1
SUPPLEMENTARY MATERIALS
‘Efficient use of energy in anoxia-tolerant plants with focus on germinating rice seedlings’
by Brian J. Atwell, Hank Greenway & Timothy D. Colmer. New Phytologist
CONTENTS
Supplementary Notes 1: Techniques used to evaluate responses to anoxia
1.1 Acclimating plant tissues prior to anoxic treatments
1.2 Assessing cell injury and survival
Supplementary Notes 2: Additional notes on PPi metabolism
2.1 The reversible glycolytic enzymes of rice are induced under anoxia
2.2 Why the reversible PPi-dependent enzymes can be viewed as a PPistat
2.3 Why does chilling not have the same effect on vacuolar H+PPiase in different experiments?
2.4 Manipulating vacuolar H+PPiase activity in vivo
2.5 Are there H+PPiases in membranes other than tonoplasts?
2.6 Is response of PPi-dependent enzymes to hypoxia useful to elucidate mechanisms of anoxia
tolerance?
Supplementary Notes 3: Additional notes on energetics and glycolysis
3.1 ATP production during sugar catabolism
3.2 ATP production during sugar catabolism depends on type of substrate and the balance
between PPi production and requirements
3.3 Assessing energy for maintenance in the rad mutant (reduced alcohol dehydrogenase
mutant)
3.4 Reduced nucleotides as an energy source?
3.5 How the rate of formation of amino acids and lipids in anoxia may influence the rate of
glycolysis
Supplementary Notes 4: Factors determining pH, the basis for using the strong ion
difference (SID)
Supplementary Notes 5: Problems with interpretation when roots are in an anoxic
medium but shoots are in air
All references to sections, figures and tables (e.g. Section I, Fig. 1, Table 1) are to material in
the published review, not in the supplementary notes.
Supplementary Notes 1: Techniques used to evaluate responses to anoxia
1.1. Acclimating plant tissues prior to anoxic treatments
The large improvement in anoxia tolerance induced during acclimating plant tissues by
exposure to hypoxia prior to anoxia (removing all O2) has been demonstrated (first by Saglio et
al., 1988) and re-emphasized by Gibbs & Greenway (2003). The type of hypoxic pre-treatment
should be established for each particular tissue or cell suspension, since internal O2 status
depends on tissue thickness, turbulence, respiration rate and temperature (Berry & Norris,
1949; Armstrong & Beckett, 1987; Gibbs & Greenway, 2003). In order to establish the
optimum exogenous O2 concentration for acclimation, an O2 response curve (O2 uptake vs
external O2 concentration) in the form of a hyperbolic curve should ideally be used to choose
an O2 concentration that achieves half-maximal activity (i.e., close to the Km). For example, in
the case of cultured cells transferred to anoxia (Mohanty et al., 1993), requirements of hypoxic
pre-treatment would depend on characteristics of the suspension such as the density and
clumping of cells, temperature, mixing (shaking speed) and O2 concentrations of the bulk
medium in the culture vessels.
2
Cases of anoxic shock, or ill-defined hypoxic pre-treatment, often appear in the
literature. For rice seedlings, the issue is at what growth stage a hypoxic pre-treatment is
required to endow the highest possible anoxia tolerance. Ellis & Setter (1999) demonstrated
that there was a large improvement in anoxia tolerance of 14-d-old rice seedlings after being
submerged in hypoxic solution for 24 h in 0.05 mM O2. In contrast, rice seeds can be
germinated anoxically without a hypoxic pre-treatment, reflecting a natural propensity to
tolerate anoxia during these first stages of growth. There are no data to indicate at what growth
stage of the rice seedling a hypoxic pre-treatment becomes desirable.
1.2 Assessing cell injury and survival
Measurement of membrane potentials with micro-electrodes is a good technique to assess cell
injury, particularly because individual cells can be assessed. As shown in a study with beetroot,
in which anthocyanins are endogenous visual markers of survival, cells which have died can be
found adjacent to cells which recover rapidly (Zhang et al., 1992). Another good criterion of
cell health, though only for tissues rather than individual cells, would be the energy charge, i.e.
(ATP+0.5ADP)/(ATP+ADP+AMP). Because of the small cytoplasmic pool of adenine
nucleotides and rapid turnover of the adenylate pool, energy charge responds rapidly to changes
in O2 supply.
