Changes in Soil Phosphorus Availability and Microbial Community Structures in Rhizospheres of Oilseed Rapes Induced by Intercropping with White Lupins.

Oilseed rape is sensitive to soil phosphorus deficiencies. In contrast, white lupin is widely used as a model plant because it has efficient phosphorus utilization. Therefore, soil fertility and microbial composition in the rhizospheres of oilseed rapes and root exudate metabolites were compared under monocropping and intercropping systems. The main purpose was to explore whether the phosphorus absorption of rapeseed can be promoted by intercropping with white lupine. In comparison with oilseed rape monoculture (RR), the results showed that the contents of soil-available phosphorus, microbial biomass and phosphorus in the rhizospheres of oilseed rapes in the intercropping system (RL) were all higher than those of RR. Meanwhile, in comparison with RR, not only phosphorus-solubilizing bacteria, such as Streptomyces, Actinomadura and Bacillus, but also phosphorus-solubilizing fungi, such as Chaetomium, Aspergillus, Penicillium, were enriched in the rhizospheres of the oilseed rape under the RL system. Moreover, more abundant soil bacterial functions, organic acids and metabolites were also detected in root exudates of the oilseed rapes under the RL system. All of the above results suggest that soil phosphorus availability in the rhizospheres of oilseed rape could be improved by intercropping with white lupin. Additionally, soil phosphorus-solubilizing microorganisms, that are enriched in the rhizospheres of oilseed rapes under RL systems, have an important function in the improvement of phosphorus absorption of rapeseed by intercropping with white lupin.

Development of an HPLC-MS/MS Method for the Determination of Alkaloids in Lupins.

Lupin alkaloids (LAs) represent a class of toxic secondary metabolites in plants, in particular in Lupinus spp.; they are produced as a defense mechanism due to their strong bitter taste and are very dangerous for human and animals. In this work, a sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analytical method for the identification and quantification of thirteen lupin alkaloids was developed and validated according to FDA guidelines. Efficient extraction and clean-up steps, carried out by solid-phase extraction, were finely tuned on the basis of the characteristics of the analytes and lupin samples, providing good selectivity with minimized matrix interference. The effectiveness of the method was proven by the satisfactory recovery values obtained for most of the analytes and a matrix effect ≤23% for all tested levels. In addition, a sensitive and reliable determination of the target compounds was obtained; LOQs were between 1 and 25 µg Kg-1, i.e., below the requested maximum levels (<200 mg Kg-1). The method was applied to evaluate the LAs profile in different batches of raw L. albus L. samples, varying in size and across farming treatments.

Elevated CO2 and biochar differentially affect plant C:N:P stoichiometry and soil microbiota in the rhizosphere of white lupin (Lupinus albus L.).

Biochar application is a potent climate change mitigation strategy in agroecosystems. However, little is known about the interactive effects of elevated CO2 (eCO2) and biochar on plant nutrient uptake and soil microbial processes. A pot experiment was conducted to investigate the effects of eCO2 and biochar addition on plant C:N:P stoichiometry and rhizobacterial community for better management of nutrient balance and use efficiency in a future climate scenario. White lupin (Lupinus albus L.) was grown for 30 days in topsoil and subsoil with or without 2% corn-stubble biochar under ambient CO2 (aCO2: 390 ppm) or eCO2 (550 ppm). Elevated CO2 increased, but biochar decreased, plant biomass and shoot N and P uptake, with no interactions in either soil layer. Elevated CO2 decreased shoot N concentration by 16% and biochar decreased shoot P concentration by 11%. As a result, eCO2 increased shoot C:N ratio by 20% and decreased the N:P ratio by 11%. Biochar decreased shoot C:N ratio by 8% in the subsoil under eCO2. However, biochar increased shoot C:P ratio by an average of 13% and N:P ratio by 23% in the subsoil. Moreover, plants grown in the subsoil showed lower shoot N (35%) and P (70%) uptake compared to the topsoil. The results indicate that N and P are the more limiting factors that regulate plant growth under eCO2 and biochar application, respectively. Elevated CO2 and biochar oppositely affected dominant rhizobacterial community composition, with the eCO2 effect being greater. The microbiota in the subsoil held a greater diversity of contrasting species than the topsoil, which were associated with nutrient cycling, hydrocarbon degradation and plant productivity. These results enrich our understanding of potential soil nutrient cycling and plant nutrient balance in future agroecosystems.

