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Role of root exudates in metal acquisition and tolerance
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ScienceDirect Role of root exudates in metal acquisition and tolerance Yi-Tze Chen, Ying Wang and Kuo-Chen Yeh Plants acquire mineral nutrients mostly through the rhizosphere; they secrete a large number of metabolites into the rhizosphere to regulate nutrient availability and to detoxify undesirable metal pollutants in soils. The secreted metabolites are inorganic ions, gaseous molecules, and mainly carbonbased compounds. This review focuses on the mechanisms and regulation of low-molecular-weight organic-compound exudation in terms of metal acquisition. We summarize findings on riboflavin/phenolic-facilitated and phytosiderophorefacilitated iron acquisition and discuss recent studies of the functions and secretion mechanisms of low-molecular-weight organic acids in heavy-metal detoxification. Address Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
I. Iron (Fe) acquisition Although Fe is highly abundant in the soil, its bioavailability is usually low at neutral or high pH because of the formation of low-soluble Fe(III) oxyhydrates [6]. Under low Fe availability, non-graminaceous plants use reduction-based Strategy-I Fe acquisition in a three-step process: Fe solubilization promoted by rhizosphere acidification, reduction of Fe(III), and Fe(II) uptake into root cells [7,8]. Upon soil acidification, free Fe released from Fe(III)-oxides can enter the root apoplasts for further reduction and transportation, but Fe uptake processes are greatly facilitated when Fe chelators are present [8]. Strategy-I plants under Fe-deficiency stress accumulate and secrete low-molecular-weight organic compounds such as flavins, phenolics, and organic acids. [9–11].
Corresponding author: Yeh, Kuo-Chen ([email protected])
Current Opinion in Plant Biology 2017, 39:66–72 This review comes from a themed issue on Cell signalling and gene regulation Edited by Tzyy-Jen Chiou and Toru Fujiwara
http://dx.doi.org/10.1016/j.pbi.2017.06.004 1369-5266/# 2017 Published by Elsevier Ltd.
Introduction Plants acquire a large number of mineral nutrients from the soil through the rhizosphere, the interface of the root and soil. To manage nutrient bio-availability and cope with environmental metal stresses, plants secrete numerous metabolites from roots into the rhizosphere to change the pH or to form metal–metabolite complexes [1,2]. The secreted metabolites are a complex mixture of inorganic ions (i.e., H+, HCO3), gaseous molecules (i.e., CO2, H2) and mainly carbon-based compounds, divided into two groups: low-molecular-weight compounds including amino acids, organic acids, phenolics and sugar and highmolecular-weight compounds including mucilage and proteins [2,3]. Under low-nutrient conditions, plants release certain metabolites to increase nutrient availability by directly binding to mineral nutrients or by changing the rhizosphere pH [4]. In heavy-metal-polluted environments, root exudation can be enhanced by non-essential metal stress to increase external detoxification [5]. Current Opinion in Plant Biology 2017, 39:66–72
Riboflavin and Fe acquisition
Riboflavin (Rbfl) was first found to be secreted from Fedeficient tobacco roots in the early 1960s, then several dicotyledonous species such as Beta vulgaris L. (sugar beet) and Hyoscyamus albus were found to secrete Rbfl when grown under Fe-deficient conditions [12,13]. Fedeficient sugar beet secretes 3’-sulfate and 5’-sulfate derivatives of Rbfl, and Medicago truncatula secretes 7hydroxy-Rbfl and 7a-hydroxy-Rbfl and 7-carboxy-Rbfl. Under Fe-deficiency, both species accumulate flavins in roots when grown at high pH, but preferentially secrete these compounds at low external pH [10,14,15]. Transcriptomic and proteomic studies revealed Rbfl biosynthesis regulated by Fe status in the species. Two key enzymes required for flavin biosynthesis, dimethyl-8ribityllumazine synthase (DMRL) and GTP cyclohydrolase II (GTPcII), were absent in Fe-sufficient roots, but de novo accumulation of DMRL was detected with Fe deficiency with or without CaCO3 (alkaline condition) treatment and GTPcII was detected with Fe deficiency plus CaCO3 treatment. The expression of these enzymes was upregulated under Fe deficiency [14,16,17,18]. Ectopically overexpressing Arabidopsis basic helix– loop–helix38 (bHLH38) and bHLH39 transcription factors in tobacco and sunflower increased Rbfl accumulation and exudation, suggesting that Rbfl synthesis and secretion is regulated by the bHLH transcription-factor network [19]. Although the exact role of secreted flavins in facilitating Fe acquisition remains unclear, they may function as a redox bridge to facilitate ferric-chelate reductase activity or mediate dissolution of low-soluble Fe(III) oxides by complexing extracellular Fe or reductive mechanisms or modifying the rhizosphere microme [20–23]. www.sciencedirect.com
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Phenolic compounds and Fe acquisition
Secretion of phenolic compounds is one of the important mechanisms in Strategy-I Fe acquisition [24]. The first clear evidence of the important role of root-secreted phenolics in response to Fe deficiency was from red clover (Trifolium pretence) [25,26], and the secretion of phenolics was stimulated by Fe deficiency. When secreted phenolics were removed from the nutrient solution, plants showed severe Fe-deficient phenotypes, including reduced shoot biomass and greatly reduced leaf-chlorophyll synthesis. Moreover, exogenously applied phenolics could enhance reutilization of apoplast Fe in red clover roots [25] suggesting that phenolics play roles in Fe uptake.
