The regulation of defence chemistry in Nicotiana

Plants have to balance their allocation of resources to growth, reproduction, and defence to survive in complex environments. To defend against a diversity of herbivores, plants produce a variety of defensive metabolites and as such Nicotiana species have evolved a cocktail of jasmonate (JA)-regulated inducible defence molecules including pyridine alkaloids and phenylpropanoid-polyamine conjugates. In several species of Nicotiana, alkaloid and phenolamide levels increase following wound- and/or herbivore-associated stresses. Studies of plant-insect interactions can improve our understanding of the regulation of plant secondary metabolites and defensive protein production. Possession of a molecular clock enables plants to control and synchronise internal rhythms to day/night cycles, thereby providing a mechanism to respond to daily stresses and modulate metabolism. The accumulation of plant defence signalling compounds, including JAs, salicylic acid (SA) and ethylene (ET), can alter under circadian control and activate biotic stress response pathways and cyclically accumulate under circadian control. The molecular systems that regulate plant metabolome dynamics and rechannel metabolic fluxes towards the production of a specific spectrum of defensive metabolites following herbivore-associated stresses remain poorly understood. Many genes associated with hormone-dependent defence response pathways are clock-regulated and a number of defence metabolites follow diurnal regulation. These include phenylpropanoids, diterpene glycosides and nicotine. Entrainment of plants by light controls JA, SA and ET rhythms as well as diurnal accumulation of defence metabolites. Clock control of hormone signalling is thought to underlie this wide spread phenomena enhancing plant resistance to herbivores amongst diverse crop species. The research presented in this thesis used molecular and biochemical tools to examine the molecular controls that underlie circadian clock-hormone controls as well as integral genes involved in putrescine-associated metabolism, which drive nitrogen defence pathways in Nicotiana species. In the present work, RNAi-mediated silencing of TIMING OF CAB EXPRESSION 1 (TOC1) demonstrates that nicotine synthesis and accumulation in N. attenuata is strongly correlated with the repression and accumulation of genes involved in ET and nicotine biosynthesis, respectively. It is also demonstrated that the control over herbivore-induced ET production by the TOC1 evening component enables plants to control induction regimes and resource input into nicotine, a costly area of nitrogen metabolism. Further exploration was undertaken of down-stream effects of complex early herbivore-induced signalling events, connecting clock-signalling networks with JA-mediated responses to herbivory using rapid patterns of accumulation of defensive phenolamides as case studies. It was determined that the transcript levels of early herbivore-induced stress signalling components, nitrogen assimilation, JA-activated regulatory genes and defence biosynthetic genes in clock-JA response networks are mediated by TOC1. TOC1-specific alterations in allocation of nitrogen in N. attenuata defensive chemistry has a major effect on the insect fitness of a specialist herbivore, Manduca sexta, and an intermediate effect on a generalist, Spodoptera littoralis. It was found that TOC1, acting with additional defence signals and molecular components, exerts independent regulatory control over branched areas of secondary metabolism. TOC1 was shown to function as a positive regulator in phenylpropanoid, polyamine and diterpene metabolism and may additionally play a role in nitrogen uptake, prioritisation or investment of nitrogenous defence resources. These findings provide insight into how plants use the clock to coordinate various defence signals, enabling optimal accumulation of clock-associated nitrogen defences, which may underlie differential resistance against herbivores. Research described in this thesis also examined ORNITHINE DECARBOXYLASE (ODC) and its role in putrescine-associated metabolism in N. tabacum. Down-regulation of ODC produced a marked effect upon the alkaloid profile, specifically reducing nicotine and concomitantly increasing anatabine levels. Additionally the role of ODC in the synthesis of anabasine in N. glauca was examined. Reduced expression of ODC in both hairy root cultures and transgenic plants of N. glauca, resulted in a diminished ability to elevate anabasine concentrations in response to wound-associated stress. It was concluded that ODC plays an important role in determining the normal amino-acid, polyamine and polyamine-associated defence profile in N. tabacum and is essential in allowing plants to increase levels in response to wound-associated stress. Additionally, ODC plays an important role in enabling plants to elevate levels of anabasine in response to wound-associated stress