Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 53, No. 367, pp. 151-173, February 1, 2002
© 2002 Oxford University Press

Review Article

pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions. II. Modifications in modes of metabolism induced by variation in the tension on the water column and by stress

A.G. Netting¹

School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, 2052, Australia

Received 10 May 2001; Accepted 19 September 2001

	Abstract

Top Abstract Introduction Photosynthesis and... Respiration Metabolic modulations induced by... Abscisic acid (ABA), the... Note added in proof References

 
The hydrolysis of ATP^4- by the plasmalemma and tonoplast H⁺/ATPases and by the tonoplast pyrophosphatase results in the export of a proton to the apoplast or vacuole with remaining in the cytoplasm. As the enzymes that synthesize ATP^4- require as a substrate it is proposed that protons are an essential substrate for ATP^4- synthesis. Thus, the entry of protons to the cytoplasm by sym- and antiports will control the rate of ATP^4- synthesis. Evidence is adduced that plants control the tension on the water column by removing water to or from the ‘cellular reservoir’ and guard cells by generating osmotic gradients. Schemes are presented that propose a series of metabolic changes that result in a seamless transition through the following states: (1) the import of K⁺, Cl^- and water from the apoplast to the vacuole, the K⁺ being admitted to the cytoplasm via a Ca²⁺-activated K⁺-H⁺ symport and the water via a Ca²⁺-activated aquaporin; (2) the continued import of K⁺ and water from the apoplast to the vacuole with the concomitant export of protons and the synthesis of malate from glucose in the cytoplasm for importation into the vacuole; (3) when the tension on the water column is optimal, respiration and photosynthesis is maximal resulting in biosynthetic reactions and growth; (4) when tension on the water column increases, K⁺, Cl^- and water are exported from the vacuole to the apoplast; (5) the continued export of K⁺ and water from the vacuole to the apoplast with malate for export being synthesized in the cytoplasm; the export of K⁺ resulting in the acidification of the vacuole; and (6) a further increase in tension results in the deactivation of the plasmalemma H⁺/ATPase by a further increase in cytoplasmic Ca²⁺ which also indirectly activates the alternative oxidase. It is suggested that mitochondrial pyruvate is partly oxidized by the TCA cycle and is partly exported to the cytoplasm where it is carboxylated to form malate^1- for continued export to the apoplast. K⁺ is transferred from the vacuole to the apoplast, the K⁺ being replaced by protons from the export of mitochondrial pyruvate. The maintenance of the tonoplast electrochemical gradient is thought to result in an increase in the pH of the apoplast which may cause the hydrolysis of abscisic acid precursors with the resulting abscisic acid opening Ca²⁺ channels so that the above events are reinforced. (7) This mode is proposed to continue by the metabolism of glucose to four phosphoenolpyruvate, three of which are carboxylated to malate^1- for continued export to the apoplast with K⁺ from the vacuole, the ‘stress-tolerant quiescent state’.

Key words: Alternative oxidase, malate, metabolic pathways, protons, stress.

	Introduction

Top Abstract Introduction Photosynthesis and... Respiration Metabolic modulations induced by... Abscisic acid (ABA), the... Note added in proof References

 
Plant cells, in contradistinction to many animal cells such as nerve, muscle and red blood cells, establish electrochemical gradients across the plasmalemma using protons (Sakarno, 1998). The principal enzyme involved, then, is the H⁺/ATPase (Netting, 2000). Similarly, the H⁺/ATPase and the pyrophosphatase establish the electrochemical gradient across the tonoplast (Netting, 2000). In these circumstances these enzymes and the distribution of protons that they control are surely absolutely central to the control of plant metabolism. Yet very little attention has been directed to attempting to quantify the distribution of protons among subcellular compartments other than in terms of the maintenance of cellular, principally cytoplasmic, pH (Davies, 1973, 1986; Smith and Raven, 1979; Sakarno, 1998). This review attempts to remedy this deficiency.

In the green alga, Eremosphaera viridis, it has been shown that the cytoplasmic pH is 7.3 (Thaler et al., 1992) and this is in the range of 7.0–7.5 given for higher plants (Smith and Raven, 1979). When guard cells are stressed the pH of the cytoplasm increases by up to 0.3 units (Irving et al., 1992; Blatt et al., 1998) probably from about 7.3 to about 7.6. It is assumed here that other plant cells behave similarly so that this range of pHs will be used here as an estimate of cytoplasmic pH.

The buffer capacity of the cytoplasm in spinach leaves has been estimated to be about 25 mM (pH unit)^-1 (Oja et al., 1999) under the assumption that the cytoplasmic pH is 7.25. Further, data on the total phosphate concentration in various subcellular compartments in spinach leaves (Gerhardt et al., 1987) suggest that this concentration for the cytoplasm is about 20 mM. This, therefore, implies that the major buffering system in the cytoplasm is HOPO₃^2-/(HO)₂PO₂^-. The pK for this equilibrium is 7.2 so that, according to the Henderson-Hasselbalch equation, [HOPO₃^2-]/[(HO)₂PO₂^-] is in the range of 1.3 at pH 7.3 to 2.5 at pH 7.6. At these pHs, then, the predominant form of phosphate is HOPO₃^2- and this formula will be used here. This is convenient in that when ATP^4- is hydrolysed by the H⁺/ATPases at the plasmalemma and the tonoplast, HOPO₃^2- remains in the cytoplasm while the proton is exported:

(001)

Conceptually, the tonoplast pyrophosphatase is similar in that the reaction that it catalyses:

(002)

is essentially:

(003)

Thus, the cytoplasmic product of establishing the electrochemical gradients across the tonoplast and plasmalemma is HOPO₃^2-. On the other hand, all of the enzymes, namely pyruvate kinase, the mitochondrial inner membrane phosphate translocator and the mitochondrial succinyl CoA synthetase and ATP synthase, that incorporate phosphate into ATP^4- utilize (HO)₂PO₂^-, or its equivalent, as a substrate (see below). That is, the hydrolysis of ATP^4-, or the equivalent phosphate esters, at membranes liberates HOPO₃^2- while the synthesis of ATP^4- requires (HO)₂PO₂^-. It can be concluded from this that H⁺ is an essential substrate for ATP^4- synthesis and that its concentration, that is the pH, may exercise some control over the rate of ATP^4- synthesis. In this situation, then, it seems likely that the phosphate buffer system will dampen fluctuations in the rate of ATP^4- synthesis, but will not appreciably alter the amount of ATP^4- synthesized in response to an influx of protons. In this review, then, phosphate will be represented by HOPO₃^2- if it is produced by a membrane H⁺/ATPase, but will be represented by (HO)₂PO₂^- (or H⁺+HOPO₃^2-) if it is a substrate for ATP^4- synthesis. Thus, in the steady-state schema of metabolism developed in this review it will be assumed that the entry of protons into the cytoplasm via various symports and antiports implies the formation of an equivalent number of (HO)₂PO₂^- ions from HOPO₃^2- so that an equivalent number of ATP^4- can be synthesized. This proposed role for protons as an essential substrate for ATP^4- synthesis is a central theme of this review so that particular attention will be devoted to the distribution of protons among subcellular compartments.

In an earlier review (Netting, 2000) it was proposed that when plants are stressed they can adopt one of two strategies. (1) Cells within the plant can ‘fight’ the stress, which apparently involves the activation of the H⁺/ATPases so that protons are pumped into the apoplast and vacuole. This might, for example, remove Na⁺ from the cytoplasm using H⁺/Na⁺ antiports. (2) Cells can adopt the stress-tolerant quiescent state in which the plasmalemma H⁺/ATPase is deactivated, so that the electrochemical gradient is reduced in magnitude, and remain in this state until the stress is removed.

The discussion in this review is directed towards the consequences for metabolism of the proposed distribution of protons and, in particular, to outlining the changes that occur in metabolism in adopting the stress-tolerant quiescent state.

	Photosynthesis and photorespiration

Top Abstract Introduction Photosynthesis and... Respiration Metabolic modulations induced by... Abscisic acid (ABA), the... Note added in proof References

 
Photosynthesis under steady-state conditions
The transformations that occur during the dark reactions of photosynthesis are summarized by the following equation (Stryer, 1995):

(004)

Thus, the light reactions of photosynthesis have to provide 18 ATP^4- and 12NADPH so that 6CO₂ can be fixed as carbohydrate. Concomitantly, the hydrolysis of 18 ATP^4- liberates 18 protons which, in due course, will be re-incorporated into 18 ATP^4-. From this view point, then, ATP^4- is a transient store for protons.

The number of protons required to flow through the ATP synthase to synthesize an ATP^4-, that is the H⁺/ATP^4- coupling ratio, has been found experimentally to be 4 (Berry and Rumberg, 1996). However, it has been observed that there are 14 c subunits arranged in a ring in the F_o complex of the thylakoid ATP synthase (Seelert et al., 2000) which may imply that the coupling ratio is 4.7 if one proton is required per c subunit (Ferguson, 2000).

Thus, as 18 ATP^4- are required either 72 or 84 protons need to pass through the ATP synthase. The following list suggests how these protons are provided with a coupling ratio of 4 and, in brackets, 4.7 (Stryer, 1995):

View this table: [in this window] [in a new window]

 
The net number of moles of protons produced or consumed, after the fixation of six moles of carbon dioxide, is therefore independent of the coupling ratio and is zero in both the thylakoid lumen and the stroma.

Thus, in spite of the flux of protons between the stroma and the lumen, the chloroplast is, under these idealized steady-state conditions, independent of the cytoplasm for the import or export of protons. This emphasizes that, in metabolic terms, the function of chloroplasts is to provide carbohydrate: under non-stressed steady-state conditions they do not provide ATP^4-, reducing equivalents or a source of or sink for protons. This result also suggests that the control of photosynthesis by the elimination of CO₂ fixation below pH 7.3 (Werdan et al., 1975) is due to an influx of protons into the stroma from the cytoplasm rather than to a redistribution of protons within the chloroplast.

The above considerations of the balance of protons between the stroma and the lumen should not be taken as a denial that proton-consuming and proton-releasing reactions occur in the stroma; only that the production of protons in the stroma may diminish the pH, if the buffering capacity of 10–20 mM (pH unit)^-1 (Oja et al., 1999) is exceeded, towards 7.3 when CO₂ fixation ceases (Werdan et al., 1975).

