No description available.

The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number of chaperone proteins were highly expressed in the nip mutant, indicating high stress in the mutant prior to infection by rhizobia. Additionally, a database containing the information regarding the nip expression profile at both 0 days post inoculation (dpi) and 10 dpi were created for screening of candidate genes as predicted from sequence in the genomic region containing NIP.

Die arbuskuläre Mykorrhiza ist die wahrscheinlich älteste Form der Wurzelsymbiosen zwischen Pflanzen und Pilzen und hat sich vor 420 Millionen Jahren entwickelt. In dieser Symbiose, die zwischen nahezu allen Landpflanzen und Pilzen des Reiches Glomeromycota ausgebildet wird, versorgt der Pilz die Pflanze mit Nährstoffen, wobei die verbesserte Versorgung mit Phosphat für die Pflanze sicher den größten Vorteil darstellt. Im Gegenzug erhält der Pilz Zucker, welche die Pflanze aus der Photosynthese bereitstellt. Zu hohe Phosphatkonzentrationen im Boden oder Dünger führen allerdings zu einer Verringerung in der Ausprägung der arbuskulären Mykorrhiza. Diese Unterdrückung der Symbiose wird nicht durch eine lokale Reaktion der Wurzeln ausgelöst, sondern in erster Linie durch einen hohen Phosphatgehalt im Pflanzenspross. Somit handelt es sich also um eine systemische, also dem Gesamtsystem „Pflanze“ betreffende Antwort. Die molekularen Mechanismen dieser Anpassung sind noch wenig bekannt und sind vor allem für die Agrarwirtschaft von besonderem Interesse. Eine Mikro-RNA (miRNA) des bereits bekannten Phosphathomöostasesignalwegs (PHR1-miRNA399-PHO2 Signalweg) akkumuliert verstärkt in mykorrhizierten Wurzeln. Das deutet daraufhin, dass dieser Signalweg und diese miRNA eine wichtige Rolle in der Regulation der arbuskulären Mykorrhiza spielen. Ziel dieser Studie war es neue Einblicke in die molekularen Mechanismen, die zur Unterdrückung der arbuskulären Mykorrhiza bei hohen Phosphatkonzentrationen führen, zu gewinnen. Dabei sollte der Einfluss von PHO2, sowie von miRNAs in dieser Symbiose genauer untersucht werden. Ein funktionelles Ortholog von PHO2, MtPho2, wurde in der Pflanze Medicago truncatula identifiziert. MtPho2-Mutanten, welche nicht mehr in der Lage waren ein funktionales PHO2 Protein zu exprimieren, zeigten schnellere Kolonisierung durch den AM-Pilz. Jedoch wurde auch in den mtpho2-Mutanten die Symbiose durch hohe Phosphatkonzentrationen unterdrückt. Dies bedeutet, dass PHO2 und somit der PHR1-miRNA399-PHO2 Signalweg eine wichtige Funktion während der fortschreitenden Kolonisierung der Wurzel durch den Pilz hat, aber und weitere Mechanismen in der Unterdückung der Symbiose bei hohen Phosphatkonzentrationen beteiligt sein müssen. Die Analyse von Transkriptionsprofilen von Spross- und Wurzeln mittels Microarrays zeigte, dass die Unterdrückung der AM Symbiose durch hohe Phosphatkonzentrationen möglicherweise auf eine Unterdrückung der Expression einer Reihe symbiosespezifischer Gene im Spross der Pflanze beruht. Um die Rolle weiterer miRNA in der AM Symbiose zu untersuchen, wurden mittels einer Hochdurchsatz-Sequenzierung 243 neue und 181 aus anderen Pflanzen bekannte miRNAs in M. truncatula entdeckt. Zwei dieser miRNAs, miR5229 und miR160f*, sind ausschließlich während der arbuskulären Mykorrhiza zu finden und weitere miRNAs werden während dieser Symbiose verstärkt gebildet. Interessanterweise führen einige dieser miRNAs zum Abbau von Transkripten, die eine wichtige Funktion in der arbuskulären Mykorrhiza und Wurzelknöllchensymbiose besitzen. Die Ergebnisse dieser Studie liefern eine neue Grundlage für die Untersuchung von regulatorischen Netzwerken, die zur zellulären Umprogrammierung während der Interaktion zwischen Pflanzen und arbuskulären Mykorrhiza-Pilzen bei verschiedenen Phosphatbedingungen führen. / AM symbiosis has a positive influence on plant P-nutrition and growth, but little is known about the molecular mechanism of the symbiosis adaptation to different phosphate conditions. The recently described induction of several pri-miR399 transcripts in mycorrhizal shoots and subsequent accumulation of mature miR399 in mycorrhizal roots indicates that local PHO2 expression must be controlled during symbiosis, presumably in order to sustain AM symbiosis development, in spite of locally increased Pi-concentration. A reverse genetic approach used in this study demonstrated that PHO2 and thus the PHR1-miR399-PHO2 signaling pathway, is involved in certain stages of progressive root colonization. In addition, a transcriptomic approach using a split-root system provided a comprehensive insight into the systemic transcriptional changes in mycorrhizal roots and shoots of M. truncatula in response to high phosphate conditions. With