The object of this paper is to compare the Tennessee green pod pole bean with other beans as to chemical composition and food value.

De la reconnaissance protéine-glucides. Une activité lectine avec une spécificité mannose/glucose a été détectée dans les graines de Platypodium elegans, une légumineuse de la sous-tribu Dalbergiae. Le gène de la lectine PELa a été cloné. Son produit est une protéine de 261 acides aminés appartenant à la famille des lectines de légumineuses et présentant des similarités avec l'agglutinine de Pterocarpus angolensis (PAL). La lectine recombinante a été exprimée dans E. coli et renaturée à partir des corps d'inclusion. L'analyse de la spécificité par Glycan Array montre une préférence très rare pour des N-glycanes de type complexe avec des branches disymmétriques. Une branche courte composée d'un résidu de mannose est préférée sur le bras 1-6 des N-glycanes, tandis que l'extension par les résidus GlcNAc et Gal et favorable sur le bras 1-3. Les affinités ont été mesurées par microcalorimétrie de titration en utilisant des heptasaccharides liés à une asparagine et obtenus par une méthode semi-enzymatique. Une très forte affinité de 5 uM a été obtenue pour deux ligands symétriques et disymmétriques. Les structures cristallographiques de PELa complexé avec le trimannose branché et l'heptasaccharide-Asn symétrique de type complexe ont été résolues respectivement à 2,1 et 1,65 Å de résolution. La lectine adopte l'organisation dimérique canonique des lectines de légumineuses. Le trimannose ponte les sites de liaison de deux dimères voisins, résultant en la formation de chaînes infinies dans le cristal. L'heptasaccharide-Asn se lie par le mannose du bras 1-6 dans le site principal de liaison et de nombreux contacts supplémentaires sont établis avec les autres résidus glucidiques. Le GlcNAc du bras 1-3 interagit avec la surface de la protéine dans une conformation contrainte qui peut expliquer la plus grande affinité que l'on observe sur les puces pour les oligosaccharides avec des bras 1-3 courts qui ne contiennent pas ce monosaccharide. / Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume from the Dalbergiae tribe. The gene of the lectin PELa has been cloned and the resulting 261 amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin (PAL) from the same tribe. The recombinant lectin has been expressed in E. coli and refolded from inclusion bodies. Analysis of specificity by Glycan Array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6- arm of the N-glycan, while extension by GlcNAc, Gal and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn- linked heptasaccharide prepared by semi-enzymatic method. Strong affinity of 5 µM was obtained for both ligands. Crystal structures of PELa complexed with branched trimannose and symmetrical complex type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighbouring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site and extensive additional contacts on both arms. The GlcNAc on the 3-arm is bound in a constrained conformation that may rationalize the higher affinity that is observed on chips for oligosaccharide with shorter 3-arm that do not present this monosaccharide.

Made available in DSpace on 2016-04-28T14:58:56Z (GMT). No. of bitstreams: 1 2007-Humberto Antao de Sousa e Silva.pdf: 1812876 bytes, checksum: fc5dddb0fe3112c5b22807ccdce3e05b (MD5) Previous issue date: 2007-02-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In a modern cattle-breeding using pastures that allows a gain of weight is an essential factor. The Arachis pintoi is an option to a good alimentary diet and to the recuperation of degraded pastures, due to its potential in biological fixation of nitrogen and resistance to acid soil with high contents of aluminium. Experiments were conducted in greenhouse and growth chamber condition, in order to study the behavior of Arachis pintoi (Amarillo cv., Belmonte cv. and accession BRA-031534) in its relation to inoculation with rhizobial strains, aluminium tolerance and Mo influence. Strains CIAT-5067, CIAT-5083, CIAT-5087, BR-1432 e BR-1433 were used in inoculation experiments. In experiments with aluminium were used: a) A simple nutritive solution, initially with rates 10μM, 20μM, 40μM, 80μM and 160μM, and later 2μM, 4μM, 8μM, 16μM e 32μM, b) A complete nutritive