Photorespiration under steady-state conditions
It can be shown that the following equation summarizes the transformations that occur during photorespiration:

(005)

Thus, the light reactions have to provide 10, 29 ATP^4- and 15NADPH. Note that in photorespiration 29 ATP^4- are hydrolysed to release 29 protons which will, in due course, be reincorporated into ATP^4- so that, again, ATP^4- may be viewed as a transient store of protons. However, photorespiration does differ from photosynthesis in one important respect: for every three moles of O₂ fixed one mole of protons from NADH+H⁺ is incorporated into glycerate in the peroxisome and then transported to the chloroplast stroma where it is, in effect, released. The implications of this release of protons in the stroma will be considered in more detail below.

The provision of 15NADPH by photosystems I and II implies that 30 protons will be released in the lumen from the splitting of water, 30 protons will be pumped in via plastoquinone and 15 H⁺ will be incorporated into NADH in the stroma. Similarly, the reduction of ferredoxin implies the release of 10 H⁺ from water and a further 10 H⁺ via plastoquinone. Thus, a total of 80 H⁺ will accumulate in the lumen. If the coupling ratio is 4 a total of 116 H⁺ need to pass through ATP synthase for the synthesis of 29 ATP^4-. Thus, 36 protons need to be provided by cyclic photophosphorylation and by the water–water cycle to give a total lumenal content of 116 H⁺. In the following summary a coupling ratio of 4 has been used with the figures in brackets corresponding to a coupling ratio of 4.7:

View this table: [in this window] [in a new window]

 
Thus, again it can be seen that the distribution of protons is independent of the coupling factor and that the net moles of protons in both the stroma and the thylakoid lumen is zero.

	Respiration

Top Abstract Introduction Photosynthesis and... Respiration Metabolic modulations induced by... Abscisic acid (ABA), the... Note added in proof References

 
Respiration under steady-state conditions
A scheme for the transformations that occur in a cell that is actively respiring is given in Fig. 1. It is assumed here that the depicted cell is in a steady-state so that ATP^4- utilized in biosynthesis and growth (‘x’ in Fig. 1) and in maintaining the tonoplast and plasmalemma electrochemical gradients (‘m’ and ‘n’, respectively) is replaced by pyruvate kinase, succinyl CoA synthetase, and ATP synthase. As the synthesis of ATP^4- by all three of these enzymes requires ADP^3-, HOPO₃^2- and H⁺ in a 1:1:1 ratio to give (HO)₂PO₂^- and ADP^3- as substrates, it is implied that the return of protons to the cytoplasm is a prerequisite for the resynthesis of the ATP^4- utilized in maintaining the electrochemical gradients. Thus the import of nutrient anions such as sulphate and nitrate via symports (Michelet and Boutry, 1996) implies the import of ‘n’ protons (Fig. 1) which, combined with nADP^3- and nHOPO₃^2- remaining in the cytoplasm from earlier cycles of plasmalemma H⁺/ATPase activity, implies the synthesis of nATP^4-. Sucrose also enters cells via an H⁺–sucrose symport (Frommer et al., 1996) presumably to be stored in starch for future biosynthetic reactions or for energy production. Thus, ‘n’ includes protons imported with sucrose. Similarly, the entrance of cations, particularly K⁺, implies, since the cell is assumed to be in a steady-state with a cytoplasm that is replete with these ions, their storage in the vacuole utilizing a K⁺/H⁺ antiport (Cooper et al., 1991) with the consequential release of ‘m’ protons (Fig. 1) into the cytoplasm. This again implies the synthesis of mATP^4-. From this viewpoint, then, cytoplasmic protons are the essential substrate for ATP^4- synthesis and therefore exert some control over the rate of turnover of ATP^4-.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Glycolysis in a cell replete with water. It is assumed that, under the steady-state conditions depicted here, the total number of protons (n) that accompany anions and sucrose from the apoplast plus those (m) that exchange with cations at the tonoplast plus those (x) released from ATP4- during growth and biosynthetic reactions minus those incorporated into ATP4- at pyruvate kinase and those accompanying malate and pyruvate into the mitochondrion (t) equals the net ATP4- synthesized (n+m+x-t) in the mitochondrion, as discussed in the text. Under the conditions depicted here it seems likely that most of the ATP4- will be utilized for growth and biosynthesis (that is, ‘x’ will be relatively large) rather than in maintaining the electrochemical gradients. The movement of anions through H+–anion symports is based on Michelet and Boutry (Michelet and Boutry, 1996) and the exchange of cations, principally K+, for protons at the tonoplast on Cooper et al. (Cooper et al., 1991). The glycolytic pathway presented here emphasizes the fate of the protons and is based on schemes in ‘Biochemistry’ (Stryer, 1995). NADH+H+ produced at glyceraldehyde-3-phosphate dehydrogenase is depicted as being reoxidized by the malate/aspartate shuttle, a reaction that requires the importation of two protons into the mitochondrion with the synthesis of a total of 31 ATP4- (glucose)-1 (Brand, 1994). Other possibilities are discussed in the text.

 
In Fig. 1 it can be seen that, per mole of glucose oxidized, a net of two moles of ATP^4- are synthesized in the cytoplasm and that two moles of protons are required to transport two moles of pyruvate into the mitochondrion. Or, from an alternative view point, one mole of protons is required for each mole of ATP^4- synthesized. Figure 1 also suggests that the NADH+H⁺ produced at glyceraldehyde-3-phosphate dehydrogenase is reoxidized by the malate/aspartate shuttle, a process that will result in the transport of two further protons into the mitochondrion and the synthesis of up to 31 ATP^4- (glucose)^-1 (Brand, 1994, see below). The malate/ aspartate shuttle is a possibility in cells that are actively growing or synthesizing secondary plant products. Alternatives are the production of NADPH+H⁺ by a transhydrogenase with the concomitant oxidation of NADH+H⁺ to NAD⁺ (this might occur during reductive biosynthetic reactions) and the oxidation of NADH+H⁺ by a dehydrogenase in the outer mitochondrial membrane (Day, 1999). The transformations in Fig. 1, including the import of two protons by the malate/aspartate shuttle, are summarized by the following equation:

(006)

Noting that in plants succinyl CoA synthetase phosphorylates ATP (Day, 1999) rather than GTP, the oxidation of pyruvate by the citric acid cycle can be represented by:

(007)

Thus, in the absence of the malate/aspartate shuttle two protons per pyruvate are required to be imported into the mitochondrion. One, depending on the ionization states of the organic acids in the mitochondrion will be incorporated into acetyl CoA and one, along with ADP^3- and HOPO₃^2-, will be required for the substrate level phosphorylation at succinyl CoA synthetase. These substrates are provided as (HO)₂PO₂, along with an additional proton, and ADP^3- when the H⁺–(HO)₂PO₂^- symport and the ADP^3-/ATP^4- antiport, acting in concert, export ATP^4-.

The oxidation of NADH+H⁺ and of FADH₂ by the respiratory chain is thought to pump 10 and six protons, respectively, across the mitochondrial inner membrane when 0.5O₂ is the electron acceptor (Hinkle et al., 1991; Brand, 1994). Thus, the following equations summarize the number of protons transferred across the mitochondrial inner membrane for the complete oxidation of two moles of pyruvate in the absence of the malate/aspartate shuttle with a coupling ratio of 4 and, in parentheses { }, in the presence of the shuttle with a coupling ratio of 3:

(008)

(009)

for the pumping of protons from the oxidation of [NADH+H⁺] and of FADH_2.in the presence of { } and absence of the malate/aspartate shuttle.

(010)

for the passage of protons through ATP synthase.

(011)

for the combined operation of the ATP^4-/ADP^3- translocase and the H⁺–(HO)₂PO₂^- symport.

The possible significance of the variable number of c subunits in the F_o complex of ATP synthase is discussed in the longer version of this paper. The distribution of protons across the inner mitochondrial membrane that these equations imply is summarized in the following lists:

(i) In the presence of the malate/aspartate shuttle with a coupling ratio of 3:

(ii) In the absence of the malate/aspartate shuttle with a coupling ratio of 4:

View this table: [in this window] [in a new window]

 

View this table: [in this window] [in a new window]

 
Under the steady-state conditions assumed here the import of 29 (i) or 20 (ii) (HO)₂PO₂^- into the mitochondrion implies that the cytoplasm has to supply 29 or 20 H⁺. Thus, in Fig. 1, m+n+x-t=29 (i) or 20 (ii) where ‘t’ equals 4 in the presence of the malate/ aspartate shuttle and 2 in its absence. It is noteworthy that, under the steady-state conditions assumed for both of these scenarios, the total H⁺ provided by the cytoplasm for incorporation into (HO)₂PO₂^- is equal to the total ATP^4- synthesized by the mitochondrion as would be expected if ATP^4- were a transitory store for protons. Also, in the cytoplasm, two protons are incorporated into two ATP^4- at pyruvate kinase, but ATP^4- synthesis is not directly coupled to the import of H⁺ by the malate/ aspartate shuttle. This latter importation may serve to increase the supply of protons in the mitochondrion thereby stimulating respiration and proton pumping.

Carbonic acid produced by the TCA cycle from the oxidation of pyruvate will diffuse across the mitochondrial membrane and then ionize in the cytoplasm, the pH there being about 7.3 while the pK₁ of carbonic acid is 6.35. This, therefore, appears to be another source of protons but, in photosynthesizing cells, the substrate for Rubisco is carbon dioxide so that bicarbonate present in the chloroplast stroma is converted to carbonic acid, utilizing carbonic anhydrase (Evans and von Caemmerer, 1999), thereby consuming these protons. In non-photosynthesizing and non-photosynthetic cells bicarbonate may tend to accumulate but it is incorporated into malate, along with accompanying protons to give malate^1-, and stored in the vacuole or exported to the apoplast as described in the next two sections.

	Metabolic modulations induced by tensional changes in the water column

Top Abstract Introduction Photosynthesis and... Respiration Metabolic modulations induced by... Abscisic acid (ABA), the... Note added in proof References

 
Decreased tension on the water column
In principle, there are two means by which a plant can respond to changes in the environment that lead to changes in the tension on the water column. Firstly, the stomatal aperture can be altered so that there is a change in the evaporation rate to compensate for the changed tension and, secondly, water can be removed from or added to the water column again to compensate for the changed tension. Results from recent experiments with barley seedlings (Passioura and Munns, 2000) suggest that both of these possibilities are exploited. Thus, when the relative humidity was increased, the elongation rate of barley seedling leaves almost doubled before settling back to close to the original value after about 20 min. These changes were accompanied by an increase in leaf thickness and an apparent increase in stomatal aperture over 20 min, as determined from the leaf temperature. This suggests that the increase in humidity decreased the tension on the water column by reducing the evaporation rate at the stomates. This decrease in tension was then transduced into an uptake of water by both guard cells and cells within the leaf so that tension increased again within the leaf and the evaporation rate increased due to an increased stomatal aperture. As the original elongation rate was restored it is, in fact, implied that the tension on the water column returned to its original, presumably optimum, value. That these changes were due to changes in the tension on the water column was demonstrated by applying a ‘balancing pressure’ on the soil in the pot so that the xylem sap from a cut petiole was kept at the point of incipient bleeding: this experimental design almost completely abolished changes in elongation rate and leaf thickness, but showed an apparent increase in stomatal aperture that compensated for a decrease in balancing pressure. These results therefore suggest that water moves freely into cells when the tension on the water column is decreased.