An additional, or alternative, criterion is the measurement of Cl- uptake and retention,
not only because methods to measure Cl- are widely available, but also because Cl- is eminently
suitable to measure membrane integrity. This suggestion is based on several facts.
i) Cl- is soluble and is not metabolised;
ii) The internal diffusion equilibrium concentrations are very low. Given that nearly all
plant cells have a plasma membrane potential of -100 mV, or more negative, the
diffusion equilibrium concentration of the anion Cl- in the cytoplasm would be at
most 1% of the external concentration (calculated from Nernst equation), i.e.
negligible. Thus, internal concentrations exceeding this must be the result of active
influx;
iii) There will be a substantial free gradient driving Cl- efflux across the plasma
membrane. In detail, the free energy gradient would consist of the electrical
component, which even at -100 mV would give a minimum of 10 kJ mol-1. This
gradient would be further complemented by a concentration component of 2.5 kJ
mol-1 for every 10 mM internal Cl- increase. Any appreciable Cl- accumulation
therefore indicates the plasma membrane has retained a low permeability to Cl-;
iv) The accumulation ratio (Ci/Co) at any given time is likely to differentiate between
tissues which are still healthy and those which already suffer some injury;
v) With an optimal rate of net Cl- uptake by aerated tissues of e.g. ~5 mol g-1 fresh
weight h-1, a time resolution of ~30 min can be achieved for net uptake
measurements providing Cl- in the external medium is 5 mM or less. Shorter
periods, or under continued anoxia, would be best evaluated with 36Cl-.
Supplementary Notes 2: Additional notes on PPi metabolism
2.1 The reversible glycolytic enzymes of rice are induced under anoxia
In aerated rice coleoptiles, the two PPi-dependent reversible glycolytic enzymes (Fig. 1b & c)
are only at very low levels, but during anoxia they are induced and reach high levels, as shown
by the high maximum catalytic activities for: a) PFK-PPi (Mertens et al., 1990; Gibbs et al.,
2000) and b) PPDK, with PPDK protein (Huang et al. 2005) and its transcripts (LasanthiKudahettige et al., 2007) reaching high levels. The presence of these enzymes, which operate in
parallel reactions with the ‘conventional’ irreversible glycolytic enzymes PFK-ATP and
3
pyruvate kinase, endow a high metabolic flexibility by switching to PPi-dependent reactions
during an energy crisis (Plaxton, 2009). The low levels of these enzymes in rice in aerated
solutions, as well as of the V-H+PPiase (Carystinos et al., 1995), indicate that under benign
aerated conditions, PPi formed in biosynthetic reactions is largely hydrolysed by
pyrophosphatases not used in energy-requiring reactions, otherwise high PPi would reduce rates
of polymer synthesis (Nelson & Cox, 2008).
As stated in the main text, the free energy of hydrolysis of ATP is substantially greater
than of PPi. Therefore, though both are energy donors, these currencies differ in the energyrequiring processes they can sustain. For example, coupling ratios of H+/ATP of 1.0 are in
theory possible for the V-H+ATPase with a free energy gradient for H+ of 43 kJ mol-1. By
contrast, a coupling ratio of 1 for H+/PPi would be possible only at a free energy gradient for H+
less than 27 kJ mol-1.
2.2 Why the reversible PPi-dependent enzymes can be viewed as a PPistat
The two reversible glycolytic enzymes depicted in Fig. 1, PPi-PFK and PPDK, can be viewed
as constituting a PPistat, either producing PPi from ATP, or substituting PPi for ATP in the PFK
reactions (Stitt, 1998). Whether these reversible enzymes work in the forward or reverse
direction would depend on the differences between PPi formed during polymer synthesis,
which are energised by NTP → NDP + PPi and PPi consumption by the key enzymes UDPG
pyrophosphorylase and the V-H+PPiase (Section II.4). When PPi formation exceeds
consumption, PPi can substitute for ATP in the PFK reactions. However, when PPi
consumption is greater than its production, the shortage of PPi has to be met by ATP exchanged
for PPi by a glycolytic enzyme working in the reverse direction (Fig. 1c). Production of PPi by
PPi-PFK remains dubious though it has some credibility, as shown by an in vitro experiment
using an ingenious coupling of the PFK-PPi to the V-H+PPiase in isolated tonoplasts (Costa dos
Santos et al., 2003). By applying a range of fructose 1,6-diphosphate, Pi and fructose-6phosphate concentrations, distinct H+ gradients were created in all cases, except when there was
inhibition of PPi-PFK by 20 mM fructose-6-phosphate (Costa dos Santos et al., 2003). On the
other hand, PPi-PFK in the glycolytic directions would be favoured by the more acidic pH in O2
deficient and anoxic tissues (Plaxton & Podesta, 2006). However, the drop in pHcyt in anoxic
rice coleoptiles was only from ~7.5 to 7.25 - 7.35 (Section III.4.1) and across this pH range,
changes in activity of PPi -PFK were very small only (Plaxton & Podesta, 2006).
