Metabolic Flexibility Helps Plants to Survive Stress - POST

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Metabolic Flexibility Helps Plants to Survive Stress - POST
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Metabolic Flexibility Helps Plants to Survive Stress
A Web-Essay for 5th Edition of Taiz & Zeiger’s Plant Physiology
by
William C. Plaxton, Professor of Biology & Biochemistry,
Queen’s University, Kingston, Ontario, Canada K7L 3N6
April 2009
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Introduction
Owing to their sessile lifestyle, plants have evolved numerous adaptations to help them cope with
unavoidable abiotic and biotic stresses that are imposed upon them in their natural environment. For
example, plants frequently alter their growth and developmental patterns so as to alleviate some of the
unfavorable environmental changes that they have been exposed to. Thus, increased allocation of
biomass to roots typically occurs in dry or mineral nutrient-deficient conditions. There is little doubt that
this type of developmental plasticity is a key aspect to the survival of plants in extreme environments.
However, it is equally apparent that certain biochemical and metabolic adaptations of plants also
facilitate their growth and/or survival under sub-optimal environmental conditions. For instance, in
contrast to animals, plants can often accomplish the same step in a metabolic pathway in a variety of
different ways. This so-called ‘metabolic flexibility’ is perhaps best exemplified by a wide variety of
genetic engineering experiments that have partially of fully eliminated an enzyme traditionally
considered to be essential, and yet the resulting transgenic plants were able to grow and develop more or
less normally (Plaxton and Podestá 2006).
Plant Respiratory Metabolism Represents a Central Feature of Plant Metabolic Flexibility
Plants utilize sucrose and starch as the principle substrates for respiration, and can fully oxidize these
fuels to CO2 and H2O via the standard pathways of glycolysis, the citric acid cycle, and the
mitochondrial electron transport chain (miETC). However, there are at least four remarkable attributes
in the organization and associated bioenergetic features of plant respiratory metabolism that are not
commonly seen in other organisms.
Remarkable Attribute #1. Plant glycolysis exists in the plastid and cytosol, with the parallel reactions
catalyzed by distinct nuclear-encoded isozymes (see textbook Figure 11.1; Plaxton and Podestá 2006).
The prime functions of glycolysis in darkened chloroplasts and non-photosynthetic plastids are to
participate in the breakdown of starch as well as to generate carbon skeletons, reductants and ATP for
anabolic pathways such as fatty acid synthesis and N-assimilation.
Remarkable Attribute #2. An extraordinary feature of plant carbohydrate metabolism is that the cytosolic
glycolytic pathway in itself is a complex network containing parallel enzymatic reactions at the level of
sucrose, fructose-6-phosphate, glyceraldehyde-3-phosphate and phosphoenolpyruvate (PEP)
metabolism (see textbook Figure 11.3). Each bypass reaction of plant cytosolic glycolysis circumvents a
classical glycolytic reaction that is dependent upon an adenine nucleotide or inorganic orthophosphate
(Pi) as a cosubstrate. As discussed below, this flexibility allows the preferential utilization of inorganic
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pyrophosphate (PPi) as an alternate energy donor, particularly when cellular ATP pools become
diminished during stresses such as anoxia and nutritional Pi starvation.
Remarkable Attribute #3. Respiratory O2 consumption by the plant miETC can be mediated by the ATPproducing cytochrome pathway or by a non-energy conserving alternative pathway that involves the
rotenone-insensitive NAD(P)H dehydrogenase bypasses to Complex I and the cyanide-resistant
alternative oxidase (AOX) bypass to Complex III and IV (see textbook Figure 11.8).
