Evaluation of a novel tool for sampling root exudates from soil-grown plants compared to conventional techniques

The release of low molecular weight (LMW) organic compounds (e.g. organic acids, amino acids, sugars, etc.) by living plant roots significantly contributes to the development of chemical, physical as well as microbial rhizosphere gradients. Suitable and accurate sampling procedures are crucial for enhancing our understanding of the dynamics of related rhizosphere processes. Here we compare common sampling techniques with a novel tool for root exudate collection that allows non-destructive and repetitive sampling from soil-grown roots. Root exudates from Zea mays L. were collected using the following techniques: (i) hydroponic growth and sampling, (ii) soil growth and hydroponic sampling and (iii) rhizoboxes fitted with a novel in situ root exudate collecting tool. Furthermore, rhizosphere soil solution for the analysis of exudates and microbial metabolites was sampled using micro-suction cups (iv). The effect of different sampling solutions (deionised water and 0.5 mM CaCl₂) on organic acid and amino acid exudation patterns was also investigated. The novel exudate collecting tool was successfully tested for root exudate sampling. Results showed that particularly amino acid exudation rates were significantly affected by growth conditions and sampling procedures, while organic acid exudation patterns varied less across the different sampling setups. Despite qualitative and quantitative differences, exudation rates were in the same order of magnitude across the different sampling procedures. Soil solution concentrations obtained from micro-suction-cup sampling at defined distance to the root surface showed no distinct gradient, highlighting the importance of soil microorganisms in regulating the soil solution concentration of LMW C compounds either via microbial degradation or the release of microbial metabolites. The exudate collector offers new opportunities to assess root exudation rates and composition from soil-grown plants and thus enhances our knowledge of fundamental rhizosphere processes.     

Composition of root exometabolites of the symbiotically effective pea cultivar triumph and its parental forms

The qualitative and quantitative composition of low-molecular exometabolites in roots of pea (Pisum sativum L.) was studied with a cultivar Triumph and its parental forms (a symbiotically effective variety k-8274 and a modern highly productive cv. Classic). A relationship between root exudation and the ability of cultivars to establish symbiosis was analyzed. In the early stages of plant growth, the roots of cv. Triumph exhibited low exudation of organic acids, sugars, and amino acids. The quantitative composition of organic acids in the root exudates of cv. Triumph was close to that of cv. k-8274, whereas the composition of sugars and amino acids was similar to that of cv. Classic. In the field experiment, the effect of inoculation with a mixture of rhizobium strains and mycorrhizal fungus on plant growth was more evident in cv. Triumph than in cvs. Classic and k-8274. The results suggest that the high symbiotic potential of cv. Triumph is related to exudation of pyruvic and succinic acids that were the major components of root exometabolites both in Triumph and k-8274 cultivars.     

Characterization of Root Exudates at Different Growth Stages of Ten Rice (Oryza sativa L.) Cultivars

Abstract: Plant root exudates play important roles in the rhizosphere. We tested three media (nutrient solution, deionized water and CaSO₄ solution) for three periods of time (2, 4 and 6 h) for collecting root exudates of soil‐grown rice plants. Nutrient culture solution created complications in the analyses of exudates for total organic C (TOC) by the wet digestion method and of organic acids by HPLC due to the interference by its components. Deionized water excluded such interference in analytical analyses but affected the turgor of root cells; roots of four widely different rice cultivars excreted 20 to 60 % more TOC in deionized water than in 0.01 M CaSO₄. Furthermore, the proportion of carbohydrates in TOC was also enhanced. Calcium sulfate solution maintained the osmotic environment for root cells and did not interfere in analytical procedures. Collection for 2 h avoided under‐estimation of TOC and its components exuded by rice roots, which occurred during prolonged exposure. By placing plants in 0.01 M CaSO₄ for 2 h, root exudates of soil‐grown traditional, tall rice cultivars (Dular, B40 and Intan), high‐yielding dwarf cultivars (IR72, IR52, IR64 and PSBRc 20), new plant type cultivars (IR65598 and IR65600) and a hybrid (Magat) were collected at seedling, panicle initiation, flowering and maturity and characterized for TOC and organic acids. The exudation rates were, in general, lowest at seedling stage, increased until flowering but decreased at maturity. Among organic acids, malic acid showed the highest concentration followed by tartaric, succinic, citric and lactic acids. With advancing plant growth, exudation of organic acids substituted exudation of sugars. Root and shoot biomass were positively correlated with carbon exudation suggesting that it is driven by plant biomass. As root exudates provide substrates for methanogenesis in rice fields, large variations in root exudation by cultivars and at different growth stages could greatly influence CH₄ emissions. Therefore, the use of high‐yielding cultivars with lowest root excretions, for example IR65598 and IR65600, would mediate low exudate‐induced CH₄ production. The screening of exciting rice cultivars and breeding of new cultivars with low exudation rates could offer an important option for mitigation of CH₄ emission from rice agriculture to the atmosphere.     

