2. MATERIALS AND METHODS
3.2. Characterisation of ACC Oxidase Expression during Water
3.2.13. Expression of TR-ACO3 in the Second Fully-expanded Leaves of White
Changes in expression of TR-ACO3 in NPS- and PS-treated Tienshan and Kopu under water-deficit in the second-fully expanded leaves was also undertaken. In this study, again, sqRT-PCR was used from total RNA isolated from leaves harvested from different SWCs. Aliquots of RT-PCR products were separated by a 1% (w/v) agarose gel electrophoresis, blotted into HybondTM-N+ membrane and
hybridised with a DIG-labelled TR-ACO3 probe (Section 3.2.2).
28.5 27.7 22.7 19.8 17.5 14.3 11.9 9.1 8.0 A B ~37 kDa ~57 kDa ~37 kDa ~57 kDa -TR-ACO2 -TR-ACO2 -CBB -CBB -SWC (%) -SWC (%) 29.4 27.9 25.2 18.9 17.8 14.7 11.7 9.0 7.6
Results 1
116 The hybridised TR-ACO3 band can be detected in the second fully-expanded leaves of NPS-treated Tienshan subjected to all water-deficit conditions (Figure 3.26). However, there was no consistent trends of TR-ACO3 expression observed in the leaves harvested from different SWC (to 10.1 %). Some higher expression of TR-ACO3 was observed at 23.2% SWC and 18.7% SWC, but less expression of
TR-ACO3 was observed at 21.5% SWC and 13.8% SWC. However, below 10.1% SWC, there was a consistent increase in expression of TR-ACO3. These patterns of TR-ACO3 expression above 13.8% SWC were consistent with the pattern of
ß-actin expression. Therefore, water deficit treatment to ca. 10.1% SWC did not alter TR-ACO3 expression, but prolonged exposure of Tienshan plants to a water deficit lower than 10.1% SWC did result in the increase of expression of
TR-ACO3.
Exposure of PS-treated Tienshan plants to an early decrease of SWC to 18.7% did not significantly alter the expression of TR-ACO3 in the second fully-expanded leaves. After this point, a decrease in expression of TR-ACO3 was observed as the SWC further declined to ca. 9.3%. When the SWC decreased below ca. 9.3%, there was an increase in the TR-ACO3 detected in leaves of PS-treated Tienshan which was in common with NPS-treated Tienshan. In these samples, expression of ß-actin was also used as internal loading control and approximately similar expression of ß-actin was seen in each sampling point.
Overall these results show that exposure of Tienshan plants to the NPS and the PS treatment to SWCs less than ca. 9.3% resulted in an increase in expression of TR- ACO3 in the second fully-expanded leaves.
Results 1
117 Figure 3.26 Expression of TR-ACO3 in the second fully-expanded leaves of
NPS-treated Tienshan (A) and PS-treated Tienshan (B) revealed by sqRT- PCR.
RT-PCR was performed using RT-generated cDNA templates from total RNA isolated from the apical structures of white clover harvested at different SWCs, as indicated. One round of PCR was performed using gene specific primers for
TR-ACO3 and the products probed with a DIG-labelled TR-ACO3 probe (upper panels). Equal loading of cDNA was assessed by RT-PCR using degenerate primers to amplify ß-actin from the same cDNA pool. RT-PCR products was separated by electrophoresis and visualised following ethidium bromide staining (lower panels).
TR-ACO3 expression, using the sqRT-PCR approach as described previously, was also observed in the second fully-expanded leaves of Kopu exposed to NPS and PS treatments (Figure 3.27). In the NPS-treated Kopu, a similar level of TR-ACO3
expression was observed in leaves harvested from all sampling points regardless of the SWC. In these samples, approximately the same expression of ß-actin was seen in most samples although slightly less expression was seen in leaves from plants exposed to ca. 8% SWC indicating that probably less mRNA was used for the RT-PCR from leaves at ca. 8% SWC. These results suggested that there was no change in the expression of TR-ACO3 in the second fully-expanded leaves of NPS-treated Kopu exposed water deficit above 8% SWC. However a water deficit
29.6 27.4 23.2 22.1 21.5 18.7 15.6 14.7 13.8 10.1 7.9 6.6 5.9 A B 29.4 25.6 23.9 21.9 21.6 18.7 16.8 14.4 12.0 9.7 9.3 7.9 5.9 -TR-ACO3 -TR-ACO3 - ß-actin - ß-actin - SWC (%) - SWC (%) ~320 bp ~320 bp ~500 bp ~500 bp
Results 1
118 at 8% SWC induced expression of TR-ACO3. In common with NPS-treated Kopu, exposure of Kopu plants to PS treatments to SWC above ca. 7.6% did not alter the expression of TR-ACO3. However, exposure of Kopu plants to a water deficit to
ca. 7.6%resulted in increase expression of TR-ACO. Using ß-actin expression as an internal loading control, a similar expression of ß-actin was seen in most of these sampling point, except for less ß-actin expression at ca. 7.6%SWC.
Figure 3.27 Expression of TR-ACO3 in the second-fully expanded leaves of
NPS-treated Kopu (A) and PS-treated Kopu (B) revealed using sqRT-PCR. RT-PCR was performed using RT-generated cDNA templates from total RNA isolated from the apical structures of white clover harvested at different SWCs, as indicated. One round of PCR was performed using gene specific primers for
TR-ACO3 and the products probed with a DIG-labelled TR-ACO3 probe (upper panels). Equal loading of cDNA was assessed by RT-PCR using degenerate primers to amplify ß-actin from the same cDNA pool. RT-PCR products was separated by electrophoresis and visualised following ethidium bromide staining (lower panels).
Results of expression analysis of TR-ACO3 in the second fully-expanded leaves of NPS and PS Tienshan and Kopu suggested that exposure of both Tienshan and Kopu plants to water deficit of less than 9% SWC did not alter the expression of
29.4 27.9 25.2 18.9 17.8 14.7 11.7 9.0 7.6 28.5 27.7 22.7 19.8 17.5 14.3 11.9 9.1 8.0 A B -TR-ACO3 - ß-actin - ß-actin -TR-ACO3 - SWC (%) - SWC (%) ~320 bp ~320 bp ~500 bp ~500 bp
Results 1
119
TR-ACO3 in the second fully-expanded leaves, but prolonged water deficit treatments to less than 9% SWC induced the expression of TR-ACO3 in the second fully-expanded leaves of Tienshan and Kopu plants.
Results 2
120