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Malate. Jack of all trades or master of a few?
Phytochemistry 70 (2009) 828–832
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Molecules of Interest
Malate. Jack of all trades or master of a few? Alisdair R. Fernie a,*, Enrico Martinoia b,* a b
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany Institute of Plant Biology, Zollikerstrasse 107, University of Zurich, CH-8008, Zurich, Switzerland
a r t i c l e
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Article history: Received 12 April 2009 Received in revised form 27 April 2009 Available online 25 May 2009 Keywords: Malate Photosynthate Stomata Root exudate
a b s t r a c t The dicarboxylic acid malate has long been thought to play important roles in plant physiology. In addition to being a major photosynthate in C4 and CAM plants and an intermediate of the tricarboxylic acid cycle it has been proposed to play essential roles in pH regulation and important roles in pathogen response, as a component of the root exudates and as a regulatory osmolyte affecting stomatal function. Recent years have seen the cloning and functional analysis of a wide range of enzymes and transporters associated with malate metabolism. Here we attempt to provide a synthesis of research in this field as well as re-evaluating the role of this metabolite in mediating guard cell function. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Malate is an important plant metabolite, it is likely present in all cell types and can accumulate to levels as high as 350 mM (Martinoia and Rentsch, 1994). It has been proposed to exhibit multiplicity of functions (see Fig. 1), however, the evidence for some of these is much stronger than for others. Despite this, malate has clearly been defined to have roles as important as photosynthate in CAM and C4 plants (Jiao and Chollet, 1991), an essential storage carbon molecule (Martinoia and Rentsch, 1994) and intermediate of the TCA cycle in all plant species (Beevers, 1961; Fernie et al., 2004). It has, furthermore, been defined as a pH regulator (Mathieu et al., 1986) and exhibits partial control over the efficacy of nutrient uptake (Weisskopf et al., 2006; Pellet et al., 1995) and over stomatal function (Lee et al., 2008; Hedrich and Marten, 1993a,b). The majority of these processes are well characterised at the molecular level, however, considerable recent developments have been made in the last two and we will thus focus our review on these advances. However, control of its intracellular transport has been purported to have two further functions. Firstly, it is believed to facilitate apoplastic NADH production that stimulates the production of the hydrogen peroxide needed to sustain lignin production (Gross et al., 1977). Secondly, the subcellular control of malate levels is used as a strategy to maintain the levels of stromal reductant to support efficient photosynthesis (Backhausen et al., 1984). In this model the chloroplastic NADP-malate dehydrogenase serves * Corresponding authors. Tel.: +49 331 5678211; fax: +49 331 5678408 (A.R. Fernie), tel.: +41 44 634 8222; fax: +41 44 634 8204 (E. Martinoia). E-mail addresses: [email protected] (A.R. Fernie), enrico.martinoia@ botinst.unizh.ch (E. Martinoia). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.04.023
as a redox valve by using excess NADPH to convert OAA to malate in order to regenerate the electron acceptor NADP. Importantly, NADPH activation is inhibited by its own product NADP and thereby switches off its own activity when NADPH is consumed for assimilatory processes in the chloroplast and as such prevents export of reducing equivalents in the form of malate (Scheibe, 2001). Given that ATP production is maintained whilst electrons are transferred to malate, this valve is a useful ‘‘indirect export system” for reducing equivalents that provide an effective means to balance ATP/NADPH stoichiometry of the chloroplast (Backhausen et al., 1998). The existence of such a valve system has been strongly supported by the identification of plastial dicarboxylate translocators which could fulfil the malate–oxaloacetate shuttle function (Menzlaff and Flügge, 1993; Tanaguchi et al., 2002; Renne et al., 2003). Moreover, given the fact that cytosolic malate has many potential cellular fates it can either (i) provide NADH for nitrate reduction, (ii) support photorespiration, (iii) be utilized by the mitochondria for ATP generation or (iv) be stored in the vacuole, the identification and biochemical characterisation of proteins involved in its metabolism and transport is of vital importance to aid our understanding of the role of this key metabolite. The recent cloning and characterisation of the vacuolar malate transporter (Emmerlich et al., 2003), as well as the previously mentioned plastidial translocators has facilitated the generation of tools to assess the relative importance of the various pool sizes with Arabidopsis knockout mutants of the vacuolar malate transporter revealing that this transporter is critical for mediating correct compartmentation of the dicarboxylates, with the mutant becoming effectively unable to store malate and that its absence severely compromises cellular pH status of the plant (Hurth et al., 2005; Emmerlich et al., 2003). The mutants, however, still
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view on the role of malate in the root exudate and in the guard cell apoplast before providing a perspective of ongoing research on this enigmatic metabolite.
