Cluster Root Formation Research Articles

Phosphorus-Use-Efficiency Gene Identification in Fabaceae and RSL2 Expansion in Lupinus albus Is Associated with Low-Phosphorus Adaptation.

Phosphorus is critical for plant growth but often becomes less accessible due to its precipitation with cations in soil. Fabaceae, a diverse plant family, exhibits robust adaptability and includes species like Lupinus albus, known for its efficient phosphorus utilization via cluster roots. Here, we systematically identified phosphorus-utilization-efficiency (PUE) gene families across 35 Fabaceae species, highlighting significant gene amplification in PUE pathways in Fabaceae. Different PUE pathways exhibited variable amplification, evolution, and retention patterns among various Fabaceae crops. Additionally, the number of homologous genes of the root hair development gene RSL2 in L. albus was far more than that in other Fabaceae species. Multiple copies of the RSL2 gene were amplified and retained in L. albus after whole genome triplication. The gene structure and motifs specifically retained in L. albus were different from homologous genes in other plants. Combining transcriptome analysis under low-phosphorus treatment, it was found that most of the homologous genes of RSL2 in L. albus showed high expression in the cluster roots, suggesting that the RSL2 gene family plays an important role in the adaptation process of L. albus to low-phosphorus environments and the formation of cluster roots.

Root growth dynamics, nutrient uptake and use efficiency of Grevillea robusta grown under nitrogen and phosphorus deficiency

Root growth, nutrient uptake, and nutrient use efficiency of a plant is influenced by the nutrient availability in its growing environment. The aim of this study was to investigate the root morphology, nutrient uptake and use efficiency of Grevillea robusta grown in nitrogen (N)- and phosphorus (P)-limited conditions. A hydroponic experiment was conducted for three months in a glasshouse with four nutrient treatments; (i) N-deficient, (ii) P-deficient, (iii) N/P-deficient (co-limitation of N and P), and (iv) control (sufficient amounts of N, P, and other nutrients in the growth medium). The composition and the concentration of each nutrient in the control solution were custom-made based on the growth requirement of G. robusta. N and P concentration in nutrient-deficient media were maintained at 2 ppm. The impact of N- or P-deficiency and their co-limitation on cluster root formation, organ-specific dry matter and nutrient accumulation, N and P uptake and their use efficiencies were measured. P-deficient conditions produced the highest number of cluster roots (24 ± 4) which was comparatively lower than those of N- and N/P-deficient conditions indicating that P-deficiency is the primary factor influencing the formation of cluster roots in G. robusta. The use efficiency of N and P in biomass formation was significantly (p < 0.05) enhanced when their uptake was reduced in response to N- and P-limitation. The efficient internal use of P by G. robusta under N- and N/P-deficiency is indicated by the significant increase in P-remobilization efficiency under these nutrient-deficient conditions.

Adenanthos species (Proteaceae) in phosphorus-impoverished environments use a variety of phosphorus-acquisition strategies and achieve high-phosphorus-use efficiency.

Soils in south-western Australia are severely phosphorus (P) impoverished, and plants in this region have evolved a variety of P-acquisition strategies. Phosphorus acquisition by Adenanthos cygnorum (Proteaceae) is facilitated by P-mobilizing neighbours which allows it to extend its range of habitats. However, we do not know if other Adenanthos species also exhibit a strategy based on facilitation for P acquisition in P-impoverished environments. We collected leaf and soil samples of Adenanthosbarbiger, A. cuneatus, A.meisneri,A. obovatus, A. sericeus and Adenanthos sp. Whicher Range (G.J. Keighery 9736) growing in their natural habitats at different locations within the severely P-limited megadiverse environment of south-western Australia. Hydroponic experiments were conducted to collect the carboxylates exuded by cluster roots. Pot experiments in soil were carried out to measure rhizosheath phosphatase activity. We found no evidence for facilitation of P uptake in any of the studied Adenanthos species. Like most Proteaceae, A. cuneatus, A. meisneri, A. obovatus, A. sericeus and Adenanthos sp. Whicher Range (G.J. Keighery 9736) expressed P-mining strategies, including the formation of cluster roots. Cluster roots of A. obovatus were less effective than those of the other four Adenanthos species. In contrast to what is known for most Proteaceae, we found no cluster roots for A. barbiger. This species probably expressed a post-fire P-acquisition strategy. All Adenanthos species used P highly efficiently for photosynthesis, like other Proteaceae in similar natural habitats. Adenanthos is the first genus of Proteaceae found to express multiple P-acquisition strategies. The diversity of P-acquisition strategies in these Proteaceae, coupled with similarly diverse strategies in Fabaceae and Myrtaceae, demonstrates that caution is needed in making family- or genus-wide extrapolations about the strategies exhibited in severely P-impoverished megadiverse ecosystems.

