Examination of changes in water relations, PER, proline accumulation as well as changes in both TR-ACO1 and TR-ACO2 gene expression as well as TR-ACO1 and TR-ACO2 protein accumulation in the leaves of the NPS Tienshan and the NPS Kopu plants have all shown differences in the responses between the more drought tolerant-Tienshan and the more drought sensitive-Kopu plants to water deficit (Section 5.2.3.1 and 5.2.3.2.). Moreover, changes in TR-ACO1 and TR-ACO2 gene expression as well as TR-ACO1 and TR-ACO2 protein accumulation in the younger tissues could also be used as an indicator of differences in the responses of white clover plants to water deficit. Therefore, TR-ACO1 and TR-ACO2 abundance may be used as a marker for the adaptation responses of white clover to water deficit. These differences were observed in both the Tienshan ecotype and the Kopu cultivar subjected to one cycle of water deficit treatment (NPS treatments). However, in this thesis, responses of these two varieties to two cycles of water deficit were also investigated. The two cycles of water deficit treatment (the PS treatments) were introduced following initial observations that only one cycle of water deficit showed that the PER in both varieties decreased at a similar SWC value (ca. 18%) and the PER also ceased at a similar SWC (at ca 6 to 8 %) in both varieties. It was suspected initially, therefore, that the Tienshan ecotype probably would not show its tolerant capability since the stock plants have been maintained in well-watered conditions and not in the conditions that mimic the original habitat of the ecotype before the
Discussion
169 water deficit experiments were undertaken. Therefore, some pre-exposure to a water deficit may be necessary for the Tienshan ecotype to display its tolerant properties. However, following prior exposure to a water deficit (PS treatment), all data obtained in this study indicated little change to the responses of Tienshan plants to the water deficit. However, the pre-stress treatment did alter the responses of the more sensitive Kopu cultivar to water deficit, as shown by alteration of the PER, TR- ACO1 and TR-ACO2 gene expression as well as TR-ACO1 and TR-ACO2 protein accumulation. Unlike the decrease in TR-ACO1 expression and TR-ACO1 abundance observed in the apical structure of NPS Kopu, the expression of TR-ACO1
and accumulation of TR-ACO1 in the apical structure of PS Kopu remained unchanged such that this response was now similar to those seen in both the NPS and the PS Tienshan (Figures 3.15 and 3.17). This suggests that following the pre-stress, meristem protection occurred in the PS Kopu. More interestingly, the expression of
TR-ACO2 and accumulation of TR-ACO2 in the first fully-expanded leaves of PS Kopu was also altered, and again followed the trends observed in the NPS and the PS Tienshan (Figure 3.17 and 3.18). When the pattern of the PER in the PS Kopu was compared, the decline in the PER also occurred more progressively after the pre- stress treatment (Figure 3.4). All of this evidence suggests that an acclimation to water deficit did occur in the more drought-sensitive Kopu cultivar after the pre- stress treatment which resulted in responses that were more similar to the drought- tolerant Tienshan ecotype.
There is now much evidence that shows the importance of acclimation of plants to environmental cues, including water deficit (Cameron et al., 2008; Chaves et al.,
2009; Nunez et al., 2009). Previous water deficit experiences have been reported to promote subsequent resistance of several tree species to a subsequent water deficit by preventing water loss through osmotic adjustment as reported in apricot (Ruiz- Sanchez et al., 2000), oak (Villar-Salvador et al., 2004) and two species of woody ornamental species: Forsythia x Intermedia cv. Lynwood and Cotinus coggygria
(Cameron et al., 2008). In these two ornamental woody species, acclimatised plants showed better control of water loss (higher leaf relative water content and a more
Discussion
170 positive LWP) as well as a higher concentration of leaf ABA (Cameron et al., 2008). In addition, Chaves et al., (2009) suggested that acclimation to a previous water deficit may change gene expression profiles resulting in the modification of plant physiology and morphology. Thus in this thesis, acclimatised Kopu plants demonstrated altered PER, TR-ACO1 and TR-ACO2 gene expression as well as altered TR-ACO1 and TR-ACO2 protein accumulation to give a more similar patterns to the more drought-tolerant Tienshan ecotype. These results suggest that either the plant hormone ethylene plays a direct role in the physiological alterations required for the acclimation process, or ethylene biosynthesis is altered as consequence of these changes.
Although ethylene evolution was not measured in this thesis, as ACO catalyses conversion of ACC into ethylene, it is likely that alteration in ACO gene expression and ACO protein accumulation could be used as an indicator of alteration in ethylene biosynthesis. Therefore, this evidence indicates that ACO, at least in part, plays some degree of regulation on ethylene biosynthesis during water deficit in white clover. Much evidence on regulation of ethylene biosynthesis by environmental cues has focused on ACS, as it is considered as the rate limiting enzyme for ethylene biosynthesis (for example Bleecker and Kende, 2000; Wang et al., 2002). Therefore, ACS involvement in regulating ethylene biosynthesis in plants exposed to stress has been intensively studied and reviewed (Wang et al., 2002; Tsuchisaka and Theologis, 2004). Studies on Arabidopsis suggested that only certain members of the ACS gene families are responsive to stress stimuli (Wang et al., 2002; Kim et al., 2003; Tsuchisaka and Theologis, 2004), and ACS activity is regulated at different levels including at the transcriptional, posttranscriptional and posttranslational levels (Wang et al., 2002; Kim et al., 2003; Tsuchisaka and Theologis, 2004).
Although the involvement of ACO has not yet been studied as extensively as ACS, evidence is now accumulating to show that ACO also plays a regulatory role in ethylene biosynthesis both during plant development and in response to hormonal and environmental cues. For example, MaACO1 in banana is expressed during fruit ripening (Kesari et al., 2007) while four members of the ZmACO gene family in
Discussion
171 maize are differentially expressed in the root (Gallie et al., 2009). Similarly, three members of MD-ACO gene family are differentially expressed in leaves and fruits of apple (Binnie and McManus, 2009). In addition, the MaACO1 transcript and MaACO1 protein in banana was shown to be upregulated by exogenously applied ethylene, but not responsive to auxin treatment and it was downregulated by wounding and cold treatments (Choudhury et al., 2008). Similar to this is the differential expression of two ACO genes in Chinese pear where expression of
PbACO1 is induced by exogenously applied ethylene while PbACO2 is induced by mechanical wounding (Yamane at al., 2007). In addition, three NaACOs transcripts in Nicotiana attenuata respond differentially to wound-induced ethylene production (von Dahl et al., 2007). Evidence shown in this thesis supports these previous findings of differential expression of ACO transcripts and ACO proteins in response to environmental cues, including water deficit.
Although it has not yet been studied at the level of gene expression and protein accumulation, it was previously suggested that ACC oxidase activity is altered during water deficit in cotton petiole (McMichael et al., 1972) and wheat (Chen et al., 2002) with no significant changes in ACC levels. Together, these results indicate that ACO may be involved in fine tuning the control of ethylene biosynthesis during a water deficit. But how such environmental cues signal to effect changes of ACO
transcripts and ACO protein abundance remains to be elucidated.