Expression of TR-ACO1 Transcripts in the Apical Structures of White

The expression of TR-ACO1 during a water deficit in the apical structures of white clover was studied using sqRT-PCR using the probe developed in the specificity and linearity assays (Figure 3.7 and 3.8). Apical structures were harvested daily at different SWCs from the two varieties of white clover plants (Tienshan ecotype and Kopu cultivar) subjected to two different water deficit treatments (see Section 2.2.1).

For the expression of TR-ACO1 in the NPS- and PS-treated Tienshan, the probe hybridised to a single transcript of ca. 320 bp but there were no consistent trends in the patterns of TR-ACO1 expression in the apical structures (Figure 3.10). In the NPS-treated Tienshan (Figure 3.10.A), a similar pattern of TR-ACO1

expression was observed in the fully-hydrated apical structures (at ca. 29.6% SWC) and apical structures harvested at and above ca. 22 % SWC. The expression of TR-ACO1 then decreased slightly in the apical structures harvested from ca. 21.5 and 18.7% SWC, and after this point expression increased again to an approximately similar level as seen in the fully-hydrated state. When SWC fell to below 7%, a slight increase in expression was again observed which was higher than that observed in the fully-hydrated apical structures. Although the trends of expression observed in the apical structures of the NPS-treated Tienshan were not consistent, the overall expression of TR-ACO1 over the decrease in SWC was quite similar, with a possible induction at ca. 6.6 and 5.9 % of SWC. Therefore, these results indicate that there were no major changes in the expression of TR- ACO1 in the apical structures of NPS Tienshan subjected to water deficit to ca.

7.9 % SWC, but there was some induction just before the PER in the first fully- expanded leaves ceases.

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89 To ensure loading of cDNA at each time point, the expression of ß-actin was also examined and used as an internal standard. The ß-actin transcript was amplified by degenerate primers (Section 2.5.5.1) from the same cDNA pool as used for TR- ACO1 expression analysis, the products were separated by 1% (w/v) agarose gel electrophoresis and stained with ethidium bromide (Figure 3.10). Over the SWC values examined, similar ß-actin expression was observed in each lane. Therefore, the differential TR-ACO1 transcript expression patterns seen were not caused by uneven loading of cDNA.

In the PS-treated Tienshan, a similar level of TR-ACO1 expression was detected in fully-hydrated apical structures (at ca. 29.4 % SWC) and in all subsequent harvest points until ca. 14.4% SWC (Figure 3.10.B). The expression of TR-ACO1

then declined as the SWC decreased to ca. 12% and 9.7% SWC, and then increased again to a level similar to that observed in the fully-hydrated plants when the SWC decreased to ca. 7.9% and 5.9%. The decrease in TR-AC01

expression observed in the apical structures at ca. 12% and 9.7% SWC was probably due to uneven loading of cDNA, since the expression of ß-actin at these sampling points had also slightly decreased. It can also be noted that expression of

ß-actin at ca. 5.9% SWC was much less than the level of expression at other SWC points. When any changes in ß-actin expression are taken into account, the expression pattern of TR-ACO1 in PS-treated Tienshan was essentially the same, except for the increase at ca. 5.9% SWC. Therefore, overall there were similar patterns of TR-ACO1 expression in the apical structures of NPS and PS Tienshan.

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Figure 3.10 Expression of TR-ACO1 in the apical structures of NPS-treated

Tienshan (A) and PS-treated Tienshan (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-ACO1 and the products were probed with a DIG-labelled TR-ACO1 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 were separated by electrophoresis and visualised, following ethidium bromide staining (lower panels).

Expression of TR-ACO1 was also examined in the NPS- and PS-treated Kopu plants (Figure 3.11). As can be seen in Figure 3.11, there was differences in TR- ACO1 expression in the apical structures between NPS- and PS-treated Kopu. In the NPS-treated Kopu, similar expression of TR-ACO1 was observed in the fully- hydrated apical structures (at ca. 28.5% SWC) and in the apical structures harvested as the SWC declined to ca. 19.8 %. After this point, expression of TR- ACO1 declined as the SWC decreased. In the PS-treated Kopu, there were no real changes in the expression of TR-ACO1 after the water-deficit imposition. An approximately similar level of TR-ACO1 expression was observed, regardless of the SWC. In these experiments, expression of ß-actin was also used as an internal standard and a similar level of expression was detected in the NPS- and PS-treated A B ~320 bp -ß-actin -ß-actin ~320 bp - SWC (%) - SWC (%) - TR-ACO1 - TR-ACO1 ~500 bp ~500 bp

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91 Kopu, thus confirming that the differences in expression of TR-ACO1 between the NPS- and PS-treated Kopu were not due to uneven loading.

Figure 3.11 Expression of TR-ACO1 in the apical structures 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 SWC, as indicated. One round of PCR was performed using gene-specific primers for

TR-ACO1 and the products probed with a DIG-labelled TR-ACO1 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).

A B - SWC (%) - SWC (%) ~320 bp ~320 bp -ß-actin -ß-actin ~500 bp -TR-ACO1 -TR-ACO1 ~500 bp

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3.2.5 Accumulation of TR-ACO1 Protein in the Apical Structures of White