White lupin: improving legume-based protein production via intercropping

White lupin: improving legume-based protein production via intercropping | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 January 2025 V1 Latest version Share on White lupin: improving legume-based protein production via intercropping Authors : Corentin Dourmap 0000-0003-3572-0887 [email protected] , Joelle Fustec , Christophe Naudin , Nicolas Carton , and Guillaume Tcherkez 0000-0002-3339-956X Authors Info & Affiliations https://doi.org/10.22541/au.173679744.40722459/v1 Published Journal of Experimental Botany Version of record Peer review timeline 454 views 301 downloads Contents Abstract Efficacy of lupin nutrient acquisition PULSAR: improving the production of plant-based protein via lupin intercropping Acknowledgements Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Climate change, increased needs for food, industry and mitigation of environmental impacts are currently driving changes in agricultural practices. Moreover, increasing demand for plant-based protein in substitution to animal protein or to reduce soybean importations is driving cultivation of high-protein crops. Legumes are such crops that play a critical role in this process. Amongst them, white lupin is a so-called orphan species, i.e. associated with relatively little cultivated surface area worldwide and limited agronomic knowledge. Lupin is nevertheless very promising since seeds contain a high content of storage proteins with interesting nutritional properties. Also, it has low fertilisation requirements since it forms root clusters allowing efficient phosphorus (P) acquisition, along with symbiotic nitrogen (N) fixation by nodules. Nevertheless, lupin cultivation faces important challenges such as yield variability, slow vegetative development or susceptibility to weeds diseases and water stress, for example. Lupin has an enormous potential for resource-saving practices such as intercropping with non-legumes, because of niche complementarity for N acquisition and facilitation of P transfer to the associated species, which can in turn mitigate weeds and pests, and ensure yield stability. To overcome several bottlenecks associated with lupin cultivation (e.g. nutrient utilisation, drought resistance or limiting the impact of weeds), genetic, metabolic, and agronomic research is required in order to define ideotypes that are particularly well-fitted to sustainable agricultural practices such as intercropping, with optimal protein yield. This is one of the purposes of the trans-disciplinary research programme PULSAR, funded by France 2030, which aims to unlock several bottlenecks in lupin utilisation in agronomy. Keywords: Lupinus albus , intercropping, metabolism, cluster root, France 2030 PULSAR project Highlights White lupin has is a legume with high potential for plant-based protein production. PULSAR is a project funded by France 2030 to study lupin unique physiological traits and overcome several challenges associated with lupin cultivation with emphasis on intercropping. Introduction Western agriculture is currently facing critical ecological and socio-economic challenges. Reducing both chemical inputs (fertilisation, fungicides, herbicides) and greenhouse gas emissions (GHG) are two of them. In effect, agriculture is responsible for around 20% of GHG emissions, in particular methane produced by bovine livestock intensive farming and nitrous oxides produced by denitrification of fertilisers (Chataut et al., 2023; Liu et al., 2019). Fertilisation may also lead to eutrophication of water ecosystems inducing green algae tides on seashores (Morand and Briand, 1996). Furthermore, fertilisers requiring fossil fuels like oil and gas for production are susceptible to market variations and thus contribute to make agricultural prices more volatile (Etienne et al., 2016). In an effort to mitigate environmental effects of farming, current recommendations include. a reduction in animal-based products as opposed to plant-based proteins for human nutrition but also a change in agricultural practices. This includes a change from conventional cultivation to intercropping in order to ( i ) increase biodiversity, ( ii ) improve nutrient efficiency, and ( iii ) fight against pests and weeds with less chemical inputs (Friel et al., 2009). Aside intercropping, crop diversification is of particular relevance in particular via co-cultivation with legumes (Magrini et al., 2016; Meynard et al., 2018; Naudin et al., 2014). However, European food industry still relies on soybean imported from both North and South America and used for animal feeding, although it is recognised that soybean cultivation contributes to deforestation in producing countries like Brazil (Boerema et al., 2016; de Visser et al., 2014; Våge and Lea, 2021). For these reasons, Western countries aim to increase the production of proteaginous crops, either as pure or intercropped species. Amongst proteinaceous crops, legumes including pea, beans, lupin, lentil, and other pulses are of particular interest (Magrini et al., 2016). First, legumes establish symbioses with Rhizobia bacteria making them capable of atmospheric dinitrogen (N 2 ) utilisation as a nitrogen source (Mus et al., 2016; Soltis et al., 1995). Consequently, legumes have low requirements for nitrogen inputs (Peoples et al., 1995). Thus, cultivating legumes is less expensive in terms of fertilisation but also emits five times less GHG per surface area unit than the average of other crops in European countries like France (Magrini et al., 2016). Second, legumes are diverse and thus there are many seed protein compositions or potential industrial utilisations, from forage (e.g. alfalfa) (Peyraud et al., 2009) to protein isolates for body