Institut für Pflanzenernährung

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Physiological, molecular, and epigenetic aspects of early transient nitrogen deprivation recovery in maize
(2024) Hernandez Pridybailo, Andres; Ludewig, Uwe
Maize is a widely cultivated crop and a primary food source for humans and livestock. Along with its substantial contribution to biomass for fuel production, maize is the most produced cereal globally. However, despite its prevalence, improving nitrogen (N) use efficiency in maize presents ongoing challenges. Understanding how maize plants utilize nitrogen is crucial for identifying traits that could aid breeders in addressing this efficiency gap. When plants experience transient stress and return to previous conditions, a recovery phase is initiated, acclimating the plants and potentially enhancing their responses to subsequent stress events. This mild or transient stress is termed "eustress," and its intentional manipulation is referred to as "crop priming." Crop priming, extensively studied in the context of drought, was explored in our previous research, which revealed that nitrogen supplementation during recovery from early transient water deficits enhances priming effects. This highlights the pivotal role of N in priming and that impaired N supply might negatively affect any acclimation to future stresses. Notably, there is limited literature on transient N deprivation, with most studies focusing on plant responses under conditions of maintained N deficiency. Furthermore, epigenetic regulation of plant mineral nutritional responses via DNA methylation has been reported, like many aspects of plant development that are regulated by such mechanism. Epigenetics is the study of phenotypic changes that can be inherited through mitosis or meiosis, which cannot be explained by changes in the DNA sequence. DNA methylation is the covalent modification of cytosines by the addition of a methyl group, which, depending on its location in the genome, can affect genomic stability and gene expression. Previous results have revealed that N deficiency modifies the methylome of maize roots, hypomethylating transposable elements in a nutrient-specific way. Weak correlations between DNA methylation and gene expression were observed, but a deeper insight into how this covalent modification of cytosines is related to plant mineral nutrition is lacking. DNA methylation patterns are heritable in a sort of “epigenetic memory” and may arise after an environmental stimulus. Here, the role of DNA methylation and its relationship with physiological responses during transient N deprivation were studied. The Fast-Flowering Mini-Maize inbred line A (FFMM-A) was chosen for this study because it can be easily grown hydroponically under controlled conditions. Maize ear leaves recovering from initial nitrogen deprivation (without water limitation) exhibited tissue-specific differences in rapidly dividing meristematic and mature photosynthetically active tissues. Through a series of experiments, a 60 h N deprivation period at the V1 stage was established as a nutritional stress that allowed plants to show mild N deprivation symptoms from which the plants fully recovered. Kinematic analysis of the recovering ear leaf revealed a slight decrease in leaf blade length due to reduced leaf meristem size and leaf number, resulting in a reduced cell production rate. Interestingly, the leaf elongation rate (LER) was maintained, causing a higher specific leaf area, which affected the leaf blade structure. Transcriptomic analysis of the cell division and maturation zones of the recovering ear leaf showed altered gene expression related to cell cycle, lipid metabolism, and secondary metabolism, including phytohormone regulation. Notably, cell cycle-related proteins are also involved in DNA repair and may be involved in somatic memory effects. If plants that had been exposed to early transient N-deficiency stress were later subjected to a second N-deprivation stress at the V5 stage, physiological measurements showed that early N deprivation affected upper leaf chlorophyll accumulation during late N-deprivation recovery. These findings suggest that early N deprivation has long-term effects, especially in dividing tissues, from which young and mature tissues are produced by replication. Furthermore, I identified that the loss of DNA methylation makes maize plants more susceptible to early N deprivation. Loss-of-function zmet2 allele was used in this study. Examination of the methylome of the zmet2 and zmet5 mutants provided insights into the relationship between DNA methylation patterns, nitrogen responses, and the expression of developmentally regulated genes in the leaf. These alleles correspond to CHROMOMETHYLASE3-like maintenance methyltransferase genes, and their mutation causes generalized genome hypomethylation, mostly in the CHG and CHH contexts. After a 96 h N deprivation period at the V1 stage, the zmet2 mutants failed to modify their root-to-shoot ratio and to maintain the LER, with respect to the isogenic B73 line. Moreover, machine learning identified interesting clusters according to their expression patterns from the leaf transcriptome of the dividing and maturation zones of FFMM-A plants. Reanalysis of publicly available datasets from B73 seedling leaf methylation patterns and those of the zmet2 and zmet5 mutants was used to compare the expression patterns of the gene clusters, their methylation patterns, and selected genome features. Interestingly, the methylation pattern of the gene body was highly correlated with the level of expression in the dividing zone of the developing ear leaf. Collectively, mutants in the methylation maintenance pathway, rather than being susceptible to N deprivation, failed to develop symptoms of recovery from such stress, which might be related to the regulation of N metabolism-related genes by DNA methylation. Additionally, the closely related gene expression pattern in the dividing zone and intron non-CpG methylation suggests an intimate role of maintenance of DNA methylation in N nutrition.
Einfluss von Phosphatmangel und erhöhter atmosphärischer CO2-Konzentration auf die Wurzelexsudation und ihre Auswirkungen auf Mobilisierung und Aufnahme von Schwermetallen durch verschiedene Lupinenarten und Tomate
(2011) Kawanishi, Ayumi; Römheld, Volker
There is an increasing awareness of a contamination of the food chain by toxic heavy metals as consequence of anthropogenic induced pollution of the environment since the industrialization in the 18. century. In addition the CO2 concentration might promote the biomass formation of plants and thus, via an increased allocation of photo-assimilates into the roots, chemical changes in the rhizosphere. These changes can promote mobility and uptake of various heavy metals by crop plants, too. Therefore it was the main objective of this Ph.D. research, to study the possible consequences of such observed increase in the atmospheric CO2 concentration on the intensification of the rhizosphere chemistry on the uptake of heavy metals by selected plant species in continuation of the research work by Egle (2003) at the University Göttingen. As plant species various lupinus species and tomato were chosen, which differ in principle in their reaction to a low phosphate nutritional status such as root growth characteristics and secretion of protons and carboxylates. As approach two nutrient solution experiments (Chapter 4 and 5) and a soil experiment with heavy metal polluted soils (Chapter 6) were conducted. In both nutrient solution experiments the well-described root-induced changes such as proton and carboxylate release could be confirmed, which were intensified at higher atmospheric CO2 concentrations (Chapter 4 and 5). Surprisingly the detected increase in proton (tomato) and caboxylate release (particularly by white lupin) with a simultaneously increased mobility of Cu and Cd in the soil did not result in an increased concentration of heavy metals in roots and shoots of the growth experimental plants. The unexpected finding in chapter 6 were discussed in the outlook of chapter 6 (6.7) and a repetition of this experiment with consideration of the discussed aspects is urgently recommended.
