Institut für Pflanzenernährung

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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).
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.
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.
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.
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.
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.
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.

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