Remarkable Attribute #4. A number of bypass reactions to the plant citric acid cycle exist and include:
(i) a NADP+-specific isocitrate dehydrogenase within the mitochondrial matrix that can circumvent the
NAD+-specific isocitrate dehydrogenase of the conventional citric acid cycle, (ii) the glyoxylate cycle of
germinating oil seeds which necessitates two key enzymes (isocitrate lyase and malate synthase) that
collectively bypass both decarboxylating reactions of the citric acid cycle, thereby allowing
gluconeogenesis from stored fats to occur (see textbook Figure 11.18), and (iii) the gamma-amino
butyric acid (GABA) shunt which may function as an alternative, NAD+-independent, mechanism for
glutamate entry into the citric acid cycle. GABA, a four carbon non-protein amino acid, may represent a
significant proportion of the free amino acid pool in plant cells subjected to abiotic or biotic stresses
(Plaxton and Podestá 2006). Various stresses initiate a signal transduction pathway in which increased
cytosolic Ca2+ activates a calmodulin-dependent glutamate decarboxylase leading to a marked elevation
in intracellular GABA levels (see textbook Figure 25.12). After the stress is relieved, GABA can be
transported into the mitochondrion and enter the TCA cycle via its conversion to succinate through the
sequential action of GABA transaminase and succinic semialdehyde dehydrogenase.
The alternative reactions of glycolysis, the citric acid cycle, and miETC are believed to endow plants
with crucial metabolic flexibility that facilitates their development and acclimation to unavoidable
abiotic stresses. An ongoing and challenging problem has been to elucidate the respective function(s),
control, and relative importance of the many alternative reactions of plant respiration.
Pyrophosphate Permits Microbes and Plants to Conserve ATP
Inorganic pyrophosphate (PPi) is a byproduct of a host of biosynthetic reactions, including the
polymerization reactions involved in the final steps of macromolecule synthesis. One dogma of cellular
bioenergetics (stemming from animal studies) is that the anhydride bond of PPi is never utilized to
perform cellular work since it is immediately hydrolyzed by a highly active soluble inorganic
pyrophosphatase (Figure 1). However, the large amounts of PPi produced during biosynthesis are not
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always wasted, but may be employed by various anaerobic microorganisms as well as the plant cytosol
to enhance the energetic efficiency of several cellular processes.
Awareness of the importance of PPi in the bioenergetics of some organisms originated from research on
energy-poor anaerobic microorganisms such as the bacteria Priopionibacterium shermanii and the
parasitic protist Entamoeba histolytica (the latter causing amoebic dysentery in humans). These species
have no ATP-dependent phosphofructokinase (ATP-PFK), but instead convert fructose-6-phosphate to
fructose-1,6-bisphosphate via a PPi-dependent phosphofructokinase (PPi-PFK) (Figure 2A). Similarly,
they lack pyruvate kinase, but instead employ PPi to convert PEP and AMP into pyruvate, ATP and Pi
via pyruvate, Pi dikinase (PPDK), thereby converting the bond energy of PPi into a high-energy
phosphate of ATP. As outlined in Figure 2B, the PPDK reaction results in the net creation of two ‘ATP
equivalents’ (i.e., two ‘high energy’ phosphoanhydride bonds). This is clearly evident when the reaction
catalyzed by PPDK is summed with that catalyzed by the ubiquitous and highly abundant adenylate
kinase (Figure 2B). Owing to their use of PPi-PFK and PPDK, these organisms are able to yield 5 ATP
per glucose oxidized to two molecules of pyruvate, with a net expenditure of 3 PPi recycled from
biosynthetic reactions (Figure 2C). This clearly represents a considerable bioenergetic advantage for
obligate anaerobes such as P. shermanii and E. histolytica, since the net ATP yield for classical
glycolysis as occurs in animals is only two ATP per glucose converted to two molecules of pyruvate.
Pyrophosphate: An Autonomous Energy Donor of the Plant Cytosol
In contrast to animal cells, the plant cytosol lacks soluble inorganic pyrophosphatase and consequently
contains PPi concentrations of up to about 0.5 mM. Moreover, the PPi level of the plant cytosol is
remarkably insensitive to abiotic stresses such as anoxia or Pi starvation, or following the addition of
respiratory poisons, which elicit significant reductions in cellular ATP pools (Plaxton and Podestá
2006). How stressed versus non-stressed plant cells maintain a relatively constant level of cytosolic PPi
remains enigmatic. Anabolism, and hence the rate of PPi production, would generally be more prevalent
under non-stressed conditions. However, even during stresses such as anoxia or Pi starvation, PPi would
continue to be generated (albeit at a lower rate) during the biosynthesis of essential macromolecules
(proteins, nucleic acids, membranes, polysaccharides) needed to support even diminished growth, and/or
to replace those that may have become damaged or worn out. This is indicated by the fact that cytosolic
PPi levels remain fairly stable in plants that have been exposed to abiotic stresses that bring about
significant reductions in cellular adenylate pools.