building and dairy supplements (e.g. lupin) (Garba and Kaur, 2014; Nichele et al., 2022). Amongst legumes, lupin ( Lupinus ) has one of the highest potentials for human food products (Arnoldi et al., 2015; Kohajdova et al., 2011; Van de Noort, 2024). In this Highlight, we provide a brief description of lupin biological peculiarities showing that this species is certainly of interest to help meet the challenges faced by modern agriculture. This is discussed in the framework of one of the biggest projects to date (about 2.7 M€) dedicated to this species, PULSAR funded by France 2030, where agronomic strategies based on intercropping are proposed. In effect, as will become apparent below, intercropping with lupin can help improve resource saving and enhance resilience to biotic and abiotic stresses. This Highlight mostly refers to white lupin, which is more represented in Western European countries like France (narrow-leafed lupin being mostly used for forage). This Highlight is also a call to promote research on lupin biology in order to obtain new varieties adapted to cropping systems guided by future societal needs. This will be instrumental to find solutions for agricultural, ecological and socio-economic challenges, which are addressed by the project PULSAR. What is lupin? The genus Lupinus is diverse and comprises about 300 species mostly originating from the Mediterranean basin and America (Drummond et al., 2012; Hughes and Eastwood, 2006; Marques et al., 2022). In Europe, lupin was originally cultivated on coastline of North-West in the 1930s, taking advantage of sandy soils (Jensen et al., 2004). Three species are of agricultural interest: Lupinus angustifolius (narrow-leafed or blue lupin), Lupinus luteus (yellow lupin) and Lupinus albus (white lupin). All have known (sequenced) genome (Duranti et al., 2008; Hane et al., 2017; Hufnagel et al., 2021; Marques et al., 2022). Lupinus albus comes from the Mediterranean basin and its genome of 451 Mb was published in 2020. It contains about 38,200 genes, possesses 60% of repeated and transposable elements, and genetic analyses of synteny showed ancestral links with 12 ancient and modern legumes (Hufnagel et al., 2021) − for a review of Lupinus domestication and species description, see (Drummond et al., 2012). Lupin seeds have a unique composition amongst legumes, with beneficial properties for human health. In fact, they are rich in proteins (32-40%, comparable to soybean), oil and secondary metabolites such as polyphenols, phytosterols, tocopherols and triterpenes (Khan et al., 2015). However, although most of these compounds have antioxidant, anti-inflammatory, anti-carcinogenic or anti-microbial activity (like polyphenols), some of them (alkaloids) are anti-nutritive (Khan et al., 2015). Thus, in the past decades, breeding has focused on their reduction, leading to low-alkaloid digestible commercial varieties (Frick et al., 2017). Gene-based molecular markers tagging low alkaloid locus in white lupin were released and further validated via identification of mutations leading to low alkaloid phenotype (Mancinotti et al., 2023; Rychel and Książkiewicz, 2019). Lupin seeds also have a low glycaemic index and twice as more fibre than soybean (Khan et al., 2015) and might have a hypoglycaemic effect (Arnoldi et al., 2015; Villarino et al., 2016) or be beneficial to reduce oxidative stress in diabetic patients (Metwally et al., 2023). Despite these benefits, lupin cultivation and utilisation is presently limited. It represents a production of slightly more than ten million t only worldwide (Fig. 1b). This paradox is explained by several factors, including potential allergenicity and cultivation constraints such as susceptibility to anthracnose (Rychel-Bielska et al., 2020) and limited tolerance to drought (Pecetti et al., 2023). Despite these issues, lupin has an acceptably good yield for a legume, of 1-1.7 t ha -1 (annual average, Fig. 1a). White lupin presents a relatively slow life cycle (spanning from October to August for winter varieties in the Northern Hemisphere) and a recent genetic study provided some avenues to address this issue (Rychel-Bielska et al., 2024). To date, lupin production mostly concerns Western countries (United States of America, Australia, Canada, and the European Union) and Russia (Fig. 1c). Lupin cultivation is rather heterogeneous within Europe, Poland, Netherlands and Germany being the most important producers. Efficacy of lupin nutrient acquisition Like other legumes, white lupin is able to fix atmospheric N 2 via the symbiosis established with Rhizobia bacteria. In addition, large and N-rich lupin seeds contain a substantial amount of protein and are thus able to sustain seedling growth for a long time at the beginning of the plant life cycle (Dayoub et al., 2022; Herdina and Silsbury, 1990). Consequently, lupin absorbs a modest fraction of its nitrogen from the soil (Carton et al., 2020) and the amount of soil nitrate captured by plants feeds seedling establishment before the onset of symbiosis. Under Mediterranean conditions, N 2 fixation has been estimated at about 300 kg N ha -1 , with fixed N preferentially accumulated into seeds (Sulas et al., 2016). Isotopic labelling has suggested that in white lupin, N found in seeds mostly comes from N 2 fixation occurring before flowering, indicating that nitrogen used to produce seeds involves massive remobilisation from vegetative parts (Schulze et al., 1999). Unlike many other legumes that down-regulate nodulation, white lupin can maintain N 2 fixing symbiosis as its main N source under moderate N fertilization, i.e., up to 5 mM soil nitrate, under controlled conditions (Génard et al., 2016; Guinet et al., 2018). This property is particularly interesting since in most cultivated soils, nitrate concentration is within 1 and 20 mM (Andrews et al., 2013) with a median about 6 mM (Crawford and Glass, 1998). That is, N metabolism of lupin could be more efficient compared to other N 2 fixing species since it does not require extra N input under intermediate situations of N availability (Guinet et al., 2018). That said, in the field, the isotopic dilution method showed that growth with ammonium nitrate decreased the proportion of N coming from fixation by 25% (Evans et al., 1987). When fertilisation is done at flowering, N 2 fixation is nearly completely arrested (Schulze et al., 1999). Furthermore, when white lupin is cultivated with fertilisation (30 kg ha -1 N, along with P and K) and pesticides, there is a noticeable increase (≈20%) in yield (Borowska et al., 2015). White lupin root system has a tap root from which numerous primary lateral roots grow and penetrate deeply in compact soils (Jensen et al., 2004). Under low P availability, lupin roots induce the development of dense root structures (Clements et al., 1993). These structures are referred to as ’cluster roots’ (or proteoid roots, alluding to root clusters observed in Proteaceae) and can be found in few legumes species: lupin, Viminaria juncea , and some species of the genus Daviesia (de Campos et al., 2013; Nge et al., 2020; Shane and Lambers, 2005). In lupin, it is interesting to note that only white lupin presents cluster-roots (narrow-leafed lupin forms proteoid-like roots) (Funayama-Noguchi et al., 2015). Cluster roots allow increased phosphorus acquisition. In fact, inorganic phosphate (Pi) absorption capacity is about 5 times higher in white lupin compared to soybean − which does not form cluster roots (Watt and Evans, 2003). Unlike most Angiosperms, white lupin has lost genes responsible for mycorrhizal symbiosis establishment (Hufnagel et al., 2020) and therefore, cluster roots are crucial for lupin phosphate acquisition. In the field, the high efficacy of cluster roots explains why white lupin has low requirements in phosphate, representing a significant advantage considering the cost of P fertilisation. The development of cluster roots is mediated by sugar signalling from the shoot, which induces phosphate-starvation response genes (Zhou et al., 2008). Moreover, a high CO 2 mole fraction (800 µmol mol -1 ) in the atmosphere is known to accelerate cluster root development (Wasaki et al., 2005). Consequently, it is believed that there is a link between photosynthetic activity and root development, which in turn sustains photosynthetic activity via phosphate provision. Cluster root development of white lupin is also mediated by auxins antagonised by flavonoids (Xiong et al., 2022) and can be modulated by root-inoculated bacterium species independently of bacterial auxin production (Lamont and Pérez-Fernández, 2016). There is an interaction between nodulation and cluster root formation, with a tendency to have a coregulation whereby conditions promoting nodule development are beneficial to cluster root formation (Pueyo et al., 2021; Thuynsma et al., 2014; Wang et al., 2019). The metabolism of cluster roots relies on exudation of organic acids like citrate (Kihara et al., 2003; Zhou et al., 2020). Organic acids allow phosphorus acquisition via anion displacement (phosphorus salt dissociation) thus enriching the soil solution in free K + , Na + and Mg 2+ . However, molecular mechanisms underlying exudation, and its regulation are still not well known. To date, studies focused on metabolic pathways associated with the production of exudated organic acids are contradictory, with either an enhanced cytosolic glycolytic flux, higher pyruvate dehydrogenase and Krebs cycle activity, or a high phospho enol pyruvate carboxylase (PEPC)-catalysed bicarbonate fixation (Johnson et al., 1996; Kihara et al., 2003; Massonneau et al., 2001). Regardless of the metabolic mechanism, organic acid production and exudation consume sugars and thus represent an important sink for carbon allocation (Massonneau et al., 2001; Pate and Herridge, 1978). White lupin seeds are very rich in proteins, which represent about 35% of total dry weight, depending on varieties and growth conditions (Annicchiarico et al., 2014; Bähr et al., 2014; Bhardwaj et al., 1998). It is one of the most elevated protein contents amongst legumes and is comparable to soybean (about 38%) or alfalfa (about 34%) and much higher than chickpea (15-20%) (Carton et al., 2020; Gueguen and Cerletti, 1994; Schroeder et al., 1974) (Fig. 2a). The elemental composition of seed proteins is also similar to soybean or alfalfa, with about 19% N and 1% S (Fig. 2b). Nevertheless, lupin seed protein presents comparatively low amounts of methionine, lysine and tryptophan (Sujak et al., 2006). Seed storage proteins are present as conglutins (α to δ, Fig. 2c), which are glutamine-, glutamate- and arginine-rich proteins like in many other legumes. Conglutins have rather contrasted amino acid compositions: for example, γ-conglutin has a much higher proportion in methionine and arginine compared to β-conglutin (Duranti et al., 2008). Interestingly, γ- and δ-conglutins are relatively enriched in cysteine (conditionally essential amino acid) and leucine (essential amino acid) (Fig. 2c). Cultivated varieties contain less β-conglutin, which correlates with increased seed vigor and lower allergenicity (Hufnagel et al., 2020). Interestingly, probably due to S abundance in seed proteins, sulphur fertilisation has been shown to improve protein content and change protein (and oil) composition (Cazzato et al., 2012). In well-studied plants like Arabidopsis, storage protein production in developing seeds has been demonstrated to be fed by protein remobilisation in leaves (Dourmap et al., 2023; Havé