Ion transport in seminal and adventitious roots of cereals ...

Journal of Experimental Botany, Vol. 62, No. 1, pp. 39–57, 2011doi:10.1093/jxb/erq271 Advance Access publication 16 September, 2010

REVIEW PAPER

Ion transport in seminal and adventitious roots of cerealsduring O2 deficiency

Timothy David Colmer* and Hank Greenway

School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley,WA 6009, Australia

* To whom correspondence should be addressed: E-mail: [email protected]

Received 14 June 2010; Revised 5 August 2010; Accepted 12 August 2010

Abstract

O2 deficiency during soil waterlogging inhibits respiration in roots, resulting in severe energy deficits. Decreased

root-to-shoot ratio and suboptimal functioning of the roots, result in nutrient deficiencies in the shoots. In N2-flushed nutrient solutions, wheat seminal roots cease growth, while newly formed adventitious roots develop

aerenchyma, and grow, albeit to a restricted length. When reliant on an internal O2 supply from the shoot, nutrient

uptake by adventitious roots was inhibited less than in seminal roots. Epidermal and cortical cells are likely to

receive sufficient O2 for oxidative phosphorylation and ion transport. By contrast, stelar hypoxia–anoxia can develop

so that H+-ATPases in the xylem parenchyma would be inhibited; the diminished H+ gradients and depolarized

membranes inhibit secondary energy-dependent ion transport and channel conductances. Thus, the presence of

two transport steps, one in the epidermis and cortex to accumulate ions from the solution and another in the stele to

load ions into the xylem, is important for understanding the inhibitory effects of root zone hypoxia on nutrientacquisition and xylem transport, as well as the regulation of delivery to the shoots of unwanted ions, such as Na+.

Improvement of waterlogging tolerance in wheat will require an increased capacity for root growth, and more

efficient root functioning, when in anaerobic media.

Key words: Aerenchyma, anoxia, Hordeum vulgare, hypoxic stele, hypoxia, ion transport, soil waterlogging, Triticum aestivum,

xylem, Zea mays.

Introduction

Waterlogged soils are usually anaerobic (Ponnamperuma,

1984). When water displaces gases in the soil, the inward

flux of atmospheric O2 is inhibited by approximately320 000 times, owing to the 104-fold slower diffusivity and

the 32-fold lower solubility of O2 in water than in air

(Armstrong and Drew, 2002). The rate of soil O2 depletion

following waterlogging depends upon the metabolic activity

of microorganisms and plant roots, so O2 depletion will be

more rapid at warmer temperatures and with high organic

matter in the soil (Drew, 1992). When O2 is lacking,

oxidative phosphorylation ceases, causing severe energy

deficits with adverse effects on growth and nutrient uptake,

and even resulting in root death. Following the onset ofanoxia, root cells of many plant species begin to die within

a few hours, or at most days (Gibbs and Greenway, 2003).

In addition to O2 deficiency, and depending on soil

type, waterlogging can also pose other challenges to roots;

Mn2+, Fe2+, S2–, carboxylic acids, CO2 and ethylene

(C2H4) can accumulate owing to soil microbial activity

(Ponnamperuma, 1984). In submerged tissues, endogenous

Abbreviations: COPR, critical O2 pressure for respiration; COPE, critical O2 pressure for root extension; Lp, hydraulic conductivity; Lpc, hydraulic conductivity of rootcortical cells; Lpr, hydraulic conductivity across roots, to the xylem; NORC, non-selective outward-rectifying channel; ROL, radial O2 loss; SKOR channel, selective K+

outward-rectifying channel.Definitions: Anoxia, zero O2. Hypoxia, O2 below the critical O2 pressure for respiration (COPR), but not zero. Anaerobiosis, strictly defined as a condition without O2, buthas been used to describe N2-flushed solutions in which O2 would be close to zero, but are not sure to be anoxic. Hypoxic–anoxic stele, stele which receivesinadequate O2 for oxidative phosphorylation, but with some uncertainties that the entire stele is truly anoxic (Darwent et al., 2003).ª The Author [2010]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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ethylene typically increases to between 0.1 and 1 Pa, partial

pressures at which maximum physiological effects are often

elicited (Jackson, 2008). In waterlogged soils CO2 can

increase to 5–40 kPa, with adverse effects on roots of some

species (Greenway et al., 2006). ‘Soil toxins’ can affect ion

transporters, however, this aspect is not discussed further

here (for a recent review see Pang and Shabala, 2010).

In addition, there may be adverse interaction betweenwaterlogging and other chemical constraints such as salinity

(Barrett-Lennard, 2003) and high Al (Setter et al., 2009).

Nevertheless, no such complexities were found in a series

of experiments on wheat (Triticum aestivum L.) lasting for

12 d: the response by wheat to anaerobic (i.e. N2-flushed)

solutions (Trought and Drew, 1980a) simulated, to a large

extent, the response to soil waterlogging in a pot experiment

(14 �C, sandy soil with 3.3% organic matter; Trought andDrew, 1980c). The similarities included slower relative

growth rates of roots and shoots, dramatically reduced

nutrient transport to the shoots resulting in nutrient

deficiencies, and premature leaf senescence (Trought and

Drew, 1980a, b, c). Similar to wheat, barley (Hordeum

vulgare L.) also soon became nutrient deficient upon soil

waterlogging (Drew and Sisworo, 1979).

Roots in anoxic media depend on internal gas-phasediffusion of O2 from the shoots, via aerenchyma, to

enable respiration (Armstrong, 1979). Aerenchyma provides

large interconnected gas channels that greatly enhance

O2 movement from the shoots into, and along, roots. The

importance of aerenchyma to enable respiration in the roots

of maize (Zea mays L.) seedlings when in an anoxic medium

was demonstrated by the adenylate energy charges in the

5 mm root tips of intact plants with roots in an anoxicmedium. The energy charges were 0.7 and 0.4 in roots with

13% and 4% gas-filled porosity, respectively, compared with

0.9 when the roots were in air (Drew et al., 1985). A gas-

filled porosity of 13% represents relatively modest aeren-

chyma formation; this value for maize compares with root

porosity of up to 41% in rice (Oryza sativa L.) (Colmer,

2003a). Even when there is some O2 in the root medium (i.e.

hypoxia¼suboptimal, but not zero O2), longitudinal gas-phase O2 diffusion within roots is an important component

of O2 supply (Armstrong and Beckett, 1987).

For roots receiving O2 via aerenchyma, the internal

concentration will decrease in a curvilinear gradient with

distance down the roots, since movement occurs via diffusion

and O2 is consumed along the diffusion pathway (Armstrong,

1979). Radial gradients in O2 also occur, the steepness of

these gradients depends upon resistances to O2 diffusion andO2 consumption rates. So, apical regions and the stele of

roots may receive a suboptimal supply of O2, since these

tissues are typically of low gas-filled porosity, both have

relatively high O2 demands, and occur at the ends of

longitudinal (gas phase) and radial (predominantly liquid

phase) diffusion paths (Armstrong, 1979; Armstrong and

Beckett, 1987; Armstrong et al., 1991; Gibbs et al., 1998a).

The focus of this review is on growth and function ofseminal and adventitious roots of wheat in O2-deficient

media, but data for maize (Zea mays L.) and barley

(Hordeum vulgare L.) are included for topics not yet well-

studied in wheat, and some comparisons are also made with

rice (Oryza sativa L.). Inhibition of nutrient uptake in

O2-deficient roots received early attention as a major

component of waterlogging damage in wheat, at least for

the first 12 d (key papers by Trought and Drew, 1980a,

1981) and other crops, including barley and maize (reviewed

by Drew, 1988). Subsequent work, as reviewed here, hasalso identified poor root functioning as a key limitation for

waterlogging tolerance in dryland crops (wheat, barley, and

maize). An overview of the responses of wheat to soil

waterlogging and associated root zone hypoxia–anoxia is

given in Fig. 1. The present review highlights the links

between root O2 supply, energy metabolism, and energy-

dependent ion transport, particularly the inhibition of xylem

loading when the stele becomes anoxic, or at least ‘severelyhypoxic’, the preferred term by Darwent et al. (2003), based

on their detailed radial O2 profiles in maize roots. Broader

information on waterlogging tolerance in wheat (and barley)

is available in the review by Setter and Waters (2003).

Recent reviews, that complement the focus here, have

discussed the influence of waterlogging on soil and plant

parameters of a nutrient uptake model (Elzenga and van

Veen, 2010) or have dealt more broadly with the adverseeffects of, and plant adaptations to, soil waterlogging and

complete submergence of the shoots (Bailey-Serres and

Voesenek, 2008; Colmer and Voesenek, 2009).

Techniques for studying plant responses tohypoxia using nutrient solutions

Most studies of the function of roots in O2-deficient

conditions have used nutrient solutions, as O2 and nutrient

levels can be controlled and measured, and roots can be

accessed and studied, more easily than in soils. ‘Anaerobic’

treatment was commonly imposed by flushing a nutrientsolution with N2 gas, but in many studies anaerobiosis

(i.e. zero O2) was not confirmed (Trought and Drew,

1980a). These solutions might still have contained some

O2 (dissolved O2 was 0.003 mM in N2-flushed solutions in

experiments by Kuiper et al., 1994); O2 supply to roots from

N2-flushed solutions will depend upon the purity of the N2

gas, pot design, and the flushing rates used. Even when

O2 concentration in the external solution is known, the O2

status of root tissues can still be uncertain, since internal O2

will depend upon respiration rates, turbulence as deter-

mined by rates of bubbling or stirring, pot size and design,

root density in the solution, root diameters, diffusion

resistances across unstirred boundary layers of various sizes

and across tissues of various diameters. As these factors

differ amongst studies and species, comparisons of results

can be difficult across experiments.An alternative to continuous N2 flushing is the use of

nutrient solutions containing 0.1% (w/v) agar, which can be

de-oxygenated by N2 flushing prior to use. The dilute agar

prevents convective movements in the solution so this

stagnant solution better mimics the changes in gas

40 | Colmer and GreenwayD

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composition found in waterlogged soils; i.e. increases in

CO2 and ethylene in addition to low O2 in the continuously

N2-flushed solutions (Wiengweera et al., 1997). The advan-

tage of using stagnant agar solution is that accumulation ofethylene enhances the development of aerenchyma

(Shiono et al., 2008), so that roots of wheat in stagnant

agar solution were of higher porosity than those in N2-

flushed solution (Wiengweera et al., 1997). On the

other hand, the N2-flushed treatment can have the advan-

tage, depending on the experimental objective, of only

changing one factor (i.e. O2 concentration) from the

condition in aerated controls. An overview of responses, ofseminal and adventitious roots of wheat to these two

methods of imposing O2 deficiency in the root zone, is given

in Fig. 2.