Plant PPi-PFK Catalyzes a Net Glycolytic Flux in the Plant Cytosol
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The discovery in 1979 of the strictly cytosolic PPi-PFK in plants, and its potent activation by low
concentrations of the regulatory metabolite fructose-2,6-bisphosphate led to a surge of research on the
role of PPi in plant metabolism. It is now evident that PPi-PFK is an adaptive plant enzyme whose
activity and subunit composition are dependent upon a variety of environmental, developmental, species
and tissue-specific cues. Of note are reports describing the production of transgenic plants exhibiting
significantly lower expression of PPi-PFK, or overexpression of mammalian 6-phosphofructo-2-kinase
(the enzyme responsible for the synthesis of PPi-PFK’s allosteric activator, fructose-2,6-bisphosphate)
(reviewed by Plaxton and Podestá 2006). Metabolite studies of these transgenic plants indicated that PPiPFK catalyzes a net glycolytic flux in vivo (although PPi-PFK theoretically catalyzes a readily reversible
reaction, in contrast to the irreversible ATP-PFK reaction).
Sucrose Synthase and the H+-Pumping Pyrophosphatase Also Employ PPi to Circumvent ATPdependent Processes of the Plant Cytosol
Apart from PPi-PFK, PPi could be employed as an alternative energy donor for two other processes of
the plant cytosol that are normally dependent upon ATP (Figure 3):
(1) Sucrose conversion to hexose-phosphates can proceed via the ATP-dependent invertase pathway or
via the PPi-dependent sucrose synthase pathway.
(2) Active transport of protons into the vacuole from the cytosol can be carried out by separate ATP- or
PPi-dependent H+-pumps of the tonoplast membrane.
Plants Upregulate Alternative PPi-dependent Enzymes in Response to Stress
Tolerance to stresses such as anoxia and nutritional Pi starvation appears to depend upon a combination
of morphological and metabolic adaptations that are both species and tissue specific. For example,
flooding-intolerant species such as pea readily succumb to anoxia, whereas other plants, notably rice, as
well as many wetland plants can germinate and grow new shoots even after several weeks of anoxia.
That PPi-powered processes may be a crucial facet of the metabolic adaptations of plants to
environmental extremes that lead to depressed ATP (but not PPi) pools is indicated by the significant
upregulation of sucrose synthase, UDP-glucose pyrophosphorylase, PPi-PFK, PPDK, and the tonoplast
H+-pyrophosphatase by anoxia or hypoxia, extended Pi-starvation, and/or cold stress (Plaxton and
Podestá 2006, Li et al. 2008). The use of PPi-dependent cytosolic bypasses is believed to help plants
survive these stresses by circumventing ATP-limited reactions while conserving diminished cellular
ATP pools.