The effect of manganese supply on exudation of carboxylates by roots of lucerne (Medicago sativa)

Some low-molecular-weight carboxylates commonly found in plant root exudates have the potential to increase the availability of Mn in the rhizosphere. Release of various compounds into the rhizosphere by plant roots may also be a mechanism by which certain species and genotypes are able to tolerate conditions of low Mn availability better than others. Lucerne (Medicago sativa L.) plants of Salado, a genotype tolerant to Mn deficiency, and Sirosal, an intolerant genotype, were grown in solution culture with 0, 5 or 500 nM Mn (Mn-0, Mn-5 and Mn-500). Exudates of whole root systems were collected at 14, 24 and 36 d and analysed by HPLC. Oxalate, tartarate, L-malate, lactate, malonate, maleate, citrate and succinate were detected and quantified in exudates under all Mn treatments. Malonate, citrate and succinate accounted for the majority of carboxylates in the exudates. Exudation increased with plant age, but amounts of individual carboxylates remained constant in proportion to the total amount exuded. A significant increase in exudation of all carboxylates other than malonate and maleate resulted from omission of Mn from nutrient solutions. Salado exuded more oxalate, tartarate, L-malate, lactate, citrate and succinate than Sirosal at Mn-0, and more citrate and succinate than Sirosal at Mn-5. Genotypic differences in carboxylate exudation under Mn-0 were associated with production of roots with diameter <100 μm. Plant Mn concentrations and growth rates suggested carboxylate exudation differences were not the sole factor responsible for differential tolerance to Mn deficiency in the lucerne genotypes.     

Citrate, Malate, and Succinate Concentration in Exudates from P-Sufficient and P-Stressed Medicago sativa L. Seedlings

Under certain stress conditions roots exude organic molecules, which may facilitate the uptake of nutrients. The objective of this research was to identify and measure the effect of low P upon the exudation of organic acids by roots of alfalfa (Medicago sativa L.) seedlings. Surface-sterilized alfalfa seeds were grown aseptically in sterile sand using an apparatus specially designed for the addition of ±P nutrient solutions and for the collection of root exudates. Citric, malic, and succinic acids were detected in the root exudates of 24-day-old alfalfa seedlings. Citrate exudation from the roots of P-stressed alfalfa was 182% that of plants receiving a complete nutrient solution. The increased release of citrate may provide a mechanism by which P-stressed plants enhance the availability of P in the rhizosphere.     