regard to the transcriptional responses of the root system, the results indicate that, although the colonization is drastically reduced, AM symbiosis is still functional at high Pi concentrations and might still be beneficial to the plant. Additionally, the data suggest that a specific root-borne mycorrhizal signal systemically induces protein synthesis, amino acid metabolism and photosynthesis at low Pi conditions, which is abolished at high Pi conditions. MiRNAs, such as miR399, are involved in long-distance signaling and are therefore potential systemic signals involved in AM symbiosis. A deep-sequencing approach identified 243 novel miRNAs in the root tissue of M. truncatula. Read-count analysis, qRT-PCR measurements and in situ hybridizations clearly indicated a regulation of miR5229a/b, miR5204, miR160f*, miR160c, miR169 and miR169d*/l*/m*/e.2* during arbuscular mycorrhizal symbiosis. Moreover, miR5204* represses a GRAS TF, which is specifically transcribed in mycorrhizal roots. Since miR5204* is induced by high Pi it might represent a further Pi status-mediating signal beside miR399. This study provides additional evidence that MtNsp2, a key regulator of symbiosis-signaling, is regulated and presumably spatially restricted by miR171h cleavage. In summary, a repression of mycorrhizal root colonization at high phosphate status is most likely due to a repression of the phosphate starvation responses and the loss of beneficial responses in mycorrhizal shoots. These findings provide a new basis for investigating the regulatory network leading to cellular reprogramming during interaction between plants, arbuscular mycorrhizal fungi and different phosphate conditions.

The aim was comparison of drought and frost resistence of selected varieties of Medicago sativa L. from domestic and foreign. The evaluation was performed by a series of laboratory experiments and observations in two replications in 2009 ? 2010. The results were evaluated as the most resistant varieties to frost variety from the Czech Republic and Slovakia. Namely variety Magda Litava, Tisa and Synthesis. As the least resistant varieties have been evaluated varieties from the warm coastal areas. Most resistant varieties to water deficit during vegetation varieties were evaluated Magda, Comete, Europe and Camilla. The climatic conditions of the Czech Republic would be most appropriate, according to the results observed varieties of Magda. It has high resistance to frost and has good results in drought. Other good varieties from Czech Republic are Pálava and Camilla. Of the foreign varieties would be best to use a French variety of Como, Europe and the Dutch variety of Capri. These varieties have good resistance to frost and high drought. The whole problem of drought and frost resistance of alfalfa is not yet explored, it would be appropriate, the results obtained confirmed by further research.

To elaborate on the function(s) of the ENOD8 gene in the nodules of M. truncatula, several different experimental approaches were used. A census of the ENOD8 genes was first completed indicating that only ENOD8.1 (nt10554-12564 of GenBank AF463407) is highly expressed in nodule tissues. A maltose binding protein-ENOD8 fusion protein was made with an E. coli recombinant system. A variety of biochemical assays were undertaken with the MBP-ENOD8 recombinant protein expressed in E. coli, which did not yield the esterase activity observed for ENOD8 protein nodule fractions purified from M. sativa, tested on general esterase substrates, α-naphthyl acetate, and p-nitrophenylacetate. Attempts were also made to express ENOD8 in a Pichia pastoris system; no ENOD8 protein could be detected from Pichia pastoris strains which were transformed with the ENOD8 expression cassette. Additionally, it was shown that the ENOD8 protein can be recombinantly synthesized by Nicotiana benthamiana in a soluble form, which could be tested for activity toward esterase substrates, bearing resemblance to nodule compounds, such as the Nod factor. Transcription localization studies using an ENOD8 promoter gusA fusion indicated that ENOD8 is expressed in the bacteroid-invaded zone of the nodule. The ENOD8 protein was also detected in that same zone by immunolocalization. Confocal immunomicroscopy with an affinity-purified anti-ENOD8 oligopeptide antibody showed that the ENOD8 protein localizes at the interface between the plant and the bacteroid-differentiated rhizobia, in the symbiosome membrane or symbiosome space. This suggests a possible link between ENOD8 protein and bacteroid differentiation, nitrogen fixation, or plant defense. These possible functions for ENOD8 could be tested with an ENOD8-RNAi transgenic line devoid of detectable ENOD8 proteins.