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Sucrose metabolism of anoxic rice seedlings was shown to proceed mainly through sucrose synthase and
UDP-glucose pyrophosphorylase with nucleoside diphosphate kinase facilitating the cycling of uridilates
needed for operation of this pathway (reaction 3 of Figure 3) (Plaxton and Podestá 2006). Assuming that
PPi is a byproduct of anabolism, no ATP is needed for the conversion of sucrose to hexose-phosphates
via the sucrose synthase/UDP-glucose pyrophosphorylase pathway, whereas 2 ATPs are needed for the
invertase/hexokinase pathway. Mertens (1991) argued that PPi-PFK functions in glycolysis in anoxic
rice seedlings, since both PPi-PFK activity and the level of its activator fructose-2,6-bisphosphate are
increased, while the activity of ATP-PFK declines. Similarly, PPDK represents a third enzyme that can
employ PPi to catalyze a glycolytic bypass reaction in the cytosol of plant cells (Figure 3). This enzyme
is highly expressed mesophyll chloroplasts of C4 leaves where it plays a key role in the C4
photosynthetic pathway in which it functions in the PPi producing direction to regenerate PEP from
pyruvate. However, PPDKs having a non-photosynthetic function are expressed as both cytosolic and
plastidic isoforms in C3 plant cells (Plaxton and Podestá 2006). Although the metabolic roles of PPDK
in C3 plants remain unclear, the cytosolic PPDK has been hypothesized to function as a glycolytic
bypass to pyruvate kinase in non-green hypoxic rice tissues (i.e., developing seed endosperm and
flooded roots), as well as Pi-starved maize roots so as to enhance the ATP yield of glycolysis (Plaxton
and Podestá 2006, Li et al. 2008). PPDK could confer a considerable bioenergetic advantage for ATPdepleted plant cells, since as discussed above and outlined in Figure 2 this enzyme catalyzes the net
creation of two ATP equivalents in the glycolytic direction, as opposed to the single ATP generated by
the pyruvate kinase reaction. Hence, the overall net yield of ATP obtained during glycolytic
fermentation of sucrose is increased from 4 to 12 if sucrose is metabolized via the sucrose
synthase/UDP-glucose pyrophosphorylase, PPi-PFK, and PPDK bypasses, relative to the
invertase/hexokinase, ATP-PFK, and pyruvate kinase pathway of classical glycolysis (Figure 3).
Alternative PPi-dependent cytosolic reactions of plant glycolysis and tonoplast H+-pumping confer a
considerable bioenergetic advantage that can extend the survival time of plant cells that have become
ATP-depleted owing to unavoidable environmental stresses such as cold temperature, anoxia/hypoxia,
salt stress, or Pi starvation. This has been corroborated by studies of mutant or transgenic plants
exhibiting altered levels of PPi-dependent enzymes and pathways. For example: (i) a 60% reduction in
root PPi levels of transgenic potato plants overexpressing a bacterial soluble inorganic pyrophosphatase
resulted in decreased plant growth and overall vitality during hypoxia stress, lower ATP levels, and an
impaired ability to resume aerobic growth following four days of hypoxia (Mustroph et al. 2005); (ii) a
crucial role for sucrose synthase in maintaining glycolysis in hypoxic maize roots was established by
examination of mutants deficient in sucrose synthase activity (Ricard et al. 1998); and (iii)
overexpression of the tonoplast H+-pumping pyrophosphatase resulted in transgenic plants that
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outperformed controls when subjected to nutritional Pi deprivation or salt stress (Gao et al., 2006, Yang
et al. 2007).
Metabolic Flexibility of Cytosolic Glycolysis and Tonoplast H+-pumping during
Phosphate Starvation
Orthophosphate (Pi) is the preferentially assimilated form of P for organisms such as plants that acquire
their minerals directly from the environment. Although Pi plays a central role in virtually all major
metabolic processes in plants, particularly photosynthesis and respiration, it is one of the least available
nutrients in most terrestrial and aquatic ecosystems. The massive use of Pi fertilizers in agriculture
demonstrates how the free Pi levels of most soils are suboptimal for plant growth. Worldwide reserves of
rock phosphate - our major source of Pi fertilizers in agriculture - have been projected to be exhausted
within the next 50 to 75 years. Thus, research on plant metabolic adaptations to Pi deficiency is of
significant practical importance. This research could lead to the development of rational strategies for
the application of biotechnology to reduce or eliminate agriculture's current over-reliance on expensive,
polluting, and non-renewable Pi fertilizers.