solution with rates 20μM, 40μM, 80μM, 160μM and 320μM at Amarillo cv. and access BRA-031534 propagated by seeds and at the Amarillo and Belmonte cv. and access BRA-031534 vegetatively propagated. In the experiment with Mo, four rates of molybdenum (0g.ha-1, 50g.ha-1, 100g.ha-1 e 200g.ha-1), three levels of pH (4,0; 5,0 e 6,0) and two collection dates (49 days and 78 days) were used. The plants inoculated with CIAT strains, presented a good response to nodulation especially in relation to Belmonte cv., and with the same effectiveness of native strain. It was observed deleterious effect of nitrogen in the number of nodules. The results in simple nutritive solution had shown reduction in growth and dry mass of the root and a reduction evaluated in 27% by the relative root length between the concentration of 16μM and the control. In these experiments were observed responses to the concentration of aluminium, with appearance of mucilage and darkening of the primary root. In studies involving complete nutritional solution, Amarillo cultivar and accession BRA-031534 propagated by seeds had had reduction in the growth of the primary root and in relative length of the root in relation to the control. In the concentrations of 320μM and 160μM, a reduction of 51% and 49% respectively, in the Amarillo cultivar and access BRA-031534. It was also observed in this study with the Amarillo cultivar, roots with tip dark in the concentrations of 160μM and 320μM, and increase in the number of secondary roots from the concentration of 80μM. Pyrocatecol violet dyes seems to be promising in allowing a visualization of aluminum effects to Amarillo cultivar and accession BRA-031534. The aluminum in vegetative propagation influenced the cultivars and accession tested. Belmonte cultivar formed a higher number of adventitious roots when compared with Amarillo cv and accession BRA-031534, and with a concentration of 80μM a reduction in the adventitious roots length of the tested cultivars and access occurred. The molybdenum application promote answer in the second harvest in the pH 6,0 being obtained a larger number of nodules in the concentration of 200g.ha-1.The pH intervened in the nodulation. / Na pecu?ria moderna, o uso de pastagens que permitem um bom ganho de peso ? um fator imprescind?vel. A leguminosa Arachis pintoi apresenta-se como uma op??o tanto para uma dieta alimentar eficiente, como para recupera??o de pastagens degradadas devido ao seu potencial em fixa??o biol?gica de nitrog?nio e resist?ncia a solos ?cidos com teor elevado de alum?nio. Com a finalidade de estudar o comportamento do Arachis pintoi em rela??o ? inocula??o com estirpes de riz?bio, toler?ncia ao alum?nio e a influ?ncia do molibd?nio, foram conduzidos experimentos em condi??es de casa de vegeta??o e c?mara de crescimento. Nos experimentos de inocula??o utilizaram-se as estirpes CIAT-5067, CIAT-5083, CIAT-5087, BR-1432 e BR-1433. Nos experimentos com alum?nio foram utilizadas: a) Solu??o nutritiva simples, (10μM, 20μM, 40μM, 80μM e 160μM) e (2μM, 4μM, 8μM, 16μM e 32μM), na cultivar Amarillo. b) Solu??o nutritiva completa (20μM, 40μM, 80μM, 160μM e 320μM) na cultivar Amarillo e acesso BRA-031534 propagadas por sementes e nas cultivares Amarillo, Belmonte e acesso BRA-031534 propagadas vegetativamente. No experimento com molibd?nio, foi utilizada a cultivar Amarillo, quatro dosagens de molibd?nio (0gha-1, 50g.ha-1, 100g.ha-1 e 200g.ha-1), tr?s n?veis de pH (4,0; 5,0 e 6,0) e duas ?pocas de coleta (49 e 78 dias). Nos experimentos de sele??o de estirpes, as plantas inoculadas com as estirpes CIAT, apresentaram uma boa resposta ? nodula??o principalmente em rela??o ? cultivar Belmonte, e com a mesma efetividade que as estirpes nativas. Na cultivar Belmonte foi observado efeito delet?rio do nitrog?nio no n?mero de n?dulos por planta. Os resultados obtidos nos experimentos com Al, em solu??o nutritiva simples mostraram uma redu??o no crescimento, redu??o na massa seca da raiz e uma redu??o avaliada em 27% pelo comprimento radicular relativo entre as concentra??es de 16μM e o controle. Ocorreram nestes experimentos respostas ?s concentra??es de alum?nio, com aparecimento de mucilagem e escurecimento da raiz prim?ria. Nos estudos envolvendo solu??o nutritiva completa, as pl?ntulas da cultivar Amarillo e do acesso