Natural Occurrence of Enantiomers of Organic Compounds Versus Phytoremediations: Should Research on Phytoremediations Be Revisited? A Mini‐review

Decontamination of polluted soils using plants is based on the ability of plant species (including transgenic plants) to enhance bioavailability of pollutants in the rhizosphere and support growth of pollutant‐degrading microorganisms via root exudation and plant species‐specific composition of the exudates. In this work, we review current knowledge of enantiomers of low‐molecular‐weight (LMW) organic compounds with emphasis on their use in phytoremediation. Many research studies have been performed to search for plants suitable for decontamination of polluted soils. Nevertheless, the natural occurrence of L‐ versus D‐enantiomers of dominant compounds of plant root exudates which play different roles in the complexation of heavy metals, chemoattraction, and support of pollutant‐degrading microorganisms were not included in these studies. D‐enantiomers of aliphatic organic acids and amino acids or L‐enantiomers of carbohydrates occur in high concentrations in root exudates of some plant species, especially under stress, and are less stimulatory for plants to extract heavy metals or for rhizosphere microflora to degrade pollutants compared with L‐enantiomers (organic acids and amino acids) or D‐carbohydrates. Determining the ratio of L‐ versus D‐enantiomers of organic compounds as a criterion of plant suitability for decontamination of polluted soils and development of other types of bioremediation technologies need to be subjects of future research. Chirality 26:1–20, 2013. © 2013 Wiley Periodicals, Inc.     

Root exudates: a pathway for short-term N transfer from clover and ryegrass

The short-term transfer of nitrogen (N) from legumes to grasses was investigated in two laboratory studies. One study was done in pots where the roots of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) were allowed to co-exist, and a second study was performed using a micro-lysimeter system designed to maintain nutrient flow from the clover to the grass, whilst removing direct contact between the root systems. The ¹⁵N-dilution technique was used to quantify the transfer of N between species. Levels of ammonia and amino acids were measured in root exudates. The amounts of N transferred were in the same order of magnitude in both the pot and micro-lysimeter experiments. In the micro-lysimeter experiment, 0.076 mg of N were transferred per plant from clover to ryegrass during the course of the experiment. Ammonium exudation was much higher than amino acid exudation. The most abundant amino acids in both clover and ryegrass root exudates were serine and glycine. However, there was no correlation between the free amino acid profile of root extracts and exudates for both plant species: Asparagine was the major amino acid in clover roots, while glutamine, glutamate and aspartate were the major amino acids in ryegrass roots. Comparison of exudates obtained from plants grown in non-sterile or axenic conditions provides evidence of plant origin of ammonium, serine and glycine.     

Atmospheric CO2 enrichment and drought stress modify root exudation of barley

Rising CO₂ concentrations associated with drought stress is likely to influence not only aboveground growth, but also belowground plant processes. Little is known about root exudation being influenced by elements of climate change. Therefore, this study wanted to clarify whether barley root exudation responds to drought and CO₂ enrichment and whether this reaction differs between an old and a recently released malting barley cultivar. Barley plants were grown in pots filled with sand in controlled climate chambers at ambient (380 ppm) or elevated (550 ppm) atmospheric [CO₂] and a normal or reduced water supply. Root exudation patterns were examined at the stem elongation growth stage and when the inflorescences emerged. At both dates, root exudates were analyzed for different compounds such as total free amino acids, proline, potassium, and some phytohormones. Elevated [CO₂] decreased the concentrations in root exudates of some compounds such as total free amino acids, proline, and abscisic acid. Moreover, reduced water supply increased proline, potassium, electric conductivity, and hormone concentrations. In general, the modern cultivar showed higher concentrations of proline and abscisic acid than the old one, but the cultivars responded differentially under elevated CO₂. Plant developmental stage had also an impact on the root exudation patterns of barley. Generally, we observed significant effects of CO₂ enrichment, watering levels, and, to a lesser extent, cultivar on root exudation. However, we did not find any mitigation of the adverse effects of drought by elevated CO₂. Understanding the multitude of relationships within the rhizosphere is an important aspect that has to be taken into consideration in the context of crop performance and carbon balance under conditions of climate change.     