As a consequence of the marked decline (up to 50-fold) in cytoplasmic Pi levels that follows severe Pi
stress, large (up to 80%) reductions in intracellular levels of ATP and related nucleoside phosphates also
occur. This would be expected to reduce carbon flux through the enzymes of classical glycolysis that are
dependent upon adenylates or Pi as co-substrates (Figure 3). Despite depleted intracellular Pi and
adenylate pools, Pi-deprived plants must continue to generate energy and carbon skeletons for key
metabolic processes. As indicated in Figure 3, at least seven Pi- and adenylate-independent glycolytic
bypass enzymes (sucrose synthase, UDP-glucose pyrophosphorylase, PPi-PFK, non-phosphorylating
NADP-glyceraldehyde-3-phosphate dehydrogenase, PEP carboxylase, PPDK, and PEP phosphatase) in
addition to the PPi-dependent H+-pump of the tonoplast membrane have been reported to be significantly
upregulated following Pi starvation of plant cells (Plaxton and Podestá 2006, Li et al. 2008). It has been
hypothesized that these enzymes represent Pi starvation-inducible bypasses to adenylate or Pi-dependent
enzymes (i.e., invertase/hexokinase, ATP-PFK, phosphorylating NAD-glyceraldehyde-3-phosphate
dehydrogenase, pyruvate kinase, and tonoplast H+-ATPase) thereby facilitating glycolytic flux and
vacuolar pH maintenance during severe Pi stress when the intracellular levels of Pi and adenylates may
be very low. Furthermore, as Pi exerts reciprocal allosteric effects on the activity of ATP-PFK (potent
activator) and PPi-PFK (potent inhibitor) (Figure 3), the large decrease in cytoplasmic Pi levels that
occurs during long term Pi starvation has also been proposed to promote the in vivo PPi-PFK activity
while curtailing that of ATP-PFK (Plaxton and Podestá 2006). Pi is also a byproduct of the reactions
catalyzed by the bypass enzymes PPi-PFK, PEP carboxylase, PPDK, PEP phosphatase, and tonoplast
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H+-pyrophosphatase (Figure 3). Thus, the upregulation of these five enzymes by Pi starvation could play
a dual role during Pi stress. They may facilitate glycolysis or tonoplast H+-pumping by bypassing
adenylate-dependent reactions, while simultaneously generating free Pi for its reassimilation into the
metabolism of the Pi-starved cells via photo- or oxidative phosphorylation.
PEP Carboxylase Plays an Important Role during Pi Starvation
PEP carboxylase is a ubiquitous and tightly regulated plant cytosolic enzyme encoded by a small gene
family that has diverse functions including: (i) the fixation of atmospheric CO2 in CAM and C4
photosynthesis, and (ii) the anaplerotic replenishment of citric acid cycle intermediates that have been
consumed in biosynthesis and N-assimilation. However, together with cytosolic malate dehydrogenase
and the mitochondrial NAD-dependent malic enzyme, PEP carboxylase can also function as a glycolytic
enzyme by indirectly bypassing the reaction catalyzed by cytosolic pyruvate kinase (Figure 3).
Enhanced levels of PEP carboxylase mRNA, protein, and/or activity during Pi-starvation have been
reported for diverse species including oilseed rape, Arabidopsis thaliana, tomato, and white lupin
(Plaxton and Podestá 2006, Gregory et al. 2009). The PEP carboxylase-malate dehydrogenase-malic
enzyme bypass of cytosolic pyruvate kinase has been hypothesized to be of particular importance during
nutritional Pi deprivation when pyruvate kinase activity may become ADP limited (Plaxton and Podestá
2006). PEPC induction has also been correlated with the synthesis and consequent excretion of large
amounts of malic and citric acids by roots during Pi stress. This increases Pi availability to the roots by
acidifying the soil to solubilize otherwise inaccessible sources of mineralized Pi. Despite considerable
biochemical and transcriptomic evidence for PEPC's participation in plant acclimation to Pi starvation,
little research has been performed to document the specific PEPC isozymes(s) upregulated during Pi
stress nor the relationship between cellular Pi nutrition and the enzyme’s in vivo phosphorylation status.
However, a recent study indicated that the parallel induction and in vivo phosphorylation-activation of
the PEP carboxylase isozyme AtPPC1 plays an important role in the metabolic adaptations of Pi-starved
Arabidopsis (Gregory et al. 2009).