2. On the role of malate as a component of the root exudates
Fig. 1. Proposed roles for malate in biochemical (energy, fatty acid, reduction equivalent production, carbon dioxide fixation and pH status) and whole plant (stomata movement, root exudation).
contained a residual malate importing activity suggesting that this gene did not encode the only vacuolar malate transporter, coupled to its biochemical characteristics, it was therefore clear that another gene encoded the biochemically well-characterised vacuolar malate channel (see Hafke et al., 2003; Martinoia et al., 1985, 1991; Pantoja and Smith, 2002; Pei et al., 1996). This channel has recently been identified and characterised at the genetic level (Kovermann et al., 2007); however, deletion mutants of the channel displayed only mild phenotypes. When taken together the results of these studies suggest that the vacuolar malate transporter and malate channel can, at least partially, compensate for the loss of one another. In addition, three dicarboxylate and one dicarboxylate/tricarboxylate carrier of the mitochondrial carrier family, capable of transporting malate, have been identified in Arabidopsis to date (Palmieri et al., 2008; Picault et al., 2002), and recent physiological studies on the malate–oxaloacetate shuttle of isolated wheat mitochondria have indicated that this antiporter limits the rate of NADH oxidation particularly when the external NADH concentration is low (Pastore et al., 2003). These studies as well as those aimed at assessing the individual functions of various enzymes participating in malate metabolism including the function of various isoforms of malate dehydrogenase (Nunes-Nesi et al., 2005; Cousins et al., 2008; Timm et al., 2008; Backhausen et al., 1998), malic enzyme (Fahnenstich et al., 2007; Maurino et al., 2009) and fumarase (Nunes-Nesi et al., 2007), and further transporter proteins such as the proton dissipating uncoupling protein (Sweetlove et al., 2006), have recently both confirmed and enhanced our knowledge in this area. Important findings of these recent studies were (i) the confirmation of the role of the mitochondria in optimising the rate of photosynthesis, (ii) the demonstration that the peroxisomal malate dehydrogenase is not essential for photorespiration (Cousins et al., 2008; Timm et al., 2008), although it is required to ensure optimal rates of photosynthesis, (iii) plants deficient in the expression of the chloroplastic malate dehydrogenase as well as those deficient in the expression of the mitochondrial uncoupling protein UCP1 provide convincing, although circumstantial, support for the operation of the malate valve, (iv) that malate (and fumarate) are required in considerable amount as respiratory substrate to stave off dark-induced senescence and finally (v) that plants deficient in the expression of fumarase display altered stomatal function. This wealth of data is allowing us a far more comprehensive idea of how malate is regulated and how it in turn regulates. That said some of the most exciting recent results concern malate’s extracellular role. Cumulative evidence also indicates that malate is one of the tricarboxylic acids found in the apoplast following elicitation of the pathogen response in both French bean and Arabidopsis (Bolwell, 1999; Bolwell et al., 2002; Bolwell and Daudi, 2009) suggesting a potential role for the metabolite in this process. In the next sections we focus our re-
Roots excrete a large number of primary and secondary compounds (Bais et al., 2006; van der Merwe et al., 2009). Among the primary compounds malate is often a predominant metabolite. Malate excretion has attracted much interest in the agronomical field, since it has been observed that it plays a role in aluminium tolerance, phosphate nutrition and in the establishment of microbial communities. Aluminium toxicity is critical in acidic soils, which comprise about 50% of the agricultural soil worldwide, since below pH 5 the highly toxic Al3+ cation is readily solubilized (Kochian et al., 2004). Al3+ very rapidly inhibits root growth and results in reduced water uptake. It has been long observed that aluminium tolerance in plants correlated with the capacity to excrete organic acids such as malate and citrate (see Delhaize et al., 2007 and references therein). Excretion of carboxylates results in chelation of Al3+, and consequently rhizo deposition, which prevents that the toxic Al3+ cation is taken up by the plant. The first molecular identification of such a trait was achieved by comparing aluminium tolerant and sensitive wheat species (Sasaki et al., 2004). The gene identified encoded a malate channel (called ALMT for aluminium-activated malate transporter) which is activated by aluminium from the apoplastic side. When the wheat gene was expressed in barley, it conferred Al3+ tolerance to this plant (Delhaize et al., 2004). The fact that the resistance is conferred just by expressing a malate channel, suggests that either cytosolic malate pools can be refilled very rapidly or that the vacuolar malate pool can be used to sustain malate excretion until the metabolism is adjusted to increase malate production. Recently, homologous genes to the wheat ALMT1 have been identified in Arabidopsis (Hoekenga et al., 2006), Brassica napus (Ligaba et al., 2006) and Secale (Fontecha et al., 2007). Interestingly in Arabidopsis ALMT1 is not only activated by Al3+ but the corresponding gene is also upregulated in response to Al3+. Detailed analysis of the wheat ALMT1 revealed that the channel is activated with a Km1/2 of 5 lM and is strongly rectifying (Pineros et al., 2008). However, the exact binding site and mechanism of Al3+ channel activation