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.

Construct Total # lines

# with X-gluc staining

Used for analysis

StPTB1prom 22 3 3

StPTB6prom 15 7 3

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.

Line 12 Line 22 Line 26 Line 2 Line 16 Line 22

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 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.

Name No. in promoter % conserved No. in promoter % conserved Sequence FactorStPTB1 Tomato1 StPTB6 Tomato2

Box I 3 4 33 3 7 33 TTTCAAA LightBox IV 3 4 33 5 2 20 ATTAAT Light

GTI 4 1 0 4 5 50 GGTTAA LightGAG-motif 1 0 0 1 1 100 GGAGATG LightGA-motif 0 0 0 1 3 100 ATAGATAA Light

CATT-motif 0 0 0 1 1 100 GCATTC LightI-box 1 0 0 6 5 33 ATGATATGA LightG-box 1 1 0 3 2 33 CACGAC Light

TCT-motif 2 1 50 5 3 20 TCTTAC LightERE 2 2 50 1 1 0 ATTTCAAA Ethylene

ABRE 1 1 0 1 1 100 GCCACGTACA ABAGARE 1 1 100 2 1 50 AAACAGA GA

TCA-element 4 1 25 4 3 50 CCATCTTTTT GATC-rich 4 1 25 2 2 50 ATTTTCTTCA DefenseW-box 0 0 0 6 8 100 TTGACC Defense

ARE 2 3 100 2 3 50 TGGTTT AerobicHSE 3 4 67 4 3 50 AAAAAATTTC HeatMBS 2 3 100 6 5 50 TAACTG Drought

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).

TSSR1.StPTB6 CTTTTAAGGAAA---AAGAATAATTA-ATAAA-GTACAA-AGAA------------AAGAAAAAAGTSSR1.Tomato2 CTTTTAAAGAAA---AAGAATAATTA-ATAAGTGTAGGA-AAAATATC-----ATTTTAAGAAAATTSSR.patatin GTGCTAAACAATTTCAAGTCTCATCACACATATATATTATATAATACTAATAAAGAATAGAAAAAG * *** ** *** * ** * * * ** * * ** **** TSSR2.StPTB6 GTTGACT-GAAATTTAATCTCTACTAAAAAAAAAAACTCACTATAATTCAAGT-TAGAATTGAAAAAGTSSR2.Tomato2 TAAACATATAAATTTAA-GAAAAAAAATATAAAATGTTGATTATAATTCAAGT-TAGAATTGAAAAAGTSSR.patatin GTGCTAAACAATTTCAA-GTCTCATCACACATATATATT-ATATAATACTAATAAAGAATAGAAAAAG ** ** ** * * * * * ****** * * * ***** *******

TSSR3.StPTB6 GTCCTAATCTCTCT---GTGAATTTCCTAATATACCCTGCACAGTTGTGGAAATGAA-ATAAAAAGTSSR3.Tomato2 GATTTATTCGATAG--GCTCAGAT--GGCGAACAGTCTTCA---TCGCAGTAAAGCA-GGGAAAAGTSSR.patatin 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.

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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 wateredStPTB6prom

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.