Understanding carotenoid esterification in cereals
Creators: name: Watkins, Jacinta
Publisher: name: The Australian National University
Year: 2019

description: Carotenoids are important micro-nutrients for humans present in fruits, vegetables and certain grains including bread wheat (Triticum aestivum). Several carotenoids including beta-carotene and beta-cryptoxanthin provide provitamin A activity, whilst xanthophylls, including lutein and zeaxanthin, are essential for ocular and brain development, maintenance of cognitive function in elderly, as well as important for the prevention of a range of degenerative diseases including age-related macular degeneration. The health benefits provided by carotenoids makes them key targets regarding biofortification of staple foods. However, due to their reactive nature, carotenoids are highly prone to oxidative degradation and have rapid turnover during the storage of crops, both in common varieties and biofortified lines. In commonly consumed foods carotenoids are often found esterified with fatty acids. Esterification positively influences stability, sequestration and accumulation of carotenoids. However, whilst the genes and regulation of carotenoid biosynthesis have been well studied, the molecular mechanisms underpinning esterification have remained unknown in all plant species to date. This thesis presents data on the identification and genetic and biochemical characterisation of Xanthophyll Acyl-Transferase (XAT), a member of the GDSL esterase/lipase gene family, responsible for the production of carotenoid esters in mature grain of bread wheat. Using heterologous expression systems, it is demonstrated that this enzyme has a broad substrate specificity. XAT can esterify lutein, beta-cryptoxanthin and zeaxanthin with multiple acyl-donor substrates, yet has a preference for triacylglycerides indicating that the enzyme acts via transesterification. A series of biochemical assays further demonstrates that XAT has activity over a broad temperature and pH range. Additionally, site-directed mutagenesis was employed to demonstrate that a conserved amino acid, Ser37, is required for activity. Intriguingly, despite carotenoids being synthesised in plastids, XAT was found to accumulate in the apoplast. The implications of this compartmentalisation and a potential alternative function for XAT in wheat are investigated by analysing substrate preferences and ester formation in vitro. It is proposed that disintegration of cellular structure during wheat grain desiccation facilitates access of the enzyme to its carotenoid substrates, thereby promoting and, in turn, controlling the timing of esterification, a novel mechanism of the regulation of carotenoid metabolism. The second chapter of this thesis begins by investigating the in vivo activity of XAT, which is confirmed via heterologous expression in carotenoid-accumulating rice callus. Results presented further establish that XAT can function when co-expressed and co-located with its substrates within the plastids of living cells as well as in species which normally accumulate exclusively un-esterified xanthophylls. The second chapter also explores the activity of XAT cereal homologs from maize, barley and rice. Expression of the maize and barley XAT homologs was found to coincide in tissues which accumulate xanthophyll esters. Further, the rice XAT homolog was observed to have xanthophyll acyl-transferase activity, producing xanthophyll esters when heterologously expressed in rice callus. Taken together, this suggests that the mechanisms of xanthophyll esterification is likely to be conserved amongst cereals. Overall, results presented in this thesis extends our understanding of carotenoid biosynthesis beyond the production of lutein and xanthophylls to the production of xanthophyll esters. Given that the accumulation of carotenoids is governed by both the rates of synthesis and degradation, understanding the mechanisms of esterification provides new opportunities for discovery and applications for biofortification to increase the nutritional status of crops.