The role of flavonoids in root-rhizosphere signalling: opportunities and challenges for improving plant-microbe interactions

The flavonoid pathway produces a diverse array of plant compounds with functions in UV protection, as antioxidants, pigments, auxin transport regulators, defence compounds against pathogens and during signalling in symbiosis. This review highlights some of the known function of flavonoids in the rhizosphere, in particular for the interaction of roots with microorganisms. Depending on their structure, flavonoids have been shown to stimulate or inhibit rhizobial nod gene expression, cause chemoattraction of rhizobia towards the root, inhibit root pathogens, stimulate mycorrhizal spore germination and hyphal branching, mediate allelopathic interactions between plants, affect quorum sensing, and chelate soil nutrients. Therefore, the manipulation of the flavonoid pathway to synthesize specifically certain products has been suggested as an avenue to improve root-rhizosphere interactions. Possible strategies to alter flavonoid exudation to the rhizosphere are discussed. Possible challenges in that endeavour include limited knowledge of the mechanisms that regulate flavonoid transport and exudation, unforeseen effects of altering parts of the flavonoid synthesis pathway on fluxes elsewhere in the pathway, spatial heterogeneity of flavonoid exudation along the root, as well as alteration of flavonoid products by microorganisms in the soil. In addition, the overlapping functions of many flavonoids as stimulators of functions in one organism and inhibitors of another suggests caution in attempts to manipulate flavonoid rhizosphere signals.     

Alfalfa Root Flavonoid Production Is Nitrogen Regulated

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.     

根黄酮在植物生长和营养中的作用

 本文简要综述了80年代以来国际上对根黄酮在调节植物根生长、完善根功能、影响氮素循环及在施加他感作用方面的研究进展,并对有关方面作了一些展望,以期引起植物营养工作者对植物次生物质的注意。     

Involvement of a soybean ATP-binding cassette-type transporter in the secretion of genistein, a signal flavonoid in legume-Rhizobium symbiosis

Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells. In this study, we performed biochemical analyses to characterize the transport mechanism of flavonoid secretion using soybean (Glycine max) in which genistein is a signal flavonoid. Plasma membrane vesicles prepared from soybean roots showed clear transport activity of genistein in an ATP-dependent manner. This transport activity was inhibited by sodium orthovanadate, a typical inhibitor of ATP-binding cassette (ABC) transporters, but was hardly affected by various ionophores, such as gramicidin D, nigericin, or valinomycin, suggesting involvement of an ABC transporter in the secretion of flavonoids from soybean roots. The K(m) and V(max) values of this transport were calculated to be 158 mum and 322 pmol mg protein(-1) min(-1), respectively. Competition experiments using various flavonoids of both aglycone and glucoside varieties suggested that this ABC-type transporter recognizes genistein and daidzein, another signaling compound in soybean root exudates, as well as other isoflavonoid aglycones as its substrates. Transport activity was constitutive regardless of the availability of nitrogen nutrition. This is, to our knowledge, the first biochemical characterization of the membrane transport of flavonoid secretion from roots.     

培养基组分对沙打旺(Astragalus adsurgens Pall)组培根增殖的影响及其培养滤液提取物的化感活性

采用L9 (315 )正交设计,研究了B5培养基营养组分对沙打旺组培根增殖的影响;并采用玻璃皿滤纸培养法,对其培养滤液提取物进行生物测定以验证沙打旺组培根的化感活性.结果显示:培养基的所有营养组分中,Fe2 对沙打旺组培根增殖的影响最大,蔗糖、H2PO 4、 Mg2 、 Mn2 、 Cu2 、 Zn2 、 BO3-3、 Co2 、 I-、C8H12ClNO3 C12H18Cl2N4OS C6H5O2N C6H12O6的影响次之,氮、Ca2 、MoO2-4 和NAA的影响最小.根据不同养分条件下沙打旺组培根干重的极差分析,筛选出适宜沙打旺组培根快速增殖的优化培养基.培养滤液提取物的生物测定结果表明沙打旺组培根培养过程中可能产生化感物质;化感作用强度的差异预示营养胁迫可能影响其化感物质的产生.研究为沙打旺组培根再生与繁殖提供一定依据,并揭示养分条件可能是该植物表达化感作用的影响因素.     

