Doctoral Thesis
Molecular mechanism regulating quinolizidine alkaloid content in vegetative tissues and seeds of narrow-leafed lupin (Lupinus angustifolius L.)
Doctor of Philosophy (PhD), Murdoch University
2026
DOI:
https://doi.org/10.60867/00000134
Abstract
Narrow-leafed lupin (NLL, Lupinus angustifolius L.) seeds are rich in protein and fibres, low in fat, making them a potential healthy food. However, their use is limited by bitter, toxic quinolizidine alkaloids (QAs), which protect the plant against pests. QAs are primarily synthesized in vegetative tissues and transported into seeds, complicating breeding efforts to separate defence from seed toxicity. To address the challenge, this thesis investigated the genetics, regulatory, and transport mechanisms underlying QA biosynthesis and allocation. Genome-wide association studies detected a major association signal of leaf QAs content that co-localized with the seed low-alkaloid (sweet) locus iucundus in NLL. Recombinant-based mapping pinpointed the transcription factor LaRAP2.7 as the candidate gene of this locus. The virus-induced gene silencing (VIGS) confirmed that LaRAP2.7 knockout decreased QAs content in leaves. β-glucuronidase (GUS), yeast one-hybrid (Y1H), and electrophoretic mobility shift assays (EMSA) showed that LaRAP2.7 bound to the promoter of QA biosynthetic genes (LaLDC and LaCAO) and activated their expression. The naturally occurring Arg196Ser mutation in LaRAP2.7 impaired its binding capacity and reduced transcription of LaLDC and LaCAO, resulting in the sweet trait. Transcriptomic and chromatin accessibility analyses suggested that LaRAP2.7 acted as a cross-tissue regulatory candidate within core QA-associated modules, which were embedded in a broader network containing QA biosynthetic enzymes and putative metabolite transporters. Further, co-expression and ortholog analyses identified purine uptake permease (PUP), nitrate/peptide transporter family (NPF), ATP-binding cassette (ABC), and multidrug and toxic compound extrusion (MATE) family members, including LaPUP1, LaPUP2, and LaNPF1, as candidates for QA source-tosink transport. Functional analysis using RT-qPCR, VIGS, and subcellular localization suggested that LaPUP1 was associated with QA export from source tissues, LaPUP2 was involved in phloem uploading, and LaNPF1 contributed to import into developing seeds.
This study established a mechanistic framework for QA biosynthesis and allocation in NLL. Functional characterization of LaRAP2.7 and three transporters provided promising targets for metabolic engineering in NLL breeding, supporting the development of a safe, pestresistant “bitter/sweet” NLL cultivar.
Details
- Title
- Molecular mechanism regulating quinolizidine alkaloid content in vegetative tissues and seeds of narrow-leafed lupin (Lupinus angustifolius L.)
- Authors/Creators
- Chunsheng Xiao
- Contributors
- Chengdao Li (Supervisor) - Murdoch University, Centre for Crop and Food InnovationGaofeng Zhou (Supervisor) - Murdoch University, School of Agricultural SciencesTianhua He (Supervisor) - Murdoch University, Centre for Crop and Food Innovation
- Awarding Institution
- Murdoch University; Doctor of Philosophy (PhD)
- Identifiers
- 991005898162307891
- Murdoch Affiliation
- Western Crop Genetics Alliance
- Resource Type
- Doctoral Thesis
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