Western blot analysis was used to examine the patterns of TR-ACO2 protein accumulation during NPS and PS treatments, in both the Tienshan ecotype and the Kopu cultivar. As an initial attempt at the protein accumulation study, 10 g aliquots of total crude protein were used in both NPS and PS Kopu and Tienshan. However, both varieties seemed to have a different TR-ACO2 abundance and therefore to be able to observe linear changes the amount of total protein loaded was altered to 5 g in the Tienshan ecotype and 7 g in the Kopu cultivar.
Aliquots of total protein, extracted from the first fully-expanded leaves harvested from different SWCs were separated by SDS-PAGE, transferred to PDVF membrane and TR-ACO2 protein detected with the anti-TR-ACO2 antibody. The anti-TR-ACO2 antibody, used in the experiments reported in this thesis, was raised against recombinant TR-ACO2 protein from white clover genotype 10-F of cultivar Grassland Challenge, reported by Hunter et al. (1999). In both varieties, the TR-ACO2 antibody recognised a protein band of ca. 37 kDa which is in the range reported from the cultivar Grassland Challenge and other plant species (36 to 41 kDa).
Accumulation of TR-ACO2 proteins in the first fully-expanded leaves of NPS- and PS-treated Tienshan are shown as Figure 3.17. In NPS-treated Tienshan, a similar level of TR-ACO2 protein abundance was seen in the well-watered plants and plants subjected to water deficit to ca. 27.4 % SWC. As the SWC decreased to ca.18.7% (at which stage the PER declined), there was an increased accumulation of TR-ACO2. However, any additional decrease in SWC did not result in a greater accumulation of TR-ACO2 protein but the TR-ACO2 protein
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101 accumulated to a lower level than was observed in the well-watered plants (Figure 3.17.A). The CBB staining of the SDS-PAGE gel of the corresponding proteins used in western blot analysis showed that approximately the same amount of proteins was loaded for each sampling point. These results suggest differential accumulation of TR-ACO2 protein seen in the first fully-expanded leaves of NPS- treated Tienshan is not due to protein loading artefact.
Figure 3.17 TR-ACO2 protein accumulation in the first fully-expanded leaves of NPS-treated Tienshan and PS-treated Tienshan (B) revealed using western analysis.
Aliquots (5 g) of total crude protein extract from apical structures harvested at different SWCs, as indicated, were separated using SDS-PAGE, electroblotted onto PDVF membrane and challenged with anti-TR-ACO1 antibodies. Protein- antibody recognition was determined using the chemiluminescent system following probing of the protein blot with anti-rabbit IgG HRP conjugated secondary antibody and exposure of the blot to an X-ray film (upper panels). CBB staining of the SDS-PAGE gel, corresponding to protein used for the western analysis (lower panels).
A B ~37 kDa ~37 kDa -CBB -CBB -SWC (%) -SWC (%) ~57 kDa -TR-ACO2 -TR-ACO2 ~57 kDa
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102 A similar trend in TR-ACO2 accumulation, in common with NPS-treated Tienshan, was also observed in PS-treated Tienshan. A similar level of TR-ACO2 protein abundance was detected in the fully-hydrated first fully-expanded leaves of PS-treated Tienshan plants subjected to early stages of water deficit treatment (to ca. 23.9 % SWC). After this, the TR-ACO2 protein accumulation increased as SWC decreased to ca. 18.7% (at which point the PER also declined). In common with NPS-treated Tienshan, accumulation of TR-ACO2 protein decreased following the further reduction in SWC to ca. 5.9% (at which point the PER had ceased) (Figure 3.17.B). The CBB staining of the SDS-PAGE gel of corresponding samples of PS-treated Tienshan showed that approximately the same amounts of proteins were loaded in each sampling point, and therefore alteration of TR-ACO2 protein accumulation was not due to uneven loading of protein.
The changes in TR-ACO2 protein accumulation were also examined in the first fully-expanded leaves of Kopu subjected to NPS and PS treatments (Figure 3.18). In the NPS-treated Kopu, there was a similar level of TR-ACO2 protein accumulation detected in the leaves of well-watered plants and plants subjected to the early stages of water deficit (to ca. 17.8 % SWC, at which point the PER declined). After this, TR-ACO2 protein accumulation increased as the SWC decreased to ca. 8% (at which point the PER ceased). CBB staining of an SDS- PAGE gel of corresponding samples confirmed that similar amounts of protein were loaded for each sample (Figure 3.18.A). In the PS-treated Kopu, accumulation of TR-ACO2 protein increased in the first-fully expanded leaves harvested from plants subjected to the early stages of water deficit (to ca. 17.8%, at which point the PER significantly decreased). After this, a decrease in the TR- ACO2 protein abundance was observed in leaves exposed to a further decrease in SWC (to ca. 7.6 % SWC, at which point the PER ceased). Again CBB staining of an SDS-PAGE gel of corresponding samples showed that approximately the same amount of protein was loaded.
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103 Figure 3.18 TR-ACO2 protein accumulation in the first-fully expanded leaves of NPS-treated Kopu (A) and PS-treated Kopu (B) revealed using western blot.