Adverse effects of O2 deficiency on growthand mineral nutrient status

Declines in shoot growth of plants in anaerobic root media

may be caused by: (i) reduced development of roots, causing

a suboptimal root-to-shoot ratio (root:shoot), and/or (ii)

impaired functioning of roots. Decreased root:shoot may

lead to an altered balance of phytohormones that might

slow shoot growth and stimulate adventitious root growth,keeping root:shoot within certain limits (Drew, 1983).

In addition, the combination of a reduced size of the root

system and suboptimal root functioning would compromise

the provision of nutrients, and perhaps water, to the

shoots. The reduced shoot and root growth is unlikely to

be associated with a shortage of sugars for catabolism, or

for incorporation into biomass, because during root

zone O2 deficiency sugars accumulate in tissues as growth

is inhibited relatively more than photosynthesis (Setteret al., 1987). In wheat with roots in N2-flushed solution

for 12–14 d, sugars increased by 20–30% in shoots and by

55–70% in roots (Barrett-Lennard et al., 1988). Sugar

accumulation was also reported in roots of barley when

growth was inhibited by low O2 supply (Limpinuntana and

Greenway, 1979). In view of these large increases in

sugar concentrations, ethanol-insoluble dry weight (i.e.

structural biomass with soluble sugars removed) rather thantotal dry weight has been used by some authors to

assess growth (Barrett-Lennard et al., 1988). Thus, respira-

tion in roots in N2-flushed solution would be inhibited by

low O2 availability (see below), rather than shortage of

substrate.

Responses of wheat to anaerobic nutrient solution at the

early tillering stage, from the key study by Trought and

Drew (1980a), are provided in Tables 1 and 2, and these arediscussed in more detail below.

O2 deficiency decreases root-to-shoot ratio and hasmore severe effects on seminal, than on adventitious,root growth

In the key study on wheat at the early tillering stage (Trought

and Drew, 1980a), ratios of root-to-shoot (root:shoot,

dry weight basis) were 0.57 and 0.18 in aerated and N2-

flushed nutrient solutions, respectively (Table 1). Other

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Fig. 1. Simplified overview of the responses to soil waterlogging of wheat seminal and adventitious roots, and resulting restrictions for

shoot growth. Intolerance of roots to anoxia, and the poor capacity for internal O2 transport impacts severely on seminal roots. Although

adventitious root numbers can increase, and these form more aerenchyma than seminal roots, suboptimal characteristics related to O2

transport result in modest growth and suboptimal functioning in waterlogged soils. ROL, radial O2 loss.

Ion transport in hypoxic roots | 41D

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experiments on wheat (e.g. Kuiper et al., 1994) also found

similar decreases in root:shoot, despite large differences incontrol plant growth and inhibitory effects of the N2

treatment on shoot relative growth rates between these

experiments conducted at different temperatures (see foot-

note of Table 1). Root:shoot also decreased, albeit to

a smaller extent, when stagnant agar was used (Watkin

et al., 1998) rather than N2 flushing to impose the

‘anaerobic’ treatment.

These decreases in root:shoot are mainly due to thecessation of seminal root growth. By contrast, adventitious

roots emerge (also termed nodal or crown roots) which at

least partially replace the damaged seminal roots (e.g. for

wheat, Trought and Drew, 1980a; for other species see

reviews by Drew, 1983; Jackson and Drew, 1984). These

adventitious roots typically develop more aerenchyma than

the pre-existing seminal roots, and so have an improved

capacity for internal O2 supply (discussed below). So, afterperiods of anaerobiosis lasting longer than 2 weeks, the

adventitious root system is the largest fraction of roots for

wheat (Trought and Drew, 1980a; Barrett-Lennard et al.,

1988). However, these roots cannot grow to their full

lengths, so the increased numbers of adventitious roots,

from 7.2 to 11 at 14 d of exposure to N2-flushed solution,

only partially compensated for the reduced rates of

elongation; the relative growth rate (ethanol-insoluble dryweight basis) of adventitious roots was 0.173 d�1 in N2-

flushed solution compared with 0.188 d�1 in aerated

solution (Barrett-Lennard et al., 1988). Increases in num-bers of adventitious roots during hypoxia are usually found

and sometimes can be quite large; for example, in a tolerant

barley cultivar adventitious root number increased from 14

in drained soil to 33 in waterlogged soil (Pang et al., 2007).

As examples of reduced root lengths, the main axes of

adventitious roots of wheat in N2-flushed nutrient solutions

only grew to 9 cm (Barrett-Lennard et al., 1988) or 12 cm

(Trought and Drew, 1980a); extension ceases when rootsreach a length beyond which internal O2 diffusion cannot

sustain growth (Armstrong, 1979). Although growth ceased,

the tips of the adventitious roots remained alive as shown

by resumed extension upon re-aeration (Trought and Drew,

1980a; Barrett-Lennard et al., 1988).

Evidence for nutrient deficiency in shoots of wheat withO2-deficient roots

A persuasive case for nutrient deficiency in shoots of wheat,

when grown with roots in N2-flushed nutrient solution, was

made in the studies by Trought and Drew (1980a). After 14 din the N2-flushed treatment, uptake of all macronutrients

was reduced (Table 2). Decreases in nutrient concentrations

in the shoots can be quite severe, as one example the N

concentration (mmol g�1 dry weight) was reduced to 24% of

that in shoots of the aerated control (Trought and Drew,

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Fig. 2. Oxygen status and associated impacts on ion transport processes in seminal and adventitious roots of wheat when in two

systems used to impose O2 deficits in nutrient solution experiments. N2-flushed solutions contain very low O2 and turbulence reduces

boundary layers so facilitates any O2 uptake from the solution, whereas 0.1% agar solutions are stagnant and can be deoxygenated prior

to use. As ethylene accumulates in stagnant agar solution, this method is more analogous to the changes in gas composition in

waterlogged soils. The contrasting responses of seminal and adventitious roots (low and moderate aerenchyma, respectively) and

treatment methods (N2-flushed versus stagnant agar) are described. ROL, radial O2 loss.


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1980a). Decreased nutrient concentrations in the shootswere not only due to reduced root:shoot, but were also

caused by poor functioning of the root system; i.e. the

inhibition of ion transport to the shoot per unit of root, as

shown by greatly reduced concentrations of macronutrients

in the xylem (Table 2).

The hypothesis that nutrient deficiencies retard the

growth of wheat in waterlogged soil can be tested by the

response to increased nutrient supply. Some success withthis approach has been achieved, though few of the experi-

ments restored the growth of plants with O2-deficient roots

to that in aerated/drained controls. Shoot N concentrations

were increased in wheat in both aerated and N2-flushed

solutions when high external NO3� (10 mM) was supplied;

but growth was only improved when all nutrients in the

medium were increased 4-fold (Trought and Drew, 1981).

Then, shoot fresh weight (the best indicator available ofgrowth in that experiment) of plants in N2-flushed solution

was reduced by 45% compared with 60% when only NO3�

was increased, however, there was no significant improve-

ment in root growth (Trought and Drew, 1981). Improved

root growth at high mineral nutrition was indicated in

another experiment with wheat in waterlogged sand culture;

when Hoagland nutrient solution was increased from half-

to full-strength (7–14 mM NO3�) the decrease in root dry

weight relative to drained controls of a tolerant variety was

less severe (50% reduction at 14mM NO3� compared with

75% reduction at 7mM NO3�; Huang et al., 1994). That N

deficiency contributed to some extent to the adverse effects

of waterlogging was shown by applying a 0.1 M foliar urea

spray to wheat seedlings during the first 3 d of a 14 d

waterlogging treatment; reductions in shoot fresh weight at

the end of 14 d were decreased from 40% to 32% and inchlorophyll concentrations from 76% to 64% (Trought and

Drew, 1980b). High nutrition may also aid recovery after

a return to aerated conditions. Even when the fresh weight

of wheat shoots was not improved in N2-flushed solution

containing 5 mM rather than 0.1 mM NO3�, transference to

aerated solution containing 0.5 mM NO3�, increased the

Table 2. Effect of N2-flushed treatment on nutrient uptake and

transport to shoots of wheat

Values were estimated based on changes in shoot content and total

transpiration between samplings, assuming no recirculation from

shoots to roots via phloem. Wheat (cv. Capelle Desprez) was raised

in aerated solution for 14 d, with aeration then continued or replaced

by N2 flushing for another 14 d, all at 14 �C. Data from Trought and

Drew (1980a). Overall, data were similar for 0–4, 4–8, and 8–14 d of

N2 flushing and the data presented here are for 8–14 d.

Element Nutrientsolution

Calculated (mM) for xylemstream of plants with rootsin

(mM) Aerated N2

flushed

Nitrogena 0.50 9.52 2.27

Phosphorus 0.25 1.28 0.24

Potassiumb 0.75 4.92 0.55

Calcium 1.00 0.51 0.26

Magnesium 0.50 0.29 0.07

Nutrient uptake rates(mmol g�1 dw root d�1) in

Aerated N2-flushed

Nitrogena 0.50 284 113

Phosphorus 0.25 57 15

Potassiumb 0.75 202 39

Calcium 1.00 22 17

Magnesium 0.50 12 4

Water uptake rates(ml g�1 dw root d�1) in

Aerated N2-flushed

47 52

a Supplied as NO3�.

b Potassium concentration in solution is uncertain as the value of0.75 in Table 3 in Trought and Drew (1980a) and reproduced here,differs from the total of 5.0 mM specified in the composition of thenutrient solution in the Materials and methods. dw, dry weight.