Influence of root exudation of white lupine (Lupinus albus L.) on uranium phytoavailability in a naturally uranium-rich soil.

Mechanisms of uranium (U) transfer from soil to plants remain poorly understood. The kinetics of supply of U to the soil solution from solid phases could be a key point to understand its phytoavailability and implications for environmental risk assessment. Root activity, particularly the continuous release of organic acids in the rhizosphere, could have an effect on this supply. We tested the impact of citrate exudation by roots of Lupinus albus, either P-sufficient (P+) or P-deficient (P-), on the phytoavailability of U from a naturally contaminated soil (total content of 413 mg U kg-1) using a rhizotest design. Combined effects of P (P-/P+ used to modulate plant physiology) and citrate (model exudate) on the solubilization of U contained in the soils were tested in closed reactors (batch). The batch experiment showed the existence of a low U available pool (0.4% total U) and high accessibility (kd' around 20 L kg-1) which was not significantly affected by P treatment or citrate concentrations. Analysis of U, Fe, Ca, P and citrate concentrations in the batches suggested a complex combination of mechanisms and factors including desorption, resorption, precipitation, co-sorption. On rhizotest, L. albus plants extracted 0.5-0.75% of the total U and between 25 and 40% of the estimated available U present in the rhizotest in 5 days. Uranium accumulation at the whole plant level (20 mg U kg-1d.w., shoot to root ratio around 10-3) seemed to be dependent neither on the plant P nutrition status nor citrate exudation level, possibly in relation with the equivalent accessibility of U whatever the growth conditions. Yet differential translocation to shoots seemed to be positively correlated to citrate exudation.

Aided phytostabilisation of As- and Cu-contaminated soils using white lupin and combined iron and organic amendments.

An aided phytostabilisation strategy consisting of several composite amendments of iron sulphate and organic materials combined with Lupinus albus L. (white lupin) was evaluated for remediation of an As- and Cu-contaminated soil. Iron sulphate was combined with lime, paper mill sludge (PS), olive mill waste compost (OMWC) or holm oak biochar (BC) and applied to a slightly acidic soil with high concentration of As (∼2200 mg kg-1) and Cu (∼150 mg kg-1). White lupin was grown for 48 days in pots containing amended and non-amended soils and the effect of soil treatments on soluble and extractable trace elements, soil fertility and plant growth and composition was evaluated. The addition of the amendments raised soil pH and reduced soluble As (50-93%) and extractable As and Cu (50-89%). Despite the reduction of As- and Cu-extractable fractions, plant As and Cu uptake was not greatly affected by the amendments. Variations in soil pH and P-Olsen seemed to have influenced As dynamics in the treated soils, although they did not provoke its mobilisation with respect to the non-amended soil. Our results suggest that the freshly formed iron oxides resulting from addition of iron sulphate controlled As dynamics in the treated soils, avoiding its mobilisation due to application of organic materials. The combination of iron sulphate with OMWC and BC is shown as appropriate for aided phytostabilisation of metal(loid)s contaminated soils, as it improved soil fertility and plant nutrition while reduced As and Cu mobility.

Light-dependent activation of phosphoenolpyruvate carboxylase by reversible phosphorylation in cluster roots of white lupin plants: diurnal control in response to photosynthate supply.