BRA-031534 propagadas por sementes tiveram redu??o no crescimento da raiz prim?ria e no comprimento relativo da raiz em rela??o ? testemunha. Nas concentra??es de 320μM e 160μM, observou-se uma redu??o de 51% e 49% respectivamente, na cultivar Amarillo e no acesso BRA-031534. Tamb?m foi observado na cultivar Amarillo, ra?zes com as extremidades escurecidas nas concentra??es de 160μM e 320μM e aumento no n?mero de ra?zes secund?rias a partir da concentra??o de 80μM. O corante violeta de pirocatecol parece promissor em permitir uma visualiza??o dos efeitos do alum?nio para a cultivar Amarillo e acesso BRA-031534. O alum?nio influenciou as cultivares e acesso testados com a cultivar Belmonte formando um maior n?mero de ra?zes advent?cias que a cv Amarillo e o acesso BRA-031534. A partir da concentra??o de 80μM, ocorreu uma redu??o no comprimento das ra?zes advent?cias das cultivares e acesso testados. A aplica??o de molibd?nio foi eficiente na segunda colheita no pH 6,0 sendo obtido um maior n?mero de n?dulos na concentra??o de 200g.ha-1. O pH interferiu na nodula??o.

Thesis (M.Sc. (Agriculture)) --University of the North, 2001

The thesis entitled “Folding studies on Peanut Agglutinin: A lectin with an unusual quaternary structure” deals with the several aspects of the folding of the tetrameric legume lectin Peanut Agglutinin (PNA). PNA is a well studied legume lectin and several interesting observations regarding its unfolding have been published from our laboratory. The present thesis is an extension of the same work to enrich our knowledge about the folding behaviour of PNA. The thesis describes both experimental as well as theoretical insight on unfolding of PNA. Chapter 1 is a general discussion on lectins. Lectins are carbohydrate binding proteins of non immune source. Lectins are generally found in all type of organisms- plants, animals as well as micro-organisms. Among the plant lectins “legume lectin” is a very well studied system. Legume lectins share a general tertiary structural fold; “jelly roll fold” while they vary in their quaternary structure. Thus they can be considered as “natural mutants” in the context of quaternary structure. The origin of the lectins, structure and sugar specificity have been discussed with emphasis on legume lectin family. Chapter 2 describes the thermodynamics related to the urea induced denaturation of PNA. PNA shows a very interesting unfolding profile, populating one molten globule like intermediate during thermal as well as chaotrope induced denaturation. The molten globule like intermediate loses most of its tertiary structure but retains sufficient secondary structure. Surprisingly, the molten globule like state retains its carbohydrate binding specificity like the native PNA. A model has been developed to fit the chaotrope induced three state denaturation profile of PNA. The model considers the tetramer to dissociate to monomeric intermediate, which in turn dissociates to complete denatured state. All the relevant thermodynamic parameters (∆G, ∆Cp, Tg) associated in the denaturation process have been extracted. The tetramer is found to be ~30 kcal/Mole more stable compared to the intermediate and the intermediate is ~8 kcal/Mole more stable compared to the denatured. The denaturation process has been followed by the changes in hydrodynamic radii by dynamic light scattering (DLS). The profile of change in hydrodynamic radius and the % intensity clearly identify the generation of two species simultaneously. The analysis shows that the intermediate is ~40 % unfolded in nature. Thus this chapter deals with the detailed study of thermodynamics and dynamic light scattering study of the urea induced denaturation of PNA. Chapter 3 deals with the effect of 2, 2, 2 - trifluoroethanol (TFE) on the structure of PNA at two different pH. TFE is a well known co-solvent and is widely used to induce α- helical structure in a protein. The secondary structures induced by TFE are assumed to reflect conformations that prevail during early stages of protein folding. Thus it was quite interesting to notice the structural changes induced by TFE. The effect of TFE has been studied at two different pH- neutral pH of 7.4 and acidic pH 2.5. The  structure of the protein is accentuated in the presence of TFE at low concentration at both the pH. TFE