Identification of regulatory factors determining nutrient acquisition in Arabidopsis
(2011) Giehl, Ricardo Fabiano Hettwer; von Wirén, Nicolaus
The acquisition and translocation of mineral nutrients involves the orchestrated action of a series of physiological and biochemical mechanisms, which are, in turn, regulated by nutrient availability and demand. Furthermore, root morphological changes play an outstanding role for nutrient acquisition, especially when the availability of a certain nutrient is low. Although for most nutrients the molecular mechanisms involved in their acquisition from soils have been described, much less is known about the regulatory pathways underlying the uptake and translocation of nutrients in plants. Thus, the main aim of the present study was to characterize root morphological responses to nutrient supply and to identify novel regulatory components. The first part of the present thesis describes the morphological response of Arabidopsis roots to the essential element iron (Fe), which has a particularly low solubility in soils. Relative to a homogenous supply of Fe, localized Fe supply to horizontally-separated agar plates doubled lateral root length without a particular effect on lateral root number. The internal tissue Fe rather than external Fe triggered the local elongation of lateral roots. In addition, the Fe-stimulated emergence of lateral root primordia and root cell elongation was accompanied by a higher activity of the auxin reporter DR5:GUS in lateral root apices. A crucial role of the auxin transporter AUX1 in Fe-triggered lateral root elongation was indicated by Fe-regulated AUX1 promoter activities in lateral root apices and by the failure of aux-1 mutants to elongate lateral roots into Fe-enriched agar patches. Furthermore, a screening was designed to identify novel regulatory components involved in the Fe-dependent stimulation of lateral roots. One member of the GATA family of transcription factors was found to play a role in the local, root-endogenous regulation of lateral root development in response to local supplies of Fe. It was concluded that a Fe sensing mechanism in roots regulates lateral root development by modulating auxin transport. The second part of the thesis describes the use of multi-elemental analyses to identify novel regulators of nutrient accumulation in Arabidopsis. Firstly, it is shown that the disruption of transcription factors expression can lead to significant alterations in the accumulation of one or more nutrients in shoots. In addition, this approach allowed the identification of a so-far uncharacterized transcription factor ? NGAL1 ? that regulates primary root elongation in response to phosphorus (P) supply. The loss of NGAL1 resulted in hypersensitive inhibition of primary root growth under low P and a P-independent increase in lateral root elongation. The results presented here indicate that NGAL1 participates in a signaling pathway that modulates meristematic activity by controlling the expression of important root patterning regulators according to the local availability of P.
Iron and ammonium sensing differentially modulate root plasticity in Arabidopsis thaliana
(2010) Lima, Joni Esrom; von Wirén, Nicolaus
Modulation of root system architecture by plants has an impact on water, nutrient acquisition and anchorage during plant development. In a given environment, root plasticity is a favorable feature to react according to abiotic and biotic factors. Under nutrient limited conditions, the root plasticity is essential for a better soil volume exploitation. This response can vary according to the plant species and the given environment in which they evolved. Moreover, nutrient mobility in the soil plays an important role for the response of plants to nutrient limitation. Thus, root plasticity is a nutrient-specific response during plant development. In fact research on the effect of nutrient availability on root system architecture is scarce. Furthermore, the mechanism how plants sense nutrients and the signaling upon nutrient availability remains a challenge. Therefore, identification of which nutrient can affect the root system architecture and investigating the molecular components involved in the signaling pathway is certainly relevant for agronomical practices. The first part of the present work aimed to identify how the root architecture is affected by iron (Fe) supply. Due to the low mobility of Fe in soils, the morphological response of lateral roots from Arabidopsis plants to localized Fe supply and its regulation were investigated. Increasing Fe concentrations in a homogenous or localized supply on separated agar plates enhanced lateral root number in a similar manner. Lateral root length, however, was twofold higher under localized relative to homogenous Fe supply. With further increasing Fe concentrations lateral root length was repressed even though shoot growth was unaffected. In the Fe uptake-defective mutant irt1, the formation of lateral roots required higher local Fe supplies, which restored wild type levels only with respect to the number but not to the length of lateral roots. Moreover, IRT1 transcript levels were strongly enhanced under localized Fe supply. In the frd3-1 mutant, which is defective in root-to-shoot translocation of Fe, lateral root development was similar to wild type plants although frd3-1 shoots were Fe deficient. These results show a differential regulation of lateral root initiation and elongation in response to localized Fe supply and that lateral root elongation is under control of a local rather than a systemic regulatory loop involving the high-affinity Fe transporter IRT1. In the second part of the thesis, a remarkable and an unknown feature of root morphology dependent on localized ammonium supply is described. Arabidopsis plants were able to increase lateral root initiation and higher-order lateral root branching. Since ammonium-stimulated lateral root number or density decreased after ammonium or glutamine supply to a separate root fraction and did not correlate with cumulative uptake of 15N-labeled ammonium, lateral root branching was not purely due to a nutritional effect but most likely a sensing event. Moreover, a detailed investigation has shown that ammonium and nitrate co-ordinate root morphology in an additive and complementary way. By a genetic approach, the ammonium-induced lateral root branching was demonstrated to be dependent on AMT1;3 activity in the root.
Interactions between non-symbiotic N2-fixing bacteria and plant roots in plant-microbial associations
(2009) Calvo Alegre, Olga-Cristina; von Wirén, Nicolaus
The development of biofertilizers on the basis of plant growth promoting rhizobacteria (PGPR) may be a promising approach to partially substitute costly and energy-consuming mineral fertilizers in agricultural plant production and to support agriculture in developing countries. A successful and competitive rhizosphere colonization of PGPR strains has been identified as a prerequisite for the expression of plant growth promoting effects. Apart from a wide range of external factors with an impact on the colonization process, such as soil properties, temperature, soil moisture and fertilization levels, in particular plant-microbial interactions may play an important role for the successful establishment of compatible associations. In this context, certain plant root exudates may act as signals to mediate bacterial responses with importance for root colonisation (e.g. motility and chemotaxis, production of extracellular polysaccharides). On the other hand, the induction of bacterial plant growth promotion may also depend on ability of the host plant to respond to the presence and the activity of the associated bacteria. It was therefore the aim of this thesis to investigate the contribution of putative PGPR to growth and N uptake in wheat plants and characterize the underlying mechanisms in root-bacterial associations. In the first part of this thesis, the contribution of various non-symbiotic diazotrophic rhizobacteria to plant growth promotion and N nutrition has been studied in a series of greenhouse pot inoculation experiments with wheat (Triticum aestivum L.). Different bacterial inoculants, plant genotypes, soil properties, water regimes and N fertilization levels have been varied as factors with potential impact on plant growth promotion by diazotrophs. The contribution of biological nitrogen fixation was assessed by the 15N dilution method. Plant growth and grain yield were influenced by the different N fertilization levels but no stimulation of growth or N uptake was note upon bacterial inoculation. These observations suggested a high degree of specificity or limiting factors, determining a successful plant-microbial association. The second part describes possible mechanisms that may be involved in the establishment of diazotrophs in the rhizosphere of suitable host plants. As an initial step of the colonization process, a targeted movement of the bacteria to the root surface is required and root exudates may act as attractants. Since dicarboxylic acids are known to exert chemotactic activity on diazotrophic bacteria, seed and root exudates of two graminaceous crops (Triticum aestivum L. and Zea mays L.) and for comparison also of a non-graminaceous plant species (Phaseolus vulgaris L.) were collected in hydroponic culture with and without N supply, and organic acid profiles in these root exudates were analysed. Bacterial motility assays were conducted with the major carboxylates detected in the root exudates of the selected plant species and compared to glucose and water, using Brevibacillus reuszeri as a model bacterium. Pure malate, which was found at high levels in root exudates of bean and wheat, and particularly malonate (bean) and t-aconitate (maize) stimulated the motility of Brevibacillus reuszeri as compared with glucose or water. A particularly intense promotion of bacterial motility was recorded in the presence of crude root exudates of wheat and maize plants grown under N limitation, which was not observed for root exudates of bean. However, this was not related with comparable changes of malate or t-aconitate concentrations in the root exudates. In wheat exudates, malate concentrations even decreased in response to N limitation. These findings suggest the presence of specific factors released in root exudates of N-deficient cereals, promoting the rhizosphere colonisation with B. reuszeri. For an identification of the respective factors, a more comprehensive profiling of the root exudates is necessary. In associations with diazotrophic bacteria, host plants are supplied with ammonium by the bacterial partner. This raised the question whether plant ammonium uptake systems have an impact on the efficiency of the association. To address this problem, an antisense approach was conducted with tomato, with characterised ammonium transporters (LeAMT1;1 and LeAMT1;2). The final goal was the inhibition of the ammonium transporters by production of LeAMT antisense lines to study their putative role in plant associations with diazotrophic bacteria. Northern blot analysis revealed a strong repression of LeAMT1;2 expression in three independent antisense lines associated with a lower ammonium uptake capacity under N-sufficient and N-deficient growth conditions. In contrast, LeAMT1,1 expression was only weakly repressed in antisense lines and there was no impact on N uptake. A faster decline of chlorophyll in older leaves indicates a physiological function of LeAMT1;1 and LeAMT1;2 in ammonium uptake and retrieval in shoot and root cells. The absence of consistent effects on N acquisition of the investigated antisense lines limited the suitability of this approach for studies on associations with diazotrophic bacteria.