南瓜组培根根系分泌物的化感效应研究

使用组织培养技术、生物测试法及室内分析相结合的方法研究了南瓜组培根在不同生长时期与不同营养谐迫下(利用正交设计调节标准B₅培养基中的大量元素、微量元素、有机质和激素)根分泌物对南瓜、萝卜和小麦3种受体的种子萌发及幼苗生长的化感作用.结果表明,南瓜具有自毒作用,南瓜组培根根系分泌物对受体幼苗生长的抑制作用呈现出高抑低促的作用表型;南瓜组培根根系分泌物产生化感作用的活性与南瓜组培根生长速度有关,南瓜组培根根系分泌物的生长抑制活性以15～17d即生长速度指数末期为最高,21d生物量最大时的南瓜组培根根系分泌物的生长抑制活性最低;营养元素的改变明显影响了南瓜根系分泌物的产生,并通过筛选出能诱导南瓜组培根对受体具有强烈抑制作用的大量元素、微量元素、维生素和激素种类和含量的最优化组合,为研究南瓜化感作用营养胁迫机理提供依据.     

Allelopathy in wheat (Triticum aestivum)

Wheat (Triticum aestivum) allelopathy has potential for the management of weeds, pests and diseases. Both wheat residue allelopathy and wheat seedling allelopathy can be exploited for managing weeds, including resistant biotypes. Wheat varieties differ in allelopathic potential against weeds, indicating that selection of allelopathic varieties might be a useful strategy in integrated weed management. Several categories of allelochemicals for wheat allelopathy have been identified, namely, phenolic acids, hydroxamic acids and short‐chain fatty acids. Wheat allelopathic activity is genetically controlled and a multigenic model has been proposed. Research is underway to identify genetic markers associated with wheat allelopathy. Once allelopathic genes have been located, a breeding programme could be initiated to transfer the genes into modern varieties for weed suppression. The negative impacts of wheat autotoxicity on agricultural production systems have also been identified when wheat straws are retained on the soil surface for conservation farming purposes. A management package to avoid such deleterious effects is discussed. Wheat allelopathy requires further study in order to maximise its allelopathic potential for the control of weeds, pests and diseases, and to minimise its detrimental effects on the growth of wheat and other crops.     

Root morphogenesis in legumes and cereals and the effect of bacterial inoculation on root development

Root morphology is both genetically programmed and environmentally determined. We have begun an analysis into the components of root development by: (a) constructing a range of transgenic clover plants to assess some of the genetic programs involved as both roots and nodules are initiated and develop. These transgenic plants report on auxin activity, flavonoid synthesis and chitinase expression and suggest a role for flavonoids as regulators of auxin levels; and (b) determining in cereals the effect of both added auxin and specific microorganisms on the initiation and development of modified root outgrowths and lateral roots. Appropriate combinations of auxin, the nitrogen fixing Acetobacter diazotrophicus, and rice variety did give rise to some plants which grew slowly for over 12 months in a nitrogen-free medium.     

Flavonoid levels in roots ofMedicago sativa are modulated by the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal fungus

Abundant data on the effect of flavonoids on spore germination, hyphal growth and root colonization by AMF are available. Moreover, the flavonoid pattern in mycorrhizal roots changes, thus flavonoids have been suggested as arbuscular mycorrhizal signalling compounds. In our work we studied the accumulation of flavonoids in roots of Medicago sativa i) after the exposure of uncolonized roots to sterile solutions containing Glomus intraradices tissue, ii) at three different stages of colonization by G. mosseae, iii) colonized by G. mosseae, G. intraradices or Gigaspora rosea.

We could show that flavonoid accumulation in M. sativa roots i) is induced before root colonization, pointing towards the presence of a fungal-derived signal, ii) depends on the developmental stage of the symbiosis and iii) depends on the root-colonizing arbuscular mycorrhizal fungus. The data presented indicate not only a time-specificity of the flavonoid accumulation during the mycorrhizal association, but also an arbuscular mycorrhizal fungal-specificity. The possible functions of the flavonoid pattern changes are discussed.     

Differential belowground allelopathic effects of leaf and root of Mikania micrantha

This study evaluates the relative contributions of leaves and roots to the belowground allelopathic effects of Mikania micrantha. The hypothesis that leaves contribute more to the allelopathic effect than roots was experimentally tested. We assessed the allelopathic effects of aqueous extracts from Mikania leaves and roots on the seed germination and seedling growth of two co-occurring woody plants in southern China, Lagerstroemia indica L. and Robinia pseudoacacia L. The results showed that the aqueous extracts from Mikania leaves and roots had inhibitory effects on the woody species. Allelopathic activity depended on the concentration of the extracts, target species, and the extract sources (i.e., leaves vs. roots of Mikania). Leaf extract showed stronger allelopathic effects than root extract on germination percentage, initial germination time, speed of germination, and shoot height; while root extract had greater allelopathic effects on roots than leaf extract. The latter phenomenon might greatly promote the invasion success of Mikania due to more direct and effective allelopathy of root. Our results suggest that allelopathy of root extract on belowground biomass might be greater than that of leaf extract for some species in contrast allelopathy of leaf extract on belowground biomass might also be greater than that of root extract for other species, at least for their effects on root growth of the target species.     