Table 1. Responses of wheat to N2-flushed nutrient solutiona

Plants (cv. Capelle Desprez) were raised in aerated solution for

13 d, with aeration then continued or replaced by N2 flushing for

another 14 d, all at 14 �C. Data from Trought and Drew (1980a);

values are means 6standard errors.

Parameter Aerated N2-flushed % ofControl

Shoot N concentration 2.9960.06 0.7260.07 24

(mmol g�1 dry weight)

Shoot fresh weight (g) 2.5760.11 0.8260.04 32

Shoot dry weight (mg) 357619 210612 59

Seminal root dry weight (mg) 175610 2061 11

Adventitious root dry

weight (mg)

2763 1762 63

Root:shoot (dry weight basis) 0.57 0.18 32

Adventitious root numberb Not given, but not

different between

treatments

Longest adventitious root (cm) 2161 1261 57

Adventitious root aerenchyma

(%)

Towards tip 0 10 –

Near base Not specified 25 –

a The example given is from the key study by Trought and Drew(1980a), with the results being considered typical for wheat. Similarinhibitory effects on roots, as described above by Trought and Drew(1980a), were also seen for cv. Gamenya grown at 15/20 �C night/dayin Kuiper et al. (1994). Reductions in shoot growth were less whenwheat was in N2-flushed solution with high nutrient concentrations(Trought and Drew, 1981; Barrett-Lennard et al., 1988), in whichseminal root growth was still severely inhibited.

b Numbers were not reported by Trought and Drew (1980a).In some studies adventitious root numbers have been reported toincrease in N2-flushed conditions (e.g. from 7 to 11 per plant after 14 dof hypoxia in cv. Gamenya; Barrett-Lennard et al., 1988).


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fresh weight of the shoots, relative to the weight at the time of

transfer, by 60% and 140% for plants exposed to 0.1 mM and

5 mM NO3�, respectively, during the preceding 14 d in N2-

flushed solution (Trought and Drew, 1981).

Experiments with foliar nutrient sprays on barley support

that nutrient deficiency contributes to decreased growth of

dryland cereals during the first 14 d of soil waterlogging.

Daily foliar sprays of complete Hoagland nutrient solutionto barley in pots of waterlogged soil improved shoot and

root growth, to varying degrees, of six genotypes; root dry

weight in one of the two tolerant genotypes became as high

as in the drained pots (Pang et al., 2007); i.e. foliar sprays

were most effective when the particular genotype has an

inherent high tolerance to waterlogging. The improvement

of root growth is particularly noteworthy and needs verifica-

tion. The foliar spray also increased N and K+ concentra-tions (% dry weight) in shoots and roots, reaching levels in

the plants in drained soil for one of the tolerant genotypes.

Foliar nutrient application also increased the number of

adventitious roots by 30–40%, while the percentage of

chlorotic leaves decreased. The experiment of Pang et al.

(2007) lacked a treatment of foliar application of nutrients to

the shoots of plants in drained soil, so whether the response

was specific to plants in waterlogged soil was not determined.Together, these experiments (Trought and Drew, 1980b,

1981; Pang et al., 2007) support the notion of nutrient

deficiency as a major factor limiting the shoot growth of

wheat and barley in waterlogged soils. Nevertheless, adverse

factors other than nutrient deficiencies probably also

contribute to the inhibition of shoot growth when roots are

in hypoxic nutrient solution. Barrett-Lennard et al. (1988)

found that wheat with high nutrient levels in solution (e.g.with NO3

� at 15 mM) still showed small (7–10%) inhibitions

of shoot relative growth rate in a N2-flushed treatment, even

though shoot tissue nutrient concentrations remained above

‘sufficiency levels’. We suggest these remaining reductions

in shoot growth of wheat in N2-flushed solution with high

nutrients may be associated with the regulation of

root:shoot, as the root system in the N2-flushed solutions

was still relatively small (see below). Possible regulation ofroot:shoot via hormonal changes in plants in O2-deficient

media, for example, inhibited production of IAA, cytoki-

nins, and gibberellins, or increased ABA transport to the

shoot, was reviewed by Armstrong and Drew (2002).

Summing up; attempts to relieve inhibitions of growth of

wheat in N2-flushed solutions by increasing nutrient supply

have only been partially successful. One interesting limita-

tion to this approach was shown in the experiments byHuang et al. (1994): an increase from half- to full-strength

Hoagland nutrients reduced the percentage of aerenchyma

in adventitious roots, in the tolerant cultivar from 30% to

24% (Huang et al., 1994); therefore improvements in

nutrition may have been partly counteracted by reduced O2

transport from shoots to roots. So, although some improve-

ment might be achieved by improved management (i.e. foliar

sprays and/or higher fertilizer use), the key approach remainsthe development of cultivars with improved tolerance to

waterlogging. At least two characteristics need attention,

growth of a reasonably-sized root system in waterlogged

soil, particularly in terms of root length and number of

adventitious roots, and improvement of root function in

O2-deficient media. In the sections below, key limitations to

root functioning in hypoxic–anoxic media, are discussed.

Aerenchyma and internal O2 supply toseminal and adventitious roots of wheat

Internal O2 supply is crucial to both growth and ion

transport in hypoxic roots. As much of this review concerns

the response of wheat to N2-flushed treatments in nutrientsolutions, a summary is given of what is known of the O2

status of the roots studied. Furthermore, limitations to

internal O2 transport are considered for seminal and

adventitious roots of wheat, as compared with rice.

In the experiments by Trought and Drew (1980a) on

wheat in N2-flushed solution, transverse sections of adven-

titious roots showed aerenchyma occupied 10% of the cross-

section near the tip and 25% near the root–shoot junction.Further information on O2 supply to adventitious roots of

wheat was obtained for intact plants grown with roots in

N2-flushed nutrient solutions (Barrett-Lennard et al., 1988),

in which adventitious root porosity was 14%. O2 concen-

trations in the cortical gas spaces of these adventitious roots

were assessed using root-sleeving O2 electrodes (Armstrong

and Wright, 1975); this technique requires transferring the

roots from the N2-flushed solution into O2-free stagnantagar. Assessed O2 partial pressures near the root tip were

1 kPa for 90 mm long roots and 2 kPa for 60 mm long

roots, whereas in basal root zones near the root–shoot

junction the levels were 4 and 8 kPa, respectively (Barrett-

Lennard et al., 1988). No O2 data for the seminal roots were

obtained, but the proximal sections had only 3% porosity,

compared with 14% in the adventitious roots (Barrett-

Lennard et al., 1988), so internal O2 concentrations wouldalmost certainly be much lower in the seminal than in the

adventitious roots.

Adventitious root elongation for wheat in stagnant agar

nutrient solution increased in response to raising O2 around

the shoots in short-term experiments, confirming root

growth in this stagnant medium was limited by low O2

supply (Wiengweera and Greenway, 2004). Raising O2

around the shoots from 20 kPa to 40 kPa for 2 h and thento 80 kPa, increased root elongation by 5-fold for 11–12 cm

long roots, and by 2-fold for both 5–6 and 1–2 cm long

roots (Wiengweera and Greenway, 2004). The large stimu-

lation in the 11–12 cm roots is consistent with the predicted

low O2 near the apex for roots of this length with 15–20%

porosity (Armstrong, 1979). Thus, when O2 around the

shoots was increased, more O2 could diffuse to the root

apex and elongation resumed. Similarly, in 5–7-d-oldseminal roots of wheat which, contrary to older seminal

roots can develop porosity (namely 12%) during hypoxia,

there were also large stimulations of elongation when O2

around the shoots was increased to 100 kPa (Thomson

et al., 1990).


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The surprising result in Wiengweera and Greenway

(2004) was the substantial stimulation of the short adventi-

tious wheat roots, indicating O2 deficiency even in the tips

of these short roots which are close to the potential O2

source at the shoot base. The crucial importance of this

limitation is highlighted by the rather slow extension of

these young roots when in stagnant agar solution, at ;50%

of aerated controls, with ambient O2 around the shoots andhence we will discuss some reasons why the growth of even

these short adventitious roots was suboptimal. O2 partial

pressure around the shoots required to stimulate root

extension in wheat exceeds by ;20-fold the 4 kPa required

around shoots of rice for optimum extension of 5–7 cm long

adventitious roots (Armstrong and Webb, 1985); i.e. with

ambient O2 concentration around the shoots, O2 provision

to the tips of rice roots in an anoxic medium was more thanample, but clearly suboptimal for roots of wheat. The

rice was grown in waterlogged potting mix prior to being

washed out and transferred into stagnant agar; the roots

were reported to be aerenchymatous, but percentage

porosity or aerenchyma was not given. Assuming porosity

in the rice roots of up to 41% (e.g. stagnant agar-raised

plants; Colmer, 2003a), the much lower porosity of 15%

for wheat adventitious roots grown in stagnant agar(Wiengweera et al., 1997) would only partially explain the

higher shoot O2 requirement to sustain extension in the

short adventitious roots of wheat. Other possible reasons

include: the 15% porosity reported by Wiengweera et al.

(1997) was for the entire adventitious root system, but short

roots might have had lower porosity than the longer roots

(TD Colmer, personal observations for young adventitious

roots of wheat). In addition to the lower amount ofaerenchyma (i.e. lower maximum porosity) formed in roots

of wheat, other factors presumably also contribute to the

spectacular differences in O2 diffusion to the root apex, as

compared with rice. Firstly, radial O2 loss (ROL) from

basal parts of wheat adventitious roots with aerenchyma

can be substantial (Thomson et al., 1992), whereas roots of

rice possess a barrier against ROL in basal zones

(Armstrong, 1971; Colmer, 2003a), although even in ricethe barrier is less pronounced in short than in long roots

(Colmer et al., 2006a). The barrier to ROL restricts radial

losses, thus promoting longitudinal diffusion towards the

apex (Armstrong, 1979). Secondly, as aerenchyma typically

terminates a few cm behind root tips (e.g. rice, Armstrong,

1971; wheat, Thomson et al., 1992), diffusion to the apex

depends on porosity in the intervening tissue, which is

almost certainly less porous in wheat, than in rice. Rootcortex cells in rice occur in a cubical arrangement (Justin

and Armstrong, 1987), whereas in wheat the pattern is

hexagonal (assessed from micrographs in Huang et al.,

1994; TD Colmer, personal observations). These different

cell packings can result in substantial differences in tissue

porosity. In rice roots with cubic packing, porosity was

;9% near the non-aerenchymatous tip (Armstrong, 1971),

whereas it can be as low as 1% in roots with hexagonal cellarrangements (Justin and Armstrong, 1987). Thus, very low

porosity in the younger tissues in adventitious roots of

wheat, resulting in high resistance to internal O2 diffusion,

would explain the finding of Wiengweera and Greenway

(2004) that, even in short roots, O2 supply from the shoot is

suboptimal for root growth in an O2-free medium.