Glyphosate use in agro-ecosystems : identification of key factors for a better risk assessment
(2010) Tesfamariam, Tsehaye; Römheld, Volker
Glyphosate ([N-phosphonomethyl] glycine) is a non-selective, post-emergence, organo-phosphorous, broad-spectrum herbicide used worldwide for controlling weeds in horticulture, agriculture, silviculture, and urban landscapes. It effectively controls most annual and perennial weed species and is the world´s biggest-selling herbicide. One reason for the popularity of glyphosate is its effect on roots and rhizome systems of weed following foliar application. After coming in contact with soil, glyphosate will be strongly adsorbed and this sorption behavior makes glyphosate unique as compared to most other herbicides and has elicited a general belief that it is rapidly adsorbed to the soil without any residual effect. However, glyphosate adsorption to the soil matrix seems a reversible process and glyphosate conserved in roots of treated target plants has been overlooked in most previous risk assessments. Therefore, in face of the increasing number of yet unexplained observations of negative side effects after glyphosate application, this thesis was initiated to identify possible risk factors associated with the frequent use of glyphosate in agro-ecosystems. For this purpose: (1) relevance of waiting time between weed desiccation by glyphosate and subsequent crop planting, (2) remobilization risk of glyphosate fixed in the soil matrix mediated by pH change in the rhizosphere, (3) glyphosate preservation in target plant roots and (4) contribution of glyphosate released from decaying weed residues for intoxication of following non-target plants were investigated in controlled greenhouse conditions using two contrasting soils: a weakly buffered acidic Arenosol (top soil) and a highly buffered calcareous Luvisol (subsoil). Furthermore, field experiment was conducted to partially confirm the found results of controlled model experiments under greenhouse conditions. These model experiments as well as the experiment in farmer´s field revealed that the residual toxicity of glyphosate increased with a declining waiting time between glyphosate weed desiccation and subsequent crop planting. In the greenhouse experiments, seedling growth and biomass production of sunflower plants were strongly impaired by pre-sowing application of glyphosate in the variants with less than 21 days waiting time. The inhibitory effects on seedling growth were associated with a corresponding increase of shikimate accumulation in the root tissue as physiological indicator for glyphosate toxicity and impairment of the manganes-nutritional status of the sunflower seedlings. Results of the field experiment at Hirrlingen/Tübingen confirmed the relevance of waiting time. Stunted development and heterogeneous emergence of winter wheat plants occurred at field plots where the wheat sowing was done 2 days, compared to plants sown 14 days after foliar application of glyphosate to weed plants. At a short waiting time (2 d), data on visual scoring showed up to 50% of the culture damage that was visually persistent still after 6 months at harvests. This was also associated with a reduced nutritional status of wheat plants Ca, Mg, Zn and Cu, particularly expressed when glyphosate application rate was elevated from 2L to 6 L ha-1. Since glyphosate shows a similar pattern of reaction like that of phosphate in soil, it has been hypothesized that rhizosphere processes responsible for P mobilization are likely to co-mobilize also glyphosate. To test this hypothesis, an experiment was conducted using the two soils with contrasting properties pre-incubated with different rates of glyphosate and supplied with stabilized NH4+-N or NO3--N to induce the different changes in rhizosphere pH. From the results of this experiment, however, it was not possible to confirm this hypothesis. No glyphosate phytotoxicity of sunflower seedlings on the Luvisol with NH4+ could be detected due to observed minor rhizosphere acidification. In agreement, also no shikimate accumulation in root was measured. However, there was a distinct decrease in biomass of the sunflower seedlings at NH4- supply, possibly due to a missing NO3- signal. In contrast in the Arenosol no difference in growth could be shown between both supplied N-forms despite a clearly expressed difference in rhizosphere pH. Root exudation of organic carboxylates has also been considered to assists the release of adsorbed phosphate in the rhizosphere from the soil matrix via exchange chelation. A similar phenomenon was expected for glyphosate. In the present study, however, supplementation of Na-citrate or citric acid to both contrasting soils, pre-incubated with different levels of glyphosate, did not show a clear evidence for an adequate glyphosate remobilization and the subsequent plant damage. On the acidic Arenosol, there was no difference in growth of sunflower seedlings between the treatments. In contrast, on the Luvisol, supplementation of Na citrate (10µmol g-1 soil) but not citric acid indicated some promotion of root growth on glyphosate free treatment. This could not be easily explained because no intracellular shikimate accumulation as bio-indicator for glyphosate could be detected in the treatments with glyphosate pre-incubated soil. In many plant species, glyphosate is not readily metabolized, but preferentially translocated to young growing tissues of roots and shoots, where it can be accumulated in millimolar concentrations. In soil-grown target plants, this inhomogeneous distribution of glyphosate within the root tissues may lead to the formation of hot spots of glyphosate containing root residues in soils. Subsequently this stored glyphosate as hot spots can be released during microbial degradation of root material. To evaluate the potential of roots of target plant in stabilization and subsequent release of glyphosate with intoxication of subsequent crop plants, model experiments were conducted with application of glyphosate either via rye grass as target weed plants or directly to the soil. Sunflower seeds were sown at different waiting times (0-21 days) for both glyphosate application modes. Toxicity of glyphosate applied shortly before sowing of sunflower as non-target was strongly dependent on the mode of glyphosate application. When glyphosate was sprayed on pre-cultured rye grass seedlings as model weed, detrimental effects on plant growth and the Mn nutritional status, as well as increased intracellular shikimate accumulation in root tissue were more strongly expressed than at a direct soil application of the same amount of glyphosate. The increased extent of toxicity after a glyphosate pre-sowing application to pre-cultured rye grass compared with a direct soil application might indicate that the root tissue of glyphosate-treated weeds represents a storage pool for glyphosate in the pots. The globally increasing adoption of no-till or reduced tillage systems are becoming a driving force for an increase of glyphosate use. In such systems, glyphosate is applied pre-sowing for weed control and glyphosate may remain in root and shoot residues. Usually in these reduced tillage systems, only a minimal soil disturbance occurs at sowing, which might lead to limited incorporation of the glyphosate contaminated straw to the upper soil layer where germination of following non-target crop will take place. To evaluate such risk, a pot experiment was conducted under controlled greenhouse conditions with the two contrasting soils. Glyphosate was supplied via glyphosate pre-treated shoot or root material of rye grass applied either as chopped plant material ?straw? or as homogenate. Analysis of physiological parameters such as intracellular shikimate accumulation as metabolic indicator for glyphosate toxicity, biomass production and micronutrient status revealed, that a detrimental effect could be only with treated rye grass shoot material as straw or homogenates incorporated into the Arenosol but not into the Luvisol. This is most probably related to the difference in soil property between the two soils. At this level of glyphosate supply, the detoxification capacity of the highly buffered calcareous subsoil might have played a primary role in preventing glyphosate toxicity, while this glyphosate supply level seems beyond the detoxification capacity of the weakly buffered acidic Arenosol. All together, the achieved results of the model pot experiments are in correspondence with that of the reported field experiments. Further, the results revealed the important role of glyphosate stored in root and shoots of weed plants as a glyphosate pool in soils for intoxication of following crops. More information on transformation of these glyphosate enriched crop residues and its glyphosate release during microbial decomposition in different soils are urgently needed for a better precaution and risk assessment of glyphosate use for weed control for farmer´s practice.