Background and Aims Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme that controls carbohydrate partitioning to organic acid anions (malate, citrate) excreted in copious amounts by cluster roots of inorganic phosphate (Pi)-deprived white lupin plants. Excreted malate and citrate solubilize otherwise inaccessible sources of mineralized soil Pi for plant uptake. The aim of this study was to test the hypotheses that (1) PEPC is post-translationally activated by reversible phosphorylation in cluster roots of illuminated white lupin plants, and (2) light-dependent phosphorylation of cluster root PEPC is associated with elevated intracellular levels of sucrose and its signalling metabolite, trehalose-6-phosphate. Methods White lupin plants were cultivated hydroponically at low Pi levels (≤1 µm) and subjected to various light/dark pretreatments. Cluster root PEPC activity and in vivo phosphorylation status were analysed to assess the enzyme's diurnal, post-translational control in response to light and dark. Levels of various metabolites, including sucrose and trehalose-6-phosphate, were also quantified in cluster root extracts using enzymatic and spectrometric methods. Key Results During the daytime the cluster root PEPC was activated by phosphorylation at its conserved N-terminal seryl residue. Darkness triggered a progressive reduction in PEPC phosphorylation to undetectable levels, and this was correlated with 75-80 % decreases in concentrations of sucrose and trehalose-6- phosphate. Conclusions Reversible, light-dependent regulatory PEPC phosphorylation occurs in cluster roots of Pi-deprived white lupin plants. This likely facilitates the well-documented light- and sucrose-dependent exudation of Pi-solubilizing organic acid anions by the cluster roots. PEPC's in vivo phosphorylation status appears to be modulated by sucrose translocated from CO2-fixing leaves into the non-photosynthetic cluster roots.

Tamanhos de unidades experimentais básicas e de parcelas em tremoço branco / Basic experimental units and plot sizes in white lupine

RESUMO: O objetivo deste trabalho foi verificar a influência do tamanho da unidade experimental básica (UEB) na estimativa do tamanho ótimo de parcela, para a avaliação da massa de matéria verde de tremoço branco ( Lupinus albus L.). Avaliou-se a massa de matéria verde aos 123, 137 e 150 dias após a semeadura. Em cada época, foram avaliadas 432UEB de 1m×1m (1m2) e formaram-se 16 planos de UEB com tamanhos entre 1 e 16m2. Para cada plano de UEB, foi determinado o tamanho ótimo de parcela, pelo método da curvatura máxima do modelo do coeficiente de variação. Em cada época de avaliação, foi calculada a diferença mínima significativa pelo teste de Tukey, em 448 cenários formados pelas combinações de 16 tamanhos ótimos de parcela, i tratamentos (i=5, 10, 15 e 20) e r repetições (r=3, 4, 5, 6, 10, 15 e 20), para o delineamento blocos ao acaso. A estimativa do tamanho ótimo de parcela depende do tamanho da unidade experimental básica. É indicado avaliar a massa de matéria verde em UEB de menor tamanho possível, para serem usadas na estimação do tamanho ótimo de parcela.

ABSTRACT: The objective of this research was to determine the influence of the basic experimental unit size (BEU) to estimate the optimum plot size, in the evaluation of white lupine ( Lupinus albus L.) fresh matter weight. Data from fresh matter weight to 123, 137 and 150 days after sowing were evaluated. In every season 432BEU 1m×1m (1m2) were evaluated and 16BEU plans of sizes from 1 to 16m2 were formed. For each BEU plan, the optimum plot size was determined by the method of maximum curvature of the coefficient of variation model. The least significant difference by Tukey's test in 448 scenarios formed by the combinations of 16 plot sizes, i treatments (i=5, 10, 15 and 20), and r replicates (r=3, 4, 5, 6, 10, 15 and 20) for randomized block designs was calculated. Estimate of optimum plot size depends of the basic experimental unit size. It is indicated to evaluate the fresh matter weight in basic experimental units smallest possible size, for use in the estimation of optimum plot size.

