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Isoxazolin-5-ones and Amino Acids in Root Exudates of Pea and Sweet Pea Seedlings.

Seeds of Pisum sativum L. cv Finale and Lathyrus odoratus L. cv Spencer were germinated aseptically in moistened sand in the dark. At several stages, the amino acid composition of the exudate and of the corresponding roots was analyzed. A number of common amino acids, including homoserine, were exuded by the growing seedling root in an early stage and were partly reabsorbed later. A number of uncommon amino acids, including several isoxazolin-5-one derivatives, uracil alanines, l-gamma-glutamyl-d-alanine, and alpha-aminoadipic acid were exuded at different rates.

Journal Article↗

Isoflavanones from the allelopathic aqueous root exudate of Desmodium uncinatum.

Three isoflavanones, 5,7,2',4'-tetrahydroxy-6-(3-methylbut-2-enyl)isoflavanone (1), 4",5"-dihydro-5,2',4'-trihydroxy-5"-isopropenylfurano-(2",3";7,6)-isoflavanone (2) and 4",5"-dihydro-2'-methoxy-5,4'-dihydroxy-5"-isopropenylfurano-(2",3";7,6)-isoflavanone (3) and a previously known isoflavone 5,7,4'-trihydroxyisoflavone [genistein (4)] were isolated and characterised spectroscopically from the root exudate of the legume Desmodium uncinatum (Jacq.) DC. We propose the names uncinanone A, B, and C for compounds 1, 2 and 3, respectively. Isolated fractions containing uncinanone B (2) induced germination of seeds from the parasitic weed Striga hermonthica (Del.) Benth. and fractions containing uncinanone C (3) moderately inhibited radical growth, the first example of a newly identified potential allelopathic mechanism to prevent S. hermonthica parasitism.

Biological Assay↗

Allelopathic substance in rice root exudates: rediscovery of momilactone B as an allelochemical.

Much research on rice allelopathy has been directed toward the selection of allelopathic rice strains and the identification of allelochemicals in rice. This paper briefly summarizes recent progress in the rice allelopathy and focuses on rediscovery of momilactone B as an allelochemical. A large number of rice varieties were found to inhibit the growth of several plant species when grown together under field and/or laboratory conditions. These findings suggest that rice probably produces and releases allelochemical(s) into the environment. The putative compound causing the inhibitory effect of rice was recently isolated from rice root exudates, and the chemical structure of the inhibitor was determined by spectral data as momilactone B. In addition, it has been found that momilactone B is released from rice roots into the neighboring environment, and the release level of momilactone B from rice may be sufficient to cause growth inhibition of neighboring plants. These findings suggest that momilactone B may play an important role in rice allelopathy.

Agriculture↗

Changes in Cytokinin Activities and Mass Spectrometric Analysis of Cytokinins in Root Exudates of Rice Plant (Oryza sativa L.) : Comparison between Cultivars Nipponbare and Akenohoshi.

Changes in exudation rate and cytokinin activities in the exudates were measured in two varieties of rice (Oryza sativa L.), cv Nipponbare (a Japanese normal cultivar) and cv Akenohoshi (a high-yielding cultivar). The exudation rates of Akenohoshi, the leaves of which remained green for a longer time, were higher than those of Nipponbare after the booting stage. Cytokinin activities in the exudates of Akenohoshi were higher than those of Nipponbare during the ripening period. Cytokinins in the exudates collected during the middle of the ripening stage were analyzed with mass spectrometry using deuterium-labeled standards. trans-Zeatin, trans-ribosylzeatin, and N(6)-isopentenyladenosine were detected as free cytokinins, and zeatin was detected in the hydrolysates of highly polar fractions ("conjugated zeatin") in the exudates of both cultivars. Conjugated zeatin was the predominant cytokinin in both cultivars. Therefore, we suggest that conjugated zeatin is an important form of cytokinin during the ripening stage. The level of each of the cytokinins in Akenohoshi was higher than that in Nipponbare. Also, we discuss the correlation between the leaf senescence and cytokinin content in root exudates.

Journal Article↗

The Rhizobium leguminosarum nodulation gene nodF encodes a polypeptide similar to acyl-carrier protein and is regulated by nodD plus a factor in pea root exudate.

