Structure, expression and evolution of QPT genes in pyridine alkaloid producing species of the Solanaceae.

Pyridine alkaloids such as nicotine, anabasine, nornicotine and anatabine are characteristic of the genus Nicotiana. They are regarded as effective but resource expensive, chemical defences that protect plants against excessive herbivory in native environments. In several species, alkaloid levels increase following wound-associated stresses such as leaf damage, apex removal and/or exposure of plants or cultured tissues to the wound hormone methyl jasmonate. Quinolinate phosphoribosyltransferase (QPT) encodes a key enzyme for NAD(P)H synthesis but is also essential in enabling adequate supply of nicotinic acid from which the pyridine ring of these alkaloids is derived. Previous work in this laboratory showed that the QPT gene family in N. tabacum contained two distinct members -QPTI and QPT2. In the present work, sequence analysis of PCR products recovered from genomic DNA of N. tabacum cv. Xanthi revealed the presence of additional QPTl and QPT2 type genes (NtQPTlb and NtQPT2b) closely related to previously identified sequences from this laboratory which were designated NtQPTla and NtQPT2a. Parsimony analysis of these QPT genes from N. tabacum and also related sequences obtained from its model progenitor species established that QPTl and QPT2 genes in N. tabacum (a and b) are orthologous to QPT genes from its model progenitor species, N. sylvestris and N. tomentosiformis. Research described in this thesis also examined activity and evolutionary relationships of both versions of QPT genes in the genus Nicotiana, and related genes in species of tribe Anthocercideae, in association with capacity for wound-associated stress stimulation of pyridine alkaloid metabolism. Transcript profiling combined with promoter reporter gene experiments in transgenic plants of N. tabacum indicated that QPT2 is the predominant member of the QPT gene family expressed in non-stressed root, leaf and floral tissues. However, complementation of a bacterial mutant showed that despite extensive divergence in non-coding sequences compared to QPT2, the QPTl gene encodes a functional protein suggesting it retains an important role in some aspect(s) of the plant life cycle. In N. tabacum, relative quantification using qPCR indicated that QPT2 transcript levels, but not QPTl, were induced in hairy root cultures following methyl jasmonate treatment and also in root tissues of plants after foliar wounding. Examination of N. sylvestris, N. tomentosiformis, and also members of the largely Australian section Suaveolentes, N. suaveolens and N. debneyi, led to similar conclusions even though some variation in capacity for wound-associated induction of pyridine alkaloids and/or related transcript levels exist in species from different sections within the genus Nicotiana. These results suggest that expression characteristics of QPT2 associated with pyridine alkaloid production may have evolved prior to speciation of the genus Nicotiana. Evolutionary and phylogenetic analyses of genomic QPT gene sequences obtained by peR from species in tribe Anthocercideae and other closely related Solanaceae genera indicated that QPTl and QPT2 genes in the genus Nicotiana are likely to be the result of a duplication of QPT in an early or pre-Nicotiana species -15 MY A after divergence from an ancestral species common with sister tribe Anthocercideae species. All tribe Anthocercideae species produce tropane alkaloids, but only the genera Cyphanthera and Duboisia have been reported previously to contain pyridine alkaloids. Hairy root cultures were successfully established from two tribe Anthocercideae species, Cyphanthera tasmanica and Anthocercis ilicifolia ssp. ilicifolia. Hairy roots of both species showed capacity to synthesise pyridine and tropane alkaloids. Treatment of these hairy root cultures with methyl jasmonate produced elevated levels of pyridine, but not tropane alkaloids however. This was associated with jasmonate induction of key pyridine alkaloid biosynthetic genes QPT, PMT and A622 in roots of both these species. This may suggest that the duplicated form of QPT may have had capacity for induction following wound-associated stress associated with pyridine alkaloid metabolism, in a lineage ancestral to both Nicotiana and tribe Anthocercideae species