induces α-helical structure from 40 % (v/v) concentration onwards at both the pH. TFE at 15 % concentration induces a molten globule like structure at low pH. The quenching of acrylamide suggests that the protein at low pH and 15 % TFE concentration has a more compact structure compared to the protein at low pH in absence of TFE as well as 6M guanidine hydrochloride (GdnHCl). Further studies of hydrodynamic radii by dynamic light scattering (DLS) also reveal that the protein undergoes some kind of compaction in presence of 15 % TFE at low pH. The induction of this type of molten globule like state at neutral pH has not been observed. Chapter 4 describes the molecular dynamics simulation of deoligomerization of PNA. The native PNA (PDB code 2PEL), excluding any ligand and metal ions has been simulated at 300 K, 400 K, 500 K and 600 K for 500 ps. The overall destabilisation has been followed by root mean square deviation (RMSD), the radius of gyration (Rg) and the solvent accessible surface area (ASA), while the atomistic details are revealed by residue wise RMSD (RRMS), hydrogen bonds and cluster analysis. The protein shows a quite a dramatic change in RMSD and radius of gyration profile at 600 K. RRMS shows that the residues belonging to the loops, mainly in the metal binding site show quite high flexibility. The relative change in average accessible surface area reveals that the primary core of the protein is exposed at 600 K while it is well buried till 500 K. The hydrogen bond analysis clearly shows that with increase in temperature number of hydrogen bonds starts decreasing. Mainly the hydrogen bonds involving side chain interactions are broken. Surprisingly, not all the monomers behave similarly. Monomers C and D are more perturbed compared to monomers A and B. The asymmetry in the interfaces of the monomers may be the key reason for it. The change in the interfaces has been probed by hydrogen bond analysis and cluster analysis. The GSIV type interfaces (A-D and B-C) have been found out to be the most dynamic in nature compared to the other two interfaces. Thus, this chapter reveals the early stage of unfolding of PNA, where perturbation in secondary and tertiary structural level is quite prominent but the interfaces are still holding weakly and are not completely dissociated. Chapter 5 is the continuation of the molecular dynamics simulation on unfolding of PNA, where the effect of metal ions has been illustrated. The monomeric PNA has been simulated to compare its dynamics with the tetramer. The metal binding loop (125-135) becomes unstable and opens up for the monomer even at 300 K after 800 ps. The monomer at 600 K is completely disorganized. The instability of the metal binding loop of the monomer triggers the urge to study the simulation in presence of metal ions (Ca2+ and Mn2+). The monomer bound with metal ions shows steady fluctuation at 300 K. Binding of metal ions seems to bring stability even at 600 K. Surprisingly binding of metal ions to the metal binding site not only stabilises the metal binding loop but also stabilises residues at back beta sheet which are involved in oligomerization. Hence, another simulation of the tetramer at 600 K bound with metal ions has been done. It has been shown that binding of metal ions increases the stability of the protein without altering the denaturation pathway. Appendix A describes a completely different study from PNA. The initial spectral and kinetic characterization of 7, 8- Diaminopelargonic acid Synthase (DAPA Synthase) has been done from Mycobacterium tuberculosis. The DAPA Synthase gene has been cloned earlier in our laboratory and the same has been used for further studies. This is a well known pyridoxal-5′ phosphate (PLP) dependent enzyme, which converts 8- Amino-7-oxopelargonic Acid (KAPA) to 7, 8-Diaminopelargonic Acid (DAPA) in the second step of biotin biosynthesis. DAPA Synthase uses S-adenosylmethionine (SAM) and KAPA as substrate. The first half of the enzymatic reaction has been followed spectroscopically, both by steady state and stopped flow. The enzyme seems to undergo change in conformation as evident from fluorescence and circular dichroism study. The Km value has been determined using bioassay technique. The detailed characterization of the enzyme has been described in this chapter.

