Periodic Reporting for period 1 - UnleashLupin (Unleashing the potential of narrow leaf lupin as locally grown protein crop)
Reporting period: 2020-03-02 to 2022-03-01
As the world population continues to rise, so does the demand for a sustainable plant-based source of protein for human consumption and animal feed. In particular, Europe is in great need of a locally produced high-protein crop as it imports 70% of its protein requirements, relying heavily on genetically modified soybean. Narrow-leafed lupin (Lupinus angustifolius L.) is a grain legume crop that represents an excellent alternative to soybean; the grain contains high protein levels (30‒40%), the crop can be used for sustainable agriculture as it fixes nitrogen in the soil (reducing fertiliser requirements), and it serves as a disease break for other crops. Most importantly, narrow-leafed lupin is a native European species and is grown as a minor grain crop in several countries throughout the European Union.
The major barrier preventing lupin from becoming a more widely cultivated grain crop is the accumulation of toxic specialised metabolites in the grain—the quinolizidine alkaloids. While modern lupin cultivars have been bred to accumulate substantially lower alkaloid levels than their wild counterparts, grain alkaloid levels vary seasonally, often exceeding the threshold for use in industry. Moreover, modern lupin cultivars are more susceptible to herbivores, which is not surprising considering the proposed role of the alkaloids in plant defense. Although little is known about how alkaloids are synthesised, it is accepted that they are synthesised in vegetative tissues and must be transported throughout the plant via the phloem to accumulate in the grain.
The main objective of this UnleashLupin was to identify and characterise transporters involved in the long-distance transport of the alkaloids into the seeds of narrow-leafed lupin. Upon achieving this objective, ‘transport engineering’ of these alkaloids was to be initiated, by knocking out selected transporters to block alkaloid accumulation in the seeds while preserving alkaloid accumulation (and insect deterrence) elsewhere in the plant. This project has achieved most of its objectives and milestones for the period.
The major barrier preventing lupin from becoming a more widely cultivated grain crop is the accumulation of toxic specialised metabolites in the grain—the quinolizidine alkaloids. While modern lupin cultivars have been bred to accumulate substantially lower alkaloid levels than their wild counterparts, grain alkaloid levels vary seasonally, often exceeding the threshold for use in industry. Moreover, modern lupin cultivars are more susceptible to herbivores, which is not surprising considering the proposed role of the alkaloids in plant defense. Although little is known about how alkaloids are synthesised, it is accepted that they are synthesised in vegetative tissues and must be transported throughout the plant via the phloem to accumulate in the grain.
The main objective of this UnleashLupin was to identify and characterise transporters involved in the long-distance transport of the alkaloids into the seeds of narrow-leafed lupin. Upon achieving this objective, ‘transport engineering’ of these alkaloids was to be initiated, by knocking out selected transporters to block alkaloid accumulation in the seeds while preserving alkaloid accumulation (and insect deterrence) elsewhere in the plant. This project has achieved most of its objectives and milestones for the period.
The work performed concerning the identification and characterisation of alkaloid transporters includes the identification of gene candidates using transcriptomic and genomic data, and cloning and testing these candidates for alkaloid transport activity using Xenopus laevis oocytes (frog eggs). The main result is the identification of several genes with alkaloid transporter activity.
To further characterise alkaloid transport in lupin, cell-specific RNA-seq data sets were generated from lupin tissues to identify where exactly these transporters were expressed and how this compares to where the alkaloid biosynthesis takes place. From this information, we could identify those genes with alkaloid transporter activity that were also likely to be facilitating the long-distance transport of alkaloids throughout the lupin plant (as opposed to short-distance). These cell-specific RNA-seq data sets are in preparation to be published so they may be exploited by other lupin researchers.
To carry out ‘transport engineering’ of these alkaloids, a mutagenized population was developed and screened to find lines with mutations in those genes likely to be involved in the long-distance transport of the alkaloids. Several lines harboring mutations in the transporters have been obtained, and their alkaloid phenotype is soon to be assessed. Project results concerning the discovery and characterization of alkaloid transporters will be published upon assessment of the mutant lines.
To further characterise alkaloid transport in lupin, cell-specific RNA-seq data sets were generated from lupin tissues to identify where exactly these transporters were expressed and how this compares to where the alkaloid biosynthesis takes place. From this information, we could identify those genes with alkaloid transporter activity that were also likely to be facilitating the long-distance transport of alkaloids throughout the lupin plant (as opposed to short-distance). These cell-specific RNA-seq data sets are in preparation to be published so they may be exploited by other lupin researchers.
To carry out ‘transport engineering’ of these alkaloids, a mutagenized population was developed and screened to find lines with mutations in those genes likely to be involved in the long-distance transport of the alkaloids. Several lines harboring mutations in the transporters have been obtained, and their alkaloid phenotype is soon to be assessed. Project results concerning the discovery and characterization of alkaloid transporters will be published upon assessment of the mutant lines.
Transport engineering is an exciting new field for the improvement of crop plants and can be used to decrease the accumulation of toxic compounds in edible tissues or to increase yields of commercially valuable compounds. However, incredibly little is known about the mechanisms underlying the long-distance transport of specialised metabolites in plants. Here, for the first time, we have identified genes involved in the transport of lupin alkaloids. Additionally, while the phenotype of mutant lines is yet to be assessed, if the transport engineering of the alkaloids is successfully achieved, this will represent only the second example of transport engineering in a crop plant (the first being the transport engineering of glucosinolates in Brassica spp.). The transport engineering of the alkaloids will greatly increase the value of lupin, promote its use in sustainable farming systems, and potentially supply Europe with a locally-grown source of protein.