Organic acid efflux
Under normal growth and development conditions, plant roots exude a wide variety of organic compounds including: simple sugars, organic acids, amino acids, phenolics, quinones (iso)-flavonoids, growth hormones, proteins, and polysaccharides (Curl & Truelove, 1986; Marschner, 1995). Exudation of organic compounds from roots can alter rhizosphere chemistry, soil microbial populations, competition, and plant growth. Exuded compounds are functionally diverse and can be involved in a wide array of processes ranging from signaling in plant–microbe interactions, to allelopathy and nutrient acquisition (Curl & Truelove, 1986; Marschner et al., 1986; Harrison, 1997).
Under the influence of environmental stress the complement of compounds found in exudates can be significantly altered in either or both quality and quantity (Marschner, 1995; Ryan et al., 2001; Neumann & Martinoia, 2002). For example, when plants are attacked by soil pathogens, the amount and type of phenolics and phytoalexins are altered, depending upon the pathogen. Similarly, rhizobia bacteria and mycorrhizal fungi induce changes in amount and types of compounds released from roots. During nutritional stress (i.e. P limitation), Al toxicity, low Fe availability, and exposure to heavy metals, roots show enhanced synthesis and exudation of several organic acids (anions) (Dinkelaker et al., 1989; Delhaize et al., 1993; Delhaize & Ryan, 1995; Ryan et al., 1995a,b, 1997; Larsen et al., 1998; Neumann et al., 2000). Of the changes in response to plant nutrition, those related to low P and excess Al are among those that have been most thoroughly documented. Convincing evidence now exists for exudation of malate and citrate as a principal mechanism in alleviating the edaphic stress of P-deficiency and Al-toxicity. The release of organic acids allows for the chelation of Al3+, Fe3+ and Ca2+, and subsequent displacement of Pi from bound or precipitated forms (Gerke, 1994; Jones, 1998; Hinsinger, 2001; Ryan et al., 2001), and may also cause organic P to become more susceptible to hydrolysis by acid phosphatases (Gerke, 1994; Braum & Helmke, 1995) (Fig. 3B). In Al-toxic soils root exudation of organic acids protects plants by chelating Al3+ ions in the rhizosphere, thus excluding their entry into the root. Because several recent excellent reviews (Jones, 1998; Lopez-Bucio et al., 2000b; Hinsinger, 2001; Ryan et al., 2001) have critically evaluated organic acid synthesis and exudation from roots, we will focus on the salient features related to the role of organic acids in alleviating P-deficiency in cluster roots of white lupin, including recent evidence for a potential efflux mechanism.
When white lupin is P deficient, cluster roots synthesize and exude striking amounts of malate and citrate (Gardner et al., 1982; Dinkelaker et al., 1989; Johnson et al., 1994; Keerthisinghe et al., 1998; Neumann et al., 1999). Grown under P-deficiency, cluster roots exude 20- to 40-fold more citrate and malate than P-sufficient roots (Fig. 3b). The amount of carbon exuded in these two compounds can range from 10% to greater than 25% of the total plant dry weight. Surprisingly, P-deficient plants do not appear to suffer any loss in either dry matter accumulation or N fixation until the reproductive stage of growth (Dinkelaker et al., 1989; J. Schulze, G. Gilbert, C. Vance, unpubl. data). Although the internal concentrations of organic acids increase in P-deficient roots, it is not proportional to the amount released as exudates. This suggests specific selective synthesis and exudation of malate and citrate in response to P-stress.
Measurements of CO2 fixation and pulse-chase labeling with 14CO2 show the carbon required for enhanced organic acid exudation from cluster roots of P-deficient white lupin is derived from both photosynthetic CO2 fixation and dark CO2 fixation by cluster roots (Johnson et al., 1994, 1996a). Photosynthetic CO2 fixation provides about 65% of the carbon exuded, while cluster root dark CO2 fixation provides about 35%. Rates of dark CO2 fixation in cluster roots are similar to those occurring in legume root nodules (Johnson et al., 1996a). The increased cluster root in vivo CO2 fixation is accompanied by enhanced specific activity of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), and citrate synthase (CS) (Johnson et al., 1996a,b; Gilbert et al., 1999; Uhde-Stone et al., 2003a).