Taking rice as a model species is useful but also has

limitations for comparisons with dryland cereals. Adapta-

tion of rice to flooded conditions involves a range of

characteristics (traits in wetland plants were reviewed byBailey-Serres and Voesenek, 2008; Colmer and Voesenek,

2009), which amplifies the challenge to achieve a large

improvement in waterlogging tolerance of dryland species

such as wheat. In any case, paddy rice may not be the best

model, since dryland cereals such as wheat would typically

experience transient waterlogging, while even 3 d water-

logging can severely retard development after drainage

occurs (Malik et al., 2002). So upland and/or rain-fed rice,in which final growth might be in drying soils, might

provide a better model for dryland cereals. Recovery upon

drainage following waterlogging would be especially impor-

tant for wheat, as rapid growth of deep roots following

drainage will be required to obtain sufficient water later in

the season in Mediterranean climates. Some traits of

importance in rice, such as a barrier to ROL (Armstrong,

1971; Colmer, 2003a) might be counterproductive forspecies faced with transient or intermittent flooding; possi-

ble reductions in water and nutrient uptake for roots of

wetland plants with a barrier to ROL were suggested by

Koncalova (1990). However, the few experiments conducted

to address this question have not found such drawbacks

(e.g. NO3� uptake by rice roots, Rubinigg et al., 2002; water

uptake by Hordeum marinum roots, Garthwaite et al.,

2006). The exception is a strong barrier induced in riceroots in response to soil toxins, then water permeability

declined and toxin entry was impeded (Armstrong and

Armstrong, 2005a). Even when the barrier in roots of rice

was induced by growth in stagnant solution, toxin (namely

Cu2+) entry was impeded (Kotula et al., 2009). One

drawback of a barrier to ROL, however, could be restricted

O2 entry into roots when the soil is not waterlogged,

causing suboptimal O2 supply to persist in some root partseven in a drained soil. Such trade-offs, if any do occur,

would be reduced in species with inducible barriers to ROL,

as compared with those that constitutively develop a barrier

(reviewed by Colmer, 2003b). Inducible barriers to ROL

occur in rice (Colmer, 2003a) and in some wild relatives of

cultivated dryland species in the Triticeae (McDonald et al.,

2001; Garthwaite et al., 2003); however, in rice once the

barrier has formed, it persists even upon re-aeration, whilenew root growth again lacks a barrier (MCH Cox and TD

Colmer, unpublished data).

Comparison of rice to wheat also emphasizes that in the

search for more tolerant wheat cultivars, a combination

of higher aerenchyma percentage and a larger root system

containing aerenchyma should be sought in wheat

(Thomson et al., 1992). Genetic diversity in capacity to

form a large system of adventitious roots was recentlyreported for barley (Pang et al., 2007); after 14 d of soil

waterlogging a tolerant genotype produced 1.7-times more


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adventitious roots than an intolerant genotype. Increased

porosity of seminal roots would also be beneficial. As stated

earlier, young seminal roots of wheat formed 12% gas-filled

porosity when raised in hypoxic nutrient solution (Thomson

et al., 1990), and it would be beneficial if that capacity was

enhanced in older seminal root systems. The capacity to

form aerenchyma in seminal roots might differ between

wheat genotypes, as aerenchyma did form in seminal rootsof wheat in sand culture when waterlogging was imposed on

14-d-old plants; with the genotype Savanna forming aeren-

chyma along the entire root length and reaching 21% near

the root–shoot junction. By contrast, in seminal roots of

a less tolerant genotype no aerenchyma formed in the

critical section near the root–shoot junction, although some

aerenchyma formed in the lower sections (Huang et al.,

1994). Yet, although during waterlogging growth in Savan-nah was better than in the second genotype, the dry weight

of the shoots of Savannah at two levels of N supply (3 and

6 mM NO3�) was still only 50% and 70% of the drained

controls, respectively (Huang et al., 1994). Experiments are

needed to test the capacity for aerenchyma formation in

seminal roots of a larger range of wheat genotypes.

Similarly, tests are required for much older plants; when

wheat in the booting stage (153-d-old) was waterlogged at1.5–2 cm above the soil surface, cross-sections showed large

aerenchyma formed within 96 h in 10–15 cm long adventi-

tious roots (Jiang et al., 2010). Furthermore, development

of constitutive aerenchyma in roots, both in adventitious

and seminal systems, might enhance the tolerance of

transient waterlogging, as aerenchyma takes time to develop

fully; 72–102 h for wheat adventitious roots (Malik et al.,

2003) and 48 h for 50–80 mm long seminal roots, as judgedby O2 transport from the shoots to root tips (Thomson

et al., 1990).

In summary, for wheat in anaerobic media, the bulk of

the seminal roots would receive very little O2 owing to low

tissue porosity, while after exceeding a certain length or age,

the seminal roots might not form aerenchyma (at least in

one genotype, Thomson et al., 1990). Adventitious roots

develop aerenchyma, so their internal O2 supply is superior

to that of pre-existing seminal roots, but the low-to-moderate

porosity and lack of a barrier against ROL (cf. Thomsonet al., 1992) means the more distal portions (i.e. further from

the root–shoot junction) of wheat adventitious roots still

suffer from O2 deficits. These O2 deficits in the distal portions

of the seminal and adventitious roots of wheat, including

towards the apex and also within the stele, as reviewed

below, will impact on functioning in anaerobic media.

O2 gradients in roots: occurrence of stelehypoxia–anoxia

O2 concentrations can differ markedly across roots. An

example of an O2 profile, from the external medium to the

centre of an excised, primary and non-aerenchymatous

maize root, is shown in Fig. 3. Even in a flowing medium,

a considerable boundary layer resistance was evident as

a steep O2 gradient between bulk solution and epidermis.

O2 also declined steeply across tissues of low porosity,namely the outer few cell layers of the root and again in the

stele, whereas within the porous cortex the gradient was

very gradual. Such concentration gradients are inherent

when supply is via diffusion between a source and sink, as

determined by resistance to diffusion and O2 consumption

along the diffusion path (Armstrong and Beckett, 1987).

The O2 concentration in the pericycle lining the protoxylem

and metaxylem vessels of the roots shown in Fig. 3 has been

Fig. 3. Profile of O2 concentration across an excised primary root of Zea mays in a flowing nutrient solution containing 0.05 mM O2, at 25

�C. An O2 microelectrode was moved in steps towards, and then through, the root (75 mm behind the apex, total length 135 mm). The flow

rate of the medium was 1.8 mm s�1, calculated using the depth of the vessel as 10 mm (H. Greenway, personal communication), rather

than the erroneous 100 mm stated by Gibbs et al. (1998). The line with symbols is the in-track, and that without symbols is the out-track.

Reproduced from Gibbs et al. (1998) in Australian Journal of Plant Physiology with kind permission of CSIRO Publishing.


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assessed at 3 lM or lower. No ready comparisons with

other tissues of high O2 demands can be made, the best

available data is for COPE of rice roots of 1.25–2.5 lM(assessed by Gibbs et al., 1998a from Armstrong and Webb,

1985). These rice root tips presumably had relatively low O2

requirements, as extension rates of 0.6 mm h�1 (;14 mm d�1)

(Armstrong and Webb, 1985) were relatively low compared

with up to 26 mm d�1 (Colmer, 2003a), as the tips mighthave been low in sugars (Gibbs and Greenway, 2003). Thus,

the low O2 concentration of 3 lM in the xylem parenchyma

indicates severe hypoxia, since the cells loading ions into the

xylem can be expected to have high rates of respiration (de

Boer and Volkov, 2003).

Gradients in O2 across roots were inferred by the classical

studies of root respiration by Berry and Norris (1949); these

authors hypothesized that as O2 was depleted from thebulk medium, hypoxia–anoxia would occur in the core of

tissues, while more peripheral cells still received adequate

O2 for oxidative phosphorylation. When the O2 concentra-

tion was below the critical O2 pressure for respiration

(COPR) of excised onion root segments, the respiratory

quotient (CO2 produced/O2 consumed) increased, implying

a switch to ethanolic fermentation in cells of the innermost

core (Berry and Norris, 1949). This interpretation of theonset of O2 deficits in the core of tissues when the external

concentration declines below the COPR is consistent with

mathematical modelling (Armstrong and Beckett, 1987;

Armstrong et al., 2009); with progressive expansion of the

volume of the anoxic core as external O2 supply decreases

below the COPR (i.e. the distance decreases to which O2 can

diffuse into the tissue before all is consumed).

The length of root over which the stele becomes O2

deficient depends upon several factors, these have already

been alluded to in the Introduction; applying these general

principles of Armstrong and Beckett (1987) to the stele, the

factors are: (i) the concentration of O2 in the cortical gas

spaces (determined by external O2 concentration, O2

diffusion in any aerenchyma and/or in the porous cortex,

rates of ROL along the root, and distance from the root–

shoot junction), (ii) the resistance to radial O2 diffusionthrough the endodermis and stelar tissues, (iii) the rate of

O2 consumption in the stele, (iv) the radius of the stele,

determining its absorbing surface at the endodermis, sub-

sequent diffusion-path distance to the centre, and total

volume. So, one adaptation to restricted O2 supply in roots

might be a narrower stele (Armstrong et al., 2000), reducing

its volume-to-surface area ratio, and diffusion-path length

for O2 to reach the parenchyma cells near the xylem. Speciesdiffer in the percentage of adventitious root cross-sectional

area occupied by the stele, with wheat at 16% (assessed

from micrographs in Huang et al., 1994) compared with

only 5% in rice (McDonald et al., 2002). Morphological

acclimation might also occur, the proportional area of stele

in cross-sections of barley adventitious roots decreased by

20% in waterlogged soil, compared with drained controls

(Pang et al., 2004).The O2 profile in Fig. 3 was for an excised maize root

immersed in solution, so there was no other O2 source than

the root medium. The O2 provision to roots of intact plants

is less well-defined than for excised roots. In studies with

intact maize, severe O2 deficiency in the stele was indicated

for the ;20 mm root tip of a 105 mm long non-

aerenchymatous root; by contrast, in roots with aeren-

chyma only the stele in the 10 mm root tip was low in O2

(Darwent et al., 2003). These authors discuss uncertainties

in interpretations of values obtained with the O2 micro-electrodes and conclude some O2 consumption might have

occurred concurrently with anaerobic catabolism, hence

they suggest referring to ‘severely hypoxic’ rather than

‘anoxic’ steles (Darwent et al., 2003). In view of these

uncertainties, data on O2 concentrations need to be

complemented with metabolic evidence.