F60 H1 and ABCG37 are upregulated by Fe deficiency [28,37,38]. The fer-like iron deficiency-induced transcription factor ( fit) mutant showed no Fe-deficiencyresponsive stimulation of F60 H1 transcription or accumulation of fluorescent phenolics, so the transcription of F60 H1 may be regulated by the FIT-bHLH transcription factor regulatory network [31]. Genes in the phenylpropanoid pathway are upregulated by Fe deficiency in A. thaliana but are unchanged or downregulated in M. truncatula, whereas Rbfl synthesis is activated in M. truncatula but not A. thaliana. Use of Rbfl or coumarin secretion may depend on the species and be mutually exclusive [17]. Phytosiderophores
In Brassica napus and Arabidopsis thaliana, secretion of the phenolic compound coumarin is crucial for Fe acquisition from alkaline substrates [27]. Low-Fe growth conditions induced synthesis and secretion of coumarin in their roots [28]. Phenolics found in the exudates of Fe-starved A. thaliana are coumarinolignans (cleomasicosins A, B, C, and D; 50 hydroxycleomiscosins A and/or B), coumarin precursors (ferulic acid, coniferyl aldehyde, and sinapyl aldehyde), and coumarins (scopoletin, scopolin, esculetin, esculin, fraxetin, isofraxidin, and fraxinol). The most prominent coumarins secreted are scopoletin and scopolin [28,29]. Fe deficiency induces coumarinolignan, coumarin, and ferulic-acid accumulation in A. thaliana roots and secretion into nutrient solution at high pH [29,30,31]. Scopoletin is the major coumarin found in root exudates, but fraxetin is the most effective coumarin mobilizing insobluble Fe(III) [29]. Coumarins can chelate and reduce Fe(III) [32]. Fraxetin was reported to mobilize Fe(III)-oxides by complexing with Fe(III) and reducing Fe(III) to Fe(II) [29]. However, phenolics were not able to complement mutation of ferric reduction oxidase 2 ( fro2) [33]. Whether phenolics facilitate Fe uptake through reduction mechanism is still debatable. Coumarins are derived from the phenylpropanoid pathway and their biosynthesis depends on key enzymes encoded by CYP98A3, caffeoyl CoA O-methyltransferase1 (CCoAOMT1), and F60 H1 (Feruloyl-CoA 60 -hydroxylase1) genes. Production of scopoletin and scopolin was greatly reduced with mutated CYP98A3, CCoAOMT1 or F60 H1 [34,35]. Coumarin secretion requires a functional ABCG37 transporter (also called pleiotropic drug resistance9; PDR9), a member of the ATP-binding cassette family. The pdr9 mutants were still able to synthesize and accumulate abundant coumarins in roots, but they barely secreted scopoletin and derivatives [28]. The acquisition of mobilized Fe by ABCG37-mediated coumarin secretion depends on the FRO2/IRT1 (Iron Regulated Transporter 1) transport system [33]. Transcriptome and proteome analyses of Arabidopsis roots revealed induction of the phenylpropanoid pathway in Fe-deficient roots [17,36,37]. Transcript and/or protein levels of www.sciencedirect.com
Graminaceous plants use a chelation-based Strategy-II Fe acquisition that involves secretion of Fe(III) chelators called phytosiderophores into the root rhizosphere [39]. Phytosiderophores are non-protein amino acids that belong to the mugineic acid (MA) family. MAs are synthesized from S-adenosyl-L-methonine via an enzymatic cascade pathway involving three essential enzymes: nicotianamine (NA) synthase for producing NA, NA aminotransferase, and deoxymugineic acid synthase for producing MA. Genes involved in MA synthesis have been isolated from barley, rice, and maize. Their expression is strongly induced by Fe deficiency [40–45]. Secretion of MAs follows a diurnal rhythm [46]. The secretion patterns were found regulated by light intensity or temperature in some graminaceous species, with exudation peak time varying among species [46–49]. Secretion of phytosiderophores is mediated by transporter of mugineic acid1 (TOM1) in rice, barley, and maize [50,51]. The secreted phytosiderophores can chelate and mobilize Fe(III). The resulting Fe(III)–phytosiderophore complexes are transported into the root by plasma membrane-localized yellow stripe1 (YS1) transporters in maize and barley and YS1-like transporters in rice [52–54].
II. Heavy-metal tolerance and low-molecularweight organic acids In addition to their essential roles in improving plant nutrient acquisition, root-secreted exudates are important for heavy-metal tolerance in plants. With industrial human activities and sewage irrigation, the greatly increased heavy-metal pollution of soils has become a severe environmental problem. Toxic-element pollutants and elevated levels of essential elements can cause toxicity in plants. Plants use several strategies including an exclusion mechanism to exclude metal ions from entering root cells by secreting metabolites into the rhizosphere [55]. Exudation of low-molecular-weight organic acids (LMWOAs) has been studied for their important roles in cadmium (Cd), aluminum (Al), gallium (Ga), copper (Cu), manganese (Mn), and lead (Pb) detoxification Current Opinion in Plant Biology 2017, 39:66–72
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[55–59,60]. LMWOAs function as metal chelators. LMWOA secretion types and patterns vary by plant species and physiological conditions and soil environment [61]. The most well-documented LMWOA-mediated detoxification mechanism is Al exclusion, which limits harmful Al 3+ taken up by plant roots [5,62]. Al and Ga detoxification
Al is the most abundant metal in the Earth’s crust, and Al toxicity is a major factor limiting agricultural production on acidic soil, which represents about 40% of arable lands worldwide [63]. Several Al-tolerant plant species can secrete organic acids from the root apex in response to Al stress and form stable harmless complexes with Al3+ to detoxify Al3+ in the rhizosphere [64]. The types of Alinduced organic-acid secretion differ by species. For example, Triticum aestivum (wheat) and A. thaliana secrete malate and citrate, Zea mays (maize) and Glycine max (soybean) mainly secrete citrate, and Fagopyrum esculentum (buckwheat) secretes oxalate [59,65–70]. Citrate was found the most effective detoxifying compound because of strong Al3+-chelating capability [5]. Organic-acid secretion in these species follows pattern I, with immediate organic-acid secretion upon Al3+ exposure, or pattern II, with release of organic acids after Al3+ treatment delayed for several hours [71]. Secretion of malate and citrate is mediated by the