The DNA sequence of approximately 3.5 kb of the nodulation (nod) region of the Rhizobium leguminosarum symbiotic plasmid pRL1JI was determined. Three open reading frames were identified; genes corresponding to these have been called nodD, nodE and nodF.nodD is adjacent to nodA and is transcribed in the opposite direction. The nodF and nodE genes are downstream of, and transcribed in the same direction as, nodD with 667 nucleotides between nodD and nodF and three nucleotides separating nodF and nodE. The induction of the nodFE operon requires the nodD gene product and a component present in plant root exudate. Regions of DNA sequence preceding nodF are similar to those preceding nodA; these sequences may be involved in the regulation of the expression of nodA and nodF. Analysis of nodD revealed an amino acid sequence similar to the predicted DNA-binding domain of known DNA-binding proteins. A protein comparison of the nodF protein showed it to be similar to the acyl-carrier protein from Escherichia coli and barley, especially around the pantothenate-binding region and on this basis it is thought that this protein may be involved in an acyl transfer reaction.

Journal Article↗

Abscisic acid and cytokinins in the root exudates and leaves and their relationship to senescence and remobilization of carbon reserves in rice subjected to water stress during grain filling.

The possible regulation of senescence-initiated remobilization of carbon reserves in rice (Oryza sativa L.) by abscisic acid (ABA) and cytokinins was studied using two rice cultivars with high lodging resistance and slow remobilization. The plants were grown in pots and either well-watered (WW, soil water potential = 0 MPa) or water-stressed (WS, soil water potential = -0.05 MPa) from 9 days after anthesis until they reached maturity. Leaf water potentials of both cultivars markedly decreased at midday as a result of water stress but completely recovered by early morning. Chlorophyll (Chl) and photosynthetic rate (Pr) of the flag leaves declined faster in WS plants than in WW plants, indicating that the water deficit enhanced senescence. Water stress accelerated starch remobilization in the stems, promoted the re-allocation of pre-fixed (14)C from the stems to grains, shortened the grain-filling period and increased the grain-filling rate. Sucrose phosphate synthase (SPS, EC 2.4.1.14) activity was enhanced by water stress and positively correlated with sucrose accumulation in both the stem and leaves. Water stress substantially increased ABA but reduced zeatin (Z) + zeatin riboside (ZR) concentrations in the root exudates and leaves. ABA significantly and negatively, while Z+ZR positively, correlated with Pr and Chl of the flag leaves. ABA, not Z+ZR, was positively and significantly correlated with SPS activity and remobilization of pre-stored carbon. Spraying ABA reduced Chl in the flag leaves, and enhanced SPS activity and remobilization of carbon reserves. Spraying kinetin had the opposite effect. The results suggest that both ABA and cytokinins are involved in controlling plant senescence, and an enhanced carbon remobilization is attributed to an elevated ABA level in rice plants subjected to water stress.

Abscisic Acid↗

The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates.

Arbuscular mycorrhizal (AM) symbiosis is an association between obligate biotrophic fungi and more than 80% of land plants. During the pre-symbiotic phase, the host plant releases critical metabolites necessary to trigger fungal growth and root colonization. We describe the isolation of a semipurified fraction from exudates of carrot hairy roots, highly active on germinating spores of Gigaspora gigantea, G. rosea, and G. margarita. This fraction, isolated on the basis of its activity on hyphal branching, contains a root factor (one or several molecules) that stimulates, directly or indirectly, G. gigantea nuclear division. We demonstrate the presence of this active factor in root exudates of all mycotrophic plant species tested (eight species) but not in those of nonhost plant species (four species). We negatively tested the hypothesis that it was a flavonoid or a compound synthesized via the flavonoid pathway. We propose that this root factor, yet to be chemically characterized, is a key plant signal for the development of AM fungi.

Daucus carota↗

Root exudate of pmi tomato mutant M161 reduces AM fungal proliferation in vitro.

Soluble factors released from roots of the pre-mycorrhizal infection (pmi) myc(-) tomato mutant M161 were analyzed and compared with normal wild-type released factors. Aseptic whole exudates from the M161 mutant retarded the proliferation of Glomus intraradices in vitro. When the whole exudate was further fractionated on a C18 SEPAK cartridge, the 50/70% methanol fraction showed an activity against hyphal tip growth of Gigaspora gigantea and Gl. intraradices. Preliminary characterization of the exudate suggests that the inhibitory moieties are heat labile, bind to PVPP (polyvinyl polypyrrolidone), and are not volatile. This is the first reported instance of the inhibition by a myc(-) plant being ascribed to inhibitory component(s) released in root exudate.

Bacterial Proteins↗

Functional significance of dauciform roots: exudation of carboxylates and acid phosphatase under phosphorus deficiency in Caustis blakei (Cyperaceae).