A hypoxic stele was indicated by the higher pyruvate

decarboxylase activity in the stele versus the cortex from theintact primary root of intact maize seedlings in anoxic

medium (Thomson and Greenway, 1991). Furthermore,

ATP:ADP in roots of intact maize in N2-flushed solution

(Table 3) are consistent with the notion that the cores of

tissues can become O2 deficient (suboptimal respiration),

whereas the outer layers receive adequate O2 to meet their

respiratory demands. Near the root–shoot junction (the

source of O2 for roots in anoxic medium), ATP:ADP inroots of ;14% porosity was more similar to that in aerated

Table 3. ATP-to-ADP ratio (i.e. ATP:ADP) in intact adventitious

roots of Zea mays pretreated with different O2 supplies so as to

obtain a range of root porosity values, prior to transfer into N2-

flushed solution, with shoots in air

Treatments were imposed on 12-d-old plants for 11 d, in solutions

bubbled with 40% O2 (;4% root porosity), 5% O2 (;10% root

porosity), or non-turbulent solution (;14% root porosity). ATP:ADP

was measured on roots 75 min after transfer of the roots into the

N2-flushed solution. Data from Drew et al. (1985).

Segments at distance(mm) from the root–shoot junction

ATP:ADPa in rootswith porosity of

;4% ;10% ;14%

0–10 (basal segment) 3.860.03 3.660.3 4.460.05

100–110 1.160.04 1.560.1 3.260.5

135–145 n.a.b n.a. 1.760.02

145–150 n.a. n.a. 2.060.15

(tips of roots with ;14%

porosity)

200–205 0.560.02 1.760.05 n.a.

205–210 0.760.02 1.560.2 n.a.

(tips of roots with ;4 and

;10% porosity)

a For comparison with the values in the table, ATP:ADP in rootsegments when excised and in aerated solution was 5.5 and when inN2-flushed was 0.66.

b n.a. ¼ not applicable. n.a. entries in the table result from rootsbeing of different lengths following pretreatments; roots from non-turbulent solution were shorter than those pretreated with 5% or 40%O2, so root tips were different distances from the root–shoot junctionas indicated in the table.


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roots, whereas in roots with ;4% and ;10% porosity

ATP:ADP had declined, and the ratio then remained rather

steady between 100 mm and 205 mm from the root–shoot

junction (Table 3). The very low values for ATP:ADP of

0.5–0.7 in the distal part of the roots with ;4% porosity

indicates these were, not unexpectedly, anoxic in the cortex

as well as in the stele. The pattern down the roots and

values in distal regions for the roots with ;10% porosity areto be expected when the porous cortex has sufficient O2 for

oxidative phosphorylation, while the adjoining stele is at

least severely hypoxic (models of Armstrong and Becket,

1987). Consistent with the O2 profiles from Darwent et al.

(2003), roots with ;14% porosity only dropped to low

ATP:ADP further than 110 mm from the root–shoot

junction. ATP:ADP of 1.7–2 in the distal parts of these

roots of ;14% porosity (Table 3) were presumably thecomposite of high values in the ‘aerobic cortex’ and low

values in the ‘hypoxic–anoxic stele’. For an anoxic stele

these values were assessed at 2.4–1.8 (see Appendix 1). An

anoxic stele would have a large effect on ATP:ADP in the

whole roots, since stelar cells are more cytoplasmic than

those of the cortex (discussed in relation to ion transport by

de Boer and Volkov, 2003; and based on differences in O2

consumption rates between stele and cortex, Armstrong andBeckett, 1987; Armstrong et al., 2000). Confirmation of

these deductions may be obtained by separating stele from

cortex for determinations of nucleotides, as was successfully

done for the active–inactive state of PDC; i.e. the separation

and liquid N2 freezing of the tissues was fast enough to

prevent the expected rapid metabolic changes (Thomson

and Greenway, 1991).

The above evidence strongly indicates that resistances todiffusion limit O2 availability to the interior of roots when

external O2 decreases below the COPR (Armstrong et al.,

2009); however, an alternative hypothesis has been pro-

posed of the down-regulation of respiration by plant tissues

‘sensing’ declines in O2 availability (Gupta et al., 2009).

This alternative hypothesis, however, appears not to have

accommodated the influence of diffusion limitations causing

declines in O2 consumption rates by tissues when externalO2 is below the COPR. In any case, the comprehensively

documented occurrence of a severely hypoxic–anoxic stele

in maize roots when external O2 declines below the COPR is

adequate to account for the observed decreases in respira-

tion of roots in hypoxic solutions (Atwell et al., 1985). The

consequences of O2 deficiency in the stele, or part thereof,

for ion transport by roots is considered in some detail later

in this review.

Water uptake and possible bypass flow ofnutrients, in O2-deficient roots

The possible inhibition of water transport is considered

before reductions in ion transport, since flow rates of xylemsap can, under certain circumstances, determine the rates of

ion transport to the shoots, for example, in plants of high

ion status a 5-fold decrease in transpiration reduced K+

flow to the shoots by 4-fold (Russell and Barber, 1960).

Effects of hypoxia–anoxia on root hydraulic conductivity(Lp)

Short-term studies, of less than 1 h exposure, show that O2

deficits reduced hydraulic conductivity of wheat root

cortical cells (Lpc) by 45%, while root hydraulic conductiv-

ity for flow to the xylem (Lpr) was reduced by 2–45%,

presumably due to partial closure of aquaporins, either in

the cortical cells or in the endodermis (Bramley et al., 2010).

The partial closures of aquaporins may be part of a more

general reduced permeability of membrane channels under

hypoxia–anoxia, particularly since some aquaporins canalso conduct several other small neutral solutes (Bramley

and Tyerman, 2010). Reduced conductivity of ion channels

has been shown for animal cells (Hochachka, 1991) and

indicated for K+ channels in anoxic rice coleoptiles (Colmer

et al., 2001). Furthermore, transcript abundances of most

aquaporin genes decreased in Arabidopsis exposed to

0.03 mM O2, but transcripts of NIP 2:1 increased (Liu

et al., 2005); of interest since the NIP 2:1 protein mighttransport small metabolites (Bramley and Tyerman, 2010).

Presumably, the NIP protein also increased in abundance,

although neither the turnover rates of aquaporins nor the

functionality of this aquaporin under hypoxic conditions is

known. Whether reductions in Lpr due to closure of

aquaporins persist over longer periods remains uncertain.

Any long-term reductions of Lpr are more likely to be

associated with any of the following factors: cell death,modifications of cell walls, and plugging of the xylem

vessels (Bramley and Tyerman, 2010).

For maize, two studies have assessed Lpr of hypoxic roots

for several hours both using excised roots. One study is

relevant to roots with low gas-filled porosity in waterlogged

soil; i.e. with time the roots became severely hypoxic or

anoxic. In this study, the excised roots showed the first

reductions in Lpr after 5 h of commencement of continuousN2 flushing, when O2 in the solution had decreased below

2 kPa; O2 would have continued to decline to very low

levels and the Lpr remained reduced by ;60% for the last

17 h (Everard and Drew, 1987). A second study is relevant

to aerenchymatous roots in waterlogged soil, since the

excised maize roots were exposed to a steady O2 concentra-

tion of 0.05 mM so that the cortex would have been aerobic

whereas the stele was hypoxic (cf. Fig. 3). These rootsinitially showed a ;25% reduction in Lpr, followed by at

least partial recovery within 1.5–4 h (Gibbs et al., 1998b).

Consistently, in the case of wheat in N2-flushed solution for

14 d, water uptake (root dry weight basis) was not reduced

when compared with aerated controls (Table 2), and was even

50% higher in the N2-flushed treatment during the first 4 d

(Trought and Drew, 1980a). Importantly, most of this flow

must have been via the, putative, hypoxia-intolerant seminalroot system, since at 4 d these roots still contributed ;90% to

the total root mass (Trought and Drew, 1980a).


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In summary, closure of aquaporins during the first few

hours of root zone hypoxia remains interesting, but at

present appears to be of little relevance to longer-term

exposures of roots to hypoxia and anoxia. Longer-term

studies of wheat are needed to confirm these deductions,

and should evaluate possible acclimation in aquaporin

regulation over a few days of hypoxic treatment, for both

seminal and adventitious roots. Furthermore, it would beinteresting to evaluate the cellular responses in the cortex

and stele of the adventitious roots, as these are expected to

differ in O2 supply when in an anoxic medium (aerobic

cortex and hypoxic–anoxic stele can occur, as described

earlier in this review).

Ion transport across roots by mass flow?

Trought and Drew (1980a) proposed that for wheat in N2-

flushed solution, water (and nutrients) might simply enter

via mass flow through damaged roots in response to

transpiration; an assessment based on comparisons of ion

concentrations estimated in the xylem with those in the

external solution (Table 2). We question this notion because

energy-dependent transport of anions is indicated by similarphosphate and higher N in the xylem than in the external

solution (Table 2). The more likely mechanism for the

persisting, albeit greatly reduced, ion uptake by roots of

wheat in N2-flushed nutrient solutions (Table 2) is second-

ary energy-dependent transport via the aerenchymatous

adventitious roots, and through the basal portions of the

seminal roots, which would still receive O2 from the shoots

(Armstrong, 1979; Barrett-Lennard et al., 1988). Applica-tion of inhibitors of respiration to roots in N2-flushed

solutions could be used to test whether the main pathway of

the nutrient uptake is by mass flow, or by energy-dependent

transport via the root portions still receiving O2 from the

shoots. Furthermore, the possible occurrence of mass flow

of external solution (also termed by some ‘by-pass flow’)

can be tested by the addition to the nutrient solution of an

apoplastic tracer such as PTS, as used so effectively instudies of rice (Yeo et al., 1987). By-pass flow was ;0.5% of

total water flow in aerated wheat roots (Garcia et al., 1997),

so any increase in such flows during root hypoxia should be

easily detected.

