Quinolizidine alkaloids (QAs) are toxic secondary metabolites found within the genus Lupinus, some species of which are commercially important grain legume crops including Lupinus angustifolius (narrow-leafed lupin, NLL), L. luteus (yellow lupin), L. albus (white lupin), and L. mutabilis (pearl lupin), with NLL grain being the most largely produced of the four species in Australia and worldwide. While QAs offer the plants protection against insect pests, the accumulation of QAs in lupin grain complicates its use for food purposes as QA levels must remain below the industry threshold (0.02%), which is often exceeded. It is not well understood what factors cause grain QA levels to exceed this threshold. Much of the early work on QA biosynthesis began in the 1970–1980s, with many QA chemical structures well-characterized and lupin cell cultures and enzyme assays employed to identify some biosynthetic enzymes and pathway intermediates. More recently, two genes associated with these enzymes have been characterized, however, the QA biosynthetic pathway remains only partially elucidated. Here, we review the research accomplished thus far concerning QAs in lupin and consider some possibilities for further elucidation and manipulation of the QA pathway in lupin crops, drawing on examples from model alkaloid species. One breeding strategy for lupin is to produce plants with high QAs in vegetative tissues while low in the grain in order to confer insect resistance to plants while keeping grain QA levels within industry regulations. With the knowledge achieved on alkaloid biosynthesis in other plant species in recent years, and the recent development of genomic and transcriptomic resources for NLL, there is considerable scope to facilitate advances in our knowledge of QAs, leading to the production of improved lupin crops.

This review summarizes the different hypotheses surrounding the biosynthesis of quinolizidine alkaloids in lupins and discusses strategies for gene discovery and pathway elucidation.

Quinolizidine alkaloids (QAs) are toxic secondary metabolites that complicate the end use of narrow-leafed lupin (NLL; Lupinus angustifolius L.) grain, as levels sometimes exceed the industry limit for its use as a food and feed source. The genotypic and environmental influences on QA production in NLL are poorly understood. Here, the expression of QA biosynthetic genes was analysed in vegetative and reproductive tissues of bitter (high QA) and sweet (low QA) accessions. It was demonstrated that sweet accessions are characterized by lower QA biosynthetic gene expression exclusively in leaf and stem tissues than bitter NLL, consistent with the hypothesis that QAs are predominantly produced in aerial tissues and transported to seeds, rather than synthesized within the seed itself. This analysis informed our identification of additional candidate genes involved in QA biosynthesis. Drought and temperature stress are two major abiotic stresses that often occur during NLL pod set. Hence, we assessed the effect of drought, increased temperature, and their combination, on QA production in three sweet NLL cultivars. A cultivar-specific response to drought and temperature in grain QA levels was observed, including the identification of a cultivar where alkaloid levels did not change with these stress treatments.

Ecosystem functionality is an increasingly important objective of ecological restoration. Despite this, a few studies have rigorously assessed reproductive functionality within restored plant populations, and it is largely assumed that pollinators follow restoration of plant communities-"build it and they will come." Here, we applied an ecological genetic approach to determine the impact of spatial separation on mating in Banksia menziesii (Proteaceae), a dominant bird-pollinated species of Banksia woodlands of Western Australia. All plants at three post-mining restored sites (n = 72 [13 years old], n = 21 [8 years old], and n = 20 [9 years old]), as well as a sample from an adjacent natural reference site (n = 42), were genotyped at nine microsatellite loci. Seed set, mating system parameters, realized pollen dispersal through the assignment of paternity to seed, and avian pollinator species composition, abundance and behavior, were assessed. All patches displayed equivalent heterozygosity (H e = 0.53-0.59) and very weak genetic divergence (F ST ≤ 0.01). Seed of plants within restored sites showed complete outcrossing and relatively high seed set, 26% of which were sired by pollen donors located beyond the local patch. Similar abundance and movement of nectar-feeding birds was observed in restored and natural sites, despite lower bird species diversity in the restored site, where a smaller, less aggressive species was dominant. Our results demonstrate the restitution of wide outcrossing in these restored Banksia patches within an active mine-site, and suggest that restored bird-pollinated Banksia populations are resilient to human impacts, due largely to their generalist pollinator requirements and highly-mobile avian pollinators.