et al., 2017; Masclaux-Daubresse et al., 2008). But unlike Arabidopsis where glutamine prevails as the export form of nitrogen, asparagine represents 50-70% of remobilised nitrogen directed to developing seeds in lupin (Atkins et al., 1975). Asparagine is likely the preferential form of N remobilisation and used as a precursor of other amino acids in the developing lupin seed, because (i) asparagine is not the prevalent amino acid in conglutins (Fig. 2c), (ii) strong arginase activity has been detected and furthermore, upon 15 N-asparagine labelling, 15 N-labelled ammonium, glutamine and alanine have been found (Atkins et al., 1975), (iii) asparagine is the major N compound in phloem sap exudates (Pate et al., 1981). Furthermore, N fixed by nodules is mostly exported in the form of asparagine in lupin (Borek et al., 2009). Lupin seeds also contain lipids, which represent a variable proportion of seed dry weight depending on the species and variety (Borek et al., 2009). In fact, they represent 7 to 14% of the seed dry weight (Borek et al., 2009) (Fig. 2d). It is worth noting that across white lupin varieties, this proportion is lower than in soybean, for example, and does not depend on protein content (Fig. 2d). That is, unlike soybean, there is no strong metabolic trade-off between lipid production and protein accumulation during seed development. Such a trade-off has been found recently in truncated alfalfa ( Medicago truncatula ) in relation to seed size regardless of genetic accession (Domergue et al., 2022). Interestingly, an effect of seed size has also been found in white lupin, heavier seeds being less rich in both oil and proteins, while the protein content appears to be negatively correlated to the total amount of antioxidant compounds (Annicchiarico et al., 2014; Spina et al., 2024; Velasco et al., 1998). These studies also suggest that seed size in white lupin depends on both the variety and the position of the inflorescence onto the plant (main stem, primary or secondary branches). Recently, a metabolomics analysis of seeds from lupin cultivars with various alkaloid content has suggested there is a balance between tocopherol, lipids and free amino acids (Mavromatis et al., 2023). Thus, seed composition of white lupin should be viewed as multidimensional and not simply binary (protein versus oil), with a balance between protein, oil, tocopherol, alkaloids and antioxidants controlled by both genotype and environment, with significant interaction effects. Agricultural practices also influence lupin seed and protein production, such as sowing date or sowing/canopy density, which in turn impact on cumulated photosynthetic carbon. Lupin yield depends mostly on photosynthetic activity and carbohydrate reserves accumulated during flowering and pod initiation, not during seed filling (Jakl and Bolhar-Nordenkampf, 1991; Pate and Herridge, 1978). In addition, isotopic labelling of narrow-leaved lupin with 14 CO 2 has shown that labelled carbon found in seed proteins originated from photosynthetic activity before and during flowering, while carbon fixed during seed filling was found in non-protein seed components (Pate et al., 1980). Intercropping with lupin to address agroecological challenges Why use lupin for intercropping? Although white lupin possesses numerous advantages due to nutrient assimilation, seed metabolism and biochemical composition, its cultivation in the field still faces numerous challenges. First, its yield is highly variable from year to year (Cernay et al., 2015) and between locations because of considerable vegetative biomass (which is sensitive to environmental conditions), late seed maturation and heterogeneity of pod development, dependence on water availability, climate, agricultural practices and geography (Borowska et al., 2017; Crochemore et al., 1994; Julier et al., 1995; Prusinski, 2021; Tobiasz-Salach et al., 2023; Valente et al., 2024). Lupin is also highly sensitive to biotic (pests, diseases, weeds) and abiotic stress, especially in winter varieties that have a long life cycle. In fact, white lupin cultivated varieties are subdivided into winter cultivars (sown during fall) and spring cultivars (sown at the end of winter) (Gladstones, 1970; Huyghe, 1997). In both winter and spring cultivars, harvesting takes place generally in late July or early August. Under optimal water availability, lupin has a high stomatal conductance and high photosynthetic activity. However, it has a generally low intrinsic water use efficiency of about 10-30 µmol CO 2 mol -1 H 2 O (Ramalho and Chaves, 1992), with strong stomatal closure under water deficit (Rodrigues et al., 1995). Therefore, lupin is considered as being highly sensitive to water availability (Jensen et al., 2004; Palta et al., 2012). That said, there are important differences between cultivars in the response to water restriction in terms of seed production (Annicchiarico et al., 2018; Carvalho et al., 2005; Pecetti et al., 2023). Also, young developing lupin leaves can acclimate to water stress, with an adjustment of the photosynthetic machinery (Palta et al., 2012). When water restriction is pronounced and leads to mature leaf loss, sugars stored in stems can be used to reform new leaves upon rewatering (Withers, 1979; Withers and Forde, 1979). To our knowledge, to date, there has been no study focused on water use efficiency and breeding based on carbon stable isotopes (δ 13 C value) in lupin unlike other legumes, such as chickpea (Ayalew et al., 2022; Lusiba et al., 2022). To save resources (including water) and alleviate detrimental effects of stress on photosynthesis and yield, intercropping can be envisaged for lupin. Intercropping refers to agroecological practices for crop diversification consisting in mixing