Transcriptional profiling of Bacillus amyloliquefaciens FZB42 in response to seed and root exudates collected under different nutrient regimes
(2010) Carvalhais Costa, Lilia; von Wirén, Nicolaus
Plant growth-promoting rhizobacteria (PGPR) live in close association with plants and improve their growth. Bacillus amyloliquefaciens strain FZB42 is a prominent plant root-colonizing bacterium that is able to stimulate the growth of maize. To decipher the molecular cross-talk between B. amyloliquefaciens and crop plants, an exploratory analysis of the effect of seed and root exudates on the transcriptome of Bacillus amyloliquefaciens FZB42 was performed. Root exudates were collected from maize plants grown in an axenic hydroponic system under nutrient sufficiency or under deficient supply of nitrogen (N), phosphorus (P), iron (Fe) or potassium (K). An analysis of primary metabolites in the exudates was carried out, compared between treatments, and correlated with the transcriptional profiles of Bacillus amyloliquefaciens FZB42 that were gained after incubation of the bacterial culture with the root exudates. Higher exudation rates of citrate were found under Fe deficiency and greater release of ã-amino butyric acid under P deficiency. Based on a negative correlation observed between the average diffusion coefficient of N, P, K, and Fe in soils and the exudation rates of primary metabolites under conditions of N, P, K, or Fe deficiency, it was hypothesized that the exudation of sugars, amino acids and organic acids may reflect the availability and mobility of plant nutrients in soils. In the presence of seed and root exudates collected from nutrient-sufficient plants, genes involved in spore germination, transport and utilization of nutrients, biosynthesis pathways, multidrug transporters, motility and competence development were differentially expressed. In comparison to P, Fe and K, N-deficient maize root exudates caused a more distinguished change in the transcriptome of bacteria when they were in the logarithmic growth phase. During this growth phase, a number of genes coding for ribosomal proteins were down-regulated by N-deficient maize root exudates, indicating that bacterial activity was repressed. Exclusively in the presence of P-deficient maize root exudates, several genes associated to bacterial motility were induced. Moreover, a gene involved in the biosynthesis of the auxin precursor tryptophan was up-regulated by all deficiency treatments. In the transitional growth phase of Bacillus amyloliquefaciens FZB42, several genes were commonly down-regulated in different deficiency treatments. This finding is in agreement with previous studies showing that quorum-sensing and starvation-sensing are integrated to regulate cell entry into the transient phase. Taken together, this is the first study comparing the effect of different nutrient deficiencies on the composition of primary metabolites in root exudates of one plant species and evaluating systematically the transcriptional response of a Gram-positive PGPR to seed and root exudates collected from plants grown under different nutrient regimes. This analysis provides new information about the early communication between plant roots and PGPR and points to involved genes and processes that merit further investigation.
Das Potenzial von Falschem Mehltau als Quelle von Omega-3-Fettsäuren für die menschliche Ernährung
(2009) Anderle, Ann-Marie; Spring, Otmar
The absolute EPA ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid) contents of the downy mildews of sunflowers (Plasmopara halstedii) and lettuce (Bremia lactucae) were quantified by means of GC-FID. The EPA content in sporangia of Bremia lactucae varied between 8 to 13 mg per dry weight. As at the institute of botany a collection of genetically identical sporangia strains of Plasmopara halstedii was established, the natural variation of EPA in four genetically different sporangia strains of Plasmopara halstedii on 10 different sunflower cultivars or -lines (15 days old seedlings) was investigated. The variance in EPA between the sporangia strains (LS-13.12.05-C6, BL-11.06.02-A4z, GG-16.10.97-A25, HE-10.01.06-A8) was only low (18-25 mg EPA per g dry weight). In contrast, the specific variation of EPA in infected sunflower seedlings (Giganteus, HA 821, HA 304, RHA 265, RHA 274, PM 13, 799-2, PM 17, 803-1, DM-2) was relatively high (0,28 to 1,10 mg EPA per g dry weight). Additionally three stages (minimum, optimum, maximum) of nitrogen fertilization were tested for their influence on the EPA content in infected sunflower seedlings. Statistical analysis was carried out by the program SAS. Analysis of variance based on F-tests and multiple t-tests. The influence of hydroponic fertilization (0.1 mM, 1 mM, 5 mM) on EPA in infected sunflower seedlings was high. The nitrogen dose of 5 mM almost doubled the EPA content in infected sunflower seedlings from 1 to almost 2 mg EPA per g dry weight. However, this content is not enough (by factor 10) to serve directly for human nutrition. Therefore at last a food chain trial with Bremia lactucae- infected lettuce was carried out in a cooperation project by several institutes of the Universtiy of Hohenheim. Infected lettuce was fed to hens. The omega-3-fatty acid content per egg was almost doubled (80 to 136 mg) if 10% lettuce was given to hen´s food. The infection with EPA containing downy mildew showed no effects.
Validation and evaluation of the DNDC model to simulate soil water content, mineral N and N₂O emission in the North China Plain
(2009) Kröbel, Roland; Römheld, Volker
Using measured datasets (various soil properties, the soil water content, daily N₂O emissions, and different crop parameters) from a multi-factorial field experiment (N fertilisation, irrigation, and straw removal) in the years 1999-2002 on the experimental site Dong Bei Wang (DBW) in the North China Plain (NCP), the ability of the process-oriented model DNDC (DeNitrification-DeComposition) was tested to simulate soil processes, and especially N₂O trace gas emissions. The soil is classified as ?calcaric cambisol? (16 % clay content), while the site itself is further characterised by the regime of a continental monsoon climate. The central hypothesis in this work was that a thorough testing of the model (using a considerable range of different datasets) will allow the identification of shortcomings or discrepancies in the model, and that, given the linear succession of model calculation steps, the model calculation can be improved step by step, starting with improvements of initial calculation steps before continuing the improvement of following calculation steps. Due to increases in the N₂O atmospheric concentration, and a lifetime of 100 to 150 years for one molecule (as well as a global warming potential 32 times that of a CO₂ molecule), N₂O is estimated to account for 7.9 % of the global warming potential. 70 % ? 90 % of the anthropogenic N₂O emissions are thought to origin from agriculture. The formation of nitrous oxide is dependent on the availability of reactive nitrogen, and, therefore, mainly influenced by the N fertilisation rate, fertiliser type, application timing and method. China, and the main cropping area NCP, are expected to contribute considerably to the anthropogenic N₂O emissions. The DNDC model consists of two compartments, which first calculate soil temperature, moisture, pH, redox potential and substrate concentration profiles from climate, soil, vegetation and anthropogenic activity datasets, and in a second step NO, N₂O, CH4 and NH3 fluxes. In accordance with the data availability, the simulation of the soil water content, the mineral nitrogen concentration, and the N₂O fluxes were investigated. An automated parameter optimisation (using the software UCODE_2005) and programmed changes in the source code were conducted to improve the model simulations. In result, neither the automated parameter optimisations, nor the programmed changes, were able to improve the unsatisfying default simulations of the DNDC model. The results of the cascade model, employed by the DNDC model to simulate soil water dynamics, suggest that conceptual errors exist in the model calculation. Also the results of the mineral nitrogen and N₂O emissions simulations suggest shortcomings in the model calculation. The best agreement between measured and simulated total cumulative N₂O fluxes was achieved using an adapted (90 cm soil depth, adjusted SOC fractioning, and added atmospheric N deposition) default model version, despite unsatisfactory simulations of soil water content, mineral nitrogen, and daily N₂O fluxes. Thus, in conclusion, the investigated DNDC model version appears to be able to give an approximation of seasonal N₂O fluxes, without being able to simulate the underlying processes accurately in detail. Therefore, caution is suggested when modelling sites on the process level.