Bradyrhizobium japonicum ist ein Bodenbakterium, welches in der Lage ist mit der bedeutenden Agrarpflanze Sojabohne zu interagieren und deren Wachstum zu fördern. In der vorliegenden Arbeit wurde das Genistein-Stimulon und verschiedenen Stressantworten von B. japonicum mittels Mikroarrayanalyse bestimmt. 101 Gene werden nach Genisteinzugabe induziert. NodW ist der Hauptaktivator der Genistein-induzierbaren Gene, welche zum Großteil kein nod-Box-Motiv in der Promotorregion aufweisen. Einzig acht Gene, wovon sieben für Transportersysteme kodieren, zeigen in der NodW-Mutante 613 nach Genisteinzugabe weiterhin ein erhöhtes Expressionsniveau. In weiterführenden Arbeiten konnte für zumindest ein Transportersystem (Bll4319/Bll4320/Bll4321) neben der Genistein-Abhängigkeit auch eine Regulation durch den Genistein-abhängigen TetR-Regulator FrrA nachgewiesen werden [Günther 2007; Bhandari 2008]. Dies zeigt, dass NodW in weiterführende Regulationskaskaden eingebunden sein muss, wobei diese in der vorliegenden Arbeit nicht näher charakterisiert wurden. Die Mikroarraydaten geben lediglich Hinweise auf mögliche Regulationskaskaden. Anhand der Mikroarraydaten liegt die Vermutung nahe, dass die Transkriptionsaktivierung des lateralen Flagellenclusters ebenfalls einer indirekten NodW-abhängigen Regulation unterliegt. Durch Pflanzentests war bekannt, dass es B. japonicum 901 möglich ist, den NodW-Defekt bei der Nodulation der Wirtspflanzen zu überwinden [Grob et al. 1993]. Dies beruht auf die Überexpression des 2-Komponentenregulators NwsB. So können Symbiose-relevante Gene wie die nod-Box-assoziierten Gene ähnlich dem Wildtyp nach Genistein-Zugabe induziert werden. NwsB ist aber nicht in der Lage, das Flagellarcluster der lateralen Flagellen (bis auf blr6846) nach Genistein-Zugabe zu induzieren. Dies bedeutet, dass trotz der hohen Ähnlichkeit zwischen den 2-Komponentenregulatoren NodW und NwsB, die Bindestellen in der Promotorregion der Genistein-induzierbaren Genen nicht identisch sind. Die NodD1-Mutante 1267 ist in der Lage, weiterhin Knöllchen mit allen Wirtspflanzen von B. japonicum zu bilden [Göttfert et al. 1992], was wahrscheinlich auf ein funktionelles NodW als Hauptaktivator der nodYABC-Operons zurückzuführen ist. Anhand der Mikroarraydaten ist festzustellen, dass kein weiteres nod-Box-assoziiertes Operon in der NodD1-Mutante 1267 verstärkt exprimiert vorliegt. Dies könnte bedeuten, dass NodW die Transkription von nodD1 und des nodYABC-Operons startet und später durch NodD1 in der Aktivierung der nod-Box-assoziierten Gene unterstützt wird. In der vorliegenden Arbeit wurde die transkriptionelle Stressantwort von B. japonicum hinsichtlich pH 4, pH 8, 80 mM NaCl, Hitzeschock und Temperaturstress analysiert. Dabei konnten sowohl Aussagen über Gene, welche in die allgemeine als auch in die spezifische Stressantwort eingebunden sind, getroffen werden. Die transkriptionelle Antwort auf pH 8 war mit 1636 differenziell exprimierten Genen die umfangreichste Stressantwort der vorliegenden Arbeit. Hierbei konnte gezeigt werden, dass B. japonicum bei pH-Stress besonders Gene der pHi-Homöostase aktiviert. Dies umfasst sowohl Transportergene als auch enzymatische Gene. Interessant waren die differenziell exprimierten Gene, welche bei pH 8 verstärkt und bei pH 4 verringert exprimiert vorlagen. Diese Gene besitzen im Promotorbereich eine RegR-Box und sind in der transkriptionellen Aktivierung von RegR abhängig [Lindemann et al. 2007]. Aufgrund der Homologie des RegSR-Systems