Embebição e qualidade fisiológica de sementes de tremoço branco tratadas com micronutrientes / Imbibition and white lupine seeds physiological quality affected by micronutrients supply

As sementes de tremoço branco, devido ao alto teor de óleo e proteína, estão sendo cada vez mais utilizadas na alimentação humana e animal. Dessa forma, o objetivo deste trabalho foi avaliar a embebição e a qualidade fisiológica de sementes de tremoço branco tratadas via imersão em soluções de micronutrientes. Os tratamentos constaram do controle (isento de imersão), imersão em água destilada e em soluções de Cu, B, Zn, Mn e Mo, em delineamento experimental inteiramente casualizado. A avaliação da embebição e qualidade fisiológica foi através de testes de germinação e de vigor (primeira contagem de germinação, condutividade elétrica e crescimento de plântulas), bem como a determinação do teor de nutrientes. A velocidade de absorção das sementes de tremoço branco com menor qualidade fisiológica inicial, na presença de Mn e Mo, diminui, enquanto o tratamento de sementes de tremoço branco via imersão em soluções de micronutrientes favorece a sua qualidade fisiológica.

White lupine seeds due to the high oil content and protein are being used in animal and human feed, so the objective was to evaluate the absorption and white lupine seeds physiological quality that were treated by immersion in micronutrients solutions. Treatments consisted of control (free immersion), immersion in distilled water and in solutions of Cu, B, Zn, Mn and Mo in a completely randomized design. Evaluation of absorption and physiological quality was through seed germination and vigor (first count, electrical conductivity and seedling growth) as well as determining the nutrient content tests. The absorption rate of white lupine seeds with lower initial quality in the presence of Mn and Mo decreases. The treatment of white lupine seeds by immersion in solutions of micronutrients favors the physiological quality.

Hormonal interactions during cluster-root development in phosphate-deficient white lupin (Lupinus albus L.).

This study addresses hormonal interactions involved in cluster-root (CR) development of phosphate (Pi)-deficient white lupin (Lupinus albus), which represents the most efficient plant strategy for root-induced mobilisation of sparingly soluble soil phosphorus (P) sources. Shoot-to-root translocation of auxin was unaffected by P-limitation, while strong stimulatory effects of external sucrose on CR formation, even in P-sufficient plants, suggest sucrose, rather than auxins, acts as a shoot-borne signal, triggering the induction of CR primordia. Ethylene may act as mediator of the sucrose signal, as indicated by moderately increased expression of genes involved in ethylene biosynthesis in pre-emergent clusters and by strong inhibitory effects of the ethylene antagonist CoCl2 on CR formation induced by sucrose amendments or P-limitation. As reported in other plants, moderately increased production of brassinosteroids (BRs) and cytokinin, in pre-emergent clusters, may be required for the formation of auxin gradients necessary for induction of CR primordia via interference with auxin biosynthesis and transport. The well-documented inhibition of root elongation by high doses of ethylene may be involved in the growth inhibition of lateral rootlets during CR maturation, indicated by a massive increased expression of gene involved in ethylene production, associated with a declined expression of transcripts with stimulatory effects (BR and auxin-related genes).

Tamanho de parcela para avaliar caracteres de tremoço branco em épocas de avaliação / Plot size to evaluate characters in white lupine in evaluation times

Os objetivos deste trabalho foram determinar o tamanho ótimo de parcela (Xo) para avaliar caracteres de tremoço branco (Lupinus albus L.) e verificar a variabilidade do Xo entre caracteres e entre épocas de avaliação. Foram realizados 36 ensaios de uniformidade de tamanho 6m×6m (36m2). Cada ensaio foi dividido em 36 unidades experimentais básicas (UEB) de 1m×1m (1m2), totalizando 1.296UEB. Foram pesadas a massa verde total, a massa verde de parte aérea e a massa verde de raízes das plantas de cada UEB. Foram realizadas três avaliações (123, 137 e 150 dias após a semeadura) e, em cada avaliação, foram avaliados 12 ensaios (432UEB). O tamanho ótimo de parcela (Xo) foi determinado por meio do método da curvatura máxima do modelo do coeficiente de variação e as comparações de médias feitas pelo teste t de Student. Há variabilidade do tamanho ótimo de parcela entre caracteres e entre épocas de avaliação. O tamanho ótimo de parcela para avaliar a massa verde de raízes é maior que o necessário para avaliar a massa verde total e a massa verde de parte aérea, independentemente da época de avaliação. Maior tamanho ótimo de parcela é necessário quando a avaliação é realizada aos 123 dias após a semeadura, em relação às avaliações aos 137 e 150 dias após a semeadura, independentemente do caractere. O tamanho ótimo de parcela de 7,48 unidades experimentais básicas de 1m2(7,48m2) é suficiente para avaliar esses três caracteres nessas três épocas de avaliação.

