Tolerance of roots to low oxygen: 'Anoxic' cores, the phytoglobin-nitric oxide cycle, and energy or oxygen sensing.

Acclimation by plants to hypoxia and anoxia is of importance in various ecological systems, and especially for roots in waterlogged soil. We present evidence for acclimation by roots via 'anoxic' cores rather than being triggered by O2 sensors. The evidence for 'anoxic' cores comes from radial O2 profiles across maize roots and associated metabolic changes such as increases in the 'anaerobic enzymes' ADH and PDC in the 'anoxic' core, and inhibition of Cl- transport to the xylem. These cores are predicted to develop within 15-20 min after sudden transfer of a root to hypoxia, so that the cores are 'anoxically-shocked'. We suggest that 'anoxic' cores could emanate a signal(s), such as ACC the precursor of ethylene and/or propagation of a 'Ca2+ wave', to other tissue zones. There, the signalling would result in acclimation of the tissues to energy crisis metabolism. An O2 diffusion model for tissues with an 'anoxic' core, indicates that the phytoglobin-nitric oxide (Pgb-NO) cycle would only be engaged in a thin 'shell' (annulus) of tissue surrounding the 'anoxic' core, and so would only contribute small amounts of ATP on a whole organ basis (e.g. whole roots). A key feature within this annulus of tissue, where O2 is likely to be limiting, is that the ratio (ATP formed) / (O2 consumed) is 5-6, both when the NAD(P)H of glycolysis is converted to NAD(P)+ by the Pgb-NO cycle or by the TCA cycle linked to the electron transport chain. The main function of the Pgb-NO cycle may be the modulating of NO levels and O2 scavenging, thus preventing oxidative damage. We speculate that an 'anoxic' core in hypoxic plant organs may have a particularly high tolerance to anoxia because cells might receive a prolonged supply of carbohydrates and/or ATP from the regions still receiving sufficient O2 for oxidative phosphorylation. Severely hypoxic or 'anoxic' cores are well documented, but much research on responses of roots to hypoxia is still based on bulk tissue analyses. More research is needed on the interaction between 'anoxic' cores and tissues still receiving sufficient O2 for oxidative phosphorylation, both during a hypoxic exposure and during subsequent anoxia of the tissue/organ as a whole.

Energy-crises in well-aerated and anoxic tissue: does tolerance require the same specific proteins and energy-efficient transport?

Many of the profound changes in metabolism that are caused by O2 deficiency also occur in well-aerated tissues when oxidative phosphorylation is partially or wholly inhibited. For these well-aerated tissues, reduction in energy formation occurs during exposure to inhibitors of oxidative phosphorylation, cold/chilling and wounding, so we prefer the term 'energy crisis' metabolism over 'anaerobic' metabolism. In this review, we note that the overwhelming body of data on energy crises has been obtained by exposure to hypoxia-anoxia, which we will indicate when discussing the particular experiments. We suggest that even transient survival of an energy crisis requires a network of changes common to a large number of conditions, ranging from changes in development to various adverse conditions such as high salinity, drought and nutrient deficiency, all of which reduce growth. During an energy crisis this general network needs to be complemented by energy specific proteins, including the so called 'anaerobic proteins' and the group of ERFVII transcription factors, which induces the synthesis of these proteins. Crucially, the difference between anoxia-intolerant and -tolerant tissues in the event of a severe energy crisis would mainly depend on changes in some 'key' energy crisis proteins: we suggest these proteins would include phytoglobin, the V-H+PPiase and pyruvate decarboxylase. A second characteristic of a high tolerance to an energy crisis is engagement of energy efficient transport. This feature includes a sharp reduction in rates of solute transport and use of energy-efficient modifications of transport systems by primary H+ transport and secondary H+-solute transport systems. Here we also discuss the best choice of species to study an energy crisis. Further, we consider confounding of the acclimative response by responses to injury, be it due to the use of tissues intolerant to an energy crisis, or to faulty techniques.

Energetics of acclimation to NaCl by submerged, anoxic rice seedlings.

BACKGROUND AND AIMS: Our aim was to elucidate how plant tissues under a severe energy crisis cope with imposition of high NaCl, which greatly increases ion fluxes and hence energy demands. The energy requirements for ion regulation during combined salinity and anoxia were assessed to gain insights into ion transport processes in the anoxia-tolerant coleoptile of rice. METHODS: We studied the combined effects of anoxia plus 50 or 100 mm NaCl on tissue ions and growth of submerged rice (Oryza sativa) seedlings. Excised coleoptiles allowed measurements in aerated or anoxic conditions of ion net fluxes and O2 consumption or ethanol formation and by inference energy production. KEY RESULTS: Over 80 h of anoxia, coleoptiles of submerged intact seedlings grew at 100 mm NaCl, but excised coleoptiles, with 50 mm exogenous glucose, survived only at 50 mm NaCl, possibly due to lower energy production with glucose than for intact coleoptiles with sucrose as substrate. Rates of net uptake of Na+ and Cl- by coleoptiles in anoxia were about half those in aerated solution. Ethanol formation in anoxia and O2 uptake in aerobic solution were each increased by 13-15 % at 50 mm NaCl, i.e. ATP formation was stimulated. For acclimation to 50 mm NaCl, the anoxic tissues used only 25 % of the energy that was expended by aerobic tissues. Following return of coleoptiles to aerated non-saline solution, rates of net K+ uptake recovered to those in continuously aerated solution, demonstrating there was little injury during anoxia with 50 mm NaCl. CONCLUSION: Rice seedlings survive anoxia, without the coleoptile incurring significant injury, even with the additional energy demands imposed by NaCl (100 mm when intact, 50 mm when excised). Energy savings were achieved in saline anoxia by less coleoptile growth, reduced ion fluxes as compared to aerobic coleoptiles and apparent energy-economic ion transport systems.