Müssen Johannisbeeren chloridfrei gedüngt werden? : Studien zur Aufnahme und Verlagerung von Chlorid durch verschiedene Johannisbeerarten
(2009) Blank, Otto-Heinrich; Römheld, Volker
Is there a need for the use of chloride-free mineral fertilizers for currant as a common berry fruit? It is a well-known practice to apply chloride-free potassium fertilizers for berry fruits since decades. Such a chloride-free fertilization with mineral fertilizers and in particular with potassium fertilizers as K2SO4 or potash magnesia (Patentkali) is also recommended in most standard text books for plant nutrition and fruit production. The aims of the present Ph.D. thesis were (1) to find out by inquiries of literature the scientific background of such a statement for a chloride-free fertilization of berry fruits as currants and strawberries (Chapter 2). Further, (2) the extent of the possible different susceptibility to chloride toxicity of various cultivars of currants and of strawberry should got evaluated by different experimental approaches (Chapter 4). In detail short-term uptake studies in nutrient solution (Chapter 6 and 7), but also pot (Chapter 8) and field experiments (Chapter 9 und 10) mainly with different cultivars of currants (black, red and white currants) but also with strawberry were conducted. Besides uptake and translocation of chloride into shoots and berries also sodium has been studied because chloride and sodium are closely linked in application of high chloride containing potassium fertilizers such as potassium chloride (muriate of potash) or kainite as a low-grade K fertilizer (Chapter 6 ? 9). In addition the possible leaching of chloride by application in autumn during the following winter months as a possible strategy to minimize chloride toxicity in currants and strawberries was evaluated in the field experiments (Chapter 9 und 10) as well as in a model experiment with soil filled columns under field conditions (Chapter 11). All these different approaches should finally allow giving a clear recommendation whether a chloride-free fertilization of berry fruits (currant and strawberry) is further needed or an application of chloride-containing potassium chloride (muriate of potash) can be used in berry fruit production by farmers in future. The inquiries of literature did not give any scientifically reasonable answer for the often cited differences in susceptibility of berry fruits or of various groups of currants (black, red or white cultivars) to chloride (Chapter 2). The nutrient solution experiments revealed the various possible mechanisms involved in the pretended different susceptibility to chloride (Chapter 6 and 7). It got obvious that black cultivars of currants had a lower uptake rate of roots for chloride than both other groups, the white and red currants (exclusion of chloride by roots). However, this exclusion of roots had only a limited capacity with only a marginal effect on chloride accumulation in the shoot in the long-term field experiments. The second possible mechanism, storage of chloride in roots (retention) as described for different cultivars of grape vine could not be observed for black currants. This missing difference in the relative translocation of chloride from roots to shoots was in contrast to sodium. Both black cultivars had a much lower sodium translocation to the shoot than red and white cultivars due to a high accumulation (retention) of sodium in roots. These various mechanisms for the described differences in susceptibility to chloride and sodium including possible differences in tissue tolerance were further studied within a pot (Chapter 8) and two field experiments (Chapter 9 and 10). All three experiments demonstrated with the determined critical chloride toxicity concentration in leaves (tissue tolerance) in accordance that currants are quite sensitive to chloride without differences between black, red and white cultivars. Crossing this critical value of about 10-15 mg Cl/g leaf dry weight necrotic symptoms on the edge of leaves could be found independent of cultivars. However such necrotic symptoms on leaves could be only found at a ?Worst-Case-Scenario? with an excessive (2 times) potassium application as the low grade K fertilizer kainite and in the pot experiment without the possibility of leach out of chloride (Chapter 8). In both field experiments with a praxis-relevant potassium application rate and the potential of chloride leaching during the winter period no such symptoms of toxicity could be observed with currants (Chapter 9) and strawberry (Chapter 10), even at a ?Worst-Case-Scenario? with the use of kainite at begin of the season. An adequate leaching of chloride in deeper soil layers during the winter could be proved by analysis of chloride in different soil horizons, but also by leaf analysis of currants and strawberries. Therefore all these above-mentioned results do not support the claim for a chloride-free potassium fertilization of berry fruits such as currant and strawberry.
The AtIREGs - characterization of a new family of metal transporters in Arabidopsis thaliana
(2009) Kirchner, Silvia; von Wirén, Nicolaus
Essential transition metals are required in all plant cells for the activities of numerous metal-dependent enzymes and proteins, but can become toxic when present in excess. For the detoxification of heavy metals and to adjust to changes in micronutrient concentrations in the environment, plants possess a tightly controlled metal homeostasis network. In this regard, transition metal transporters are of central importance. Many metal transporters have already been identified, but a large number of candidates for heavy metal transport proteins still have to be analyzed at the biochemical level and within the plant metal homeostasis network. Based on the description of the animal IREG1 metal transporter as an iron exporter in vertebrates, a phylogenetic analysis of eukaryote and prokaryote sequences with similarity to IREG1 showed three homologous genes in Arabidopsis, which were named AtIREG1, AtIREG2 and AtIREG3. As these AtIREG family members were candidates for yet uncharacterized metal transporters, the main objective of this thesis was to investigate the physiological function of this newly identified transporter family in plants.