The role of root exudates and allelochemicals in the rhizosphere

Plant roots serve a multitude of functions in the plant including anchorage, provision of nutrients and water, and production of exudates with growth regulatory properties. The root–soil interface, or rhizosphere, is the site of greatest activity within the soil matrix. Within this matrix, roots affect soil structure, aeration and biological activity as they are the major source of organic inputs into the rhizosphere, and are also responsible for depletion of large supplies of inorganic compounds. Roots are very complicated morphologically and physiologically, and their metabolites are often released in large quantities into the soil rhizosphere from living root hairs or fibrous root systems. Root exudates containing root-specific metabolites have critical ecological impacts on soil macro and microbiota as well as on the whole plant itself. Through the exudation of a wide variety of compounds, roots impact the soil microbial community in their immediate vicinity, influence resistance to pests, support beneficial symbioses, alter the chemical and physical properties of the soil, and inhibit the growth of competing plant species. In this review, we outline recent research on root exudation and the role of allelochemicals in the rhizosphere by studying the case of three plants that have been shown to produce allelopathic root exudates: black walnut, wheat and sorghum     

Quantitative and qualitative effects of phosphorus on extracts and exudates of sudangrass roots in relation to vesicular-arbuscular mycorrhiza formation

A comparison was made of water-soluble root exudates and extracts of Sorghum vulgare Pers. grown under two levels of P nutrition. An increase in P nutrition significantly decreased the concentration of carbohydrates, carboxylic acids, and amino acids in exudates, and decreased the concentration of carboxylic acids in extracts. Higher P did not affect the relative proportions of specific carboxylic acids and had little effect on proportions of specific amino acids in both extracts and exudates. Phosphorus amendment resulted in an increase in the relative proportion of arabinose and a decrease in the proportion of fructose in exudates, but did not have a large effect on the proportion of individual sugars in extracts. The proportions of specific carbohydrates, carboxylic acids, and amino acids varied between exudates and extracts. Therefore, the quantity and composition of root extracts may not be a reliable predictor of the availability of substrate for symbiotic vesicular-arbuscular mycorrhizal fungi. Comparisons of the rate of leakage of compounds from roots with the growth rate of vesicular-arbuscular mycorrhizal fungi suggest that the fungus must either be capable of using a variety of organic substrates for growth, or be capable of inducing a much higher rate of movement of specific organic compounds across root cell membranes than occurs through passive exudation as measured in this study.     

Studies on the rhizosphere mycoflora of broad bean and cotton

Summary Seed and root exudates from three varieties of both broad bean and cotton grown aseptically were analyzed for amino acids and sugars. Broad bean varieties were found to excrete considerable amounts of amino compounds with 15 different amino acids, while cotton varieties excreted less consisting of 12 amino acids. Five sugars were identified in the seed and root exudates from broad bean varieties, while three were present in the seed and root exudates from cotton varieties. Seed and root exudates stimulated spore germination and growth of fungi isolated from the rhizosphere.     

The effect of the phenolic compounds exuded by pea roots in darkness on the reproduction of <Emphasis Type="Italic">Rhizobium</Emphasis>

The exudation, composition, and biological activity of the phenolic compounds (PC) of pea (Pisum sativum L.) roots in the light and darkness were studied. The roots of leguminous plants grown for 5 days in darkness exuded a smaller amount of PC that displayed a weaker stimulation of Rhizobium reproduction. Moreover, the root exudates contained antimicrobial compounds, stilbenes. It is assumed that a lower PC exudation by roots and the specific features of PC composition influencing the biological activity are among the reasons causing a delayed nodulation of legumes grown in darkness.     

Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS

Root exudates released into soil have important functions in mobilizing metal micronutrients and for causing selective enrichment of plant beneficial soil micro-organisms that colonize the rhizosphere. Analysis of plant root exudates typically has involved chromatographic methods that rely on a priori knowledge of which compounds might be present. In the research reported here, the combination of multinuclear and 2-D NMR with GC-MS and high-resolution MS provided de novo identification of a number of components directly in crude root exudates of different plant types. This approach was applied to examine the role of exudate metal ion ligands (MIL) in the acquisition of Cd and transition metals by barley and wheat. The exudation of mugineic acids and malate was enhanced by Fe deficiency. which in turn led to an increase in the tissue content of Cu, Mn, and Zn. The presence of elevated Cd maintained at a free activity pCd of 8.8 (10(-8.8) M), resulted in reduced phytosiderophore production by Fe deficient plants. The buffer morpholinoethane sulfonate (MES), which is commonly used in chelator-buffering nutrient solutions, was detected in the root exudate mixture, suggesting uptake and re-secretion of this compound by the roots. The ability to detect this compound in complex mixtures containing organic acids, amino acids, and other substances suggests that the analytical methods used here provide an unbiased method for simultaneous detection of all major components contained in root exudates.     

Root Secretion of Defense-related Proteins Is Development-dependent and Correlated with Flowering Time

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.     

Effect of Pseudomonas fluorescens on the root exudates of two tomato mutants differently sensitive to Fe chlorosis

Plants with different Fe-mobilization properties are known to differ in the amount and kind of Fe-reducing and Fe-chelating compounds exuded by their roots. Although rhizosphere bacteria are known to affect the exudation of organic compounds by the plant roots, their effect on the root exudates of plants differing in Fe-mobilization properties is not known. We studied the effect of Pseudomonas fluorescens, on the exudation of sugars and organic and amino acids by roots of an iron chlorosis-resistant (T3238FER) and a chlorosis-susceptible (T3238fer) tomato mutant. Under sterile conditions two tomato mutants grew equally well and did not differ in the total amount of sugars and organic acid exuded by their roots. More amino acids, however, were exuded by the roots of T3238FER than T323fer. Mutants differed in the amount of oxalic acid and the amino acids Ala, Asp, Gaba, Gln, Gly, His, Hyl, Ile, Leu, Lys, Phe, Pro, and Val exuded by their roots into sterile rooting media. Addition of P. fluorescens to the rooting medium did not affect the growth of T3238FER but stimulated the root growth of chlorosis-susceptible T3238fer, reduced the amounts of glucose, arabinose and fructose but increased the amount of sucrose, reduced the amounts of fumaric, malic and oxalic acid but increased the amounts of citric and succinic acid in the rooting media of both mutants. P. fluorescens resulted in the following changes in the amino acids in the rooting media: reduced the amounts of Gly, Leu, and Lys in T3238FER, and of Asp, Gln, Hyp, and Ile in T3238fer, and increased the amounts of Cys, Glu, His, Hyp, Ile, Phe and Tyr in T3238FER and of Ala, Glu, His, Phe, and Ser in T323fer—in cases more than 40-fold. These differential effects of P. fluorescens in altering the pattern of organic and amino acids compounds with some Fe-chelating properties detected in the rooting medium of these two mutants may indicate that the differences in Fe-chlorosis susceptibility of these tomato mutants may be the result of, or modified by, the interactions between plant roots and rhizosphere microorganisms. We postulate that the Fe-chlorosis susceptibility in plants may be the product of the interactions between soil microorganisms and plant roots, and may not be solely related to the plant per se.     

Low-level herbivory by root-knot nematodes (Meloidogyne incognita) modifies root hair morphology and rhizodeposition in host plants (Hordeum vulgare)