two or more arable crop species together in the same field (Willey, 1979). It has long been recognized to improve sustainability of crop production, in particular in low-input systems and under sub-optimal agronomic conditions (Bedoussac et al., 2015; Li et al., 2010; Martin-Guay et al., 2018). Intercropping legumes with cereals has been used to obtain higher total grain yield or produce cereals with higher protein content while reducing the use of N fertilisers (Bedoussac et al., 2015). It has also been used to facilitate cultivation of a legume (lentil) and improve its profitability in terms of mean marketable gross margin (Viguier et al., 2018). Intercropping can be used for lupin as a cash crop (Carton et al., 2018; Carton et al., 2020) along with a service crop which provides ‘ecosystemic services’ (e.g., weed control). Although the major role of the service crop is not yielding, it can also be harvested to help securing production under severe climatic events (Mousavi and Eskandari, 2011). There are several intercropping strategies depending on service crop agronomy (Gardarin et al., 2022): ( i ) synchronous intercropping where the life cycle of the two cultivated species coincide; ( ii ) semi-permanent or permanent-living mulch where the service crop is not an annual plant and plays the role of field covering including when the cash crop is sown; and ( iii ) relay intercropping where the service crop is sown in time windows between two cash crops. Intercropping may use several cash crops simultaneously (Malagoli et al., 2020; Naudin et al., 2010). Legumes and grasses represent the majority of crops used as service crops (Duchene et al., 2017; Gardarin et al., 2022). Intercropping where the legume is the cash crops is less common (Cheriere et al., 2023; Dayoub et al., 2022). Notable examples where lupin has been used in intercropping are summarised in Table 2. Pros and cons of the use of lupin for intercropping are discussed below. A key advantage of successful intercropping is that it improves productivity and stability of yield while decreasing the use of chemical inputs (Brooker et al., 2015; Gaba et al., 2014; Weih et al., 2021). Cereal-legumes intercropping can increase yield and seed quality, especially the protein content (Jensen et al., 2020; Naudin et al., 2010; Pelzer et al., 2012). With intercropping, the yield of legume crops decreases by 39-44% on average compared to legume cultivated as sole crop (also often referred to as ‘monoculture’), while total yield (cereal + legume) increases by 26-37% compared to legume sole crop (Carton et al., 2020; Jensen et al., 2020; Layek et al., 2018). Quite understandably, the yield gain provided by intercropping is higher using low-yield legume species and even more when resources are limited. With triticale-lupin intercropping, lupin yield is generally lower than that obtained with lupin sole crop but higher when triticale contribution is included (Table 2). The efficacy of cereal-legume intercropping is often calculated on the basis of land equivalent ratio (LER) (de Wit and van den Bergh, 1965; Willey and Osiru, 1972). LER values higher than 1 indicate a per-area advantage of intercropping compared to sole crops, usually driven by an improved use of environmental resources. LER for cereal-legume intercrops is often higher than 1 and can reach 1.5 when low yield levels are observed in one or several sole crops (Salinas-Roco et al., 2024; Yu et al., 2015). Intercropping lupin (var. Magnus ) with wheat (var. Rubisko ) leads to higher protein yield than wheat or lupin sole crops (Carton et al., 2018; Carton et al., 2020). Probable reasons to explain the potential yield improvement with lupin intercropping are provided below. Lupin to improve resource use In intercropping, as opposed to sole crops, each companion species can benefit from two major positive interactions between them: facilitation and complementarity for available resources. Under abiotic or biotic yield-limiting conditions, these interactions are beneficial to agrosystem resilience, leading to a better growth rate for both cash and service crops, due to nutrient transfers and nutritional specialisation (niche separation) (Brooker et al., 2008; Rao and Willey, 1980). For instance, when a legume is intercropped with a non N 2 -fixing species, niche separation for N utilisation is maximized, i.e. with a differential use of available resource (seed, soil N pools, atmosphere) (Corre-Hellou et al., 2006). Different root distributions may also result in better exploration of the whole soil volume compared with sole crops (Corre-Hellou and Crozat, 2005; Hinsinger et al., 2011). In addition, as the non N 2 -fixing species depletes the soil in N near their roots, the percentage of N derived from fixation of the legume is increased by about 10% in intercropping compared to sole legume crop (Bedoussac et al., 2015; Naudin et al., 2010). Similarly, niche separation can occur between a legume and a non N 2 -fixing species for phosphorus utilisation. Facilitation involving plant-to-plant P transfer has also been demonstrated in legume-cereal intercrops (Hinsinger et al., 2011; Xue et al., 2016). Nevertheless, under optimal, non-limiting conditions, a compromise has to be found between ecosystemic services and production (of the cash crop) when selecting species and varieties, because competition of the two species for resources, rather than complementarity or facilitation, might prevail (Cheriere et al., 2020; Corre-Hellou and Crozat, 2005). White lupin is a relatively large plant, with long stems and many leaves and thus it can be viewed as competitive for light, with strong variation in light interception depending on populations, sowing date and sowing density (Duthion et al., 1994; Herbert, 