von B. japonicum mit dem pH-abhängigen ActSR-System von S. meliloti besteht die Möglichkeit, dass RegSR ebenfalls in die pH-abhängige Regulation dieser Gene eingebunden ist. Neben solch spezifischen Stressantworten konnte auch eine allgemeine Stressantwort ermittelt werden. So weisen fünf Gene ein gleiches Expressionsmuster in den untersuchten Stressanalysen dieser Arbeit auf. blr5264 kodiert hierbei für einen 2-Komponenten-Hybridsensor und -Regulator und könnte als Regulator in die allgemeine Stressantwort von B. japonicum eingebunden sein. Aus diesem Grund wurde Blr5264 in GscR (general stress control regulator) umbenannt. Mittels Mutagenese von gscR wurde B. japonicum D826 erzeugt, dessen Transkriptom bezüglich Salzstress und Hitzeschock verifiziert wurde. Es wurden 87 Gene identifiziert, welche bei Stresseinfluss von GscR abhängig sind. Hierunter sind sieben verringert exprimierte Gene, welche bei Stress im Wildtyp verstärkt exprimiert vorliegen. Diese scheinen demzufolge bei Stress durch GscR transkriptionell aktiviert zu werden. Das interessanteste Gen war hierbei blr7881, welches für einen ArsR-Typ-Regulator kodiert und einen weiteren Schritt in der allgemeinen Stressantwort von B. japonicum sein könnte.

A fundamental goal in ecology and evolutionary biology has been to understand how microevolutionary forces affect the origin and maintenance of mutualisms over ecological and evolutionary time scales. Mutualistic partners vary in the reciprocal benefits they provide, yet the role of partner variation on microevolutionary forces that impact the maintenance of mutualisms is unclear. Using the mutualism between legumes and nitrogen fixing symbionts, my dissertation investigated the ecological and evolutionary consequences of variation in partner quality. In the first experiment, I demonstrate how insect herbivory can change the costs and benefits of associating with exploiters, and that some degree of exploitation from non-beneficial rhizobia can reduce insect herbivory, thus removing the fitness advantage of associating purely with beneficial rhizobia. In the second study, I examine how rhizobia genotype modifies competition between hosts grown in kin and non-kin groups. I show that lower fitness in plant kin groups can simply be a by-product of genetic variance in plant size and non-linear relationships between plant size and fitness. I further show that the symbiotic community can change difference in fitness between kin and non-kin groups independent of these by-product effects. In my last chapter, I provide the first empirical evidence that an important mechanism for mutualism stability-- the ability for hosts to preferentially associate with beneficial rhizobia-- is genetically variable and can evolve in response to exploitation. I also show that host preference for beneficial rhizobia can be maintained in legume populations, even in the absence of exploitation. My dissertation provides insight into the potential evolutionary dynamics of stabilizing mechanisms by suggesting that the agents of selection that affect the level of host exploitation can come from biotic factors other than the exploiters themselves. My dissertation has also shown that inclusion of other ecological interactions, such as herbivory, can provide valuable perspective on fitness effects of symbionts on their hosts, and can even change our fundamental assumptions about the effects of exploitation on host fitness, which has formed the backbone of mutualism theory.