Figure 2 offers a scenario for the metabolic changes that might occur so that water can be drawn into cells under these conditions. A mechanosensitive Ca²⁺ channel is shown linking the cell wall to the plasmalemma so that relative movement between them, or between the former and the cytoskeleton, could open the channel (Garrill et al., 1996). Elevated cytoplasmic Ca²⁺ is known to open an aquaporin (Johansson et al., 1996, 1998) so that water may flow out of or into the cell (Schäffner, 1998) depending on the osmotic gradients between the compartments (Netting, 2000). Elevated cytoplasmic Ca²⁺ is also shown as activating the K⁺–H⁺-symport so that both protons and K⁺ are admitted to the cytoplasm. This is based on the observation that Ca²⁺ appears to be required to assay the properties of this transporter, HKT1 (Schachtman and Schroeder, 1994). Interestingly, HKT1 has the type of distribution that might be expected if it is involved in the uptake of ions to control the osmotic potential: it is particularly prevalent in the root cortex and layers of cells surrounding the vascular tissue in leaves (Schachtman and Schroeder, 1994). The admission of protons could explain the apparent activation of the plasmalemma H⁺/ATPase by Ca²⁺ (Kinoshita et al., 1995) because these protons would be stored transiently in ATP^4- before the latter formed a substrate for the plasmalemma H⁺/ATPase. If the water that is taken up by the Ca²⁺-activated aquaporin is to be transferred through the tonoplast, presumably by another aquaporin, the admission of K⁺ to the vacuole is required. Thus, it is proposed here that cytoplasmic K⁺ is transferred to the vacuole by a K⁺/H⁺ antiport (Cooper et al., 1991) so that a second proton is admitted to the cytoplasm to serve as a substrate for ATP^4- synthesis. An additional ‘r’ (where ‘r’ is zero or greater) H⁺ and K⁺ are also shown (Fig. 2) as being admitted to the cytoplasm so that the plasmalemma (and tonoplast) electrochemical gradient is maintained. The ‘r’ K⁺ ions would be re-exported by the K⁺ outward rectifying channel and the protons would form a substrate for the synthesis of ‘r’ ATP^4- so the ‘r’ H⁺ can be re-exported by the plasmalemma H⁺/ATPase. The recycling of ‘r’ protons is therefore independent of the uptake of ions to form the osmotic gradient. The import of K⁺ into the vacuole also implies the import of anions so that the charge balance is maintained. Cl^- ions are shown as performing this role because they, unlike NO₃^- and SO₄^2-, are not essential nutrients and yet should be plentiful in the soil water available to most plants. The import of Cl^- implies the import of two protons so that the Cl^- ions can utilize the plasmalemma electrochemical gradient resulting in the synthesis of a further two ATP^4-. However, the import of Cl^- into the vacuole should be able to proceed via an anion channel as the tonoplast electrochemical gradient is positive on the vacuolar side. It can be seen, therefore, that the result of the transformations in Fig. 2 is:

(012)

View larger version (33K): [in this window] [in a new window]   Fig. 2. Water uptake utilizing KCl. In this scheme Cl- is taken up by a 2 H+–Cl- symport and then secreted into the vacuole by an anion channel. The charge balance is maintained by importing K+ by the K+–H+ symport and exchanging K+ for H+ at the tonoplast via an antiport. The plasmalemma (and tonoplast) electrochemical gradient is maintained by importing ‘r’ H+ with ‘r’ K+ by the K+–H+ symport and utilizing these protons for ATP synthesis so that a total of (r+3) H+ can be exported by the plasmalemma H+/ATPase. One H+ is also provided by the tonoplast H+/ATPase to replace the H+ exported on the import of K+. The broad-shafted arrows at the plasmalemma K+–H+-symport and aquaporin indicate that these carriers may be activated by Ca2+.

 
Thus, providing the opening of the aquaporins is co-ordinated with the admission of these ions, water should flow into the vacuole.

Within a plant there is likely to be a surplus of inorganic cations over free inorganic anions since NO₃^- and SO₄^2- would be reduced and fixed in nucleotides, proteins and secondary plant products while only a very small proportion of the cations, such as Fe²⁺, Fe³⁺ and Cu²⁺, would be so fixed. This implies that there may be insufficient free anions, in relation to the free inorganic cations, in the apoplast for the required transport of ions to the vacuole to provide the osmotic potential to draw in water. A scenario in which these anions are provided as malate^1- from the cytoplasm is presented in Fig. 3. This differs from the scheme presented in Fig. 2 in that, as there are now no anions available in the apoplast, the incoming K⁺ are replaced by exported H⁺ and that the energy is provided by the oxidation of one of the four moles of malate^1- derived from the oxidative cleavage of two moles of glucose. If, in this scheme, the synthesis of ATP^4- is dependent on the availability of protons as proposed above it is possible to track the fate of protons and deduce the number of ATP^4- synthesized. Thus, during glycolysis from two glucose (Fig. 3) four protons are produced during the synthesis of fructose-1,6-bisphosphate. These are reincorporated and lost again at 3-phosphoglycerate kinase, and then one of them is proposed to be imported into the mitochondrion by the dicarboxylate translocator with three remaining in the cytoplasm. There is no direct evidence for the participation of this carrier, but such a scheme provides a proton for incorporation into acetyl CoA and a bicarbonate for condensation with phosphoenolpyruvate. The other three bicarbonates incorporated into oxaloacetate are, of course, derived from the oxidation of pyruvate by the citric acid cycle This scheme, therefore, results in (9+r+x) protons remaining in the cytoplasm: three are imported from the apoplast by three cycles through the K⁺–H⁺-symport, three are exported from the vacuole by three cycles through the K⁺/H⁺-antiport, ‘r’ are imported by the K⁺–H⁺-symport in maintaining the plasmalemma (and tonoplast) electrochemical gradient and ‘x’ are released from ATP^4- during maintenance reactions (Fig. 3), besides the three generated by glycolysis. That is, there are (9+r+x) (HO)₂PO₂^- available for ATP^4- synthesis by ATP synthase and by succinyl CoA synthetase. The oxidation of pyruvate by the TCA cycle is represented by the equation given above so that 10 and six protons would be pumped across the mitochondrial inner membrane by the oxidation of NADH+H⁺ and FADH₂, respectively. These transformations can, therefore, be summarized by the following equations:

(013)

View larger version (34K): [in this window] [in a new window]   Fig. 3. Water uptake utilizing K+ malate1-. In this scheme it is assumed that K+ malate1- needs to be secreted into the vacuole so that sufficient osmotic potential is developed to draw water into the vacuole. It is further assumed that three protons required to suppress the ionization of one of the carboxyl groups of malate are provided by the export of carbonic acid from the mitochondrial oxidation of pyruvate derived from imported malate. Under steady-state conditions, the number of protons released in the cytoplasm is (3+r) from the plasmalemma K+–H+-symport plus three from the tonoplast K+/H+ antiport plus four from the ionization of 3-phosphoglycerate minus one imported into the mitochondrion by the dicarboxylate exchanger plus ‘x’ from ATP4- required for the maintenance of cellular integrity. Thus, it is assumed that the (3+3+4-1+r+x)H+ imported into the mitochondria by the H+–(OH)2PO2- symport is equal to the ATP4- synthesized as discussed in the text.

 
Omitting the maintenance of the plasmalemma and tonoplast electrochemical gradients for clarity, the transfer of malate^1- and K⁺ to the vacuole can be represented by:

(014)

The total for the above reactions including the citric acid cycle is:

(015)

Thus, the overall effect of these reactions is that, for each malate^1- stored in the vacuole, one K⁺ is transferred from the apoplast to the vacuole and one proton generated in the cytoplasm is exported to the apoplast. This exchange of one H⁺ for one K⁺ in the apoplast is in agreement with data for guard cells from bean leaves (Raschke and Humble, 1973) and the acidification of the apoplast is in agreement with observation for guard cells in opening stomata (Edwards et al., 1988) and would provide the acidic apoplastic milieu required for cell expansion (Cosgrove, 1998). Also of interest is the observation that the solute content of Vicia faba guard cells increased by 4.8x10^-12 Osmol per stoma when the stomatal aperture increased from about 2 µm to 12 µm (Humble and Raschke, 1971). During stomatal opening an average of 4.0x10^-12 g equivalents K⁺ entered a guard cell pair which, if accompanied by a dibasic organic anion, would be sufficient to produce the required increase in osmotic pressure (Humble and Raschke, 1971) to draw water into the guard cell vacuole. These authors suggested that malate, synthesized from stored starch, was a possibility for the dibasic anion. Further, when blue light induces stomatal opening by activating the plasmalemma H⁺/ATPase intracellular malate is increased and the activity of phosphoenolpyruvate carboxylase, which is involved in malate synthesis (Fig. 3), also increases, an activation that may depend upon increased K⁺ in the cytoplasm (Assmann and Shimazaki, 1999).

The oxidation of malate in the mitochondrion will provide 5NADH+5 H⁺ and one FADH₂ for oxidation by the respiratory chain. Thus, 56 H⁺ will be pumped out of the mitochondrion with a coupling ratio of 4{3}, 44{41.25}H⁺ will pass through ATP synthase to generate 11{13.75}ATP^4- and a total of 12{14.75}H⁺ will accompany (HO)₂PO₂^- with ADP^3- in exchange for 12{14.75}ATP^4-. This transfer of protons is summarized in the following list for a coupling ratio of 4 and, in parentheses { }, 3:

View this table: [in this window] [in a new window]

 
It was concluded above that (9+r+x) (HO)₂PO₂^- were available for ATP^4- synthesis so that the above figures suggest that 9+r+x=12–14.75, i.e. x+r=3–5.75.

If this scenario does indeed reflect reality these calculations emphasize that the uptake of water utilizing K⁺ malate^1- to drive cell expansion and stomatal opening is energetically quite expensive: apparently nine of the 12, or perhaps 14.75, ATP^4- available from the oxidation of one-quarter of the malate are devoted to secreting the remaining three-quarters into the vacuole as K⁺ malate^1-. On the other hand, three ATP^4- per two glucose oxidized might be more than sufficient for the maintenance of cellular integrity and of the plasmalemma and tonoplast electrochemical gradients.