Soilborne bacterium Klebsiella pneumoniae promotes cluster root formation in white lupin through ethylene mediation.

Cluster roots of white lupin are induced by low phosphorus (LP) to efficiently access unavailable P, but how soilborne microbes are associated with cluster root formation (CRF) is unclear. We investigated the roles of soilborne bacteria in CRF response to LP by high-throughput sequencing and root-bacteria interactions. Cluster root number was significantly decreased in plants grown in sterilized soil compared with nonsterilized soil. Proteobacteria was enriched in CR, as shown by microbiome analysis of soil (bulk, rhizosphere, and rhizosheath) and roots (main, lateral, and CR). Large-scale gene expression level implicated ethylene mediation in CRF. Klebsiella pneumoniae (P7), a soilborne bacterium belonging to Proteobacteria, was isolated from CR. Among 11 isolated strains, P7 exhibited the highest 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity; this enzyme inhibits the biosynthesis of ethylene in plants by the cleavage of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and promotes CRF under LP. We constructed an ACCD-deficit mutant accd in the P7 genetic background. The loss-of-function mutation failed to promote CRF under LP conditions. Also, auxin responses may be involved in K. pneumoniae-ethylene-mediated CRF. Overall, we propose that the soilborne bacterium K. pneumoniae promotes CRF of white lupin in response to LP by ethylene mediation.

The vacuolar transporter LaMTP8.1 detoxifies manganese in leaves of Lupinus albus.

Manganese (Mn) is an essential microelement, but overaccumulation is harmful to many plant species. Most plants have similar minimal Mn requirements, but the tolerance to elevated Mn varies considerably. Mobilization of phosphate (P) by plant roots leads to increased Mn uptake, and shoot Mn levels have been reported to serve as an indicator for P mobilization efficiency in the presence of P deficiency. White lupin (Lupinus albus L.) mobilizes P and Mn with outstanding efficiency due to the formation of determinate cluster roots that release carboxylates. The high Mn tolerance of L. albus goes along with shoot Mn accumulation, but the molecular basis of this detoxification mechanism has been unknown. In this study, we identify LaMTP8.1 as the transporter mediating vacuolar sequestration of Mn in the shoot of white lupin. The function of Mn transport was demonstrated by yeast complementation analysis, in which LaMTP8.1 detoxified Mn in pmr1∆ mutant cells upon elevated Mn supply. In addition, LaMTP8.1 also functioned as an iron (Fe) transporter in yeast assays. The expression of LaMTP8.1 was particularly high in old leaves under high Mn stress. However, low P availability per se did not result in transcriptional upregulation of LaMTP8.1. Moreover, LaMTP8.1 expression was strongly upregulated under Fe deficiency, where it was accompanied by Mn accumulation, indicating a role in the interaction of these micronutrients in L. albus. In conclusion, the tonoplast-localized Mn transporter LaMTP8.1 mediates Mn detoxification in leaf vacuoles, providing a mechanistic explanation for the high Mn accumulation and Mn tolerance in this species.