Plants have many nitrate transporters; in the model legume Medicago truncatula, MtNPF1.7 is among them. MtNPF1.7 is important for M. truncatula growth and it has been established that MtNPF1.7 is a high affinity nitrate transporter. M. truncatula plants with mutations in MtNPF1.7 gene show defects during plants growth, with striking abnormalities in nodule development and root architecture. Nitrogen fixation is an energy expensive process; when legumes have sufficient bioavailable nitrogen like nitrate available, it suppresses nodulation and nitrogen fixation. Previous preliminary results in our lab showed that plants constitutively expressing MtNPF1.7 have a growth phenotype in the absence of nitrate, but no data was available on how M. truncatula plants constitutively expressing MtNPF1.7 are affected by the presence of nitrate. For my research, I confirmed the preliminary results on the growth of M. truncatula plants overexpressing NPF1.7 and examined these plants' phenotypes when nitrate was not provided in the growth media and when it was provided at two different concentrations. Compared with wild type A17, plants constitutively expressing MtNPF1.7 gene grow larger, have more lateral roots and more nodules when grown in the absence of nitrate and when 0.2 mM KNO3 was provided. At 1 mM KNO3, there are fewer differences between wild type A17 and plants constitutively expressing the MtNPF1.7 gene. Compared with wild type A17, plants constitutively expressing the MtNPF1.7 gene flower earlier, which indicates MtNPF1.7 gene may have a function in plant flowering.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Annual medic pastures play an important role in conservation agriculture (CA) practices in the Western Cape, because of the beneficial role it plays in rotation systems and the fact that it can re-establish on its own. In the Overberg medic pastures are the main pasture short rotation crop, but farmers in recent years shifted away from including medics. This was due to unsuccessful re-establishment and a visible decrease in dry matter production. This trend started after CA practices were implemented for a few years. A field study conducted during 2013 investigated medic re-establishment and production following a wheat, barley, oat and medic pasture production year ( WM, BM, OM and MM) of which residues were left on the soil surface at different cover percentage levels (100%, 75%, 50%, 25% and 0%). The objective of this study was to determine what the effect of different amounts of residues was on annual medic re-establishment and production. Data from this study suggest that management of annual medic pastures should aim to re-sow the medic pasture if plant count drops below 78 pants per square meter. Weed management is of cardinal importance as it competes for resources, light and space and decrease medic pasture re-establishment and production. The data also indicates that the wheat/medic sequence is the best option when applying a short cash crop/annual pasture cropping system. Producers should manage their animals to ensure that a 50% to 75% cover is left on top of the soil following the grazing of residues during the summer months. The study in 2013 should have been replicated, but due to the low levels of re-establishment and production a decision was made to re-plant the trial sites. The field study conducted during 2014 investigated the medic/clover establishment and production following a re-plant. Medics were replanted following a W, B, O and M season, respectively. Residues again were manipulated to different cover percentages (100%, 75%, 50%, 25% and 0%). The objective was again to look at the amount and type of residues on medic/clover establishment and production following re-plant. Data from this study indicated that it might be advisable for annual medic/clovers to be re-sown after a cereal production year rather than a medic pasture year. With the production of medic/clover pastures not being affected by the residue cover percentage, a 100% residue cover following re-plant is best in rotations, if the optimal effect of CA wants to be observed. If animals are included in the production cycle, grazing of residues during summer months can occur until 50% cover is left. Soils will take longer to reach its potential, but by including animals the gross margin is more stable year on year. Two supplementary studies were conducted to investigate the germination of annual medics under controlled conditions. The objectives of the first supplementary study was to investigate the physical barrier effect of residues at different percentage cover (100%, 75%, 50%, 25% and 0%) and a possible allelopathic action from different types of residues (wheat, barley, oat and medic) on the annual medic cultivar Cavalier (one of the cultivars used during 2014 field re-plant). The different amounts of residue had no significant effect on percentage emergence of Cavalier. The 0% residue cover having the slightly higher germination could be because there are no physical