Figure 4.

Molecular Analysis of Transgenic Hybrid Aspen Lines with Altered Auxin Signaling.

(A) Amino acid sequence of mutated Ptt IAA3m protein. In the mutated Ptt IAA3m cDNA, the codon encoding the conserved Pro residue (highlighted in gray) was changed to a codon encoding a Ser residue.

(B) Transcript levels of the mutated version of Ptt IAA3 cDNA (Ptt IAA3m) under the control of the 35S CaMV promoter in three independent transgenic lines (Ptt IAA3m) and the wild type by RT-PCR. A total of three replicates were used for each RT-PCR experiment. Bars shows averages (n = 3) of the ratio of the intensity of the fragment of interest relative to that of 18S. Error bars represent sd.

(C) Comparison of the height of transformed and wild-type plants.

(D) Transgenic hybrid aspen plants expressing mutated Ptt IAA3 cDNA display auxin-resistant root growth. Root growth of wild-type hybrid aspen shoots (T89) was compared with that of three independent transgenic lines (35S:PttIAA3m) expressing mutated Ptt IAA3 cDNA (lines 6, 12, and 15) on a medium supplemented with 0.5 μM IBA.

(E) and (F) Auxin inducibility of Ptt IAA6 and Ptt PIN1 transcripts in wild-type (T89) and 35S:Ptt IAA3m transgenic trees. Stem tissues of T89 and three independent transgenic lines were depleted of auxin for 16 h before the addition of auxin. Ptt IAA6 and Ptt PIN1 are auxin-inducible in wild-type plants, but their auxin induction is reduced in the three independent lines expressing Ptt IAA3m cDNA. The difference in transcript levels between the wild type and the Ptt IAA3m lines was significant (P < 0.01) according to t tests.