Phosphorus supply affects seedling growth of mycorrhizal but not cluster-root forming jarrah-forest species

AimsFertiliser is often used to kick-start ecological restoration despite growing evidence of the potentially negative impacts on plant diversity. Jarrah (Eucalyptus marginata) forest species growing on nutrient (especially phosphorus) impoverished soils in southwestern Australia have a suite of adaptations for phosphorus (P) acquisition, including the formation of cluster roots, and associations with mycorrhizal fungi. Here we investigated how escalating P supply, along with a stoichiometric adjustment of nitrogen (N) supply, impacted the growth and nutrition of a wide range of jarrah forest seedlings.MethodsIn a pot experiment, we measured seedling biomass and nutritional responses of 12 jarrah forest species to a gradient of P supply in relation to N supply, and for the mycorrhizal species, inoculation with arbuscular mycorrhizal fungi.ResultsThree cluster-root forming species did not respond to increasing P, probably because they were reliant on seed P. Generally, mycorrhizal species showed a positive biomass response to increasing P when N was available. Mycorrhizas benefited seedling growth at low P (9 mg P added per kg of jarrah forest soil) when N was also available, and were parasitic to seedling growth at high P (243 mg P/ kg soil) without additional N.ConclusionsThese results highlight importance of P and N supply in determining the nature of the symbiosis between plants and mycorrhizal fungi. Since P supply has the potential to reduce plant growth, for a range of species, our results suggest careful consideration of fertiliser amounts for ecological restoration of ecosystems adapted to nutrient poor soils.

Adaptive Mechanisms Make Lupin a Choice Crop for Acidic Soils Affected by Aluminum Toxicity.

Almost half of the world’s agricultural soils are acidic, and most of them present significant levels of aluminum (Al) contamination, with Al3+ as the prevailing phytotoxic species. Lupin is a protein crop that is considered as an optimal alternative to soybean cultivation in cold climates. Lupins establish symbiosis with certain soil bacteria, collectively known as rhizobia, which are capable of fixing atmospheric nitrogen. Moreover, some lupin species, especially white lupin, form cluster roots, bottlebrush-like structures specialized in the mobilization and uptake of nutrients in poor soils. Cluster roots are also induced by Al toxicity. They exude phenolic compounds and organic acids that chelate Al to form non-phytotoxic complexes in the rhizosphere and inside the root cells, where Al complexes are accumulated in the vacuole. Lupins flourish in highly acidic soils where most crops, including other legumes, are unable to grow. Some lupin response mechanisms to Al toxicity are common to other plants, but lupin presents specific tolerance mechanisms, partly as a result of the formation of cluster roots. Al-induced lupin organic acid secretion differs from P-induced secretion, and organic acid transporters functions differ from those in other legumes. Additionally, symbiotic rhizobia can contribute to Al detoxification. After revising the existing knowledge on lupin distinct Al tolerance mechanisms, we conclude that further research is required to elucidate the specific organic acid secretion and Al accumulation mechanisms in this unique legume, but definitely, white lupin arises as a choice crop for cultivation in Al-rich acidic soils in temperate climate regions.

Flavonoids are involved in phosphorus-deficiency-induced cluster-root formation in white lupin.

Initiation of cluster roots in white lupin (Lupinus albus) under phosphorus (P) deficiency requires auxin signalling, whereas flavonoids inhibit auxin transport. However, little information is available about the interactions between P deficiency and flavonoids in terms of cluster-root formation in white lupin. Hydroponic and aeroponic systems were used to investigate the role of flavonoids in cluster-root formation, with or without 75 μm P supply. Phosphorus-deficiency-induced flavonoid accumulation in cluster roots depended on developmental stage, based on in situ determination of fluorescence of flavonoids and flavonoid concentration. LaCHS8, which codes for a chalcone synthase isoform, was highly expressed in cluster roots, and silencing LaCHS8 reduced flavonoid production and rootlet density. Exogenous flavonoids suppressed cluster-root formation. Tissue-specific distribution of flavonoids in roots was altered by P deficiency, suggesting that P deficiency induced flavonoid accumulation, thus fine-tuning the effect of flavonoids on cluster-root formation. Furthermore, naringenin inhibited expression of an auxin-responsive DR5:GUS marker, suggesting an interaction of flavonoids and auxin in regulating cluster-root formation. Phosphorus deficiency triggered cluster-root formation through the regulation of flavonoid distribution, which fine-tuned an auxin response in the early stages of cluster-root development. These findings provide valuable insights into the mechanisms of cluster-root formation under P deficiency.