Growth and dark respiration rates of leaves of transgenic tobacco lacking leaf cytosolic pyruvate kinase
were largely unaffected (relative to wild type controls), which proves that plants (unlike animals or
yeast) have metabolic bypasses to cytosolic pyruvate kinase (Grodzinski et al. 1999). These results
reflect the amazing flexibility of plant PEP metabolism, which is probably an evolutionary adaptation to
the stresses that plants are frequently exposed to in their natural environment.
Alternative Pathways of Plant Mitochondrial Electron Transport Also Contribute to
the Survival of Pi-Starved Plants
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Respiratory O2 consumption by plant mitochondria can be mediated by the energy conserving
cytochrome pathway (as in animals) or by the non-energy conserving alternative pathway (Figure 3)
(also see textbook Figure 11.8, and Web Topics 11.1 and 11.2). The significant reductions in cellular Pi
and ADP levels that follow severe Pi deprivation will impede respiratory electron flow through the
cytochrome pathway at the sites of coupled ATP synthesis. However, the presence of non-proton motive
pathways of electron transport could provide a mechanism whereby respiratory flux is maintained under
conditions when the availability of ADP and/or Pi are restrictive (i.e., during severe Pi deprivation).
Plants acclimate to Pi stress by increased engagement of the non-energy conserving (i.e., rotenone- and
cyanide-insensitive) alternative pathways of the miETC (Figure 3) (Plaxton and Podestá 2006).
Moreover, increased levels of the AOX protein have been reported to occur following Pi stress of some
plant species. This allows continued functioning of the citric acid cycle and respiratory electron
transport chain with limited ATP production and may thereby contribute to the survival of Pi-deficient
plants. By preventing severe respiratory restriction, the AOX prevents both undesirable redirections in
carbon metabolism as well as the excessive generation of harmful reactive O2 species in the
mitochondrion of Pi starved plants (see Web Essay 11.4). This has been corroborated by the impaired
growth and metabolism of Pi-starved transgenic tobacco suspension cells that cannot synthesize a
functional AOX protein (Plaxton and Podestá 2006). Northern blot and proteomic analyses indicated
that the absence of AOX led to the increased expression of proteins normally associated with oxidative
stress in the Pi-deprived transgenic tobacco cells. By draining excess electrons, AOX also helps to
minimize the production of damaging ROS that otherwise arises when ubiquinone becomes overreduced.
High-throughput Transcript and Proteome Profiling Indicates that Pi starvation
Inducibility of Glycolytic and Mitochondrial Electron Transport Bypass Proteins is
Widespread in Plants
The recent application of high throughput transcript and proteome profiling technologies has allowed
researchers to simultaneously catalogue the effects of Pi deficiency on the expression of many genes and
proteins in plants such as Arabidopsis, rice, maize, and white lupin. Interestingly, the enhanced
expression of alternative glycolytic enzymes (such as sucrose synthase, UDP-glucose
pyrophosphorylase, PPi-PFK, and PPDK) and miETC proteins (such as AOX) that do not require Pi or
adenylates as co-substrates has been observed (Plaxton and Podestá 2006, Li et al. 2008).
Conclusions
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This web essay considered how the unique flexibility of plant carbohydrate and energy metabolism may
help plants to cope with unavoidable environmental stresses that may be imposed upon them. Much
research has focused on the molecular biology of plant stress responses, and this has revealed the ability
of plants to respond to stress by altered gene expression leading to the synthesis of various stressinducible proteins. Somewhat less attention has been devoted to the organization and control of
intermediary metabolism as pertains to the acclimation of plants to stress. However, studies of plant
metabolic responses to extreme environments have revealed some remarkably adaptive mechanisms that
may serve to limit the deleterious consequences of stress. Although these adaptations are by no means
identical in all plants, certain aspects are conserved in a wide variety of species from very different
environments. A better understanding of the extent to which changes in flux through alternative
enzymes and pathways influences plant stress tolerance is of significant practical interest. This
knowledge is relevant to the applied efforts of agricultural biotechnologists to engineer transgenic crops
exhibiting an improved resistance to abiotic stress, including nutritional Pi deprivation.
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Literature Cited
Gao, F., Gao, Q., Duan, X., Yue, G., Yang, A., and Zhang, R. (2006) Cloning of an H+-PPase gene from
Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance. J.
Exp. Bot. 57: 3259-3270.
Gregory, A. L., Hurley, B. A., Tran, H. T., Valentine, A. J., She, Y-M., Knowles, V. L, and Plaxton, W.
C. (2009) In vivo regulatory phosphorylation of the phosphoenolpyruvate carboxylase AtPPC1 in
phosphate-starved Arabidopsis thaliana. Biochem. J. 420: 57-65.
Grodzinski, B., Jiao, J., Knowles, V. L., and Plaxton, W. C. (1999) Photosynthesis and carbon
partitioning in transgenic tobacco plants deficient in leaf pyruvate kinase. Plant Physiol. 120:
887-895.
Li, K., Xu, C., Li, Z., Zhang, K., Yang, A., and Zhang, J. (2008) Comparative proteome analyses of
phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant
reveal root characteristics associated with phosphorus efficiency. Plant J. 55: 927-939.
Mertens, E. (1991) Pyrophosphate-dependent phosphofructokinase: an anaerobic glycolytic enzyme?
FEBS Lett. 285: 1-5.
Mustroph, A., Albrecht, G., Hajirezaei, M., Brimm, B., and Biemelt, S. (2005) Low levels of
pyrophosphate in transgenic potato plants expressing E. coli pyrophosphatase lead to decreased
vitality under oxygen deficiency. Annals of Botany 96: 717-726.
Plaxton, W. C., and Podestá, F. E. (2006) The functional organization and control of plant respiration.
Crit. Rev. Plant Sci. 25: 159-198.
Ricard, B., Van Toai, T., Chourey, P., and Saglio, P. (1998) Evidence for the critical role of sucrose
synthase for anoxic maize roots using a double mutant. Plant Physiol. 116: 1323-1331.
Yang, H., Knapp, J., Koirala, P., Rajagopal, D., Peer, W. A., Silbart, L. K., Murphy, A., and Gaxiola, R.
A. (2007) Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorusresponsive type I H+-pyrophosphatase. Plant Biotech. J. 5: 735-745.
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FIGURE LEGENDS
Figure 1 Pyrophosphate hydrolysis is catalyzed by inorganic pyrophosphatase. In all animals and many
microbes the large amount of PPi that is generated as a byproduct of anabolism is immediately
hydrolyzed by an abundant inorganic pyrophosphatase in a highly exergonic reaction. However, in some
microbes and the plant cytosol, PPi produced during biosynthesis is not wasted since: (1) there is little or
no inorganic pyrophosphatase present (this allows PPi to accumulate), and (2) PPi-dependent enzymes
exist that can use the PPi instead of ATP to do useful cellular work.
Figure 2 Nucleoside phosphate and pyrophosphate metabolism in the glycolytic pathway of a
eukaryotic anaerobic amoeba Entamoeba histolytica. A. The glycolytic pathway of E. histolytica. B.
PPDK uses PEP and PPi to catalyze the conversion of AMP to ATP (i.e., the production of two high
energy phosphoanhydride bonds = two ‘ATP equivalents’). C. The ATP and PPi balance sheet for E.
histolytica glycolysis. Abbreviations are as described in the text or as follows: 1,3-DPGA, 1,3diphosphoglycerate; Fru-1,6-P2, fructose-1,6-bisphosphate; Fru-6-P, fructose-6-phosphate; Glu-6-P;
glucose-6-phosphate; 3-PGA, 3-phosphoglycerate.