Nutrient uptake into roots, and transport intothe xylem, during hypoxia

A much larger effect of hypoxia on ion transport to the

xylem than on net uptake into the root cells will be

discussed using the scheme in Fig. 4 and also the crucial

information discussed earlier that, depending on a number

of root characteristics, low external O2 concentrations maylead to severe hypoxia in the stele over much of the root

length, while the cortex and epidermis still receive sufficient

O2 for oxidative phosphorylation.

In fully-aerobic roots, ions taken up from the external

solution are loaded into the xylem with energy provided by

their plasma membrane H+-ATPases with subsequent ion

release into the xylem (de Boer and Volkov, 2003). For

example, K+ flux into the xylem is through SKOR channels;

i.e. highly K+-selective outward rectifying channels (de Boer

and Volkov, 2003; Pang and Shabala, 2010). This process is

also dependent on the accumulation of high K+ in the

xylem parenchyma cells (de Boer and Volkov, 2003), as

supported by X-ray microanalysis, showing particularlyhigh K+ concentrations in the xylem parenchyma cells

bordering the protoxylem and early-maturing metaxylem

vessels (Drew et al., 1990). During stelar hypoxia–anoxia,

H+-ATPases in the xylem parenchyma would be inhibited,

so the sophisticated regulatory mechanism of optimizing

transport to the xylem is disrupted, as SKOR channels

would close. We suggest that ions accumulated by the

epidermis and cortex from the external solution might stillflow via plasmodesmata to the xylem parenchyma by

diffusion, facilitated by cytoplasmic streaming, while ions

in the xylem parenchyma could still be released through

non-selective ion channels, most likely via NORC (non-

selective outward-rectifying channels, de Boer and Volkov,

2003; Pang and Shabala, 2010); these outward rectifying

channels are strongly voltage dependent (Pang and Shabala,

2010). This, albeit reduced ion movement to the xylem,would be facilitated by the depolarization of the membranes

of the xylem parenchyma, favouring passive cation flow

into the xylem vessels. Overall, both the total flow of ions to

the xylem and its K+/Na+ selectivity will be reduced in

hypoxic roots.

Electrochemical gradients for both anions and K+ trans-

fer to the xylem vessels are likely to be favourable. Data on

the effect of decreased O2 in the medium on trans-rootelectrical potential (i.e. electrical potential difference be-

tween external bathing medium and xylem sap) are avail-

able for Plantago, when O2 was lowered to 10 kPa the trans-

root electrical potential became more negative from –20 mV

in aerated roots to –60 mV in the hypoxic roots (de Boer

et al., 1983), presumably because the stele became hypoxic.

During hypoxia, the xylem parenchyma cells are likely to

depolarize to a value less negative than –100 mV; asmeasured for cortical cells of seminal roots of wheat

(–90 mV at 0.008 mM O2, Buwalda et al., 1988b; –80 mV

at 0.02–0.03 mM O2, Zhang and Tyerman, 1997). So the

potential difference still favours anion release, but main-

tains, albeit less pronounced, an electrical gradient favour-

ing K+ fluxes from the xylem into the parenchyma.

Nevertheless, the free energy gradient for K+ fluxes may be

from the parenchyma to the xylem, since the electricalcomponent of the free energy can be more than balanced by

a steep concentration gradient across the xylem parenchyma–

xylem interface. The free energy gradient for diffusive ion

movement between xylem parenchyma and xylem vessels can

be assessed from the flux ratio equation (equation 3.24 in

Nobel, 1974; Jjin/Jj

out¼cjo/(cj

iezjFEM/RT), where Jjin is the influx

of ion j, Jjout is the efflux of ion j, cj

o and cji are outside and

inside concentrations of ion j, e is the exponential, zj is thevalence of ion j, F is Faraday, EM is the membrane potential,

R is the gas constant, and T is the temperature in oK).


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Assuming –100 mV, in the xylem parenchyma cells and –60 mV

in the vessels, this equation shows the free energy gradient for

K+ fluxes would be into the xylem provided the concentration

in the parenchyma cells is at least five-times greater than in the

xylem. That requirement can easily be achieved even at a high

xylem K+ concentration of 10 mM, since K+ in the cytoplasmof xylem parenchyma would probably be 100 mM or higher

(10-fold greater than in the xylem; Drew et al., 1990). The rate

of delivery via this mechanism in roots with a hypoxic–anoxic

stele would, however, be substantially lower than in aerobic

roots, which have xylem loading. Consistent with the view that

transport to the xylem can still occur, even though the loading

process in the xylem parenchyma is inhibited, comes from

a SKOR-minus mutant (i.e. SKOR being a central componentof the loading mechanism); the mutant retained 50% of K+

transport to the shoot compared with the wild type (de Boer

and Volkov, 2003).

The available data for tissue ion concentrations, ion

uptake and xylem transport in roots of cereals (discussed

below) support the argument that xylem loading is inhibited

more than net ion uptake by O2-deficient roots. After 14 d

of N2-flushed treatment, there were only 0–15% decreases of

P or N concentrations in either adventitious or seminal

roots, compared with reductions of about 30% in the shoots

(Buwalda et al., 1988a). These favourable nutrient concen-

trations in the hypoxic roots are not unique for the highexternal concentrations used in the experiment by Buwalda

et al. (1988a). At a lower external K+ of 0.4 mM, K+

concentration in the shoots was reduced by 45% whereas

the reduction was only 20% in adventitious roots, while in

seminal roots K+ increased by ;25% (Kuiper et al., 1994).

Effects on K+:Na+ selectivity will be discussed in a later

section.

Similarly to the reduction for other ions, wheat tissue Cl–

concentrations were also decreased by ;50% in the shoots,

but increased by 20–30% in the seminal roots and by 10–

30% in the adventitious roots, as compared with plants in

aerated solution (Buwalda et al., 1988a, Cl– external at

1 mM). The, albeit, small increases of Cl– concentration in

the adventitious roots are particularly noteworthy since the

Epidermis and cortex

PTA

Xylem parenchyma

Xylem

H+PTA

H+

H+

noitaC

noinA

noinA

ot 021-Vm 061-

Vm 021- ot 02-Vm 04-

Epidermis xetroc dna

PTA

melyXamyhcnerap

melyX

H+

noitaC

noinA

Vm 001-Vm 021- Vm 06-

A Fully-aerobic root B Aerobic epidermis/cortex and anoxic stele

H+noI

aN decudeR +

laveirteraN +

laveirter

]noi[ ]noi[

melyXmelyX melyXamyhcnerap

melyXamyhcnerap

simredipExetroc dna

simredipExetroc dna

.txE.txE

]noi[ lacitehtopyHstoor ssorca stneidarg

snoIsnoI

Endodermis

Endodermis

snoitaCsnoinA &detaG

K+ROKS

CRONCRON

TKHTKH

ROKS

aN +

H+

aN +

H+H+

noinA

H+PTA

H+noI

detaG

Fig. 4. Diagram of ion movements through roots to the xylem, based on de Boer and Volkov (2003), Munns and Tester (2008), and

Pang and Shabala (2010), simplified by us to postulate on the consequences of a hypoxic–anoxic stele (see text). (A; Left half) In aerobic

roots, ATP produced in respiration is available to membrane H+-ATPases, ‘energized membranes’ enable ion transport, so ions are

accumulated to high concentrations in xylem parenchyma and released into the xylem (‘active loading’). (B; Right half) In roots with

anoxic stele but aerobic cortex, active xylem loading is inhibited, and ion movement to the xylem depends upon ion levels accumulated

in the aerobic cortex, with diffusion across the symplast to the xylem facilitated by cytoplasmic streaming. Below each main diagram,

graphs show hypothetical K+ gradients across roots (note: Ext., external solution). Numerous ion transporters and channels are not

shown in this simplified scheme. Channels of particular interest are: (i) SKOR which releases K+ into the xylem (highly selective for K+

over Na+) as part of the loading system, but which would close owing to the inhibition of the H+-ATPase and thus inhibition of energy-

dependent transport in the hypoxic–anoxic stele, and (ii) NCOR a non-selective channel, transporting K+, Na+, and anions, that might be

gated in aerobic cells but could open when membranes depolarize during hypoxia–anoxia. Na+ retrieval from the xylem is also shown via

a HKT-type Na+ uniport (Munns and Tester, 1980); in a hypoxic–anoxic stele although retrieval would be reduced, some could possibly

still occur, but Na+ would then need to move to the aerobic cortex and be compartmentalized in vacuoles (dependent upon trans-

tonoplast H+ difference and Na+/H+ antiport; Munns and Tester, 2008).


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relative growth rates of these roots was only 10% lower

than in the aerated treatment (Barrett-Lennard et al., 1988).

The decreased Cl– concentration in the shoots, despite

increases in the roots, again support the notion that net ion

uptake by the roots is less vulnerable to hypoxia than

transport to the shoots.

The above-described observations have the defect that the

transport to the shoot was through a combination ofadventitious and seminal roots, of different gas-filled

porosity, and the internal O2 concentrations are either

unknown, or based on deductions from measurements

with cylindrical O2 electrodes. Such defects were avoided in

studies using excised primary roots of maize in a flowing

solution at 0.05 mM O2 (Gibbs et al., 1998a; Fig. 3 for

radial O2 profile). Cl– was at 0.4 mM, a suitable concentra-

tion to study energy-dependent anion uptake. Anion uptakefrom low external concentrations must require energy since

both the root cells and the xylem have negative electrical

potentials relative to the medium.