Increased tension on the water column
When the relative humidity was decreased in the experiments with barley seedlings (Passioura and Munns, 2000) the barley leaf elongation rate dropped to zero over about 6 min, but recovered to close to the original value by 20 min. At the same time the leaf thickness decreased and the stomatal aperture decreased and, again, all of these changes were almost completely abolished by the application of a balancing pressure. As these changes are in the opposite direction to those observed with an increase in humidity these results imply that water moved out of cells within the leaf and out of the guard cells.

A case has been made (Netting, 2000) that increased tension on the water column opens mechanosensitive Ca²⁺ channels (Garrill et al., 1996) so that aquaporins could be activated to allow the outflow of water to the apoplast. This is similar to the scenario offered in the previous section in which the Ca²⁺-activated aquaporins allowed the inflow of water and Ca²⁺ activated the import of K⁺ and H⁺ via the K⁺–H⁺ symport. In the present scenario (Fig. 4) it is proposed, therefore, that the aquaporin is activated to allow the outflow of water and that this is driven by an osmotic gradient generated by the outflow of K⁺ and Cl^- from the vacuole. This is essentially the reverse of the scenario presented in Fig. 2, the principal difference being that only two ATP^4- are required to replace the two protons utilized to export Cl^- from the vacuole rather than four ATP^4- (Fig. 2). Cl^- can be exported from the cytoplasm by the anion channel and K⁺ from the vacuole via the tonoplast K⁺ channel (Allen and Sanders, 1997) and from the cytoplasm by the outward rectifying channel. Thus, the summation of the reactions in Fig. 4 is:

(016)

An additional ‘r’ ATP^4-, where ‘r’ is zero or greater, have been included (Fig. 4) so that the plasmalemma and tonoplast electrochemical gradients can be maintained.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Water export utilizing KCl. Cl- is proposed to be exported from the vacuole via a 2 H+–Cl- symport and then to be released into the apoplast via an anion channel. The charge balance is maintained by exporting K+ via the tonoplast K+ channel and the outward rectifying channel. The plasmalemma and tonoplast electrochemical gradients are maintained, respectively, by the hydrolysis of ‘r’ ATP4- by the plasmalemma H+/ATPase and two ATP4- by the tonoplast H+/ATPase.

 
If the supply of Cl^- in the vacuole is exhausted it is proposed that malate^1- synthesized in the cytoplasm is used as a counter ion in its place (Fig. 5). Thus, K⁺ flows out of the vacuole via the tonoplast K⁺ channel as above, with the charge balance in the vacuole being maintained by the hydrolysis of ATP^4- by the tonoplast ATPase, and the K⁺ exits the cell by the K⁺ outward rectifying channel. Concurrently with the export of K⁺, malate^1- is synthesized in the cytoplasm and is transported to the apoplast via the anion channel to maintain the charge balance in the latter compartment. The outflow of water should be readily accomplished by these transformations because the volume of the apoplast has been reported to be only 5% of the volume of the symplast (Hoffmann and Kosegarten, 1995).

View larger version (32K): [in this window] [in a new window]   Fig. 5. Water export utilizing K+ malate1- (or ‘malate export/cytochrome oxidase’ mode of metabolism). In this scheme it is assumed that K+ malate1- needs to be exported to the apoplast so that sufficient osmotic potential is developed to draw water out of the cell. It is further assumed that the three protons required to suppress the ionization of one of the carboxyl groups of malate are provided by the export of carbonic acid from the mitochondrial oxidation of pyruvate. Under steady-state conditions, the number of protons released in the cytoplasm is ‘r’ from the plasmalemma K+–H+-symport for the maintenance of the plasmalemma and tonoplast electrochemical gradients, plus four from the ionization of 3-phosphoglycerate minus one imported into the mitochondrion by the dicarboxylate exchanger, plus ‘x’ from ATP4- required for the maintenance of cellular integrity. Thus, it is assumed that the (r+4-1+x) H+ imported into the mitochondria by the H+–(OH)2PO2- symport is equal to the ATP4- synthesized as discussed in the text.

 
As before, the following equation describes the synthesis of malate:

(017)

Transfer of malate^1- and K⁺ to the apoplast (Fig. 5):

(018)

The oxidation of pyruvate by the citric acid cycle is given in the previous section so that the total for these reactions is:

(019)

Thus, the overall result of these transformations is the transfer of one malate^1- to the apoplast with an accompanying K⁺ being transferred from the vacuole to the former compartment. Concurrently, one H⁺ that was generated during glycolysis is secreted in the vacuole. In this case only (3+r)ATP^4- are required to maintain electrochemical gradients at the plasmalemma and the tonoplast so that, according to the scenario proposed in the previous section for the oxidation of malate^1-, up to 9 or 11.75 ATP^4- remain for cellular maintenance. As the process described here is essentially the reverse of that proposed in the previous section it seems unlikely that more than three ATP^4- are required for this purpose.

On the whole there seems to have been less work published on ion fluxes during stomatal closure than with stomatal opening but it has been suggested that guard cells release as much K⁺ during closure as they take up during opening (Blatt et al., 1998). These authors also state that the flux of ions across the plasmalemma is considerable and consists mostly of KCl and in some cases potassium malate. Malate^1- has been shown to be released into the bathing solution from isolated epidermal strips from both Vicia faba and Commelina communis leaves when stomata close (van Kirk and Raschke, 1978). Also, it has been shown that voltage-dependent anion channels of guard cells are activated by increased cytoplasmic Ca²⁺ and that these anion currents are carried by chloride and malate (Hedrich et al., 1990). Thus, although there is no direct evidence for the export of K⁺ Cl^-/malate^1- from cells contiguous with the water column within the plant it does seem that these ions are released from guard cells when the stomatal aperture is reduced, a response that would decrease the tension on the water column by reducing the evaporation rate at the stomata.

The scenarios presented in this and the previous section leave an impression that plant cells ‘breathe’ water so that, like lungs, they are expanded during growth by taking in the appropriate fluid and they use that fluid to ensure that the amount of the literally vital component, water or oxygen, is optimal in the conducting tissue.

	Abscisic acid (ABA), the alternative oxidase and the toleration of stress

Top Abstract Introduction Photosynthesis and... Respiration Metabolic modulations induced by... Abscisic acid (ABA), the... Note added in proof References

 
The roles of ABA and Ca²⁺ in transforming metabolism at the onset of stress
Elevated cytoplasmic Ca²⁺ is known to deactivate the plasmalemma H⁺/ATPase (Kinoshita et al., 1995; Xing et al., 1996) presumably at a higher concentration than that which opens the plasmalemma aquaporin and the K⁺–H⁺-symport. This latter proposition is lent some support by the observation that cytoplasmic Ca²⁺ activates the Ca²⁺-dependent slow vacuolar channel (Allen and Sanders, 1997) thereby increasing the concentration of Ca²⁺ in the cytoplasm. If the plasmalemma H⁺/ATPase is deactivated but the K⁺–H⁺-symport continues to transport H⁺ and K⁺ into the cytoplasm so that ATP^4- continues to be synthesized and the tonoplast electrochemical gradient is maintained (Figs 6, 7), the pH of the apoplast will increase. This increase in pH could then hydrolyse a precursor of ABA to liberate ABA in the apoplast (Netting, 2000; Duffield and Netting, 2001) and, as ABA is known to open Ca²⁺ channels at least in guard cells (Schroeder and Hagiwara, 1990), this will result in an increase in cytoplasmic Ca²⁺ (McAinsh et al., 1990). There would, therefore, with increasing tension on the water column, be a spiral of events that would lead to increased cytoplasmic Ca²⁺, increased apoplastic pH and ABA, as was found with increasing pressure on cotton leaves (Hartung et al., 1988), as well as an increased outflow of water with the osmotic gradient created by the outflow of K⁺ malate^1- (Figs 6, 7). Perhaps this, then, is the reason that leaves wilt, that is become flaccid, when they are severely stressed: most of the water flows out of cells into the vascular system in an attempt to maintain the integrity of the water column.

View larger version (28K): [in this window] [in a new window]   Fig. 6. The transition from the malate export/cytochrome oxidase mode to the alternative oxidase mode. To account for the activation of pyruvate orthophosphate dikinase (PPDK) at the onset of stress (Moons et al., 1998) it is proposed that the dicarboxylate translocator is deactivated and that some of the mitochondrial pyruvate is exported to the cytoplasm. This pyruvate was presumably synthesized from mitochondrial malate, which accumulated because the NAD-dependent malic enzyme was rate-limiting, by the NADP-dependent malic enzyme. Thus, on the activation of the alternative oxidase pyruvate can either ‘a’ be exported and phosphorylated or can ‘b’ be oxidized by the TCA cycle. NADP+ produced by the cytoplasmic malate dehydrogenase in the synthesis of malate is proposed to be reduced by the Ca2+-dependent NADP+ dehydrogenase that reduces, working in the reverse direction, external NADPH+ (Rasmusson and Møller, 1991). It is also suggested that NADP+H+ produced in the matrix by the NADP+-dependent malic enzyme is reoxidized by the Ca2+-dependent NADPH dehydrogenase (Agius et al., 1998). Malate is then exported to the apoplast via an anion channel and the charge balancing ‘a’ K+ ions are exported from the vacuole in response to the import of ‘a’ H+ by the tonoplast K+-activated pyrophosphatase. The tonoplast electrochemical gradient is maintained by the provision of ‘r’ H+ by the K+–H+ symport for the synthesis of ‘r’ ATP4-. As the plasmalemma H+/ATPase has been deactivated by the influx of Ca2+ the apoplastic pH will rise. This increase in apoplastic pH may hydrolyse apoplastic precursors of ABA (Netting, 2000; Duffield and Netting, 2001).

 

View larger version (29K): [in this window] [in a new window]   Fig. 7. The alternative oxidase mode. It is proposed that in this mode pyruvate is provided by glycolysis for subsequent ATP4- generation and also provides phosphoenolpyruvate for the synthesis of malate. Bicarbonate is again fixed in malate which is then exported to the apoplast along with three charge balancing K+ from the vacuole as in Fig. 5. The tonoplast electrochemical gradient is maintained by importing ‘r’ H+ via the K+–H+ symport to provide ‘r’ ATP4- to the tonoplast H+/ATPase. As the plasmalemma H+/ATPase has been deactivated by the influx of Ca2+ the apoplastic pH will rise. This increase in apoplastic pH may hydrolyse apoplastic precursors of ABA (Netting, 2000; Duffield and Netting, 2001). Of the additional three ATP4- required, two are provided by the mitochondrion from the two H+ remaining in the cytoplasm from glycolysis after the import of pyruvate into the mitochondrion, and one ATP4- is provided by pyruvate kinase. A quarter of the NADH+H+ generated by 3-phosphoglyceraldehyde dehydrogenase is assumed to be reoxidized by the Ca2+-dependent NADH dehydrogenase that oxidizes external NADH+H+ (Rasmusson and Møller, 1991).

 
Further, elevated cytoplasmic calcium appears to inhibit respiration. Thus, when protoplasts from cells close to the basal intercalary meristem of light-grown barley were treated with ABA respiration was only inhibited if Ca²⁺ ions were present (Owen et al., 1987a). This inhibition was similar to that induced by the Ca²⁺ channel agonist, BAY K8644, so it was concluded that apoplastic ABA opened a Ca²⁺ channel. This increased cytoplasmic Ca²⁺ was then suggested to act as a second messenger and regulate mitochondrial respiratory activity. These results were extended (Owen et al., 1987b) by treating similar protoplasts with the Ca²⁺ blockers lanthanum, verapamil and nifedipine which all reduced the Ca²⁺-dependent inhibition of respiration by ABA. The ionophore, A 23187, was proposed to inhibit protoplast respiration perhaps because it, like ABA, increased the plasmalemma permeability to apoplastic Ca²⁺. The calmodulin antagonists, trifluoperazine and compound 40/80, decreased the Ca²⁺-dependent inhibition of protoplast respiration by ABA and gibberellic acid markedly increased the rates of protoplast respiration apparently independently of apoplastic Ca²⁺. These observations suggest that ABA opens a plasmalemma Ca²⁺ channel and that the resulting elevated cytoplasmic Ca²⁺ increases the mitochondrial Ca²⁺ concentration (Askerlund and Sommarin, 1996) which then inhibits respiration.

Activation of the alternative oxidase
The discussion in the section on respiration under steady-state conditions implies that up to 31 ATP^4-/glucose will be synthesized in actively growing cells using the cytochrome oxidase pathway. This was presumably the situation in 4-d-old roots from soybean seedlings where respiration proceeded almost entirely by the cytochrome oxidase system (Millar et al., 1998). However, by day 17 more than 50% of the respiratory flux occurred by the alternative oxidase (Millar et al., 1998) so that a considerable proportion of the cells had at least partly engaged this enzyme. This implies a change in metabolism so that the discussion in the next section is directed towards characterizing the ‘transition from the cytochrome oxidase mode to the alternative oxidase mode’ while the section after that focuses on the ‘pyruvate oxidation/alternative oxidase mode’.

The alternative oxidase appears to withdraw reducing equivalents from the ubiquinol pool so that it is capable of eliminating proton pumping from the oxidation of succinate in the presence of cytochrome oxidase inhibitors (Vanlerberghe and McIntosh, 1997) and can oxidize ubiquinol within Complex I. As two protons are believed to be pumped across the membrane in Complex I via FMNH₂ (Walker, 1992) this implies that a maximum of two protons/(NADH+H⁺) could be pumped if the alternative oxidase were to be engaged to the exclusion of the cytochrome oxidase branch. This would provide insufficient protons for ATP^4- synthesis by ATP synthase so that the alternative oxidase is unlikely to become fully engaged to the exclusion of the cytochrome oxidase pathway. Rather, a balance is likely to be struck between the two pathways so that sufficient ATP^4- is generated and yet the cytochromes are not exposed to over-reduction.

Deactivation of the H⁺/ATPase by elevated cytoplasmic Ca²⁺ implies the possibility of the accumulation of protons in the cytoplasm because less ATP^4- is hydrolysed at the plasmalemma. It appears, however, that these protons are stored in the vacuole via the K⁺-activated pyrophosphatase (Colombo and Cerana, 1993), a process that, at least in guard cells, causes the cytoplasmic pH to rise by 0.04 to 0.3 units (Irving et al., 1992) or 0.2–0.3 units (Blatt et al., 1998). Thus, under these conditions the plasmalemma electrochemical gradient will be reduced in magnitude, and the concentration of protons in the cytoplasm could be almost halved (about 5.0x10^-8 M to 2.5x10^-8 M). The availability of (HO)₂PO₂^- for the mitochondrial H⁺–(HO)₂PO₂^- symport will, therefore, be diminished so that the rate of oxygen uptake will be reduced since the respiratory control ratio has dropped. This suggests that the proportion of ubiquinol, that is the QH₂/(Q+QH₂) ratio, within the respiratory chain would increase because the rate of electron flow is dependent on the rate of pumping of protons across the membrane which is, in turn, dependent on the rate of flow of protons through the ATP synthase. If the QH₂/(Q+QH₂) ratio exceeds about 0.5 the alternative oxidase is engaged (Day et al., 1995) apparently to protect respiratory chain components from becoming over-reduced and therefore liable to attack by reactive oxygen species (Day, 1999). In the scenarios presented here this is likely to occur when cells are stressed because, due to the combined actions of the plasmalemma K⁺–H⁺ symport and tonoplast pyrophosphatase, the availability of (HO)₂PO₂^- at the mitochondrial inner membrane will be reduced. A corollary of this is that, although the alternative oxidase is inherently wasteful of the energy available in NADH+H⁺ and FADH₂ the low metabolic turnover implied by a low rate of ATP^4- synthesis would allow the conservation of oxidizable carbon compounds. However, this does presuppose that there are controls on alternative oxidase activity. These do, in fact, appear to be numerous: it is activated by pyruvate and other 2-oxo acids, by reduction from an inactive covalently linked dimer to an active non-covalently linked dimer and perhaps by the amount of enzyme synthesized. It is not known if the plasmalemma K⁺–H⁺ symport and the tonoplast pyrophosphatase are activated as cells mature but one suspects that this might be the case since, as mentioned above, more than 50% of respiration occurred by the alternative oxidase in 17-d-old soybean roots compared to almost none in 4-d-old roots (Millar et al., 1998).

It has been shown that if pyruvate accumulates in the mitochondrion the alternative oxidase is activated (Millar et al., 1996), as it is on the addition of exogenous pyruvate (Umbach et al., 1994; Day et al., 1995). In vivo this pyruvate could either be provided by glycolysis or from mitochondrial malate. The former seems unlikely, at least in the scenarios presented here for cells coping with changes in tension in the water column, because glycolytic pyruvate is not available. The latter requires a change in mitochondrial metabolism so that the NAD-dependent malic enzyme, which needs to be rate-limiting in mildly stressed cells in this scenario, could be replaced by some alternate sink for reducing equivalents. The mitochondrial NADP-dependent malic enzyme seems to be a possibility since its activation appears to be dependent on declining mitochondrial pH (Agius et al., 1998), a consequence of importing malate with an accompanying proton with limited subsequent metabolism. The reducing equivalents from this NADP-dependent malic enzyme could then be transferred to ubiquinone by the rotenone insensitive, Ca²⁺-dependent NADPH dehydrogenase that is present in potato tuber (Melo et al., 1996) and pea leaf mitochondria (Agius et al., 1998). The ubiquinol would then be available to both the cytochrome oxidase branch (Melo et al., 1996; Agius et al., 1998) and the alternative oxidase depending on the redox poise of the ubiquinol pool (Fig. 6). It appears that this NADP-dependent malic enzyme competes with its NAD-dependent counterpart that feeds reducing equivalents into Complex I so that, in the absence of Ca²⁺, the latter enzyme is two to three time more active (Agius et al., 1998). However, after a stress-induced influx of Ca²⁺ the NADP-dependent enzyme is likely to be more active because the reducing equivalents will be passed on to ubiquinone by the Ca²⁺ and NADPH+H⁺-dependent dehydrogenase. It is also possible that the NADPH+H⁺ generated by this NADP-dependent malic enzyme could activate the alternative oxidase by reducing the inactive oxidized form to the active reduced form (Vanlerberghe and McIntosh, 1997). This scenario implies a burst of pyruvate, as well as of NADPH+H⁺, synthesis in the mitochondrion at the onset of stress, precisely what is required to activate the alternative oxidase. It is also noteworthy that there is a Ca²⁺-independent NADH+H⁺-dependent dehydrogenase present in potato tuber and pea leaf mitochondria (Agius et al., 1998) that feeds its reducing equivalents into the ubiquinone pool. Does this imply that, if there is an excess of NADH+H⁺ in the mitochondrion, the first FMNH₂ proton pump of Complex I is bypassed? Perhaps components of this pump are also prone to over-reduction.

