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Lipoxygenase-dependent degradation of storage lipids Ivo Feussner, Hartmut Kühn and Claus Wasternack Oilseed germination is characterized by the mobilization of storage lipids as a carbon source for the germinating seedling. In spite of the importance of lipid mobilization, its mechanism is only partially understood. Recent data suggest that a novel degradation mechanism is initiated by a 13-lipoxygenase during germination, using esterified fatty acids specifically as substrates. This 13-lipoxygenase reaction leads to a transient accumulation of ester lipid hydroperoxides in the storage lipids, and the corresponding oxygenated fatty acid moieties are preferentially removed by specific lipases. The free hydroperoxy fatty acids are subsequently reduced to their hydroxy derivatives, which might in turn undergo β-oxidation.

Several developmental processes during the life cycle of plants are characterized by changes in the composition and turnover of intracellular lipids. The life cycle of seed plants begins with germination and, in oilseeds, this process is characterized by the mobilization of the storage lipids, which serve as major carbon source for growth of seedlings1. During seed maturation, the storage lipids are deposited in special organelles, the so-called lipid or oil bodies. These storage organelles are intracellular droplets of triacylglycerols surrounded by a phospholipid monolayer2. The lipid bodies are degraded during early stages of germination and a new set of proteins become detectable at the phospholipid monolayer (Fig. 1). Among them are a 13-lipoxygenase (13-LOX) and at least two ester-lipid-cleaving enzymes, a phospholipase and a triacylglycerol lipase3. This 13-LOX exhibits substrate specificity for esterified fatty acid residues. LOX pathway Ivo Feussner Dept Molecular Cell Biology, Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Corrensstr. 3, Germany. e-mail: [email protected] Claus Wasternack Dept Natural Product Biotechnology, Leibniz Institute for Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany. Hartmut Kühn Institute for Biochemistry, Universitätsklinikum Charité, Humboldt University Berlin, D-10115 Berlin, Hessische Str. 3–4, Germany.

LOXes (linoleate:oxygen oxidoreductase, EC 1.13.11.12) form a family of non-heme-iron-containing fatty acid dioxygenases that are widely distributed in plants and animals4,5. They catalyse the regio- and stereospecific dioxygenation of polyenoic fatty acids containing a (1Z,4Z)-pentadiene system6. Because arachidonic acid is a minor polyenoic fatty acid in the plant kingdom, plant LOXes are classified according to their positional specificity of linoleic acid oxygenation. This substrate is oxygenated at either carbon atom 9 (9-LOX) or carbon atom 13 (13-LOX) of the hydrocarbon backbone of the fatty acid7. A more comprehensive classification of plant LOXes has been proposed that is based on the comparison of their primary structure8. According to their overall sequence similarity (~70%), most plant LOXes can be grouped into one gene family. The encoded enzymes lack a putative chloroplast transit

peptide and are designated type-1 LOXes. Several LOX cDNAs have been isolated from various sources (Arabidopsis, rice, wheat, barley, potato, tomato, and tobacco) that carry a putative chloroplast transit peptide sequence. Based on this N-terminal extension and because these enzymes show only a moderate overall sequence similarity of ~40% among each other, they have been classified as type-2 LOXes (Ref. 8). Although plant LOXes have been studied extensively in the past, there is no general concept of their biological importance5. However, during the past few years, physiological functions for distinct 9and 13-LOXes have been found. It has been generally accepted that LOXes produce hydroperoxy fatty acids that are subsequently metabolized via several secondary pathways to form bioactive compounds such as 12-oxo-phytodienoic acid and jasmonate9. Four major metabolic routes for the metabolism of hydroperoxy fatty acids have been characterized (Fig. 2)10,11: • The hydroperoxide lyase (HPL) pathway, in which the hydrocarbon backbone of fatty acid hydroperoxides is cleaved under rearrangement of the hydroperoxide, leading to the formation of short chain aldehydes (C6- or C9-) and the corresponding C12- or C9-ω-keto fatty acids12. • The allene oxide synthase (AOS) pathway, in which allene oxide synthase converts hydroperoxy fatty acids to unstable allene oxides, which form nonenzymatically racemic derivatives of 12-oxophytodienoic acid or undergo non-enzymatic hydrolysis to form α- and γ-ketols. In presence of an allene oxide cyclase, they are metabolized to (9S,13S)-12-oxo-phytodienoic acid13. • The peroxygenase pathway, in which intramolecular oxygen transfer converts fatty acid hydroperoxides to epoxy- or dihydrodiol polyenoic fatty acids10,14. • The divinyl ether synthase (DES) pathway15, which converts fatty acid hydroperoxides into cytotoxic divinyl ethers16. Hydroperoxy fatty acids do not exhibit a major biological function but products of these four metabolic pathways (including the leaf aldehydes, the jasmonates and other oxygenated unsaturated fatty acids named oxylipins) are bioactive10 and some can be released from plant tissues as volatiles. A few of these compounds are known to function as signalling molecules and others are phytoalexins16,17.

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Fig. 1. Composition of lipid bodies during seed germination. The lipid body is composed of a phospholipid monolayer surrounding the storage triglycerols (TAGs). In the dormant seed, two structural proteins are found in its membrane, the oleosin and the caloleosin. Upon germination, at least three additional proteins became detectable at its membrane: a trilinoleate 13-lipoxygenase, a patatin-type phospholipase and a triacylglycerol lipase. The germination-induced attachment of the later proteins and their concerted enzymatic action initiate storage lipid mobilization.

In addition to these four major pathways, there are other reactions for hydroperoxide metabolism that are not as well characterized (Fig. 2). The first of these is the hydroperoxidase activity of LOXes (ketodieneforming pathway). Under certain conditions, such as low oxygen pressure, LOXes can catalyse the homolytic cleavage of the O–O bond, forming alkoxy radicals that rearrange to ketodienes6. The second is the epoxy alcohol synthase (EAS) pathway18. Epoxy hydroxy fatty acids are formed by intramolecular rearrangement of hydroperoxy fatty acids catalysed by EAS. Finally, there is the reductase pathway. The corresponding enzyme has not been characterized in plants in detail but a protein harbouring such a hydroperoxide-reducing activity has been described19,20. Hydroxylated fatty acids containing a conjugated diene system were found to increase plant defense reactions21 and were detected as intermediate metabolites in the degradation of storage lipids, which is the main focus of this article. LOX-dependent degradation of storage lipids

At the phospholipid monolayer of lipid bodies of various oilseed seedlings, including cucumber seedlings, a special linoleate 13-LOX can be detected22 that is capable of oxygenating esterified linoleate residues without the preceding action of a lipid hydrolysing enzyme23. During early stages of germination, this reaction leads to a strong increase http://plants.trends.com

in the hydroperoxide content of the storage lipids. This oxygenated fatty acid fraction contains mainly (13S,9Z,11E)-13-hydro(pero)xy-9,11-octadecadienoic acid [(13S)-H(P)OD], which is preferentially released from the lipid bodies to undergo β-oxidation. According to this scenario, mobilization of the storage lipids might be initiated not by a lipase but by the special 13-LOX (Ref. 24). The cDNA coding for the lipid body 13-LOX from cucumber has been cloned. It is not expressed in resting seeds but is strongly induced during the early stages of germination25. Immunocytochemical studies of cucumber cotyledons revealed a specific localization of the enzyme at the membrane of lipid bodies during early stages of germination22. Similar results were obtained when the subcellular location of the enzyme was analysed in developing seeds of transgenic tobacco. Here, also, the transgenic cucumber 13-LOX was mainly located at the lipid body membrane26. Synthesized at free ribosomes, the enzyme originally appears in the cytosol but is then transferred to the lipid body membrane, where it is attached to the phospholipid monolayer. The N-terminal β-barrel domain of the LOX appears to contain a targeting signal, which might be important for the translocation process27. Binding of the enzyme to the lipid body membrane augmented its linoleate-oxygenating activity approximately fourfold11. Lipid body LOXes have a higher molecular weight (~100 kDa) than other type-1 LOXes and are unique in being capable of directly oxygenating trilinolein to single-, double- and triple-oxygenated triacylglycerol derivatives. The cucumber enzyme exhibits an alkaline pH optimum and an interesting positional specificity11: linoleic acid is specifically converted to its (13S)-hydroperoxy derivative [(13S)-HPOD]. By contrast, arachidonic acid, which is of minor

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Fig. 2. Secondary metabolism of lipoxygenase-derived hydroperoxy fatty acids in plants. The first reaction steps are shown for the conversion of α-linolenic acid by a 13-lipoxygenase. Abbreviations: AOS, allenoxide synthase; DES, divinyl ether synthase; α-DOX, α-dioxygenase; EAS, epoxy alcohol synthase; HPL, hydroperoxide lyase; LOX, 13-lipoxygenase; POX, peroxygenase; PUFA, polyunsaturated fatty acid.

physiological importance in plants, is oxygenated to a more complex product mixture consisting of 15S-, 12Sand 8-hydroperoxy-eicosatetraenoic acid (15S-, 12Sand 8-HPETE)11. Although the molecular determinants that control the position of oxygen insertion within this special LOX have been isolated, the structural determinants for the unique substrate specificity against triacylglycerols are unknown28. In vitro, the purified recombinant enzyme is capable of oxygenating trilinolein to single-, double- and tripleoxygenated triacylglycerol derivatives6. Because these compounds have also been detected in cucumber, sunflower (Helianthus spp.), marigold (Calendula officinalis) and flax (Linum usitatissimum) seedlings, this enzyme must be involved in the germination process23. Accordingly, about 20% of the linoleic acid residues within the triacylglycerol fraction were detected as oxygenated derivatives in cucumber seedlings at the fourth day of germination24. The protein-chemical characteristics, the subcellular location, the enzymatic properties and the distinct function of the particulate 13-LOX indicate that this enzyme differs from other LOXes present in seedlings. Fractionation studies of the storage lipids as well as analysis of lipid bodies of different germination stages suggested the following degradation cascade of the http://plants.trends.com

lipid bodies3,29. As a first step disruption of the integrity of the lipid body membrane is required. This might be facilitated by two sequential or simultaneous reactions (Fig. 3): (1) proteolytic digestion of the membrane-shielding cytosolic domains of structural proteins of the lipid bodies (oleosins), which might protect the storage lipids against degradation until they are needed for germination2,30; and (2) the degradation of the phospholipid monolayer of this organelle, presumably by a patatin-type phospholipase31. However, it should be stressed that these early events of the degradation cascade are not well investigated and remain a matter of discussion. After rupture of the lipid body membrane (step 1), the storage triacylglycerols are accessible to the 13-LOX and the enzyme can oxygenate unsaturated storage lipids (step 2), as indicated by the large amounts of LOX products detected in the triacylglycerol fraction of lipid bodies23. Preferential cleavage of the oxygenated storage lipids then occurs (step 3). Among the free fatty acid derivatives released from the storage lipids of sunflower or cucumber seedlings, (13S)-H(P)OD was mainly detected23,24. Linoleic acid was present only in trace amounts. Moreover, a lipase activity responsible for the liberation of the fatty acids from the storage lipid derivatives exhibited a high specificity for oxygenated fatty acid derivatives. These observations were made in various oilseeds such as cucumber, castor bean (Ricinus communis), pumpkin (Cucurbita spp.), marigold, sunflower, Vernonia and flax32,33. In all cases, only oxygenated fatty acids were liberated by a lipid-body lipase activity. The amount of linoleic acid residues in the storage lipid fraction (Fig. 4) fall from the first day of germination, paralleled by the occurrence of LOX protein at the lipid body membrane and a transient increase in the amount of 13-HPOD in the storage lipids (Fig. 4). A day later, a transient increase in free 13-H(P)OD is found in the seedling tissue, resulting from the liberation of 13-H(P)OD from the storage lipids. Taken together, these data prompted us to propose a new model for the breakdown of the storage lipids during germination of oilseeds29 (Fig. 3) that differs from the ‘classical’ scheme of peroxisomal or glyoxysomal degradation of linoleic acid1,34. In this new model, the LOX-catalysed oxygenation of the storage lipids precedes the hydrolysis of triacylglycerols. After disruption of the lipid body membrane, the 13-LOX oxygenates the storage triglycerides to their corresponding hydroperoxy derivatives, which are cleaved by a lipase. The resulting free hydroperoxy polyenoic fatty acids undergo reduction to the hydroxy compounds. The reducing enzyme has not yet been identified. Although little is known about this enzyme, two principal mechanisms are possible: (1) a glutathioneS-transferase acting as glutathione peroxidase under certain conditions20; or (2) specific glutathione peroxidases19. In addition, it should be mentioned

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Fig. 3. Metabolic pathways involved in the breakdown of storage lipids during plant germination. The green pathway displays the classical glyoxysomal or peroxisomal degradation of linoleic acid1. The blue pathway shows the 13-lipoxygenase-dependent degradation of polyenoic fatty acids and the red pathway indicates a hypothetical fatty acid CoA-synthetase-independent pathway for the degradation of storage lipids. Abbreviations: 13-LOX, 13-lipoxygenase; DAGAT, acyl-CoA–diacylglycerol acyltransferase.

that convincing data are lacking for an involvement of 2-cysteine peroxiredoxins in the reduction of lipid peroxides, as has been suggested before35. The final steps in the catabolism of the oxidized storage lipids are glyoxysomal β-oxidation. These steps provide reducing equivalents and acetyl-CoA for synthetic processes in seedling growth. Interestingly, recent experiments showed the capacity of isolated glyoxysomes from etiolated sunflower cotyledons for degrading (13S)-HOD-CoA was comparable with that of linoleic acid-CoA (B. Gerhardt and I. Feussner, unpublished). http://plants.trends.com

The metabolic scheme presented for the lipid mobilization in germinating oilseeds suggests a novel biological role for a special particulate 13-LOX. This enzyme appears to ‘label’ the storage lipids, making them prone to degradation by specific triacylglycerol lipases. In this way, the plant is able to discriminate between lipid turnover steadily occurring in seedling development and the maturational lipid breakdown that takes place as an essential part of the germination process. Which is the preferred pathway of storage lipid degradation?

Based on data available for mobilization of storage lipids, both the classical LOX-independent and the novel LOX-dependent pathway exist in parallel (Fig. 3). This raises the question of whether one of the two metabolic routes is used preferentially during germination of oilseeds. To date, there is no clear

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Acknowledgements We are grateful to Christine Kaufmann for the preparation of the figures, and for the constructive help of four anonymous reviewers. Our work was supported by the Deutsche Forschungsgemeinschaft. This article is dedicated to Prof. Helmut Kindl (Marburg, Germany) and Prof. Bernt Gerhardt (Münster, Germany) on the occasion of their retirement, who initiated and stimulated the work presented in this article.

Fig. 4. Temporal appearance of metabolites involved in the breakdown of storage lipids during germination. (a) The temporal appearance of 13-lipoxygenase (lipid body LOX) protein in the lipid body fraction during the germination of cucumber. (b) The accumulation of breakdown products during the first five days of germination. The red line shows the decrease of linoleic acid in the storage lipid fraction. The green line shows the transient accumulation of esterified (13Shydroperoxy octadecadienoic acid, 13S-HPOD) in the storage lipid fraction and the blue line represents the transient accumulation of free 13-hydroperoxy linoleic acid liberated from the lipid bodies. The storage lipid fraction contains linoleic acid, esterified 13-hydroperoxy linoleic acid and free 13-hydro(peroxy) linoleic acid at a ratio of about 100:10:1 in cucumber seedlings.

answer but it seems that different plant species prefer different pathways. In the case of corn and rape (Brassica napus), the classical LOX-independent pathway seems to be the preferred one34,36, whereas the situation in soybean seedlings is still a matter of debate37–39. However, the data discussed in this article support the preferential activity of the LOX-dependent pathway in cucumber, sunflower and flax. First, special LOXes are detectable at the phospholipid monolayer of

References 1 Gerhardt, B. (1993) Catabolism of fatty acids (αand β-oxidation). In Lipid Metabolism in Plants (Moore, J.T.S., ed.), pp. 527–565, CRC Press 2 Huang, A.H.C. (1996) Oleosins and oil bodies in seeds and other organs. Plant Physiol. 110, 1055–1061 3 Kindl, H. (1997) The oxygen-dependent modification of triacylglycerols and phospholipids, the different way of initiating lipid body mobilization. Z. Naturforsch. 52c, 1–8 4 Kühn, H. and Thiele, B.J. (1999) The diversity of the lipoxygenase family. FEBS Lett. 449, 7–11 5 Rosahl, S. (1996) Lipoxygenases in plants – their role in development and stress response. Z. Naturforsch. 51c, 123–138 6 Feussner, I. and Kühn, H. (2000) Application of lipoxygenases and related enzymes for the preparation of oxygenated lipids. In Enzymes in Lipid Modification (Bornscheuer, U.T., ed.), pp. 309–336, Wiley–VCH http://plants.trends.com

lipid bodies, and these enzymes can oxidize esterified polyenoic fatty acids6. Second, germination of each of these plants is correlated with an abundant and transient increase in LOX-derived esterified polyenoic fatty acids23. Third, the substrate specificity of the triacylglycerol lipases isolated from the lipid bodies of germinating oilseeds show a remarkable preference for oxygenated fatty acids32. Fourth, lipid bodies isolated from cucumber and from sunflower seedlings show a preferential release of the LOX-derived oxygenated polyenoic fatty acids. By contrast, non-oxygenated polyenoic fatty acids were only liberated in small amounts24. These data suggest that the breakdown of polyenoic fatty acid-containing storage lipids occurs in these plants via the LOX-dependent pathway. Although saturated fatty acids such as palmitate are also abundant constituents of storage lipids, they could not be oxygenated by any LOX, thereby excluding a LOX-dependent mobilization. However, at least monoenoic fatty acids such as oleic acid might enter the LOX-dependent pathway by a recently detected ∆12-desaturase activity at the membrane of sunflower lipid bodies40. This converts esterified oleic acid to linoleic acid, which can then enter the LOXdependent degradation pathway. A second LOX-independent pathway of storage lipid mobilization can be envisaged that involves a recently isolated acyl-CoA–diacylglycerol acyltransferase41. This enzyme is known to catalyse the last step in the biosynthesis of triacylglycerols42. Interestingly, the highest levels of transcript coding for this enzyme were found in germinating seeds and not, as expected, in developing seeds, where storage lipid biosynthesis takes place41. Therefore, it is tempting to speculate that this special acyl-CoA–diacylglycerol acyltransferase is involved in the degradation of storage lipids by catalysing the reverse reaction. This might lead to the direct formation of acyl-CoAs from triacylglycerols (Fig. 3). Indeed, in the case of purified lipid bodies from sunflower seedlings, the ATPindependent formation of acyl-CoAs has recently been observed (B. Gerhardt and I. Feussner, unpublished).

7 Brash, A.R. (1999) Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate. J. Biol. Chem. 274, 23679–23682 8 Shibata, D. et al. (1994) Lipoxygenases. Plant Mol. Biol. Rep. 12, S41–S42 9 Creelman, R.A. and Mullet, J.E. (1997) Biosynthesis and action of jasmonates in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 355–381 10 Blee, E. (1998) Phytooxylipins and plant defense reactions. Prog. Lipid Res. 37, 33–72 11 Feussner, I. and Wasternack, C. (1998) Lipoxygenase catalyzed oxygenation of lipids. Fett/Lipid 100, 146–152 12 Matsui, K. (1998) Properties and structures of fatty acid hydroperoxide lyase. Belg. J. Bot. 131, 50–62 13 Ziegler, J. et al. (2000) Molecular cloning of allene oxide cyclase. J. Biol. Chem. 275, 19132–19138 14 Hamberg, M. (1995) Hydroperoxide isomerases. J. Lipid Mediators Cell Signal. 12, 283–292

15 Itoh, A. and Howe, G.A. (2001) Molecular cloning of a divinyl ether synthase: identification as a CYP74 cytochrome P450. J. Biol. Chem. 276, 3620–3627 16 Weber, H. et al. (1999) Divinyl ether fatty acid synthesis in late blight-diseased potato leaves. Plant Cell 11, 485–493 17 Croft, K.P.C. et al. (1993) Volatile products of the lipoxygenase pathway evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae pv phaseolicola. Plant Physiol. 101, 13–24 18 Hamberg, M. (1999) An epoxy alcohol synthase pathway in higher plants: biosynthesis of antifungal trihydroxy oxylipins in leaves of potato. Lipids 34, 1131–1142 19 Eshdat, Y. et al. (1997) Plant glutathione peroxidases. Physiol. Plant. 100, 234–240 20 Weiler, E.W. (1997) Octadecanoid-mediated signal transduction in higher plants. Naturwissenschaften 84, 340–349

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21 Weichert, H. et al. (1999) Metabolic profiling of oxylipins upon salicylate treatment in barley leaves. FEBS Lett. 464, 133–137 22 Feussner, I. et al. (1996) Lipid-body lipoxygenase is expressed in cotyledons during germination prior to other lipoxygenase forms. Planta 198, 288–293 23 Feussner, I. et al. (1997) Structural elucidation of oxygenated storage lipids in cucumber cotyledons. J. Biol. Chem. 272, 21635–21641 24 Feussner, I. et al. (1995) Lipoxygenasecatalyzed oxygenation of storage lipids is implicated in lipid mobilization during germination. Proc. Natl. Acad. Sci. U. S. A. 92, 11849–11853 25 Höhne, M. et al. (1996) Lipid body lipoxygenase characterized by protein fragmentation, cDNA sequence and very early expression of the enzyme during germination of cucumber seeds. Eur. J. Biochem. 241, 6–11 26 Hause, B. et al. (2000) Expression of cucumber lipid body lipoxygenase in transgenic tobacco. Planta 210, 708–714 27 May, C. et al. (2000) The N-terminal β-barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies. Eur. J. Biochem. 267, 1100–1109 28 Hornung, E. et al. (1999) Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating

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species by site-directed mutagenesis. Proc. Natl. Acad. Sci. U. S. A. 96, 4192–4197 Feussner, I. et al. (1997) Do specific linoleate 13lipoxygenases initiate β-oxidation? FEBS Lett. 406, 1–5 Matsui, K. et al. (1999) Cucumber cotyledon lipoxygenase during postgerminative growth. Its expression and action on lipid bodies. Plant Physiol. 119, 1279–1287 Noll, F. et al. (2000) Phospholipid monolayer of plant lipid bodies attacked by phospholipase A2 shows 80 nm holes analyzed by atomic force microscopy. Biophys. Chem. 86, 29–35 Balkenhohl, T. et al. (1998) A lipase specific for esterified oxygenated polyenoic fatty acids in lipid bodies of cucumber cotyledons. In Advances in Plant Lipid Research (Sánchez, J. et al., eds), pp. 320–322, Secretariado de Publicaciones de la Universidad de Sevilla, Spain Adlercreutz, P. et al. (1997) Vernonia lipase: a plant lipase with strong fatty acid selectivity. Methods Enzymol 284, 220–232 Huang, A.H.C. (1993) Lipases. In Lipid Metabolism in Plants (Moore, J.T.S., ed.), pp. 473–503, CRC Press Baier, M. and Dietz, K.J. (1999) Alkyl hydroperoxide reductases: the way out of the

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oxidative breakdown of lipids in chloroplasts. Trends Plant Sci. 4, 166–168 Fuchs, C. et al. (1996) Purification and characterization of the acid lipase from the endosperm of castor oil seeds. J. Plant Physiol. 149, 23–29 Feussner, I. and Kindl, H. (1992) A lipoxygenase is the main lipid body protein in cucumber and soybean cotyledons during the stage of triglyceride mobilization. FEBS Lett. 298, 223–225 Fuller, M.A. et al. (2001) Activity of soybean lipoxygenase isoforms against esterified fatty acids indicates functional specificity. Arch. Biochem. Biophys.388, 146–154 Wang, C.X. et al. (1999) Subcellular localization studies indicate that lipoxygenases 1 to 6 are not involved in lipid mobilization during soybean germination. Plant Physiol. 120, 227–235 Sarmiento, C. et al. (1998) Oleate desaturation and acyl turnover in sunflower (Helianthus annuus L.) seed lipids during rapid temperature adaptation. Planta 205, 595–600 Zou, J. et al. (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J. 19, 645–653 Millar, A.A. et al. (2000) All fatty acids are not equal: discrimination in plant membrane lipids. Trends Plant Sci. 5, 95–101

Aluminium tolerance in plants and the complexing role of organic acids Jian Feng Ma, Peter R. Ryan and Emmanuel Delhaize The aluminium cation Al3+ is toxic to many plants at micromolar concentrations. A range of plant species has evolved mechanisms that enable them to grow on acid soils where toxic concentrations of Al3+ can limit plant growth. Organic acids play a central role in these aluminium tolerance mechanisms. Some plants detoxify aluminium in the rhizosphere by releasing organic acids that chelate aluminium. In at least two species, wheat and maize, the transport of organic acid anions out of the root cells is mediated by aluminium-activated anion channels in the plasma membrane. Other plants, including species that accumulate aluminium in their leaves, detoxify aluminium internally by forming complexes with organic acids.

Jian Feng Ma Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kita-gun, Kagawa 761-0795, Japan. e-mail: maj @ag.kagawa-u.ac.jp Peter R. Ryan Emmanuel Delhaize CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia.

Aluminium (Al) is a light metal that makes up 7% of the earth’s crust and is the third most abundant element after oxygen and silicon. Plant roots are therefore almost always exposed to Al in some form. Fortunately, most of this Al occurs as harmless oxides and aluminosilicates. However, when soils become acidic as a result of natural processes or human activities, Al is solubilized into the toxic trivalent cation, Al3+. Aluminium toxicity has been recognized as a major limiting factor of plant productivity on acidic soils, which now account for ~40% of the earth’s arable land.

Micromolar concentrations of Al3+ can inhibit root growth within minutes or hours in many agriculturally important plant species1. The subsequent effects on nutrient and water acquisition result in poor growth and productivity. The molecular mechanisms underlying Al toxicity are not known, but because Al forms strong bonds with oxygen-donor compounds2, it can interact with multiple sites in the apoplasm and symplasm of root cells. The binding of Al with these substances is probably an important factor in its toxicity. Some plant species have evolved mechanisms to tolerate Al stress, which helps them to grow on acid soils. Understanding the nature of these tolerance mechanisms has been the focus of ongoing research in the area of stress physiology. Much of the current evidence points to a central role for certain organic acids that detoxify Al3+ by complexing these cations in the cytosol or at the root–soil interface. Organic acids detoxify Al3+ external to the root

Over a dozen Al-tolerant plant species are known to secrete organic acids from their roots in response to Al treatment3,4. Citrate, oxalate and malate are some of

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