Efficient use of energy in anoxia-tolerant plants with focus on germinating rice seedlings.

Anoxia tolerance in plants is distinguished by direction of the sparse supply of energy to processes crucial to cell maintenance and sometimes to growth, as in rice seedlings. In anoxic rice coleoptiles energy is used to synthesise proteins, take up K(+) , synthesise cell walls and lipids, and in cell maintenance. Maintenance of electrochemical H(+) gradients across the tonoplast and plasma membrane is crucial for solute compartmentation and thus survival. These gradients sustain some H(+) -solute cotransport and regulate cytoplasmic pH. Pyrophosphate (PPi ), the alternative energy donor to ATP, allows direction of energy to the vacuolar H(+) -PPi ase, sustaining H(+) gradients across the tonoplast. When energy production is critically low, operation of a biochemical pHstat allows H(+) -solute cotransport across plasma membranes to continue for at least for 18 h. In active (e.g. growing) cells, PPi produced during substantial polymer synthesis allows conversion of PPi to ATP by PPi -phosphofructokinase (PFK). In quiescent cells with little polymer synthesis and associated PPi formation, the PPi required by the vacuolar H(+) -PPi ase and UDPG pyrophosphorylase involved in sucrose mobilisation via sucrose synthase might be produced by conversion of ATP to PPi through reversible glycolytic enzymes, presumably pyruvate orthophosphate dikinase. These hypotheses need testing with species characterised by contrasting anoxia tolerance.

Rice alcohol dehydrogenase 1 promotes survival and has a major impact on carbohydrate metabolism in the embryo and endosperm when seeds are germinated in partially oxygenated water.

BACKGROUND AND AIMS: Rice (Oryza sativa) has the rare ability to germinate and elongate a coleoptile under oxygen-deficient conditions, which include both hypoxia and anoxia. It has previously been shown that ALCOHOL DEHYDROGENASE 1 (ADH1) is required for cell division and cell elongation in the coleoptile of submerged rice seedlings by means of studies using a rice ADH1-deficient mutant, reduced adh activity (rad). The aim of this study was to understand how low ADH1 in rice affects carbohydrate metabolism in the embryo and endosperm, and lactate and alanine synthesis in the embryo during germination and subsequent coleoptile growth in submerged seedlings. METHODS: Wild-type and rad mutant rice seeds were germinated and grown under complete submergence. At 1, 3, 5 and 7 d after imbibition, the embryo and endosperm were separated and several of their metabolites were measured and compared. KEY RESULTS: In the rad embryo, the rate of ethanol fermentation was halved, while lactate and alanine concentrations were 2·4- and 5·7- fold higher in the mutant than in the wild type. Glucose and fructose concentrations in the embryos increased with time in the wild type, but not in the rad mutant. The rad mutant endosperm had lower amounts of the α-amylases RAMY1A and RAMY3D, resulting in less starch degradation and lower glucose concentrations. CONCLUSIONS: These results suggest that ADH1 is essential for sugar metabolism via glycolysis to ethanol fermentation in both the embryo and endosperm. In the endosperm, energy is presumably needed for synthesis of the amylases and for sucrose synthesis in the endosperm, as well as for sugar transport to the embryo.

Tolerance of submerged germinating rice to 50-200 mM NaCl in aerated solution.

This paper concerns tolerance to 50-200 mM NaCl of submerged rice (Oryza sativa cv. Amaroo) during germination and the first 138-186 h of development in aerated solution. Rice was able to germinate and the seedlings even tolerated exposure to 200 mM NaCl, albeit with severe growth restrictions. After return to 0.3 mM NaCl, growth increased, indicating that even at 200 mM NaCl there was no irreparable injury. Osmotic adjustment was achieved by using Na⁺ and Cl⁻ as the major osmotica. At 200 mM NaCl commenced at sowing, the shoot Na⁺ and Cl⁻ concentrations between 50-110 h were about 210 and 260 mM, respectively, i.e. above the external concentration. Thus, there was a high tissue tolerance to NaCl. The internal concentrations declined subsequently, concurrent with a decline in growth. At 50-200 mM NaCl, the contributions from ions to πsap were 81-92% in roots and 62-74% in shoots. The assessed turgor pressures at 200 mM NaCl were 0.33 MPa in shoots and 0.15 MPa in roots, compared to 0.62 and 0.43 MPa at 0.3 mM NaCl. In the General Discussion section, we compare the different responses of submerged seedlings to the responses of transpiring rice plants, reported in the literature, and suggest that the submerged system is useful to evaluate effects of NaCl on turgor pressure and particularly to establish whether there are specific effects of Na⁺ and Cl⁻ in tissues.

pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NOFormula.

