Response to NAA and interaction with CPPU

Early application of another synthetic auxin, NAA, to A. deliciosa fruit also failed to induce an increase in fresh weight of fruit. In contrast to the 3,5,6- TPA results though a late application resulted in a significant increase in the fresh weight of fruit. Also in contrast to the results of the 3,5,6-TPA experiment application of NAA did not increase percentage dry matter at either date. Application of CPPU on the late application date to A. deliciosa in the same experiment also resulted in a greater increase in fruit weight than when it was applied on the early application date, although it must be noted that the concentration used was only 1 mgL-1 compared to the more common 5 or 10 mgL-1. An additive response was achieved by applying both CPPU and NAA at either the early or late application date. However, perhaps the most interesting result was observed when CPPU was applied early and NAA was applied late. This combination of treatments resulted in a synergistic interaction whereby the increase in weight induced by applying CPPU early plus the increase induced by applying NAA late on separate fruit was less than that induced by the application of CPPU early plus NAA late on the same fruit. This interaction cannot be tested statistically as there was no internal replication due to a limitation of canes and the large nature of this experiment, but this could be tested in future experiments. The significance of this result may be related to the timing of application and the activity of natural cytokinins and auxin. The early application of CPPU, the synthetic cytokinin-like compound, was during the period of cell division and cytokinins are predominantly associated with cell division in fruit (Atwell, et al., 1999).

Chapter four: General discussion

Furthermore the late application of NAA, the synthetic auxin, was during the period of cell expansion and auxin is predominantly associated with cell expansion (Atwell, et al., 1999). These results do not support or disprove any one of the theories behind the potential mechanisms of action put forward for CPPU over any others. That is, the protective role of CPPU for endogenous cytokinins perhaps by interfering with the activity of cytokinin oxidase, or the direct role of CPPU acting like a naturally occurring cytokinin, or the indirect role whereby CPPU acts by forming a positive feedback loop where the concentration of naturally occurring cytokinins is increased. These results do not correspond with those obtained by Patterson et al. (1993) and Lewis et al. (1996) which suggest that CPPU causes an increase in cell expansion, nor do they support the hypothesis put forward in this thesis that CPPU application causes an increase in fresh weight simply by altering the concentration of endogenous IAA. However the results can be tentatively interpreted in terms of optimal auxin/cytokinin ratios at specific stages of fruit growth.

4.4. Summary

Both the natural plant extract Benefit® and the synthetic plant growth regulator CPPU increased the fresh weight of fruit when applied to A. chinensis (Figure 2.1), as found in previous studies (Woolley & Cruz-Castillo, 2006). There was a statistically significant (p < 0.05) interaction between Benefit® and CPPU when they were applied together, whereby the fruit treated with both were significantly heavier than when either was applied alone (Table 2.4). However

Chapter four: General discussion

each individually was added together. While this result could suggest that both Benefit® and CPPU induce an increase in fruit weight by the same means this is not necessarily the case. There have been conflicting results as to whether application of CPPU increases fresh weight by way of cell division (Kurosaki & Mochizuki, 1990; and Neri et al., 1993) or cell expansion (Lewis et al., 1996; and Patterson et al., 1993) or both (Woolley et al., 1991). Although, given that the fresh weight has increased without a proportional increase in percentage dry matter, it may be reasonable to conclude that the increase is at least partially due to cell expansion. However, Benefit® has been shown to have little or no cytokinin activity. It would, therefore, seem unlikely that Benefit® and CPPU induce an increase in fruit weight by means of the same mechanism. Benefit® was also shown to decrease the percentage dry matter of fruit significantly when applied to A .chinensis, while application of CPPU did not (Table 2.2).

Not all treatments applied to A. deliciosa increased the fresh weight of fruit. Application of CPPU increased fresh weight in both of the experiments it was tested in (Figure 2.3 and Table 2.6), thus supporting previous results (Iwahori

et al., 1988; Lawes et al., 1991; Patterson et al., 1993; Lewis et al., 1996; Cruz-Castillo et al., 1999; Woolley & Cruz-Castillo, 2006; Woolley & Currie, 2006). However application of Benefit® did not affect fresh weight, proportions of pericarps, or percentage dry matter, in any way (Table 2.4 and 2.5). This results supported of one previous study (Woolley & Cruz-Castillo, 2006) but not another (Costa et al., 2002). It could be suggested that A. deliciosa is less sensitive to Benefit® than A. chinensis and thus requires a higher

Chapter four: General discussion

concentration. The differing outcome of Benefit® application between A. chinensis and A. deliciosa fruit suggests a difference in their physiology, either in terms of their limiting factors or their ability to metabolise Benefit®. Application of 3,5,6-TPA also failed to induce an increase in A. deliciosa fruit weight (Figure 2.4) which was in contrast to previous studies (Bregoli et al., 2006). Percentage dry matter was, however, significantly altered and concentration and application date affected this (Figure 2.5). The effect of NAA application was dependent on date with an early application having a negative effect on fresh fruit weight and a late application date significantly increasing the fresh weight (Table 2.6). Reaction to CPPU was also found to be affected by application date. The most intriguing result came from the application of CPPU early plus NAA late as this resulted in a synergistic interaction. However, none of the NAA/CPPU combinations resulted in an increase in percentage dry matter (Table 2.6).

A large amount of method development was required for the extraction and purification of both endogenous auxin and cytokinins from A. chinensis fruit. The method, at this stage, is by no means absolute and requires improvement due to high losses of the internal standard and the putative IAA peak did not reach baseline in all of the traces from the fluorescence detector. Also it has not been determined that the putative IAA peak does in fact contain solely IAA. Nonetheless there does appear to be a correlation between treatment of

A. chinensis fruit with CPPU and concentration of endogenous IAA (Figure 3.11 and 3.12). However, results at this point in time are limited and inconclusive. Though the endogenous IAA in the control fruit does appear to

Chapter four: General discussion

follow a similar pattern over the development of fruit as has been previously determined for A. deliciosa fruit, peach, and blackcurrant berries (Ohara et al., 1997; Millar et al., 1987; and Wright, 1956 as cited by Wareing & Phillips, 1970 respectively) and this supports the validity of the results.