Figure 3 A model suggesting several adaptative metabolic processes (indicated by bold arrows) that
may promote the survival of Pi-starved vascular plants. Alternative pathways of cytosolic glycolysis,
mitochondrial electron transport, and tonoplast H+ pumping may facilitate respiration and vacuolar pH
maintenance by Pi-deficient plant cells because they negate the dependence on adenylates and Pi, the
levels of which can become markedly depressed during severe Pi starvation. Organic acids produced by
PEP carboxylase can also be excreted by roots to increase the availability of mineral bound Pi (by
solubilizing Ca-, Fe- and Al-phosphates). A key component of this model is the critical secondary role
played by 'metabolic Pi recycling systems' during Pi deprivation. Enzymes that catalyze the numbered
reactions are as follows: 1, hexokinase; 2, fructokinase; 3, nucleoside diphosphate kinase; 4,
phosphoglucose mutase; 5, phosphoglucose isomerase; 6, NAD-dependent G3P dehydrogenase
(phosphorylating); 7, 3-PGA kinase. Abbreviations are as described in the legend for Figure 2 or as
follows: DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; Glu-1-P; glucose-1phosphate; MDH, malate dehydrogenase; OAA, oxaloacetate; PK, pyruvate kinase; UGPase, UDPglucose pyrophosphorylase; UQ, ubiquinone.
Fig. 1
‘High Energy’
Phosphoanhydride
bond
O
O
-O - P- O - P- O+ H2O
OOInorganic
Pyrophosphate (PPi)
Mg2+
O
2 -O - P- OH
OInorganic
Phosphate (Pi)
ΔGo’ = -19 kJ/mol
Fig. 2
A.
Glucose
ATP
Hexokinase
ADP
Glu-6-P
B.
PPDK catalyzes the production of 2 ATP ‘equivalents’…
PEP + AMP + PPi
1) PPDK:
2 ADP
+ 2) Adenylate Kinase:
Fru-6-P
PPi
Pi
PPi-PFK
= 3) Net Reaction:
PEP + 2 ADP + PPi
Pyruvate + ATP + Pi
ATP + AMP
Pyruvate + 2 ATP + Pi
Fru-1,6-P2
1,3-DPGA
ADP
3-PGA
ATP
Kinase
3-PGA
PEP
PPDK
C.
ATP & PPi Expenditures & Yields (per Glucose
Hexokinase: - 1 ATP
3-PGA Kinase: + 2 ATP
PPi
AMP
ATP
Pyruvate
Pi
2 Pyruvate)
PPDK: + 4 ATP
Net ATP Yield:
+ 5 ATP
PPi-PFK: - 1 PPi
PPDK: - 2 PPi
Net PPi Expenditure:
- 3 PPi
Fig. 3
SUCROSE
Sucrose Synthase
Invertase
Glucose
UDP
Fructose
ATP
ATP
1
3
2
ADP
UDP-Glucose
PPi UGPase
UTP
ADP
Glu-1-P
Glu-6-P
4
5
5
Fru-6-P
ATP
Pi
+
Glu-6-P
PPi
PPi-PFK
Pi
ATP-PFK
ADP
- Pi
+
-
Fru-1,6-P2
CYTOSOL
DHAP
NAD+ + Pi
NADH
NADP+
Non-phosphorylating
1,3-DPGA
NADP-G3P
Dehydrogenase
ADP NADPH
7
VACUOLE
ATP
H -ATPase
+
+
H+
H
ATP
3-PGA
PEP
?
ADP + Pi
PPi
H+-PPiase
H+
HCO3-
PPi
Pi
-
PEP
Phosphatase
H2O
AMP
ATP
Pi
H+ Pyruvate
2Pi
Pi
?
CO2
Pi
PPDK PK
ADP
MDH
Biosynthesis
NAD+
Pyruvate
ADP + Pi
Excretion to
Environment
Malate
Pyruvate
Malic Enzyme
ATP
Complex I
Citric Acid Cycle
NADH
ATP
CO2 NADH
Acetyl-CoA
OAA
PEP PEP Carboxylase
MITOCHONDRION
2CO2
Inhibition
G3P
6
Tonoplast
Activation
Malate
NAD+
ADP + Pi
Rotenone
X
UQ
2e-
ADP + Pi
ATP
Complex III
Cytochrome
Oxidase
X
Cyanide
2H+ + ½O2
Rotenone Insensitive
NADH Dehydrogenase
ATP
Alternative
Oxidase
H2O

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