Flavonoid content and antioxidant activity of Artemisia vulgaris L. “hairy” roots

We investigated the effect of Agrobacterium rhizogenes-mediated transformation on antioxidant activity of Artemisia vulgaris “hairy” roots. It appeared that transformation may increase flavonoid content as well as DPPH-scavenging activity and ability to reduce Fe³⁺ as compared to the non-transformed plants. Some “hairy” roots accumulated flavonoids up to 73.1?±?10.6?mg RE/g DW (while the amount of flavonoids in the leaves of non-transformed plants was up to 49.4?±?5.0?mg RE/g DW). DPPH-scavenging activity of some “hairy” root lines was 3–3.8 times higher than such one of the roots of the control plants. The Fe³⁺-reducing power of most transgenic root extracts exceeded such power of the extracts of the roots of the control plants. The decrease in SOD activity was found in the most “hairy” root lines compared to the control roots. The increase of flavonoid content correlated with the increase of ability of extracts to scavenge DPPH*- radical and Fe³⁺ - reducing power. No correlation between SOD activity of extracts and concentration of flavonoids was found (p?≥?0.2).Thus, transformation has led to the alteration in flavonoid accumulation and antioxidant activity in A. vulgaris “hairy” roots. Transgenic roots with high-antioxidant properties can be selected after A. rhizogenes-mediated transformation.     

Facilitation and inhibition: changes in plant nitrogen and secondary metabolites mediate interactions between above-ground and below-ground herbivores

To date, it remains unclear how herbivore-induced changes in plant primary and secondary metabolites impact above-ground and below-ground herbivore interactions. Here, we report effects of above-ground (adult) and below-ground (larval) feeding by Bikasha collaris on nitrogen and secondary chemicals in shoots and roots of Triadica sebifera to explain reciprocal above-ground and below-ground insect interactions. Plants increased root tannins with below-ground herbivory, but above-ground herbivory prevented this increase and larval survival doubled. Above-ground herbivory elevated root nitrogen, probably contributing to increased larval survival. However, plants increased foliar tannins with above-ground herbivory and below-ground herbivory amplified this increase, and adult survival decreased. As either foliar or root tannins increased, foliar flavonoids decreased, suggesting a trade-off between these chemicals. Together, these results show that plant chemicals mediate contrasting effects of conspecific larval and adult insects, whereas insects may take advantage of plant responses to facilitate their offspring performance, which may influence population dynamics.     

Bioactive chemicals and biological—biochemical activities and their functions in rhizospheres of wetland plants

Wetland soils provide anoxia-tolerant plants with access to ample light, water, and nutrients. Intense competition, involving chemical strategies, ensues among the plants. The roots of wetland plants are prime targets for root-eating pests, and the wetland rhizosphere is an ideal environment for many other organisms and communities because it provides water, oxygen, organic food, and physical protection. Consequently, the rhizosphere of wetland plants is densely populated by many specialized organisms, which considerably influence its biogeochemical functioning. The roots protect themselves against pests and control their rhizosphere organisms by bioactive chemicals, which often also have medicinal properties. Anaerobic metabolites, alkaloids, phenolics, terpenoids, and steroids are bioactive chemicals abundant in roots and rhizospheres in wetlands. Bioactivities include allelopathy, growth regulation, extraorganismal enzymatic activities, metal manipulation by phytosiderophores and phytochelatines, various pest-control effects, and poisoning. Complex biological-biochemical interactions among roots, rhizosphere organisms, and the rhizosphere solution determine the overall biogeochemical processes in the wetland rhizosphere and in the vegetated wetlands. To comprehend how wetlands really function, it is necessary to understand these interactions. Such understanding requires further research.     

Flavonoids are differentially taken up and transported long distances in Arabidopsis

Flavonoids are synthesized in response to developmental and environmental signals and perform many functions in plants. Arabidopsis (Arabidopsis thaliana) roots grown in complete darkness do not accumulate flavonoids since the expression of genes encoding enzymes of flavonoid biosynthesis is light dependent. Yet, flavonoids accumulate in root tips of plants with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain, and localized aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating uptake and movement systems. This was confirmed by time-course fluorescence experiments and high-performance liquid chromatography. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application to midroot or cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol, and dihydroquercetin were taken up at the root tip, midroot, or cotyledons and traveled long distances via cell-to-cell movement to distal tissues, followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Using ATP-binding cassette (ABC) transporter and H(+)-ATPase inhibitors suggested that a multidrug resistance-associated protein ABCC transporter facilitated flavonoid movement away from the application site.     