1977; López-Bellido et al., 1994; Tobiasz-Salach et al., 2023). In terms of water, because of the relatively poor water use efficiency, lupin is a rather poorly competitive crop. In other words, depending on the intended intercropping strategy and available resources, lupin can be used either as a cash crop or a service crop. The service crop used in intercropping can be used to control weeds, typically via rapid utilisation of nutrients, water and light that are no longer available for weeds (Liebman and Staver, 2001). Nevertheless, the efficiency of weed control via the use of a service crop depends on the cash crop itself and is generally not as efficient as the use of herbicides (Gardarin et al., 2022). Intercropping is of interest for legumes since they are not very competitive compared to weeds, leading to yield losses, N consumption by weeds at the expense of crops, and weed persistence via seed spreading (Knott and Halila, 1988; Seymour et al., 2012). Intercropping legumes with a non-legume species is always beneficial for weed management compared to legume sole crop. Reciprocally, weeds tend to be less abundant when legumes are intercropped with cereals, as compared to cereal monoculture (Carton et al., 2020; Corre-Hellou et al., 2011).When lupin was intercropped with triticale, a substantial reduction in weed biomass (but not weed species richness) compared to lupin sole crop has been found, perhaps due to allelopathic effects and nutrient utilisation by triticale which outcompetes weeds (Table 2) (Carton et al., 2020). This result could be particularly interesting with winter lupin because weeds that take advantage of slow lupin development during seedling establishment would be inhibited by the fast vegetative development of triticale (Carton et al., 2018; Carton et al., 2020). Service crops can also help control pests. Pests and associated damages on crops are generally less numerous when plant diversity is high (Iverson et al., 2014; Risch and Risch, 1983). The first effect of the service crop is to produce additional biomass that offers alternative resources for pests (Malézieux et al., 2009). It can also be regarded as more attractive for pests than cash-crops. It has been demonstrated that intercropping of onion with phacelia and buckwheat reduces attacks of Thrips but lowered onion yield due to high growth competitiveness (Trdan et al., 2006). Similar intercropping could be useful for lupin since it is very sensitive to Thrips species and has a larger biomass than onion thus able to compete with phacelia or buckwheat. Service crops can also have a repulsive effect on pests (Beizhou et al., 2012) or attract natural predators of pests. To our knowledge, to date, such intercropping strategies have not been documented with lupin. Service crops can protect soil form erosion thereby preventing up to 50% of nutrient content from leaching out (Valkama et al., 2015). In addition, if service crop biomass is let down onto the soil and decomposes, it participates to increasing soil organic matter. This strategy has been suggested with lupin (Carranca et al., 2009a; Carranca et al., 2009b). This may be of high interest to increase soil nutrient availability, and thus benefit the yield of the subsequent crop. When legumes are chosen as service crops, fixed nitrogen is transferred to the soil via rhizodeposition and decomposition of legume residues (Génard et al., 2016). With lupin, there is an improvement of total (legume + cereal) soil-derived N acquisition of about 70% (Rodriguez et al., 2020). Significant rhizodeposition by lupin has been suggested via isotopic labelling with 15 N-urea and might represent up to 17% of N absorbed by lupin (Russell and Fillery, 1996). As stated above, interspecific competition for soil resources between intercropped species can be significantly reduced through complementarities resulting in niche separation in time, space and nutrient pools. That is, intercropped species may exhibit contrasting phenologies and/or growth periods, which may result in differential nutrient requirements over time. Direct or indirect N transfers from the legume to another, non-fixing crop generally are believed to occur but in annual crops, their contribution to the performance of the companion cash crop is controversial because it appears to be highly dependent on the degradability of legume organic matter, root architecture, microbial community activity and composition, as well as climate and soil properties (Génard et al., 2016; Jamont et al., 2013; Li et al., 2007; Lorin et al., 2015; Thilakarathna et al., 2016). Intercropping with white lupin led to variable results. In the field, when intercropped with triticale, N provided by lupin via both rhizodeposition and residues mineralisation represented 22% of N needs and thus was insufficient to arrest fertilisation (Carton et al., 2020). That said, N fixed by the legume and eventually neither available nor used by the intercropped companion species may enter longer term agrosystems N cycle and thus, should be available for subsequent crops (Kuzyakov and Xu, 2013). Unlike nitrogen, P transfer to the companion species has been more clearly demonstrated at least with annual intercrops (Hinsinger et al., 2011; Li et al., 2007). Lupin has been shown to have a beneficial effect on phosphorus available to rapeseed (Chen et al., 2023). This beneficial effect is particularly interesting since rapeseed has relatively high N and P needs and is very sensitive to phosphorus starvation (Girondé et al., 2015). Moreover, white lupin had no detrimental effect on rapeseed growth (unlike lentil or pea) and increased soil microbial diversity, especially in phosphorus-solubilising bacteria and fungi (Chen et al., 2023). It has been shown that wheat-intercropped