Al-associated efflux transporters aluminum-activated malate transporter1 (ALMT1) and multidrug and toxin efflux (MATE), respectively. ALMT1 was first isolated in wheat and later reported in A. thaliana, Secale cereale L (rye), and Brassica napus (rapeseed) [72,73–75]. Heterologous expression of TaALMT1 in barley and rice increased malate efflux and enhanced tolerance to Al3+, for an important role for the malate transporter in crop tolerance to Al stress [72,76]. Citrate transporters are members of the MATE protein family. The expression of MATE genes was found highly correlated with Al-activated citrate secretion, leading to Al3+ tolerance in many species [70,77,78–80]. Citrate transporter OsFRDL4 (AtFRD3-like 4) is an essential Altolerance component in rice. OsFRDL4 expression remained low in roots in the absence of Al but was highly induced with Al treatment. The plasma membrane-localized OsFRDL4 protein showed citrate efflux activity. Knockout of OsFRDL4 reduced citrate efflux and aggravated Al-induced growth inhibition [80]. In Arabidopsis, AtALMT1 and AtMATE both function in Al tolerance, but AtALMT1-mediated malate efflux is the major contributor [78]. Although oxalate secretion is associated with Al tolerance, transporters responsible for Al-regulated oxalate secretion and mechanisms regulating the secretion have yet to be identified. Ga is a non-essential element for living organisms, and Ga pollutants are largely produced by the semiconductor industry, which might threaten the environment and Current Opinion in Plant Biology 2017, 39:66–72
human health. Intriguingly, the Ga-stress-triggered responses in Arabidopsis are similar to those triggered by Al stress. Similar to Al detoxification, Arabidopsis detoxifies Ga externally by secreting citrate and malate potentially by the formation of nontoxic Ga(III)-organicacid complexes in the rhizosphere or apoplastic space, Gainduced secretion of organic acids and the expression of both AtALMT1 and AtMATE transporters. Furthermore, exogenous application of citrate alleviated Ga toxicity in Arabidopsis seedlings [60]. Zn/Cd detoxification
Root exudation is potentially involved in Zn accumulation in the Zn/Cd hyperaccumulator Arabidopsis halleri and in Cd accumulation in the hyperaccumulator ecotype (HE) of Sedum alfredii. Exudation of NA, the intermediate component in phytosiderophore biosynthesis, was greater from roots of A. halleri than the non-accumulator A. thaliana, and secretion was induced by excess Zn. Secreted NA is a major metal chelator that forms stable complexes with Zn to prevent rapid Zn uptake into roots and therefore alleviates Zn toxicity to roots [81]. The HE Sedum alfredii secreted more dissolved organic carbon (DOC)/LMWOAs, especially Cd-induced oxalate, than the non-hyperaccumulator ecotype (NHE) [82,83]. The dissolved organic matter (DOM) in exudate had higher Zn, Cd, and Pb complexation and extraction activity in the HE than NHE [82,84]. With the high abundance and high activity of DOM, the HE could accumulate more Zn/Cd via formation of soluble DOM–metal complexes in soil than the NHE [85] and grow normally in highly polluted environment as compared with the inhibited growth of the NHE [82,83,85]. A role for LMWOA secretion in Cd resistance was also found in non-hyperaccumulator plant species. Certain cultivars of Lycopersicon esulentum (tomato) were found to secrete oxalate from the root apex in response to Cd stress. The secreted organic acids prevented Cd from entering root cells and therefore led to Cd resistance in plants [55].
Conclusion Plants use root exudation to adapt to various soil environments (Figure 1). The root-secreted compounds can alter the soil environment for a more beneficial condition for plant growth and development. Findings from numerous studies have provided much knowledge of the molecular mechanisms and regulation of root exudation in terms of metal acquisition. Root exudation facilitating Fe acquisition is the most well-documented mechanism. Graminaceous plants secrete phytosiderophores and non-graminaceous plants secrete Rbfls and phenolics to enhance Fe acquisition from soil. Although Rbfls and phenolics have a known role in facilitating Fe uptake, their exact biochemical roles are still not fully understood. Enhanced Fe uptake by these secreted compounds involves chelation and reduction processes [26,27]. However, further investigation is needed to www.sciencedirect.com
Plant root secretion Chen, Wang and Yeh 69
Figure 1
Fe deficiency Transporter for secretion Extudate
Possible mechanism
ABCG37 PDR9
Rbfl
Rbfl-Fe ? Fe(lI)
Fe(III)
Excess metal TOM1
Phe
Phe-Fe ? Fe(lI)
ALMT
PS
PS-Fe
Fe(III)
MATE OsFRDL4
Malate
Citrate
OA-AI OA-Ga
OA-AI OA-Ga
Microbes
Enhance Fe uptake
Species
N. Tabacum H. albus B. vulgaris
T. pretence B. napus A. thaliana
Oxalate
NA
OA-AI OA-Zn OA-Cd OA-Pb
NA-Zn
Prevent rapid metal uptake (detoxification)
O. sativa H. vulgare Z. mays
T. aestivum A. thaliana
O. sativa A. thaliana Z. mays G. max
F. esculentum T. turgidum L. esulentum S. alfredii (HE)
A. halleri (HE)
Current Opinion in Plant Biology
Summary of root exudates in iron acquisition and metal detoxification. Iron deficiency enhances secretion of riboflavin (Rbfl), phenolic compounds (Phe) and phytosiderophores (PS) from roots. These exudates may facilitate iron uptake by complexing Fe, increasing ferric chelate reductase activity or modifying rhizosphere microme. On the other side, secretion of organic acids (OA, such as malate, citrate, oxalate) and nicotianamine (NA) are induced by excess metals (Al, Ga, Zn, Cd or Pb). OA and NA may play as chelators which form stable complexes with metals to prevent rapid uptake and therefore alleviated toxicity to roots. Exudates found in different species mentioned in this review are listed in lower panel. Full name of each species: N. tabacum, Nicotiana tabacum; H. albus, Hyoscyamus albus; B. vulgaris, Beta vulgaris (sugar beet); T. pretence, Trifolium pretence (red clover); B. napus, Brassica napus; A. thaliana, Arabidopsis thaliana; O. sativa, Oryza sativa (rice); H. vulgare, Hordeum, vulgare (barley); Z. mays, Zea mays (maize); T. aestivum, Triticum aestivum (wheat); G. max, Glycine max (soybean); F. esculentum, Fagopyrum esculentum (buckwheat); T. turgidum, Triticum turgidum var. durum L. (durum wheat); L. esulentum, Lycopersicon esulentum (tomato); S. alfredii, Sedum alfredii; A. halleri, Arabidopsis halleri; HE, hyperaccumulator ecotype.
unravel how these compounds work with other components such as IRT1 and FRO2 to orchestrate the acquisition process.
Acknowledgements
In recent years, the beneficial role of root exudation in metal acquisition has been exploited for biofortification and phytoremediation. Several transgenic rice lines overexpressing Fe-deficiency-responsive genes were generated to increase Fe acquisition [86–88]. The intercropping system is used to improve crop nutrient acquisition or to remediate heavy-metal pollution. Intercropping strategy-I with strategy-II crops or intercropping crops with metal hyperaccumulators can enhance nutrient uptake in crops, remediate farmland and result in safer agriculture products [89,90]. Therefore, a better understanding of the comprehensive root-exudation mechanism will be advantageous for agricultural and environmental development.
References and recommended reading
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This work was financially supported by Academia Sinica and Ministry of Science and Technology, Taiwan.
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51. Nozoye T, Nakanishi H, Nishizawa NK: Characterizing the crucial components of iron homeostasis in the maize mutants ys1 and ys3. PLoS One 2013, 8:pe62567. 52. Curie C et al.: Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 2001, 409:346349. 53. Murata Y et al.: A specific transporter for iron(III)phytosiderophore in barley roots. Plant J 2006, 46:563-572. 54. Inoue H et al.: Rice OsYSL15 is an iron-regulated iron(III)deoxymugineic acid transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings. J Biol Chem 2009, 284:3470-3479. 55. Zhu XF et al.: Cadmium-induced oxalate secretion from root apex is associated with cadmium exclusion and resistance in Lycopersicon esulentum. Plant Cell Environ 2011, 34:1055-1064. 56. Meier S et al.: Influence of copper on root exudate patterns in some metallophytes and agricultural plants. Ecotoxicol Environ Safety 2012, 75:8-15. 57. Yang YY et al.: Identification of rice varieties with high tolerance or sensitivity to lead and characterization of the mechanism of tolerance. Plant Physiol 2000, 124:1019-1026. 58. Mora MD et al.: Differential tolerance to Mn toxicity in perennial ryegrass genotypes: involvement of antioxidative enzymes and root exudation of carboxylates. Plant Soil 2009, 320:79-89. 59. Zheng SJ, Ma JF, Matsumoto H: High aluminum resistance in buckwheat — I. Al-induced specific secretion of oxalic acid from root tips. Plant Physiol 1998, 117:745-751. 60. Chang HF, Wang SL, Yeh KC: Effect of gallium exposure in Arabidopsis thaliana is similar to aluminum stress. Environ Sci Technol 2017, 51:1241-1248. The authors revealed that the Ga-stress-triggered responses in Arabidopsis are similar to those triggered by Al stress. Arabidopsis utilize exudation of organic acids to detoxify Ga externally in the rhizosphere. The authors also showed that excess Ga is accumulated and precipitated as the form of Ga-rich granular structures in the intercellular spaces, which is suggested as one of the main Ga detoxification mechanisms. 61. Agnello AC et al.: Enhanced phytoremediation: a review of low molecular weight organic acids and surfactants used as amendments. Crit Rev Environ Sci Technol 2014, 44:2531-2576. 62. Delhaize E, Ma JF, Ryan PR: Transcriptional regulation of aluminium tolerance genes. Trends Plant Sci 2012, 17:341-348. 63. Kochian LV: Cellular mechanisms of aluminum toxicity and resistance in plants. Ann Rev Plant Physiol Plant Mol Biol 1995, 46:237-260. www.sciencedirect.com
70. Ryan PR et al.: A second mechanism for aluminum resistance in wheat relies on the constitutive efflux of citrate from roots. Plant Physiol 2009, 149:340-351. 71. Ma JF, Furukawa J: Recent progress in the research of external Al detoxification in higher plants: a mini review. J Inorg Biochem 2003, 97:46-51. 72. Sasaki T et al.: A wheat gene encoding an aluminum-activated malate transporter. Plant J 2004, 37:645-653. The study identified Al-activated malate transporter ALMT1 in wheat. ALMT1 expression co-segregates with Al tolerance. Heterologous expression of ALMT1 in rice and cultured tobacco cells increased malate efflux and enhanced tolerance to Al. These findings suggest an important role for the malate transporter in Al tolerance. 73. Hoekenga OA et al.: AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. Proc Natl Acad Sci U S A 2006, 103:9738-9743. 74. Ligaba A, Matsumoto H, Katsuhara M: Cloning and functional analysis of an aluminum-induces malate transporter gene from rape (Brassica napus). Plant Cell Physiol 2006, 47:S121-S121. 75. Collins NC et al.: An ALMT1 gene cluster controlling aluminum tolerance at the Alt4 locus of rye (Secale cereale L.). Genetics 2008, 179:669-682. 76. Delhaize E et al.: Engineering high-level aluminum tolerance in barley with the ALMT1 gene. Proc Natl Acad Sci U S A 2004, 101:15249-15254. 77. Magalhaes JV et al.: A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nat Genet 2007, 39:1156-1161. The study identified the gene encoding Al-activated citrate transporter, MATE, in sorghum (Sorghum bicolor). This gene plays a major role in sorghum Al tolerance. The Al-induced expression of SbMATE is associated with elevated citrate exudation, which results in enhanced Al tolerance. 78. Liu JP et al.: Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance. Plant J 2009, 57:389-399. 79. Yang XY et al.: A de novo synthesis citrate transporter. Vigna umbellata multidrug and toxic compound extrusion, implicates in Al-activated citrate efflux in rice bean (Vigna umbellata) root apex. Plant Cell Environ 2011, 34:2138-2148. 80. Yokosho K, Yamaji N, Ma JF: An Al-inducible MATE gene is involved in external detoxification of Al in rice. Plant J 2011, 68:1061-1069. 81. Tsednee M et al.: Root-secreted nicotianamine from Arabidopsis halleri facilitates zinc hypertolerance by regulating zinc bioavailability. Plant Physiol 2014, 166:839U553. Current Opinion in Plant Biology 2017, 39:66–72
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This study demonstrated that Zn-induced secretion of nicotianamine (NA) is an important mechanism for Zn hypertolerance in hyperaccumulator Arabidopsis halleri. The secreted NA forms a stable complex with Zn(II) to regulate Zn uptake rate into A. halleri roots. Under excess Zn, secretion of NA is also important for the maintenance of Fe homeostasis. 82. Li T et al.: Complexation with dissolved organic matter and mobility control of heavy metals in the rhizosphere of hyperaccumulator Sedum alfredii. Environ Pollut 2013, 182:248255.
86. Takahashi M et al.: Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes. Nat Biotechnol 2001, 19:466-469. 87. Ishimaru Y et al.: Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil. Proc Natl Acad Sci U S A 2007, 104:7373-7378.
83. Tao Q et al.: Oxalate secretion from the root apex of Sedum alfredii contributes to hyperaccumulation of Cd. Plant Soil 2016, 398:139-152.
88. Banakar R et al.: The expression of heterologous Fe (III) phytosiderophore transporter HvYS1 in rice increases Fe uptake, translocation and seed loading and excludes heavy metals by selective Fe transport. Plant Biotechnol J 2017, 15:423-432.
84. Li TQ et al.: Rhizosphere characteristics of zinc hyperaccumulator Sedum alfredii involved in zinc accumulation. J Hazard Mater 2011, 185:818-823.
89. Xiong HC et al.: Molecular evidence for phytosiderophoreinduced improvement of iron nutrition of peanut intercropped with maize in calcareous soil. Plant Cell Environ 2013, 36:1888-1902.
85. Li TQ et al.: Effects of dissolved organic matter from the rhizosphere of the hyperaccumulator Sedum alfredii on sorption of zinc and cadmium by different soils. J Hazard Mater 2011, 192:1616-1622.
90. Zhan FD et al.: Cadmium and lead accumulation and lowmolecular-weight organic acids secreted by roots in an intercropping of a cadmium accumulator Sonchus asper L. with Vicia faba L.. RSC Adv 2016, 6:33240-33248.
Current Opinion in Plant Biology 2017, 39:66–72
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ScienceDirect Role of root exudates in metal acquisition and tolerance Yi-Tze Chen, Ying Wang and Kuo-Chen Yeh Plants acquire mineral nutrients mostly through the rhizosphere; they secrete a large number of metabolites into the rhizosphere to regulate nutrient availability and to detoxify undesirable metal pollutants in soils. The secreted metabolites are inorganic ions, gaseous molecules, and mainly carbonbased compounds. This review focuses on the mechanisms and regulation of low-molecular-weight organic-compound exudation in terms of metal acquisition. We summarize findings on riboflavin/phenolic-facilitated and phytosiderophorefacilitated iron acquisition and discuss recent studies of the functions and secretion mechanisms of low-molecular-weight organic acids in heavy-metal detoxification. Address Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
I. Iron (Fe) acquisition Although Fe is highly abundant in the soil, its bioavailability is usually low at neutral or high pH because of the formation of low-soluble Fe(III) oxyhydrates [6]. Under low Fe availability, non-graminaceous plants use reduction-based Strategy-I Fe acquisition in a three-step process: Fe solubilization promoted by rhizosphere acidification, reduction of Fe(III), and Fe(II) uptake into root cells [7,8]. Upon soil acidification, free Fe released from Fe(III)-oxides can enter the root apoplasts for further reduction and transportation, but Fe uptake processes are greatly facilitated when Fe chelators are present [8]. Strategy-I plants under Fe-deficiency stress accumulate and secrete low-molecular-weight organic compounds such as flavins, phenolics, and organic acids. [9–11].
Corresponding author: Yeh, Kuo-Chen ([email protected])
Current Opinion in Plant Biology 2017, 39:66–72 This review comes from a themed issue on Cell signalling and gene regulation Edited by Tzyy-Jen Chiou and Toru Fujiwara
http://dx.doi.org/10.1016/j.pbi.2017.06.004 1369-5266/# 2017 Published by Elsevier Ltd.
Introduction Plants acquire a large number of mineral nutrients from the soil through the rhizosphere, the interface of the root and soil. To manage nutrient bio-availability and cope with environmental metal stresses, plants secrete numerous metabolites from roots into the rhizosphere to change the pH or to form metal–metabolite complexes [1,2]. The secreted metabolites are a complex mixture of inorganic ions (i.e., H+, HCO3), gaseous molecules (i.e., CO2, H2) and mainly carbon-based compounds, divided into two groups: low-molecular-weight compounds including amino acids, organic acids, phenolics and sugar and highmolecular-weight compounds including mucilage and proteins [2,3]. Under low-nutrient conditions, plants release certain metabolites to increase nutrient availability by directly binding to mineral nutrients or by changing the rhizosphere pH [4]. In heavy-metal-polluted environments, root exudation can be enhanced by non-essential metal stress to increase external detoxification [5]. Current Opinion in Plant Biology 2017, 39:66–72
Riboflavin and Fe acquisition
Riboflavin (Rbfl) was first found to be secreted from Fedeficient tobacco roots in the early 1960s, then several dicotyledonous species such as Beta vulgaris L. (sugar beet) and Hyoscyamus albus were found to secrete Rbfl when grown under Fe-deficient conditions [12,13]. Fedeficient sugar beet secretes 3’-sulfate and 5’-sulfate derivatives of Rbfl, and Medicago truncatula secretes 7hydroxy-Rbfl and 7a-hydroxy-Rbfl and 7-carboxy-Rbfl. Under Fe-deficiency, both species accumulate flavins in roots when grown at high pH, but preferentially secrete these compounds at low external pH [10,14,15]. Transcriptomic and proteomic studies revealed Rbfl biosynthesis regulated by Fe status in the species. Two key enzymes required for flavin biosynthesis, dimethyl-8ribityllumazine synthase (DMRL) and GTP cyclohydrolase II (GTPcII), were absent in Fe-sufficient roots, but de novo accumulation of DMRL was detected with Fe deficiency with or without CaCO3 (alkaline condition) treatment and GTPcII was detected with Fe deficiency plus CaCO3 treatment. The expression of these enzymes was upregulated under Fe deficiency [14,16,17,18]. Ectopically overexpressing Arabidopsis basic helix– loop–helix38 (bHLH38) and bHLH39 transcription factors in tobacco and sunflower increased Rbfl accumulation and exudation, suggesting that Rbfl synthesis and secretion is regulated by the bHLH transcription-factor network [19]. Although the exact role of secreted flavins in facilitating Fe acquisition remains unclear, they may function as a redox bridge to facilitate ferric-chelate reductase activity or mediate dissolution of low-soluble Fe(III) oxides by complexing extracellular Fe or reductive mechanisms or modifying the rhizosphere microme [20–23]. www.sciencedirect.com
Plant root secretion Chen, Wang and Yeh 67
Phenolic compounds and Fe acquisition
Secretion of phenolic compounds is one of the important mechanisms in Strategy-I Fe acquisition [24]. The first clear evidence of the important role of root-secreted phenolics in response to Fe deficiency was from red clover (Trifolium pretence) [25,26], and the secretion of phenolics was stimulated by Fe deficiency. When secreted phenolics were removed from the nutrient solution, plants showed severe Fe-deficient phenotypes, including reduced shoot biomass and greatly reduced leaf-chlorophyll synthesis. Moreover, exogenously applied phenolics could enhance reutilization of apoplast Fe in red clover roots [25] suggesting that phenolics play roles in Fe uptake.
F60 H1 and ABCG37 are upregulated by Fe deficiency [28,37,38]. The fer-like iron deficiency-induced transcription factor ( fit) mutant showed no Fe-deficiencyresponsive stimulation of F60 H1 transcription or accumulation of fluorescent phenolics, so the transcription of F60 H1 may be regulated by the FIT-bHLH transcription factor regulatory network [31]. Genes in the phenylpropanoid pathway are upregulated by Fe deficiency in A. thaliana but are unchanged or downregulated in M. truncatula, whereas Rbfl synthesis is activated in M. truncatula but not A. thaliana. Use of Rbfl or coumarin secretion may depend on the species and be mutually exclusive [17]. Phytosiderophores
In Brassica napus and Arabidopsis thaliana, secretion of the phenolic compound coumarin is crucial for Fe acquisition from alkaline substrates [27]. Low-Fe growth conditions induced synthesis and secretion of coumarin in their roots [28]. Phenolics found in the exudates of Fe-starved A. thaliana are coumarinolignans (cleomasicosins A, B, C, and D; 50 hydroxycleomiscosins A and/or B), coumarin precursors (ferulic acid, coniferyl aldehyde, and sinapyl aldehyde), and coumarins (scopoletin, scopolin, esculetin, esculin, fraxetin, isofraxidin, and fraxinol). The most prominent coumarins secreted are scopoletin and scopolin [28,29]. Fe deficiency induces coumarinolignan, coumarin, and ferulic-acid accumulation in A. thaliana roots and secretion into nutrient solution at high pH [29,30,31]. Scopoletin is the major coumarin found in root exudates, but fraxetin is the most effective coumarin mobilizing insobluble Fe(III) [29]. Coumarins can chelate and reduce Fe(III) [32]. Fraxetin was reported to mobilize Fe(III)-oxides by complexing with Fe(III) and reducing Fe(III) to Fe(II) [29]. However, phenolics were not able to complement mutation of ferric reduction oxidase 2 ( fro2) [33]. Whether phenolics facilitate Fe uptake through reduction mechanism is still debatable. Coumarins are derived from the phenylpropanoid pathway and their biosynthesis depends on key enzymes encoded by CYP98A3, caffeoyl CoA O-methyltransferase1 (CCoAOMT1), and F60 H1 (Feruloyl-CoA 60 -hydroxylase1) genes. Production of scopoletin and scopolin was greatly reduced with mutated CYP98A3, CCoAOMT1 or F60 H1 [34,35]. Coumarin secretion requires a functional ABCG37 transporter (also called pleiotropic drug resistance9; PDR9), a member of the ATP-binding cassette family. The pdr9 mutants were still able to synthesize and accumulate abundant coumarins in roots, but they barely secreted scopoletin and derivatives [28]. The acquisition of mobilized Fe by ABCG37-mediated coumarin secretion depends on the FRO2/IRT1 (Iron Regulated Transporter 1) transport system [33]. Transcriptome and proteome analyses of Arabidopsis roots revealed induction of the phenylpropanoid pathway in Fe-deficient roots [17,36,37]. Transcript and/or protein levels of www.sciencedirect.com
Graminaceous plants use a chelation-based Strategy-II Fe acquisition that involves secretion of Fe(III) chelators called phytosiderophores into the root rhizosphere [39]. Phytosiderophores are non-protein amino acids that belong to the mugineic acid (MA) family. MAs are synthesized from S-adenosyl-L-methonine via an enzymatic cascade pathway involving three essential enzymes: nicotianamine (NA) synthase for producing NA, NA aminotransferase, and deoxymugineic acid synthase for producing MA. Genes involved in MA synthesis have been isolated from barley, rice, and maize. Their expression is strongly induced by Fe deficiency [40–45]. Secretion of MAs follows a diurnal rhythm [46]. The secretion patterns were found regulated by light intensity or temperature in some graminaceous species, with exudation peak time varying among species [46–49]. Secretion of phytosiderophores is mediated by transporter of mugineic acid1 (TOM1) in rice, barley, and maize [50,51]. The secreted phytosiderophores can chelate and mobilize Fe(III). The resulting Fe(III)–phytosiderophore complexes are transported into the root by plasma membrane-localized yellow stripe1 (YS1) transporters in maize and barley and YS1-like transporters in rice [52–54].
II. Heavy-metal tolerance and low-molecularweight organic acids In addition to their essential roles in improving plant nutrient acquisition, root-secreted exudates are important for heavy-metal tolerance in plants. With industrial human activities and sewage irrigation, the greatly increased heavy-metal pollution of soils has become a severe environmental problem. Toxic-element pollutants and elevated levels of essential elements can cause toxicity in plants. Plants use several strategies including an exclusion mechanism to exclude metal ions from entering root cells by secreting metabolites into the rhizosphere [55]. Exudation of low-molecular-weight organic acids (LMWOAs) has been studied for their important roles in cadmium (Cd), aluminum (Al), gallium (Ga), copper (Cu), manganese (Mn), and lead (Pb) detoxification Current Opinion in Plant Biology 2017, 39:66–72
68 Cell signalling and gene regulation
[55–59,60]. LMWOAs function as metal chelators. LMWOA secretion types and patterns vary by plant species and physiological conditions and soil environment [61]. The most well-documented LMWOA-mediated detoxification mechanism is Al exclusion, which limits harmful Al 3+ taken up by plant roots [5,62]. Al and Ga detoxification
Al is the most abundant metal in the Earth’s crust, and Al toxicity is a major factor limiting agricultural production on acidic soil, which represents about 40% of arable lands worldwide [63]. Several Al-tolerant plant species can secrete organic acids from the root apex in response to Al stress and form stable harmless complexes with Al3+ to detoxify Al3+ in the rhizosphere [64]. The types of Alinduced organic-acid secretion differ by species. For example, Triticum aestivum (wheat) and A. thaliana secrete malate and citrate, Zea mays (maize) and Glycine max (soybean) mainly secrete citrate, and Fagopyrum esculentum (buckwheat) secretes oxalate [59,65–70]. Citrate was found the most effective detoxifying compound because of strong Al3+-chelating capability [5]. Organic-acid secretion in these species follows pattern I, with immediate organic-acid secretion upon Al3+ exposure, or pattern II, with release of organic acids after Al3+ treatment delayed for several hours [71]. Secretion of malate and citrate is mediated by the Al-associated efflux transporters aluminum-activated malate transporter1 (ALMT1) and multidrug and toxin efflux (MATE), respectively. ALMT1 was first isolated in wheat and later reported in A. thaliana, Secale cereale L (rye), and Brassica napus (rapeseed) [72,73–75]. Heterologous expression of TaALMT1 in barley and rice increased malate efflux and enhanced tolerance to Al3+, for an important role for the malate transporter in crop tolerance to Al stress [72,76]. Citrate transporters are members of the MATE protein family. The expression of MATE genes was found highly correlated with Al-activated citrate secretion, leading to Al3+ tolerance in many species [70,77,78–80]. Citrate transporter OsFRDL4 (AtFRD3-like 4) is an essential Altolerance component in rice. OsFRDL4 expression remained low in roots in the absence of Al but was highly induced with Al treatment. The plasma membrane-localized OsFRDL4 protein showed citrate efflux activity. Knockout of OsFRDL4 reduced citrate efflux and aggravated Al-induced growth inhibition [80]. In Arabidopsis, AtALMT1 and AtMATE both function in Al tolerance, but AtALMT1-mediated malate efflux is the major contributor [78]. Although oxalate secretion is associated with Al tolerance, transporters responsible for Al-regulated oxalate secretion and mechanisms regulating the secretion have yet to be identified. Ga is a non-essential element for living organisms, and Ga pollutants are largely produced by the semiconductor industry, which might threaten the environment and Current Opinion in Plant Biology 2017, 39:66–72
human health. Intriguingly, the Ga-stress-triggered responses in Arabidopsis are similar to those triggered by Al stress. Similar to Al detoxification, Arabidopsis detoxifies Ga externally by secreting citrate and malate potentially by the formation of nontoxic Ga(III)-organicacid complexes in the rhizosphere or apoplastic space, Gainduced secretion of organic acids and the expression of both AtALMT1 and AtMATE transporters. Furthermore, exogenous application of citrate alleviated Ga toxicity in Arabidopsis seedlings [60]. Zn/Cd detoxification
Root exudation is potentially involved in Zn accumulation in the Zn/Cd hyperaccumulator Arabidopsis halleri and in Cd accumulation in the hyperaccumulator ecotype (HE) of Sedum alfredii. Exudation of NA, the intermediate component in phytosiderophore biosynthesis, was greater from roots of A. halleri than the non-accumulator A. thaliana, and secretion was induced by excess Zn. Secreted NA is a major metal chelator that forms stable complexes with Zn to prevent rapid Zn uptake into roots and therefore alleviates Zn toxicity to roots [81]. The HE Sedum alfredii secreted more dissolved organic carbon (DOC)/LMWOAs, especially Cd-induced oxalate, than the non-hyperaccumulator ecotype (NHE) [82,83]. The dissolved organic matter (DOM) in exudate had higher Zn, Cd, and Pb complexation and extraction activity in the HE than NHE [82,84]. With the high abundance and high activity of DOM, the HE could accumulate more Zn/Cd via formation of soluble DOM–metal complexes in soil than the NHE [85] and grow normally in highly polluted environment as compared with the inhibited growth of the NHE [82,83,85]. A role for LMWOA secretion in Cd resistance was also found in non-hyperaccumulator plant species. Certain cultivars of Lycopersicon esulentum (tomato) were found to secrete oxalate from the root apex in response to Cd stress. The secreted organic acids prevented Cd from entering root cells and therefore led to Cd resistance in plants [55].
