Hints of rhizobia induced alleviation of tungsten stress in Glycine max via increased levels of primary and secondary metabolites

Die Koordinationschemie des Übergangsmetalls Wolfram (W) und des essentiellen pflanzlichen Mikronährstoffs Molybdän (Mo) sind in struktureller und funktioneller Hinsicht ähnlich. Aus diesem Grund liegt es nahe, daß W die enzymatische Aktivität von Molybdoenzymen wie der pflanzlichen Nitratreduktase hemmt, indem Mo im Molybdopterin-Co-Faktor (Mo-Co) ersetzt wird. Dies würde die N-Assimilation stark beeinträchtigen und einen N-Mangel bei Pflanzen verursachen. Einige Studien legen nahe, dass ein anderes Enzym, die Nitrogenase von symbiotisch lebenden Wurzelbakterien, die einen Fe-Mo-Co-Faktor für die N2-Fixierung aufweist, ihre Funktionalität nach W-Exposition beibehalten kann, da sie von einer Symbiosomenmembran geschütz werden. Das würde die N-Versorgung der Pflanzen sichern. Darüber hinaus gibt es Hinweise darauf, dass Wurzeln und Knöllchen von symbiotisch gewachsenen Hülsenfrüchten in Gegenwart hoher Konzentrationen von W einen höheren Anteil an Proteinen aufweisen, die an der Hormon- und Flavonoid-Biosynthese beteiligt sind, also Verbindungen, die an der Stressregulation beteiligt sind. Um zu klären, ob und wie die symbiotische N2-Fixierung die Metabolitenhäufigkeit während der W-Exposition im Vergleich zu nicht-symbiotischen Pflanzen verändert und ob dies mit einer erhöhten Toleranz gegenüber Schwermetalltoxizität zusammenhängt, habe ich eine integrative Analysemethode (Sequentielle Extraktion) zum Nachweis von Primär- und Sekundärmetaboliten ausgearbeited und angewendet. Mit Bradyrhizobium japonicum (Nfix) oder Nitrat (Nfed; 10 mM KNO3) vertilisierte Sojabohnenpflanzen wurden in einem halbhydroponischen Aufbau angezogen. Drei Wochen nach vollständiger Ausbildung der Symbiose, wurden die Pflanzen zur Untersuchung der W- und Mo-spezifischen Reaktion zwei Wochen lang hohen Konzentrationen an W (0,5 mM Na2WO4) oder Mo (0,5 mM Na2MoO4) ausgesetzt und anschließend zur metabolischen Analyse geerntet. Meine Ergebnisse zeigten, dass Nfix- im Vergleich zu Nfed-Pflanzen, sowohl in Gegenwart von W als auch Mo eine stärkere metabolische Reaktion aufwiesen. Während bei beiden, Nfix- und Nfed-Pflanzen, nach W-Verabreichung eine Abnahme der Sproßbiomasse beobachtet wurde, wiesen symbiotisch gewachsene Sojabohnen eine höhere Wurzel- und Knöllchenbiomasse / -zahl auf. Ich fand zudem eine Zunahme von Phenolverbindungen, Flavonoiden und löslichen Zuckern in Nfix-Wurzeln und Blättern, was auf eine höhere Antioxidationskapazität im Vergleich zu Nfed-Pflanzen hindeutet. Darüber hinaus konnte ich in Nfix-Pflanzen einen Anstieg von organischen Säuren, Polyaminen (z.B. Putrescin, Spermidin) und Aminosäuren (z.B. Prolin, Alanin) als Reaktion auf 0,5 mM W nachweisen. Im Vergleich dazu behielten Molybdän-exponierte Pflanzen ihre Biomasse auf Kontrollniveau. Dennoch zeigten Wurzeln von Nfix-Pflanzen hier höhere Werte in Aminosäuren, löslichen Zuckern, Phenolen und Flavonoiden. Meine Ergebnisse deuten auf eine Linderung des Wolframstresses durch verbesserten Osmoseschutz, sowie erhöhte radikalfangende und metallchelatisierende Kapazität bei symbiotischen Pflanzen hin.The coordination chemistries of the transition metal tungsten (W) and the essential plant micro nutrient molybdenum (Mo) are similar in structural and functional aspects; for this reason, W is proposed to inhibit enzymatic activity of molybdoenzymes such as nitrate reductase, by replacing Mo in the molybdopterin co-factor (Mo-Co). This would severely effect N-assimilation, causing N-deficiency. A few studies suggest that another enzyme, the nitrogenase of symbiotically plant living rhizobacteria, which presents a different Fe-Mo co-factor for N2 fixation, can retain its functionality after W exposure. Moreover, there is some evidence that roots and nodules of symbiotically grown leguminous plants exhibit higher levels of proteins involved in hormone and flavonoid biosynthesis, compounds involved in stress regulation, in the presence of high concentrations of W. The aim of my thesis was to develop an integrative analytical method for comprehensive primary and secondary metabolite detection in order to clarify if and how symbiotic N2 fixation changes metabolite abundances during tungsten exposure, compared to non-symbiotic plants and if this relates to enhanced tolerance against heavy metal toxicity. Soybean plants inoculated with Bradyrhizobium japonicum (Nfix) and a non-symbiotic control supplied with nitrate (Nfed – 10 mM KNO3) were grown in a semi hydroponic setup. After three weeks, when symbiosis was fully established, in order to investigate the W and Mo specific response, plants were exposed to W (0.5 mM Na2WO4) or high Mo (0.5 mM Na2MoO4) for two weeks and subsequently harvested for metabolomic analysis applying a novel sequential extraction procedure. My results showed that Nfix plants exhibit a stronger metabolic response compared to their non-symbiotic counterparts in presence of both W and Mo. While a decrease in biomass was observed in both Nfix and Nfed treatments when exposed to tungsten, symbiotically grown soy beans were able to retain shoot growth, healthier roots, and nodule mass/count. I found an increase in phenolic compounds, flavonoids, and soluble sugars in Nfix roots and leaves exposed to tungsten which resulted in a higher antioxidant capacity in comparison to Nfed plants. Furthermore, I could show an increase in organic acids, polyamines (i.e. putrescine, spermidine) and amino acids (i.e. Proline, Alanine) in Nfix plants in response to 0.5 mM W. Plants exposed to molybdenum retained their biomass to control level, however, roots of Nfix plants exhibited higher values in amino acids, soluble sugars, phenols and flavonoids. My results suggest a symbiont induced alleviation of tungsten stress via enhanced osmo-protective, radical-scavenging and metal-chelating capacity

