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It has been repeatedly demonstrated that phosphate (P) and the herbicide glyphosate compete for adsorption sites in soils. Surprisingly, the potential consequences of these interactions for plants e.g. re-solubilisation of phytotoxic glyphosate residues in soils by application of P fertilisers or by root-induced mechanisms for P mobilization have not been investigated so far. In model experiments under greenhouse conditions, the potential for glyphosate re-mobilisation by P-fertiliser application was evaluated by bio-indication with soybean (Glycine max L.) cultivated on five contrasting soils with or without glyphosate application at 10–35 days before sowing. Different levels of P-fertilisation (0, 20, 40, 80, 240 mg P kg−1 soil) were supplied at the date of sowing. Visual symptoms of glyphosate toxicity, plant biomass, intracellular shikimate accumulation as physiological indicator for glyphosate toxicity and the plant nutritional status were determined. On glyphosate-treated soils, P application induced significant plant damage. Expression of damage symptoms declined in the order Arenosol > Acrisol ≈ Ferralsol > Luvisol subsoil > Regosol. On the Arenosol, Ferralsol and Luvisol subsoil plant damage was associated with increased shikimate accumulation in the root tissue. On the Acrisol decline of germination and plant damage in absence of shikimate accumulation indicate toxicity of AMPA (aminomethylphosphonic acid) as the main metabolite of glyphosate in soils. On the Regosol, a growth-stimulating effect of glyphosate soil application (hormesis) was detected. The results suggest that re-mobilisation of glyphosate may represent an additional transfer pathway for glyphosate to non-target plants which is strongly influenced by soil characteristics such as P fixation potential, content of plant-available iron, pH, cation exchange capacity, sand content and soil organic matter.

Elevated atmospheric CO2 treatments stimulated biomass production in Fe-sufficient and Fe-deficient barley plants, both in hydroponics and in soil culture. Root/shoot biomass ratio was increased in severely Fe-deficient plants grown in hydroponics but not under moderate Fe limitation in soil culture. Significantly increased biomass production in high CO2 treatments, even under severe Fe deficiency in hydroponic culture, indicates an improved internal Fe utilization. Iron deficiency-induced secretion of PS in 0.5 to 2.5 cm sub-apical root zones was increased by 74% in response to elevated CO2 treatments of barley plants in hydroponics but no PS were detectable in root exudates collected from soil-grown plants. This may be attributed to suppression of PS release by internal Fe concentrations above the critical level for Fe deficiency, determined at final harvest for soil-grown barley plants, even without additional Fe supply. However, extremely low concentrations of easily plant-available Fe in the investigated soil and low Fe seed reserves suggest a contribution of PS-mediated Fe mobilization from sparingly soluble Fe sources to Fe acquisition of the soil-grown barley plants during the preceding culture period. Higher Fe contents in shoots (+52%) of plants grown in soil culture without Fe supply under elevated atmospheric CO2 concentrations may indicate an increased efficiency for Fe acquisition. No significant influence on diversity and function of rhizosphere-bacterial communities was detectable in the outer rhizosphere soil (0-3 mm distance from the root surface) by DGGE of 16S rRNA gene fragments and analysis of marker enzyme activities for C-, N-, and P-cycles.

Increased root exudation and a related stimulation of rhizosphere-microbial growth have been hypothesised as possible explanations for a lower nitrogen- (N-) nutritional status of plants grown under elevated atmospheric CO2 concentrations, due to enhanced plantmicrobial N competition in the rhizosphere. Leguminous plants may be able to counterbalance the enhanced N requirement by increased symbiotic N2 fixation. Only limited information is available about the factors determining the stimulation of symbiotic N2 fixation in response to elevated CO2. In this study, short-term effects of elevated CO2 on quality and quantity of root exudation, and on carbon supply to the nodules were assessed in Phaseolus vulgaris, grown in soil culture with limited (30 mg N kg � 1 soil) and sufficient N supply (200 mg N kg � 1 soil), at ambient (400mmol mol � 1 ) and elevated (800mmol mol � 1 ) atmospheric CO2 concentrations. Elevated CO2 reduced N tissue concentrations in both N treatments, accelerated the expression of N deficiency symptoms in the Nlimited variant, but did not affect plant biomass production. 14 CO2 pulse-chase labelling revealed no indication for a general increase in root exudation with subsequent stimulation of rhizosphere microbial growth, resulting in increased N-competition in the rhizosphere at elevated CO2. However, a CO2-induced stimulation in root exudation of sugars and malate as a chemo-attractant for rhizobia was detected in 0.5–1.5 cm apical root zones as potential infection sites. Particularly in nodules, elevated CO2 increased the accumulation of malate as a major carbon source for the microsymbiont and of malonate with essential functions for nodule development. Nodule number, biomass and the proportion of leghaemoglobin-producing nodules were also enhanced. The release of nod-gene-inducing flavonoids (genistein, daidzein and coumestrol) was stimulated under elevated CO2, independent of the N supply, and was already detectable at early stages of seedling development at 6 days after sowing. r 2007 Elsevier Ltd. All rights reserved.

