Analysis of T-DNA insertion lines

3.3.2.1 Segregation analysis of T-DNA insertion lines

To examine the T-DNA insertion phenotypes of all four genes of the AtARO gene family, three T-DNA insertion lines with different insertion sites for AtARO1 (aro1-1 to aro1-3) and one insertion line for each AtARO2 (aro2), AtARO3 (aro3) and AtARO4 (aro4) were obtained from the SALK institute (Alonso et al., 2003). Plants were analyzed by PCR for their genetic background as described in section 2.7. For all insertion lines, except aro1-3, homozygous plants could be identified (Fig. 3.12). In the fourth insertion ordered for AtARO1 (aro1-4), the T-DNA integration was shown to be elsewhere than in the AtARO1 gene by PCR and was excluded from further work.

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Legend for Fig. 3.12. AtARO1 to -4 T-DNA insertions. Seeds of corresponding plant lines were obtained from the SALK collection. (A) Genomic structure of AtARO1. The ORF of AtARO1 is encoded by a single exon, containing six complete and three incomplete ARM repeats (red boxes). The positions of T-DNA integrations in lines aro1-1, aro1-2 and aro1-3, determined by sequencing, are indicated by yellow arrowheads. (B-H) Genotypic analysis of T-DNA insertion lines by PCR. Plants from all lines were tested with two gene specific primers flanking the T-DNA insertion (upper panel) as well as one gene and a T-DNA specific primer (lower panel), respectively. WT plants show a signal with gene specific primers only, while in plants homozygous for the T-DNA insertion (marked by asterisks), a signal is solely detected with a combination of gene specific and T-DNA specific primer.

Heterozygous plants show amplification products in both PCR reactions. (B) PCR of progenies of line aro1-1.

Progenies of plants aro1-1 1, aro1-1 4 and aro1-1 6 are homozygous for the insertion, while five descendants of plant aro1-1 2 seem to be heterozygous for the T-DNA insertion. (C) Segregating progenies of line aro1-2. From 9 plants tested, only aro1-2-8 is homozygous. (D) Segregating progenies of line aro1-3. No homozygous progeny could be obtained for the allele aro1-3. 7 of 16 plants are heterozygous. For plant aro1-3 7-2-1 no PCR product was obtained in this PCR reaction. (E) Segregating progenies of line aro2. Three WT, one heterozygous and 11 homozygous plants were identified from 16 analyzed progeny plants of insertion line aro2. (F) Progenies of line aro3. Only homozygous progeny were obtained. (G) Segregating progenies of line aro4. One homozygous plant (aro4-4) could be detected among 16 tested plants.

59 Fig. 3.12 Legend see previous page

The mutant allele aro1-3 (insertion at +791 bp, corresponding to amino acid position 263) segregated in a ration of 1:1.02 (+/+ : aro1-3/+) and no homozygous plant was detected amongst 178 aro1-3 progenies, indicating a gametophytic effect (Fig. 3.12D). Insertion lines aro1-1 (insertion -360 bp 5´

upstream of the start codon) and aro1-2 (insertion -327 bp 5´ upstream of the start codon) in the potential promoter region of AtARO1 on the other hand, did have homozygous progeny (Fig. 3.12B, C). It is therefore very likely that neither the T-DNA insertion of aro1-1 nor of aro1-2 affect the expression of AtARO1 to an extent that reproduction processes are hampered.

3.3.2.2 Phenotypic analysis of T-DNA insertion lines

Heterozygous plants from line aro1-3 as well as homozygous individuals from lines aro1-1, aro1-2, aro2, aro3 and aro4 were examined for their phenotypes during vegetative and generative development. All lines examined showed normal growth, flower development and seed set, indistinguishable from the WT. In Fig. 3.13, phenotypes of a heterozygous aro1-3 (aro1-3/+) plant and homozygous plants of the lines aro2 and aro3 are shown in comparison to WT plants. Closer examination of flowers and siliques revealed normal development and full seed set (Fig. 3.13 B-K).

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Fig. 3.13. Phenotypic analysis of AtARO1 to -3 T-DNA insertion lines in comparison to WT. The plant of line aro1-3/+ is heterozygous, while the plants of lines aro2 and aro3 are homozygous for the T-DNA insertion. (A) All insertion lines grew at same rates and without any obvious changes in overall morphology compared to WT. (B-E) Phenotypic comparison of flowers from the T-DNA insertion lines and WT. All flowers show the same morphology, similar to WT flowers. (B) WT flower. (C) Heterozygous aro1-3/+ flower. (D) Flower of homozygous aro2 plant. (E) Flower of homozygous aro3 plant. (F-K) Phenotypic analysis of opened siliques from the T-DNA insertion lines and WT. All siliques are normally developed and show full seed set. (F) WT silique, showing a full row of developed seeds. (G) Silique from a heterozygous aro1-3/+ line. (H) Silique of homozygous aro2 (J) aro3 silique from a homozygous plant. (K) Siliques of WT and T-DNA insertion lines have the same length. From left to right:

WT, aro1-3/+, aro2, and aro3.

61 3.3.2.3 Promoter analysis of AtARO1

As the T-DNA in aro1-2 is integrated 327 bp upstream of the start codon, all promoter elements important for development- and tissue-specific expression of AtARO1 are very likely to be located downstream of this insertion site. The 711 bp AtARO1 promoter region used for promoter-GUS studies was analyzed in silico using the plant cis-acting regulatory DNA elements database PLACE (Fig.

3.14). The transcription start point (TSP) of the AtARO1 mRNA was predicted to be located 162 bp upstream of the start codon. Several general as well as tissue-specific binding sites for transcription factors and enhancer elements were found mainly up to 480 bp upstream of the start codon. 54 bp upstream of the TSP, a TATA-box and a TATA-like motif were found on the (+) and (-) strands of the promoter sequence, respectively. Besides these general promoter motifs, several putative (C)CAAT-boxes and Initiator elements were found before and after the TSP, but none of them was located at typical distances from the transcription start. (C)CAAT-boxes are usually found about 75 bp upstream of the TSP, while Initiator elements are found at -1 to +6 from the TSP. Some elements for transcriptional activators, namely the ARR1-binding element and a CT-rich motif, which can enhance gene expression, were found throughout the length of the putative promoter sequence and 5´UTR.

Two promoter elements of late pollen specific genes (GTGA-motif and Pollen1 LAT52-motif) were also present in several copies on the (+) and (-) strands of the AtARO1 promoter and 5´ UTR. Further, three copies of a motif for seed-specific expression were found at 218 to 209 bp upstream of the TSP.

Many other binding sites for transcription factors could be identified, like the core site for plant-specific Dof transcription factors (Yanagisawa and Schmidt, 1999) or several Myb transcription factor recognition sites. Additionally, several motifs for stress-induced and light regulated transcriptional activation were found, but for better overview not included in figure 3.14.

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Fig. 3.14. 711 bp promoter fragment and 5´ UTR of AtARO1 displaying some putative cis-acting elements.

Putative cis-acting elements are in colored boxes and each colored box is described beneath. Angled arrow indicates the putative transcription star point (TSP). Yellow arrows indicate the position of the T-DNA insertions of aro1-1 and aro1-2.