In these excised maize roots the amounts of Cl– trans-

ported to the xylem were, in % of aerated roots, 60% and

30% at 0.05 and 0.02 mM O2, respectively (Gibbs et al.,

1998a). Despite these reductions in total Cl– transport, the

remaining flux still required at least one energy-dependenttransport step between external solution and the xylem, as

shown by the ratios of [Cl–]xylem:[Cl–]medium in the root

exudate at a range of O2 concentrations in the medium of:

93 at 0.27 mM O2, 70 at 0.05 mM O2, and 48 at 0.02 mM

O2, but only 0.6 in anoxia (Gibbs et al., 1998a). These

data are consistent with the development of, at least,

a severely hypoxic stele (Fig. 3), which would inhibit the

xylem loading step in the xylem parenchyma, but maintainCl– accumulation in the epidermis and cortex. For these

excised roots with the only O2 source in the external

solution, sufficient O2 supply to the epidermis is ensured

(Fig. 3).

The above data for Cl– transport in excised maize roots

(Gibbs et al., 1998a) also allow an estimate of residual

transport to the xylem when H+-ATPase activity in the

stele is diminished; i.e. xylem loading is inhibited.A conservative estimate of this residual ion movement is

30% of that in fully aerobic roots; i.e. the value given in the

preceding paragraph for Cl– transport to the xylem at

external O2 of 0.02 mM (as % of aerated roots). High ion

concentrations in the cortex would provide a substantial

diffusion gradient towards the xylem, and since plasmodes-

mata remain conductive in hypoxic roots (Cleland et al.,

1994), cell-to-cell ion movement could occur in the sym-plast, facilitated by cytoplasmic streaming. Further testing

of the hypothesis on the changes in uptake by the root cells

and transport to the xylem, when the stele becomes

hypoxic–anoxic, would include measuring the initial unidi-

rectional Cl– influx over the first 60 min after supply of36Cl– as this represents the influx into the cytoplasm (cf.

Tregeagle et al., 2010); the hypothesis predicts this flux

would not be greatly affected by changing O2 between 0.27mM and 0.02 mM in the flowing medium bathing excised

roots.

K+:Na+ selectivity

Stelar hypoxia might, in addition to decreasing nutrient ion

fluxes to the shoots, also impact on K+:Na+ selectivity of

transport to the shoots. The present review only considers

K+:Na+ selectivity for external Na+ at 10 mM or lower (i.e.

low concentrations not considered as saline). In hypoxia–

anoxia, net K+:Na+ selectivity to shoots is reduced and, as

will be shown shortly, this is partly due to a substantial

increase of Na+ flow to the shoots. This increase occurs,

even though Na+ in aerated solutions is thought to be

transported to the xylem by a SOS1 antiport (Munns and

Tester, 2008), which presumably gets inhibited during O2

deficiency. We suggest any decrease in intake via this

antiport is more than compensated for by opening of the

non-selective NORC channels, as these open due to the

strong membrane depolarization of xylem parenchyma cells

(Pang and Shabala, 2010). Transport to the shoot would be

mitigated by Na+ retrieval from the xylem and this

possibility will also be discussed.

For wheat in external K+ and Na+ both at 0.4 mM, shoot

K+:Na+ decreased from 150 to 4.5 after 18 d in N2-flushed

solution, while adventitious root K+:Na+ decreased from 10

to 2; both these declines were mainly due to increased Na+

(Kuiper et al., 1994). Similar decreases in K+:Na+ were

found for 10–15-d-old maize; for example, at 0.025 mM O2

in the external solution with K+ at 1 mM and Na+ at

10 mM, the ratio decreased from 96 to 7 in the shoots, but

only from 19 to 9.5 in the roots (Drew and Lauchli, 1985).

At first sight it seems enigmatic that substantial decreases

in K+:Na+ occur for the aerenchymatous roots of wheat, as

described in Kuiper et al. (1994); i.e. for ion pools which are

presumably mainly located in the ‘aerobic cortex’. However,

it is possible that a substantial portion of the Na+ pool in

the cortical cells was associated with retrieval of Na+ from

the xylem. Since the cortical cells would have sufficient

energy to sequester the Na+ into vacuoles, the crucial

question is whether retrieval from the xylem sap is credible

in these hypoxic roots. In roots with a substantial length of

‘aerobic’ stele towards the root–shoot junction (as indicated

by models, Armstrong and Beckett, 1987; and shown in

Darwent et al., 2003), Na+ retrieval from the xylem would

presumably be important in mitigating Na+ flow to leaves

(Johanson and Cheeseman, 1983), most likely via HKT

transporters of subfamily 1 which function as a Na+ uniport

(Munns and Tester, 2008). In most cases, where the root

portions near the root–shoot junction still receives sufficient

O2 to maintain oxidative phosphorylation in the stele as

well as in the cortex, the driving force for Na+ retrieval

would be very strong; taking –120 mV in the stelar

parenchyma cells and –20 mV in the xylem of these upper

portions receiving O2, the flux ratio equation (Nobel, 1974)

indicates that Na+ retrieval would stop only when the Na+

concentration in the xylem parenchyma was 52 times higher

than in the xylem. With an anoxic stele, potential for Na+

retrieval from the xylem via a HKT uniport is weaker; yet

still likely, since the negative potential relative to the xylem

of –40 mV (de Boer et al., 1983), will still permit a 5-fold


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higher Na+ concentration in the xylem parenchyma than in

the xylem before retrieval would cease. In xylem sap of

barley, expressed from the cut end of the shoots of control

plants grown in supported hydroponics without Na+ added

(i.e. ‘background’ Na+ not defined), Na+ concentrations

were about 2–3 mM (Shabala et al., 2010) and these

concentrations would presumably decrease when there

was rapid transpiration. So, taking reasonable concentra-tions of 0.5–2.5 mM Na+ in the xylem, Na+ retrieval

would cease only when the symplast concentration reached

2.5–12.5 mM, these values are above the expected level if

Na+ withdrawn from the xylem moves to the cortical cells

and is sequestered into vacuoles. This suggestion needs

further testing with focused experiments to elucidate for

hypoxic roots whether the Na+ accumulated is derived

straight from the medium, or by entry and subsequentretrieval of Na+ from the xylem.

Summing up, the data reviewed here on O2 profiles,

respiration, and ion transport in roots, some of which still

need more rigorous testing, are all consistent with the

dominant influence of development of a hypoxic–anoxic

stele under hypoxia, rather than the result of down-

regulation of metabolism involving an O2-sensing system

(as suggested by Geigenberger, 2003; Gupta et al., 2009).The one observation which does not fit well with the model

of an ‘aerobic cortex’ and ‘hypoxic–anoxic stele’ is the

depolarization by 34 mV in the cortical cells of wheat upon

exposure to 0.02–0.03 mM O2 (Zhang and Tyerman, 1997),

when these cells can be expected to be still ‘aerobic’, i.e.

depolarization in the still aerobic cortex could be used as an

argument in favour of the down-regulation of metabolism.

However, depolarization in the cortex might result from‘electrical coupling’, as the expected depolarization in the

stele might be conveyed to cortical cells. This is possible

even though the coupling ratio between cortical cells in

the wheat roots was only 6% (Zhang and Tyerman, 1997),

as the possible quantitative importance of coupling can not

be readily gauged from the coupling ratio alone (Solocar,

1977).

Ion uptake by seminal and adventitious rootsof wheat in O2-deficient media

Ion uptakes by seminal and adventitious roots of wheat inN2-flushed versus aerated solutions were measured sepa-

rately, in split-root experiments (Kuiper et al., 1994). The

net uptakes (root fresh weight basis), expressed as a percent-

age of the aerated controls, were 50% for K+ and 35% for

NO3� in seminal roots, and 82% and 55% in adventitious

roots (average for 26 d and 33 d in N2-flushed solution;

Kuiper et al., 1994). This difference between root types is

less than expected in view of the much larger gas-filledporosity of 14% in the adventitious roots than the 3% in

seminal roots (reported for the same cultivar by Barrett-

Lennard et al., 1988). The seminal roots presumably

benefited from the low O2 concentration of 0.003 mM in

the N2-flushed solution, probably enabling respiration at

least in the epidermis and thus ion uptake by this outermost

cell layer. By contrast, when in stagnant agar nutrient

solution, the lack of turbulence would negate O2 supply

from the nutrient solution and hence the internal O2 supply

will solely depend on diffusion via the aerenchyma, then

net uptake of K+ and P by seminal roots (root fresh

weight basis), never exceeded 20% of the aerated controls

(Wiengweera and Greenway, 2004). This low uptakeoccurred even though K+ concentration in the shoots would

have been 35% lower than in the aerated plants

(Wiengweera et al., 1997). By contrast, in the adventitious

roots, O2 supply to the epidermis was demonstrated by

ROL (Barrett-Lennard et al., 1988) and, accordingly, net

K+ uptakes by the adventitious roots, as a percentage of the

aerated controls, were 70% between 4–8 d and 115% during

the final 8–12 d (Wiengweera and Greenway, 2004). Thus,overall, in media without any O2 supplied externally,

adventitious roots are capable of greater ion transport than

seminal roots, presumably owing to the higher porosity

enabling more O2 movement from the shoot to the root to

sustain respiration in the adventitious roots.

Surprisingly, measurements of O2 and ion uptake rates

for the same roots, at various O2 supply, are generally

lacking. The exception being the pioneering study of barleyseedling roots in hypoxia (0.02 mM O2, 0.05% agar

solution) by Pang et al. (2006) using ion-specific and O2

microelectrodes. At 10 mm behind the tip of 70 mm long

roots, a position just behind the elongation zone, O2 influx

was reduced by 80% in a hypoxia-tolerant cultivar and by

93% in a hypoxia-intolerant cultivar, as compared with

rates when bulk medium O2 was 0.28 mM (Pang et al.,

2006). It is not clear why the varieties differed in O2 uptake;root lengths and diameters were the same, and although

root porosity values were not given, potential differences in

internal O2 supply from the shoots were probably not

a factor, since excised roots showed similar responses as

intact roots (Pang et al., 2006). No response curve was

determined for O2 uptake versus O2 concentration.

