Biochemical and immunocytochemical localization of a BAPAase in developing and mature lupin cotyledons

Summary Activity measurements and specific antibodies were used to detect and localize in developing and mature cotyledons ofLupinus albus seeds an endopeptidase, active on BAPA, previously isolated from the same seeds. Total activity and enzyme amount were highest at full seed maturation and then declined during germination. Protein bodies were isolated from mature dry cotyledons under anhydrous conditions with a yield of intact organelles of about 80% as assessed by dot blotting with antibodies to lupin legumin-like storage globulin. Activity assays on the isolated protein bodies indicated that 72% of BAPAase activity was associated with these organelles. Quantitative immunocytolocalization with antibodies to the enzyme on thin sections of mature lupin cotyledons confirmed that 75% of the enzyme was located inside the protein bodies. The possible involvement of the BAPAase in the proteolytic processing of the storage proteins during seed ontogeny is discussed.Abbreviations BAPA N-benzoyl-L-arginine-4-p-nitroanilide - DAF days after flowering - EM electron microscopy - NaPi sodium phosphate buffer - LRW London resin white - SDS sodium dodecylsulphate - PAGE polyacrylamide gel electrophoresis     

Investigating the potential of Andean lupin as a lignocellulosic feedstock for Europe: First genome-wide association study on Lupinus mutabilis biomass quality

The development of multipurpose crops will drive the transformation of agricultural “waste” into added-value products, helping to meet biomass demands without competing with food production or increasing environmental pressure. Lupinus mutabilis, has been proposed not only as a valid source of protein and oil for Europe but also as a possible source of lignocellulosic feedstock for the biorefinery industry. In this study, the quality of L. mutabilis lignocellulosic biomass and its genetic architecture are investigated for the first time, using a panel of 223 accessions planted across three locations in two different European cropping conditions. Biomass quality was evaluated based on the estimation of neutral detergent fiber, cellulose, hemicellulose and acid detergent lignin fractions, and on the basis of the monosaccharide composition of cell wall polysaccharides. The broad variation in yield and composition of biomass encountered in the panel confirms the potential of L. mutabilis as lignocellulosic feedstock and points out the value of this panel as a breeding tool for the improvement of biomass quality. A genome-wide association study was conducted to identify single-nucleotide polymorphisms (SNPs) associated with biomass quality, both across locations and per specific location. Scanning of 16,781 SNPs across the whole genome identified 46 unique quantitative trait loci for biomass quality, 4 of which were detected as common either among traits or GWAS models. For each of the traits analyzed, between 3 and 10 SNPs were detected, explaining 2.7%–15.9% of the phenotypic variation. Underlying these loci, 28 genes were proposed as candidate genes for biomass quality. Important genes involved in cellulose and sucrose synthesis (CESA4, SPP1,WRKY33, GONST2), monolignol biosynthesis (SKIP31, WAT1, CCR-SNL6) and pectin degradation (RAV1, PE) were identified and will require validation to confirm their value for application in L. mutabilis breeding.     

The role of exo-(1-->4)-beta-galactanase in the mobilization of polysaccharides from the cotyledon cell walls of Lupinus angustifolius following germination