The transition from the cytochrome oxidase mode to the alternative oxidase mode
At the onset of stress pyruvate orthophosphate dikinase (PPDK), which catalyses the synthesis of phosphoenolpyruvate from pyruvate in the roots of stressed rice seedlings, is activated (Moons et al., 1998). This enzyme produces pyrophosphate as a by-product, a possible substrate for the tonoplast pyrophosphatase in these cells with their stress-induced restrictions on ATP^4- synthesis. It seems unlikely that the pyruvate that is required as a substrate would be generated by glycolysis since this would involve a PPDK/pyruvate kinase futile cycle so it is proposed here that the pyruvate is exported, together with a proton, from the mitochondrion. This pyruvate was presumably generated from malate in the mitochondrion when the alternative oxidase was activated. The scenario depicted in Fig. 6 requires the deactivation of the dicarboxylate translocator on the perception of stress so that a futile cycle of synthesizing malate from pyruvate in the cytoplasm and oxidizing the same malate to pyruvate in the mitochondrion does not become established. Activation of the NADP⁺-dependent malic enzyme would then remove the excess equimolar concentrations of malate and protons present, providing the NAD⁺-dependent enzyme had been rate-limiting, and generate equimolar concentrations of NADPH+H⁺, bicarbonate and pyruvate. The protons could then either (‘a’) accompany pyruvate during its transport out of the mitochondrion or could (‘b’) be incorporated into acetyl CoA if the pyruvate were to be oxidized. Bicarbonate is required for condensation with phosphoenolpyruvate so that ‘a’ carbonic acid would be exported from the mitochondrion with the accompanying proton being available to suppress the ionization of malate^2- to form malate^1-. Some of the remaining ‘4b’ carbonic acid (Fig. 6) may diffuse out of the mitochondrion, but this would probably not go to completion because it implies an increase in the pH (a decrease in [H⁺] proportional to the ‘a+b’ bicarbonate generated by the NADP⁺-dependent malic enzyme) of the mitochondrion. The pyruvate (‘a’) that is exported from the mitochondrion will require ‘a’ ATP^4- to give ‘a’ phosphoenolpyruvate and ‘a’ P₂O₇^4- and a further ‘a’ ATP^4- will be required for the regeneration of ‘2a’ ADP^3- from ‘a’ AMP^2-. If the phosphoenolpyruvate produced is to be reduced to malate via oxaloacetate a source of reducing equivalents such as NADPH+H⁺ will be required. One possibility is that they could be generated by the pentose phosphate pathway (R Locy, personal communication), in which case some of the ‘4b’ bicarbonate from the mitochondrion might be fixed as malate after the 6-phosphono-gluconate is metabolized to phosphoenolpyruvate. Alternatively, the mitochondrial rotenone-insensitive Ca²⁺-dependent NADPH dehydrogenase that is capable of oxidizing external NADPH and feeding reducing equivalents into the ubiquinone pool (Rasmusson and Møller, 1991), but working in the reverse direction, is a candidate for this role (Fig. 6). A reduction of oxaloacetate does seem likely because both phosphoenolpyruvate (PEP) carboxylase and total malate dehydrogenases were activated under the same stress conditions as those that activated PPDK (Moons et al., 1998). As ‘a’ oxaloacetate (Fig. 6) in the cytoplasm would require ‘a’ ubiquinol for reduction to ‘a’ malate^2-, some of the ubiquinol generated by the matrix, Ca²⁺-dependent NADPH+H⁺ dehydrogenase mentioned in the last paragraph of the previous section would, in effect, be available to reduce the cytoplasmic oxaloacetate. If ‘a’ malate^1- is exported to the apoplast ‘a’ K⁺ will need to be exported from the vacuole to maintain the charge balance. This vacuolar ‘a’ K⁺ will be replaced by ‘a’ H⁺ transported into the vacuole by the tonoplast pyrophosphatase. Although the plasmalemma H⁺/ATPase is not active the tonoplast electrochemical gradient needs to be maintained. Thus, r(K⁺+H⁺) are admitted by the K⁺–H⁺-symport so that ‘r’ H⁺ can be liberated into the vacuole with the total of (r+a)K⁺ leaving the vacuole by the tonoplast K⁺ channel giving a total of (2r+a)K⁺ to be exported by the outward rectifying channel. The overall result of these transformations is that ‘a’ pyruvate and ‘a’ carbonic acid are exported from the mitochondrion to the apoplast as ‘a’ malate^1- and ‘a’ H⁺ are transferred from the mitochondrion to the vacuole. This allows ‘a’ K⁺ to be transferred from the vacuole to the apoplast. In energy terms these transformations cost (r+2a) ATP^4-. If ‘a’ is assumed to be equal to ‘b’, so that equal amounts of pyruvate are being phosphorylated and oxidized, a calculation indicated in the longer version of this paper suggests that the cytochrome oxidase branch needs to be at least 9% engaged to supply ‘2a’ ATP^4-.

Thus, the scheme in Fig. 6 is proposed primarily to account for the activation of PPDK on the imposition of stress but it also shows how some of the bicarbonate produced by the NADP⁺-dependent malic enzyme could be fixed and how K⁺ malate^1- may continue to be exported to the apoplast. In general terms, this scheme (Fig. 6) suggests that the plant cell has a good deal of flexibility in adjusting to stressful conditions: the ratio of pyruvate oxidized to pyruvate exported from the mitochondrion can be modified; the proportions of reducing equivalents from NADH+H⁺, produced by the citric acid cycle, passed to Complex I or to the NADH-dependent dehydrogenase can be adjusted; the degree of engagement of the alternative oxidase can be varied.

The pyruvate oxidation/alternative oxidase mode
In stressed rice roots it has been observed (Moons et al., 1998) that the PPDK activity peaked at about 48 h after the onset of stress and then fell away to close to its original value over the next 24 h. Total protein for both PEP carboxylase and the malate dehydrogenases also rose to a maximum at 48 h but it was not determined if this trend continued or if the total protein in these enzymes then declined. The former observation implies that PPDK activity was no longer required after 72 h presumably because only sufficient pyruvate remained in the mitochondrion to maintain the alternative oxidase in its active state. As many plants are able to tolerate stress for several weeks it seems likely that some low level of metabolism continues after the pyruvate derived from malate in the mitochondrion has been utilized. Figure 7 offers a scenario in which one pyruvate, derived from the metabolism of two glucose molecules, is oxidized leaving three phosphoenolpyruvate for condensation with bicarbonate so that three malate^1- can be exported to the apoplast. This again implies the export of three K⁺ from the vacuole to the apoplast. Again, the tonoplast H⁺/ATPase maintains the vacuolar electrochemical gradient by the hydrolysis of a total of (3+r) ATP^4-. Figure 7 also shows the production of four protons from the ionization of 3-phosphoglycerate with one of these being incorporated into ATP^4- at pyruvate kinase and one accompanying pyruvate into the mitochondrion. The two remaining protons, along with two ADP^3- and two HOPO₃^2- from the tonoplast hydrolysis of two ATP^4-, would then form substrates for the mitochondrial synthesis of two ATP^4-. Thus, with the inclusion of the ATP^4- produced by pyruvate kinase, there is a total of three ATP^4- available so that three K⁺ can be exported from the vacuole. In the scheme presented in Fig. 7 there is a surplus of a pair of reducing equivalents from NADH+H⁺, produced by glyceraldehyde-3-phosphate dehydrogenase, which will need to be reoxidized. It is proposed here that these two reducing equivalents are reoxidized by the mitochondrial Ca²⁺ and NADH-dependent dehydrogenase that oxidizes external NADH+H⁺ (Rasmusson and Møller, 1991) apparently using ubiquinone as an acceptor. Figure 7 suggests that for every pyruvate that enters the mitochondrion one NADH+H⁺ will require reoxidation by this enzyme so that the equivalent of one-quarter of the NADH+H⁺ provided by pyruvate dehydrogenase and the TCA cycle will be available for reoxidation by ubiquinone. Again, a calculation in the longer version suggests that the provision of two ATP^4- by the mitochondrion requires that the cytochrome oxidase branch will need to be engaged to at least 8%.

It can be seen (Fig. 7) that the consequences of this scheme are very similar to those from the scheme presented in Fig. 6: for each malate^1- produced in the cytoplasm and exported to the apoplast, one K⁺ is transported from the vacuole to the apoplast and two ATP^4- are synthesized in the mitochondrion.

The transformations outlined in Fig. 7 might provide a definition for the ‘stress-tolerant quiescent state’ (Netting, 2000) for a plant organ or indeed for a whole plant in that the rate of metabolic turnover would be minimal and will remain so until tension on the water column is relieved.

In this scenario (Fig. 7) the plasmalemma H⁺/ATPase has been deactivated by cytoplasmic Ca²⁺, but protons are still being imported by the K⁺–H⁺-symport so that the tonoplast electrochemical gradient can be maintained. The pH of the apoplast will therefore rise. Thus, as the apoplastic pH approaches about 7.5, pHs of up to 8.3 have been recorded from Tradescantia leaf epidermal cell apoplasts in the dark (Edwards and Bowling, 1986), the plasmalemma electrochemical gradient will disappear so that it will no longer be possible to export malate^1-. This suggests that if a stress is not relieved, malate^1-, and therefore K⁺, will tend to accumulate in the cytoplasm. Thus, it is likely that, under these conditions, cellular metabolism will slowly come to an almost complete stop.

Malate oxidation versus pyruvate oxidation
In the subsection entitled ‘Increased tension on the water column’ and in the preceding section (‘Abscisic acid, the alternative oxidase and the toleration of stress’) it has been proposed that there are two pathways that lead to the cytoplasmic synthesis of malate and its export to the apoplast (Figs 5, 7). The former was proposed to import one mole of malate into the mitochondrion and use some of the ATP^4- from its oxidation by the cytochrome oxidase pathway to export three moles of malate to the apoplast. The latter was proposed to import one mole of pyruvate into the mitochondrion and use the ATP^4- from its oxidation by an appropriate balance between the cytochrome oxidase branch and the alternative oxidase to export three moles of malate. The interesting questions then arise as to why there should be two different pathways and why the former should import malate into the mitochondrion and the latter, pyruvate. The following observations suggest some plausible reasons for the two pathways and for the assignments given here:

(1) The mitochondrial NAD-dependent malic enzyme appears to control the rate of entry of pyruvate into the TCA cycle in the malate synthesis/cytochrome oxidase mode (Fig. 5) so that if cytoplasmic pyruvate were to be oxidized in this mode this control point would be lost. In fact, the result would be similar to that depicted in Fig. 1 where ATP^4- synthesis, presumably to provide intermediates for growth, is maximal.

(2) The coupled import of malate into and export of bicarbonate from the mitochondrion by the dicarboxylate exchanger proposed here suggests that the mitochondrion would have some control over the rate of synthesis of malate in the cytoplasm, a control that may be important if malate is being used as an osmoticum. If the relatively rapid synthesis of cytoplasmic malate utilized the pyruvate pathway (Fig. 7) the mitochondrion could only influence the rate of malate synthesis via the Ca²⁺-dependent mitochondrial NADH dehydrogenase on one-quarter of the NADH+H⁺ produced.

(3) When roots suffer anoxia glucose is metabolized to ethanol (Roberts et al., 1992) apparently by a classical fermentation pathway. If pyruvate is already being produced for importation into the mitochondrion as proposed in Fig. 7 it is easy to see how carbon skeletons might be diverted from pyruvate into ethanol. On the other hand, as the mitochondrial NAD-malic enzyme appears to be rate-limiting a build-up of pyruvate and its conversion to ethanol in the cytoplasm seems unlikely if malate had been entering the mitochondrion for oxidation.

(4) Transgenic tobacco plants that lacked alternative oxidase activity accumulated ethanol (Vanlerberghe et al., 1995) in the presence of antimycin A, an inhibitor of the cytochrome oxidase pathway. This suggested that fermentation to ethanol was the only remaining option if cytoplasmic pyruvate built up when the mitochondrion was unable to oxidize pyruvate by either oxidase pathway. Thus, the entry of malate into the mitochondrion for oxidation again appears unlikely.

(5) To account for the incorporation of pyruvate into phosphoenolpyruvate on the imposition of stress (Moons et al., 1998) it was proposed above that pyruvate is exported from the mitochondrion (Fig. 6). If the ATP^4- for the phosphorylation of pyruvate were to be provided by the importation of malate for oxidation, the conditions for a futile cycle of carboxylating phosphoenolpyruvate in the cytoplasm and decarboxylating malate in the mitochondrion would have been set up.