White lupine (Lupinus albus L.) was used as a phosphorus (P)-efficient model plant to study the effects of elevated atmospheric CO(2) concentrations on (i) P acquisition, (ii) the related alterations in root development and rhizosphere chemistry, and (iii) the functional and structural diversity of rhizosphere microbial communities, on a P-deficient calcareous subsoil with and without soluble P fertilization. In both +P (80 mg P kg(-1)) and -P treatments (no added P), elevated CO(2) (800 micromol mol(-1)) increased shoot biomass production by 20 to 35% and accelerated the development of cluster roots, which exhibit important functions in chemical mobilization of sparingly soluble soil P sources. Accordingly, cluster root formation was stimulated in plants without P application by 140 and 60% for ambient and elevated CO(2) treatments, respectively. Intense accumulation of citrate and increased activities of acid and alkaline phosphatases, but also of chitinase, in the rhizosphere were mainly confined to later stages of cluster root development in -P treatments. Regardless of atmospheric CO(2) concentrations, there was no significant effect on accumulation of citrate or on selected enzyme activities of C, N, and P cycles in the rhizosphere of individual root clusters. Discriminant analysis of selected enzyme activities revealed that mainly phosphatase and chitinase contributed to the experimental variance (81.3%) of the data. Phosphatase and chitinase activities in the rhizosphere might be dominated by the secretion from cluster roots rather than by microbial activity. Alterations in rhizosphere bacterial communities analyzed by denaturing gradient gel electrophoresis (DGGE) were related with the intense changes in root secretory activity observed during cluster root development but not with elevated CO(2) concentrations.

A possible contribution of alterations in metabolic sequences involved in citrate catabolism, to intracellular accumulation and subsequent release of citrate was investigated in cluster roots of phosphorus (P)-deficient white lupin (Lupinus albus L.). Citrate accumulation during maturation of root clusters was associated with decreased levels of intracellular soluble Pi and ATP, and with reduced rates of respiration. Inhibitor studies with KCN and salicylhydroxamic acid (SHAM) suggest a reduced capacity of both the cytochrome pathway and of the alternative respiration with a concomitant decrease of immunochemically detectable protein levels of the alternative oxidase. Reduced respiration seems to be related to a general impairment of the respiratory system, rather than to limitation of respiratory substrates such as Pi and adenylates, as indicated by the absence of stimulatory effects of the uncoupler CCCP. The citrate/malate ratio in juvenile root clusters with high rates of respiration and low inherent levels of citrate accumulation was increased by short-term application (4–8 h) of azide and SHAM as respiration inhibitors. During maturation of root clusters, a shift from intracellular malic acid to citric acid accumulation was associated also with down-regulation of ATP citrate lyase (ACL), which catalyzes cleavage of citrate into acetyl-CoA and oxaloacetate with a putative function as anapleurotic source for the production of acetyl-CoA under P-deficient conditions. Inhibition of nitrate uptake and assimilation is a general response to P limitation in many plant species including white lupin. Reduced consumption of the amino acceptor 2-oxoglutaric acid as a product of citrate turnover may therefore contribute to increased citrate accumulation. Accordingly, artificial inhibition of nitrate reduction by localized application of tungstate significantly increased the citrate/malate ratio in juvenile root clusters. Lowering the cytosolic pH by external application of propionate stimulated citrate and malate exudation in non-cluster lateral roots and in developing root clusters. This effect was reverted by preincubation with phosphonate to buffer the cytosol. The results suggest that acidification of the cytosol may be an important factor, triggering the transient release of citrate and protons from mature root clusters in P-deficient white lupin.