BACKGROUND AND AIMS: The cotyledons of Lupinus angustifolius contain large amounts of cell wall storage polysaccharide (CWSP) composed mainly of (1-->4)-beta-linked D-galactose residues in the form of branches attached to a rhamnogalacturonan core molecule. An exo-(1-->4)-beta-galactanase with a very high specificity towards (1-->4)-beta-linked D-galactan has been isolated from L. angustifolius cotyledons, and shown to vary (activity and specific protein) in step with CWSP mobilization. This work aimed to confirm the hypothesis that galactan is the main polymer retrieved from the wall during mobilization at the ultrastructural level, using the purified exo-galactanase as a probe. METHODS: Storage mesophyll cell walls ('ghosts') were isolated from the cotyledons of imbibed but ungerminated lupin seeds, and also from cotyledons of seedlings after the mobilization of the CWSP. The pure exo-(1-->4)-beta-galactanase was coupled to colloidal gold particles and shown to be a specific probe for (1-->4)-beta-D-galactan. They were used to localize galactan in ultrathin sections of L. angustifolius cotyledonary mesophyll tissue during CWSP mobilization. KEY RESULTS: On comparing the morphologies of isolated cell walls, the post-mobilization 'ghosts' did not have the massive wall-thickenings of pre-mobilization walls. Compositional analysis showed that the post-mobilization walls were depleted in galactose and, to a lesser extent, in arabinose. When pre-mobilization ghosts were treated with the pure exo-galactanase, they became morphologically similar to the post-mobilization ghosts. They were depleted of approximately 70% of the galactose residues that would have been mobilized in vivo, and retained all the other sugar residues originally present. Sharply defined electron-transparent wall zones or pockets are associated with CWSP mobilization, being totally free of galactan, whereas wall areas immediately adjacent to them were apparently undepleted. CONCLUSIONS: The exo-(1-->4)-beta-galactanase is the principal enzyme involved in CWSP mobilization in lupin cotyledons in vivo. The storage walls dramatically change their texture during mobilization as most of the galactan is hydrolysed during seedling development.     

Influence of growth environment on coaggregation between freshwater biofilm bacteria

AIM: To characterize the expression of coaggregation between Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 following growth in liquid culture, on agar and in an artificial biofilm matrix composed of poloxamer hydrogel. METHODS AND RESULTS: The ability of B. natatoria 2.1 and M. luteus 2.13 to coaggregate with one another was assessed following growth in liquid culture as colonies on agar or within a poloxamer hydrogel matrix. In all these environments a cycle of gain and loss of coaggregation occurred when the two cell types were aged simultaneously, with optimum expression occurring in early stationary phase. Blastomonas natatoria 2.1 cells only coaggregated maximally after entry into stationary phase. Conversely, M. luteus 2.13 cells only coaggregated in exponential phase and early stationary phase and coaggregation ability was lost in late stationary phase. Maximal coaggregation therefore only occurred between the two strains if both were in early stationary phase, when the surface properties of the two cell types were optimal for coaggregation. CONCLUSION: In addition to occurring between cells grown in liquid culture, coaggregation between aquatic bacteria occurs after growth as a biofilm on agar and in an artificial biofilm matrix in poloxamer. Under all conditions, the B. natatoria 2.1 coaggregation adhesin and complementary receptor on M. luteus 2.13 were only expressed simultaneously during early stationary phase.     

Fine-scale genetically based differentiation of life-history traits in the perennial shrub Lupinus arboreus

Across large spatial scales, plants often exhibit genetically based differentiation in traits that allow adaptation to local sites. At smaller spatial scales, sharp boundaries between edaphic conditions also can create strong gradients in selection that counteract gene flow and result in local adaptation. Few studies, however, have examined the degree to which continuous populations of perennial plants exhibit genetically based differentiation in life-history traits over small spatial scales. We quantified the degree of genetically based differentiation in adaptive traits among bush lupine (Lupinus arboreus) from nearby dune and grassland sites (sites separated by < 0.75 km) that formed part of a larger continuous population of L. arboreus. We also investigated the spatial genetic structure of bush lupine by examining how genetic structure differed between seeds and juvenile plants that were less than two years old. We calculated F-statistics from gel electrophoresis of 10 polymorphic loci. We then used these values to infer levels of gene flow. To examine differentiation in adaptive traits, we created full-sibling/half-sibling families of lupine within each area and established reciprocal common gardens at each site. Across two years, we measured canopy volume, flowering time, seed set, and mortality of progeny planted in each garden. Spatial genetic structure among seeds was virtually nonexistent (F(ST) = 0.002), suggesting that gene flow between the three areas could be quite high. However, genetic structure increased 20-fold among juvenile plants (F(ST) = 0.041). We found strong evidence for fine-scale genetically based differentiation and local adaptation in adaptive traits such as plant size, flowering phenology, fecundity, and mortality. Thus, it is likely that strong but differing selection regimes within each area drive spatial differentiation in lupine life-history traits.     