obstructions preventing seedlings to establish. The different types of residue cover had no significant effect on the germination of annual medics, as the germination under wheat, barley, oats and medic residues did not differ from the control. The control had a slightly higher germination percentage (85%), while germination under residues was just below the recommended germination rate of 80-85%. This could be an indication of allelopathy from residues. The objective of the second supplementary study was to investigate the allelopathic effects of different residue leachates (wheat, barley, oat and medic) at different levels of concentrations (100% leachate, 75%, 50%, 25% and distilled water being the control) on Cavalier germination. The interaction between leachate type and concentration were significant. Low levels of leachate concentration did not have a significant impact on medic germination when compared between each other and the control. When the concentration percentage was increased differences were detected. Cavalier germination decreased drastically when medic leachate concentration increased, indicating allelopathic effects. Cavalier germination followed the same trend, just not as drastic, when wheat leachate concentration increased. This indicates that wheat could also have a negative allelopathic effect. With oat leachate Cavalier germination did not decrease except when 100% concentrate was used, which could indicate a small allelopathic effect. Cavalier germination following barley leachate showed no effect as concentration increased, even showing the odd increase. Depending on repeatability or follow-up studies of these experiments, data suggest that re-plant of medic pastures is beneficial if plant count drops below sustainable levels. Management of weeds during the medic pasture year improves production. Annual medic pastures should be re-planted following a cereal production year rather than a previous pasture year. Thus single medic rotations are preferred, for example WMWM rotation. Greater amounts of residues are beneficial for CA effects, but allelopathic effects of wheat and oat residues should be taken in consideration during re-establishment and residue levels should be lowered. / AFRIKAANSE OPSOMMING: Eenjarige medic weidings speel ʼn belangrike rol in bewaring landbou (CA) praktyke in die Wes Kaap, vanweë sy vermoë om jaarliks op sy eie te hervestig en sy voordelige rol in rotasie stelsels. Eenjarige medic weidings is tans die hoof kort rotasie gewas in die Overberg, maar boere is tans besig om dit uit die rotasie uit te sluit. Dit is as gevolg van lae hervestiging sowel as die opvallende afname in produksie. Hierdie waarnemings het na ʼn paar jaar na die toepassing van CA praktyke begin. Gedurende 2013 is daar ʼn veldstudie voltooi rakende medic weiding hervestiging en produksie wat na ʼn koring, gars, hawer en medic weiding produksie jaar volg (WM, BM, OM en MM). Gedurende die studie is stoppels by verskillende persentasie vlakke van bedekking op die grond gelaat (100%, 75%, 50%, 25% en 0% bedekking). Die doel van die studie was om die invloed van verskillende tipes en hoeveelhede stoppels op die hervestiging en produksie van eenjarige medic weidings vas te stel. Data van hierdie studie dui aan dat jaarlikse medic weidings so bestuur moet word dat medics in die Overberg area se plant telling nie laer as 78 plante per vierkante meter daal nie. Onkruid bestuur is van kardinale belang, omdat dit kompeteer met medics en veroorsaak ʼn verlaging in hervestiging en opbrengs. Data dui ook aan dat ʼn koring/medic stelsel die beste opsie is wanneer ʼn kort kontant gewas/eenjarige weiding gewas stelsel toegepas word. Produsente moet hul vee so bestuur dat ʼn 50 tot 75% stoppel bedekking gedurende die somer maande oorgelaat word na beweiding. Die herhaling van die 2013 veld studie was van plan, maar ag gevolg van lae hervestiging en produksie was die proef kampe oor geplant. Die veldstudie in 2014 was medic/klawer vestiging en produksie na herplanting ondersoek. Die medic/klawer saad is geplant na ʼn koring, gars, hawer en medic weiding seisoen onderskeidelik. Stoppels is weereens na verskillende bedekking persentasies verander (100%, 75%, 50%, 25% en 0% bedekking). Die doel was om te kyk wat die effek van verskillende tipes en hoeveelhede stoppels op eenjarige medic/klawer weiding is na herplant. Data wys dat medic/klawer weidings verkieslik herplant moet word na ʼn graan produksie jaar as ʼn medic weiding produksie jaar. Die medic/klawer weiding is nie geaffekteer deur die hoeveelheid stoppels op die grond oppervlakte nie, dus is ʼn 100% stoppel bedekking verkieslik vir optimale CA effekte. As diere in die sisteem teenwoordig is, kan stoppels bewei word gedurende die somer maande tot ʼn 50% bedekking bereik word. Grond sal langer vat om sy potensiaal te bereik, maar die jaarlikse bruto marge sal meer stabiel wees. Twee aanvullende studies is onderneem en ontkieming van eenjarige medics is ondersoek onder beheerde toestande. Die doelwit van die