awaits elucidation. Phosphate is often tightly bound to soil particles or on rocks; however, its availability is increased in most plants by the formation of the mycorrhiza symbiosis (Bonfante and Genre, 2008). That said some plants, such as Brassicacae or most members of the Proteacea and some other plants from diverse families do not form mycorrhiza. In order to improve their phosphate nutrition they excrete carboxylates. Some plants, mainly those of the Proteacea family form specialized roots to excrete large amounts of carboxylates, so called cluster roots (Neumann and Martinoia, 2002; Shane and Lambers, 2005). These plants excrete citrate predominantly, but malate also plays an important role. The molecular nature of the malate release protein remains elusive; however, it is tempting to speculate that an ALMT is involved in this process. In white lupin, young roots excrete mainly malate, older roots and mature cluster roots, mainly citrate. Since it was observed that ATP-citrate lyase is highly expressed in young roots than in older roots, it was postulated that this enzyme could be responsible for the switch of root excretion from malate to citrate and has an impact on the nature of the carboxylate excreted (Langlade et al., 2002). Recently it has been shown that the excretion of malate attracts beneficial soil bacteria (Rudrappa et al., 2008). Leaves treated with the pathogen DC3000 induce root excretion of malate. This effect was much weaker when a mock solution or a non-pathogen was
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used. DC3000 induced also malate excretion when applied to the roots, most probably through AtALMT1, since DC3000 had a similar effect on the induction of this ALMT as Al3+. Malate excreted by the roots in turn attracted beneficial bacteria such as Bacillus subtilis.
(ABA) and redox signals invoked by shifts in light quality, have additionally been demonstrated to be important in stomatal control (Schroeder and Hagiwara, 1989; Kearns and Assmann, 1993; Pei et al., 2000; Li et al., 2000; Chen and Gallie, 2004) and there was in fact considerable support for these factors having higher importance than malate. It should therefore be borne in mind that malate is only one of a range of cellular factors which is capable of regulating stomatal function. That said a series of recent papers have provided fresh support for the importance of malate in regard to the normal operation of stomata (Lee et al., 2008; Negi et al., 2008; Vahisalu et al., 2008). These studies focussed on either the ABC transporter AtABCB14 or the SLAC1 transporters both of which mediate transfer of malate between the apoplast and the cytosol. Plants lacking the ABCB14 transporter exhibited elevated closure on transition to elevated carbon dioxide levels in comparison to wild type controls (see Fig. 2A). Moreover, in isolated epidermal
3. The role of malate in stomatal function Similar to the situation described above for root exudation, malate has long been discussed as an important regulator of stomatal opening and is thought to be an integral part of the mechanism by which guard cells adjust their action in response to external CO2 concentrations (Roelfsema et al., 2002; van Kirk and Raschke, 1978; Hedrich and Marten, 1993a,b; Hedrich et al., 1994; Raschke, 2003). However, in contrast to root exudation malate is the only carboxylic acid proposed to play a major role in this process. That said both K+ and Cl , as well as the phytohormone abscisic acid
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strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the ABCB14 transporter and slower in plants overexpressing it indicating that this transporter catalyses the transport of apoplastic malate into the guard cells. Furthermore, heterologous expression of ABCB14 in Escherichia coli and HeLa cells resulted in increases in malate transport confirming the role of the transporter (Lee et al., 2008). Similarly, the function of the SLAC1, slow S-type anion, channel was recently characterised (Negi et al., 2008; Vahisalu et al., 2008). This channel has been demonstrated to mediate CO2 sensitivity to gas exchange (see Fig. 2B). The SLAC1 protein is a distant homolog of bacterial and fungal dicarboxylate transporters and is specifically localised to the plasma membrane of guard cells. A loss of function mutation in this protein was paralleled with an over accumulation of osmoregulatory anions in guard cell protoplasts; however, guard-cellspecific expression of SLAC1 or its close homologs complemented the phenotype (Negi et al., 2008; Vahisalu et al., 2008). Intriguingly, mutations in SLAC1 were detailed to impair slow anion channel currents that are activated by cytosolic Ca2+ and ABA but do not affect either rapid anion channels or Ca2+ channel function (Vahisalu et al., 2008). Further, albeit indirect evidence for the importance of malate in stomatal function was provided by the study of tomato plants deficient in the expression of fumarase, which catalyses its production in the mitochondrial, these plants exhibited a dramatic growth impediment and reduced rate of photosynthesis that could be attributed to a defective stomatal function with the rate of opening of the stomata being dramatically compromised in the transgenic lines (Nunes-Nesi et al., 2007). Whilst in these plants the overall malate levels were not dramatically altered, the subcellular compartmentation of malate was not studied. When taken together, the sum findings of these studies identify the molecular mechanisms by which malate can affect guard cell functioning and likely represent the most convincing evidence, presented as yet, of its physiological role in plants.