Relative genome size variation in the African agroforestry tree Parkia biglobosa (Fabaceae: Caesalpinioideae) and its relation to geography, population genetics and morphology

Variation in genome size and in chromosome number can be linked to genetic, morphological and ecological characteristics, and thus be taxonomically significant. We screened the relative genome size (RGS) and counted the number of mitotic chromosomes in the African agroforestry tree Parkia biglobosa, a widely distributed savannah species that shows conspicuous morphological clinal variation and strong genetic structure, and tested for linkage of RGS variation to geography, leaf morphology, and population genetic variation. An improved protocol for the preparation of chromosomes was developed. The study is based on 58 individuals from 15 populations covering most of the distribution range of the species. We observed differences in RGS among individuals of up to 10.2 %, with some of the individuals differing statistically in RGS from the bulk of screened individuals. Most of the RGS variation was within populations whereas variation was unrelated to any of the tested features of the species. Those chromosome numbers which could be exactly established were invariable 2n = 2x = 26. In conclusion, there was no evidence from the karyological data for structured intra-specific taxonomic heterogeneity.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Yolk granule fusion and microtubule aster formation regulate cortical granule translocation and exocytosis in zebrafish oocytes.

Dynamic reorganization of the cytoplasm is key to many core cellular processes, such as cell division, cell migration, and cell polarization. Cytoskeletal rearrangements are thought to constitute the main drivers of cytoplasmic flows and reorganization. In contrast, remarkably little is known about how dynamic changes in size and shape of cell organelles affect cytoplasmic organization. Here, we show that within the maturing zebrafish oocyte, the surface localization of exocytosis-competent cortical granules (Cgs) upon germinal vesicle breakdown (GVBD) is achieved by the combined activities of yolk granule (Yg) fusion and microtubule aster formation and translocation. We find that Cgs are moved towards the oocyte surface through radially outward cytoplasmic flows induced by Ygs fusing and compacting towards the oocyte center in response to GVBD. We further show that vesicles decorated with the small Rab GTPase Rab11, a master regulator of vesicular trafficking and exocytosis, accumulate together with Cgs at the oocyte surface. This accumulation is achieved by Rab11-positive vesicles being transported by acentrosomal microtubule asters, the formation of which is induced by the release of CyclinB/Cdk1 upon GVBD, and which display a net movement towards the oocyte surface by preferentially binding to the oocyte actin cortex. We finally demonstrate that the decoration of Cgs by Rab11 at the oocyte surface is needed for Cg exocytosis and subsequent chorion elevation, a process central in egg activation. Collectively, these findings unravel a yet unrecognized role of organelle fusion, functioning together with cytoskeletal rearrangements, in orchestrating cytoplasmic organization during oocyte maturation

A hydraulic feedback loop between mesendoderm cell migration and interstitial fluid relocalization promotes embryonic axis formation in zebrafish

Interstitial fluid (IF) accumulation between embryonic cells is thought to be important for embryo patterning and morphogenesis. Here, we identify a positive mechanical feedback loop between cell migration and IF relocalization and find that it promotes embryonic axis formation during zebrafish gastrulation. We show that anterior axial mesendoderm (prechordal plate [ppl]) cells, moving in between the yolk cell and deep cell tissue to extend the embryonic axis, compress the overlying deep cell layer, thereby causing IF to flow from the deep cell layer to the boundary between the yolk cell and the deep cell layer, directly ahead of the advancing ppl. This IF relocalization, in turn, facilitates ppl cell protrusion formation and migration by opening up the space into which the ppl moves and, thereby, the ability of the ppl to trigger IF relocalization by pushing against the overlying deep cell layer. Thus, embryonic axis formation relies on a hydraulic feedback loop between cell migration and IF relocalization

Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization

Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole—a protuberance of the zygote’s vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces