Abstract
The leaves of Pseudowintera colorata (Winteraceae) are well known for their bright displays of red colour, and their strong peppery flavour. The variable patterns of red and green colour seen across the leaves of this species are caused by pigments called anthocyanins, while the peppery flavour is caused by the presence of two colourless sesquiterpene dialdehydes, polygodial and 9-deoxymuzigadial. Anthocyanins, and to a lesser extent sesquiterpene dialdehydes, are widespread in nature, occurring across a large number of plant species. Anthocyanins are known for their functionality against a wide variety of biotic and abiotic stresses, including light stress and herbivory. The rarer sesquiterpene dialdehydes have strong anti-herbivore, anti-fungal and anti-bacterial properties. In this thesis I explored the significance of these compounds in P. colorata leaves, by investigating two possible functions of anthocyanins that are previously untested on this species: aposematism and photoprotection. One of these functions, aposematism, links anthocyanin functionality to that of sesquiterpene dialdehydes.
Aposematism is the phenomenon where organisms produce signals, such as bright colours, that warn potential predators of their defences, such as toxic compounds. In this study it was determined whether anthocyanins could function as the bright colours of an aposematic signal, and sesquiterpenes as the defence component. Anthocyanins could also function as a photoprotectant, protecting the leaves of P. colorata against high irradiances of natural sunlight by absorbing excess light or mitigating the effective of oxidative damage caused by such exposures. Chilling temperatures have the potential to increase photoinhibition, and therefore were also investigated because P. colorata grows in a climate where such temperatures are common in the winter. Three other properties of P. colorata leaves were also examined as they had the potential to influence the functional significance of these compounds. Firstly, the effects of leaf wounding were explored as this species is known to produce anthocyanins when wounded, but it was unknown how sesquiterpenes would respond. Secondly, sesquiterpenes are stored in specialised oil cells in Tasmannia lanceolata, another member of the Winteraceae. These structures were present in P. colorata leaves, but their chemical content was unknown. The chemical content, development and density of oil cells in P. colorata leaves had the potential to explain the chemical responses of these leaves to wounding. Thirdly, leaf age could affect the functionality of anthocyanins, and possibly sesquiterpenes, if younger leaves are redder than older leaves. During the course of investigating these attributes of P. colorata I developed novel methods for analysing leaf colour and responses to wounding, and for extracting and analysing oil cell contents.
The results of this study did not support an aposematic function of anthocyanins in relation to the presence of sesquiterpene dialdehydes. Instead this study demonstrated that anthocyanin and sesquiterpene dialdehyde production are largely decoupled in P. colorata leaves. Anthocyanins were found to be positively correlated with sesquiterpene dialdehydes in naturally occurring fully expanded leaves, but this relationship was weakened by leaf wounding. Wounding decreased the concentration of polygodial and 9-deoxymuzigadial by an average of 3.3 mg/g and 1.3 mg/g respectively in new leaves, and 3.2 mg/g and 2.5 mg/g respectively in mature leaves. In contrast, wounding increased the concentration of anthocyanins by an average of 0.52 mg/g in new leaves, and 0.18 mg/g in mature leaves.
The loss of sesquiterpene dialdehydes was explained by their presence in the oil cells. Oil cells were found to occur across the entire leaf surface area at densities ranging from 23 to 205 cells per mm2, and therefore some were destroyed during leaf wounding. Leaves only produced oil cells during their period of expansion, so the loss of oil cells in fully expanded leaves meant that they could not be replaced. The positive relationship between anthocyanins and sesquiterpene dialdehydes was also found to be a function of leaf age. Fully expanded new leaves had an average anthocyanin concentration of 0.88 mg/g, which was significantly higher than that of the mature leaves, 0.52 mg/g. The average concentration of polygodial for new leaves was 29.41 mg/g, and was significantly higher than that of the mature leaves, 9.99 mg/g. Average levels of 9-deoxymuzigadial in the new leaves were 19.14 mg/g dry leaf, and were not significantly different to that of the mature leaves, 17.59 mg/g. Within these age groups, there was no consistent relationship between the concentrations of these compounds.
The results of this study did support a photoprotective function of anthocyanins. Anthocyanins were suitably located to intercept light before it reached the photosynthetic tissues, being located in the uppermost tissue layers. Most notably, red leaf tissue was better protected against photoinhibition than green leaf tissue. This protection was evident after the leaves were exposed to 1500 μmol m-2 s-1 white light for 4 hours, which caused a smaller decline in Fv/Fm in the red regions compared to the green regions, a difference of approximately 0.07 units. This protection was not enhanced at chilling temperatures, as commonly observed in some other plant species. Younger leaves are redder than older leaves and also likely to be more exposed to light in a natural environment because of their position on the stems, therefore anthocyanins probably function to protect these leaves from high light over long exposures.
Previous studies investigating aposematism in plants have focused on anthocyanins in the autumn leaves of deciduous trees, with few focusing on young developing leaves. The findings of this thesis build upon the knowledge concerning anthocyanin function in young leaves. Furthermore, it is the first study to demonstrate the importance of the cellular distribution of toxic compounds, and the responses of leaves to wounding, when investigating foliar aposematism.