4. Concluding remarks Whilst the central importance of malate in plant metabolism and physiological processes has long been recognised, research in the post genomic era has allowed us far greater insight into mechanistic aspects of the biochemical and molecular mechanisms that underlie both the regulation of subcellular malate concentrations and the effects which it, in turn, has on diverse cellular processes. Due to space constraints we chose to focus on root exudation and stomatal function. However, there is also a wealth of important literature on its role in C4 and CAM metabolism as well as on fruit quality and development for which we refer the interested reader to Kalt et al. (1990), Cushman and Bohnert (1999), Brautigam et al. (2008), Carrari et al. (2006) and Hawker (1969) for further details. To summarize we think our article is fair to say that great advances have been made in understanding the control of, and by, malate in the last few years. There remains, however, still much to be elucidated. Even in Arabidopsis there are many open questions concerning both cellular and whole plant partitioning of malate, precise details of its control of stomates and its function in delaying senescence as well as its contribution to cellular pH regulation. Despite its breadth of function we believe that malate can be described as a master tradesman in C3 plants. The release of the first genome sequence for a C4 plant (sorghum; Paterson et al., 2009), as well as the nascent plant sequencing projects for maize and CAM species such as Kalanchoe fedtschenko (http://www.liv.ac.uk/researchintelligence/issue35/pdfs/ri35pp4-5.pdf) suggests that we will shortly be able to address similar questions in plant species which utilize malate in a radically different manner. A more detailed knowledge about root malate excretion should not only help to im-
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Pei, Z.M., Ward, J.M., Harper, J.F., Schroeder, J.I., 1996. Magnesium sensitizes slow vacuolar channels to physiological cytosolic calcium and inhibits fast vacuolar channels in Fava bean guard cell vacuoles. EMBO J. 15, 6564–6574. Pei, Z.M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen, G.J., Grill, E., Schroeder, J.I., 2000. Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase. Nature 406, 731–734. Pellet, D.M., Grunes, D.L., Kochian, L.V., 1995. Organic-acid exudation as an aluminium-tolerance mechanism in maize (Zea mays L.). Planta 196, 788–796. Picault, N., Palmieri, L., Pisano, I., Hodges, M., Palmieri, F., 2002. Identification of a novel transporter for dicarboxylates and tricarboxylates in plant mitochondria – bacterial expression, reconstitution, functional characterisation and tissue distribution. J. Biol. Chem. 277, 24204–24211. Pineros, M.A., Cancado, G.M.A., Kochian, L.V., 2008. Novel properties of the wheat aluminum tolerance organic acid transporter (TaALMT1) revealed by electrophysiological characterization in Xenopus oocytes: functional and structural implications. Plant Physiol. 147, 2131–2146. Raschke, K., 2003. Alteration of the slow with the quick anion conductance in whole guard cells effected by external malate. Planta 217, 651–657. Renne, P., Dreßen, U., Hebbeker, U., Hille, A., Flügge, U.I., Westhoff, P., Weber, A.P.M., 2003. The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate transporter DiT2. Plant J. 35, 316–331. Roelfsema, M.R.G., Hanstein, S., Felle, H.H., Hedrich, R., 2002. CO2 provides an intermediate link in the red light response of guard cells. Plant J. 32, 65–75. Rudrappa, T., Czymmek, K.J., Paré, P.W., Bais, H.P., 2008. Root-secreted malic acid recruits beneficial soil bacteria. Plant Physiol. 148, 1547–1556. Sasaki, T., Yamamoto, Y., Ezaki, B., Katsuhara, M., Ahn, S.J., Ryan, P.R., Delhaize, E., Matsumoto, H., 2004. A wheat gene encoding an aluminum-activated malate transporter. Plant J. 37, 645–653. Scheibe, R., 2001. Redox-modulation of chloroplast enzymes. A common principle for individual control. Plant Physiol. 96, 1–3. Schroeder, J., Hagiwara, S., 1989. Cytosolic calcium regulates ion channels in the plasma membrane of Vicia faba guard cells. Nature 338, 427–430. Shane, M.W., Lambers, H., 2005. Cluster roots: a curiosity in context. Plant Soil 274, 101–125. Sweetlove, L.J., Lytovchenko, A., Morgan, M., Nunes-Nesi, A., Taylor, N.L., Baxter, C.J., Eickmeier, I., Fernie, A.R., 2006. Mitochondrial uncoupling protein is required for efficient photosynthesis. Proc. Natl. Acad. Sci. USA 103, 19587–19592. Tanaguchi, M., Tanaguchi, Y., Kawasaki, M., Takeda, S., Kato, T., Sato, S., Tabata, S., Miyake, H., Sugiyama, T., 2002. Identifying and characterizing plastidic 2oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana. Plant Cell Environ. 