In light of these observations and the modest body of in vivo evidence, PPDK remains
the most plausible enzyme for exchange of ATP for PPi. This cycle are energised by ATP →
PPi + AMP (Fig. 1c), so the exchange of ATP for PPi would need re-phosphorylation of the
AMP formed by PPDK in Cycle 2 (Table 1), where ATP is spent using adenylate kinase.
Interestingly, in rice coleoptiles and endosperm, anoxia increased the activity of the required
adenylate kinase (enzyme 6 in Table 1), with increases on a protein basis by 2- to 3-fold
(Kawai et al., 1998, no recovery checks).
2.3 Why does chilling not have the same effect on vacuolar H+PPiase in different experiments?
Widely differing effects of chilling on the V-H+PPiase were reported by Carystinos et al.
(1995) and Liu et al. (2010). Carystinos and colleagues reported chilling-induced increases in
protein levels and enzyme activity of the V-H+PPiase, while Liu and colleagues reported during
chilling no change in activity of the promoter region of an anoxia-inducible V-H+PPiase. Liu et
al. (2010) acclimated seedlings to chilling (i.e. stepped changes over time) but Carystinos et al.
(1995) did not. Further, different plant organs were tested in the two studies: roots and leaves of
20-d-old rice plants by Liu et al. (2010), but 4-d-old rice seedlings by Carystinos et al. (1995).
Finally, Carystinos et al. (1995) used an indica rice and Liu et al. (2010) a japonica rice, the
latter are generally more cold-tolerant.
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2.4 Manipulating vacuolar H+PPiase activity in vivo
Evidence that V-H+PPiase activity affects membrane potential has now been obtained using
transgenic plants in which the V-H+PPiase in the moderately anoxia-tolerant japonica cultivar,
Calrose, was knocked out or over-expressed (Liu, 2009). The plasma membranes of 10 - 15
mm long primary roots de-polarised, upon transfer to anoxia, from their resting potential of 120 mV, by ~65 mV in the knockout line, ~35 mV in the wild type, and only ~25 mV in the
over-expression line; the V-H+-PPiase is purported to maintain tighter sub-cellular
compartmentation of H+ and would ipso facto help maintain a more negative plasma membrane
potential. Notably, upon transfer to air, cells of the over-expression line re-polarized by ~50
mV more than the wild-type controls. Overall, these results demonstrate that in these rice root
tips the V-H+PPiase has a crucial role during O2 deficits. There was no evidence that overexpression of V-H+-PPiase improves growth of the highly anoxia-tolerant rice coleoptile tissues
in anoxia. An improvement was demonstrated vividly in the anoxia-intolerant species,
Arabidopsis, where transgenic plants in which the V-H+PPiase was over-expressed (AVP1)
suffered 15% mortality after 48 h of anoxia while 90% of the controls died (Liu, 2009). This
adaptive advantage of a single-gene introduction emphasises that transport across the tonoplast
is one of the most crucial processes which needs to be maintained to achieve anoxia tolerance
(Sections II.3.2 & III.3).
2.5 Are there H+PPiases in membranes other than tonoplasts?
H+PPiase activity has been detected in membranes other than tonoplasts (Stitt, 1998). A recent
review has highlighted that in Arabidopsis, the Golgi apparatus contained H+PPiase, though in
low abundance relative to the tonoplast (Ferjani et al., 2014). A relevant case for anoxic tissues
is a H+PPiase associated with the sieve elements of the phloem (Long et al., 1995). H+PPiase
activity may be associated with the plasma membranes of Ricinus communis cotelydons
(Robinson et al., 1996). The free energy gradient was calculated from the free energy of
hydrolysis and assuming a stoichiometry of 1:1 (H+:PPi) (Davies et al., 1997). Taking -110 mV
for the plasma membrane of anoxic rice coleoptiles (Zhang & Greenway, 1995), then this
energy is inadequate to drive H+ extrusion (using Davies et al., 1997). So, in general Davies et
al. (1997) suggested that, if indeed a H+PPiase resides in the plasma membrane, it is more
likely to produce PPi than to export H+. The alternative during anoxia would be a H+:PPi with a
coupling ratio of 1:2, which would be energetically expensive, yet if energy needs to be
preferentially directed to the plasma membrane under anoxia, it remains a reasonable
proposition. Both Robinson et al. (1996) and Long et al. (1995) took great care to test whether
the detected H+PPiase protein may have been a contamination from the tonoplast, using
immunology and membrane fractions collected by density gradient centrifugation (cf. Long et
al., 1995). These authors very plausibly argued the case that there is negligible contamination,
although the possibility remains (Long et al., 1995).