eerste aanvullende studie was om te kyk na die fisiese versperring effek van stoppels by verskillende persentasie bedekking (100%, 75%, 50%, 25% en 0%) en ʼn moontlike allelopatiese effek van verskillende tipe stoppels (koring, gars, hawer en medic) op die eenjarige medic kultivar Cavalier. Verskillende hoeveelhede stoppels het geen beduidende uitwerking op die vestiging van Cavalier gehad nie. Die 0% stoppel bedekking het ʼn effens hoër vestiging gehad. Dit kon wees as gevolg van geen fisiese versperring wat die saailing verhoed om te vestig nie. Die verskillende tipes stoppels het geen beduidende uitwerking op die ontkieming van eenjarige medics nie, die vestiging onder koring, gars, hawer en medic stoppels het nie statisties verskil van die kontrole nie. Die kontrole het wel ʼn effense hoër persentasie vestiging gehad (85%), terwyl die vestiging onder die stoppels onder die aanbevole koers van 80-85% was. Dit kan dalk ʼn allelopatiese effek van die stoppels aandui. Die doel van die tweede aanvullende studie was om die allelopatiese effek van die verskillende tipes stoppels (koring, gars, hawer en medic) by verskillende vlakke van konsentrasie (100%, 75%, 50%, 25% van die onverdunde loogsel en gedistilleerde water as kontrole) op Cavalier ontkieming. Daar was ʼn beduidende interaksie tussen tipe en konsentrasie loogsel. Met lae konsentrasie vlakke van loogsel was daar nie ʼn werklike impak op Cavalier ontkieming tussen die verkillende tipes en die kontrole nie. Slegs wanneer die konsentrasie persentasie verhoog is, is verskille waargeneem. Cavalier ontkieming het drasties af geneem soos die medic loogsel konsentrasie toegeneem het, wat ʼn negatiewe allelopatiese en verhoogde osmolaliteit effek wys. Cavalier ontkieming het dieselfde tendens gewys wanneer koring loogsel konsentrasie verhoog was, maar nie so drasties soos medic loogsel. Dit dui daarop dat koring ook ʼn negatiewe allelopatiese effek wys. Met hawer loogsel het Cavalier ontkieming slegs by die 100% konsentrasie pyl afgeneem, wat op ʼn lae allelopatiese effek dui. Cavalier ontkieming onder gars loogsel het geen verandering gewys as konsentrasies toegeneem het nie, en het selfs ʼn toename in ontkieming in party gevalle ondergaan. Afhangend van herhaling of op-volg studies van hierdie eksperimente, wys die data dat dit voordelig is om medic weidings te herplant as plant telling onder 78 plante per vierkante meter daal. Die bestuur van onkruid tydens die medic weidings jaar verbeter opbrengs. Eenjarige medic weidings moet herplant word na ʼn graan produksie jaar liewer as ʼn vorige weidings jaar. Medics moet dus in ʼn eenjarige rotasie stelsel wees, byvoorbeeld WMWM rotasie. Meer stoppels is voordelig vir CA promosie, maar allelopatiese stowwe van koring en hawer stoppels moet in ag geneem word en stoppels moet verlaag word vir hervestiging.

No description available.

The Arbuscular mycorrhiza (AM) symbiosis is a mutual association formed by plant roots and soil fungi. Most vascular flowering plants have the ability to form AM associations, which show significant impact on ecosystem function and plant health. This association is based on the mutual exchange of nutrients between plant and fungus. Therefore, AM association leads to increased demands for photosynthesis. The main aim of this study was to investigate the pathway used by plants during AM to increase the photosynthetic performance. To achieve this aim, we used the model legume Medicago truncatula. We have found out that AM symbiosis develops in roots, where AM fungi colonize the roots, leading to better plant growth and more biomass. Furthermore, AM symbiosis increases chlorophyll content and photosynthetic electron transport rate in leaves. Based on these results we suggest that AM symbiosis increases both efficiency and capacity of photosynthetic apparatus in Medicago truncatula

This research investigated early transcriptional responses of the model legume, Medicago truncatula, to herbivory by caterpillars of the beet armyworm, Spodoptera exigua. Differentially expressed genes were identified by the differential display technique, cDNA-amplified fragment length polymorphism (cDNA-AFLP). To distinguish between caterpillar-specific responses and general wound responses, a subset of plants was mechanically damaged. Furthermore, to identify responses to salivary elicitors, plants were subject to herbivory by caterpillars with normal salivary secretions and those that had their spinneret, the appendage through which labial saliva is secreted, cauterized shut. Eighteen differentially expressed gene fragments, representing 16 genes, were identified. The expression pattern of 5 of these genes was analyzed by Northern analysis, confirming a caterpillar-specific reduction in transcripts encoding rubisco activase and glyceraldehyde-3-phosphate dehydrogenase. This research shows that plants are able to differentiate between caterpillar herbivory and mechanical damage and that transcriptional response are initiated within one hour after caterpillar infestation.