White lupin (Lupinus albus L.) is an annual crop cultivated for its protein-rich seeds. It is adapted to poor soils due to the production of cluster roots, which are made of dozens of determinate lateral roots that drastically improve soil exploration and nutrient acquisition (mostly phosphate). Using long-read sequencing technologies, we provide a high-quality genome sequence of a cultivated accession of white lupin (2n = 50, 451 Mb), as well as de novo assemblies of a landrace and a wild relative. We describe a modern accession displaying increased soil exploration capacity through early establishment of lateral and cluster roots. We also show how seed quality may have been impacted by domestication in term of protein profiles and alkaloid content. The availability of a high-quality genome assembly together with companion genomic and transcriptomic resources will enable the development of modern breeding strategies to increase and stabilize white lupin yield.

We report the first transcript profiling of a high-alkaloid lupin variety and identify a new alkaloid biosynthetic gene, thus accelerating the efforts to develop alkaloid-free 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.

Faba bean ( Vicia faba L.) is a widely adapted and high-yielding legume cultivated for its protein-rich seeds ( 1 ). However, the seeds accumulate the pyrimidine glucosides vicine and convicine, which can cause hemolytic anemia (favism) in 400 million genetically predisposed individuals ( 2 ). Here, we use gene-to-metabolite correlations, gene mapping, and genetic complementation to identify VC1 as a key enzyme in vicine and convicine biosynthesis. We demonstrate that VC1 has GTP cyclohydrolase II activity and that the purine GTP is a precursor of both vicine and convicine. Finally, we show that cultivars with low vicine and convicine levels carry an inactivating insertion in the coding sequence of VC1 . Our results reveal an unexpected, purine rather than pyrimidine, biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free of these anti-nutrients.

25Faba bean is a widely adapted and high-yielding legume cultivated for its protein-rich seeds 1 . 26 However, the seeds accumulate the anti-nutritional pyrimidine glucosides vicine and convicine, which 27 can cause haemolytic anaemia-favism-in the 400 million individuals genetically predisposed by a 28 deficiency in glucose-6-phosphate dehydrogenase 2 . Here, we identify the first enzyme associated with 29 vicine and convicine biosynthesis, which we name VC1. We show that VC1 co-locates with the major QTL 30 for vicine and convicine content and that the expression of VC1 correlates highly with vicine content 31 across tissues. We also show that low-vicine varieties express a version of VC1 carrying a small, frame-32 shift insertion, and that overexpression of wild-type VC1 leads to an increase in vicine levels. VC1 33 encodes a functional GTP cyclohydrolase II, an enzyme normally involved in riboflavin biosynthesis from 34 the purine GTP. Through feeding studies, we demonstrate that GTP is a precursor of vicine both in faba 35 bean and in the distantly related plant bitter gourd. Our results reveal an unexpected biosynthetic origin 36 for vicine and convicine and pave the way for the development of faba bean cultivars that are free from 37 these anti-nutrients, providing a safe and sustainable source of dietary protein. 38 39 Main Text 40 According to the UN's Intergovernmental Panel on Climate Change (IPCC), switching to a plant-based 41 diet can reduce carbon emissions, especially in the West 3 . The suggested change in diet will require a wider 42and more varied cultivation of locally adapted protein crops. On a worldwide basis, faba bean ( Fig. 1a) has 43 the highest yield of the legumes after soybean (1.92 Mg/ha in 2013-2017) 4 and the highest seed protein 44 content of the starch-containing legumes (29% dry-matter basis) 5 . Furthermore, faba bean is adapted to 45 cool climates such as Mediterranean winters and northern European summers, where soybean performs 46 poorly 6 . The main factor restricting faba bean cultivation and consumption is the presence of the anti-47 2, Supplementary File 6). Using seed coat cDNA and PCR primers able to distinguish between VC1 and vc1, 132

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.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report the assembly of an improved N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated 3-4 times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and opens the possibility that BBLs are the sought-after coupling enzymes in nicotine biosynthesis.

White lupin (Lupinus albus L.) is a legume that produces seeds recognized for their high protein content and good nutritional value (lowest glycemic index of all grains, high dietary fiber content, and zero gluten or starch) 1-5 . White lupin can form nitrogen-fixing nodules but has lost the ability to form mycorrhizal symbiosis with fungi 6 . Nevertheless, its root system is well adapted to poor soils: it produces cluster roots, constituted of dozens of determinate lateral roots that improve soil exploration and phosphate remobilization 7 . As phosphate is a limited resource that comes from rock reserves 8 , the production of cluster roots is a trait of interest to improve fertilizers efficiency. Using long reads sequencing technologies, we provide a high-quality genome sequence of a modern variety of white lupin (2n=50, 451 Mb), as well as de novo assemblies of a landrace and a wild relative. We describe how domestication impacted soil exploration capacity through the early establishment of lateral and cluster roots. We identify the APETALA2 transcription factor LaPUCHI-1