Net K+ uptake from 0.2 mM external K+ concentration

(also at 10 mm behind the root tip) was reduced by hypoxiaby 35–65% in the intolerant cultivar, but was not affected in

the tolerant cultivar (Pang et al., 2006). K+ net uptake by

this tolerant cultivar at 0.02 mM O2 was 2.1 lmol g�1 fresh

weight h�1, essentially the same as for seminal roots of

wheat in turbulent solutions at 0.003 mM O2 (Kuiper et al.,

1994); the Pang et al. (2006) data were converted by us from

surface area to a fresh weight basis (average of Figs 6 and 7

in Pang et al., 2006). The persistence of a reasonably highnet uptake in the barley was despite a reduction of 80% of

O2 uptake in stagnant solution at 0.02 mM external O2

compared with that by roots in stagnant air-saturated

solution. Net H+ efflux in the mature zone was changed

into a net influx in the presence of vanadate, the inhibitor of

the plasma membrane H+-ATPase. Pang and Shabala

(2010) discussed these findings as being consistent with the

possible engagement of the H+-ATPase under anoxia, citingthe case for anoxic maize root tips (Xia and Roberts, 1996).

However, the two situations are quite different, the barley


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roots received O2 (see above) whereas the maize root tips

were in anoxia. O2 uptake by the barley roots would have

enabled oxidative phosphorylation in the epidermis and

perhaps also in the outer cortical cells, so accordingly, ATP

should have been available to the H+-ATPases in the root

periphery. Although the outer cell layers would have

received some O2, cells towards the interior would pre-

sumably have been severely hypoxic or even anoxic.

Recovery of nutrient transport inO2-deficient roots upon re-aeration

Recovery of nutrient uptake and transport to the xylem

upon re-aeration following anaerobiosis has been evaluated

on only a few occasions. To our knowledge, only one

experiment has evaluated net ion uptake in the first hours

after re-aeration of previously hypoxic wheat roots (Kuiper

et al., 1994); in this experiment it took ; 4 h of re-aeration

before net K+ uptake accelerated for both seminal and

adventitious roots. These lags indicate that the membranetransport processes in the roots exposed to anaerobic

solution were impaired, and not simply suffering a simple

energy limitation, so that repairs were needed; whether the

lags were associated with general metabolism (e.g. lag in

recovery of mitochondria), to membranes, or to the ion

uptake machinery, remains to be elucidated. The lag in re-

covery following re-aeration might be associated with the

production of oxygen radicals after re-aeration, for exam-ple, 1–10 mM H2O2 caused pronounced membrane de-

polarization and massive K+ effluxes in roots of four barley

cultivars (Chen et al., 2007). Kuiper et al. (1994) also

assessed the recovery of K+ and NO3� net uptakes during

the first 23 h of re-aeration; rates were 1.3–3.5-fold higher

for both seminal and adventitious roots, compared with

roots that continued in the N2-flushed solution.

In contrast to wheat, roots of rice grown in N2-flushedsolution had, at most, minor reductions in ion uptake

compared with continuously aerated plants (John et al.,

1974). Somewhat unexpectedly, upon transfer of rice from

N2-flushed to aerated solutions during the first 2 h there

were substantial ‘overshoots’ (i.e. increases above rates in

continuously aerated plants) for P, Cl–, and K+ uptakes by

roots and transport to the shoots (John et al., 1974; rates

determined by tracer uptake). Confirmation of theseresponses is needed using more refined techniques, but

the ‘overshoots’ imply that, in rice, there may be acclimative

changes in nutrient-transport systems, in addition to the

well-known enlargement of aerenchyma.

More studies of recovery following re-aeration are

needed, using techniques with high temporal resolution

(e.g. unidirectional influxes using isotopes cf. Davenport

et al., 2005; ion-specific microelectrodes, cf. Pang et al.,2006). Use of elevated O2 around the shoots would enable

recovery to be assessed with time-course measurements

continuing in stagnant agar (i.e. maintaining the same

conditions external to the roots, especially the unstirred

medium for the microelectrode net flux measurements). No

experiments to examine the influence of improved internal

aeration on nutrient uptake by roots of cereals are avail-

able, but in young alder trees illumination of the stem

improved O2 supply to the roots (Armstrong and

Armstrong, 2005b); and the improved O2 status restored

net uptake of NO3�, P and K+ by roots in N2-flushed

solution above the rates in aerated solution (Grosse and

Meyer, 1992). The improvements due to improved O2

supply in this experiment with alder seem in little doubt,

but the extent of the restoration of nutrient uptake remains

uncertain because of the high variability of the data by

Grosse and Meyer (1992).

Conclusions and perspectives

Improved root growth and functioning will be required to

alleviate shoot nutrient deficiencies, a main cause for

reduced growth of wheat, barley, and maize in anaerobic

solutions and very likely also in many waterlogged soils.

Improved root functioning should also aid tolerance of ‘soiltoxins’ in anaerobic soils, such as reduced metal ions and

salinity, as well as promote recovery following transient

waterlogging. Priority traits for improving the performance

of wheat roots in waterlogged soils would be an ability to

develop aerenchyma in seminal roots to prevent their death,

as well as a larger volume of more efficient aerenchyma in

adventitious roots. Another beneficial trait would be cubic

cell packing in the root cortex to increase porosity of non-aerenchymatous tissue, as occurs in rice, whereas the three

dryland cereals have hexagonal cell packings (few genotypes

examined). Stelar hypoxia, which inhibits the sophisticated

system of xylem loading, may not be alleviated merely by

more aerenchyma but could be avoided with a smaller

radius stele. In addition to improved internal aeration,

other traits might also improve the functioning of hypoxic

roots, such as protection against free radicals of oxygen.Oxidative damage might occur after cell damage (Smirnoff,

1995), so lipid peroxidation associated with membrane

damage is possibly a secondary effect, nevertheless, studies

of animal cells indicate that radicals formed can further

accelerate the pathology (Arthur et al., 2008). So antioxi-

dant systems should have ameliorating effects and might be

important during recovery, which would be particularly

relevant to survival following the transient waterlogging ofseminal roots of low gas-filled porosity. Genetic differences

in reactive-oxygen scavenging systems were important

during the recovery of rice following de-submergence (Ella

et al., 2003). A broad programme exploiting differences

within and across species is needed to combine the necessary,

complex characteristics; trait screening, physiological-based

breeding and selection including development and use of

molecular markers (Setter and Waters, 2003), and, wherepossible, wide hybridizations and cytogenetic approaches to

make use of tolerant ‘wild’ relatives (Colmer et al., 2006).

One of the exciting challenges for future physiological

research is to elucidate more fully the functioning of roots

with the intriguing combination of an aerobic cortex


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(epidermis and cortex with sufficient O2 for oxidative

phosphorylation) and a hypoxic–anoxic stele. The persis-

tence of substantial, albeit reduced, ion transport to the

xylem of roots with a hypoxic–anoxic stele might be viewed

as a primitive system before evolution of the sophisticated

system of xylem loading. The postulated different concen-

tration gradients between epidermis and xylem may be

tested using X-ray microanalysis (e.g. for salinity, seeLauchli et al., 2008). Further, physiological co-operation of

the aerobic cortex and hypoxic–anoxic stele seems likely,

such as consumption in the cortex of products of anaerobic

metabolism in the stele and, crucially, a possible transfer of

high energy compounds produced in the aerobic cortex to

the hypoxic–anoxic stele (suggested by Gibbs and Green-

way, 2003). Physiological experiments need to be more

focused and less descriptive, using some techniques whichhave already shown promise, such as measurements of both

net uptake by the root cells and transport to the xylem,

while measuring the trans-root electrical potential and O2

profiles across roots. Further net flux measurements along

roots, using ion-selective microelectrodes with concurrent

O2 fluxes also measured, would contribute to the elucida-

tion of ion transport and K+:Na+ selectivity as affected by

hypoxia. Recovery following re-aeration (or elevation of O2

supply around shoots) should also be included in future

experiments, with measurements taken as soon as possible

after the O2 supply is increased. Such experiments will

address whether transport activity is merely limited by low

energy associated with low O2 supply, or also by other

lesions in metabolic capacity including transport capacity or

loss of membrane integrity.

Acknowledgements

For incisive criticisms on a final draft: Sergey Shabala for

the whole review; Helen Bramley for the water transport

section; Bill Armstrong on the hypoxic–anoxic stele; and

Natasha Teakle for useful comments on the whole review.The Grains Research and Development Corporation in

Australia is thanked for supporting research by TDC on

waterlogging tolerance in wheat and wild species in the

Triticeae.

Appendix

Here we assess that the ratio of ATP-to-ADP (i.e. ATP:ADP) ina mature root segment of maize, with an anoxic stele and ‘aerobiccortex’ that still receives sufficient O2 for oxidative phosphoryla-tion, will be in the range 2.4 to 1.8. This assessment is based on thefollowing values for maize: (i) proportion of the root occupied bystele and cortex are 0.24 and 0.76 (Gibbs et al., 1998); (ii)ATP:ADP in aerobic tissues of 5.5 and in anoxic tissues of 0.66(Drew et al., 1985). These values are for roots exposed to 1 mMNaF, since Drew et al. (1985) used this inhibitor of glycolysis inaddition to an anoxic medium in their studies with roots of intactplants; (iii) assumed values for proportions of the nucleotide poolcontained in the cortex and stele, based on their proportionalvolumes and expected differences in cytoplasm content. As anexample, we assume 5.5-times higher nucleotide concentration in

the stele than in the cortex plus epidermis, a value based on theratio of O2 uptake rates in the stele versus the cortex (Armstrongand Beckett, 1987). Then, the fraction of the nucleotide pool in thecortex plus epidermis¼(0.7631)/[(0.7631)+(0.2435.5)]¼0.36, andfor the stele¼(0.2435.5)/[(0.7631)+(0.2435.5)]¼0.64. Thus theATP:ADP in a root segment as a whole with a hypoxic–anoxicstele, becomes (0.3635.5)+(0.6430.66)¼2.4. The assessment of thenucleotide concentration in stele versus cortex is not sure,however, even if we take the unlikely high value of 10-times higherconcentration in the stele than cortex, and repeating the calcula-tion above, then the ATP:ADP is 1.8; i.e., not all that much lowerthan when assuming a 5.5-times difference.

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