Conclusion Plants use root exudation to adapt to various soil environments (Figure 1). The root-secreted compounds can alter the soil environment for a more beneficial condition for plant growth and development. Findings from numerous studies have provided much knowledge of the molecular mechanisms and regulation of root exudation in terms of metal acquisition. Root exudation facilitating Fe acquisition is the most well-documented mechanism. Graminaceous plants secrete phytosiderophores and non-graminaceous plants secrete Rbfls and phenolics to enhance Fe acquisition from soil. Although Rbfls and phenolics have a known role in facilitating Fe uptake, their exact biochemical roles are still not fully understood. Enhanced Fe uptake by these secreted compounds involves chelation and reduction processes [26,27]. However, further investigation is needed to www.sciencedirect.com
Plant root secretion Chen, Wang and Yeh 69
Figure 1
Fe deficiency Transporter for secretion Extudate
Possible mechanism
ABCG37 PDR9
Rbfl
Rbfl-Fe ? Fe(lI)
Fe(III)
Excess metal TOM1
Phe
Phe-Fe ? Fe(lI)
ALMT
PS
PS-Fe
Fe(III)
MATE OsFRDL4
Malate
Citrate
OA-AI OA-Ga
OA-AI OA-Ga
Microbes
Enhance Fe uptake
Species
N. Tabacum H. albus B. vulgaris
T. pretence B. napus A. thaliana
Oxalate
NA
OA-AI OA-Zn OA-Cd OA-Pb
NA-Zn
Prevent rapid metal uptake (detoxification)
O. sativa H. vulgare Z. mays
T. aestivum A. thaliana
O. sativa A. thaliana Z. mays G. max
F. esculentum T. turgidum L. esulentum S. alfredii (HE)
A. halleri (HE)
Current Opinion in Plant Biology
Summary of root exudates in iron acquisition and metal detoxification. Iron deficiency enhances secretion of riboflavin (Rbfl), phenolic compounds (Phe) and phytosiderophores (PS) from roots. These exudates may facilitate iron uptake by complexing Fe, increasing ferric chelate reductase activity or modifying rhizosphere microme. On the other side, secretion of organic acids (OA, such as malate, citrate, oxalate) and nicotianamine (NA) are induced by excess metals (Al, Ga, Zn, Cd or Pb). OA and NA may play as chelators which form stable complexes with metals to prevent rapid uptake and therefore alleviated toxicity to roots. Exudates found in different species mentioned in this review are listed in lower panel. Full name of each species: N. tabacum, Nicotiana tabacum; H. albus, Hyoscyamus albus; B. vulgaris, Beta vulgaris (sugar beet); T. pretence, Trifolium pretence (red clover); B. napus, Brassica napus; A. thaliana, Arabidopsis thaliana; O. sativa, Oryza sativa (rice); H. vulgare, Hordeum, vulgare (barley); Z. mays, Zea mays (maize); T. aestivum, Triticum aestivum (wheat); G. max, Glycine max (soybean); F. esculentum, Fagopyrum esculentum (buckwheat); T. turgidum, Triticum turgidum var. durum L. (durum wheat); L. esulentum, Lycopersicon esulentum (tomato); S. alfredii, Sedum alfredii; A. halleri, Arabidopsis halleri; HE, hyperaccumulator ecotype.
unravel how these compounds work with other components such as IRT1 and FRO2 to orchestrate the acquisition process.
Acknowledgements
In recent years, the beneficial role of root exudation in metal acquisition has been exploited for biofortification and phytoremediation. Several transgenic rice lines overexpressing Fe-deficiency-responsive genes were generated to increase Fe acquisition [86–88]. The intercropping system is used to improve crop nutrient acquisition or to remediate heavy-metal pollution. Intercropping strategy-I with strategy-II crops or intercropping crops with metal hyperaccumulators can enhance nutrient uptake in crops, remediate farmland and result in safer agriculture products [89,90]. Therefore, a better understanding of the comprehensive root-exudation mechanism will be advantageous for agricultural and environmental development.
References and recommended reading
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This work was financially supported by Academia Sinica and Ministry of Science and Technology, Taiwan.
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