2.6 Is response of PPi-dependent enzymes to hypoxia useful to elucidate mechanisms of anoxia
tolerance?
In a recent review, Mustroph et al. (2014) presented a table comparing induction of transcripts
of key PPi-dependent enzymes by various low O2 treatments/conditions in seven different
species, including germinating rice seedlings. This comparison has value in terms of ecology
and physiology and indicates that there are several adverse conditions during which transcripts
of PPi-dependent enzymes increase though not uniformly over the species, or type of condition;
increases in PPi-dependent enzymes under various ‘stresses’ was earlier discussed by Plaxton
& Podesta (1996).
5
The relevance to the present paper is whether the comparison provides firm conclusions
on the mechanisms endowing anoxia tolerance. In our view that is not so, since the techniques
used in the different investigations vary widely and often change several factors at the same
time. The complexities are summarised in the following table.
Treatments applied in various studies of different species (listed in the table
below) which were used by Mustroph et al. (2014) to make comparisons of species
for the induction of transcripts of PPi-dependent enzymes.
Species
Treatment
Complications Shoots
Reference
environment
Arabidopsis
On plates
Few
Argon
Branco-Price et al.
flushed with
(2005).
Argon
Maize
stagnant
Aerenchyma
Air
Rahji et al. (2011)
solution
formation, not
well defined
O2 deficits
Rorippa
Submerged , CO2 and
Submerged Sasidharan et al.
roots in soil, ethylene and
(2013)
plants
soil chemical
completely
changes
submerged
Arabidopsis
In soil
Air
Lee et al. (2011)
submerged
Soybean
Waterlogged
Nanjo et al. (2010)
sand
Cotton
Soil
Christianson et al.
(2010)
Poplar
Soil
Kreuzwieser et al.
(2009)
Germinating N2 gassing
No
Exposure to Lasanthi rice seedlings
confounding
anoxia only Kudahettige et al.
conditions
with good
(2007) & Narsai et
ventilation
al. (2009)
One frequent complication is to impose hypoxia on the roots while the leaves remain in
air. This technique will result in a complex pattern in the root with O2 varying from relatively
high near the shoot-root junction and low towards the tips of the roots (see also Notes S5).
Since the whole root systems were extracted, the extracts would have consisted of a mixture of
cells that had experienced widely different O2 regimes (Armstrong & Beckett, 1987). Another
major complication is use of flooded soil, which imposes a complex pattern of O2 decrease,
CO2 and ethylene accumulation and chemical changes in the anoxic soil (Kirk et al., 2014). For
the elucidation of mechanisms of anoxia tolerance per se, bubbling high-purity N2 or argon gas
through solutions in appropriate chambers would be best way to obtain a tight comparison
between putative anoxia tolerant and intolerant species under well-defined conditions.
Supplementary Notes 3: Additional notes on energetics and glycolysis
3.1 ATP production during sugar catabolism
6
To assess ATP production during sugar catabolism in anoxia we need to estimate the difference
between PPi produced in reactions involving NTP → NDP + PPi and the requirements of
UDPG pyrophosphorylase and V-H+PPiase (Sections II.2 & II.3). The amount of PPi formed
during polymer synthesis can be assessed, albeit tediously, by incorporation of substrates into
polymers and hence PPi production can be deduced using known stoichiometric relationships.
Only formation of PPi via reactions which are energised by NTP → NDP + PPi yields a net
flow into the PPi pool, such PPi-forming polymerisations are cell wall and RNA synthesis
(Sections II.2 & IV.7). As already emphasised in the main text, protein and lipid synthesis
produce PPi via NTP → AMP + PPi (see also Notes S2.2); these reactions need expenditure of
an ATP to re-phosphorylate the AMP formed and thus rebalance the nucleotide pool.