Root morphology and rhizosheath acid phosphatase activity in legume and graminoid species respond differently to low phosphorus supply

Given the worldwide effort to improve crop phosphorus (P) efficiency, it is important to understand the adaptive strategies of crops with different root types and properties under limited P availability. The present study examined root morphological and physiological responses of two graminoids (wheat and maize), and three legumes (soybean, white lupin and narrow-leafed lupin) to low P supply under two difference growth conditions. Two experiments using non-destructive root observation systems (Expt.1, semi-hydroponic system; Expt.2, rhizoboxes) were conducted under low P (Expt.1: 2 μM; Expt.2: 9 mg kg−1 soil) and optimal P (Expt.1: 200 μM; Expt. 2: 60 mg kg−1 soil). Plants were assessed 40 days after transplanting (Expt. 1) and 35 days after sowing (Expt. 2). Results showed that low P stress generally suppressed plant shoot and root growths of all crops and significantly increased root-shoot ratio in graminoid plants in both experiments. Root length vertical distribution in each 10 cm layer was relatively lower in low P than in optimal P treatments with the majority roots distributed in 10–40 cm layers in both experiments. Under low P, all plants accumulated significantly less P in plant tissues. Maize, soybean and white lupin plants had larger dry weights and P contents than wheat and narrow-leaf lupin under respective P treatment under both experiments except high P content in wheat cv Zhongyu 6 under optimal P in Expt. 2. Low P stress significantly increased plant P-use efficiency in both experiments with more profound increase in maize and white lupin in Expt. 1. Low P stress stimulated cluster roots formation in white lupin. Both lupin species had significantly higher rhizosheath acid phosphatase activity than other crops under low P supply. In conclusion, five crop species differed in their adaptive strategies to low P availability. Graminoid species had strong response by modifying their root morphology while legumes especially lupin species used the limited P more efficiently through enhancing rhizosheath acid phosphatase activity to cope with soil P deficit.

Phosphorus uptake is associated with the rhizosheath formation of mature cluster roots in white lupin under soil drying and phosphorus deficiency

Phosphorus (P) deficiency largely restricts plant growth and lead to severe yield losses. Therefore, identification of novel root traits to improve P uptake is needed to circumvent yield losses. White lupin (Lupinus albus) is a legume crop that develops cluster roots and has the high phosphorus use efficiency in low P soils. We aimed to investigate the association between cluster roots (CR) rhizosheath formation and P uptake in white lupin. Rhizosheath formation and P concentration were evaluated under four soil treatments. CR increased up to 2.5-fold of overall plant dry weight under SD–P compared to WW + P (control), partly attributable to variations in CR development. Our data showed that SD–P significantly increase rhizosheath weight in white lupin. Among the root segments, MCR showed improved P accumulation in the root which is associated with increased MCR rhizosheath weight. Additionally, a positive correlation was observed between MCR rhizosheath weight and P uptake. Moreover, high sucrose content was recorded in MCR, which may contribute in CR growth under SD–P. Expression analysis of genes related to sucrose accumulation (LaSUC1, LaSUC5, and LaSUC9) and phosphorus uptake (LaSPX3, LaPHO1, and LaPHT1) exhibited peaked expression in MCR under SD-P. This indicate that root sucrose status may facilitate P uptake under P starvation. Together, the ability to enhance P uptake of white lupin is largely associated with MCR rhizosheath under SD–P. Our results showed that gene expression modulation of CR forming plant species, demonstrating that these novel root structures may play crucial role in P acquisition from the soil. Our findings could be implicated for developing P and water efficient crop via CR development in sustainable agriculture.

Petroleum hydrocarbon rhizoremediation and soil microbial activity improvement via cluster root formation by wild proteaceae plant species

