Metabolic regulation of pH in plant cells: Role of cytoplasmic pH in defense reaction and secondary metabolism

Metabolic Regulation of pH in Plant Cells: Role of Cytoplasmic pH in Defense Reaction and Secondary Metabolism KatsuhiroSakano Department of Plant Physiology, National Institute of Agrobiological Tsukuba, Ibaraki 30.58602 Japan

Resources,

A new biochemical pH-stat hypothesis that revised the classic hypothesis is presented to understand the metabolic regulation of intracellular pH in plant cells. Alternative pathway glycolysis, alternative pathway respiration and malate-derived lactic and alcoholic fermentation (alternative pathway fermentation), all unique to plants, are integrated into a regulatory mechanism of pH in the cytoplasm. Its uniqueness to plant kingdom is discussed from the evolutionary viewpoint: it is suggested that when the ancestors of extant terrestrial plants expanded their habitat from oceans to freshwater, they abandoned a “sodium system” and adopted a “proton system” for nutrient uptake. Validity of the new hypothesis is examined with available data on a secondary active transport, anoxia and other experimental evidence. The hypothesis predicts that biotic and abiotic stress-induced cytoplasmic acidification triggers synthesis of phytoalexins and other secondary metabolites. Possible roles of cyanide-resistant alternative pathway respiration in the secondary metabolite production, metabolic switching between primary and secondary metabolisms, and defense reactions are proposed. KEY WORDS: Alternative oxidase, Cytoplasmic acidification, Defense reaction, Evolution of plants, Oxidative burst, Phytoalexin, Secondary metabolism, Secondary active transport. 0 2001 Academic press.

Intemtio~l Review of Cytology, Vol. 206 0074-7696/01 $35.00

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KATSUHIROSAKANO

I. Introduction Intracellular pH regulation is one of the most basic processes of all living organisms (bacteria, protista, algae, plants, animals, etc.) on earth. Despite their different habitats (terrestrial, ocean, freshwater, etc.) and independent evolution, cytoplasmic pH is kept in a similar, narrow range, 7.0-7.5, which enabled the organisms to use essentially the same and common basic primary metabolisms (glycolysis, pentose phosphate pathway, Krebs cycle, protein and other macromolecule syntheses, etc.). However, this does not mean the regulatory mechanisms for cytoplasmic pH is also the same among organisms of different habitat. As discussed later, plant cells that use H+ pump as the primary pump to energize the plasma membrane mainly employ organic acid metabolism for the cytoplasmic pH regulation when the pump is inhibited. In contrast, animal cells that use Na+/K+ pump generally utilize Na+/H+ antiport instead of organic acid metabolism for the same purpose. Why are they different? As far as I know, no one has ever discussed this question. Almost a decade ago, Guern et al. (1991) presented an exhaustive review on intracellular pH regulation of plant cells. They discussed intracellular pH regulation in relation to environmental factors (light, temperature, anoxia, etc.) and its experimental manipulations. They pointed out the primary importance of pH control by biophysical processes at the plasma membrane and tonoplast (Hf pumping and ion fluxes through channels), and further mentioned that control by metabolism (biochemical pH stat) is a fine-tuning mechanism of the cytoplasmic pH that works in coordination with the membrane processes (coarse control). It is not likely that any report appearing during the last decade upset the aspects presented in the previous review, except that the classic biochemical pH stat proposed by Davies (1973, 1986) was substantially revised to conquer its inherent shortcomings (Sakano, 1998). In the present review, the main subject is a new aspect of metabolic regulation of the cytoplasmic pH in plant cells. In addition, I describe possible roles of cytoplasmic pH, as an early component of the signal transduction pathway, in the defense reaction of plant cells against pathogens. Possible involvement of alternative pathway respiration in the plant defense reaction is a recent concern in plant pathology (Murphy et al., 1999). However, the role of alternative pathway respiration in the defense reaction is unknown. In this review, I try to implicate oxidative burst that is induced by infection or elicitor treatment to cytoplasmic acidification, activation of pentose phosphate pathway and alternative pathway respiration, and synthesis of phytoalexins and other secondary metabolites including phenylpropanoids and terpenoids. The rationale that explains switching from the primary to secondary metabolism through activation of alternative pathway respiration is also presented.

METABOLICREGULATIONOFpHlN PLANTCELLS

II. An Unquestioned Question: Why Proton System in Plants? A. From Sodium System to Proton System Sakano (1998) assumed that the first cells born in the ancient ocean collected external nutrients by means of a “sodium system.” At the expense of the Na+ electrochemical potential gradient across the plasma membrane created by Na+ pump, nutrients were taken up by Na+ symport. This is probably because extrusion of Na+ was prerequisite to avoid Naf accumulation in the cell and, at the same time, it was the most efficient way to energize the plasma membrane to drive active transport in the saline environment as well. Therefore, we can speculate that all the outset-oceanic lives utilized, in order to carry out secondary active transport, the Na+ electrochemical potential gradient across the plasma membrane that was created by Na+ pump. However, when some of these cells expanded their habitat via brackish water into the freshwater range, only those that developed a transport system that could work in the sodium-free environment were allowed to enter the “New World.” The plant ancestor cell was the first to advance into freshwater. Unless autotrophic plants (green algae) could thrive in the New World, animals that are heterotrophic (by definition) had no motive to enter that world. Freshwater animals seem to have created a sodium system inside their cells (protozoa, mesozoa) or inside their bodies (metazoa). Being obligate heterotroph, animal cells do not absorb nutrients directly from external medium like plant cells. Primitive animal cells caught food, say, tiny nutritious algal cells, by endocytosis. After internalization of the endocytotic vesicle and its fusion to vacuoles, the algal inclusion is digested in the vesicle (digestive vacuole). The resultant digest, containing amino acids, sugars, nucleotides, inorganic ions, etc., is then taken up into the cytosol, probably utilizing the sodium electrochemical potential gradient across the vesicle membrane. As far as the author knows, there seems to be no direct evidence for sodium. However, Baldwin (1949) described the interior of the digestive vacuole at the beginning of digestion as acidic, but in the later stage it turns alkaline. The alkalinization suggests an establishment of a sodium system at the vesicle membrane. Thus, in their adaptation to freshwater, the external high water potential was all the organisms could manage in order to avoid osmolarity fall of the cytoplasm by penetrating water. The contractile vacuoles, seen in every primitive freshwater animal cell, solved the problem: they collected excess intracellular water and extruded it outside the cells. Extant metazoan animals inherited a sodium system from their ancestors that succeeded in being effective by incorporating “ancient ocean” in their interstitial fluid as the internal environment (Baldwin, 1949). Thus, animal cells are compared to being immersed in the

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internal ocean. As long as the daily loss of interstitial Na+ is replenished, their sodium system is effective in freshwater and ashore as well. In the digestion and absorption of nutrients, they developed the intestine, a specialized extracellular mechanism based on sodium system. Digestive juice secreted by pancreas to the intestine contains not only hydrolytic enzymes such as protease, lipase, and glucosidase, but also sodium chloride and sodium bicarbonate. The alkalinity of the juice promotes enzymatic digestion and Na+ in it facilitates nutrient absorption by the epithelium cells that utilize sodium system. In contrast, green algal cell, the putative ancestor of extant terrestrial plants (Graham, 1985), must have advanced into the freshwater in a different way. Being obligate autotroph, the ancestor was forced to abandon the sodium system that was now useless in freshwater to absorb nutrients such as K+, NO;, Pi-, etc. Endocytosis (pinocytosis), like that in heterotrophic primitive animal cells, was not employed for nutrient acquisition, because it was too inefficient in freshwater. Instead, the plant ancestor cell invented a “proton system,” in which the H+ electrochemical potential gradient (which is the composite of electric potential of membrane and pH gradient) across the plasma membrane that is generated by H+ pump was the driving force for the secondary active transport. The decisive merit of the proton system adopted by plants is that H+ can be produced internally by metabolism. This is a great advantage to plants that are immobile, and sharply contrasts with animals: they must seek for Na+, an essential nutrient, in the external environment. There is evidence that in certain aquatic angiosperms and charophytic species, secondary active transport is energized by sodium electrochemical potential gradient mediate K+ uptake (Maathuis et al., 1996). In characeae, Na+ symport is reported to be the main uptake mechanism of Pi (Mimura, 1999), in which the symport may be coupled to Na+/H+ antiport energized by proton pump. Such Na+/Pi symport may be a remnant of an intermediary step in the evolution of the proton system. Freshwater green algae and characeae are reported to be the intermediary step in the evolution of extant terrestrial plants (Graham, 1985).

6. Why Plants Have Unique Biochemical

pH Stat

Though protected by the cell wall, plant cells that replaced the sodium system with the proton system are essentially “naked” in relation to the external environment. The physical strength of the cell wall against turgor restricts water from unlimited influx into cells under a high-water-potential environment, and prevents dilution of cytoplasmic constituents. The cell wall functions as an immediate external environment. Its cation exchange capacity functions as a “buffer” against rapid changes in the external conditions, such as ionic strength and pH. Despite that, the function of the cell wall is far inferior to that of the interstitial fluid in animals: the cell wall is more or less in passive equilibrium to the external environment (Grignon and Sentenac, 1991).

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METABOLICREGULATIONOFpHlN PLANTCELLS

In the proton system, secondary active transport, such as H+/glucose symport and Ca+/H+ antiport, utilizes the electrochemical potential gradient of Hfs across the plasma membrane. It depends on a coordination of ATP-dependent H+ efflux and accompanying passive influx of K+ across the plasma membrane. Under favorable conditions where H+ efflux (driven by Hf pump) balances H+ influx (accompanied by symport and/or antiport), there may be no disturbance in the cytoplasmic pH. Under stressconditions (limited oxygen supply, low temperature, etc.), however, an ATP shortage and other factors including inactivation (inhibition) of the H+ pump itself, would limit H+ pumping and lead to cytoplasmic acidification. This can also happen under normal conditions if the H+ influx accompanied by secondary active transport exceeds the capacity of H+ extrusion by H+ pump (Sakano et al., 1992). How can plant cells cope with such an inherent potential hazard, cytoplasmic acidification, under stress conditions? At this point, the plant-specific existence of biochemical pH stat is justified as the security mechanism of the proton system.

III. Metabolic

Regulation

of Intracellular

A. Classic Biochemical pH-Stat and Its Shortcomings

pH

Hypothesis

Davies (1973, 1986) proposed his (classic) “biochemical pH-stat” hypothesis as a fine-tuning mechanism of cytoplasmic pH, in which a set of carboxylating and decarboxylating enzymes with different pH optima worked in coordination to regulate the cytoplasmic pH. Of the models proposed (Davies, 1986), the combination of phosphoenolpyruvate (PEP) carboxylase and malic enzyme has been the most popular and realistic. As shown in the inset in the lower left block of Fig. 1, when the cytoplasmic pH shifts toward the alkaline range, PEP carboxylase @, having its optimum pH on the alkaline side of the target pH to which the cytoplasm is to be adjusted, produces more oxaloacetate (OAA), which in turn is transformed to malate by malate dehydrogenase 0. Because the malate is a strong acid, the newly formed carboxyl group dissociates and the pH shift is nullified (Smith and Raven, 1979). (This is not correct. The actual production of H+ associated with malate synthesis is a result of the early reactions of glycolysis prior to PEP carboxylase. See next section.) In contrast, when the pH shift is toward the acid range, malic enzyme 8, which has its optimum pH in the acid range, would decarboxylate more malate and result in a correction of the pH shift. Thus, cytoplasmic pH regulation would be established through the synthesis and degradation of malate by coordination of the two enzymes.

.. .

FIG. 1 Structure and function of the revised biochemical pH stat in a plant cell. (Structure) Heavy and dotted arrows indicate the main flow of the metabolism related to the function of the pH stat and its regulation (activation: $; inhibition: a) by metabolites, respectively. Arrows from H+ point to the H+-consuming reactions, whereas (2e- + H’) indicates electron-plus-proton equivalent of NADH. The pH stat consists of four functional units of metabolism. The left block (from glucose to pyruvate via malate, on white plate) denotes the alternative glycolytic pathway, which functions as a H+ source and pH-sensitive trigger of the pH-stat (shaded plate). The protonogenic reactions are hexokinase 0, phosphofructokinase (PFK) a, and glyceraldehyde phosphate dehydrogenase 0. The pH-sensitive trigger unit of the pH stat is essentially the same as that in the classic biochemical pH stat (Davies, 1986) except that the pyruvate kinase @ reaction is drawn isolated from the main route. The upper right block (alternative pathway respiration @I, on shaded plate and cytochrome pathway respiration @, on white plate) and the lower right block (alternative pathway fermentation, on white plate) are the H+ sink units of the pH stat under aerobic and anaerobic conditions, respectively. (Function) In response to alkaline-pH stimulus, activity of PEP carboxylase @ increases according to the pH-activity curve shown in the inset, resulting in promotion of the carbonic anhydrase reaction 0 (protonogenic) and activation (deinhibition) of PFK Q due to consumption of PEP and production of Pi by PEP carboxylase (Plaxton, 1996). OAA (oxaloacetate) is reduced to malate by malate dehydrogenase @ (H+ consuming). By the conversion of 1 mol of glucose to 2 mol of malate, 4 Eq of H+s are produced. Upon acid-pa stimulus, activity of malic enzyme @ increases according to the pH-activity curve (inset) and produces more pyruvate, NADH, and CO2, of which, under aerobic conditions, pyruvate (Millar et al., 1996; Hoefnagel et al., 1997) and NADH (Umbach and Siedow, 1993) activate the alternative pathway respiration @I and CO2 inhibits the cytochrome pathway respiration @ (Gonzalez-Meler et al., 1996) resulting in diversion of more electrons (and, hence, more H+) to the alternative pathway. Although the cytochrome pathway 0 is subject to control by energy charge, the alternative pathway 8 is not. Thus, any excess H+ that acidify the cytoplasm and activate malic enzyme are destined to quick disposal, preferentially through the alternative pathway respiration irrespective of the energy charge of the cell. Under anaerobic conditions (lower right block), pyruvate and NADH, derived from the malic enzyme reaction, are transformed either to lactate by lactate dehydrogenase @ or to ethanol by way of pyruvate decarboxylase 0 and alcohol dehydrogenase @I (Roberts et al., 1992). All of these reactions consume H’. Note that the origin of lactate and ethanol when produced for pH control is malate, not glucose via pyruvate kinase @. (From Sakano, 1998.)

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The classic biochemical pH-stat hypothesis (Davies, 1973, 1986), together with the biophysical pH-stat hypothesis (Smith and Raven, 1979), has been widely accepted, because it explains well the changes in endogenous malate level in response to experimental treatments that were expected to cause cytoplasmic pH shifts (Haschke and Ltittge, 1975; Johnson and Rayle, 1976; Stout et al., 1978; Marre, 1979; Romani et al., 1983; Mathieu et al., 1986; Sakano et al., 1997; Smith and Raven, 1979; Kurkdjian and Guern, 1989). However, examination of component reactions of the classic biochemical pH-stat mechanism revealed two critical shortcomings: Shortcoming I: Malate synthesis through PEP carboxylase @ (Maruyama et al., 1966) and malate dehydrogenase 0 does not produce but, on the contrary, consumes a H+ near neutral pH. PEP carboxylase @ : PEP3- + HCO; + OAA’- + Pi* Malate dehydrogenase 0: OAA2- + NADH + H+ + malate*- + NAD+ Shortcoming 2: Malate decarboxylation by malic enzyme @ (Macrae, 1971) does not consume a H+. [In the original model, Davies (1973) employed NADP malic enzyme (EC. 1.1.1.40) of the cytoplasm, but in his revised model (Davies, 1986) he suggested NAD malic enzyme (EC. 1.1.1.39) in the mitochondria because its response to pH change is much sharper than that of NADP malic enzyme.] Malic enzyme @: malate’- + NAD+ + pyruvate- + NADH + CO2

6. Revision of Biochemical

pH Stat

The inherent inconsistency of the classic theory went unrecognized for longer than two decades before Sakano (1998) pointed it out. In his revised hypothesis (Fig. l), he placed the classic biochemical pH-stat mechanism within the whole flow of the metabolism that precedes (glycolysis) and follows it (respiration), and reassigned the roles of PEP carboxylase 0 and malic enzyme @ as pH-sensitive triggers of H+-generating and H+-consuming metabolisms, respectively. 1. Metabolic Flow (glycolysis) Preceding Malate Synthesis When PEP is consumed by PEP carboxylase @, it is replenished through glycolysis, which is H+ producing. If the starting material is glucose, the reactions leading

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KATSUHIROSAKANO

to malate via PEP carboxylase @ produce net H+. The protonogenic reactions are: Hexokinase ($ : Glucose + ATP‘- + G6P2- + ADP3- + H+ Phosphofructokinase (PFK) 0: F6P2- + ATP+- -+ F1,6BP‘- + ADP3- + H+ Glyceraldehyde phosphate dehydrogenase 0: glycelaldehyde-3-P*- + NAD+ + Pi2- -+ 1, 3-diphosphoglycerate4+ NADH + H+ Carbonic anhydrase 0: CO2 + H20 -+ HCO, + H’ The rest of the glycolytic reactions leading to PEP neither consume nor produce a Hf (Busa and Nuccitelli, 1984). In the production of malate, we must take the carbonic anhydrase 0 reaction (H+ producing) that provides HCO, to the PEP carboxylase (Raven and Newman, 1994) and malate dehydrogenase 0 reaction (H+ consuming, as described above) into account. Then, the balance sheet of H+ during metabolic conversion of glucose (plus CO;?)to malate is the net production of 4 H+ per one glucose consumed (or 2 H+ per one malate produced). Thus malate synthesis from glucose is protonogenic. Malate synthesis from glucose : glucose + 2 CO2 -+ 2 malate2- + 4 H+ 2. Alternative Pathway of Glycolysis: The Proton Source of pH Stat It is noteworthy that plant glycolysis has unique features that are not seen in other organisms (Plaxton, 1996). One of them is the alternative route to pyruvate that branches at PEP. In addition to the ordinary route via the pyruvate kinase @ reaction (the only path in nonplant organisms), plants have another route via PEP carboxylase a,, malate dehydrogenase 0, and malic enzyme @ reactions. One of the physiological functions of this route has been assigned to bypassing the pyruvate kinase @ reaction during Pi starvation (Theodorou and Plaxton, 1995). Another unique feature of plant glycolysis is in its mode of control. In nonplant systems, glycolytic flux is controlled in a “feed-forward’ fashion: activation and deinhibition of the first key enzyme phosphofructokinase PFK 0 by various effectors (activators: AMP, fructose-2,6-biphosphate, etc.; inhibitors: ATP, citrate, etc.) lead to an increased level of fructose- 1,6-biphosphate (F 1,6BP), which in turn activates the second downstream key enzyme pyruvate kinase 8, and glycolysis is allowed to proceed. In contrast, in the plant system, the regulation is a feedback process: consumption of PEP either by pyruvate kinase @ or by PEP carboxylase @ relieves the PEP inhibition of upstream key enzyme PFK 0. Moreover, Pi,

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METABOLlCREGULATlONOFpHIN PLANTCELLS

another product of the PEP carboxylase reaction, also activates PFK 0 by relieving the PEP inhibition of PFK. From the viewpoint of intracellular pH regulation, feedback is the control mode suitable for avoiding overproduction of Hf, because the protonogenic glycolysis should be allowed only when the cytoplasm is alkaline enough to stimulate PEP carboxylase @. Such situations may be achieved under aerobic conditions when H+ extrusion is active in the presence of external K’ (Sakano et al., 1992, 1997) and under salt stress conditions (Katsuhara et al., 1997). In this respect, it is paradoxical that pyruvate kinase @ is activated by acid pH under conditions such as anaerobiosis and operation of active transports (Plaxton, 1996). This may indicate that, where there is an urgent requirement for energy, a plant cell dares to accept cytoplasmic acidification that may be compensated by the operation of biochemical pH stat as described below.

3. Role of PEP Carboxylase in Revised Biochemical pH Stat In the revised pH-stat mechanism, PEP carboxylase @ is characterized as the alkaline-pH-sensitive trigger of H+-generating machinery: glycolysis. When the cytoplasmic pH shifts toward the alkaline range, e.g., through H+ pumping in the presence of external Kf under aerobic conditions (Sakano et al., 1997), the resultant increasing consumption of PEP and simultaneous production of Pi by PEP-carboxylase would open the upstream gate of glycolysis at PFK 0 by releasing it from PEP inhibition (Plaxton, 1996). In the classic theory, this was incorrectly assigned as the H+ generator itself.

4. Metabolic Flow (Respiration) Following Malate Decarboxylation Although the malic enzyme @ reaction itself consumes no Hf, subsequent respiratory oxidation (8, @) of NADH (one of the reaction products of malic enzyme) consumes H+. If the oxidation is through cytochrome pathway respiration @, extra H+ will be consumed by accompanying oxidative phosphorylation @Y. Respiration 8, @: NADH + H+ + 1/202 --+ NAD+ + HZ0 Oxidative phosphorylation 0’: ADP3- + Pi2- + H+ + energy + ATI“-

+ Hz0

As described below, all products of the malic enzyme reaction @ are feed-forward regulators of the respiratory pathways. Pyruvate is destined to be metabolized in mitochondria through the Krebs cycle. But its role is more than a simple substrate of the cycle. The same is true of the role of NADH in respiration; even CO2 has its own regulatory function.

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5. Alternative Pathway Respiration: The Aerobic Proton Sink Cyanide-resistant alternative pathway respiration is a common feature of plant cells and tissues (Laties, 1982; Lambers, 1985; Lance et al., 1985; Lance, 1991; Day et al., 1995; Siedow and Umbach, 1995; Vanlerberghe and McIntosh, 1997) and seems as unique as the proton system is to the plant kingdom. Differing from cyanide-sensitive cytochrome pathway respiration, the electron transport from malic enzyme-derived NADH to molecular oxygen catalyzed by rotenoneinsensitive NADH dehydrogenase (Rasmusson and M@ller, 1991) and alternative oxidase (AOX) is not coupled to energy conservation (Rustin et al., 1980, Lance et al., 1985) and, therefore, is free from control by energy charge. This apparently futile respiration has been shown to increase under stress conditions: aging (Solomos, 1977), wounding, microbial infection (Uritani and Asahi, 1980), low temperature (Purvis and Shewfelt, 1993), and nutrient deficiency (Rychter and Mikulska, 1990; Rychter et al., 1992; Hoefnagel et al., 1993, 1994; Weger, 1996; Parsons et al., 1999), treatment with chemicals (Klerk-Kiebert et al., 1982; Morohashi et al., 1991; Gallerani and Romani, 1996; Padua et al., 1999). Its involvement is also reported in thermogenesis (Meeuse, 1975), CAM metabolism (Rustin and Queirotz-Claret, 1985, Robinson et al., 1992), and defense reaction (Chivasa et al., 1997; Chivasa and Carr, 1998). Since inhibition or suppression of alternative pathway respiration generates reactive oxygen species (ROS), it is proposed that the respiration removes excess reducing equivalents so that ROS are not produced (Wagner and Krab, 1995; Wagner and Moore, 1997). Recent extensive investigations have revealed that the partitioning of electrons between the cytochrome and alternative pathways is under the regulation of AOX activity, which is subject to further regulation by a sulfbydryl-disulfide redox system (Umbach and Siedow, 1993) and by allosteric activation by o-keto acids, especially by pyruvate (Millar et al., 1996, Hoefnagel et al., 1997). Although “energy overflow” function seems to have been the consensus (Lambers, 1985; Day et al., 1996, Vanlerberghe and McIntosh, 1997), no convincing explanation has been presented for its universal occurrence in plants. However, because reducing equivalents in biological systems occurs mostly in the form of NAD(P)H + H+, overflooding of reducing equivalents can be regarded as a state of cytoplasmic acidification. This is discussed in Section 1V.A. From the foregoing overview, it is now clear that alternative pathway respiration 8, rather than the cytochrome pathway @, is an integral part of the revised biochemical pH stat. Not merely just an electron donor to respiration, NADH also activates, in feed-forward fashion, AOX through the sulfhydryl-disulfide redox system. An NADPH-dependent mitochondrial sulthydryl-disulfide redox system, which is supposed to be responsible for disulfide bond reduction of AOX, has been suggested (Vanlerberghe and McIntosh, 1997; Moller and Rasmusson, 1998). Although NADP isocitrate dehydrogenase (that is decarboxylating) produces NADPH, malate dehydrogenase in the matrix cannot (Agius et al., 1998).

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As long as NADP-dependent and NAD-dependent dehydrogenases in the matrix share a respiratory chain as the final sink of reducing equivalents, flooding of NADH owing to stimulated malic enzyme reaction would increase the NADPH concentration in the matrix, which in turn would provide reducing equivalents to the redox system to activate AOX. Otherwise, transhydrogenase (Carlenor et al., 1988) may contribute to increases in the NADPH level in the matrix. Moreover, pyruvate, another reaction product, should also activate the same enzyme in an allosteric manner. Furthermore, one of the products of the enzyme reaction, CO*, preferentially inhibits cytochrome pathway respiration at low concentration (Palet et al., 1991; Gonzalez-Meler et al., 1996), resulting in promoted linking of the malic enzyme reaction with an alternative pathway. Collectively, all of these features of malic enzyme reaction products indicate that, when low pH stimulates malic enzyme, an alternative pathway is reserved exclusively for the oxidation of its products. Independence from the energy-charge control of this alternative pathway is another favorable feature of the pH stat, because the situations that require pH regulation are usually urgent. The observation of acid-pH-dependent malate oxidation by malic enzyme in plant mitochondria (Macrae, 1971; Tobin et al., 1980), together with its close association to alternative pathway respiration (Rustin et al., 1980), suggests that malic enzyme and alternative pathway respiration responded to acid pH as a pH stat. Later, this was indeed suggested by Lance and Rustin (1984). In Section III.C.3, involvement of alternative pathway respiration in H+/Pi symport by Cutharunthus cells is shown. 6. Lactic and Alcoholic Fermentation:

The Anaerobic Proton Sink

Synthesis of lactate or ethanol has generally been regarded as the means to regenerate NAD+ required for sustaining glycolytic energy production under anaerobic conditions. Lactic acid fermentation has been attributed to the cause of cytoplasmic acidification in maize root tips under hypoxia (Roberts et al., 1984a). Hochachka and Mommsen (1983) pointed out that lactate formation from glucose through glycolysis via the pyruvate kinase reaction @ produces no Hf, but ethanol formation from glucose via the same reactions consumes a H+. These stoichiometries assume that synthesized ATP is not used: l/2 Glucose + ADP3- + Pi*- + lactate- + ATP4l/2 Glucose + ADP3- + Pi*- + Hf + ethanol + CO2 + ATP4However, if the ATP is used (i.e., hydrolyzed to produce energy, a more likely situation in vivo, which we will assume in the following discussion), a H+ is generated: ATP4- + H20 -+ ADP3- + Pi*- + H+ + energy

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KATSUHIRO SAKANO

Then, l/2 Glucose + lactate- + H+ + energy l/2 Glucose -+ ethanol + CO2 + energy Therefore, under more realistic conditions, the stoichiometry from glucose will be one Hf production in lactic fermentation and no H+ production in ethanol fermentation, respectively. Despite that, the revised biochemical pH-stat hypothesis claims that lactate synthesis is not a cause of cytoplasmic acidification, but a result of pH regulation by the stat mechanism. The hypothesis assumes that malate, but not glucose, is the precursor of lactate or ethanol. Although lactate synthesis from glucose produces a H+ (under ATP utilizing conditions), its synthesis from malate via the malic enzyme reaction @ consumes a H+ (below, @ + @). Similarly, ethanol synthesis from malate via the same reaction consumes two H+s (8 + @$+ 0) (lower right block of Fig. 1). Malate2- + NADf

+ pyruvate- + NADH + CO2

(8)

Pyruvate- + NADH + H+ -+ lactate- + NAD+

m

Pyruvate- + H+ + acetaldehyde + CO2

CD)

Acetaldehyde + NADH + Hf + ethanol + NAD+

(0)

Malate*- + H+ + lactate- + CO2

@+a3

Malate2- + 2Hf + ethanol + 2CO2

(@+@l+@o,

Indeed, stimulation of malic enzyme (Edwards et al., 1998) and formation of lactate and ethanol from malate (Roberts et al., 1992) have been demonstrated in corn root tips under anaerobic conditions that acidified the cytoplasm. The induction of lactate dehydrogenase by hypoxic treatment of barley root tissues (Hoffman et al., 1986, Hondred and Hanson, 1990) is apparently not physiological if it is to synthesize more lactate from glucose that should intensify the cytoplasmic acidification (Ratcliffe, 1995). However, if the induction is for lactate synthesis from malute via the malic enzyme reaction, it should help ameliorate the cytoplasmic acidification under hypoxia. Ethanol accumulation on treatment with antimycin A (AA; inhibitor of cytochrome pathway) in the transgenic tobacco leaf that lacked AOX (Vanlerberghe er al., 1995) is consistent with the proposed pH-stat mechanism under anaerobic conditions. If respiration is not available for H+ consumption, alcoholic fermentation will take over the role. 7. Role of Malic Enzyme in the Revised Biochemical pH Stat Malic enzyme @ is characterized as the acid-pH-sensitive trigger of the H+consuming machinery: the alternative pathway respiration (under aerobic

METABOLlCREGULATlONOFpHIN

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PLANTCELLS

conditions) and lactic and/or alcoholic fermentation (under anaerobic conditions). In the classic hypothesis, this was wrongly defined as the H+-consuming reaction of the pH stat.

8. Evolutionary

Aspect of “Protozoan”

Alternative Oxidase

The exceptional distribution of AOX is trypanosome, a protist that is believed to belong to a group of primitive parasitic animals (protozoa). This organism has an AOX similar to plant enzyme (Clarkson et al., 1989), but its function does not seem to be pH regulation (Sakano, 1998). However, a survey of publications thereafter led the author (KS) to a finding that trypanosome has a proton system, i.e., its primary pump at the plasma membrane is an ATP-driven H+ pump (Vieira, 1998). This suggests that the ancestor of trypanosome that challenged the freshwater habitat succeeded in inventing the proton system, and abandoned the sodium system. In other words, trypanosome is not an animal but more likely to be a plant-like organism that shares a common ancestor with extant plants. Plasmodium, another protist that is also reported to have AOX (Murphy et al., 1997) may have Hf pump in the plasma membrane. The proton system is the basic mechanism used by a plant cell to adapt to nonsaline environments. From the recent viewpoint of plant evolution, all extant terrestrial plants originated from an algal cell that succeeded in entering the freshwater range from saline ocean (Mishler, 1999). As discussed in the preceding section, establishment of the proton system should have been a prerequisite to accomplishing this advancement. It is likely that only a cell (or a lineage) could achieve it, because molecular evolution of a proton pump from a sodium pump requires more than one amino acid replacement and, therefore, the probability of invention of a functional H+ pump (and proton system) is very low (Axelsen et al., 1998).

C. Role of Biochemical

pH Stat in Cstherenthus

roseus Cells

The revised biochemical pH-stat hypothesis is exemplified by available evidence of Cutharunthus cells that were subjected to anoxia or Pi-feeding under aerobic and anoxic conditions (Sakano, 1990, 1998; Sakano et al., 1992, 1997, 1998).

1. Role at Alkaline Range: Aerobic Expansion of Endogenous Organic Acid Pool Sakano et al. (1997) observed that aerobic Cutharunthus cells acidified external medium, absorbed K+, and expanded the endogenous pool of organic acids (Fig. 2). They considered these aerobic processes to be indicating that H+ pumping at the plasma membrane induced alkalinization of the cytoplasm and promoted malate synthesis, because PEP carboxylase has optimum pH in an alkaline pH

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KATSLIHIROSAKANO -

Air --N,--Air

6

s 5.

4.

Is

I

I

.

I

I

.

,

0123456 Time

(hour)

FIG. 2 Changes

in medium pH (solid line) and K+ concentration (dotted line) during culture of C. IOSCUS cells in 10 mM CaCl2, I m&f KCl, and 2% glucose under aerobic and anoxic conditions. Note that the medium pH and K+ concentration rose quickly on nitrogen gas flushing in a reversible manner. A to E denote the time points between which fluxes of H+ and K+ were compared. Ratios of H+ (efflux) to Kf (influx) under aerobic conditions or that of H+ (influx) to K+ (efflux) under anoxic conditions are not constant after initiation of measurement, indicating that fluxes of ions other than K+ are also involved in the medium pH change. (From Sakano et al., 1997.)

range (Davies, 1973). In exchange for the H+ extruded, external K+ entered the cell and balanced the electric charge across the plasma membrane. They interpreted these to be neutralization processes of newly synthesized organic acids in the cells to avoid cytoplasmic acidification (Sakano et al., 1997).

2. Role of pH Regulation at Acid Range: Anoxia and Hypoxia Roberts et al. (1984a) observed, in hypoxic maize root tissue, that the cytoplasmic pH dropped to 6.8 and stabilized throughout the hypoxic period. They attributed the hypoxia-induced acidification to lactic acid accumulation, and the subsequent stabilization was due to the metabolic shift to alcoholic fermentation (Roberts et al., 1984a). They considered that initial lactic acid accumulation acidified the cytoplasm, and this stimulated pyruvate decarboxylase @$ having optimum pH in the acid range, resulting in redirection of carbon flow to ethanol synthesis, which accompanies no H+ production. In fact, hypoxia caused seedlings of a corn mutant line that lacked an alcohol dehydrogenase 0 isozyme to experience

15

METABOLICREGULATIONOFpHIN PLANTCELLS

severe cytoplasmic acidification and subsequent death (Roberts et al., 1984b). Despite the apparently reasonable interpretation, this view has been questioned as a result of many conflicting observations. Using similar anoxic maize root tips, Saint-Ges et al. (1991) also observed a limited cytoplasmic acidification that was associated with lactate accumulation. However, the lactate accumulation continued even after the cytoplasmic pH had stabilized at pH in acid range. Similar limited cytoplasmic acidification (ca. 0.2 pH unit) was detected in an anoxic shoot of rice plant (Menegus et al., 1991) that produced negligible amounts of lactate (Menegus et al., 1989, 1991; Rivoal et al., 1989). Also in Catharanthus (Sakano et al., 1997), despite the continued Hf influx into the cells (Fig. 2) the cytoplasmic acidification (0.2 pH unit) leveled off soon after onset of anoxia (data not shown), accompanying an increase in endogenous lactate content and decreases in malate and citrate contents throughout the anoxia (Fig. 3). Quantitatively, the increment of lactate could not account for the observed cytoplasmic acidification: in terms of carboxylate equivalent, the accompanying decrease in malate and citrate was larger than the increment of lactate. These observations led them to attribute the anoxia-induced cytoplasmic acidification to the Hf influx from external medium (Sakano et al., 1997). They further suggested that the precursor of lactate was

I-Air-l-N&-Air

Incubation

Time (h)

FIG. 3 Change in endogenous organic acids during culture of C. rn~eus cells in 10 mM CaC12, 1 mM KCl, and 2% glucose under aerobic and anoxic conditions, Note that contents of citrate and malate increased to a stable level during the first 6 hr of aerobic conditions, but they decayed rapidly under anoxic conditions, while lactate which was the minor component in the aerobic cells, rapidly increased throughout the period. Also note that these changes were reversed when aerobic conditions were restored. (From Sakano et al., 1997.)

16

KATSIJHIROSAKANO

malate, because the overall reaction of malate-lactate (or -ethanol) conversion via malic enzyme reaction is H+ consuming (Section III.B.6). The nature of H+ influx under anoxia is not known. This can be due to “H+ channels” in the plasma membrane (Saint-Ges et al., 1991), or can be a reflection of some secondary active transports, probably of glucose: even though at slower rates, Catharanthus cells can take up glucose (data not shown) and Pi (see below) under anoxia.

3. Role of pH Regulation in Secondary Active Transport: Pi Uptake Absorption of external nutrients by plant cells depends on the electrochemical potential gradient of H+ across the plasma membrane. When Catharanthus cells were fed with Pi under aerobic conditions, they took up Pi accompanying external alkalinization, and as soon as medium Pi was exhausted, the pH shift turned to acidification (Fig. 4). The stoichiometry of the transport (H+/Pi) was calculated to be 4 (Sakano, 1990). The external alkalinization was accompanied by a concomitant acidification of the cytoplasm (Fig. 5; Sakano et al., 1992), indicating that H+/Pi symport is the uptake mechanism. It is worth noting that the acidification stopped soon after initiation of Pi uptake while Hf influx was still continuing, suggesting a delayed activation of H+ pump and metabolic consumption of H+.

0

10 20 Incubation

30 40 50 Time(min)

FIG. 4 Time courses of H+ and inorganic phosphate uptake by phosphate (40 p,mol) was added at zero time and the phosphate in intervals. Net changes in the concentrations of H+ and phosphate (peak time of pH shift) zero. Note that the peak time of medium time of Pi exhaustion. (From Sakano, 1990.)

60

cultured cells of C. roseus. Inorganic the medium was determined at 5-min are shown as their changes at 42 min pH shift (42 min) coincides with the

METABOLlCREGULATlONOFpHIN

17

PLANTCELLS

-5.2

-4.4

FIG. 5 Inorganic phosphate-induced changes in pH of external medium (straight line) and in cytoplasmic pH as measured by BCECF-fluorescent intensity of C. roseus cells (fluctuated line) suspended in unbuffered medium (10 mM CaCl2, 1 mM KC1 and 2% glucose) as a function of time. Prior to experiment, the cells were loaded with acetoxymethylester of BCECF (2’, 7’-bis(2-carboxymethyl)-5 (and -6) carboxyfluorescein), which was hydrolyzed in the cytoplasm to BCECF, the fluorescent pH indicator. Pi (0.2 pmol, final concentration: 100 l&f) was added at the time indicated by an arrowhead. Note that the decrease in fluorescence intensity (cytoplasmic acidification) stopped soon after Pi addition even though Hf influx was continuing (external pH was still increasing). When exhaustion of Pi by cells was complete, fluorescent intensity began to increase (cytoplasmic alkalinization) along with external acidification (From Sakano et al., 1992.)

Restoration toward normal cytoplasmic pH was initiated when external Pi was exhausted. The H+/Pi symport also brought about transient K+ efflux (Fig. 6, left). The situations seemed quite similar to that of the cells subjected to anoxia: The Pi-induced cytoplasmic acidification decreased contents of endogenous malate and citrate although no lactate accumulated (Fig. 7a). All of these changes were reversed when medium Pi was exhausted: the malate (but not citrate) level began to increase, the external pH began to decrease, and concomitantly (Fig. 4) the cytoplasmic pH started to increase (Fig. 5). Furthermore, Pi uptake switched the respiration of Cutharunthus cells from cytochrome to alternative pathway respiration, which was reversed upon Pi exhaustion as examined by the stable oxygen isotope discrimination method (Guy et al., 1989), direct evidence that alternative pathway respiration is involved in H+ consumption (Fig. 8). The possible mechanism of pH regulation in relation to biochemical pH stat is shown in Fig. 9, upper. Of the 4 H+s that entered the cytoplasm by H+/Pi symport and acidified it, 3 H+ are extruded by H+ pump, but the remaining one is consumed by the biochemical pH-stat mechanism: the acidification stimulates malic enzyme, and its reaction products (NADH and pyruvate) in turn activate AOX. When AOX oxidizes NADH that is derived from the malic enzyme reaction, one H+ is consumed. Of the K+ that have been the

18

KATSUHIROSAKANO

I

Air

3.6,

0.7

0.65 R 0.4 '

Time (min) FIG. 6 Changes in the fluxes of H+ and K+ during Pi uptake by C. roseus cells under aerobic (air, left) and anaerobic (N2 gas, right) conditions. Pi (20 t.tmol) was added at zero time. Values for pH are that of medium, and those for K+ are medium concentration (in mM). The average rate of anaerobic Pi uptake (right) was about l/6 that of aerobic conditions (left). Note that exhaustion of Pi always coincides with the peak pH shift, and that the K+ efflux is temporary under aerobic conditions, while it continued until Pi exhaustion under anoxia, suggesting that the activation of H+ pump is delayed (aerobic) or almost completely inhibited (anaerobic).

countercation of the decarboxylated malate, some now serve as that of the Pi anion taken up, and the rest leave the cell as K+ efflux. Catharunthus cells could absorb Pi even under anoxia (Fig. 6, right). The average rate of anoxic Pi uptake was about l/6 of that under aerobic conditions. Medium

METABOLIC

REGUL .ATlONOF

19

ptl IN PLANTCELLS

-

(a)

organic

(DJ medium

acids

pH

3.5v-2 Time after Pi addition

(h)

FIG. 7 Changes in endogenous contents of malate and citrate (a) and medium pH (b) as a function of time after addition of 50 pmol Pi (final concentration: 1 mA4) to the suspension of C. roseus cells at zero time. The peak time of pH shift (30 min after Pi addition) corresponds to the time when medium Pi was exhausted, as shown in Fig. 4 (Sakano et al., 1998). Note that malate and citrate contents decreased during H+/Pi symport (medium alkalinization), but malate (but not citrate) content began to increase as soon as H+ efflux was initiated upon Pi exhaustion.

alkalinization and K+ efflux continued until Pi uptake ceased by exhaustion. The mechanism of anoxic Pi uptake may be similar to that under aerobic conditions, except that H+ pump is not active and, therefore, all the H+ that entered the cytoplasm in Pi symport must be consumed by the anaerobic biochemical pHstat mechanism: lactic (and/or alcoholic) fermentation of malic enzyme reaction products consumes H+ (Fig. 9, lower). In terms of electrophysiology, the driving force of anoxic Pi uptake is explained by the diffusion potential of K+: H+/Pi symport, which carries positive charge into the cell, depolarizes the plasma membrane. Accompanying K+ efflux reflects it. From the viewpoint of pH regulation, the pH gradient across the plasma membrane maintained by pH stat drives the anoxic symport, and the K+ efflux that support the membrane potential is the reflection of malate decarboxylation.

20

KATSUHIROSAKANO

Time

(min)

(b)

I

0.25 -In f

0

FIG.8 Time courses of (a) changes in 02 concentration in headspace and medium pH, and(b) changes in respiratory oxygen isotope discrimination before, during, and after absorption of Pi. Cutharanthus roseus cell suspension (1 g fresh weight in 50 ml of medium composed of 10 mM Cat& 2 mM KCI, and 2% glucose) was shaken under aeration in a vessel with 40 ml of headspace air for about 4 hr when the system was closed (SC, zero time). After SC, headspace gas (1 ml) for analysis was collected at 30-min intervals. The cells were allowed to respire for 90 min and then 20 pmol of Pi was applied. At the peak time of pH shift (153 min), medium Pi was exhausted (see Fig. 4). Pi uptake increased not only (a) the rate of 02 consumption, but also (b) the respiratory discrimination against heavy oxygen isotope (‘*02). The slopes of the three lines, which are connected to each other, indicate the discrimination factor (D values, in %) for before, during, and after Pi uptake, respectively. The D value was calculated according to Guy et al. (1989) as a slope of the linear regression obtained when 1000 times the natural logarithm of the ratio of isotope ratio (‘sO/‘6O) at a given time (R) to that at initial (Ro), (i.e., 1000 x In R/Ro) were plotted against the negatively signed natural logarithm of

21

METABOLICREGULATlONOFpHINPLANTCELLS

In either way, the role of the endogenous pool of K+ salt of malate (or of other organic acids that are capable of decarboxylation and accepting of reducing equivalents in response to cytoplasmic acidification) may be compared to that of a battery that supplies electric power during an emergency. Under normal (aerobic) conditions, the battery is charged (K+ salt of malate is accumulated) at the expense of ATP consumption (in H+ extrusion and K+ influx). Even under emergency power failure (anoxia), the battery can drive electric machineries (H+/Pi symport) as long as the battery can provide electricity (K+ salt of malate). This is achieved at the expense of malate decarboxylation, K+ efflux, and successive fermentation reaction of the decarboxylation products (Fig. 9, lower).

IV. Production and Consumption in Plant Metabolisms A. Respiratory

of Protons

Metabolism

Generally, in a dehydrogenase reaction (nondecarboxylating), one H+ is produced as part of the reducing equivalents in the forward reaction and is consumed in the reverse reaction: RH2 + NAD(P)+ ts

R + NAD(P)H + H+

This implies that dehydrogenase reactions are deeply concerned with cytoplasmic pH regulation. During aerobic catabolism of glucose via glycolysis and the Krebs cycle, reducing equivalents (Hz) taken from intermediary substrates in the form of NAD(P)H + H+ by dehydrogenases, otherwise acidifying the cytoplasm, are oxidized to Hz0 by respiration. This indicates that no net H+ is produced in the steady-state metabolism. NAD(P)H + H+ + l/2 02 + NAD(P)+ + Hz0 + energy

(respiration)

a fraction of unreacted 02, -1n f, where f is the ratio of 02 concentration at a given time [02] to that at initial [02]0, (i.e., f = [02]/[02]a). It has been reported that both AOX and cytochrome oxidase discriminate heavy 02 isotope (IgO) from light (t60), but the former discriminates more than the latter (Guy et al., 1989). Therefore, if cells in a sealed vessel were allowed to respire, the ‘8O/16O ratio of the 02 remaining in the vessel would increase as 02 is consumed; and the larger the contribution of AOX in the total respiration, the greater the increase in the ‘8O/‘6O ratio. The D values for cytochrome oxidase and AOX are reported to be in the range of 17-20%, and 23-26%, respectively (Guy et al., 1989). Note that D values increased upon Pi feeding and were restored almost the same value after Pi exhaustion, indicating increased engagement to the alternative pathway respiration during Pi uptake. (Sakano, Noguchi, Kiyota, Yazaki, and Ueda, unpublished data, 1999.)

22

KATSUHIRO

SAKANO

Plasma membmne

(OUT)

(IN) I

Pathway

Respiration

Plasma membrane (OUT)

(IN) I

FIG. 9 Hypothesis of cytoplasmic pH regulation during Pi uptake under aerobic and anaerobic conditions. Upper: Under aerobic conditions, H+/Pi sympott (stoichiometry of H+/Pi = 4; Sakano, 1990) acidifies the cytoplasm and activates H+ pump at the plasma membrane and biochemical pH stat as well. Typically, of the four H+ that are carried into the cytoplasm by H+/Pi symport, three are extruded by H+ pump (if it is in an active state), and the remaining one is consumed by respiration when the malic enzyme-derived NADH is oxidized by alternative pathway respiration. Of the 2 K+, which have been the countercations of malate before its decarboxylation by malic enzyme, one K+ will serve as the countercation of Pi anion, and the other, as that of pyruvate anion. Therefore, no K+ efflux is required to neutralize the total ion fluxes. However, at least in Catharanthus cells, since the H+ pump activation is significantly preceded by symport-induced acidification (Sakano, 1990) most of the early H+ are consumed entirely by biochemical pH stat, and thus temporary K+ effhrx takes place (Fig. 6, left). Lower; Under anaerobic conditions, 4 H+ that are brought about by H+/Pi symport will be consumed entirely by the anaerobic biochemical pH stat. For example, if we assume lactic fermentation is the exclusive mechanism of anaerobic H+ consumption, the consumption of 4 H+ would accompany efflux of 3 K+ in order to neutralize the total ion fluxes. Stoichiometry of K+ efflux in Pi uptake will not change even if ethanol, instead of lactic acid, is the fermentation product, although the amount of malate decarboxylation is reduced to half that of lactic fermentation (see Section III.B.6). Note, however, that K+ may not be the sole ion that balances the total ion fluxes in a real cell (Fig. 2).

23

METABOLlCREGULATlONOFpHINPLANTCELLS

Similarly, another steady-state H+ balance is also associated with metabolism: the metabolic turnover of high-energy phosphate bonds (-P), as in ATP, accompanies production and consumption of H +. For example, hexokinase reaction in the glycolysis produces one Hf: Glucose + ATP4- + G6P2- + ADP3- + H+

(hexokinase)

In the steady state, this reaction is coupled to other phosphorylating reactions that consume Hfs such as; PEP3- + ADP3- + H+ -+ pyruvate- + ATP‘-

(pyruvate kinase)

ADP3- + Pi2- + H+ + energy + ATP’- + Hz0

(oxidative phosphorylation)

Therefore, in the steady-state metabolisms of both reducing equivalents and -P, the generation and consumption of H+ are balanced, and they do not contribute to any change in the cytoplasmic pH. It is important to note that this balance does not necessarily specify a “normal aerobic pH of the cytoplasm around 7.5.” In the presence of a weak auxin NAA (naphthalene acetic acid), the apparent (steadystate) cytoplasmic pH of plant cells is in the acid range (Hagendoorn et al., 1994). Under the metabolic steady-state conditions, H+ pump is out of work. The pump is required when the steady state is broken and resultant extra H+ are to be extruded; e.g., expansion of the endogenous organic acid pool, assimilation of ammonia, and H’ symports at the plasma membrane. Therefore, if respiration is hampered for some reason, the steady-state balance is lost and NAD(P)H + H+ will accumulate in the cytoplasm. This will make the cytoplasm acidic and reducing. Anoxia is a case that applies to this condition. Fox et al. (1995) showed that cytoplasmic acidification of maize root tips took place under anoxia even though medium pH was raised as high as 10, where no H+ influx was expected. Respiratory oxidation of reducing equivalents as the H+-consuming mechanism is, thus, significant and cannot be overlooked. Similar cytoplasmic acidification due to “flooding” of reducing equivalents should also be expected under aerobic conditions. In the next sections, some basic metabolisms are examined with special reference to production and consumption of reducing equivalents and, therefore, of H+.

E3. Primary

Metabolism

In animals and plants, protein synthesis is one of the representatives of primary metabolism that supports cell growth and proliferation. Although it seems selfevident that synthesis of protein from its precursors, glucose, nitrate, and sulfate, requires phosphate-bond energy (-P) of ATP, GTP, etc., and reducing equivalents (NAD(P)H + H+) as well, its stoichiometry is not available in common textbooks. Therefore, I examine how much -P and reducing equivalents are required

24

KATSUHIROSAKANO

to synthesize a protein consisting of n amino acid residues. For simplicity and comprehensiveness, both reducing equivalents represented by NADH + H+ and NADPH + H+ are expressed as HZ(NADH)and HZ(NADPH), respectively, and are assumed equal to reducing equivalents, HZ. For the same reason, 1 -P is assumed to be equivalent to 0.3 Hz, which is expected if NAD(P)H + H+ is oxidized through the cytochrome pathway. By this expedience, the stoichiometry of the -P and H2 requirement for an amino acid synthesis is calculated and expressed collectively as the stoichiometry of total Hz equivalents (the Total Hz Eq. columns in Table I). (In Table II, the procedures for glutamate and glutamine are illustrated as examples to show the calculation method.) In Table I, the summaries of stoichiometry are shown for the syntheses of 19 amino acids from the precursors glucose, nitrate, and sulfate. Here, nitrate (not ammonia) and sulfate are assumed to be the precursors for N and S in the amino acids. The results (Total Hz Eq.) indicate that syntheses of all amino acids except leucine and tyrosine from these precursors require a net supply of reducing equivalents. [However, if the N source is ammonia, the stoichiometry (number in the column of Total Hz Eq.) for each amino acid should be smaller by 4 per one nitrogen, the cost of nitrate reduction to ammonia; see Table I.] Calculations for spinach Rubisco small subunit protein consisting of 123 amino acid residues (Taylor and Andersson, 1997) indicated that about 500 Hz are required for the synthesis of component amino acids (Table I). In the next step, the stoichiometry of energy (-P) requirement for a synthesis of protein (consisting of y1amino acid residues) on ribosome is examined (see Table III). Activation of amino acid (2n - P), elongation (n - P) and translocation (n - P) of peptide on ribosome, and initiation complex formation (indefinite number, i - P) require at least 4n - P in the forms of ATP or GTP, or, in terms of Total H2 Eq., 1.3n HZ. If this stoichiometry is applied to the spinach Rubisco small subunit protein that consists of 123 amino acids (n = 123), about 660 H2 are required (about 500 H2 for the constituent amino acid synthesis and 1.3 x 123 = 160 Hz for their polymerization). On average, about 5.4 Hz are required per one amino acid residue that is integrated in the Rubisco small subunit protein molecule. Similarly, stoichiometry for DNA synthesis (from glucose, nitrate, sulfate and Pi) is calculated for a tetramer DNA (as model) composed of dAMP, dTMP, dCMP, and dGMP (see Table IV): 6.75 Glucose + 15 HN03 + 4 Pi2- + 44.5 - P + 41 Hz(~~o(r)n) + 4H+ + (dXMPp)4 + 0.5 CO? Again, DNA synthesis consumes large amounts of -P and HZ: in terms of Total H2 Eq., an average of 14 H2 per one nucleotide residue of DNA are required. Such rough calculations confirm the initial confidence that the syntheses of amino acids and their subsequent polymerization to protein require large amounts of high-energy bond (-P) and reducing equivalents. The same is true for DNA synthesis.

25

METABOLICREGULATIONOF pH IN PLANT CELLS

TABLE I Summary of Stoichiometry in Consumption and Generation of ATP and Reducing Equivalents in Amino Acid Synthesesaand Rubisco Small Subunit Protein* Rubisco ATP

Glutamate

0

Total HzC

Total Hz Eq.d

+4

-3

+1

+1

9

+9

HZ(NADPH)

NO.

SSf

HZ(NADH)

Total H2 Eq.

Glutamine

+1

+7

-2

+5

+5.3

5

+26.5

Proline

+1

16

-3

+3

+3.3

9

+29.7

Arginine

+7

-1

f15

+1.3

5

+86.5

Alanine

0

+4

+4

5

+20 +22.2

tl6

0

+4

Valine

-1

+5

-1

+4

+3.1

6

Leucine

-3

+5

-4

+I

0

12

0

Isoleucine

-3

+6

+1

+7

t2

6

+12

Aspartic

acid

+1

+4

+4

+4.3

6

+25.8

Asparagine

12

+7

+1

+8

+8.7

7

+60.9

Threonine

t3

+5

+1

+6

+7

6

4-42

Lysine

+2

+9

+1

f10

t10.7

7

t74.9

Methionine

+6

+9

-2

+I

+9

3

+27

+4

-1

+3

+3

4

+12

+2

-1

+1

+0.7

6

+4.2

+8 e

4

Serine

0

Glycine

-1

Cysteine

0

Histidine

+3 c

+8 e

-1 r

Phenylalanine

+3

t-1

-1

Tyrosine

+3

+I

-2

Tryptophan

+5

+3

-3

+1

Rubisco

+7 e 0

0

SS

HNO3

+

NH3

0

+3

H2SO4

+

H2S

+2

+4

+1

-1

0

3

+32 -

6

+6

0

10

0

+1.7

4

+6.8

Sum

123

+497.5

+4

+4

+4

+4.7

Key: +, Synthesis requires substrate; -, synthesis overproduces as by-product. ‘Amino acid synthesis and its stoichiometry accomplished according to textbook by Held (1997). bfrom glucose, HNOs and H2SO4. For details of calculation, see examples for glutamate and glutamine in Table II. ‘Total Hz assumes that reducing equivalents from both NADPH and NADH are equivalent. dTotal H2 Eq assumes that 3 moles of ATP (or equivalent) is equivalent to NAD(P)H, which is expected if NAD(P)H is oxidized through cytochrome pathway respiration. eNo data available because of complexity of biosynthetic pathway (Miflin, 1980). fData of spinach Rubisco small subunit from Taylor and Andersson (1997).

KATSIJHIROSAKANO TABLE II Stoichiometry of Energy Requirement in Syntheses of Glutamic Acid and Glutamine from Glucose and HN03 I. Nitrate

assimilation:

Hi-h

+ HZ(NADH)

2. Glutamic

+

HZ(NADPH)

+

Pyr + CoASH Acetyl

Co.4

Citrate

+

Pyr + ‘-P + H~(NADH) +

Acetyl

+ OAA

CoA + CO2 + H2(NADH)

+ Hz0

+

Citrate

+

2-oxo-Glutarate

2-oxo-Glutarate (GS)

+ CO2 + H~(NADH)

+ H~(NA~PH)

Glutamic

+ HZ(NADH)

PEP + CO2 +

+ NH1 +

acid + NH3 + -P

Glutamine

(GOGAT)

+

OAA

3

+

-

HZ(NADPH)

PEP + H~(NADH)

(Sum) Glucose

+ HNO3

+ 4

acid + Hz0

(GS + GOGAT)

+ H20 +

2 Glutamic

NH3 + 3 H20 + H20 +

H~(NADPH)

Glutamic

acid + CO;! + 3 H~(NA~H)

+ 3 H20

synthesis: acid + NH3 + -P

+ H2(NADH)

Glucose

Glutamic

Glutamine

+ Pi

+ Pi +

3. Glutamine

- P + -

H~(NADPH)+ 2-oxo-Glutarate

0.5 Glucose

Glutamic

+ CoASH

Isocitrate

kOCitrate

HN03

NH3 + 3 Hz0

acid synthesis:

0.5 Glucose

HNO-I

3

+

+ HNO3

(SW Glucose

+

3

+

+ 4 HZ(NADPH)

+ 2 HNO3

Glutamine

H2(NADPH)

+

+ H20

NH3 + 3 H20

--f Glutamic

+ 7 H2(NADPH) + -P

acid + Co2 + 3 H~(NA,,,,) +

Glutamine

+ 3 H20

+ Co2 + 2 H~(NADH)

+ 7 H20

In the cell that is in the midst of growth and proliferation, there must be no redundant reducing equivalents, and a cytochrome pathway, but not an alternative pathway, should be the main source of respiration in order to supply ATP and other nucleotide triphosphates required for growth and proliferation. As long as the demands by the primary metabolism “pull” the production of -P and reducing equivalents, steady-state H+ balance would be toward its consumption (cytoplasmic alkalinization), which is favorable for macromolecule syntheses (see Section 1V.D)

C. Secondary

Metabolism

Production of secondary metabolites such as phenylpropanoids, flavonoids, lignin, terpenoids, and alkaloids is generally limited to plants and microorganisms, and

27

METABOLICREGULATIONOF pH IN PLANT CELLS TABLE Ill Stoichiometry of Energy Requirement in Protein Synthesis from Amino Acids (1) Activation Amino

of amino acid0 Acid

AA-AMP

+ 2 -P

+ tRNA

(2) 40s Initiation

+ AMP +

+

AA-AMP

(Amino

acyl AMP)

(Amino

acyl tRNA)

+ AMP

AA-tRNA

complex

formation

mRNA + Cap-binding protein + eIF4 + eIF3 + eIF2-GTP-Met-tRNA (initiation RNA) + 40 (S ribosome) + Initiation complex (on mRNA) (3) 80 S Initiation

complex

formation

Initiation complex + i -P + 60 S subunit --f 80 S Initiation complex + eIF2-GDP-Met-tRNA (4) SUM

in initiation

Components

complex

[i, number of ATP required (AUG) on mRNA, i z 01

Complex

+ -P(oTp)

for the Initiation

+ eIFs

+ i -P

Complex

+

80 S Initiation

to move from

complex

CAP site to the initiation

site

on 80 S ribosome

80 S Initiation complex + AA-tRNA --f 80 S Complex with elongated (6) Translocation

+ EFs (eEFlu, eEF1 l3r) + -P(GTP) peptide + tRNA

on 80 S ribosome

80 S Complex (7) SUM:

Complex)

formation

of 80 S Initiation

(5) Elongation

(Initiation

Peptide

with elongated

peptide

formation

n (number)

from

+ - P(~rp)

+

80 S Complex

with translocated

peptide

AAs (polymerization)

~AAs+~~--P+~-P+~~-P(GTP)~(AA), or nAAs+4n

-P+i

-P+

(AA),

(8) Summary Synthesis

(-P)

of a polypeptide

“Amino acid activation in PPi was assumed

composed

of n amino acids requires

(4n + i) ATP, or at least 4n ATP.

with ATP produces acyl AMP and PPi. Here, the high energy to be hydrolyzed to 2 Pi. EIF, eukaryotic elongation factor.

bond energy

is an important field of research in plant science and technology. Secondary metabolism, which is responsible for the synthesis of such metabolites, branches from various pathways of the primary metabolism (glycolysis, pentose phosphate pathway, shikimate pathway, etc.). However, it is likely that secondary metabolism is not always “on” during a cell’s life. In plant cell culture, secondary metabolites usually accumulate in the stationary phase, but not in the log phase. Despite intensive research, the mechanism for switching from primary to secondary metabolism has not been elucidated.

28

KATSUHIROSAKANO

TABLE IV Stoichiometry of Energy Requirement in Syntheses of Deoxyribonucleoside Monophosphate from Glucose, Pi and HNOs, and Their Polymerization into DNA (1) dAMP 1.75 Glucose + 5 HNO3 + Pi*+ dAMP*+0.5CO2+15HzO

+ 14.5 -P

+ 5 &(NAD”)

+ 8.5 H~(NADPH)

+ 5 H~(NA~H)

+ 9.5 HZcNADHI

(2) dGMP

I.75 Glucose +

i-

5 HN03

+ Pi*- + 13.5 -P + 0.5 CO2 + 15 H20

dGMP*-

(3) dCMP 1.5 GluSe + 3 HNOs + C0z + Pi*--f dCMP*+ 9 Hz0

+ 5 -P

+ 3 H~(NADH)

+ 6 Hz(~,,~pu)

(4) dTMP 1.75 Glucose + 2 HN03 + 4.5 -P + Pi’+ dTMP*+ 0.5 CO2 + 6 HZ0

+ 2 HZ(NADH)

+ 2 Hz(~~npu)

(5)Sum(l+2+3+4) 6.75 +

Glucose + 15 HNO3 + 4 Pi*- + 37.5 -P + 15 H2(NADH) + 26 H2(NADPH) dAMP*-

(6) Polymerization dAMP*(7) Synthesis

+ dGMP2-

+ dCMP*-

+ dTMP*-

+ 0.5 CO2 + 45 H20

of dAMP*-

+ dGMP*-

+ dTMP2-

+ dCMP*

+ dGMP*of DNA

+ dTMP*-

(tetramer)

6.75Glucose 3

+ dCMP2-

from Glucose

+ 15 HN03 + 4 Pi*(dXMP-)4 + 0.5 CO2

+ 4H+

(HNOs

+ 45.5 -P

+ 8 -P

as nitrogen

+ 15 H~(N~~~)

--f (dXMP-)‘, source)

(sum:

5 + 6)

+ 26 Hz(NADpH)

+ 4 H+

To compare the characteristics of secondary metabolism to that of primary metabolism (Section IVB), I examine the stoichiometry of syntheses of some secondary metabolites from the precursor glucose (Table V). First example is the salicylic acid (SA) that is regarded as a signal molecule in systemic acquired resistance in plant-pathogen interaction (Lamb and Dixon, 1997). Although phenylalanine is the immediate precursor of SA, its intracellular content is usually so low (order of pM) that net synthesis from glucose is required when a significant amount of SA is synthesized. In short, synthesis of 1 mol SA requires 2 and 3 mol each of erythrose-4-phosphate (E4P) and PEP, each derived from the pentose phosphate pathway and glycolysis, respectively. In the shikimate pathway, they are condensed and transformed into phenylalanine, which in turn is converted to truns-cinnamic acid (t-CA) by phenylalanine ammonia lyase. The side chain of t-CA is shortened by B-oxidation, then the resulting benzoic acid is hydroxylated at position 2 by a monooxygenase to form SA. The overall stoichiometry of the synthesis is: 2Glucose+3 +

SA

+

-P+G2 H~(NADPH)

+

6

Hz(~~nu) + Hz(FAon)+ 5 CO2 + H2G

29

METABOLICREGULATIONOFpH IN PLANT CELLS

TABLE V Summary of Synthesis of Some Phenolics and Terpenoids from Glucose and Their Stoichiometry of Consumption and Generation of ATP and Reducing Equivalents Quinic

acid

1.5 Glucose r-Cinnamic

+ -P

+ Hz0

2 Glucose

+

Chlorogenic

QA + 4 H~(NADPH) + 2 CO2

3 -P

+

t-CA

+ 2 HZ(NADH)

+ 2 H~(N.,+,DPHJ + 3 CO2 + 4 Hz0

acid

3.5 Glucose

+ 5 --P

i- 2 02

Isochlorogenic

acid

5.5 Glucose Salicylic acid

+ 8 - p + 4

2 Glucose -+

+

acid

02

+

ChA

+ 2 HZ(NADH)

--f IsoCh.4

+ 4

+ 4 HZ(N.03)

HZ(NADPH) + 6 Hz0 + 5 CO2

+ 4 H~(N..,DPH) + 8 Co2 + 13 H20

+ 3 m P- + 02

salicYliC

Isopentenyl

acid

+ H2(NADPH)

+

6

H2(NADH)

+ H2(FADH)

+

5 co2

+ Hz0

PP

1.7 Glucose

+ 3.2 - P + 2 Pi +

isopentenyl

PP + 6 H2cNADHj + 5 CO2 + Hz0

Ipomeamarone

Summary

of stoichiometry ATP

NADPH

NADH

Quinate

+1

-4

r-Cinnamate

+3

-2

-2

-4

-3

Chlorogenate

+5

-4

-2

-6

-4.3

-10

-7.7

HZ Eq.

0

Total Hz Eq.

-4

-3.7

Isochlorogenate

+7

-8

-2

Salicylate

+3

-1

-6

-7.7”

-6.7 -5

Isopentenyl

PP

Ipomeamarone

+3.2

0

-6

-6

+9.7

0

-18

-18

Key: +, Synthesis requires substrate; -, synthesis overproduces as by-product. ‘Production of FADH (by-product, +0.7 HZ equivalents) during the synthesis

-14.7

is included.

Similarly, the overall stoichiometry of chlorogenic acid (ChA), one of the predominant phenolic compounds in plants, is expressed as: 3.5 Glucose

+ 5 -P

-I 202

-+ ChA

+

2 &(NADH)

+ 4

HZ(NA~PH)+ 6 Hz0 + 5 COz

In these stoichiometries, somewhat unexpected was the relatively little requirement of-P and abundant production of NAD(P)H + Hf. Since oxidation of only a part (1 or 2 mol) of the by-product NADH + H+ by cytochrome pathway respiration can afford -P required for the reactions, both reactions are considered to overproduce reducing equivalents. This means that, unless the by-product NAD(P)H + H+

30

KATSUHIROSAKANO

are rapidly oxidized to regenerate NAD(P)+, synthesis of SA or ChA would not proceed, because the reactions leading to SA and ChA require NAD(P)+. The same applies to quinic acid and isochlorogenic acid (Table V). Similarly, the overall stoichiometry of synthesis of a sesquiterpenoid phytoalexin ipomeamarone (Ip), which accumulates in large quantities in the sweet potato root tissue infected by CeratocystisJimbriata (Schneider et al., 1984) is: 4.5 Glucose i- 8 H~(NADPH)

+ 9 -P + 3 02

+

Ip +

18

H~(NADH)

+12COZ+6Hz0 However, if the 8 H2(~~orn) required for the synthesis (mainly in hydroxylation reactions) are assumed to be provided by the pentose phosphate pathway (Glucose + -P + 6 Hz0 -+ 12 HZ(NADPH) + 6 COZ), then: 5.2 Glucose + 9.7 -P + 3 02 + Ip + 18 H~(N,$DH)+ 16 CO2 + 2H20 Again, Ip synthesis overproduces reducing equivalents. In general, terpenoid synthesis produces a large amount of reducing equivalents as by-product. For example, stoichiometry of synthesis of isopentenyl PP,a common intermediary precursor of terpenoids is: 1.7 Glucose + 3.2 k P + 2Pi + isopentenyl PP + 6 HZ(NADH)+ 5 CO2 + Hz0 There are several reasons why secondary metabolism produces excess reducing equivalents. First, secondary metabolites generally contain no nitrogen or sulfur, except for alkaloids and some N-containing secondary products like betacyanin. (These compounds are out of the scope of this review.) Assimilation of nitrate and sulfate requires large amount of -P and reducing equivalents (Table I). Second, polymerization is not a common feature of secondary products and, therefore, requires less -P. Although lignin is a polymer of phenolic precursors, the polymerization is a peroxidase-mediated radical reaction and consumes no -I? This is in sharp contrast to protein and DNA syntheses, which require a large amount of -P as described above. In summary, synthesis of secondary metabolites from glucose overproduces reducing equivalents that are energetically in excess of -P required for the synthesis. Thus consumption of excess reducing equivalents is the key to secondary metabolite synthesis. From the viewpoint of cell economy, it is desirable for the synthesis to be coupled to other energy-consuming processes such as protein and DNA syntheses. However, as discussed later, there seems to be little coupling between these metabolisms. Provision of -P to protein synthesis is achieved by oxidation of glucose through glycolysis, the Krebs cycle, and cytochrome pathway respiration, and not by secondary metabolism even if it overproduces reducing equivalents. The converse may be also true: excess reducing equivalents produced in the secondary metabolism may be oxidized through alternative pathway respiration without conserving energy. As discussed in the next section, the invariable

METABOLlCREGULATlONOFpHIN PLANTCELLS

31

association of cytoplasmic acidification with secondary-product-synthesizing cells (Hagendoom et al., 1994) strongly suggests that alternative pathway respiration that is triggered by acid pH is deeply involved in the oxidation of excess reducing equivalents. In addition, because secondary metabolism overproduces reducing equivalents and requires its removal, its steady-state H+ balance must be toward its accumulation (cytoplasmic acidification), which is unfavorable for macromolecule synthesis, but favorable for stimulating alternative pathway respiration to consume excess reducing equivalents.

D. Switching Control between Primary and Secondary Metabolisms Reversible switching from primary to secondary metabolism has been observed under various conditions, Hagendoorn et al. (1994) showed that production of secondary metabolites (lignin, anthraquinone, coniferin) by cultured plant cells could be controlled by manipulating phytohormones or microelement (Fe3+) in the culture medium. They found that production of secondary metabolites was always associated with cytoplasmic acidification as detected by fluorescent pH probe. The cytoplasmic pH of Morindu cells that were grown in the presence of NAA was low and the cells synthesized anthraquinone. Addition of 2,bdichlorophenoxy acetic acid (2,4-D) increased cytoplasmic pH and stopped anthraquinone synthesis. In petunia cells with low concentrations of NAA in the medium, cytoplasmic pH was low and lignin production was enhanced. But, with higher NAA concentrations, pH increased and lignin production was inhibited. We can speculate that, even at low concentration, strong auxin 2,4-D could stimulate plasma membrane H+ pump strong enough to maintain a normal high cytoplasmic pH (value not reported), whereas weak auxin NAA could not. In the same cells, omission of Fe3+ from culture medium induced cytoplasmic acidification and lignin production, both of which were reversed with Fe3+ supply. The lignin production could be stimulated also by treatment of cells with orthovanadate, an inhibitor of plasma membrane H+ pump that would acidify the cytoplasm (Hagendoom et al., 1991). Similar correlations between cytoplasmic acidification and phytoalexin synthesis upon elicitor application are reported in Phaseolus vulgaris (Ojalvo et al., 1987), and Eschscholtzia californica cells (Roos et al., 1998). Their results strongly suggest that cytoplasmic acidification is closely related to switching to secondary metabolism. In contrast with secondary metabolism, primary metabolism, such as protein and nucleic acid syntheses, is susceptible to cytoplasmic acidification. Walker et al. (1998) showed that cytoplasmic pH in K+-deficient barley roots (pH 7.0) was significantly lower than that in K+ sufficient roots (pH 7.3-7.4). The rate of protein synthesis declined as a function of decrease in internal K+ concentration and,

32

KATSUHIROSAKANO

therefore, of the cytoplasmic acidification. However, the protein synthesis was more sensitive to low cytoplasmic pH. In the Kf-sufficient cell, butyrate-induced cytoplasmic acidification inhibited its protein synthesis, but in the K+-deficient cell, procaine-induced cytoplasmic alkalinization stimulated it. Webster et al. (1991) also demonstrated that in vitro translation by the system from maize root tissue exhibited optimum pH around 7.5 and was sensitive to acid pH except for translation of some minor proteins. Moreover, it is noteworthy that optimum pH of DNA synthesis resides in the alkaline range in a number of experimental materials (Busa and Nuccitelli, 1984). Ojalvo et al. (1987) noted that elicitor-induced cytoplasmic acidification was associated with a decrease in intracellular ATP content. Because cytoplasmic acidification triggers stimulation of alternative pathway respiration through biochemical pH-stat (Fig. 6; Sakano, 1998), this would divert fewer electrons to the cytochrome pathway, resulting in a downshift of ATP production. Although acid pH inhibits macromolecule syntheses, their inhibition is likely to induce cytoplasmic acidification: inhibition of macromolecule synthesis would result in reduced consumption of -P and, hence, in an accumulation of reducing equivalents (accompanying cytoplasmic acidification) in the cell. Therefore, it is no wonder that an increase in the alternative pathway capacity occurred after treatment of plant tissues and cells with inhibitors of macromolecule synthesis: cycloheximide (Morohashi et al., 1991; Gallerani and Romani, 1996) chloramphenicol (Klerk-Kiebert et al., 1982), actinomycin D, and D-MDMP (Gallerani and Romani, 1996). Another condition is the limitation of nutrition (especially of nitrogen and phosphorus), which generally leads plant cells to cease growth and proliferation (primary metabolism) and further to initiation of senescence accompanying secondary metabolite production. Indeed, Tabata (1977) showed that synthesis and accumulation of secondary metabolites occurs in the stationary phase of plant cell culture, and they are induced by nutrient depletion. Yamakawa et al. (1983) reported that anthocyanin accumulation in the stationary phase of V&is cell culture was due to depletion of Pi from the medium, and Pi supplement recovered cell proliferation and, at the same time, inhibited anthocyanin synthesis. Hirose et al. (1990) further demonstrated that addition of aphidicolin (DNA polymerase inhibitor) and cycloheximide (protein synthesis inhibitor) to the Vitis cells in their log phase induced anthocyanin synthesis. These studies indicate that the primary and the secondary metabolisms are mutually exclusive (Sakuta et al., 1987a). However, not all secondary metabolite productions are associated with stationary phase metabolism. In contrast to anthocyanin, which accumulates in the stationary phase in other plant species as described above, betacyanin, a nitrogen-containing pigment, accumulates in the log phase of Phytolacca americana cell cultures. The responses of betacyanin production to medium Pi and aphidicolin were also opposite to those of anthocyanin (Sakuta et al., 1986; Hirose et al., 1990). Sakuta et al. (1987b) noted that betacyanin synthesis was promoted by rather high nitrogen concentration, especially, of nitrate in medium. The author (KS) speculates that the contrasting difference might come from the fact that nitrate assimilation requires

33

METABOLICREGULATlONOFpHIN PLANTCELLS

a large amount of reducing equivalents and this may be associated with synthesis of the carbon skeleton of betacyanin in the proplastid, which produces reducing equivalents. Such coupling may not take place in the stationary phase, where there is little nitrate assimilation even if reducing equivalents are abundantly available. Although no one has ever compared the cytoplasmic pH in the stationary cells with that in the log phase cells, it is very likely that the former is more acidic than the later. A similar relationship between Pi (or N) deficiency and AOX expression has been reported for cell cultures of tobacco (Parsons et al., 1999), Cutharunthus (Hoefnagel et al., 1993, 1994), and bean roots (Rychter and Mikluska, 1990; Rychter et al., 1992). In summary, it is very likely that cytoplasmic acidification is the key signal that switches primary to secondary metabolism, in which alternative pathway respiration seems to play a central role. Cytoplasmic acidification not only inhibits the primary metabolism (DNA and protein syntheses, etc.) directly, but also initiates secondary metabolism indirectly by stimulating alternative pathway respiration. In addition, it is possible that cytoplasmic acidification is a signal responsible for expression of new genes (including those of AOX and enzymes of secondary metabolism) that are required to respond to the new situation that caused the acidification. For example, treatment of plant cell with Cu2+ induces AOX gene expression (Padua et al., 1999), promotes production of secondary metabolites (Fujita et al., 1981), inhibits Hf pump (Demidchik et al., 1997), and induces cytoplasmic acidification in Cuthurunthus cells (Yazaki, unpublished data, 1999).

V. Intracellular pH as Signal Transduction in Plant Cell-Pathogen Interaction A. Salicylic Acid and Cytoplasmic

pH in Defense

Component

Reaction

Salicylic acid (SA) has been recognized as one of the central signal compounds in plant-pathogen interaction (Yalpani et al., 1991; Raskin, 1992). Infection of tobacco mosaic virus (TMV) to tobacco plant induces hypersensitive response accompanying SA accumulation in resistant (NN) strains, but not in susceptible (nn) strains. Pretreatment of the susceptible strain with SA affected TMV replication after inoculation (Chivasa et al., 1997). They found that SHAM (salicylhydroxamic acid, an inhibitor of AOX) antagonized both SA-induced resistance to TMV in the susceptible strain and SA-induced acquired resistance in the resistant strain. However, SHAM did not inhibit SA-induced accumulation of PR- 1 pathogenesis related protein and resistance to Erwiniu carotovora or Botrytis cinerea. Conversely, treatment of plants with antimycin A and KCN (inhibitors of cytochrome pathway respiration) induced AOX transcript and resistance to TMV without inducing PR-1 accumulation (Chivasa and Carr, 1998). Transgenic plant of resistant tobacco strain (NN) expressing bacterial n&G gene encoding salicylate

34

KATSUHIROSAKANO

hydroxylase no longer accumulated SA and was susceptible to TMV. KCN treatment again restored the resistance and SHAM antagonized the effect. These observations led them to hypothesize that, in defense signal transduction, the pathway divides downstream of SA into two: one leading to induction of PR proteins and resistance to fungi and bacteria, and the other to induction of resistance to virus (Murphy et al., 1999). Since inhibitors of cytochrome pathway respiration (antimycin A, KCN) activated the latter virus-specific pathway, and SHAM antagonized it, they thought that AOX might play a role in the induction of systemic acquired resistance @AR) to viruses. The observed induction of AOX protein and elevated level of its transcript in tobacco tissue at SAR is consistent with their hypothesis. Despite that, the role of AOX in the hypothesis remains unknown. Xie and Cheng (1999) reported that, in suspension cultured tobacco cells, SA application (20-500 p,M) induced drastic decreases in both endogenous ATP level and oxygen uptake rate. Of the SA analogs tested, only biologically active analogs capable of inducing PR-protein genes could mimic the effects. In isolated mitochondria, however, both rate of respiration (with NADH as substrate) and ATP level were comparable to those of normal mitochondria from untreated plants, and direct incubation of mitochondria with SA exhibited no significant effects. Thus, the factor(s) that was induced in the SA-treated cells was missing in the isolated mitochondria. What is the underlying mechanism that implicates SA, AOX, and resistance to pathogen? So far, since most of the treatments that are reported to stimulate AOX transcription (Cu2+, propionate, KCN, elicitors, etc.) induced cytoplasmic acidification, we wondered if SA could acidify the cytoplasm of C. YOS~US cells using 31P-NMR spectrometry. SA concentrations of lo-50 @4 indeed acidified the cytoplasm (0.2-0.3 pH unit) and alkalinized the vacuole (Yazaki et al., unpublished data, 1999). Therefore, it is likely that SA acts not only as a signal molecule that induces PR proteins, but also as a direct inducer of cytoplasmic acidification that elicits defense reactions. Because SA acidifies the cytoplasm, through the action of biochemical pH stat, it would stimulate AOX resulting in a lowered ATP level. If stimulation of AOX and the resulting low ATP level are general features of SA-treated cells, it is tempting to speculate that SA is the signal upstream of AOX that is absolutely required in the defense reaction, probably in the synthesis of antipathogenic secondary products (phytoalexin), phenylpropanoids, terpenoids, lignins, SA itself, etc. Thus, SA-induced cytoplasmic acidification can be the missing link that connects the experiments at the levels of cell and mitochondria.

8. Function

of Oxidative Burst

in Defense

Reaction

Oxidative burst after elicitation of plant cells by biotic and abiotic elicitors has been regarded as one of the earliest responses of plant cells to pathogen infection

METABOLIC

REGULATlONOFpHIN

35

PLANTCELLS

(Doke, 1983; Levine et al., 1994). Superoxide anion (0;) and other ROS derived from it are produced in the external medium by NADPH oxidase at the plasma membrane. ROS are expected not only to attack pathogens directly, but also to act indirectly as a substrate of the cell wall peroxidases that form cross-linkages between cell wall components. ROS are also shown to work as a central signal that leads to hypersensitive response (HR) (Levine ef al., 1994). From the viewpoint of pH regulation, it is worth noting that oxidative burst induces cytoplasmic acidification (Mathieu et al., 1991; Kuchitsu ef al., 1997; Pugin et al., 1997; He et al., 1998). In oxidative burst, NADPH oxidase at the plasma membrane catalyzes transfer of electrons from internal NADPH which is provided by pentose phosphate pathway to external oxygen (reduction) and results in internal acidification and external alkalinization (Pugin etal., 1997): (NADPH + H’)in + (2 @)out + (NADP+ + 2 H+)i” + (2 O,),“,

(NADPH oxidase)

(2 0, + 2 HzO)out + (Hz02 + 02 + 2 OH-),,,

(superoxide dismutase)

(2

K+hn+

(2

K+)out

(K+channel)

(Overall reaction) (NADPH + 2 H+ + 2 K+)in+ (02 + 2 H20)out + (NADP+ + 2 H+)i” + (Hz02 + 2 OH- + 2 K+),,t Since NADPH oxidase reaction transfers only electrons, the plasma membrane depolarizes severely and, as a consequence, potassium efflux (2K+) takes place through the Kf channel in order to neutralize the electrogenic process. This results in an internal (cytoplasmic) acidification. Although many authors have noted extracellular alkalinization, so far little attention has been paid to the intracellular acidification during oxidative burst. And, of course, we should expect activation of alternative pathway respiration that would play a crucial role in the production of secondary metabolites including phytoalexins as a part of defense reactions. Oxidative-burst-induced cytoplasmic acidification may have at least two important functions. First is the induction of defense genes: some (including PAL) are expressed simply by acidifying the cytoplasm with propionic acid (He et al., 1998). This indicates that cytoplasmic acidification is one of the components in signal transduction. Second, it triggers the pentose phosphate pathway that is essential for secondary metabolism as it provides E4P, an indispesable precursor of many secondary metabolites leading to synthesis of phenylpropanoids, lignin, flavonoids, anthocyanins, etc. The pathway is usually inhibited at the first step (glucose-6-phosphate dehydrogenase, G6PDH) under the strong constraint of NADPH, an allosteric inhibitor of G6PDH (Ashihara and Komamine, 1974, 1976; Turner and Turner, 1980). When cells are elicited by pathogen infection or by elicitors, NADPH oxidase is activated and oxidizes NADPH to NADP+.

36

KATSUHIROSAKANO

Once the NADPH/NADP+ ratio is lowered, G6PDH is released from the inhibition by NADPH and the pathway initiates to produce E4P. C. Signal Transduction from Cytosolic Acidification to Alternative Pathway Respiration Since cytoplasm is not a single compartment of a cell, the initial cytoplasmic acidification induced by, e.g., Hf symport or oxidative burst should be limited to cytosol in the close vicinity of plasma membrane where the symport or elicitor binding takes place. The acidification would eventually prevail to mitochondrial matrix, where it is expected to stimulate malic enzyme and AOX. What is the mechanism that introduces cytosolic Hf into the mitochondrial matrix? Using isolated mitochondria from mung bean hypocotyl, Neuburger and Deuce (1980) observed that matrix pH could be varied by changing the pH of external medium: the lower the medium pH, the lower the pH of the matrix space and vice versa. This suggests the existence of H+ transporter(s), such as Kf/Hf antiporter, K+ channel, in the inner mitochondrial membrane. Despite the mitochondrial NAD-dependent malic enzyme (EC 1.1.1.39) adopted in the revised biochemical pH stat (Fig. l), we should not neglect the universal presence of cytosolic NADP-dependent malic enzyme (EC 1.1.1.40), expression of which gene is reported to be stimulated in tobacco plants by wounding, and pathogen-defense related stimuli such as reduced-form glutathione, fungal elicitor, and cellulase (Schaaf et al., 1995). Upon cytosolic acidification, this NADPdependent malic enzyme will respond to it, because it has optimum pH in acid-pH range (Davies, 1973,1986). We may expect that the reaction products of the cytosolit NADP malic enzyme (pyruvate, NADPH, CO*) will promote the engagement of alternative pathway respiration. In the oxidation of cytosolic pyruvate, it must be taken up first into the matrix across the inner mitochondrial membrane. Since it depends on the H+/pyruvate symport mechanism, we should expect matrix acidification. But the oxidation of pyruvate to CO2 and Hz0 is a H+-consuming process: Pyruvate- + H+ + 2.5 02 + 3 CO2 + 2 Hz0 + (energy) Therefore, steady-state oxidation of pyruvate balances the entrance of and consumption of H+, resulting in no piling up of H+ in the matrix. On the other hand, non-steady-state sudden increment of cytosolic pyruvate as derived from the malic enzyme reaction would increase its symport into the matrix and acidify it, which in turn would either directly or indirectly (through stimulation of mitochondrial malic enzyme) activate AOX. As to oxidation of NAD(P)H of cytosolic origin, Meller (1997) reported that plant mitochondria have rotenone-insensitive, Ca2+-dependent NAD(P)H

METABOLlCREGULATlONOFpHINPLANTCELLS

37

dehydrogenases located in the inner membrane facing the intermembrane space. Under stress conditions such as oxidative burst that accompany cytoplasmic acidification, Ca2+ influx is one of the essential conditions that leads to a defense reaction (Felle, 1988; Bach er al., 1993; Tavernier et al., 1995; Jabs et al., 1997; Ebel and Mithiifer, 1998). Increase in cytosolic Ca2+ concentration associated with cytoplasmic acidification would help enable the oxidation of external NAD(P)H by these dehydrogenases, which in turn, would provide reducing equivalents to reduce disulfide bond of AOX. Otherwise, external NAD(P)H + H+ (i.e., reducing equivalents) could be transferred into matrix by way of the malate-oxaloacetate shuttle system (Held, 1997).

VI. Concluding

Remarks

and Prospects

In the previous paper, Sakano (1998) pointed out that the metabolisms that constitute the revised biochemical pH stat (i.e., the alternative pathway glycolysis, alternative pathway fermentation and alternative pathway respiration) are common and unique, if not exclusive, to the plant kingdom and not seen in nonplant organisms. Two features of the plant alternative pathway respiration, (1) energycharge independent Hf-consuming function and (2) activation by the reaction products of malic enzyme (pyruvate and NAD(P)H) in a feed-forward manner, were taken as an indication that its basic function is pH regulation of the cytoplasm. Uniqueness and commonality among the plant kingdom of the biochemical pH-stat mechanism (including alternative pathway respiration) was characterized as the “security mechanism” of the H+ system adopted by ancestors of plant during evolution. In contrast, recent aspects of the physiological role of alternative pathway respiration are overwhelmingly an overflow of excess reducing equivalents caused by metabolic imbalance (Lambers, 1985; Day et al., 1996; Vanlerberghe and McIntosh, 1997; Simons et al., 1999) to prevent reactive oxygen generation (Wagner and Krab, 1995; Wagner and Moore, 1997). The two seemingly opposing views are reconciled because the reducing equivalents in biological system are usually in the form of NAD(P)H + H+ and, therefore, respiratory metabolism (glycolysis, Krebs cycle, and respiration) is deeply involved in H+ metabolism (Section 1V.A). This reconciliation led to the new thought that cytoplasmic acidification is a signal that triggers consumption of excess reducing equivalents. Thus, metabolisms that cogenerate excessreducing equivalents, such as biotic and abiotic stress-induced secondary metabolite production, are triggered through cytoplasmic acidification. In future studies, measurement of cytoplasmic pH and application of biochemical pH-stat hypothesis to secondary metabolism will be fruitful in understanding plant defense reactions. This will provide a comprehensive view that connects a number of apparently complex and conflicting observations to each other.

38

KATSUHIROSAKANO

In the present review, H+ metabolism in the alkaline-pH range was discussed only scarcely. NaCl induces cytoplasmic alkalinization in a salt stress-tolerant barley root tissue (Katsuhara et al., 1997). This would in turn stimulate PEP carboxylase and glycolysis that is protonogenic (Section III.B.2). The author (KS) speculates that this can contribute to production of osmoticum-like sugar alcohols (Lambers, 198.5), glycerol (Goyal et al., 1987), proline (Samaras et al., 1995), etc. Retardation of xylem differentiation in soybean roots under saline stress (Hilal et al., 1998) may be explained by a possible alkalinization-induced disengagement (or inhibition of gene expression) of alternative pathway respiration that is required in the synthesis of phenylpropanoids (precursors of lignin). The proposed roles of the biochemical pH stat in the alkaline-pH range are also subject to future examination.

Acknowledgments The author expresses his encouragement throughout University, and Professor during the course of this cooperation as coworkers

gratitude to Professor Emeritus M. Tazawa of University of Tokyo for his this work. He is also grateful to Professor Emeritus I. Uritani of Nagoya T. Minamikawa of Tokyo Metropolitan University for helpful discussions study. The author also thanks Dr. Y. Yazaki and Mr. S. Kiyota for their in the studies cited in this review.

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