Elucidating the molecular basis of dihydroxyacetone (DHA) production in Leptospermum nectar

Abstract

Mānuka (Leptospermum scoparium) nectar contains variable amounts of dihydroxyacetone (DHA), a triose sugar that is the precursor to methylglyoxal (MGO), the antimicrobial compound underpinning the high value of mānuka honey and forming the basis of the Aotearoa-New Zealand honey industry. This trait is unique to some species within the Myrtaceae. The molecular basis of this high-value trait is unknown and identifying it would allow development of gene-based tools to identify high value germplasm to inform replanting and breeding programmes. Despite the fundamental importance of nectar to crop pollination and honey industries, the genetic control of nectary function is poorly understood, especially in non-model species. This thesis aims to elucidate the molecular basis of DHA production in Leptospermum nectar, through identifying associated genes and genomic regions, followed by further exploration of gene regulation and enzyme characterisation in vitro and in vivo. RNAseq analysis identified nectary-associated genes differentially expressed between high and low nectar-DHA genotypes of L. scoparium, and a mānuka high-density linkage map and quantitative trait loci (QTL) mapping population revealed genetic regions associated with nectar DHA content. Expression and QTL analyses both pointed to the involvement of a phosphatase gene, LsSgpp2, as its expression correlated with nectar DHA accumulation and it co-located with a QTL on chromosome 4. To investigate Sgpp2 and its complex locus further, we produced high-quality genomes and hypanthium transcriptomes for two further Leptospermeae species with contrasting DHA phenotypes – Leptospermum morrisonii and Gaudium laevigatum. Expression patterns of Sgpp2 again correlated with nectar DHA in these species, and comparative analyses of Sgpp sequences within the Myrtaceae indicated that high Sgpp2 expression was likely ancestral in DHA producing taxa, followed by repeated loss of the trait. Comparison within and between Sgpp genes with differing expression profiles identified regions unique to the highly expressed Sgpp2 genes, which contained two C-box motifs, and a bZIP11 transcription factor predicted to bind to these motifs was identified as significantly differentially expressed in the RNAseq dataset. The bZIP11 gene was subsequently found to co-locate with another of the QTLs identified, further supporting its involvement. The L. morrisonii promoter drove strong nectary-specific expression in transgenic lines of Petunia and Nicotiana, showing elements essential for Sgpp2 expression are within that region. However, the promoter in 13 DHA producing L. scoparium genotypes was found to be similar, indicating variation in nectar DHA amounts may be conferred by further complex transcriptional regulation fine tuning Sgpp2 expression from beyond the promoter region analysed here. Functional analyses demonstrated that LmSGPP2 can dephosphorylate dihydroxyacetone-phosphate (DHAP) to produce DHA and phosphate in vitro. Transgenic Petunia and Nicotiana lines were created to characterise LmSGPP2 activity in vivo, but results were inconclusive due to low and mis-located expression of the transgene. Together our results suggest Sgpp2 may contribute to maintaining phosphate homeostasis in a photosynthesising nectary, potentially evolving due to low phosphate availability – which is common in Australia where this tribe originates. This work advances our understanding of nectar biology and reports some of the first QTLs and genes linked to a low abundance nectar compound. The genes and genomic regions identified here provide a foundation for developing tools to identify high-value germplasm – ensuring biodiversity can be maintained while increasing high-value honey production.