Rhizoremediation potential of different wild plant species for total (aliphatic) petroleum hydrocarbon (TPH)-contaminated soils was investigated. Three-week-old seedlings of Acacia inaequilatera, Acacia pyrifolia, Acacia stellaticeps, Banksia seminuda, Chloris truncata, Hakea prostrata, Hardenbergia violacea, and Triodia wiseana were transplanted in a soil contaminated with diesel and engine oil as TPH at pollution levels of 4,370 (TPH1) and 7,500 (TPH2) mg kg−1, and an uncontaminated control (TPH0). After 150 days, the presence of TPH negatively affected the plant growth, but the growth inhibition effect varied between the plant species. Plant growth and associated root biomass influenced the activity of rhizo-microbiome. The presence of B. seminuda, C. truncata, and H. prostrata significantly increased the TPH removal rate (up to 30% compared to the unplanted treatment) due to the stimulation of rhizosphere microorganisms. No significant difference was observed between TPH1 and TPH2 regarding the plant tolerance and rhizoremediation potentials of the three plant species. The presence of TPH stimulated cluster root formation in B. seminuda and H. prostrata which was associated with enhanced TPH remediation of these two members of Proteaceae family. These results indicated that B. seminuda, C. truncata, and H. prostrata wild plant species could be suitable candidates for the rhizoremediation of TPH-contaminated soil.

Leaf Phosphorus Concentration Regulates the Development of Cluster Roots and Exudation of Carboxylates in Macadamia integrifolia.

Phosphorus (P) deficiency induces cluster-root formation and carboxylate exudation in most Proteaceae. However, how external P supply regulates these root traits in Macadamia integrifolia remains unclear. Macadamia plants were grown hydroponically with seven P levels to characterize biomass allocation, cluster-root development, and exudation of carboxylates and acid phosphatases. Plant biomass increased with increasing P supply, peaking at 5 μM P, was the same at 5–25 μM P, and declined at 50–100 μM P. Leaf P concentration increased with increasing P supply, but shoot biomass was positively correlated with leaf P concentration up to 0.7–0.8 mg P g–1 dry weight (DW), and declined with further increasing leaf P concentration. The number of cluster roots declined with increasing P supply, with a critical value of leaf P concentration at 0.7–0.8 mg P g–1 DW. We found a similar trend for carboxylate release, with a critical value of leaf P concentration at 0.5 mg g–1 DW, but the activity of acid phosphatases showed a gradually-decreasing trend with increasing P supply. Our results suggest that leaf P concentration regulates the development and functioning of cluster roots, with a critical P concentration of 0.5–0.8 mg g–1, above which macadamia growth is inhibited.

Gevuina avellana and Lomatia dentata, two Proteaceae species from evergreen temperate forests of South America exhibit contrasting physiological responses under nutrient deprivation

Gevuina avellana and Lomatia dentata are Proteaceae species from evergreen temperate forests, but only G. avellana can colonize nutrient deprived volcanic depositions. We hypothesized that under nutrient deprivation, G. avellana would present higher survival, physiological performance, and adjustment regarding cluster root (CR) formation and carboxylate exudation rate than L. dentata. We evaluated relative growth rate, maximal photosynthetic rate, photochemical performance, nitrogen (N) and phosphorus (P) leaf concentrations, specific leaf area, photosynthetic P and N use efficiency (PPUE and PNUE), CR formation and carboxylate exudation rate. Plants were grown in a greenhouse, using recent volcanic substrate and watered with different nutrient Hoagland (H) solutions: modified full Hoagland, without P, without N, or tap water. Both species showed high survival. G. avellana exhibited higher growth rates (RGR), and higher CR number and biomass allocation under nutrient deprivation, but lower carboxylate exudation rates than L. dentata. Malate, oxalate and succinate were detected in root exudates of G. avellana but only oxalate in L. dentata. However, PPUE and PNUE were higher in L. dentata than in G. avellana . Our hypothesis was not entirely accepted, CR formation was more “constitutive” in G. avellana, and composition of carboxylates was more diverse, with lower exudation rates than L. dentata. Moreover, L. dentata showed higher PNUE and PPUE, partly explained by thinner leaves. Different responses are related with edaphic conditions, where both species inhabit: more diverse to G. avellana and restricted to developed soils to L. dentata.