white lupin depleted two distinct inorganic P fractions, resulting in better phosphorus use efficiency (Cu et al., 2005). Nevertheless, beneficial effects of lupin on nutrient release and availability depend on soil physicochemical properties. For example, intercropping barley with lupin led to an accumulation of rare earth elements in barley when it was grown on alkaline soil whereas the opposite was found when cultivation was conducted on acidic soil (Monei et al., 2022). This soil effect was probably due to cluster roots involving acidification via organic acids exudation in base/cation-rich soils thus being of limited efficacy under already acidic, cation-poor conditions. Interestingly, cluster roots are known to accumulate heavy metals (Tian et al., 2009). Thus, intercropping with lupin could also be useful for bioremediation. Collectively, available data suggest that lupin intercropping has considerable agronomic interest because yield loss (as compared to pure lupin cultivation) should, in principle, be compensated by gains obtained by overcoming abiotic and biotic stress, or advantages provided to the associated species (Carton et al., 2020). It should be kept in mind that lupin intercropping may imply modifications of agronomic practices. For example, fertilisation of intercrops where the cereal is the cash crop should be adjusted to meet cereal requirements while not being detrimental to lupin itself (Naudin et al., 2010). Also, sowing density has consequences on yield in a non-linear manner, i.e. with a plateau effect (Hauggaard-Nielsen et al., 2008). In the case of lupin intercropping, such a dependency has been shown too (Borowska et al., 2017; Fayaud et al., 2014; Prusinski, 2021; Tobiasz-Salach et al., 2023). The choice of the partner species intercropped with lupin is also an important aspect to consider because there is a compromise between developmental rate, light interception, weed-control ability, etc. For example, oat has higher capacity to reduce weeds than wheat, barley, or triticale but may have a stronger light interception effect (Deveikyte et al., 2009). As already suggested before, a balance between plant traits must be found to make interspecific competition weaker than intra-specific competition (Vandermeer et al., 1998) (Carton et al., 2018; Carton et al., 2020). Unfortunately, to date, despite local initiatives in France, Sweden and Germany, there is limited published information available to determine the best partner species to intercrop with lupin adapted to searched objectives (i.e. ecosystemic services that are looked for), soil and climate conditions. PULSAR: improving the production of plant-based protein via lupin intercropping Taken as a whole, lupin appears to be a legume species with a good potential not only to produce plant-based proteins, but also, to implement intercropping as an ecofriendly agricultural practice. There are, however, fundamental questions that remain unanswered, justifying the use of the term ‘orphan’ legume species for lupin. In particular, physiological determinants of seed protein content, selection and breeding of water-stress resistant cultivars, conditions to implement intercropping have been poorly if at all addressed. Research topics associated with lupin agrobiology are illustrated in Fig. 3. To address them, several labs have formed a consortium to improve our knowledge in lupin biology, agronomy, and food science and find solutions to increase the range of white lupin varieties available for resilient and efficient cropping systems. This confederative project, called PULSAR, is funded by France 2030, a program aiming to bring an answer to socio-economic, ecological, and agricultural challenges raised by the lack of plant-based protein culture in Europe. It will involve genomic exploration of 1,500 accessions of white lupin to form a core-collection of 150 accessions representative of genetic and phenotypic diversity. Using this core-collection, intercropping will be conducted with different partner crops, and agronomic performance will be assessed. Development and physiology will also be investigated; in particular, cluster roots mechanisms will be studied to understand the crossed regulation between nodulation and cluster formation. This aspect is particularly important for agronomic applications since P and N nutrition interact in lupin development. In fact, lupin is believed to balance nodulation and formation of cluster roots depending on both genotypes and growth conditions (Pueyo et al., 2021). Source-sink relations and seed metabolomics will also be examined to better understand the regulation of seed storage protein accumulation. Genome-wide association studies will be conducted to determine genetic markers associated with beneficial traits, should they be related to plant-specific properties, seed quality traits or whole-plant scale traits revealed by intercropping. Importantly, PULSAR also includes biochemical and immunological analysis of lupin protein isolates focused on digestibility and allergenicity and how the latter might be alleviated by fermentation. Future research is thus warranted to provide significant advance to draw a genetic and metabolic lupin ideotype adapted to present agroecological and food industry challenges. Beyond the scientific questions addressed by PULSAR, intercropping development also needs to overcome socio-economical and agricultural practices issues that have been discussed previously in (Magrini et al., 2016). Acknowledgements PULSAR project is funded by the Agence Nationale de la Recherche through the program France 2030 (funding number ANR-23-PLEG-0003). Competing interests The authors declare that they have no conflicts of interest in relation to this work. Author contribution G.T., C.N. and J.F. designed the research and wrote the proposal for funding. C.D. and G.T. made the bibliographical review and wrote the manuscript with contributions of C.N., J.F. and N.C. All authors approved the final version of the article. References Andrews, M., Raven, J. A., and Lea, P. J. (2013). Do plants need nitrate? 