Caustis blakei produces an intriguing morphological adaptation by inducing dauciform roots in response to phosphorus (P) deficiency. We tested the hypothesis that these hairy, swollen lateral roots play a similar role to cluster roots in the exudation of organic chelators and ectoenzymes known to aid the chemical mobilization of sparingly available soil nutrients, such as P. Dauciform-root development and exudate composition (carboxylates and acid phosphatase activity) were analysed in C. blakei plants grown in nutrient solution under P-starved conditions. The distribution of dauciform roots in the field was determined in relation to soil profile depth and matrix. The percentage of dauciform roots of the entire root mass was greatest at the lowest P concentration ([P]) in solution, and was suppressed with increasing solution [P], while in the field dauciform roots were predominantely located in the upper soil horizons, and decreased with increasing soil depth. Citrate was the major carboxylate released in an exudative burst from mature dauciform roots, which also produced elevated levels of acid phosphatase activity. Malonate was the dominant internal carboxylate present, with the highest concentration in young dauciform roots. The high concentration of carboxylates and phosphatases released from dauciform roots, combined with their prolific distribution in the organic surface layer of nutrient-impoverished soils, provides an ecophysiological advantage for enhancing nutrient acquisition.

Acid Phosphatase↗

Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates.

The distribution of flavonoids, isoflavonoids, and their conjugates in developing soybean (Glycine max L.) seedling organs and in root and seed exudates has been examined. Conjugates of the isoflavones daidzein and genistein are major metabolites in all embryonic organs within the dry seed and in seedling roots, hypocotyl, and cotyledon tissues at all times after germination. Primary leaf tissues undergo a programmed shift from isoflavonoid to flavonoid metabolism 3 days after germination and become largely predominated by glycosides of the flavonols kampferol, quercetin, and isorhamnetin by 5 days. Cotyledons contain relatively constant and very high levels of conjugates of both daidzein and genistein. Hypocotyl tissues contain a third unidentified compound, P19.3, also present in multiple conjugated forms. Conjugates of daidzein, genistein, and P19.3 are at their highest levels in the hypocotyl hook and fall off progressively down the hypocotyl. These isoflavones also undergo a programmed and dramatic decrease between 2 and 4 days in the hypocotyl hook. All root sections are predominated by daidzein and its conjugates, particularly in the root tip, where they reach the highest levels in the seedling. Light has a pronounced effect on the distribution of the isoflavones; in the dark, isoflavone levels in the root tips are greatly reduced, while those in the cotyledons are higher. Finally, the conjugates of daidzein and genistein and several unidentified aromatic metabolites are selectively excreted into root and seed exudates. Analysis of seed exudates suggests that this is a continuous, but saturable event.

Journal Article↗

Alfalfa Root Exudates and Compounds which Promote or Inhibit Induction of Rhizobium meliloti Nodulation Genes.

Using a plate induction assay, we demonstrate that alfalfa exudes inducer of Rhizobium meliloti nodulation genes. The inducer is exuded from the infectible zone of the root, accumulates to at least 1 micromolar, and is not affected by 10 millimolar nitrate. No zones of inhibition are observed. A nodulation minus mutant line of alfalfa, MN-1008, exudes normal levels of inducer. R. meliloti grown in rich medium requires ten-fold higher concentrations of luteolin to achieve half-maximal induction as compared to cells grown in a minimal medium. Flavonoids other than luteolin are found to have activity in R. meliloti nodulation gene induction assays. The compounds apigenin and eriodictyol have activities two-fifths and one-seventh that of luteolin, respectively. Several of the flavonoids tested (morin = naringenin > kaempferol = chrysin > quercetin = fisetin = hesperitin) demonstrate antagonistic activity toward induction by luteolin. The most effective antagonist is the coumarin, umbelliferone.

Journal Article↗

Role of plant root exudate and Sym plasmid-localized nodulation genes in the synthesis by Rhizobium leguminosarum of Tsr factor, which causes thick and short roots on common vetch.

In a previous paper it was shown that cocultivation of Rhizobium leguminosarum with the plant Vicia sativa subsp. nigra on solid medium causes a changed mode of growth of the plant roots, resulting in thick and short roots (Tsr). The Sym plasmid present in the bacterium appeared to be essential for causing Tsr (A. A. N. van Brussel, T. Tak, A. Wetselaar, E. Pees, and C. A. Wijffelman, Plant Sci. Lett. 27:317-325, 1982). In the present paper, we show that a role in causing Tsr is general for Sym plasmids of R. leguminosarum and Rhizobium trifolii. Moreover, mutants with transposon insertions in the Sym plasmid-localized nodulation genes nodA, B, C, and D are unable to cause Tsr, in contrast to nodulation mutants localized in other parts of the Sym plasmid. The observation that Tsr could also be brought about in liquid medium enabled us to show that Tsr is caused by a soluble factor. Experiments in which plants and bacteria were grown separately in the sterile supernatant fluids of each other resulted in establishing the following sequence of events. (i) The plant produces a factor, designated as factor A. (ii) Factor A causes the Sym plasmid-harboring bacteria to produce Tsr factor. (iii) Growth of young plants in the presence of Tsr factor results in the Tsr phenotype. Models explaining this example of molecular signalling between bacteria and plants are discussed.