Contrasting responses of cluster roots formation induced by phosphorus and nitrogen supply in Embothrium coccineum populations from different geographical origin

Embothrium coccineum is a pioneer tree that produces cluster roots (CR) induced by phosphorus (P) or nitrogen (N) deficiency, but the role which both N and P play in CR formation for different E. coccineum populations is still unknown. We hypothesized that in E. coccineum, N influences the CR formation response, primarily induced by P limitation. However, this response depends on local adaptations to their specific conditions of geographic origin. Seedlings from three contrasting edaphoclimatic conditions (Northern, Central and Southern populations) were grown in hydroponic culture under high and low N supply and at five different P supply. Morphophysiological responses, including CR number, growth, biomass, P and N plant concentration, were studied. Our results showed that at high N supply, CR formation was stimulated with increased P supply, but just until it reaches a maximum of 5 μM, above this concentration CR formation decreases. However, at low N supply, a constitutive response was observed regardless of P supply. Moreover, each of these responses depends on the populations. Our study suggests that N drives CR formation induced by P supply and that a differential response exists among E. coccineum populations, that could be related to their origin edaphoclimatic conditions.

The genome evolution and low-phosphorus adaptation in white lupin

White lupin (Lupinus albus) is a legume crop that develops cluster roots and has high phosphorus (P)-use efficiency (PUE) in low-P soils. Here, we assemble the genome of white lupin and find that it has evolved from a whole-genome triplication (WGT) event. We then decipher its diploid ancestral genome and reconstruct the three sub-genomes. Based on the results, we further reveal the sub-genome dominance and the genic expression of the different sub-genomes varying in relation to their transposable element (TE) density. The PUE genes in white lupin have been expanded through WGT as well as tandem and dispersed duplications. Furthermore, we characterize four main pathways for high PUE, which include carbon fixation, cluster root formation, soil-P remobilization, and cellular-P reuse. Among these, auxin modulation may be important for cluster root formation through involvement of potential genes LaABCG36s and LaABCG37s. These findings provide insights into the genome evolution and low-P adaptation of white lupin.

Coupling zymography with pH mapping reveals a shift in lupine phosphorus acquisition strategy driven by cluster roots

Phosphorus (P) availability affects the spatial and temporal distribution of phosphatase activity and acidification in the rhizosphere: two strongly interactive key strategies of nutrient acquisition by roots. Zymography was coupled with pH planar optode mapping to reveal the effects of P-availability (P deficiency, or with phytate or Ca(H2PO4)2 amendment) on the spatial distribution of phosphatase activity, pH and rhizosphere extent around the taproot of lupine before and after cluster root formation. Before cluster root formation, phosphorus deficiency increased acid phosphatase activities by 20%, decreased pH by 0.8 units and broadened the rhizosphere extent by 0.4 mm across the taproot. Phytate (80 mg kg−1) addition dampened these changes before cluster root formations. In contrast, the rhizosphere extent of phosphatase activity after cluster root formation was 0.2 mm narrower under P-deficiency than with Ca(H2PO4)2 amendment. Due to cluster root formation, the hotspot areas of alkaline phosphatase activity were 40% larger for lupine grown under P-deficiency than amended with Ca(H2PO4)2. Lupine rhizosphere strategies shifted during growth: increasing phosphatase activity, acidifying soil and broadening the rhizosphere around the taproot are dominant mechanisms before cluster root formation. After cluster root development, the main mechanism is increasing the area of phosphatase activity hotspots around cluster roots to enlarge the exploited soil volume.

Cluster root formation and function vary in two species with contrasting geographic ranges

Southern South American Proteaceae can occupy soils that are rich in total phosphorus (P) but poor in available P (for example volcanic soils) thanks to their cluster roots (CR), which mine soil P. However, some southern South American Proteaceae occur in a wide range of soil nutrition. We hypothesized that CR formation and function are more responsive to nutrient soil availability in the widely-distributed Embothrium coccineum than in the narrowly-distributed Orites myrtoidea, which exclusively occurs in recent volcanic depositions. Survival, growth rate, CR formation (number, biomass) and function (carboxylate exudation, phosphatase activity) were evaluated in seedlings of both species after five months of growth in either a volcanic or organic substrate. E. coccineum exhibited full survival in both substrates, but had significantly lower growth, higher CR formation, higher CR citrate and malate exudation, and higher phosphatase activity in the volcanic substrate. By contrast, O. myrtoidea had similar growth rate in both substrates but 73% lower survival and null CR formation in the organic compared to the volcanic substrate. Variation in soil nutrient availability caused variation in growth and CR formation and function in a southern South American Proteaceae species of wider distribution, but not in a narrowly-distributed counterpart.

