTB1-like); 3,593
nt for StPTB6; and 3,508 nt for Tomato2 (PTB6-like).
Name
Box I
Box IV
GTI
GAG-motif
GA-motif
CATT-motif
I-box
G-box
TCT-motif
ERE
ABRE
GARE
TCA-element
TC-rich
W-box
ARE
HSE
MBS
No. in promoter
StPTB1
Tomato1
3
4
3
4
4
1
1
0
0
0
0
0
1
0
1
1
2
1
2
2
1
1
1
1
4
1
4
1
0
0
2
3
3
4
2
3
% conserved
33
33
0
0
0
0
0
0
50
50
0
100
25
25
0
100
67
100
No. in promoter
StPTB6
Tomato2
3
7
5
2
4
5
1
1
1
3
1
1
6
5
3
2
5
3
1
1
1
1
2
1
4
3
2
2
6
8
2
3
4
3
6
5
% conserved
Sequence
Factor
33
20
50
100
100
100
33
33
20
0
100
50
50
50
100
50
50
50
TTTCAAA
ATTAAT
GGTTAA
GGAGATG
ATAGATAA
GCATTC
ATGATATGA
CACGAC
TCTTAC
ATTTCAAA
GCCACGTACA
AAACAGA
CCATCTTTTT
ATTTTCTTCA
TTGACC
TGGTTT
AAAAAATTTC
TAACTG
Light
Light
Light
Light
Light
Light
Light
Light
Light
Ethylene
ABA
GA
GA
Defense
Defense
Aerobic
Heat
Drought
TSSR1.StPTB6
TSSR1.Tomato2
TSSR.patatin
CTTTTAAGGAAA---AAGAATAATTA-ATAAA-GTACAA-AGAA------------AAGAAAAAAG
CTTTTAAAGAAA---AAGAATAATTA-ATAAGTGTAGGA-AAAATATC-----ATTTTAAGAAAAT
GTGCTAAACAATTTCAAGTCTCATCACACATATATATTATATAATACTAATAAAGAATAGAAAAAG
* *** **
*** * ** * * *
** * * **
****
TSSR2.StPTB6
TSSR2.Tomato2
TSSR.patatin
GTTGACT-GAAATTTAATCTCTACTAAAAAAAAAAACTCACTATAATTCAAGT-TAGAATTGAAAAAG
TAAACATATAAATTTAA-GAAAAAAAATATAAAATGTTGATTATAATTCAAGT-TAGAATTGAAAAAG
GTGCTAAACAATTTCAA-GTCTCATCACACATATATATT-ATATAATACTAATAAAGAATAGAAAAAG
** ** **
* * * *
*
****** * * * ***** *******
TSSR3.StPTB6
TSSR3.Tomato2
TSSR.patatin
GTCCTAATCTCTCT---GTGAATTTCCTAATATACCCTGCACAGTTGTGGAAATGAA-ATAAAAAG
GATTTATTCGATAG--GCTCAGAT--GGCGAACAGTCTTCA---TCGCAGTAAAGCA-GGGAAAAG
GTGCTAAACAATTTCAAGTCTCATCACACATATATATTATATAATACTAATAAAGAATAGAAAAAG
*
** * *
*
*
* *
* *
*
** * *
*****
Figure S3. Alignment of conserved TSSR elements within potato and tomato PTB gene promoters. Three functional TSSR
elements have been reported from the analysis of the patatin promoter with locations starting -143, -226 and -930 bp
upstream of the transcriptional start site (Grierson et al. 1994). Similarly, three TSSR elements were discovered within the
promoters of StPTB6, at locations -192, -291 and -992 bp, and its tomato ortholog, Tomato2, at locations -218, -291 and 1,003 bp. Each site was numbered based on its relative location to the transcriptional start site and aligned with the
proximal -226 bp TSSR element from pat-1. The boxed sequences on the left and right correspond to cis-elements known
to confer tuber-specificity and sucrose responsiveness, respectively, with the intervening bolded sequence designating a
third element responsible for transcriptional repression in other organs. Asterisks indicate positions of identity match among
all three sequences.
1.8
700
StPTB1
% GUS activity
600
500
1.2
400
300
*
*
0.6
200
100
0
0
Dark 0h Dark 24h Light 24h Heat 24h Cold 24h
Dark 24h Light 24h Heat 24h Cold 24h
700
StPTB6
% GUS activity
600
1.8
*
500
400
*
*
1.2
*
300
*
200
0.6
100
0
0
Dark 24h Light 24h Heat 24h Cold 24h
MUG assay
Dark 0h Dark 24h Light 24h Heat 24h Cold 24h
RT-PCR
Figure S4. MUG assay for PTB promoter activity in transgenic lines and qRT-PCR for PTB1 and -6 transcript levels for WT plants under the same
environmental conditions for light, heat, and cold treatments for 24 h. Control levels were measured before treatment (0h) or after 24h of
darkness. The same leaflet assay described in the legend of Figure 7 was used. Terminal leaflets of wild-type plants were harvested for RNA
extraction and One-step RT-PCR (Invitrogen) was performed with 90 ng RNA and 30 and 32 cycles for StPTB1 and -6, respectively, using the
primer pairs 5´-CTCCCTTTCCTCCATTTCTGAAG-3´ (forward) and 5´-CGTAAACATTCCTCCCCCTTATGC-3´ (reverse) for StPTB1 and 5´CTGGTCTTTCAAGCCCTAATTCTC-3´ (forward) and 5´-CACTTCTCCCCCTTATGCTG-3´ (reverse) for StPTB6. PCR products were quantified using
ImageJ (Abramoff et al., 2004) and normalized using 18S rRNA values (y-axis). Relative values are shown. Standard errors of the means of three
biological replicates are shown with asterisks indicating significant differences (p<0.05) from comparable controls using a student’s t-test.
20 d drought
StPTB6prom
20 d watered
StPTB6prom
Figure S5. Effect of drought on plant growth of a StPTB6prom transgenic line. For the drought treatment (Fig. 7), whole
plants were not watered for 20 days before harvesting undamaged terminal leaves. Control plants were watered every
three days. Plants were grown in growth chambers (Percival Scientific) set to a 16-h-light (fluence rate of 300 µmol m-2 s-1)
and 8-h-dark cycle with a 22 °C day temperature and a 18 °C night temperature. Both drought-treated WT and StPTB1prom
transgenic lines exhibited similar phenotypes.