Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Maps of genomic structure, T-DNA insertion and DNA constructs for GUS, RNAi and GFP. (A) Genomic organisation of LHT1 (At5g40780) and location of T-DNA insertions. Exons are indicated as black boxes. Positions of T-DNA insertion were verified by sequencing. (B) Promoter LHT1-GUS construct. 2740 bp upstream of the ATG, followed by the first exon, the first intron and 28 bp of second exon and a linker sequence were translationally fused to the uid A gene. (C) P_35S-LHT1-RNAi construct. CaMV 35 S promoter was fused with 374 bp of the open reading frame of LHT1 (base 49-422) in sense orientation followed the second intron of AAP6 (At5g49630) and the 374bp in antisense orientation. (D) P_35S-LHT1-GFP construct. CaMV 35S promoter was fused with the coding region of LHT1 followed by a linker sequence and a translational fusion to the GFP5 sequence and the rbcs terminator.
• Supplemental Figure 2 - Verification of T-DNA insertion and reduced RNA-level in heterozygous plants. (A) Plants used for isolation of genomic DNA and RNA were grown in greenhouse and a strong phenotype was visible in the homozygous insertion lineslht1/lht1. (B) Control PCR on genomic DNA isolated from Col 0 and insertion lines with primers on both sides of the insertion (lht1-1) and LB-primer and a downstream primer confirmed that the insertion destroyed the locus. (C) RNA gel blot analysis of the expression of LHT1 in Col 0, heterozygous and homozygous insertion lines. A reduced mRNA level is already detectable in the heterozygous lines. (D) Ribosomal RNA was used as loading control
• Supplemental Figure 3 - Absence of LHT1 transcripts in lht1/lht1 plants compared to Col-0 determined by RT-PCR. (A) Control reaction was performed with actin specific primers (15 cycles). (B) RT PCR with primers amplifying the open reading frame of LHT1 (35 cycles). In the lht1/lht1 plants no mRNA of LHT1 is detectable.