(6) When maize seedlings were stressed with salt (Spickett et al., 1993) it was proposed that, after the plasmalemma H⁺/ATPase had essentially ceased pumping protons in exchange for cytoplasmic Na⁺ by an antiport, the pyrophosphatase commenced pumping protons into the vacuole to exchange these for cytoplasmic Na⁺. Finally, the tonoplast H⁺/ATPase was thought to be activated to continue the exchange of Na⁺ thus again reducing the cytoplasmic concentration of Na⁺ (Netting, 2000). The order of activation of these proton pumps is the same as that implied by the pathways proposed in Figs 5, 6, 7.

The trigger for the transition to the alternative oxidase mode
It was concluded above that cytoplasmic Ca²⁺ enters the mitochondrion so it would be expected to activate the following Ca²⁺-dependent enzymes: the mitochondrial NADPH dehydrogenase (Rasmusson and Møller, 1991) that has been proposed to reduce extra-mitochondrial NADP⁺; the mitochondrial NADH dehydrogenase (Rasmusson and Møller, 1991) that possibly oxidizes cytoplasmic NADH+H⁺ (Fig. 7), and the NADPH dehydrogenase (Agius et al., 1998) that is thought to reoxidize the NADPH+H⁺ produced by the mitochondrial malic enzyme (Fig. 6). If these assignments are indeed correct it seems that elevated mitochondrial Ca²⁺ would be quite capable of switching metabolism from the malate synthesis/cytochrome oxidase mode through the mitochondrial pyruvate export phase and into the pyruvate oxidation/alternative oxidase mode. Elevated mitochondrial Ca²⁺ is therefore a prime candidate for the initial trigger for plant cells to move from active metabolism to the stress-tolerant quiescent state (Netting, 2000).

Photorespiration and the alternative oxidase mode
It was suggested above that one proton was released in the chloroplast stroma for every three oxygen molecules fixed during photorespiration. The pH of the stroma needs to be greater than 7.3 for CO₂ to be fixed and it has been suggested that the pH-dependent step is catalysed by fructose-1,6-bisphosphatase (Werdan et al., 1975). As this step is also essential in photorespiration it seems likely that this influx of protons would deactivate photorespiration. From this viewpoint then, photorespiration, although it removes a considerable amount of oxygen, is perhaps a switch from fully active photosynthesis to an oxygen stress-tolerant mode of metabolism. If this is true a knowledge of the source of the peroxisomal protons becomes important if the integration of metabolism under these conditions is to be understood.

As well as pyruvate the following organic acids activate the alternative oxidase: hydroxypyruvate, glyoxylate, 2-oxoglutarate, and oxaloacetate (Day et al., 1995). It is of considerable interest that the first three of these occur as intermediates in peroxisomes during photorespiration. Thus, if any of these accumulate and can be transferred to the mitochondrion there is a chance that the alternative oxidase could be activated (Wiskich, 1999). If the scenarios presented in this paper have any validity one might suspect that the activation of photorespiration in some way increases cytoplasmic Ca²⁺ so that the plasmalemma K⁺–H⁺ symport is opened, as a source of protons, and mitochondrial Ca²⁺ and -keto acid levels increase to activate the alternative oxidase mode of metabolism. Under these conditions these photosynthetic cells are likely to adopt the stress-tolerant quiescent state until the excess oxygen is removed by some other pathway.

Recovery from stress and its effect on long-distance transport
Very little is known about how plant cells respond to the removal of a stress, but some of the events that may occur can be surmized from the foregoing. One of the earliest, if not the first, would have to be the removal of cytoplasmic Ca²⁺ because it was proposed above that it not only deactivates the plasmalemma H⁺/ATPase but also activates the switch in the mitochondrion from the cytochrome oxidase mode to the alternative oxidase mode. One suspects that there is a continual turnover of cytoplasmic Ca²⁺ so that if the plasmalemma Ca²⁺ channel (Figs 6, 7) is closed when the tension on the water column is reduced, Ca²⁺ will be removed from the cytoplasm and the mitochondrion probably by plasmalemma and tonoplast Ca²⁺/ATPases (Askerlund and Sommarin, 1996) and/or the tonoplast H⁺/Ca²⁺ antiport (Hirschi, 2001). This would presumably allow the synthesis of more ATP^4- to provide a substrate for the now active plasmalemma H⁺/ATPase. This scenario also implies that if apoplastic ABA is present from the earlier stress event it will need to be removed since ABA appears to open Ca²⁺ channels. It has been observed that uptake of [³H]ABA by isolated leaf epidermal and mesophyll tissue was greater at pH 6.0 than at pH 7.0 (Wilkinson and Davies, 1997) so perhaps ABA is taken up at lower pHs and catabolized to remove it from the apoplast. By re-establishing the plasmalemma electrochemical gradient the active H⁺/ATPase should allow the import of K⁺ and protons with the concomitant import of Cl^- or the synthesis of malate^1- as outlined in Figs 2 and 3. Secretion of K⁺Cl^-/malate^1- into the vacuole would then create the osmotic potential to draw water into the cells from the now replete apoplast so that the cells would again approach optimal metabolism (Fig. 1).

When a plant recovers from stress protons will be liberated into the apoplast and, because water will be evaporating from the stomata, they will accumulate in the leaves, particularly close to the guard cells. K⁺Cl^-/malate^1- is also likely to accumulate near the stomata as cells upstream are unlikely to take up all of these ions when a stress is removed. Here, K⁺ and H⁺ are likely to be taken up by the guard cell K⁺–H⁺ symport so that the cycle of events described above (‘Decreased tension on the water column’) continues and results in increased stomatal aperture. Closer to the vascular tissue, protons will also be available from the activity of the plasmalemma H⁺/ATPase, either upstream of or near to the vascular tissue, so that protons will be available for the loading of sucrose into the phloem by the H⁺-sucrose symporter (Frommer et al., 1996). Thus, high levels of protons in the apoplast might act as a signal to load more sucrose into the phloem so that glucose metabolized in the roots can be replaced. It seems clear that phloem can take up both K⁺ (Frommer et al., 1996) and malate^1- (Pate et al., 1974) so that K⁺ malate^1- is probably recycled, perhaps to stelar cells, because there are early indications that at least K⁺ may be involved in the uptake of water by the stele from the cortex (Tester, 1999).

Supplementary information
An expanded version of this review can be found at JXB online. Animations of the schema can be viewed at: www.life.unsw.edu.au/edtech/bioctuts/netting/index.htm

	Note added in proof

Top Abstract Introduction Photosynthesis and... Respiration Metabolic modulations induced by... Abscisic acid (ABA), the... Note added in proof References

 
It has recently been shown convincingly (Kwak et al., 2001) that the inward rectifying K⁺ channel makes a significant contribution to the uptake of K⁺ by light-stimulated guard cells in Arabidopsis. In terms of the schema presented here, this is similar to Fig. 2 except that the inward rectifying K⁺ channel would be substituted for the K⁺–H⁺ symport with the recycling of one less ATP^4-.

	Acknowledgments

 
I am grateful to Gerald Berkowitz, David Day, Harvey Millar, Rana Munns and, in particular, John Raven for critical reviews of earlier versions of this manuscript. I also thank the reviewers for their constructive comments and encouragement.

	Notes

 
¹ Fax: +61 2 9385 1483. E-mail: A.Netting{at}unsw.edu.au

	References

Top Abstract Introduction Photosynthesis and... Respiration Metabolic modulations induced by... Abscisic acid (ABA), the... Note added in proof References

 
Agius SC, Bykova NV, Igamberdiev AU, Møller IM. 1998. The internal rotenone-insensitive NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria. Physiologia Plantarum 104, 329–336.

Allen GJ, Sanders D. 1997. Vacuolar ion channels of higher plants. Advances in Botanical Research 25, 217–252.

Askerlund P, Sommarin M. 1996. Calcium efflux transporters in higher plants. In: Smallwood M, Knox JP, Bowles DJ, eds. Membranes: specialized functions in plants. Oxford: Bios, 281–299.

Assmann SM, Shimazaki KI. 1999. The multisensory guard cell. Stomatal responses to blue light and abscisic acid. Plant Physiology 119, 809–815.[Free Full Text]

Berry S, Rumberg B. 1996. H⁺/ATP coupling ratio at the unmodulated CF₀CF₁-ATP synthase determined by proton flux measurements. Biochimica et Biophysica Acta 1276, 51–56.

Blatt MR, Leyman B, Grabov A. 1998. Cellular responses to water stress. In: Shinozaki K, ed. Cold, drought, heat and salt stress: molecular responses in higher plants. R.G. Landes Co. 99–124.

Brand MD. 1994. The stoichiometry of proton pumping and ATP synthesis in mitochondria. The Biochemist 20–24.

Colombo R, Cerana R. 1993. Enhanced activity of tonoplast pyrophosphatase in NaCl-grown cells of Daucus carota. Journal of Plant Physiology 142, 226–229.

Cooper S, Lerner HR, Reinhold L. 1991. Evidence for a highly specific K⁺/H⁺ antiporter in membrane vesicles from oil-seed rape hypocotyls. Plant Physiology 97, 1212–1220.[Abstract/Free Full Text]

Cosgrove DJ. 1998. Cell wall loosening by expansins. Plant Physiology 118, 333–339.[Free Full Text]

Davies DD. 1973. Control of and by pH. In: Rate control of biological processes. Symposia of the Society for Experimental Biology XXVII. Cambridge University Press, 513–529.

Davies DD. 1986. The fine control of cytosolic pH. Physiologia Plantarum 67, 702–706.

Day DA. 1999. Respiration and energy generation. In: Atwell BJ, Kriedemann PE, Turnbull CGN, eds. Plants in action. Adaption in nature, performance in cultivation. Melbourne: MacMillan Education Australia, 67–82.

Day DA, Whelan J, Millar AH, Siedow JN, Wiskich JT. 1995. Regulation of the alternative oxidase in plants and fungi. Australian Journal of Plant Physiology 22, 497–509.

Duffield PH, Netting AG. 2001. Methods for the quantitation of abscisic acid and its precursors from plant tissues. Analytical Biochemistry 289, 251–259.[Web of Science][Medline]

Edwards MC, Bowling DJF. 1986. The growth of rust germ tubes towards stomata in relation to pH gradients. Physiological and Molecular Plant Pathology 29, 185–196.