Aliquots (7 g) of total crude protein extract from apical structures harvested at different SWCs, as indicated, were separated using SDS-PAGE, electroblotted onto PDVF membrane and challenged with anti-TR-ACO2 antibodies. Protein- antibody recognition was determined using the chemiluminescent system following probing of the protein blot with anti-rabbit IgG HRP conjugated secondary antibody and exposure of the blot to an X-ray film (upper panels). CBB staining of the SDS-PAGE gel, corresponding to protein used for the western analysis (lower panels).
3.2.9. Expression of TR-ACO3 Transcripts in the First Fully-Expanded Leaves Subjected to a Water Deficit
To investigate if water deficit induced the expression of TR-ACO3, sqRT-PCR as described previously (Section 3.2.2.) was used. RT-PCR was performed from total RNA isolated from the first fully-expanded leaves harvested from white clover plants experiencing different SWCs. From previous studies (Hunter et al., 1999), expression of TR-ACO3 was predicted to be low in mature green tissues and therefore more PCR cycles were used to amplify the TR-ACO3 gene (Section A B ~37 kDa ~37 kDa -CBB -CBB kDa -SWC (%) -SWC (%) ~57 kDa ~57 kDa -TR-ACO2 -TR-ACO2
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104 3.2.2.). Aliquots of PCR products were separated by 1% (w/v) agarose gel eletrophoresis, blotted onto HybondTM-N+ membrane and probed with a DIG-
labelled TR-ACO3 probe, before being exposed to X-ray film for visualisation. In the first fully-expanded leaves of NPS-treated Tienshan, a blot probed with a DIG-labelled TR-ACO3 probe showed hybridisation to an approximately 320 bp band. The hybridisation was detectable at all sampling points and similar expression of TR-ACO3 was seen in leaves harvested from full–hydrated leaves (ca. 29.6 % SWC) and leaves exposed to a water deficit to ca. 5.9% SWC. Slightly lower expression of TR-ACO3 was seen in some samples i.e. at ca. 23.2 %, 21.5 % and 18.7 % SWC, and slightly higher expression was observed in other samples i.e. at ca. 13.8%, 10.1 and 5.9 % SWC. However, overall these changes did not follow a consistent pattern. They were also not due to uneven loading of cDNA in these samples, as indicated from the expression of ß-actin of the same samples (Figure 3.19.A). These results suggested that water deficit treatment did not significantly alter the expression of TR-ACO3 in the first fully-expanded leaves of NPS-treated Tienshan.
A similar pattern of TR-ACO3 expression was also observed in PS-treated Tienshan (Figure 3.19.B). In the first fully-expanded leaves of PS-treated Tienshan, a blot probed with DIG-labelled TR-ACO3 also showed hybridisation to an approximately 320 bp band and there was a similar expression of TR-ACO3
observed in all sampling point regardless of the SWC. In these samples, the expression of ß-actin was again used as an cDNA loading control and a similar expression of ß-actin was seen in all sampling points, suggesting that the similar expression is not due to cDNA loading artefacts. Therefore, both NPS and PS treatments did not significantly induce expression of TR-ACO3 in the first-fully expanded leaves of Tienshan plants when exposed to a water deficit.
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105 Figure 3.19 Expression of TR-ACO3 in the first 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 SWC, 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 ß- actin 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).
SqRT-PCR analysis was also used to examine the expression of TR-ACO3 in the first fully-expanded leaves of NPS- and PS-treated Kopu (Figure 3.20). Blots hybridised with a DIG-labelled TR-ACO3 probe could be detected in the first fully-expanded leaves and these blots showed hybridisation to a ca. 320 bp band. In the first fully-expanded leaves of NPS-treated Kopu plants, a similar level of
TR-ACO3 expression was seen in all sampling point regardless of the SWC (Figure 2.20.A.). In these samples, a similar expression of ß-actin was also observed suggesting that the similar level of TR-ACO3 seen was not due to uneven loading of cDNA. Thus water deficit to ca. 8% SWC (the point at which the PER ceased) did not significantly alter the expression of TR-ACO3 in the first fully-expanded leaves of NPS-treated Kopu. In PS-treated Kopu, in common with the NPS-treated Kopu, a similar level of TR-ACO3 expression was also seen in the
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 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 ~320 bp --actin ~500 bp -TR-ACO3 -SWC (%) ~320 bp ~500 bp -TR-ACO3 --actin -SWC (%)
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106 first fully-expanded leaves harvested from plants grown at all SWCs (Figure 3.20.B). Again, using ß-actin as a loading control of RNA, similar expression was also seen in all samples.
Figure 3.20 Expression of TR-ACO3 in the first fully-expanded leaves of NPS-
treated Kopu (A) and PS-treated Kopu (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
Overall, the results of expression analysis of TR-ACO3 in the first fully-expanded leaves of NPS- and PS-treated Tienshan and Kopu showed that the two water deficit treatments did not significantly induce the expression of TR-ACO3 in the first fully-expanded leaves in either variety.
29.4 27.9 25.2 18.9 17.8 1 4.7 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 --actin -SWC (%) -SWC (%) --actin -TR-ACO3 ~320 bp ~320 bp ~500 bp ~500 bp
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107
3.2.10. Changes in Chlorophyll Concentration in the Second Fully-expanded