The PPi requirement of the V-H+PPiase is particularly hard to assess, since the rate of
+
H pumping across the tonoplast can be estimated only indirectly by flux analysis of ions like
K+ (Luettge & Higinbotham, 1979), to assess the rates of H+-solute co-transport. These flux
analyses across the tonoplast are tedious and not very exact, requiring flux equilibrium, which
may only be feasible in anoxia-tolerant tissues; as far as we know such flux analysis has never
been done during anoxia.
Due to these uncertainties, there remain three possibilities on how much PPi can affect
the ATP yield during sucrose mobilisation, leading to widely different values for the amount of
ATP formed during catabolism of sucrose:
i) The minimal case would 2.5 mol ATP per mol hexose flowing through glycolysis
(see Notes S3.2), which applies when PPi production during polymer synthesis is
very low, while the PPi requirements of UDPG pyrophosphorylase and the VH+PPiase remains substantial. A likely case is in acclimated non-growing tissues.
Then the PPi for UDPG pyrophosphorylase may have to be derived by exchange of
ATP for PPi via a reversible PPi-dependent glycolytic enzyme (Fig. 1c), as argued
in the main text most likely PPDK. The ATP required to sustain PPi production is
spent in the adenylate kinase reaction (Fig. 1c);
ii) If there is more net flow of PPi into the PPi pool derived from polymer synthesis,
there would be sufficient PPi for UDPG pyrophosphorylase, yielding 3 mol ATP
per mol of hexose flowing through glycolysis. We chose this option (Table 2)
because the PPi produced during cell wall synthesis was about equal to the amount
of PPi required by UDPG pyrophosphorylase;
iii) Yet two imponderables remain: Firstly, we have no information on the rate of RNA
synthesis during anoxia, so our assessment of the reactions which are energised by
NTP → NDP + PPi is an underestimate. Secondly, there are no good estimates on
PPi requirements of the V-H+PPiase. The cost of H+ pumping depends on both the
rate of H+ transport required to maintain compartmentation, but also on the
coupling ratio H+/PPi. As an example, the coupling ratio H+/ATP of the V-type H+pump of yeast can be as high as 4, as long as the pH is small (Kettner et al.,
2003). So, since the free energy of PPi hydrolysis of ~ 27 kJ mol-1 (Section II.1)
exceeds the free energy requirement for H+ transport of ~ 7 kJ through the
tonoplast (Section III.4.1) by more than 3 fold, there may be a coupling ratio of
H+/PPi of 3.
If the net flow of PPi derived from polymer synthesis is high, the PPi formed in the
reactions, which include NTP → NDP + PPi, may exceed the combined needs of the VH+PPiase and UDPG pyrophosphorylase. Then ATP production via sucrose catabolism can be
increased by substitution of PPi in the PFK reactions. This option has been assumed by
Igamberdiev & Kleczkowski (2011), Edwards et al. (2012) and Huang et al. (2008), who
assumed formation of 4 mol ATP per mol of hexose flowing through glycolysis.
7
3.2 ATP production during sugar catabolism depends on type of substrate and the balance
between PPi production and requirements
The substrate of glycolysis is a critical factor in energy efficiency. The effect of type of
substrate on ATP production can be deduced relatively accurately. When sucrose is the
substrate, as in intact seedlings, UDPG pyrophosphorylase converts PPi to UTP, thereby
increasing energy yield from glycolysis by 25% or 50% above that when hexoses are the
substrates for glycolysis (Ricard et al., 1998).
The low yield of ATP derived from glycolysis when hexoses are the substrate is
relevant to many studies with excised tissues, which are usually supplied with exogenous
glucose (Huang et al., 2005; Greenway et al., 2011). Provision of sucrose, rather than glucose,
would not solve this issue since cell wall invertases will cleave sucrose. An additional
disadvantage of excised tissues may be a requirement of energy for sugar uptake, but that
remains uncertain as usually high external glucose concentrations are used. Even so the lower
ATP yield in these excised tissues, than in the coleoptiles of intact seedlings, makes it all the
more interesting that despite lower energy production, the excised rice coleoptiles in exogenous
glucose still have a high anoxia tolerance, as shown by their survival in anoxia for at least 5 d,
and even when exposed to a combination of anoxia and pH 3.5 external for at least 30 h
(Greenway et al., 2011).