Enhanced exudation of organic acids may not only occur because of upregulation of selected reactions leading to greater synthesis but also may be due to either reduced degradation or utilization of citrate. The activity of aconitase (AC) is reduced in cluster roots (Neumann & Römheld, 1999; Neumann et al., 1999). Similarly, respiration rates are also reduced in P-deficient cluster roots (Johnson et al., 1994; Neumann et al., 1999). Both phenomena could result in more citrate available for exudation.
Organic acid anion (OA−) exudation from cluster roots of white lupin coincides with a marked acidification of the rhizosphere (Marschner et al., 1987; Dinkelaker et al., 1989; Neumann & Martinoia, 2002). This is consistent with reports of a correlation of OA− exudation and the release of protons from white lupin and chickpea roots (Neumann & Römheld, 1999; Sas et al., 2001). Sas et al. (2001) have shown that extrusion of protons and organic acids in white lupin was highly dependent upon P supply. On an equimolar basis, H+ extrusion in P-deficient plants was two- to three-fold greater than organic acid exudation. Similarly, a recent report by Yan et al. (2002) showed substantially enhanced proton release from cluster roots of P-deficient white lupin. Proton release occurred primarily from young clusters and decreased significantly as cluster roots aged. During prolonged P deficiency in white lupin plants, OA− exudation from cluster roots increased while the release of protons decreased, indicating that different mechanisms are involved in proton release and OA− exudation (Sas et al., 2001). A comparison with tomato (Lycopersicon esculentum) showed an increase of the net release of protons from roots of P-starved tomato while OA− excretion from tomato roots decreased under P deficiency (Neumann & Römheld, 1999). Wheat (Triticum aestivum) displayed a decrease in OA− exudation under P deficiency and no proton extrusion (Neumann & Römheld, 1999).
Proton release into the rhizosphere is a common response of plant cells to counter intracellular acidity (Raven & Smith, 1976). Generally, proton release results from the activity of a plasma membrane H+ ATPase. This enzyme uses ATP to pump protons out of the cell, thereby creating pH and electrical potential differences across the plasmalemma. Yan et al. (2002) reported enhanced catalytic activity of a plasma membrane H+ ATPase of P-deficient cluster roots in white lupin that might be responsible for the increase in H+ extrusion. Characterization of the H+ ATPase from P-deficient cluster roots showed a threefold increase in Vmax and a fourfold increase in H+ ATPase enzyme protein compared with P-sufficient roots. Only the youngest P-deficient cluster roots had significant proton extrusion and H+ ATPase activity. It is noteworthy that Sakano (2001) addresses the potentially problematic generation of protons during OA− synthesis via glycolysis in his revised pH-stat hypothesis of the plant cell. By contrast to the ‘feed-forward’ fashion of the nonplant system, plant glycolysis is regulated by a feedback process. This feedback regulation only permits glycolytic activity when the cytoplasm is alkaline enough to stimulate PEPC, an enzyme with an alkaline pH optimum (Sakano, 2001). As Sakano points out, active H+ extrusion is a possible response of the plant cell to prevent the inhibiting effect of cytosolic acidification.
While neither the specific cell types for nor the mechanism of organic acid exudation have been characterized for cluster roots of white lupin, studies on Al tolerance in wheat (Ryan et al., 1997) and maize (Zea mays) (Kollmeier et al., 2001; Pineros & Kochian, 2001) have identified the root apex as the zone of organic acid exudation. Moreover, these patch-clamp and inhibitor studies indicate that Al may activate a malate/citrate-permeable anion channel to facilitate the exudation of large quantities of organic acids. Zhang et al. (2001) showed that anion channel frequency and rapidity of activation was greater in an Al3+-tolerant wheat line compared with a near-isogenic Al33+-sensitive line. Neumann et al. (1999) showed that exposure of P deficiency induced cluster roots to the anion channel-blockers ethacrynic- and anthracene-9-carboxylic acids inhibited citrate exudation by 40–60%. Moreover, we have recently shown through in situ hybridization (Uhde-Stone et al., 2003a) that PEPC and MDH transcripts are localized to cluster root apices and elongation zones, indicating the enzymes necessary for malate and perhaps citrate synthesis occur in the root tips (Fig. 5). Our data coupled with that of Neumann et al. (1999) suggest that P deficiency induced rapid exudation of organic acids, similar to Al3+-tolerance, may involve selective anion channel proteins at sites of exudation. Since malate and citrate are fully dissociated in the cytosol and because cell membranes are in essence impermeable to ions, it is not surprising that the release of organic acids from root tips in either P deficiency or Al toxicity might involve some type of channel protein.
Figure 5. In situ localization of phosphoenolpyruvate carboxylase (PEPC) in P-deficient cluster roots of white lupin. Sections A–J were hybridized with 35S-labeled antisense RNA derived from LaPEPC1 cDNA, whereas section K was hybridized with the corresponding sense RNA probe. The dark-field (a, c, e, g, i and k) and bright-field (b, d, f and j) images correspond to longitudinal and transverse sections through cluster rootlets 9 d after emergence (DAE) (a, b) and 12 DAE (c–h) as well as through 12 DAE +P normal roots (i, j, k). Arrows in d indicate the approximate location of the two transverse sections shown in e–h. The areas that are white in a, c, e, g and i indicate the presence of PEPC mRNA. Sense control (K) showed no reaction. Bars are 50 µm (e, g), 100 µm (a) and 200 µm (c, i, k); pi, proteoid initials; vb, vascular bundle; c, cortex; e, epidermis, am, apical meristem, rc, rootcap. (Derived from Uhde-Stone et al., 2003a.)
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Although a large number of cation channels and transporters belonging to a multiplicity of gene families have been identified over the past decade (Cao et al., 1995; Maathuis & Sanders, 1999; Barbier-Brygoo et al., 2000; Howitt & Udvardi, 2000; Theodoulou, 2000; Maser et al., 2001), the molecular events involved in organic acid (anion) excretion remain elusive. While anion channels are probably ubiquitous in plants, few have been characterized (Schroeder, 1995; Barbier-Brygoo et al., 2000). Mechanisms of anion transport potentially could include chloride channels (CLC), aquaporins, and multidrug extrusion proteins (MATE). To date, only putative anion channel genes related to the CLC family have been characterized (Hechenberger et al., 1996; Lurin et al., 1996; Geelen et al., 2000). These genes encode messages of approximately 2.4 kb giving rise to proteins of approximately 785 amino acids with Mr of c. 85 kDa. The Arabidopsis CLC-d gene could functionally substitute for a yeast CLC protein involved in iron-limited growth, while the CLC-a gene appeared to be related to nitrate acquisition. Anion channel genes encoding transmembrane proteins involved in organic acid efflux have yet to be characterized.
During our annotation of ESTs from cluster roots we found that several proteins with putative transport-related functions were highly expressed in P-stressed conditions. One of these ESTs was redundant (found 10 times) and showed a ninefold induction in -P cluster roots (Uhde-Stone et al., 2003b). This EST displayed high homology to a putative integral membrane protein in Arabidopsis belonging to the MATE protein family. MATE proteins are a large family of putative antiporters that are thought to be involved in excretion of a variety of drugs and toxins (Brown et al., 1999; Debeaujon et al., 2001; Diener et al., 2001). Around 55 putative members of the MATE family have been predicted in Arabidopsis, two distantly related MATE proteins have been predicted in yeast. To date, only a few MATE proteins have been cloned (Debeujon et al., 2001; Diener et al., 2001; Rogers & Guerinot, 2002). An Arabidopsis MATE has been shown to control response to iron deficiency. It represents either an Fe sensor or transporter of small organic molecules (Rogers & Guerinot, 2002). Analysis of the white lupin P-stress induced MATE revealed a 2.1-kb cDNA encoding a deduced amino acid sequence of 531 amino acids having 10–12 transmembrane helices, depending upon the program used for analysis. These findings make this putative MATE EST an extremely interesting candidate for a role in organic acid excretion. Functional studies in yeast and Xenopus oocytes will allow us to examine the role of the MATE type protein in organic acid excretion.
Alternative glycolytic pathways
Besides increased acquisition of soil P, conservation of internal pools of acquired Pi is considered an important adaptation for growth on low P. As several enzymes of the glycolytic pathway depend on Pi or adenylates as cosubstrates, metabolic processes may be impaired by severe P deprivation. Yet the generation of energy and the production of carbon skeletons continues during conditions of P limitation, as evident in the excretion of significant amounts of organic acids. In response to limited P, many plants show remarkable flexibility in adjusting metabolic rates and utilizing alternative metabolic pathways. Alternative glycolytic reactions can bypass Pi- or ATP-requiring steps of glycolysis under environmental stress conditions such as Pi starvation or anoxia (Duff et al., 1989b; Mertens, 1991; Theodorou et al., 1992; Theodorou & Plaxton, 1996). While ATP and ADP-levels have been reported to decline under P deficiency (Ashihara et al., 1988; Duff et al., 1989b), pyrophosphate (PPi) concentrations appear to be buffered during Pi stress (Duff et al., 1989b; Dancer et al., 1990). Despite an earlier assumption of cellular bioenergetics that PPi anhydride bonds were not utilized in cellular metabolism, studies have shown that under certain environmental stresses PPi can serve as an energy donor in the plant cytosol (Duff et al., 1989b; Plaxton, 1996; Plaxton & Carswell, 1999). A well-documented alternative glycolytic pathway is catalysed by a PPi-dependent phosphofructokinase (PFP) that, under P deficiency, can bypass the ATP-dependent phosphofructokinase (PFK), generating fructose 1,6-biphosphate (Theodorou et al., 1992; Theodorou & Plaxton, 1996; Plaxton & Carswell, 1999). Other processes that might use PPi are the cleavage of sucrose by a PPi-dependent sucrose synthase pathway and the active transport of protons into the vacuole by a PPi-dependent H+ pump in the tonoplast (Plaxton & Carswell, 1999). PPi has been assumed to be a byproduct of secondary metabolism and anabolism, therefore use of PPi as an energy donor helps to conserve limited ATP pools (Duff et al., 1989b; Theodorou et al., 1992; Plaxton, 1996; Theodorou & Plaxton, 1996; Plaxton & Carswell, 1999).
Another alternative glycolytic pathway known in plants is catalysed by the action of a nonphosphorylating NADP-dependent glyceraldehyde-3P dehydrogenase (NADP-G3PDH) that bypasses Pi-dependent NAD-G3PDH and phosphoglycerate kinase (Duff et al., 1989b; Theodorou et al., 1992; Plaxton & Carswell, 1999). A third bypass of the glycolytic pathway can be catalysed by the combined activities of PEPC, MDH and NAD-malic enzyme (Theodorou & Plaxton, 1993). Phosphorus stress can severely limit the activity of pyruvate kinase (PK), an enzyme requiring Pi and ADP. The PEPC, MDH and NAD-malic enzymes can bypass PK and thus maintain the flow of carbon from glycolysis to the TCA cycle by avoiding the use of ADP but generating free Pi (Theodorou et al., 1992; Plaxton & Carswell, 1999).
The described metabolic adaptations to Pi stress appear to be plant specific. P-deficiency induced PFP activity was found in plants that are adapted to infertile soils and contain very low PFP activity when grown under sufficient P, e.g. Arabidopsis, rape (Brassica napus), sugar beet (Beta vulgaris), and buckwheat (Fagopyrum esculentum) (reviewed in Plaxton & Carswell, 1999). Murley et al. (1998) reported that P-stress inducible PFP activity is not found in plants that form symbiotic associations with mycorrhizal fungi. In plants that typically contain abundant PFP activity when grown under sufficient P, PFP activity stayed unchanged or declined under P deficiency (in tobacco (Nicotiana tabacum) seedlings, bean seedlings and tomato root cultures; Plaxton & Carswell, 1999). Analysis of ESTs from P-deficient cluster roots of white lupin indicated the induction of genes encoding for PFP and the PEPC/MDH bypass of PK, but did not reveal evidence for the involvement of a nonphosphorylating NADP-dependent G3PDH (Fig. 6).
Figure 6. Schematic representation of the glycolytic pathway. Expressed sequence tags (ESTs) with induced expression in P-deficient cluster roots, compared with P-sufficient normal roots, are represented in gray boxes. The average of gene induction, as determined by two independent macroarrays is indicated at the corresponding arrows (e.g. 2×). Enzymes of the glycolytic pathway that were not found in a collection of 1250 ESTs are shown in white boxes and are represented by dotted arrows. The majority of ESTs with possible function in the glycolytic pathway displayed increased expression in P-deficient cluster roots compared with P-sufficient normal roots. (Derived from Uhde-Stone et al., 2003a.)
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Alternative mitochondrial respiration
Phosphorus limitation impairs mitochondrial respiration in many plants. Considering the adaptive flexibility of plant responses to P deficiency that is displayed in the glycolytic pathway, adaptations which allow alternative mitochondrial electron transport under P starvation have been hypothesized (Rychter & Mikulska, 1990; Theodorou & Plaxton, 1993).
The significant reduction in cellular ADP and Pi levels in plants undergoing P limitation is thought to inhibit the cytochrome (Cyt) pathway of electron transport. Low ADP and Pi can result in a high ATP : ADP ratio which inhibits respiration, a phenomenon well known as adenylate control of respiration (Bryce et al., 1990). Nonphosphorylative pathways that can bypass energy-requiring sites include rotenone-insensitive NADH dehydrogenase (Rasmusson & Moller, 1991) and the alternative oxidase (AOX) (Lambers, 1985; Lance et al., 1985; Day & Wiskich, 1995). The cyanide-resistant AOX catalyses the oxidation of ubiquinol and the reduction of oxygen to water but bypasses the last two energy-conserving sites normally associated with the Cyt pathway. Alternative oxidase activity is subject to regulation by a sulfhydryl–disulfide redox system (Siedow & Umbach, 2000) and by allosteric activation via α-keto acids, especially pyruvate (Millar et al., 1993; Hoefnagel et al., 1995). It was proposed that induction of AOX during P deficiency might be a consequence of elevated PEPC activity.
In roots of P. vulgaris, P deficiency led to decreased activity of the respiratory cytochrome pathway, while alternative, cyanide-resistant respiration and the NADH : NAD ratio increased (Rychter & Mikulska, 1990; Rychter et al., 1992). Indication for a possible link between AOX respiration and P starvation has also been found in Catharanthus roseus (Hoefnagel et al., 1993), Chlamydomonas reinhardtii (Weger & Dasgupta, 1993) and tobacco cell cultures (Parsons et al., 1999). However, oxidative respiration rates under P deficiency can vary greatly among different plants. Respiratory O2 consumption in the green alga Scenedesmus obtusiusculus was stimulated during the first 96 h of P starvation (Tillberg & Rowley, 1989), while pea (Pisum sativum) roots displayed no effect of P starvation on respiration (reviewed by Theodorou & Plaxton, 1996). The respiration rates of P. vulgaris, Selenastrum minutum, Lemna gibba and suspension-cultured C. roseus, however, declined under P deprivation (Theodorou & Plaxton, 1996). In P-deficient cluster roots of white lupin, up to 60% reduction in mitochondrial respiration, as measured by O2 consumption, has been reported (Johnson et al., 1994; Neumann et al., 1999), accompanied by an increased NADH : NAD ratio. These results indicate that in some plants inhibition of the CN-sensitive cytochrome pathway of respiration might not be fully compensated by AOX activity.
Enhanced glycolysis in P-deficient white lupin was accompanied by significant induction of genes encoding for an alcohol dehydrogenase (Massonneau et al., 2001) and a formate dehydrogenase (fdh) (Uhde-Stone et al., 2003b), indicating a possible role of fermentative processes during P deficiency. In the green algae C. reinhardtii, phosphate starvation resulted in the accumulation of an mRNA with homology to pyruvate formate-lyase, another enzyme of fermentative metabolism (Dumont et al., 1993). Taken together, these findings indicate that the increase of glycolytic activity and the resulting organic acid synthesis under P deprivation in white lupin could be, in part, a consequence of severely impaired oxidative respiration.
A switch from primary to secondary metabolism is a common response of plants undergoing nutrient limitation. Phosphorus deficiency typically results in the accumulation of secondary metabolites such as flavonoids and indole alkaloids (reviewed in Plaxton & Carswell, 1999). In rice culture, accumulation of the polyamine putrescine is thought to mediate growth inhibition in response to P depletion (Shih & Kao, 1996). Yamakawa et al. (1983) reported that P depletion induced anthocyanin accumulation in Vitis cell culture, while P supplementation induced primary metabolism and inhibited anthocyanin synthesis. Increased synthesis of anthocyanins is a frequent response of plants to P deficiency and presumably functions to ameliorate photoinhibitory damage to chloroplasts (Takahashi et al., 1991).
In addition to the possible involvement of phenolics in senescence, phenolic compounds can be exuded into the rhizosphere in response to P starvation and might act as chelators and/or reductants increasing the release of bound Pi. Examples of phenolics that are exuded into the rhizosphere are piscidic acid from roots of pigeon pea (Cajanus cajan) (Ae et al., 1990), alfafuran from alfalfa (Medicago sativa) roots (Masaoka et al., 1993), and isoflavonoids from P-deficient cluster roots of white lupin (Neumann et al., 2000).
Sclerophylly, characteristic of many of the Proteaceae, is also thought to be an adaptation involving enhanced phenolic metabolism by many species that occupy low P habitats. Sclerophylls are typically considered to be hard and stiff leaves that are tough (Turner, 1994). They are characterized by a thick cuticle and outer epidermal cell wall with abundant sclerification (Loveless, 1961, 1962). Bundle sheaths and leaf margins in particular show sclerification. The secondary wall of sclerids are heavily lignified, hence the relationship to phenolic secondary metabolism. Loveless (1962) postulated that sclerophylly is an adaptation to conserve nutrients particularly P and N. Turner (1994) concluded that sclerophylly is also related to protection against herbivory.
Enzymes in the phenolic and flavonoid pathways have been reported to be upregulated in Pi-deficient plants (Plaxton & Carswell, 1999). Several white lupin ESTs with high similarity to enzymes involved in secondary metabolism were represented in our cDNA library derived from mature cluster roots of P-deficient plants, including DAHP (3-deoxy-d-arabino-heptulosonate-7-phosphate synthase), phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase, isoliquiritigenin 2′-O-methyltransferase, 4-coumarate-CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase (http://www.home.earthlink.net/~whitelupinacelimation). Whether these enzymes are involved in either anthocyanin or lignin biosynthesis remains to be established. By contrast to primary metabolism, secondary metabolism in general does not consume as much Pi but can serve to recycle significant amounts of Pi from phosphate esters (Plaxton & Carswell, 1999; Sakano, 2001). However, secondary metabolism produces an excess of reducing equivalents. The resulting cytosolic acidification could lead to the activation of AOX and other pathways that can relieve the accumulation of reducing equivalents (Sakano, 2001).