A fundamental goal in ecology and evolutionary biology has been to understand how microevolutionary forces affect the origin and maintenance of mutualisms over ecological and evolutionary time scales. Mutualistic partners vary in the reciprocal benefits they provide, yet the role of partner variation on microevolutionary forces that impact the maintenance of mutualisms is unclear. Using the mutualism between legumes and nitrogen fixing symbionts, my dissertation investigated the ecological and evolutionary consequences of variation in partner quality. In the first experiment, I demonstrate how insect herbivory can change the costs and benefits of associating with exploiters, and that some degree of exploitation from non-beneficial rhizobia can reduce insect herbivory, thus removing the fitness advantage of associating purely with beneficial rhizobia. In the second study, I examine how rhizobia genotype modifies competition between hosts grown in kin and non-kin groups. I show that lower fitness in plant kin groups can simply be a by-product of genetic variance in plant size and non-linear relationships between plant size and fitness. I further show that the symbiotic community can change difference in fitness between kin and non-kin groups independent of these by-product effects. In my last chapter, I provide the first empirical evidence that an important mechanism for mutualism stability-- the ability for hosts to preferentially associate with beneficial rhizobia-- is genetically variable and can evolve in response to exploitation. I also show that host preference for beneficial rhizobia can be maintained in legume populations, even in the absence of exploitation. My dissertation provides insight into the potential evolutionary dynamics of stabilizing mechanisms by suggesting that the agents of selection that affect the level of host exploitation can come from biotic factors other than the exploiters themselves. My dissertation has also shown that inclusion of other ecological interactions, such as herbivory, can provide valuable perspective on fitness effects of symbionts on their hosts, and can even change our fundamental assumptions about the effects of exploitation on host fitness, which has formed the backbone of mutualism theory.

The symbiosis between plants of the legume family and nitrogen-fixing rhizobia underpins global food security. Legume crops are a major source of protein in human diets, either directly or indirectly as feed for livestock. Application of inoculant rhizobial strains is common practice in many areas, as plant growth is often nitrogen limited and the symbiosis can significantly enhance yields. However, rhizobial strains and outcomes of the symbiosis vary widely. This variation has also been studied by evolutionary biologists interested in the stability of mutualisms. They proposed that plants may prevent establishing symbioses with ineffective strains (partner choice), or provide them with fewer resources (sanctioning). I studied both mechanisms, combining modelling and experimental approaches. Mathematical modelling was used to predict how plants should allocate resources to maximise growth rates, depending on rhizobial nitrogen provision and carbon requirements and on soil nitrogen conditions. The use of marked mutant strains – easily distinguishable and differing in a single rhizobial characteristic – overcame previous experimental difficulties. It was found that pea (Pisum sativum L.) plants are not able to exert partner choice, but do sanction in a more complex way than was previously established. In line with model predictions, resources were preferentially allocated to the single – best available – strain, so that resources allocated to an intermediate-fixing strain depended on whether or not a strain providing more nitrogen was available. Contrary to model predictions, there was no indication of discrimination based on rhizobial carbon requirements. The results cannot be explained by resource allocation in proportion to nitrogen received, and indicate systemic integration of information from different nodules. I formulate a hypothesis about the underlying plant regulatory mechanisms, and discuss implications of the results for selecting inoculant strains and enhancing yields in the field. Future work will rely on further integration of theoretical and applied methods and perspectives.

This diploma work was assessed effects of different seed treatment (untreated flour from whole seed, flour from the uterus after soaking seeds, flour from the seeds sprouted cotyledon) by three species of legumes (Pisum sativum conv. Sativum L., Vicia faba L., Lupinus angustifolius L.) yield and composition of protein isolate obtained by isoelectric precipitation. It was found that the seeds of change most affects yield precipitated N (mg) and 45%. When determining the value of the yield of protein was affected by significant interactions (treatment and the type of legume seeds). The largest share was, however, precipitate in untreated and germinated lupine seeds, in which values are around 57%. Furthermore, the thesis was to reverse the precipitation of proteins, using which we obtained protein concentrates from different species of legumes. The yields of protein concentrates reach values in the range of 60-80%. Spectra of soluble proteins is clearly visible high concentration of isolated proteins. Variation of sprouted seeds and soaked that occurred during treatment of seeds for the collapse of proteins with higher molecular weight. Furthermore, these grafts are transferred protein extraction at pH 9.0 in the later produced protein isolates. After acid precipitation is observed on a spectrum that is re-soluble protein isolates.