Consumo de antibioticospor subgrupos terapéuticos.

As a comparison, five Arabidopsis transgenic 35S:AtAOS were also assessed for their responses to wound treatment. The five transgenic lines were identified as AtAOS-11, AtAOS-13, AtAOS-14, AtAOS-16 and AtAOS-18. The assessment was carried out under similar treatments and conditions as for the transgenic 35S:VvAOS plants. However, for this assessment, qRT-PCR primers were not able to be designed to differentiate between the endogenous and transgene AtAOS. This experiment was designed to investigate the effect of increasing overall AOS transcripts and, therefore, presumably AOS activity in JA the biosynthetic pathway. Transcript abundance of the AtAOS gene in six Arabidopsis 35S:AtAOS transgenic lines is shown in figure 4.6. The results indicated that AtAOS transcript abundance in 35S:AtAOS transgenic plants were exceptionally low compared to the total copy number

101

Figure 4.5 Transcript abundance of VvAOS and AtAOS genes in Arabidopsis 35S:VvAOS transgenic lines

Eight homozygous Arabidopsis 35S:VvAOS transgenic lines, i.e. VvAOS-6, VvAOS-7, VvAOS-8, VvAOS-9, VvAOS- 11, VvAOS-12, VvAOS-14 and VvAOS-15, were screened for their responses to mechanical wounding. Gene transcripts of VvAOS and AtAOS genes were quantified at six hours after wounding. A gene transcript of AtAOS from a wild type was used as a comparison. Bars represent standard deviation of the mean from the three biological replicate samples collected. Letters represent statistical significant differences among the multiple data collected.

102 of AOS transcripts in the 35S:VvAOS transgenic plants. The vast differences in the total level of AOS transcript abundance between 35S:VvAOS transgenic plants (Figure 4.5) and 35S:AtAOS transgenic plants (Figure 4.6) was quite a surprising result considering that both transgenes were regulated by the same gene promoter. Generally, AtAOS gene expression of transgenic 35S:AtAOS lines can be categorized into three groups, i.e. low – AtAOS-13; medium – AtAOS-16, AtAOS-18; and high – AtAOS- 11, AtAOS-14. For further investigation of the effect of AtAOS gene overexpression in the Arabidopsis JA biosynthesis pathway, three transgenic lines were selected based on this primary response result; 1. AtAOS-11 – Highest AtAOS gene expression among the transgenic 35S:AtAOS lines

2. AtAOS-13 – Lowest and most stable AtAOS gene expression among the five transgenic 35S:AtAOS lines

3. AtAOS-18 – Medium and showing more stable gene expression among three biological replicates compared to AtAOS-16 (low SD value)

Figure 4.6 Transcript abundance of AtAOS genes in Arabidopsis 35S:AtAOS transgenic lines

Five homozygous Arabidopsis 35S:AtAOS transgenic lines, i.e. AtAOS-11, AtAOS-13, AtAOS-15, AtAOS-16 and AtAOS-18 and a WT were screened for their responses to wound treatment. AtAOS gene expressions were quantified at six hours after wounding. Bars represent standard deviation of the mean from three biological replicate samples. Letters represent statistical significant differences among the multiple data collected

104

Figure 4.7 Transcript abundance of transgene VvAOS and endogenous AtAOS in three lines of Arabidopsis transgenic 35S:VvAOS plants

Transcript abundance of VvAOS and AtAOS genes were quantified from three selected transgenic 35S:VvAOS plants (VvAOS-6, VvAOS-9 and VvAOS-15) after a six-hour time course of wound treatment via a qRT-PCR approach. Bars represent the standard deviation of the mean from three biological replicate samples.

105

Figure 4.8 Transcript abundance of AtLOX2 and AtVSP2 in three lines of Arabidopsis transgenic 35S:VvAOS plants

Transcript abundance of AtLOX2 and AtVSP2 genes were quantified from three selected transgenic 35S:VvAOS plants (VvAOS-6, VvAOS-9 and VvAOS-15) after a six-hour time course of wound treatment via a qRT-PCR approach. Bars represent the standard deviation of the mean from three biological replicate samples.

106 By comparison, expression of the AtAOS gene in each transgenic line pre-wounding was at a comparable level to AtAOS expression in the Arabidopsis wild type (WT). After wound treatment, AtAOS expression in all transgenic lines increased and peaked at one hour but exhibited different expression magnitudes before declining to near pre-wound condition levels six hours after wounding. AtAOS gene transcripts in transgenic VvAOS-6 increased from 0.2 to 4.3 x 107_{, from 0.2 to 4.3 x 10}7 _in

VvAOS-9 and 0.4 to 6.5 x 107 _{in VvAOS-15 copies in 4 µL of cDNA samples synthesized from 500 ng total}

RNA. In comparison, AtAOS expression in the WT increased and peaked at three hours, during which gene transcripts increased from 0.7 to 7.6 x 107 _{copies in 4 µL of cDNA samples synthesized from 500}

ng total RNA before declining to near pre-wound conditions six hours after wounding. An interesting observation worth mentioning from this comparison was that the time of AtAOS expression in 35S:VvAOS transgenic plants declined two hours earlier compared to AtAOS expression in WT might suggest an early suppression on endogenous AOS (Figure 3.7B).

Both JA-responsive genes, AtLOX2 and AtVSP2 in all three transgenic lines showed increased gene expression as a response to wound treatment (Figure 4.8). Interestingly, at the pre-wound condition, both JA-responsive genes in 35S:VvAOS transgenic lines exhibited approximately similar levels to their respective genes' expression in the WT (Figures 4.8A and 4.8B). This was a clear indication that high levels of VvAOS gene expression in pre-wound conditions did not elevate the transcription of JA-responsive genes. Generally, over the course of the six-hour wound treatment, AtLOX2 gene expression exhibited in all transgenic 35S:VvAOS lines were below to the transcript level in WT. This was a surprising result because we expected that AtLOX2 gene expression in the transgenic lines will be higher than the expression in WT because of the high turnover of overall AOS gene transcripts in transgenic lines. Whereas, in AtVSP2, the transcript level only showed an increase six hours after wounding and exhibited the highest expression level of VvAOS-15. Again, our expectation was AtVSP2 gene expression will be much higher in the transgenic lines compared to WT, as reported by Park et al (2002). Although AtVSP2 transcript abundance in transgenic VvAOS-15 was higher compared to the WT (only a 0.5-fold difference), this result alone was not strong enough to conclude that AtVSP2 expression in transgenic plant was significantly high compared to the gene expression in WT. Furthermore, AtVSP2 transcript abundances of VvAOS-6 and VvAOS-9 were below the transcript abundance in WT.