Functional analysis of the cis-elements within the EsALDH7B4 promoter in response to

3.2.2.5.1 Generation of EsALDH7B4 promoter deletion lines

To experimentally identify regulatory regions involved in the transcriptional control of the gene expression, promoter deletion analysis was performed. Taking TP0 plasmid that harbors the EsALDH7B4_promoter::GUS-nos_terminator cassette as template, EsALDH7B4 promoter fragments of different lengths were amplified using GUS-Start primer and primers with introduced restriction enzyme sites. Fragments were amplified using the primer GUS-Start combined with primers TsA7pro2 (EcoRI), TsA7pro3 (EcoRI), ThA7pro-3de (SpeI), TsA7pro8 (EcoRI) and TsA7pro6 (SpeI) were digested with XhoI and the corresponding

Fig. 24 Activities of the AtALDH7B4 and EsALDH7B4 promoters under different stress conditions. 6-week-old wild-type (Col-0) A. thaliana and independent transgenic A. thaliana plants harboring AtALDH7B4::GUS and EsALDH7B4::GUS cassettes were subjected to different stresses. Salt stress was given using 250 mM NaCl for 10 days; drought stress was given by withholding watering for 14 days;

dehydration was applied by removing plants from pots and drying them for 16 h; wound stress was given by cutting leaves with scissors or by treating the leaf surface area with abrasive sandpaper; well watered plants served as control. (A) In situ detection of the GUS activity. (B) Salt-, drought-, dehydration-, and wounding-induced activities of A. thaliana and E. salsugineum ALDH7B4 promoters measured as enzymatic activities of the GUS reporter protein.

95 restriction enzymes (EcoRI, EcoRI, SpeI, EcoRI and SpeI, respectively) and then cloned into the pBT10-GUS vector resulting in TP2, TP3, TP4, TP5, TP6 plasmids, respectively (Fig. 25).

The TP7 plasmid (Fig. 25) was generated directly by digesting TP0 plasmid with ClaI and XbaI and ligated to the pBT10-GUS vector digested with the same enzyme.

The alignment of the EsALDH7B4 and AtALDH7B4 promoters showed a 38 bp “TC” rich motif does not exist in A. thaliana but is present in E. salsugineum. To analyze the relevance of the

“TC” motif, the 38 bp “TC” motif was deleted by substituting a single nucleotide adenine “A”.

To delete the “TC” motif from the EsALDH7B4 promoter, fragments upstream and downstream of the “TC” motif were amplified from the TP0 plasmid using primer combinations 5’-pBT10-GUS-fw/ThA7pro-5de and GUS-Start/ThA7pro-3de. These fragments were digested with restriction enzymes EcoRI/SpeI and SpeI/NcoI, respectively.

The digested upstream “TC” fragment was first cloned into the EcoRI/SpeI digested pBT10-GUS vector resulting in plasmid 5LpBT10-pBT10-GUS. The 5LpBT10-pBT10-GUS plasmid was further digested with SpeI/NcoI and ligated with the SpeI/NcoI digested downstream “TC” fragment and finally resulted in the plasmid No“TC”. The schematic representation of all mutated versions of the EsALDH7B4 promoter is shown in Fig. 25.

Fig. 25 Distribution of putative cis-acting regulatory elements within the promoter of EsALDH7B4 and schematic representation of the mutated versions of the EsALDH7B4 promoter region. TP0 denotes the 0.75 kb wild-type promoter fragment. No“TC” denotes the “TC” region deleted TP0. TP2, TP3, TP4, TP5, TP6, and TP7 derived from sequential 5’ end deletions of the wild-type promoter. The deletion positions are shown in Fig. 18B.

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The EsALDH7B4_promoter::GUS-nos_terminator cassette from the plasmids described above were isolated using restriction enzymes EcoRI and BglII for plasmids No“TC”, TP2, TP3, TP4, TP5 and TP6 and HindIII and BglII for plasmid TP7. The isolated cassettes were then sub-cloned into the EcoRI/BamHI or HindIII/BamHI digested binary vector pBIN19 to yield the corresponding E. coli DH10B clones. Plasmids from positive E. coli DH10B clones were transformed into A. tumefaciens cells separately. Recombinant Agrobacterium clones were used to transform wild-type A. thaliana (ecotype Col-0) and wild-type E. salsugineum (ecotype Shandong) plants to generate transgenic plants harboring promoter GUS fusion constructs.