43, 706–717. Timm, S., Nunes-Nesi, A., Pamik, T., Morgenthal, K., Wienkoop, S., Keerberg, O., Weckwerth, W., Kleczkowski, L.A., Fernie, A.R., Bauwe, H., 2008. A cytosolic pathway for the conversion of hydroxypyruvate to glycerate during photorespiration in Arabidopsis. Plant Cell 20, 2848–2859. Vahisalu, T., Kollist, H., Wang, Y.F., Nishimura, N., Chan, W.Y., Valerio, G., Lamminmäki, A., Brosche, M., Moldau, H., Desikan, R., Schroeder, J.I., Kangasjärvi, J., 2008. SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling. Nature 452, 487–493. van der Merwe, M.J., Osorio, S., Moritz, T., Nunes-Nesi, A., Fernie, A.R., 2009. Decreased activities of malate dehydrogenase and fumarase in tomato lead to altered root growth and architecture via diverse mechanisms. Plant Physiol. 149, 653–669. van Kirk, C.A., Raschke, K., 1978. Release of malate from epidermal strips during stomatal closure. Plant Physiol. 61, 474–475. Weisskopf, L., Abou-Mansour, E., Fromin, N., Santelia, D., Edelkott, I., Neumann, G., Aragno, M., Tabacchi, R., Martinoia, E., 2006. White lupin has developed a complex strategy to limit microbial degradation of secreted citrate required for phosphate acquisition. Plant Cell Envrion. 29, 919–927.
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Molecules of Interest
Malate. Jack of all trades or master of a few? Alisdair R. Fernie a,*, Enrico Martinoia b,* a b
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany Institute of Plant Biology, Zollikerstrasse 107, University of Zurich, CH-8008, Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 12 April 2009 Received in revised form 27 April 2009 Available online 25 May 2009 Keywords: Malate Photosynthate Stomata Root exudate
a b s t r a c t The dicarboxylic acid malate has long been thought to play important roles in plant physiology. In addition to being a major photosynthate in C4 and CAM plants and an intermediate of the tricarboxylic acid cycle it has been proposed to play essential roles in pH regulation and important roles in pathogen response, as a component of the root exudates and as a regulatory osmolyte affecting stomatal function. Recent years have seen the cloning and functional analysis of a wide range of enzymes and transporters associated with malate metabolism. Here we attempt to provide a synthesis of research in this field as well as re-evaluating the role of this metabolite in mediating guard cell function. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Malate is an important plant metabolite, it is likely present in all cell types and can accumulate to levels as high as 350 mM (Martinoia and Rentsch, 1994). It has been proposed to exhibit multiplicity of functions (see Fig. 1), however, the evidence for some of these is much stronger than for others. Despite this, malate has clearly been defined to have roles as important as photosynthate in CAM and C4 plants (Jiao and Chollet, 1991), an essential storage carbon molecule (Martinoia and Rentsch, 1994) and intermediate of the TCA cycle in all plant species (Beevers, 1961; Fernie et al., 2004). It has, furthermore, been defined as a pH regulator (Mathieu et al., 1986) and exhibits partial control over the efficacy of nutrient uptake (Weisskopf et al., 2006; Pellet et al., 1995) and over stomatal function (Lee et al., 2008; Hedrich and Marten, 1993a,b). The majority of these processes are well characterised at the molecular level, however, considerable recent developments have been made in the last two and we will thus focus our review on these advances. However, control of its intracellular transport has been purported to have two further functions. Firstly, it is believed to facilitate apoplastic NADH production that stimulates the production of the hydrogen peroxide needed to sustain lignin production (Gross et al., 1977). Secondly, the subcellular control of malate levels is used as a strategy to maintain the levels of stromal reductant to support efficient photosynthesis (Backhausen et al., 1984). In this model the chloroplastic NADP-malate dehydrogenase serves * Corresponding authors. Tel.: +49 331 5678211; fax: +49 331 5678408 (A.R. Fernie), tel.: +41 44 634 8222; fax: +41 44 634 8204 (E. Martinoia). E-mail addresses: [email protected] (A.R. Fernie), enrico.martinoia@ botinst.unizh.ch (E. Martinoia). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.04.023
as a redox valve by using excess NADPH to convert OAA to malate in order to regenerate the electron acceptor NADP. Importantly, NADPH activation is inhibited by its own product NADP and thereby switches off its own activity when NADPH is consumed for assimilatory processes in the chloroplast and as such prevents export of reducing equivalents in the form of malate (Scheibe, 2001). Given that ATP production is maintained whilst electrons are transferred to malate, this valve is a useful ‘‘indirect export system” for reducing equivalents that provide an effective means to balance ATP/NADPH stoichiometry of the chloroplast (Backhausen et al., 1998). The existence of such a valve system has been strongly supported by the identification of plastial dicarboxylate translocators which could fulfil the malate–oxaloacetate shuttle function (Menzlaff and Flügge, 1993; Tanaguchi et al., 2002; Renne et al., 2003). Moreover, given the fact that cytosolic malate has many potential cellular fates it can either (i) provide NADH for nitrate reduction, (ii) support photorespiration, (iii) be utilized by the mitochondria for ATP generation or (iv) be stored in the vacuole, the identification and biochemical characterisation of proteins involved in its metabolism and transport is of vital importance to aid our understanding of the role of this key metabolite. The recent cloning and characterisation of the vacuolar malate transporter (Emmerlich et al., 2003), as well as the previously mentioned plastidial translocators has facilitated the generation of tools to assess the relative importance of the various pool sizes with Arabidopsis knockout mutants of the vacuolar malate transporter revealing that this transporter is critical for mediating correct compartmentation of the dicarboxylates, with the mutant becoming effectively unable to store malate and that its absence severely compromises cellular pH status of the plant (Hurth et al., 2005; Emmerlich et al., 2003). The mutants, however, still
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view on the role of malate in the root exudate and in the guard cell apoplast before providing a perspective of ongoing research on this enigmatic metabolite.
2. On the role of malate as a component of the root exudates
Fig. 1. Proposed roles for malate in biochemical (energy, fatty acid, reduction equivalent production, carbon dioxide fixation and pH status) and whole plant (stomata movement, root exudation).
contained a residual malate importing activity suggesting that this gene did not encode the only vacuolar malate transporter, coupled to its biochemical characteristics, it was therefore clear that another gene encoded the biochemically well-characterised vacuolar malate channel (see Hafke et al., 2003; Martinoia et al., 1985, 1991; Pantoja and Smith, 2002; Pei et al., 1996). This channel has recently been identified and characterised at the genetic level (Kovermann et al., 2007); however, deletion mutants of the channel displayed only mild phenotypes. When taken together the results of these studies suggest that the vacuolar malate transporter and malate channel can, at least partially, compensate for the loss of one another. In addition, three dicarboxylate and one dicarboxylate/tricarboxylate carrier of the mitochondrial carrier family, capable of transporting malate, have been identified in Arabidopsis to date (Palmieri et al., 2008; Picault et al., 2002), and recent physiological studies on the malate–oxaloacetate shuttle of isolated wheat mitochondria have indicated that this antiporter limits the rate of NADH oxidation particularly when the external NADH concentration is low (Pastore et al., 2003). These studies as well as those aimed at assessing the individual functions of various enzymes participating in malate metabolism including the function of various isoforms of malate dehydrogenase (Nunes-Nesi et al., 2005; Cousins et al., 2008; Timm et al., 2008; Backhausen et al., 1998), malic enzyme (Fahnenstich et al., 2007; Maurino et al., 2009) and fumarase (Nunes-Nesi et al., 2007), and further transporter proteins such as the proton dissipating uncoupling protein (Sweetlove et al., 2006), have recently both confirmed and enhanced our knowledge in this area. Important findings of these recent studies were (i) the confirmation of the role of the mitochondria in optimising the rate of photosynthesis, (ii) the demonstration that the peroxisomal malate dehydrogenase is not essential for photorespiration (Cousins et al., 2008; Timm et al., 2008), although it is required to ensure optimal rates of photosynthesis, (iii) plants deficient in the expression of the chloroplastic malate dehydrogenase as well as those deficient in the expression of the mitochondrial uncoupling protein UCP1 provide convincing, although circumstantial, support for the operation of the malate valve, (iv) that malate (and fumarate) are required in considerable amount as respiratory substrate to stave off dark-induced senescence and finally (v) that plants deficient in the expression of fumarase display altered stomatal function. This wealth of data is allowing us a far more comprehensive idea of how malate is regulated and how it in turn regulates. That said some of the most exciting recent results concern malate’s extracellular role. Cumulative evidence also indicates that malate is one of the tricarboxylic acids found in the apoplast following elicitation of the pathogen response in both French bean and Arabidopsis (Bolwell, 1999; Bolwell et al., 2002; Bolwell and Daudi, 2009) suggesting a potential role for the metabolite in this process. In the next sections we focus our re-
Roots excrete a large number of primary and secondary compounds (Bais et al., 2006; van der Merwe et al., 2009). Among the primary compounds malate is often a predominant metabolite. Malate excretion has attracted much interest in the agronomical field, since it has been observed that it plays a role in aluminium tolerance, phosphate nutrition and in the establishment of microbial communities. Aluminium toxicity is critical in acidic soils, which comprise about 50% of the agricultural soil worldwide, since below pH 5 the highly toxic Al3+ cation is readily solubilized (Kochian et al., 2004). Al3+ very rapidly inhibits root growth and results in reduced water uptake. It has been long observed that aluminium tolerance in plants correlated with the capacity to excrete organic acids such as malate and citrate (see Delhaize et al., 2007 and references therein). Excretion of carboxylates results in chelation of Al3+, and consequently rhizo deposition, which prevents that the toxic Al3+ cation is taken up by the plant. The first molecular identification of such a trait was achieved by comparing aluminium tolerant and sensitive wheat species (Sasaki et al., 2004). The gene identified encoded a malate channel (called ALMT for aluminium-activated malate transporter) which is activated by aluminium from the apoplastic side. When the wheat gene was expressed in barley, it conferred Al3+ tolerance to this plant (Delhaize et al., 2004). The fact that the resistance is conferred just by expressing a malate channel, suggests that either cytosolic malate pools can be refilled very rapidly or that the vacuolar malate pool can be used to sustain malate excretion until the metabolism is adjusted to increase malate production. Recently, homologous genes to the wheat ALMT1 have been identified in Arabidopsis (Hoekenga et al., 2006), Brassica napus (Ligaba et al., 2006) and Secale (Fontecha et al., 2007). Interestingly in Arabidopsis ALMT1 is not only activated by Al3+ but the corresponding gene is also upregulated in response to Al3+. Detailed analysis of the wheat ALMT1 revealed that the channel is activated with a Km1/2 of 5 lM and is strongly rectifying (Pineros et al., 2008). However, the exact binding site and mechanism of Al3+ channel activation awaits elucidation. Phosphate is often tightly bound to soil particles or on rocks; however, its availability is increased in most plants by the formation of the mycorrhiza symbiosis (Bonfante and Genre, 2008). That said some plants, such as Brassicacae or most members of the Proteacea and some other plants from diverse families do not form mycorrhiza. In order to improve their phosphate nutrition they excrete carboxylates. Some plants, mainly those of the Proteacea family form specialized roots to excrete large amounts of carboxylates, so called cluster roots (Neumann and Martinoia, 2002; Shane and Lambers, 2005). These plants excrete citrate predominantly, but malate also plays an important role. The molecular nature of the malate release protein remains elusive; however, it is tempting to speculate that an ALMT is involved in this process. In white lupin, young roots excrete mainly malate, older roots and mature cluster roots, mainly citrate. Since it was observed that ATP-citrate lyase is highly expressed in young roots than in older roots, it was postulated that this enzyme could be responsible for the switch of root excretion from malate to citrate and has an impact on the nature of the carboxylate excreted (Langlade et al., 2002). Recently it has been shown that the excretion of malate attracts beneficial soil bacteria (Rudrappa et al., 2008). Leaves treated with the pathogen DC3000 induce root excretion of malate. This effect was much weaker when a mock solution or a non-pathogen was
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used. DC3000 induced also malate excretion when applied to the roots, most probably through AtALMT1, since DC3000 had a similar effect on the induction of this ALMT as Al3+. Malate excreted by the roots in turn attracted beneficial bacteria such as Bacillus subtilis.
(ABA) and redox signals invoked by shifts in light quality, have additionally been demonstrated to be important in stomatal control (Schroeder and Hagiwara, 1989; Kearns and Assmann, 1993; Pei et al., 2000; Li et al., 2000; Chen and Gallie, 2004) and there was in fact considerable support for these factors having higher importance than malate. It should therefore be borne in mind that malate is only one of a range of cellular factors which is capable of regulating stomatal function. That said a series of recent papers have provided fresh support for the importance of malate in regard to the normal operation of stomata (Lee et al., 2008; Negi et al., 2008; Vahisalu et al., 2008). These studies focussed on either the ABC transporter AtABCB14 or the SLAC1 transporters both of which mediate transfer of malate between the apoplast and the cytosol. Plants lacking the ABCB14 transporter exhibited elevated closure on transition to elevated carbon dioxide levels in comparison to wild type controls (see Fig. 2A). Moreover, in isolated epidermal
3. The role of malate in stomatal function Similar to the situation described above for root exudation, malate has long been discussed as an important regulator of stomatal opening and is thought to be an integral part of the mechanism by which guard cells adjust their action in response to external CO2 concentrations (Roelfsema et al., 2002; van Kirk and Raschke, 1978; Hedrich and Marten, 1993a,b; Hedrich et al., 1994; Raschke, 2003). However, in contrast to root exudation malate is the only carboxylic acid proposed to play a major role in this process. That said both K+ and Cl , as well as the phytohormone abscisic acid
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strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the ABCB14 transporter and slower in plants overexpressing it indicating that this transporter catalyses the transport of apoplastic malate into the guard cells. Furthermore, heterologous expression of ABCB14 in Escherichia coli and HeLa cells resulted in increases in malate transport confirming the role of the transporter (Lee et al., 2008). Similarly, the function of the SLAC1, slow S-type anion, channel was recently characterised (Negi et al., 2008; Vahisalu et al., 2008). This channel has been demonstrated to mediate CO2 sensitivity to gas exchange (see Fig. 2B). The SLAC1 protein is a distant homolog of bacterial and fungal dicarboxylate transporters and is specifically localised to the plasma membrane of guard cells. A loss of function mutation in this protein was paralleled with an over accumulation of osmoregulatory anions in guard cell protoplasts; however, guard-cellspecific expression of SLAC1 or its close homologs complemented the phenotype (Negi et al., 2008; Vahisalu et al., 2008). Intriguingly, mutations in SLAC1 were detailed to impair slow anion channel currents that are activated by cytosolic Ca2+ and ABA but do not affect either rapid anion channels or Ca2+ channel function (Vahisalu et al., 2008). Further, albeit indirect evidence for the importance of malate in stomatal function was provided by the study of tomato plants deficient in the expression of fumarase, which catalyses its production in the mitochondrial, these plants exhibited a dramatic growth impediment and reduced rate of photosynthesis that could be attributed to a defective stomatal function with the rate of opening of the stomata being dramatically compromised in the transgenic lines (Nunes-Nesi et al., 2007). Whilst in these plants the overall malate levels were not dramatically altered, the subcellular compartmentation of malate was not studied. When taken together, the sum findings of these studies identify the molecular mechanisms by which malate can affect guard cell functioning and likely represent the most convincing evidence, presented as yet, of its physiological role in plants.
4. Concluding remarks Whilst the central importance of malate in plant metabolism and physiological processes has long been recognised, research in the post genomic era has allowed us far greater insight into mechanistic aspects of the biochemical and molecular mechanisms that underlie both the regulation of subcellular malate concentrations and the effects which it, in turn, has on diverse cellular processes. Due to space constraints we chose to focus on root exudation and stomatal function. However, there is also a wealth of important literature on its role in C4 and CAM metabolism as well as on fruit quality and development for which we refer the interested reader to Kalt et al. (1990), Cushman and Bohnert (1999), Brautigam et al. (2008), Carrari et al. (2006) and Hawker (1969) for further details. To summarize we think our article is fair to say that great advances have been made in understanding the control of, and by, malate in the last few years. There remains, however, still much to be elucidated. Even in Arabidopsis there are many open questions concerning both cellular and whole plant partitioning of malate, precise details of its control of stomates and its function in delaying senescence as well as its contribution to cellular pH regulation. Despite its breadth of function we believe that malate can be described as a master tradesman in C3 plants. The release of the first genome sequence for a C4 plant (sorghum; Paterson et al., 2009), as well as the nascent plant sequencing projects for maize and CAM species such as Kalanchoe fedtschenko (http://www.liv.ac.uk/researchintelligence/issue35/pdfs/ri35pp4-5.pdf) suggests that we will shortly be able to address similar questions in plant species which utilize malate in a radically different manner. A more detailed knowledge about root malate excretion should not only help to im-
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