Low amounts of root infestation by plant parasitic nematodes are suggested to increase nutrient supply and in turn enhance microbial activity and net mineralization rate in the rhizosphere. These effects are generally related to “leakage” of plant-derived metabolites from damaged roots. Besides leakage, the present study examines other nematode–host interactions such as alterations in root exudation and morphology, which were almost not considered yet. This includes undamaged root parts in order to assess systemic plant response. The root-knot nematode Meloidogyne incognita (Kofoid and White 1919; Chitwood 1949) and barley (Hordeum vulgare L. cv. Europa) was used as model system. Host plants were grown in mini-rhizotrons inoculated with 0, 2,000, 4,000 or 8,000 M. incognita for 4 weeks. Root morphology, rhizodeposition (sugars, carboxylates, amino acids), and rhizosphere microbial communities (PLFAs) were assessed. In treatments with 4,000 nematodes, shoot biomass, total N and P content increased by the end of the experiment. Generally, an enhanced release of plant metabolites (sugars, carboxylates, amino acids) from the apical root zone occurred 1 week after inoculation with 4,000 and 8,000 M. incognita, indicating root leakage. Low levels of root herbivory stimulated root hair elongation in both infected and uninfected roots. These systemic changes in root morphology likely contributed to the increased sugar exudation in uninfected roots in all nematode treatments at 3 weeks after inoculation. Root-knots formed a separate microhabitat within the root-system. They were characterised by decreased rhizodeposition and increased fungal to bacterial ratio in the adhering rhizosphere soil. The present study provides the first evidence that, apart from leakage, nematode root herbivory at background levels induces local and systemic effects on root morphology and exudation, which in turn may affect plant performance.     

Root specific elicitation and exudation of fluorescent β-carbolines in transformed root cultures of Oxalis tuberosa

Stable transformation was achieved in oca (Oxalis tuberosa L.) using an Agrobacterium rhizogenes-mediated system. Transformation frequencies varied with the use of different types of strains of A. rhizogenes and the age of explants. The transfer of rol A gene into the oca genome was confirmed by PCR analysis. In vitro transformed root cultures of oca grown in sterile liquid media induced purplish-blue fluorescence of the culture flask medium when irradiated with UV light. We have previously observed a similar phenomenon, the exudation of fluorescent compounds by the roots of in vitro and field-grown oca plants. Hairy root cultures of O. tuberosa transformed with A. rhizogenes (ATCC-15834) exuded constitutive levels of harmine (7-methoxy-1-methyl-β-carboline) and harmaline (3,4-dihydroharmine), the main fluorescent compounds detected from oca’s root exudates. Transformed roots showed better growth and exudation of harmine and harmaline compared to the untransformed normal roots. Upon elicitation with fungal cell wall elicitors from Phytophthora cinnamoni, the production and exudation of harmine/harmaline was enhanced in both transformed and non-transformed roots. Harmine and harmaline showed a wide range of antimicrobial activity against soil-borne microorganisms. Biologically, these findings suggest that in nature β-carbolines are constitutive antimicrobial compounds released into the rhizosphere upon microbial challenge. Transformed root cultures of oca make a simple, reliable and well-defined model system to investigate the molecular and metabolic exudation of fluorescent β-carboline biosynthesis, and to evaluate the biological significance of the phenomenon of root exudation of fluorescent metabolites.     

Exudation of amino acids by intact and damaged roots of wheat and peas

Summary Wheat and pea seedlings were grown aseptically in solution-culture and the total free amino nitrogen released by the roots was determined by a quantitative ninhydrin test. Amino nitrogen from wheat plants after 14 days growth was not detected by the test, indicating the release of less than 3 µg of amino nitrogen from a culture of 15 plants. Pea plants of the same age released from 2 to 7 µg per plant. Paper chromatograms of highly concentrated undisturbed solution-cultures revealed up to 13 amino compounds from wheat and 11 from pea. The pattern of amino acids in exudates was similar to that in crushed roots, except for an unidentified amino compound which was detected only in exuded material. The total amino nitrogen and relative proportions of several amino acids in the root exudates of sand-grown peas was influenced by several ratios of oxygen and carbon dioxide supplied to the root zone. Roots, experimentally damaged by swirling and rinsing in sand, released in 1 hour amino nitrogen of from 73 to 120 per cent of that released by normal exudation over a 2-week period. Our findings suggest that experimental and environmental root damage may be responsible for a large proportion of organic materials released by growing plant roots.Trade names are used in this publication to provide specific information. Their use does not constitute a guarantee of the products named and does not signify that they are approved by the U.S. Department of Agriculture to the exclusion of others of suitable composition.