The objectives of this research were to determine the optimum plot size (Xo) to evaluate characters of white lupine (Lupinus albus L.) and verify the variability of the Xo among characters and evaluation times. It was carried 36 uniformity assays of size 6m×6m (36m2). Each assay was divided in 36 basic experimental units (UEB) 1m×1m (1m2), totaling 1,296 UEB. The total fresh mass, fresh mass of shoots and fresh mass roots of plant of each UEB was weighed. Three evaluations were carried (123, 137 and 150 days after sowing) and at each assessment were evaluated 12 assays (432UEB). The optimum plot size was determined by the method of maximum curvature of the model coefficient of variation and the means compared by Student's t test. There is variability in the optimum plot size among characters and evaluation times. The optimum plot size for evaluating the fresh mass of roots is greater than necessary to evaluate the total fresh mass and the fresh mass of shoots, independently of the evaluation time. Higher optimum plot size is necessary when the evaluation is carried out at 123 days after sowing in relation to evaluations for 137 and 150 days after sowing, independently of character. The optimum plot size of 7.48 basic experimental units of 1m2 (7.48m2) is sufficient to evaluate these three characters in these three evaluation times.

Mitigation of Cu stress by legume-Rhizobium symbiosis in white lupin and soybean plants.

The effect of Bradyrhizobium-legume symbiosis on plant growth, toxicological variables and Cu bioaccumulation was studied in white lupin and soybean plants treated with 1.6, 48, 96 and 192 µM Cu. In both species, those plants grown in the presence of root nodule-forming symbiotic Bradyrhizobium showed less root and shoot growth reduction, plus greater translocation of Cu to the shoot, than those grown without symbiotic Bradyrhizobium. The effective added concentrations of Cu that reduced shoot and root dry weight by 50% (EC50), and the critical toxic concentration that caused a 10% reduction in plant growth (CTC10%), were higher in plants grown with symbiotic Bradyrhizobium, and were in general higher in the roots whether the plants were grown with or without these bacteria. The production of malondialdehyde and total thiols was stimulated by Cu excess in the shoots and roots of white lupin grown with or without symbiotic Bradyrhizobium, but mainly in those without the symbionts. In contrast, in soybean, the increases in malondialdehyde and total thiols associated with rising Cu concentration were a little higher (1.2-5.0 and 1.0-1.6 times respectively) in plants grown with symbiotic Bradyrhizobium than without. Finally, the organ most sensitive to Cu excess was generally the shoot, both in white lupin and soybean grown with or without symbiotic Bradyrhizobium. Further, Bradyrhizobium-legume symbiosis appears to increase the tolerance to Cu excess in both legumes, but mainly in white lupin; plant growth was less reduced and CTC10% and EC50 values increased compared to plants grown without symbiotic Bradyrhizobium. Bradyrhizobium N2 fixation in both legumes would therefore seem to increase the phytoremediation potential of these plants when growing on Cu-contaminated sites.

Relações lineares entre caracteres de nabo forrageiro e de tremoço branco / Linear relations among characters of forage turnips and of white lupine

O objetivo deste trabalho foi avaliar as relações entre caracteres de nabo forrageiro (Raphanus sativus L.) e de tremoço branco (Lupinus albus L.), e identificar caracteres para a seleção indireta. Foi conduzido um experimento com nabo forrageiro e outro com tremoço branco, em Santa Maria, Estado do Rio Grande do Sul (latitude 29°42'S, longitude 53°49'W e 95m de altitude). Na colheita, foram mensurados a altura de planta, o diâmetro de caule, o número de folhas e as massas verde e seca de parte aérea em 300 plantas de nabo forrageiro e a altura de planta, o diâmetro de caule, o número de vagens e as massas verde e seca de parte aérea em 400 plantas de tremoço branco. Foi investigada a relação entre os caracteres por meio de diagramas de dispersão e análises de correlação e de trilha. Na cultura de nabo forrageiro, o diâmetro de caule tem relação linear positiva com as massas verde e seca e pode ser utilizado para seleção indireta. Na cultura de tremoço branco, o número de vagens por planta tem relação linear positiva com as massas verde e seca e pode ser utilizado para seleção indireta.

The objective of this paper was to evaluate the relationships among characters in forage turnips (Raphanus sativus L.) and white lupine (Lupinus albus L.), and identify character for indirect selection. An experiment was conducted with forage turnips and another with white lupine, in Santa Maria, Rio Grande do Sul State, Brazil (latitude 29°42'S, longitude 53°49'W and 95m altitude). At harvest, were measured plant height, stem diameter, leaf number and fresh and dry masses of shoots in 300 plants of forage turnips and plant height, stem diameter, number of pods and the fresh and dry masses of shoots in 400 plants of white lupine. It was studied the relationship among the traits by scatter plots, correlation and path analysis. In the culture of forage turnips the stem diameter has a positive linear relationship with the fresh and dry masses of shoots and can be used for indirect selection. In the culture of white lupine the number of pods has a positive linear relationship with the fresh and dry masses of shoots and can be used for indirect selection.

Growth conditions determine different melatonin levels in Lupinus albus L.

Melatonin, an indoleamine, which has recently been assigned several roles in plant physiology as a growth promoter, as rooting agent, and as antioxidant in senescence delay and cytoprotection, seems to have a relevant function in plant stress situations. The presence of melatonin increases the resistance of lupin plant tissues (Lupinus albus L.) against natural or artificially induced adverse situations. In this work, we studied the response of lupin plants in controlled stress situations (drought-, anaerobic-, pH-, and cold stress and using ZnSO4 , NaCl, and H2 O2 as chemical stressors) and measured the changes in endogenous melatonin levels in lupin plants. Also, the effect of abscisic acid, ethylene, and natural environmental conditions were evaluated. In general, nearly all stressful factors caused an increase in melatonin in the investigated organs. The chemical stress provoked by ZnSO4 or NaCl caused the most pronounced changes in the endogenous level of melatonin, followed by cold and drought stressors. In some cases, the level of melatonin increased 12-fold with respect to the levels in control plants, indicating that melatonin biosynthesis is upregulated in common stress situations, in which it may serve as a signal molecule and/or as a direct antistress agent due to its well-known antioxidative properties.

Sugar signalling mediates cluster root formation and phosphorus starvation-induced gene expression in white lupin.

Cluster root (CR) formation contributes much to the adaptation to phosphorus (P) deficiency. CR formation by white lupin (Lupinus albus L.) is affected by the P-limiting level in shoots, but not in roots. Thus, shoot-derived signals have been expected to transmit the message of P-deficiency to stimulate CR formation. In this study, it is shown that sugars are required for a response to P starvation including CR formation and the expression of P starvation-induced genes. White lupin plants were grown in vitro on P-sufficient or P-deficient media supplemented with sucrose for 4 weeks. Sucrose supply stimulated CR formation in plants on both P-sufficient and P-deficient media, but no CR appeared on the P-sufficient medium without sucrose. Glucose and fructose also stimulated CR formation on the P-sufficient medium. On the medium with sucrose, a high concentration of inorganic phosphate in leaves did not suppress CR formation. Because sorbitol or organic acid in the media did not stimulate CR formation, the sucrose effect was not due to increased osmotic pressure or enriched energy source, that is, sucrose acted as a signal. Gene transcription induced by P starvation, LaPT1 and LaPEPC3, was magnified by the combination of P limitation and sucrose feeding, and that of LaSAP was stimulated by sucrose supply independently of P supply. These results suggest that at least two sugar-signalling mediating systems control P starvation responses in white lupin roots. One system regulates CR formation and LaSAP expression, which acts even when P is sufficient if roots receive sugar as a signal. The other system controls LaPT1 and LaPEPC3 expression, which acts when P is insufficient.