White lupin was grown in a quartz sand–soil mix with poorly available Ca phosphate. The plants were harvested on days 21, 35 and 51 and DNA was extracted from the non-cluster roots, the young, mature and senescent cluster roots with adhering soil. Bacterial community structure was examined by PCR-DGGE of 16S rDNA, digitisation of the band patterns and multivariate analyses. In all root zones the bacterial community structure changed with plant age. The communities in the rhizosphere of the non-cluster roots were always different from those of the cluster roots. The bacterial communities of the cluster roots were cluster age and plant age dependent. The differences in bacterial community structure between the cluster root age classes were significant on days 35 and day 51 but not on d 21. A separate experiment, in which root exudates and samples for PCR-DGGE were collected simultaneously, showed that both bacterial and eukaryotic (18S rDNA) community structures change with organic acid exudation. While eukaryotic community structure of the cluster roots was correlated with citric acid exudation, bacterial community structure was correlated with cis-acconitic, citric and malic acid exudation.

Lupinus albus serves as model plant for root-induced mobilization of sparingly soluble soil phosphates via the formation of cluster-roots (CRs) that mediate secretion of protons, citrate, phenolics and acid phosphatases (APases). This study employed next-generation sequencing to investigate the molecular mechanisms behind these complex adaptive responses at the transcriptome level. We compared different stages of CR development, including pre-emergent (PE), juvenile (JU) and the mature (MA) stages. The results confirmed that the primary metabolism underwent significant modifications during CR maturation, promoting the biosynthesis of organic acids, as had been deduced from physiological studies. Citrate catabolism was downregulated, associated with citrate accumulation in MA clusters. Upregulation of the phenylpropanoid pathway reflected the accumulation of phenolics. Specific transcript expression of ALMT and MATE transporter genes correlated with the exudation of citrate and flavonoids. The expression of transcripts related to nucleotide degradation and APases in MA clusters coincided with the re-mobilization and hydrolysis of organic phosphate resources. Most interestingly, hormone-related gene expression suggested a central role of ethylene during CR maturation. This was associated with the upregulation of the iron (Fe)-deficiency regulated network that mediates ethylene-induced expression of Fe-deficiency responses in other species. Finally, transcripts related to abscisic acid and jasmonic acid were upregulated in MA clusters, while auxin- and brassinosteroid-related genes and cytokinin receptors were most strongly expressed during CR initiation. Key regulations proposed by the RNA-seq data were confirmed by quantitative real-time polymerase chain reaction (RT-qPCR) and some physiological analyses. A model for the gene network regulating CR development and function is presented.

In cluster roots of P-deficient white lupin, intense exudation of citrate, and the contemporaneous acidification of the rhizosphere, are an adaptation to mobilise sparingly soluble soil P sources. Transport processes involved in citrate exudation were investigated through a membrane physiological approach, using isolated root plasma membrane (PM) vesicles. Exogenous application of citrate [100–250 μM] to root PM vesicle preparations stimulated H+-ATPase, activity. However, a strong inhibition occurred at concentrations >2 mM, independently of Mg supply. No such effects were observed when malate was applied. Accordingly, application of 5 mM citrate to inside-out PM vesicles decreased ATP-dependent intravesicular H+ accumulation, which was increased after the application of 5 mM malate. In contrast, lower citrate concentrations [2 mM] stimulated vesicle acidification even in the absence of Mg-ATP. Acidification was further enhanced by the addition of Mg-ATP, and was most evident in vesicles isolated from cluster roots of P-deficient plants. Moreover, the uptake of 14C citrate in the presence of ATP and PM H+-ATPase activity were particularly enhanced in PM vesicles obtained from cluster roots. The results provide evidence for a P deficiency-induced transport mechanism for citrate anions at the PM of cluster root cells, coupled with the activity of the PM H+-ATPase, On the other hand, PM H+-ATPase, activity may be modulated by concentration levels of citrate characteristic for the cytosol.

The release of high amounts of citrate and protons confined to proteoid roots of P deficient white lupin is an efficient strategy for mobilization of sparingly soluble P forms in soils. A membrane physiological approach was employed to study transport mechanisms involved in citrate exudation by use of highly purified root plasma membrane (PM) vesicles. Exogenous application of citric acid-BTP (pH 6.5) to root PM vesicles stimulated H+-ATPase activity at low concentrations of 100...250 μm, but strong inhibition occurred at concentration levels > 2 mm, which could not be attributed to Mg2+ complexation by citrate. No such effects were observed by malate application. Accordingly, the application of 5 mm citrate diminished intravesicular H+ accumulation, detected with the pH probe acridine orange, whereas proton influx was increased by application of 5 mm malate. Lower citrate concentrations (2 mm) stimulated PM vesicle acidification even in the absence of ATP, which was further enhanced by the addition 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 root-PM vesicles of P-starved white lupin compared with vesicles of P-sufficient control plants.