Grain hormone changes

Consistent with previous results, the metabolites of ABA detected in the dry de- embryonated grain tissue all appear to be from the 8’-OH pathway. In agreement with the noted (but not statistically significant) trend of increased trans-ABA in the embryo, grain trans-ABA content has increased in the transgenic lines com- pared to controls. The increase in embryo PA is not reflected by a statistically significant difference in grain but, in a similar situation as embryotrans-ABA, a possible trend of increased PA could be seen; indeed, the large error bar associ- ated with line G4.7N in Figure 5.9 for PA is essentially due to one datapoint. If we consider the ABA andtrans-ABA results separately from the metabolite con- tent and consider this as an indication of bioactive ABA (as trans-ABA appears to be produced by UV light exposure of ABA, rather than enzymatically), a sig- nificant increase of 17-23% can be seen in the transgenic lines (F value of 6.029,p = 0.0303; Figure 5.17). This could indicate an increased bioactive ABA content in these grains as the conversion oftrans-ABA is not ’intentional’ on the part of the plant and is rather simply a result of environment; however, how relevant this is in reality is questionable. Searching of literature was unsuccessful in identify- ing iftrans-ABA is converted by hydroxylation in the same way as ABA, but can be conjugated to glucose. No ABA-glucose ester products were detected in any of the samples here. This potential modification agrees with the findings of the previous embyro section.

The role of ABA in the developing endosperm are in the phases of highest seed growth, when storage reserves are accumulating (Srivastava, 2002). ABA re- sponse elements are found in grain protein promoters (such as gliadin and glutenin wheat storage proteins) which control the grain specific expression of these genes∗. There is also considerable evidence to support the notion in maize that sugars and ABA co-regulate starch synthesis genes and AGPase, with sugars positively in- fluencing and ABA negatively influencing these genes (Chen et al., 2011). It has ∗_{A good example is the Bx17 high molecular weight glutenin promoter used in the construct for} this study.

Figure 5.17– Grain ABA+trans-ABA levels. Average hormone levels indicated as calculated by adding ABA totrans-ABA content, with error bars indicating Standard Errors. Significant difference due to transgene presence indicated by stars in top right corner (*p< 0.05).

also been found that barley mutants with low endosperm starch synthesis rates (many with lesions in starch related genes) also have a low grain dormancy char- acteristics, suggesting a feedback mechanism at play.

During early grain filling (initial starch accumulation) ABA levels are low and genes such as sucrose synthase and AGPase are highly expressed. As ABA levels increase towards and during maturation these activities are reduced and stor- age protein accumulation takes precedence, as does essentially programmed cell death of the cells of the developing endosperm. Chen et al. (2013) provide evi- dence that this is in some way mediated by the action of SnRK1 and SnRK2 (a closely related kinase family), with SnRK1 activity high early in development and influencing starch and sugar related genes such as those previously men- tioned. As ABA levels increase SnRK1 levels are reduced and SnRK2 becomes more prominent, encouraging desiccation and cell death in the endosperm. The ABA-SnRK interactions are also implicated heavily in reproductive stage stress responses (Jiet al., 2011).

A pattern of increased GA19levels in transgenic grain tissue compared to controls was seen but is outside of the usualp< 0.05 significance cut-offs, instead having ap= 0.069. This general pattern is interesting given the previous results showing a reduction of embryo GA53 levels. Both of these GA species fall on the early GA13ox side of the synthesis pathway. Here we have an indication of an increase in GA content in the endosperm of the transgenic lines.

The previous work reported on the lines examined here noted an increase in α- amylase activity of mature grain in the transgenic GWD RNAi lines compared to controls (Ral et al., 2012). Although we did not detect a consistentα-amylase activity increase in our material, this may be due to age differences in the mate- rial being examined. GA is known to be involved in the expression ofα-amylase genes in grain, specifically from the aleurone layer (Woodgeret al., 2010). In addi- tion to GA, sugars and the SnRK1 regulatory system interact to controlα-amylase expression (Laurieet al., 2003; Chenet al., 2006; Luet al., 2007). Someα-amylase promoters contain a sugar response complex which blocks expression. At the same time, sugars (depending on type) reduce the activity level of SnRK1, which are responsible in part for the induction of factors that can induceα-amylase ex- pression via a GA responsive element. SnRK1 and GA work together in this respect. Loss of SnRK1 is known to reduce AMY2 expression in wheat embryos, and SnRK1A relieves glucose suppression of rice AMY3 expression (Laurieet al., 2003; Luet al., 2007). Wheat lines with constitutive late maturityα-amylase (high

α-amylase activity in mature grain) were found to have greatly increased GA19 levels compared to controls in a study by Barreroet al.(2013), although the levels found in that report were much higher than reported here. α-amylase activity level changes were seen during germination in the results reported here, raising interesting questions as to potential GA level changes post imbibing.

The combination of ABA and GA potential flux changes in endosperm found in these results certainly require further research to understand. Measurement of hormones and sugar levels during development would be greatly beneficial.