Edwards MC, Smith GN, Bowling DJF. 1988. Guard cells extrude protons prior to stomatal opening: a study using fluorescence microscopy and pH micro-electrodes. Journal of Experimental Botany 39, 1541–1547.[Abstract/Free Full Text]

Evans JR, von Caemmerer S. 1999. Leaf anatomy, light interception and gas exchange. In: Atwell BJ, Kriedemann PE, Turnbull CGN, eds. Plants in action. Adaption in nature, performance in cultivation. Melbourne: MacMillan Education Australia, 24–34.

Ferguson SJ. 2000. ATP synthase: What dictates the size of the ring? Current Biology 10, R804–R808.[Web of Science][Medline]

Frommer WB, Hirner B, Kühn C, Harms K, Martin T, Riesmeier JW, Schulz B. 1996. Sugar transport in higher plants. In: Smallwood M, Knox JP, Bowles DJ, eds. Membranes: specialized functions in plants. Oxford: Bios, 319–335.

Garrill A, Findlay GP, Tyerman SD. 1996. Mechanosensitive ion channels. In: Smallwood M, Knox JP, Bowles DJ, eds. Membranes: specialized functions in plants. Oxford: Bios, 247–260.

Gerhardt R, Stitt M, Heldt HW. 1987. Subcellular metabolite levels in spinach leaves. Regulation of sucrose synthesis during diurnal alterations in photosynthetic partitioning. Plant Physiology 83, 399–407.[Abstract/Free Full Text]

Hartung W, Radin JW, Hendrix DL. 1988. Abscisic acid movement into the apoplastic solution of water-stressed cotton leaves. Role of apoplastic pH. Plant Physiology 86, 908–913.[Abstract/Free Full Text]

Hedrich R, Busch H, Raschke K. 1990. Ca²⁺ and nucleotide-dependent regulation of voltage-dependent anion channels in the plasma membrane of guard cells. The EMBO Journal 9, 3889–3892.[Web of Science][Medline]

Hinkle PC, Kumar MA, Resetar A, Harris DL. 1991. Mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Biochemistry 30, 3576–3582.[Medline]

Hirschi K. 2001. Vacuolar H⁺/Ca²⁺ transport: who's directing the traffic? Trends in Plant Science 6, 100–104.[Web of Science][Medline]

Hoffmann B, Kosegarten H. 1995. FITC-dextran for measuring apoplast pH and apoplastic pH gradients between various cell types in sunflower leaves. Physiologia Plantarum 95, 327–335.

Humble GD, Raschke K. 1971. Stomatal opening quantitatively related to potassium transport. Evidence from electron probe analysis. Plant Physiology 48, 447–453.[Abstract/Free Full Text]

Irving HR, Gehring CA, Parish RA. 1992. Changes in cytosolic pH and calcium of guard cells precede stomatal movements. Proceedings of the National Academy of Sciences, USA 89, 1790–1794.[Abstract/Free Full Text]

Johansson I, Karlsson M, Shukla VK, Chrispeels, MJ, Larsson C, Kjellbom P. 1998. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation. The Plant Cell 10, 451–459.[Abstract/Free Full Text]

Johansson I, Larsson C, Ek B, Kjellbom P. 1996. The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca²⁺ and apoplastic water potential. The Plant Cell 8, 1181–1191.[Abstract]

Kinoshita T, Nishimura M, Shimazaki K. 1995. Cytosolic concentration of Ca²⁺ regulates the plasma membrane H⁺-ATPase in guard cells of fava bean. The Plant Cell 7, 1333–1342.[Abstract]

McAinsh MR, Brownlee C, Hetherington AM. 1990. Abscisic acid-induced elevation of guard cell cytosolic Ca²⁺ precedes stomatal closure. Nature 343, 186–188.[Web of Science]

Melo AMP, Roberts TH, Møller IM. 1996. Evidence for the presence of two rotenone-insensitive NAD(P)H dehydrogenases on the inner surface of inner membrane of potato tuber mitochondria. Biochimica et Biophysica Acta 1276, 133–139.

Michelet B, Boutry M. 1996. Proton-translocating ATPases of the plasma membrane: biological functions, biochemistry and molecular genetics. In: Smallwood M, Knox JP, Bowles DJ, eds. Membranes: specialized functions in plants. Oxford: Bios, 261–279.

Millar AH, Atkin OK, Menz RI, Henry B, Farquhar G, Day DA. 1998. Analysis of respiratory chain regulation in roots of soybean seedlings. Plant Physiology 117, 1083–1093.[Abstract/Free Full Text]

Millar AH, Hoefnagel MHN, Day DA, Wiskich JT. 1996. Specificity of the organic acid activation of alternative oxidase in plant mitochondria. Plant Physiology 111, 613–618.[Abstract]

Moons A, Valcke R, Van Montagu M. 1998. Low-oxygen stress and water deficit induce cytosolic pyruvate orthophosphate dikinase (PPDK) expression in roots of rice, a C₃ plant. The Plant Journal 15, 89–98.[Web of Science][Medline]

Netting AG. 2000. pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implications for plant water relations. Journal of Experimental Botany 51, 141–158.

Oja V, Sauchenko G, Jakob B, Heber U. 1999. pH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves. Planta 209, 239–249.[Web of Science][Medline]

Owen JH, Hetherington AM, Wellburn AR. 1987a. Inhibition of respiration in protoplasts from meristematic tissues by abscisic acid in the presence of calcium ions. Journal of Experimental Botany 38, 498–505.[Abstract/Free Full Text]

Owen JH, Hetherington AM, Wellburn AR. 1987b. Calcium, calmodulin and the control of respiration in protoplasts isolated from meristematic tissues by abscisic acid. Journal of Experimental Botany 38, 1356–1361.[Abstract/Free Full Text]

Passioura JB, Munns R. 2000. Rapid environmental changes that affect leaf water status induce transient surges or pauses in leaf expansion rate. Australian Journal of Plant Physiology 27, 941–948.[Web of Science]

Pate JS, Sharkey PJ, Lewis OAM. 1974. Phloem bleeding from legume fruits, a technique for study of fruit nutrition. Planta 120, 229–243.[Web of Science]

Raschke K, Humble GD. 1973. No uptake of anions required by opening stomata of Vicia faba: guard cells release hydrogen ions. Planta 115, 47–57.[Web of Science]

Rasmusson AG, Møller IM. 1991. Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria. European Journal of Biochemistry 202, 617–623.[Web of Science][Medline]

Roberts JKM, Hooks MA, Miaullis AP, Edwards S, Webster C. 1992. Contribution of malate and amino acid metabolism to cytoplasmic pH regulation in hypoxic maize root tips studied using nuclear magnetic resonance spectroscopy. Plant Physiology 98, 480–487.[Abstract/Free Full Text]

Sakarno K. 1998. Revision of biochemical pH-stat: involvement of alternative pathway metabolisms. Plant and Cell Physiology 39, 467–473.[Abstract/Free Full Text]

Schachtman DP, Schroeder JI. 1994. Structure and transport mechanism of a high-affinity potassium uptake transporter from higher plants. Nature 370, 655–658.[Medline]

Schäffner AR. 1998. Aquaporin function, structure and expression: are there more surprises to surface in water relations? Planta 204, 131–139.[Web of Science][Medline]

Schroeder JI, Hagiwara S. 1990. Repetitive increases in cytosolic Ca²⁺ of guard cells by abscisic acid activation of non-selective Ca²⁺ permeable channels. Proceedings of the National Academy of Sciences, USA 87, 9305–9309.[Abstract/Free Full Text]

Seelert H, Poetsch A, Dencher NA, Engel A, Stahlberg H, Müller D. 2000. Proton-powered turbine of a plant motor. Nature 405, 418–419.[Medline]

Smith FA, Raven JA. 1979. Intracellular pH and its regulation. Annual Review of Plant Physiology 30, 289–311.[Web of Science]

Spickett CM, Smirnoff N, Ratcliffe RG. 1993. An in vivo nuclear magnetic resonance investigation of ion transport in maize (Zea mays) and Spartina anglica roots during exposure to high salt concentrations. Plant Physiology 102, 629–638.[Abstract]

Stryer L. 1995. Biochemistry, 4th edn. New York: W.H. Freeman & Co.

Tester M. 1999. The control of long-distance K⁺ transport by ABA. Trends in Plant Science 4, 5–6.

Thaler M, Simonis W, Schönknecht G. 1992. Light-dependent changes in the cytoplasmic H⁺ and Cl^- activity in the green alga Eremosphaera viridis. Plant Physiology 99, 103–110.[Abstract/Free Full Text]

Umbach AL, Wiskich JT, Siedow JN. 1994. Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulphide bond redox status in soybean seedling mitochondria. FEBS Letters 348, 181–184.[Web of Science][Medline]

van Kirke CA, Raschke K. 1978. Release of malate from epidermal strips during stomatal closure. Plant Physiology 61, 474–475.[Abstract/Free Full Text]

Vanlerberghe GC, Day DA, Wiskich JT, Vanlerberghe AE, McIntosh L. 1995. Alternative oxidase activity in tobacco leaf mitochondria. Dependence on tricarboxylic cycle-mediated redox regulation and pyruvate activation. Plant Physiology 109, 353–361.[Abstract]

Vanlerberghe GC, McIntosh L. 1997. Alternative oxidase: from gene to function. Annual Review of Plant Physiology and Plant Molecular Biology 48, 703–734.[Web of Science]

Walker JE. 1992. The NADH:ubiquinone oxidoreductase of respiratory chains. Quarterly Review of Biophysics 25, 253–324.

Werdan K, Heldt HW, Milovancev M. 1975. The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO₂ fixation in the light and dark. Biochimica et Biophysica Acta 396, 276–292.[Medline]

Wilkinson S, Davies WJ. 1997. Xylem sap pH: a drought signal received at the apoplastic face of the guard cell that involves the suppression of saturable abscisic acid uptake by the epidermal symplast. Plant Physiology 113, 559–573.[Abstract]

Wiskich JT. 1999. Thermogenesis. In: Atwell BJ, Kriedemann PE, Turnbull CGN, eds. Plants in action. Adaption in nature, performance in cultivation. Melbourne: MacMillan Education Australia, 79–81.

Xing T, Higgins VJ, Blumwald E. 1996. Regulation of plant defense response to fungal pathogens: two types of protein kinases in the reversible phosphorylation of the host plasma membrane H⁺-ATPase. The Plant Cell 8, 555–564.[Abstract]

Kwak JM, Murata Y, Baizabal-Aguirre VM, Merrill J, Wang M, Kemper A, Hawke SD, Tallman G, Schroeder JI. 2001. Dominant negative guard cell K⁺ channel mutants reduce inward rectifying currents and light-stimulated stomatal opening in Arabidopsis. Plant Physiology 127, 473–485.[Abstract/Free Full Text]

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