3.3 Assessing energy for maintenance in the rad mutant (Section III.1 – reduced alcohol
dehydrogenase mutant)
Ethanol formation by coleoptiles excised from anoxic seedlings of the rad mutant was 2.3 μmol
g-1(fresh weight) h-1 (Edwards et al., 2012). So, there would be ATP production of 2.9- 3.5
μmol g-1(fresh weight) h-1. The 2.9 μmol ATP g-1(fresh weight) h-1 is the more likely value,
since cellulose synthesis, the reaction which would maintain synthesis of PPi, is likely to be low
in rad coleoptiles which are energy-deficient and slow growing in anoxia. These values can be
converted to a protein basis by using protein/fresh weight ratio in anoxic rice coleoptiles (21
mg g-1(fresh weight, calculated from Edwards et al., 2012), yielding a maintenance cost of
0.14-0.17 mol ATP mg-1 protein h-1.
3.4 Reduced nucleotides as an energy source?
It should be noted there is no detailed discussion given here on the consumption of reduced
nucleotides formed during anaerobic catabolism, since overall the issue is not their use but the
need to recycle NADH to NAD+ to sustain glycolysis. Though traditionally this recycling was
thought to be achieved mainly by ethanolic fermentation, Fox et al. (1994) showed that for the
rice weed Echinochloa, that the main reconversion of NADH to NAD+ occurred during lipid
synthesis. This importance of disposing most of the NADH generated during carbohydrate
catabolism does not negate the possibility that NADH may be sometimes a valuable energy
source (e.g. in formation of glutamate; Section IV.5).
3.5 How the rate of formation of amino acids and lipids in anoxia may influence the rate of
glycolysis
In elongating rice coleoptiles under anoxia (Edwards et al., 2012), alanine and lipid synthesis
accounted for 14% and ~ 6% respectively of the demand for total energy donor (ED) (Table 2).
How much the generation of carbon skeletons via sugar catabolism destined for these pathways
contributes to ATP synthesis depends on what factors limit the rate of glycolysis. Two
scenarios are: (i) if glycolysis was limited by carbohydrate supply, there would be no increase
in glycolysis, merely a diversion of ethanol to alanine and lipids; (ii) if glycolysis could be
8
stimulated by the higher demand for pyruvate required for alanine and lipid synthesis, more
hexoses would be catabolised via glycolysis and the cell would produce more ATP.
Supplementary Notes 4: Factors determining pH, the basis for using the strong ion
difference (SID)
The pH is a dependent variable determined by the SID, which is: i) the difference between
(cations- H+) and (anions- OH-), ii) the total concentration of weak ions, iii) the partial pressure
of CO2 (Stewart, 1983). Changes in all these independent variables need to be established
before the causes of the pH changes can be elucidated (Gerendas & Schurr, 1999). All the
same, Ullrich & Novacky (1990) gave persuasive explanations for some short-term changes in
pHcyt in terms of changes in SID. Some examples where the concept helped in understanding
the plant response for anoxia are in Greenway & Gibbs (2003). Briefly, an increase of a cation
will tend to increase pH, while increase of an anion will tend to decrease pH. So, a larger
increase in cations than anions will increase pH, with the magnitude of the change in cells
being dependent on the buffering capacity of the fluids as determined by the other contents
(e.g. the capacity of carboxyl and amino groups of organic molecules to dissociate), vice versa
a larger increase in anions than cations would decrease pH.
Supplementary Notes 5: Problems with interpretation when roots are in an anoxic
medium but shoots are in air
The experiment by Manusco & Marras (2006), which used vine cuttings with shoots in air is
valuable, since it provides excellent physical data showing that decreased K+ permeability
caused the reduced K+ fluxes during severe hypoxia and by extrapolation to anoxia. The key
problem with interpretation is that the shoots were in air, so the O2 provision to the roots
remains quite uncertain. This complex and intriguing situation was studied extensively by
Armstrong and coworkers (Armstrong & Beckett, 1987; Armstrong et al., 2000). These papers
demonstrate that both theory and measurements with O2 micro-electrodes indicate the O2
concentration in the roots will vary both vertically and radially and will greatly depend on the
porosity of the roots. The redeeming aspect of the experiments by Manusco & Marras (2006) is
that the roots had very high rates of ethanol formation, demonstrating large parts of the roots
suffered at least very severe hypoxia, if not anoxia. All the same, the valuable K+ permeability
measurements would come better to their right, either in anoxia, or in well controlled
experiments, in which at the same location of the root there were complementary measurements
with O2 micro-electrodes.
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