3.2.2.5.2 Characterization of EsALDH7B4 promoter deletion GUS expression lines in response to different stress factors

Due to the difficulty in transforming E. salsugineum, four transgenic E. salsugineum plants were firstly obtained and analyzed (two harboring TP0, one harboring No “TC” and one harboring TP6). The activities of the deleted promoters were compared with the TP0 promoter in transgenic A. thaliana lines to analyze the effect of the deletions. In addition, the barley promoter GUS fusion transgenic lines were also analyzed to compare the monocot

Fig. 26 Activities of the deleted EsALDH7B4 promoters in 2-week-old A. thaliana seedlings. 2-week-old transgenic A. thaliana seedlings grown under control conditions were taken from MS medium and then incubated in GUS staining buffer. Photos were taken at different time points to compare the GUS activity in different lines. TP0, No“TC”, TP2, TP3, TP4, TP5, TP6 and TP7 represent different EsALDH7B4 deletion GUS expression lines (details see Fig. 24). Ba represents the HvALDH7B4 promoter GUS expression line.

97 HvALDH7B4 promoter activity. Three independent T2 generation transgenic plants were analyzed for each promoter GUS deletion line of the transgenic A. thaliana plants. GUS activity was firstly detected in 2-week old seedlings using in situ GUS staining. There was no significant difference in TP0, No“TC”, TP2, TP3 and TP4 except for TP4 that showed a slightly stronger GUS activity than TP3. Lower GUS activity was observed for TP5. GUS activity was nearly abolished in TP6 and no GUS activity was detected in TP7 (Fig. 26). Only very slight GUS activity was detected in HvALDH7B4::GUS fusion lines in 2-week-old seedlings (Fig. 26).

Fig. 27 Activities of the deleted EsALDH7B4 promoters under different stress conditions. 6-week-old wild-type (Col-0) and independent transgenic A. thaliana plants harboring different EsALDH7B4 promoter fragments fused to the GUS reporter gene were subjected to different stresses. Salt stress was given using 250 mM NaCl for 10 days; drought stress was given by withholding watering for 14 days; wound stress was given by cutting leaves with scissors or by treating the leaf surface with abrasive sandpaper; well watered plants served as control. TP0, No“TC”, TP2, TP3, TP4, TP5, TP6 and TP7 represent different EsALDH7B4 deletion GUS expression lines (details see Fig. 24) (A) In situ detection of the GUS activity. (B) Activity of EsALDH7B4 promoter deletion constructs measured as enzymatic activities of the GUS reporter protein under non-stress, salt, drought, and wound stress conditions.

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The deleted promoter activities in response to different stress factors were then compared in 6- week-old transgenic A. thaliana plants. Stress treatments were made as described in “3.2.2.4”.

Consistent with the result of 2-week-old seedlings, GUS activity was detected in the deletion lines TP2 to TP5. Only slight GUS activity was detected in TP6 and no GUS activity was detected in TP7 lines. Higher GUS activity was detected in the TP5 lines in response to stress treatments (Fig. 27A, B). This indicates that the region which was deleted from TP5 plays a crucial role for gene expression and stress responsiveness. The TP3 lines showed a weaker GUS activity than TP4, which is particularly obvious after wounding stress (Fig. 27A).

Quantitative GUS assays confirmed that the TP3 lines have lower GUS activity than TP4 lines in all treatments (Fig. 27B). The No“TC” lines also showed significant lower GUS activity than TP0 lines except for the drought stress. The decreased GUS activity in lines with the deleted “TC” motif was observed in all the treatments by comparing the TP0 and no “TC” lines, and TP3 and TP4 lines.

The same stress treatments had also been applied to the transgenic E. salsugienum lines. In contrast to the results from transgenic A. thaliana lines, the TP6 promoter still shows a high activity in transgenic E. salsugineum. However, only one TP6 transgenic E. salsugienum line

Fig. 28 In situ detection of GUS activity of the deleted EsALDH7B4 promoters in transgenic E.

salsugineum under different stress conditions. 6-week-old wild-type (Shandong) and transgenic E.

salsugineum plants harboring different EsALDH7B4 promoter fragments fused with the GUS reporter gene were subjected to different stresses. Salt stress was given using 250 mM NaCl for 10 days; drought stress was given by withholding watering for 14 days; wound stress was given by cutting leaves with scissors or by treating the leaf surface with abrasive sandpaper; well watered plants served as control. TP0, No“TC” and TP6 represent different EsALDH7B4 deletion GUS expression lines (details see Fig. 24), WT represents the wild-type E. salsugineum plants.

99 was analyzed (Fig. 28). The “TC” motif which showed a repressor effect in transgenic A.

thaliana did not show this effect in E. salsugineum because TP0 and No“TC” lines did not show a difference in the GUS staining. The EsALDH7B4 promoter activity as reflected by GUS expression in all the treatments especially under non-stress conditions was lower in transgenic E. salsugineum than in transgenic A. thaliana.