Purine Catabolism in Wheat: Source of Nutrients and Protective Metabolites

Abstract

Purine catabolism is known to have a dual function in recycling nitrogen (N) and carbon (C) atoms present in the heterocyclic purine ring and participating in stress signalling through the stimulation of ABA metabolism by allantoin, an intermediate in the pathway. However, little information was available of the functions in cereals and, in particular, bread wheat. The aims of this PhD thesis was to investigate the role of purine catabolism and allantoin in Australian bread wheat (Triticum aestivum) genotypes (RAC875 and Mace) grown under N deficiency and water deficit. Firstly, the purine catabolic genes, coding for seven enzymes in total, were identified and annotated in the hexaploid bread wheat genome of cv. Chinese Spring. The analysis revealed 24 loci associated with the enzyme genes. Interestingly, there was a duplication of the xanthine dehydrogenase gene, namely TaXDH1 and TaXDH2. Sequence analysis of the TaXDH2 homeologs located on chromosome group 6 appeared to be either non-functional (TaXDH2-6AS/6BS) or with an inactive xanthine substrate binding site (TaXDH2-6DS). Protein structure modelling and a unique expression pattern under stress indicated that, TaXDH2-6DS may have a novel function in wheat. Characterisation of allantoin and transcription of purine catabolic genes under N and water restrictions, revealed that allantoin levels were reduced (22-fold) when N was limiting, whilst it tended to accumulate in large amounts under drought (up to 30-fold compared to well-watered plants). The latter may suggest allantoin is used as a temporary N sink as the N assimilatory pathway (GS/GOGAT cycle) is likely to have a reduced capacity in plants growing in water deficit conditions. This would prevent the accumulation of ammonium that is toxic at high concentrations and reduce ammonia emissions from plants leaves. The reduction or accumulation of allantoin under drought and N stress appeared transcriptionally regulated by purine catabolic genes. In particular, transcription of TaALN, coding for the allantoin-degrading enzyme allantoinase, oppositely reflected the levels of allantoin in the tissue. Further growth studies showed that wheat seedlings, when re-supplied with xanthine or allantoin as their sole N source after short-term N starvation, had growth rates which were equivalent to plants grown with inorganic nitrogen. This suggests that the N recycled through the purine catabolic pathway can support the growth of wheat. The data also provided evidence that allantoin takes part in N remobilisation during natural senescence. Sequence analysis of TaALN homeologs in a large number of bread wheat accessions highlighted substantial genetic variability when compared to the reference genome of Chinese Spring. Candidate accessions with nucleotide polymorphisms in regulatory elements or in the coding sequence were identified and represent valuable material for future studies. The outcomes of this PhD project provide the ground work for future fundamental research in wheat focussing on the dual role of purine catabolism in N recycling and abiotic stress. In addition, the candidate accessions identified, besides representing useful experimental material, could ultimately be used for breeding purposes. Additional strategies will include genetic engineering of target genes in the pathway that may lead to improved wheat growth and yields in unfavourable environments.