Background Whilst information regarding small RNAs within agricultural crops is increasing, the miRNA composition of the nutritionally valuable pulse narrow-leafed lupin ( Lupinus angustifolius ) remains unknown. Results By conducting a genome- and transcriptome-wide survey we identified 7 Dicer-like and 16 Argonaute narrow-leafed lupin genes, which were highly homologous to their legume counterparts. We identified 43 conserved miRNAs belonging to 16 families, and 13 novel narrow-leafed lupin-specific miRNAs using high-throughput sequencing of small RNAs from foliar and root and five seed development stages. We observed up-regulation of members of the miRNA families miR167, miR399, miR156, miR319 and miR164 in narrow-leafed lupin seeds, and confirmed expression of miR156, miR166, miR164, miR1507 and miR396 using quantitative RT-PCR during five narrow-leafed lupin seed development stages. We identified potential targets for the conserved and novel miRNAs and were able to validate targets of miR399 and miR159 using 5′ RLM-RACE. The conserved miRNAs are predicted to predominately target transcription factors and 93% of the conserved miRNAs originate from intergenic regions. In contrast, only 43% of the novel miRNAs originate from intergenic regions and their predicted targets were more functionally diverse. Conclusion This study provides important insights into the miRNA gene regulatory networks during narrow-leafed lupin seed development. Electronic supplementary material The online version of this article (10.1186/s12864-019-5521-8) contains supplementary material, which is available to authorized users.

SUMMARY Narrow‐leafed lupin (NLL; Lupinus angustifolius) is a key rotational crop for sustainable farming systems, whose grain is high in protein content. It is a gluten‐free, non‐genetically modified, alternative protein source to soybean (Glycine max) and as such has gained interest as a human food ingredient. Here, we present a chromosome‐length reference genome for the species and a pan‐genome assembly comprising 55 NLL lines, including Australian and European cultivars, breeding lines and wild accessions. We present the core and variable genes for the species and report on the absence of essential mycorrhizal associated genes. The genome and pan‐genomes of NLL and its close relative white lupin (Lupinus albus) are compared. Furthermore, we provide additional evidence supporting LaRAP2‐7 as the key alkaloid regulatory gene for NLL and demonstrate the NLL genome is underrepresented in classical NLR disease resistance genes compared to other sequenced legume species. The NLL genomic resources generated here coupled with previously generated RNA sequencing datasets provide new opportunities to fast‐track lupin crop improvement.

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.

Quinolizidine alkaloids (QAs) are toxic secondary metabolites produced in lupin species that protect the plant against insects. They form in vegetative tissues and accumulate to a different extent in the grains: high levels in ‘bitter’ narrow-leafed lupin (NLL) and low levels in ‘sweet’ NLL. Grain QA levels vary considerably, and sometimes exceed the industry limit for food and feed purposes. We hypothesised that jasmonates regulate QA biosynthesis in response to environmental stresses such as wounding and aphid predation, which may explain non-genetic variability in grain QA levels. Methyl jasmonate (MeJA)-inducible genes were identified and verified in NLL. Exogenous MeJA application-induced expression of QA biosynthetic genes and QA levels for bitter, but not sweet NLL. Although MeJA-inducible genes responded to wounding, the expression of QA biosynthetic genes was not induced for bitter and sweet NLL. We assessed the effect of aphid predation on QA production for two cultivars – one moderately resistant and one susceptible to aphid predation. Although MeJA-inducible genes responded to aphid predation, no change in QA levels was found for either cultivar. These findings offer insights into the regulation of QA biosynthesis in bitter and sweet NLL and concludes that aphids are not a concern for increasing grain QAs in NLL cultivars.