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Overview of advantages and disadvantages of intercropping in relation to lupin agronomic research Advantages Control of weeds Capture of nutrients, water and light Control of pests Increasing plant diversity Biomass dilution effect Attracting pests more than cash crops Repulsive effect for pests Attracting pest natural predators Improvement of soil quality Protection from erosion Increasing organic matter content Transfer of nutrients to cash crop Disadvantages and drawbacks Complexity of management Compromises between ecosystemic services and profitability Competition for resources between partner species Using herbicides may destroy service crop Yield variability depending on the partner species Actions needed for implementation with lupin Find the most suitable species for lupin intercropping Identify metabolic and developmental traits associated to high intercropping performance Find genotypes with faster growth Understand nutrient transfer regulation to cash crop Maximise yield in crop management with low inputs Table 2 . Summary of major effects studied in intercropping studies using white lupin as a crop Article Intercropping type Lupin type Partner species Was yield improved? Were weed reduced? Soil nutrients Biotic interactions Carton et al., (2020) Synchronous Winter Triticale Yes (merged)/No (compared to sole crop) yes N needs decreased N/A Jensen et al., (2004) Relay Spring Winter Barley Yes N/A N needs decreased N/A Almeida-Garcia et al., (2022) Relay N/A Wheat Yes yes N/A N/A Chen et al., (2023) Synchronous Spring Rapeseed N/A N/A P availability increased Soil microbial diversity enriched Carruthers et al., (2000) Synchronous Spring Corn No N/A N/A N/A Fig. 1. Overview of lupin production at the world scale . (a) Yield (tons per ha) and (b) Global production (millions of tons), including a comparison with other legumes. In this figure, the generic term “beans” refers to Phaseolus . (c) Geographical distribution of lupin production. Data shown here refer to 2022 production reported by FAOSTAT (www.fao.org) and include all lupin species (mostly white and narrow-leaved lupin). Fig. 2. White lupin seed composition and comparison with two other legumes of similar seed biochemistry . (a) Overall average seed composition in major fractions (at 10% moisture): proteins, oil, ashes, fibres, non-nitrogenous compounds (incl. starch and soluble sugars). (b) Average elemental composition of stored proteins. (c) Amino acid composition of major storage proteins, ordered from most to least abundant. (d) Relationship between protein and oil content in lupin (grey; meta-regression with n = 32, R² = 0.04). For comparison, the meta-regression line found by (Wilson, 2004) with n = 36 cultivars of soybean is shown in green. In lupin, the regression explains only 4% of variance and is not significant ( P = 0.23), while it explains nearly 80% of variance in soybean. Fig. 3. Summary of advantages, difficulties and unknowns in the use of white lupin as a crop for plant-protein production and intercropping . See main text for further details. This figure does not include details on genetic aspects, in particular the present lack of genetic characterisation of most accessions, or the lack of calibrated varieties for cultivation. See also Table 1 for open questions to implement intercropping with lupin. Supplementary Material File (image1.emf) Download 1.15 MB File (image2.emf) Download 8.95 MB File (image3.emf) Download 6.56 MB Information & Authors Information Version history V1 Version 1 13 January 2025 Peer review timeline Published Journal of Experimental Botany Version of Record 19 Mar 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cluster root france 2030 pulsar project highlights intercropping. lupinus albus metabolism Authors Affiliations Corentin Dourmap 0000-0003-3572-0887 [email protected] Université d'Angers, INRAE, Institut Agro, Institut de Recherche en Horticulture et Semences, Beaucouzé, France View all articles by this author Joelle Fustec USC LEVA Légumineuses, Ecophysiologie Végétale, Agronomie, Ecole Supérieure des Agricultures (ESA), INAE, Angers, France View all articles by this author Christophe Naudin USC LEVA Légumineuses, Ecophysiologie Végétale, Agronomie, Ecole Supérieure des Agricultures (ESA), INAE, Angers, France View all articles by this author Nicolas Carton USC LEVA Légumineuses, Ecophysiologie Végétale, Agronomie, Ecole Supérieure des Agricultures (ESA), INAE, Angers, France View all articles by this author Guillaume Tcherkez 0000-0002-3339-956X View all articles by this author Funding Information Agence Nationale de la Recherche ANR-23-PLEG-0003 Metrics & Citations Metrics Article Usage 454 views 301 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Corentin Dourmap, Joelle Fustec, Christophe Naudin, et al. White lupin: improving legume-based protein production via intercropping. Authorea . 13 January 2025. DOI: https://doi.org/10.22541/au.173679744.40722459/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); Cited by Roberta Rossi, Daniele Cavalli, Tommaso Notario, Luciano Pecetti, Early belowground interactions in lupin–wheat intercropping assessed by a simple root phenotyping approach, Plant and Soil, (2025). https://doi.org/10.1007/s11104-025-07693-z Crossref Loading... View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. 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