Characterisation of natural and synthetic nitrification inhibitors and their potential use in tomato cultivation
(2008) Souri, Mohammad Kazem; Römheld, Volker
Summary Besides commercial NIs, many chemicals could also inhibit nitrification. In our study (Chapter 3) regarding efficiency of chloride compared to 3,4-Dimethylpyrazole phosphate (DMPP), it was found that chloride at applied concentration of 30.5 mg per 100g dry soil, could effectively inhibit nitrification. Despite a lag period of 3 weeks in detectable net nitrification, inhibitory effect of chloride continued to persist even after 7 weeks of soil incubation compared to control. Nevertheless, DMPP particularly with higher concentration (2 % of N-NH4+ instead of 1%) stabilized ammonium more strongly than Cl-1. The extent of nitrification inhibition after 5 and 7 week of incubation was in order of: (2 % of N-NH4+) DMPP > (1 % of N-NH4+) DMPP> NH4Cl > KCl > control. The residue ammonium in the soil as well as the produced nitrate concentrations in samples showed a significant NI activity of chloride in both forms NH4Cl and KCl. Nitrification-induced pH decrease, however, showed a better correlation with measured nitrate than ammonium in this experiment. In a second series of experiments undertaken to identify whether the reported NI release by Brachiaria humidicola accession 26159 is an active or passive phenomenon, root exudates of plants grown under various treatments, have been collected in distilled water or in 1 mM NH4Cl. Under various pre-culture conditions such as N form (NH4+ versus NO3-), N concentrations (1, 2, 4 mM), light intensities (180, 240, 350 µmol m-2 s-1), plant age (3-weeks old versus 7-weeks old) and collecting periods (24 versus 6 h), there was no significant NI activity when root exudates were collected in distilled water. However, NI activity was detectable in root washings when the plants were exposed to extended collection times (24 h) in combination with NH4+ supply, but not after short term collection (6 h) or with NO3- in the collection solution. This observation is consistent with the results of Subbarao et al., (2006, 2007), but it also strongly suggests that the observed release of NI compounds was rather a consequence of membrane damage (passive phenomena) due to inadequate collection conditions, than mediated by controlled exudation from undamaged roots. It has been assumed that supplying only ammonium (1 mM) in distilled water as root washing medium over extended time periods (24 h) could lead to rapid ammonium uptake and medium acidification associated with the risk of Ca2+ desorption, which is an important element required for membrane stabilisation and integrity. To test the hypothesis that NI compounds are released from damaged plant cells of Brachiaria, the NI potential of fresh root and shoot homogenates was measured after soil incorporation and incubation. Surprisingly, NI potential was detected in shoot but not in root homogenates. The NI effect of soil-incorporated shoot tissues lasted for at least 8 d, while root tissue even stimulated nitrification with increasing incubation time. This NI effect was independent of the N form. However, the variability of data increased with NO3- form, higher light intensity or higher N concentrations during plant pre-culture. Independent of N forms, further extraction and characterisation of NI compounds in shoot tissue of Brachiaria plants revealed a particularly high activity in the ethanol-soluble fraction, both in plants with NH4+ and NO3- pre-culture. In a third experiment, the role of Ca2+ ions on improvement of tomato growth under ammonium nutrition was investigated. In this experiment root damage, probably by membrane damage and cytosolic sensitivity were hypothesised to be the main cause of toxicity symptoms of NH4+ on tomato plants. At application of 2 mM N as NH4+, plant biomass, number of lateral shoots, and transpiration were strongly inhibited and an increased Ca2+ application into the nutrient solution counteracted these observed negative effects. Transpiration or water consumption was found to be a good indicator of plant performance under NH4+ nutrition. Plants grown under nitrate nutrition had the highest transpiration rates, as well as the best growth characteristics. There was a positive correlation between nitrate concentrations and transpiration rates. On the other hand, plants grown in ammonium (as control, or 3 and 6 split applications of NH4+ during 4 days) showed severe toxicity symptoms including growth inhibition and leaf abscission. However, when ammonium was applied together with 10 mM Ca2+ (as CaSO4), or in a buffered solution of pH 6.6 with CaCO3 (pH or/and Ca2+ effect), transpiration and other growth factors (e.g. root and shoot dry matter, number of lateral shoots), as well as the nutrients especially N concentrations in the biomass were significantly improved. In other words, shoot and particularly root growth inhibited when NH4+ treated plants (control and split applications) did not received CaSO4 or CaCO3. Micro molar concentrations of NH4+ in 6 split applications also could not prevent ammonium toxicity symptoms.
Regulation of phosphate deficiency-induced carboxylate exudation in cluster roots of white lupin (Lupinus albus L.)
(2005) Kania, Angelika; Römheld, Volker
In many tropical and subtropical areas crop production is severely limited by a deficiency of plant-available phosphorus (P) in the soils. Therefore plant mechanisms to mobilize the sparingly soluble P fraction are of high interest. One such mechanism of P-deficient plants is the exudation of carboxylates and protons from roots. White lupin (Lupinus albus L.) was chosen as a model system to investigate plant metabolism under P deficiency which enable the plant to release ex-traordinarily high amounts of citrate and protons from its cluster roots (bottlebrush-like clusters of short rootlets of determinate growth which form along secondary lateral roots). The aim of this work was to determine the reasons for the high citrate acccumulation observed in mature cluster roots of P-deficient white lupin and to characterize the regulation of citrate release. A threshold citrate concentration is seen as a prerequisite for the transient pulse of intense citrate exudation associated with rhizosphere acidification which occurs over a time period of 2-3 days. Biochemical changes on the anabolic side of citrate metabolism such as increased activities of phosphoenolpyruvate carboxylase (PEP-C) or malate dehydrogenase (MDH) cannot solely ex-plain the very high citrate accumulation observed during cluster root development, although these reactions supply the cluster roots with citrate precursors. In addition, pyruvate concentra-tions decrease in developing cluster roots, probably in relation to the decreasing malic enzyme activities in the respective clusters. Citrate accumulation might also be caused by an impaired citrate turnover. Aconitase, the en-zyme catalyzing the turnover of citrate via cis-aconitate to isocitrate, showed decreasing activi-ties during cluster root development. NADP-isocitrate dehydroganase (NADP-ICDH) activities, as the next metabolic reaction which oxidizes isocitrate to 2-oxoglutarate, paralleled aconitase activities in all the different root segments investigated, although on a two- to threefold higher level. For this, aconitase rather than NADP-ICDH activities seem to limit citrate turnover. Spe-cific activities of aconitase and NADP-ICDH were the same in all the root segments investi-gated. Aconitase is rapidly inactivated by H₂O₂, which can be produced at increased rates under P limitation. However, neither H₂O₂ concentrations nor malondialdehyde concentrations as a marker for lipid peroxidation under oxidative stress were increased in clusters with low aconitase activities. Artifical inhibition of aconitase by incubating young cluster roots with high amounts of externally applied H₂O₂ did not change citrate and malate concentrations in these root seg-ments. However, a strong increase in citrate concentrations and a strong decrease in malate con-centrations in young cluster roots, together with high citrate exudation rates, could be observed when monofluoroacetate (MFA) as another aconitase inhibitor was applied. Inhibition of the aconitase enzyme therefore forced still young clusters to react like mature ones. This hints to aconitase as a key metabolic step in citrate turnover. High rates of carboxylate exudation were measured even from seedling root tips when incubated with MFA. Decreasing dehydrogenase activities as found during cluster root development by in situ staining with formazan were independent of the substrate supplied (citrate, aconitate, isocitrate, succinate, malate). This is in accordance with the decreasing enzyme activities measured in the different root segments such as aconitase, NADP-ICDH or malic