Biosynthesis of quinolizidine alkaloids in lupins: mechanistic considerations and prospects for pathway elucidation.

Covering: up to 2022Quinolizidine alkaloids (QAs) are a class of alkaloids that accumulate in a variety of leguminous plants and have applications in the agricultural, pharmaceutical and chemical industries. QAs are notoriously present in cultivated lupins (Lupinus spp.) where they complicate the use of the valuable, high-protein beans due to their toxic properties and bitter taste. Compared to many other alkaloid classes, the biosynthesis of QAs is poorly understood, with only the two first pathway enzymes having been discovered so far. In this article, we review the different biosynthetic hypotheses that have been put forth in the literature (1988-2009) and highlight one particular hypothesis (1988) that agrees with the often ignored precursor feeding studies (1964-1994). Our focus is on the biosynthesis of the simple tetracyclic QA (-)-sparteine, from which many of the QAs found in lupins derive. We examine every pathway step on the way to (-)-sparteine and discuss plausible mechanisms, altogether proposing the involvement of 6-9 enzymes. Together with the new resources for gene discovery developed for lupins in the past few years, this review will contribute to the full elucidation of the QA pathway, including the identification and characterization of the missing pathway enzymes.

Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin.

BACKGROUND: Lupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. The new resources facilitate the in silico identification of candidate genes controlling important agronomic traits. However, a major bottleneck for lupin research and crop improvement is the in planta characterization of gene function. Here, we present an efficient protocol for virus-induced gene silencing (VIGS) to down-regulate endogenous genes in narrow-leafed lupin (NLL) using the apple latent spherical virus (ALSV). RESULTS: We identified ALSV as an appropriate VIGS vector able to infect NLL without causing a discernible phenotype. We created improved ALSV vectors to allow for efficient cloning of gene fragments into the viral genome and for easier viral propagation via agroinfiltration of Nicotiana benthamiana. Using this system, we silenced the visual marker gene phytoene desaturase (PDS), which resulted in systemic, homogenous silencing as indicated by bleaching of newly produced tissues. Furthermore, by silencing lysine decarboxylase (LaLDC)-a gene likely to be involved in toxic alkaloid biosynthesis-we demonstrate the applicability of our VIGS method to silence a target gene alone or alongside PDS in a 'PDS co-silencing' approach. The co-silencing approach allows the visual identification of tissues where silencing is actively occurring, which eases tissue harvesting and downstream analysis, and is useful where the trait under study is not affected by PDS silencing. Silencing LaLDC resulted in a ~ 61% or ~ 67% decrease in transcript level, depending on whether LaLDC was silenced alone or alongside PDS. Overall, the silencing of LaLDC resulted in reduced alkaloid levels, providing direct evidence of its involvement in alkaloid biosynthesis in NLL. CONCLUSIONS: We provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.