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Base ABA levels have not changed in embryo tissues of transgenic lines compared to controls measured in this chapter, but a metabolite of ABA, PA has increased in the lines. The next level metabolite∗, diphaseic acid, has not been altered. A trend of increasedtrans-ABA could be seen in the results but was not significantly different in nested ANOVA. Total measured ABA levels also do not indicate any large changes between lines.

The vast majority of ABA metabolites measured in the embryos of these lines were inactive compounds. Unfortunately, 8’-OH ABA, an intermediate metabo- lite in the pathway used for ABA inactivation seen here, was not able to be mea- sured in this assay, which may have provided additional information about the total amount of ABA stored as 8’OH metabolites. However, the lack of neopha- seic acid and 7’-OH ABA indicates that this 8’-OH ABA pathway is responsible for all ABA inactivation in the wheat embryos we have studied.

Given these results it is not possible to confirm that embryo ABA content is re- sponsible for the delayed germination phenotype exhibited by the positive lines (See §3.3.1), as bioactive ABA levels have not changed, nor has total ABA mea- sured at maturity in after-ripened embryos. Phaseic acid is known to have an inhibitory effect on germination, but at much lower levels than ABA (Dashek et al., 1979). Even though this affect is 0.5% of that of ABA, it is interesting to note that PA levels were increased by up to 50%. This may be of interest with respect

to the germination delay found in transgenic lines compared to controls in this study∗_{, but cannot be considered as a conclusive cause.}

No differences in bioactive IAA levels were detected in these samples, but IAA- Asp content was increased in transgenic lines compared to controls (marginally statistically significant result). The overall trend of the data suggests an increase in total auxin content of the transgenic lines compared to controls.

There is strong evidence of a role for auxins in early embryo development. Aux- ins are highly important in the correct formation of embryos, as mediated by the activity of auxin transporters, the pin-formed (PIN) proteins (Möller and Weijers, 2009). Several different PIN proteins are arranged in specific orientations in em- bryos, and the changing of polarity, or direction of auxin transport, alters the local auxin concentration in the developing embryo. The localisation at different stages of development mediate the precise steps required for the correct formation of the embryo. Given that there appears to have been some alteration of auxin content evident in the mature embryos of transgenic lines it is interesting to hypothesise that this could be linked to the altered embryo size found in §2.3.1.

Additionally, the altered IAA-Asp levels of the transgenic lines may be related to sugar status of the lines in question. Glucose is known to increase the concen- trations of IAA precursors and also IAA metabolites such as IAA-Asp (LeClere et al., 2010; Sairanen et al., 2012). This is negatively regulated by phytochrome- interacting factor, which itself demonstrates increased expression in response to sucrose. This is of note even though we have not identified a major carbohydrate modification of the transgenic lines that could be associated with this result. Our previous carbohydrate quantification was unable to measure all of the potential signalling compounds involved in such pathways.

The conclusion to be drawn from the results on embryo GA levels is that the vast majority of gibberellins detected were from the early GA13ox part of GA synthesis†. The smallest fraction of GA detected was GA24, which could not be detected with great confidence, which is the only component of the GA20ox GA synthesis pathway.

No bioactive GAs were detected in the dry embryo tissue examined in this study; rather, GA and precursors appears to have been produced in the embryos to the GA19 form and no further, potentially as a pool for quick conversion to active

∗_{See §3.3.1.}

GAs upon imbibition (GA19 →GA20→ GA1 or GA3). The first sign of a change in potential GA flux is in the reduction of GA53 content in transgenic lines com- pared to controls seen in these results. This is the first product formed in the early GA13ox pathway portion. The role of GAs in seed development are still not understood; it has been suggested that its role is probably in late development when both the embryo and endosperm are enlarging, which is understandable given GAs stimulation of cell division and elongation (Srivastava, 2002).

The Arabidopsissex1mutant, which is deficient in GWD, demonstrates reduced total GA content at the end of night (starch degradation phase) (Paparelliet al., 2013). During this phase of growth the components measured from the early GA13ox section of GA synthesis all appear reduced, whereas this is not the case by the end of day. During this phase of starch accumulation the opposite is true, with reduction of the components of the GA20ox side (reflected by a large in- crease of the GA51 metabolite) and the GA13ox side has no difference to wild type or, in the case of GA3, mildly increased. Paparelliet al.(2013) also found that the dwarfism of thesex1line could be reversed by addition of GA4 and GA7 (al- though they still exhibited signs of starvation). They attributed the changes in GA to inhibition of early GA synthesis, but the interesting switching of flux direction was not examined in this work. The inhibition of GA synthesis was attributed to low sugar levels during night stages due to the inability to degrade starch, which altered the expression of copalyl diphosphate synthase, ent-kaurene oxidase and, most significantly, ent-kaurene synthase. The sugar-starvation response was con- firmed by examining expression markers such as dark inducible 6 and trehalose 6-phosphate synthase; both of these are regulated via the previously mentioned SnRK1 regulatory system. The reversal of this inhibition during starch accumula- tion periods is understandable given the sugar-excess noted insex1plants in light conditions by Casparet al.(1991).

There is some evidence presented here that hormone regulation has been altered in these GWD RNAi lines when compared to the controls. The mechanism by which these changes arise is still uncertain. A modification of an ABA metabolite with potential for low-level inhibition of germination has been found, which coin- cides with our previous results. Circumstantial evidence suggests that transgenic lines have an increase in total detectable auxin content, which may be of interest given the previous indications that embryo size has increased in the transgenic lines. Finally, early GA precursors appear to be reduced in the transgenic embryo tissue. Although a conclusive need for GA in germination of cereals is (not α-

amylase production) has not been found, a potential for delay of GA production in the transgenic lines as a result of this modification may be of interest in this study.