The LaCEP1 peptide modulates cluster root morphology in Lupinus albus.

White lupin cluster roots are specialized brush-like root structures that are formed in some species under phosphorus (P)-deficient conditions. They intensely secrete protons and organic acid anions for solubilization and acquisition of sparingly soluble phosphates. Phytohormones and sucrose modulate cluster root number, but the molecular mechanisms of cluster root formation have been elusive. Here, a novel peptide phytohormone was identified that affects cluster root development. It belongs to the C-TERMINALLY-ENCODED PEPTIDE (CEP) family. Members of that family arrest root growth and modulate branching in model species. LaCEP1 was highly expressed in the pre-emergence zone of clusters. Over-expression of the gene encoding the LaCEP1 propeptide resulted in moderate inhibition of cluster root formation. The primary and lateral root lengths of lupin were little affected by the overexpression, but LaCEP1 reduced cluster rootlet and root hair elongation. Addition of a 15-mer core peptide derived from LaCEP1 similarly altered root morphology and modified cluster activity, suggesting that a core sequence of the propeptide is functionally sufficient. Stable overexpression in Arabidopsis confirmed the LaCEP1 function in root growth inhibition across species. Taken together, the root inhibitory effects of the LaCEP1 phytohormone suggest a role as of a regulatory module involved in cluster root development in white lupin.

Nodulation promotes cluster-root formation in Lupinus albus under low phosphorus conditions

Phosphorus deficiency often limits crop productivity, while phosphate rock, which is used to produce phosphorus fertilisers, is a non-renewable resource. Formation of cluster roots is an adaptation to nutrient-poor soils in Lupinus species, including L. albus. Lupinus species also produce nodules, which require a large investment of phosphorus. Our aim is to test whether nodulation promotes cluster-root formation in L. albus. Seedlings of L. albus, either inoculated with rhizobia or non-inoculated, were grown in nutrient solution with a low phosphorus supply. Non-inoculated plants were provided with the same amount of nitrogen in the form of nitrate as the nodulated ones acquired, from both air and nutrient solution, based on preliminary experiments. We measured biomass, phosphorus and nitrogen concentrations as well as photosynthesis just prior to each harvest. Nodulated plants and non-nodulated control plants produced the same amount of biomass. Nodulated plants had, on average, 86% more cluster roots than non-nodulated ones at the four harvests. As hypothesised, nodulation significantly promoted cluster-root formation; it also enhanced rates of photosynthesis. Nodulation promoted cluster-root formation and photosynthesis, presumably because nodules are significant sinks for phosphorus and photosynthates. Our results do not provide evidence for a trade-off between investment of resources in nodules and cluster roots.

Mapping the footprint of nematodes in the rhizosphere: Cluster root formation and spatial distribution of enzyme activities

Nematodes are among the most important pathogens in agriculture, greatly reducing crop biomass and yield. The direct effects of nematodes on above– and belowground plant parts are well known, but the broad range of indirect effects, especially on carbon (C) and phosphorus (P) cycles underground, remains unknown. For the first time, using soil zymography, we analyzed the indirect effects of Meloidogyne incognita cellobiohydrolase and phosphatase. The rhizosphere of lupine (Lupinus polyphyllus L.), a species sensitive to pathogens with high P demand, was selected to study the activity, distribution and localization of two enzymes responsible for C and P cycling: The distribution patterns of cellobiohydrolase and phosphatase demonstrated that M. incognita induced the formation of knots as well as cluster roots, which corresponded to hotspot locations on zymogram images for both enzymes. Increased C release by nematode–infected roots into the soil led to a decrease in the overall activity of cellobiohydrolase and especially at hotspots (by ∼ 20 times). In contrast, the increased P demand of infected plants raised the phosphatase activity, leading to an increase in the rhizosphere extent around the roots and especially of the hotspot area (by 6 times). Remarkably, this 1 mm increase of rhizosphere extent in 2D equals a 2-fold increment in soil volume (3D) for nutrient mobilization.We conclude that nematode infection not only has direct effects by changing root morphology, but also induces a number of subsequent biochemical changes (e.g. enzyme activities and consequently nutrient mobilization) in the rhizosphere, affecting C and P cycling.