enzyme in vitro. A reduced nitrate re-ductase (NR) activity under P deficiency, resulting in a lower drainoff of 2-oxoglutarate for N assimilation, seems not to play an important role for citrate accumulation, since an artificial NR inhibition with tungstate did not significantly increase citrate concentrations in young cluster roots. The change from malate to citrate accumulation during cluster root development is paralleled by a reduction in ATP-citrate lyase (ACL) activity, an enzyme cleaving citrate to oxaloacetate and acetyl-CoA. The good correlation between the citrate/malate ratio in root exudates and ACL ac-tivities indicates that ACL plays a key role as a metabolic switch between malate and citrate ac-cumulation during cluster root development under P deficiency. The enzyme might prevent high citrate concentrations under less severe P deficiency, when ACL activity is not limited by ATP availability. The attempt to inhibit the ACL enzyme by application of hydroxycitrate (HC) did not show any effect on citrate or malate concentrations in the young cluster roots. However, HC was probably not taken up into the root cells and could therefore not exert any inhibitory effects. Decreasing total respiration rates as found for developing cluster roots might affect citrate accu-mulation directly by reduced consumption of citrate in the TCA cycle or indirectly by H₂O₂-induced inhibition of aconitase activity. However, a reduced respiration rate did not result in higher H₂O₂ concentrations in white lupin. Cytochrome pathway capacity decreased parallel to total respiration, suggesting that the cytochrome pathway determines total respiration. An in-crease in alternative oxidase (AOX) capacity did take place in cluster roots, but was not high enough to compensate for the decreased cytochrome capacity. The AOX enzyme often occurs under P deficiency or under oxidative stress, probably to bypass a limiting Pi-and ADP-dependent cytochrome pathway. The amount of the AOX protein, determined by immunodetec-tion, paralleled AOX capacity. However, the availability of Pi and adenylates was not limiting for total respiration, since uncoupling oxidative phosphorylation with CCCP did not increase the respiration rate. The citrate/malate ratio in young clusters with high rates of respiration and low inherent levels of citrate accumulation was only slightly increased by short-term application (4 -8 h) of azide and SHAM as respiration inhibitors. The concomitant release of citrate and protons from mature cluster roots of P-deficient white lupin plants hints to a common regulation of citrate exudation and H+-ATPase activity in this specific root zone. Highly purified inside-out plasma membrane (PM) vesicles were isolated in a membrane-physiological approach to determine H+-ATPase characteristics involved in citrate exudation under P deficiency. Increased hydrolytic activity of the PM H+-ATPase derived from P-deficient plants parallels an increase in rhizosphere acidification and citrate exudation and hints to a causal relationship. Western blot analysis revealed a higher H+-ATPase protein amount under P deficiency. The op-timum pH of the H+-ATPase was shifted towards more acidic conditions under P-deficiency, which might be an adaptation to the supposedly decreased cytosolic pH brought about by the pH stat mechanism when carboxylates accumulate. Lower citrate concentrations (2 mM) stimulated PM vesicle acidification even in the absence of ATP, which was further enhanced by the addi-tion of Mg-ATP, and particularly expressed in PM vesicles isolated from roots of P-deficient plants. Accordingly, 14C-citrate was taken up at higher rates into vesicles derived from P-deficient white lupin compared with vesicles of P-sufficient control plants. Therefore citrate transport predominantly occurs in roots of P-deficient plants, and is linked with the activity of the PM H+-ATPase to maintain the electrochemical potential gradient which is reduced by citrate export out of the cell. Citrate exudation combined with an increase in H+-ATPase activity seems to prevent citrate accumulation up to concentrations which might exert inhibitory effects on the PM H+-ATPase. Such an inhibition was seen by diminished intravesicular proton accumulation, detected with the pH probe acridine orange, when 5 mM citrate were applied to the vesicle preparation. No such inhibitory effects were observed by malate application, which hints to a citrate-specific reaction. Lowering the cytosolic pH by external application of propionate stimulated citrate and malate exudation in non-cluster laterals and in young clusters. Therefore a causal relationship might exist between citrate accumulation and exudation by acidification of the cytosol. The threshold citrate concentration at which citrate exudation is triggered perhaps is reached when citrate ac-cumulation leads to acidification of the cytosol. Carboxylate exudation in young cluster roots and seedling root tips hints to a putative anion channel which already exists in young tissue and might be regulated in relation with H+-ATPase activity and cytosolic pH. Protoplasts, isolated from mature cluster roots, did only give very low yield and were not viable for seals high enough for patch-clamp studies. This might be due to the fast senescence in the developing clusters which also seems to change membrane integrity. High yields could only be gained from seedling root tips, or cotyledons. Similarly, protoplast isolation from root hairs also was only possible from seedling root tips or non-cluster lateral root tips, but even not from just emerging root hairs of young cluster roots. To determine the influence of a second growth factor in addition to P deficiency on citrate me-tabolism, white lupin was cultivated in nutrient solution and in rhizoboxes at ambient (400 μmol mol-1) and at elevated (800 μmol mol-1) atmospheric CO₂ concentrations. Plant development was accelerated at elevated CO₂ concentrations, and P deficiency and senes-cence symptoms such as yellowing, wilting, and abscission of leaves could be seen much earlier. When cultivated in nutrient solution, shoot growth was rather unaffected by the CO₂ concentra-tion, whereas root growth was much faster at elevated CO₂. Quite contrary, shoot growth was slightly higher at elevated CO₂ concentrations in plants in rhizobox culture, but root growth was unchanged. However, the harvest of a higher amount of cluster roots from plants at elevated CO₂ or the calcareous soil might have reduced root growth of the plants grown in rhizoboxes. Higher root/shoot ratios under P deficiency were further increased at elevated CO₂ concentra-tions. The amount of clusters was higher in plants grown in nutrient solution at 800 μmol mol-1 CO₂ from day 21 to day 33 after sowing, but thereafter the differences disappeared. No signifi-cant differences between CO₂ treatments were observed for the proportion of cluster roots rela-tive to the whole root system. Independent of the cultivation method, root exudation per cluster or per cluster root weight was unchanged by the elevated CO₂ concentration. The distribution of citrate and malate exudation in different cluster root segments with decreasing malate exudation and a peak of citrate exudation in mature clusters was also confirmed at 800 μmol mol-1 CO₂. The increased carbon distribution into the root at 800 μmol mol-1 CO₂, seen in a higher root/shoot ratio, was not transformed into higher exudation rates from the single cluster. Acid and alkaline phosphatase activities in the rhizosphere of L. albus continually increased during cluster root development independent of the CO₂ supply. Phosphatase activities and carboxylate accumulation and exudation rates were essentially un-changed by different atmospheric CO₂ concentrations. This might be due to also unaltered Pi concentrations in the respective root segments, because internal P concentrations seem to deter-mine these parameters. Since citrate accumulation and exudation probably depends on citrate degradation, which is not influenced by the amount of carbon supplied for anabolic processes, elevated CO₂ concentrations do rather not change citrate concentration and exudation. Accord-ingly, no significant effects of different CO₂ concentrations were seen on microbial diversity in the rhizosphere of white lupin. So far, no single cause or mechanism was found to be responsible for the high citrate concentra-tions measured in mature cluster roots, although citrate degradation seems to be important and aconitase probably plays a key role. A general impairment of metabolism due to decreasing con-centrations of Pi, adenylates, RNA, and proteins rather seems to bring about decreasing enzyme activities and reduced respiration. Various regulatory mechanisms via phosphorylation/ dephos-phorylation, phytohormones, nitric oxide, or others also have to be considered.