人参皂苷对4种栽培作物早期根系发育的化感效应

人参、西洋参能通过根系分泌三萜皂苷等化感物质,严重影响后茬人参的生长,但对人参以外的植物是否具有化感效应尚不清楚。本实验研究了不同质量浓度的人参皂苷对小麦、白菜、黄瓜及绿豆4种常见栽培作物早期根系发育的影响,结果发现人参皂苷处理液（25、50和100mg·L-1）对4种作物主根及不定根的发育影响不尽相同。随着处理质量浓度的升高,小麦、白菜、黄瓜根系活力分别比同组CK明显降低,根长、根鲜重也呈降低趋势。各浓度人参皂苷处理对黄瓜和绿豆下胚轴不定根的数量、根长、根鲜重及根系活力的影响均未达到显著水平,但二者抗氧化酶的活性都微有升高。总之,人参皂苷对4种栽培作物的主根发育均有抑制作用,尤其对小麦、黄瓜主根生长的抑制作用较强;但对黄瓜和绿豆不定根发育的影响不明显。     

Roles of Allelopathy in Plant Biodiversity and Sustainable Agriculture

Allelopathic compounds are metabolites released from plants that might be beneficial or detrimental to the growth of receptor plants. These compounds are involved in the environmental complex of managed or natural ecosystems. Allelopathic compounds have been shown to play important roles in the determination of plant diversity, dominance, succession, and climax of natural vegetation and in the plant productivity of agroecosystems. The overuse of synthetic agrochemicals often causes environmental hazards, an imbalance of soil microorganisms, nutrient deficiency, and change of soil physicochemical properties, resulting in a decrease of crop productivity. The incorporation of allelopathic substances into agricultural management may reduce the use of synthetic herbicides, fungicides, and insecticides and lessen environmental deterioration. Scientists in many different habitats around the world have demonstrated the above examples previously. It is known that most volatile compounds, such as terpenoids, are released from plants in drought areas. In contrast, water-borne phytotoxins, such as phenolics, flavonoids, or alkaloids, are released from plants in humid zone areas. Both allelopathy and autointoxication play an important mechanism in regulating plant biodiversity and plant productivity. A unique case study of a pasture-forest intercropping system, which is particularly emphasized here, could be used as a model for forest management. After the deforestation of coniferous or hardwood forests, a pasture grass, kikuyu grass (Pennisetum clandestinum), was transplanted onto the land. The grass was quickly established within 6 months. Significant suppression of weed growth by the kikuyu grass was found; however, the growth of coniferous or hardwood plants was not suppressed but stimulated. This example as well as others described in this text clearly indicate that allelopathy plays a significant role in sustainable agriculture. Nevertheless, room for allelopathic research in the next century is available for biologists, biochemists, biotechnologists, and chemists. Future allelopathic research should focus on the following tasks: (1) a continuous survey of potential allelochemicals from natural vegetation or microorganisms, (2) the establishment of practical ways of using allelochemicals in the field, (3) to understand the mode of action of allelopathic chemicals in receptor organisms, (4) to understand the role of allelopathic chemicals in biodiversity and ecosystem function, (5) to explore advanced biotechnology for allocating allelopathic chemical genes in plants or microorganisms for biological control, and (6) to challenge the natural product chemists to develop a better methodology for isolating allelopathic compounds or their degraded compounds from the environment, particularly the soil environment.     

人参皂苷对4种栽培作物早期根系发育的化感效应

人参、西洋参能通过根系分泌三萜皂苷等化感物质,严重影响后茬人参的生长,但对人参以外的植物是否具有化感效应尚不清楚.本实验研究了不同质量浓度的人参皂苷对小麦、白菜、黄瓜及绿豆4种常见栽培作物早期根系发育的影响,结果发现人参皂苷处理液(25、50和100 mg· L-1)对4种作物主根及不定根的发育影响不尽相同.随着处理质量浓度的升高,小麦、白菜、黄瓜根系活力分别比同组CK明显降低,根长、根鲜重也呈降低趋势.各浓度人参皂苷处理对黄瓜和绿豆下胚轴不定根的数量、根长、根鲜重及根系活力的影响均未达到显著水平,但二者抗氧化酶的活性都微有升高.总之,人参皂苷对4种栽培作物的主根发育均有抑制作用,尤其对小麦、黄瓜主根生长的抑制作用较强;但对黄瓜和绿豆不定根发育的影响不明显.