Phenotype↗

A plasmid of Rhizobium meliloti 41 encodes catabolism of two compounds from root exudate of Calystegium sepium.

Our objectives were to identify substances produced by plant roots that might act as nutritional mediators of specific plant-bacterium relationships and to delineate the bacterial genes responsible for catabolizing these substances. We discovered new compounds, which we call calystegins, that have the characteristics of nutritional mediators. They were detected in only 3 of 105 species of higher plants examined: Calystegia sepium, Convolvulus arvensis (both of the Convolvulaceae family), and Atropa belladonna. Calystegins are abundant in organs in contact with the rhizosphere and are not found, or are observed only in small quantities, in aerial plant parts. Just as the synthesis of calystegins is infrequent in the plant kingdom, their catabolism is rare among rhizosphere bacteria that associate with plants and influence their growth. Of 42 such bacteria tested, only one (Rhizobium meliloti 41) was able to catabolize calystegins and use them as a sole source of carbon and nitrogen. The calystegin catabolism gene(s) (cac) in this strain is located on a self-transmissible plasmid (pRme41a), which is not essential to nitrogen-fixing symbiosis with legumes. We suggest that under natural conditions calystegins provide an exclusive carbon and nitrogen source to rhizosphere bacteria which are able to catabolize these compounds. Calystegins (and the corresponding microbial catabolic genes) might be used to analyze and possibly modify rhizosphere ecology.

Alkaloids↗

Carboxylate composition of root exudates does not relate consistently to a crop species' ability to use phosphorus from aluminium, iron or calcium phosphate sources.

* The relationship between carboxylate release from roots and the ability of the species to utilize phosphorus from sparingly soluble forms was studied by comparing Triticum aestivum, Brassica napus, Cicer arietinum, Pisum sativum, Lupinus albus, Lupinus angustifolius and Lupinus cosentinii. * Plants were grown in sand and supplied with 40 mg P kg(-1) in the sparingly soluble forms AlPO(4), FePO(4) or Ca(5)OH(PO(4))(3), or as soluble KH(2)PO(4); control plants received no P. * The ability to utilize sparingly soluble forms of P differed between forms of P supplied and species. Pisum sativum and C. arietinum did not access AlPO(4) or FePO(4) despite releasing carboxylates into the rhizosphere. * Species accessed different forms of sparingly soluble P, but no species was superior in accessing all forms. We conclude that a single trait cannot explain access to different forms of sparingly soluble P, and hypothesize that in addition to carboxylates, rhizosphere pH and root morphology are key factors.

Aluminum↗

[Allelopathic effects of cultured Cucurbita moschata root exudates].

By using the techniques of tissue culture, bio-assay and laboratory analysis, this paper studied the effects of the allelopathic chemicals from pumpkin (Cucurbita moschata) roots on the seed germination and seedling growth of pumpkin, wheat (Triticum aestivum), and radish (Raphanus sativus). The pumpkin root was cultured on a sterile B5 media, and the concentrations of macro- and microelements, organic supplements and hormones in the media were adjusted by using an orthogonal design. After culturing, the culture media was filtered and used in a bioassay to test the autotoxicity and allelopathic effects. The results showed that the pumpkin had both autotoxic and allelopathic effects, and the media having been used to culture the pumpkin roots contained the chemicals that significantly inhibited the seedling growth of wheat and radish. The allelopathic effect decreased when the culture media was diluted. The production of allelochemicals seemed to be related to the growth rate of the pumpkin roots. When the root growth was rapid, the concentration of allelochemicals was high. The allelopathic effect was stronger on radish than on wheat. The optimum concentrations of macro- and microelements, vitamins and hormones for culturing pumpkin root were determined, and the effect of pumpkin root nutrition on the production of allelochemicals was tested. The results indicated that pumpkin root nutrition had a significant effect on the production of allelochemicals.

Cucurbita↗

Production of recombinant proteins in plant root exudates.

The large-scale production of recombinant proteins in plants is limited by relatively low yields and difficulties in extraction and purification. These problems were addressed by engineering tobacco plants to continuously secrete recombinant proteins from their roots into a simple hydroponic medium. Three heterologous proteins of diverse origins (green fluorescent protein of jellyfish, human placental alkaline phosphatase [SEAP], and bacterial xylanase) were produced using the root secretion method (rhizosecretion). Protein secretion was dependent on the presence of the endoplasmic reticulum signal peptide fused to the recombinant protein sequence. All three secreted proteins retained their biological activity and, as shown for SEAP, accumulated in much higher amounts in the medium than in the root tissue.

Alkaline Phosphatase↗