Emission von Ammoniak (NH₃) und Lachgas (N₂O) von landwirtschaftlich genutzten Böden in Abhängigkeit von produktionstechnischen Maßnahmen
(2003) Leick, Barbara Cornelia Elisabeth; Engels, Christof
The goal of this research was to quantify event-based NH₃ and N₂O emissions in various farming systems and to propose emission-avoidance strategies. Emission measurements were made on pasture land (Allgaeu, Hohenheim) and on cultivated fields (Hohenheim, Biberach). These measurements were made after applying organic and mineral fertilizers, after incorporating crop residues, and after freeze / thaw cycles; furthermore, experiments were conducted using container plants of different species (leguminous, and non-leguminous) and different fertilizers. NH3 emissions data was gathered under field conditions using the wind tunnel method and the IHF method (Integrated Horizontal Flux). In the container experiments, data was gathered by taking photo-acoustic measurements. N₂O emissions data was compiled using closed chambers (Hohenheim measuring chambers) and using an open-chamber system in which an exchange occurred between the air in the chambers and the ambient air. N₂O levels were determined using a gas chromatograph or by photo-acoustic measurements. The NH₃ emissions after applying liquid manure to pasture land varied between 11 and 40% of the total nitrogen applied. Emission levels of less than 20% occurred when it rained shortly after spreading liquid manure causing it to be washed into the soil. The application technique (splash plate, surface banding and liquid manure injection) had no apparent influence on NH₃ emissions under these conditions. The N₂O emissions after liquid manure fertilization on pasture land in Hohenheim were 0.16% of the total NH4+-N. In comparison, the emissions in the Allgäu were between 1.7 and 2.3% of the total NH4+-N applied. Liquid manure injection led to higher emissions as did application using a splash plate. In the Allgäu, the N₂O emissions after mineral-nitrogen fertilization were markedly lower (0.3 to 0.8% of applied N) than after liquid manure application. In Hohenheim, the nitrogen form had no distinct influence on the emissions (<0.16% of applied N). Definitive differences between the two locations were observed during the experiments. These differences were based on N₂O losses due to the respective soil and weather conditions (precipitation, temperature). The higher emissions after applying liquid manure compared to those after applying mineral nitrogen fertilizer are explainable in that aside from the nitrogen compounds found in liquid manure, carbon compounds which promote the microbial formation of N₂O were also entering the soil. The NH3 emissions after liquid manure fertilization on cultivated fields using a splash plate varied between 25 and 35% of the applied NH4+-N. By using a slurry cultivator which combines application with immediate incorporation, the NH3 emissions can be clearly reduced to 6% of the applied NH4+-N. Application with a drag hose, in comparison to using a splash plate, did not always result in an emission reduction; however, in taller plants, a readable emission reduction was measured. The N₂O emissions after liquid manure application on cultivated fields varied between 0.1 and 2.2% of the applied NH4+-N whereby the emissions after guided application with the drag hose were always higher than after using a splash plate. Mineral fertilizer had lower N2O emissions (<0.13% of applied N), especially when ammonium fertilizer was brought out in combination with a nitrification inhibitor. The incorporation of green manure crops notedly increased N₂O emissions. N₂O emission after the incorporation of legumes was especially high. In the container experiments, a diurnal rhythm of the N₂O and NH₃ flows in growing rape and vetch was observed. This indicated a stomatal flow of these gaseous nitrogen forms. N₂O emissions also occurred outside of the vegetation period at temperatures between 0 and 5°C, with the N₂O emissions from the nitrogen fertilized parcels being greater than the emissions from the unfertilized parcels. In container experiments, the N₂O emissions after freeze / thaw cycles were greater from white clover than from perennial rye grass. In fallow soil columns, the N₂O emissions after freeze / thaw cycles were especially high if the content of nitrate and water-soluble organic carbon in the soil was large. The results of this research show that the emission of nitrogen-containing compounds after organic and inorganic fertilization can be reduced through application methods (immediate incorporation), appropriate fertilization technology (addition of nitrification inhibitors), but also through fertilizer application under favourable weather conditions to include seasonal and volume adjustment of the fertilizer based on the growth requirements of the plants. Because high N₂O emissions can also occur at low temperatures, cultivation practices that influence the availability of mineral nitrogen and easily degradable organic substances in the soil during cold weather have a large impact on the N₂O emissions from agricultural land.
Abgabe von bodenbürtigem Lachgas über Pflanzen
(2003) Ferch, Norbert-Jakob; Römheld, Volker
The aim of this work was to explore and to rank the different ways and forms of transition of N2O through plants (dissolved in water and transported with the transpiration or gaseous through aerenchyma). To achieve this goal an experimental set-up had to be realized that allowed the determination of possible N2O emissions by plants, the determination of different ways of transition of N2O through the plant and the determination of different influencing factors (e.g. N2O concentration) on the N2O emissions. In the beginning experiments with closed chambers and with ?controlled opened chambers? were conducted in comparison to each other. In the experiments with closed chambers samples were drawn by means of molecular sieves and vacutainers. N2O concentrations of the samples were measured with a GC (gas chromatograph type HP 5890) equipped with an ECD (electron capture detector). Besides the two methods mentioned above in order to determine the N2O concentrations within the experiments with the ?controlled opened chambers? a third method was used for N2O measurement by means of a photo acoustic online measuring machine. The accuracy of the photo acoustic measurement was evaluated with the GC. For the questions of interest the photo acoustic measurement showed to be the best to determine differences of N2O emissions between different experimental treatments. The experiments that were taken in consideration were conducted in a ?controlled opened system? because in closed chambers CO2 concentration decreased rapidly. Additionally, the air in the closed chambers became saturated in water vapour within a few minutes. These two factors lead to inhibited growth of the plants and to undesired influences on the N2O measurements. The ?controlled opened system? consisted of a root and a shoot compartment. Both compartments were separated airtight from each other and from the surroundings. The root compartments were enriched with a definite amount of N2O. The N2O concentrations measured in the shoot compartments of the systems with N2O enrichment in the root compartment were compared with measurements of systems without N2O enrichment and measurements of ambient air. The necessity to divide the root compartment from the shoot compartment airtight was realised with a material on the basis of silicone that is usually used to make prints of teeth (Optosil, from Haereus) and a sealing mass (Prestik AE hellgrau, from Bostik GmbH). To determine the different factors potentially influencing the N2O emission through plants a hydroponical culture system was established that allowed controlling the following factors: concentration of nutrients, pH-factor, concentration of different water soluble gases (e. g. N2O, CO2) and the ratio between water and gas filled space in the root compartment. As experimental plants sunflower (Helianthus annuus cv. Frankasol), barley (Hordeum vulgare cv. Scarlet), rice (Oryza sativa cv. 94D-22) and corn (Zea mays cv. Helix) were used. For the experiment with sunflower (no aerenchyma, N2O dissolved in water available only) a relationship between N2O concentration in the root compartment, the emitted amount of N2O by the shoots and the intensity of transpiration in a diurnal pattern was found. In systems with gaseous availability of N2O in the root compartment the observed emissions were higher than in systems with availability of N2O dissolved only in water. From this it could be concluded, that gaseous N2O is better available for plants than N2O dissolved in water. Similar results were obtained from experiments with barley. The only difference was that the highest N2O emissions were observed in systems with availability of N2O dissolved in water only. The possible N2O emission through aerenchyma was checked with rice plants. In these experiments a pronounced diurnal pattern of the N2O emissions was also found. This lead to the conclusion that aerenchyma only have a small influence on the N2O emissions out of the root compartment through rice plants. Because the N2O emission in the three experiments described above followed the diurnal pattern of the transpiration, it was concluded that N2O was transported with the transpiration water flow from the root (compartment) to the shoot (compartment). The experiments with corn showed for all treatments (control and availability of N2O in gaseous form or dissolved in water) a net N2O depletion in the shoot compartment for night (darkness) and day (light) respectively, thus leading to the conclusion that N2O can be metabolised and used as a nitrogen source. All in all the experiments showed that the main way of transition of N2O through plants is water dissolved with the transpiration water flow and not gaseous (through aerenchyma).

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