The effects of nutrient supply,predominantly addition of iron,and rhizobial inoculation on the tolerance of Lupinus pilosus genotypes to a calcareous soil

Two glasshouse experiments were conducted to examine the effects of nutrient supply and rhizobial inoculation on the performance of Lupinus pilosus genotypes differing in tolerance to calcareous soils. In experiment 1, plants were grown for 84 days in a calcareous soil (50% CaCO₃; soil water content 90% of field capacity) at four nutrient treatments (no-added nutrients, added nutrients without Fe, added nutrients with soil applied FeEDDHA, added nutrients with foliar applied FeSO₄). In experiment 2, plants were grown for 28 days with supply of NH₄NO₃ without inoculation or inoculated with Bradyrhizobium sp. (Lupinus). Chlorosis in the youngest leaves was a good indicator of the relative tolerance of the genotypes to the calcareous soil in both experiments, except the treatment with FeEDDHA at 5 mg kg^–1 soil which was toxic to all genotypes. Chlorosis scores correlated with chlorophyll meter readings and chlorophyll concentrations. The foliar application of FeSO₄ did not fully alleviate chlorotic symptoms despite concentrations of active or total Fe in the youngest leaves being increased. Adding nutrients and chemical nitrogen did not change the severity of chlorosis or improve the growth of the plant. The nutrient supply did not alter the ranking of tolerance of genotypes to the calcareous soil. The results suggest that nutrient deficiency or poor nodulation was not a major cause of poor plant growth on calcareous soils and that bicarbonate may exert a direct effect on chlorophyll synthesis. The mechanism for tolerance is likely to be related to an ability to exclude bicarbonate or prevent its transport to the leaves.     

The effect of abscisic acid and K+ on xylem exudation from excised roots of Lupinus luteus

External application of abscisic acid (ABA) induces a relatively high rate of xylem exudation in excised roots of Lupinus luteus L. cv. Weiko III. The response is relatively slow with a lag period of ca 1 h. It is also slowly, but reversibly, abolished by application of 3.6 or 36 μ M cycloheximide (CHX). Contrary to expectation, K⁺ is not a significant factor in maintaining flow rates in lupin roots as no response was measured after adding K⁺ to root systems, from which K⁺ had been withheld for periods ranging from 3 h to several days. In fact, excised roots obtained from seedlings raised in the absence of K⁺ failed to respond to added K⁺. Total depletion of K⁺ is difficult to achieve, because of initial seed reserves, and prolonged exudation in lupins can be maintained utilising only a small proportion of the K⁺ originally present in the root tissue. Nevertheless, the data cast doubt on the general applicability of the concept that volume flow is maintained by an osmotic gradient with K⁺ as the principal mineral ion.     

Organospecific responses of lupin seedlings to lead Localization of hydrogen peroxide and peroxidase activity

In an earlier work using tissue printing method, we found that the PR-10 stress protein was observed in leaf petiole of lupin seedling where lead was not detected (Przymusiński et al. 2001). These results suggested the presence of substance(s) mediating a signal transduction from directly affected cells to distant organs. As the hydrogen peroxide was found to be involved in signal transduction pathway, in the present paper, we analysed the level of H₂O₂ in the organ of lupin seedlings exposed to Pb²⁺ with spectrophotometric method and tissue printing technique. It was unequivocally demonstrated that the level of H₂O₂ and the activity of peroxidase increased in every tested organ of lead-treated lupin seedling. Both the level of H₂O₂ and the activity of POX were correlated with amount of Pb²⁺ ions in the cells (Przymusiński et al. 2001) and decreased in tissues more and more distant from the site of metal application. On the other hand, there was no correlation between the histological localization of H₂O₂ and peroxidase. Our results seem to confirm the hypothesis that H₂O₂ may act as a signalling substance involved in the induction of PR protein synthesis. It was indicated that there is high degree of correlation between the localization of H₂O₂ and the histological localization of PR-10 proteins (Przymusiński et al. 2001) in every tested organ of lupin seedling. The presented hypothesis is also supported by the fact that H₂O₂ and PR-10 proteins are detected in organs and tissues where Pb²⁺ was not found at all.