During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H(+) influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K(+) efflux was continuous. Further experiments used excised coleoptile tips (7-10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO(3)(-), which distinguished two processes involved in pH regulation. Net H(+) influx (µmol g(-1) fresh weight h(-1)) for coleoptiles with NO(3)(-) was ∼1.55 over the first 24 h, being about twice that in the absence of NO(3)(-), but then decreased to 0.5-0.9 as net NO(3)(-) uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H(+)-NO(3)(-) symports. NO(3)(-) reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K(+) balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO(3)(-) supply. Thus, biochemical pHstats and reduced net H(+) influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5.

Ion transport in seminal and adventitious roots of cereals during O2 deficiency.

O(2) 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 N(2)-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 O(2) 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 O(2) 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 nutrient acquisition 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.

Regulation of intracellular pH during anoxia in rice coleoptiles in acidic and near neutral conditions.

Rice coleoptiles, renowned for anoxia tolerance, were hypoxically pretreated, excised, 'healed', and then exposed to a combination of anoxia and pH 3.5. The putative acid load was confirmed by net effluxes of K(+) to the medium, with concurrent net decreases of H(+) in the medium, presumably mainly due to H(+) influx. Yet the coleoptiles survived the combination of anoxia and pH 3.5 for at least 90 h, and even for at least 40 h when the energy crisis, inherent to anoxia, had been aggravated by supplying the coleoptiles with 2.5 mM rather than 50 mM glucose. Even in the case of coleoptiles with 2.5 mM glucose, an accumulation ratio of 6 for Cl(-) was attained at 4 h after the start of re-aeration, implying plasma membrane integrity was either maintained during anoxia, or rapidly restored after a return to aerated conditions. Cytoplasmic pH and vacuolar pH were measured using in vivo (31)P nuclear magnetic resonance spectroscopy with 50 mM glucose in the basal perfusion medium. After 60 h in anoxia, external pH was suddenly decreased from 6.5 to 3.5, but cytoplasmic pH only decreased from 7.35 to 7.2 during the first 2 h and then remained steady for the next 16 h. During the first 3 h at pH 3.5, vacuolar pH decreased from 5.7 to 5.25 and then stabilized. After 18 h at pH 3.5, the initial values of cytoplasmic pH and vacuolar pH were rapidly restored, both upon a return to pH 6.5 while maintaining anoxia and after subsequent return to aerated solution. Summing up, rice coleoptiles exposed to a combination of anoxia and pH 3.5 retained pH regulation and cellular compartmentation, demonstrating tolerance to anoxia even during the acid load imposed by exposure to pH 3.5.

Conditions leading to high CO2 (>5 kPa) in waterlogged-flooded soils and possible effects on root growth and metabolism.

AIMS: Soil waterlogging impedes gas exchange with the atmosphere, resulting in low P(O2) and often high P(CO2). Conditions conducive to development of high P(CO2) (5-70 kPa) during soil waterlogging and flooding are discussed. The scant information on responses of roots to high P(CO2) in terms of growth and metabolism is reviewed. SCOPE: P(CO2) at 15-70 kPa has been reported for flooded paddy-field soils; however, even 15 kPa P(CO2) may not always be reached, e.g. when soil pH is above 7. Increases of P(CO2) in soils following waterlogging will develop much more slowly than decreases in P(O2); in soil from rice paddies in pots without plants, maxima in P(CO2) were reached after 2-3 weeks. There are no reliable data on P(CO2) in roots when in waterlogged or flooded soils. In rhizomes and internodes, P(CO2) sometimes reached 10 kPa, inferring even higher partial pressures in the roots, as a CO2 diffusion gradient will exist from the roots to the rhizomes and shoots. Preliminary modelling predicts that when P(CO2) is higher in a soil than in roots, P(CO2) in the roots would remain well below the P(CO2) in the soil, particularly when there is ventilation via a well-developed gas-space continuum from the roots to the atmosphere. The few available results on the effects of P(CO2) at > 5 kPa on growth have nearly all involved sudden increases to 10-100 kPa P(CO2); consequently, the results cannot be extrapolated with certainty to the much more gradual increases of P(CO2) in waterlogged soils. Nevertheless, rice in an anaerobic nutrient solution was tolerant to 50 kPa CO2 being suddenly imposed. By contrast, P(CO2) at 25 kPa retarded germination of some maize genotypes by 50%. With regard to metabolism, assuming that the usual pH of the cytoplasm of 7.5 was maintained, every increase of 10 kPa CO2 would result in an increase of 75-90 mM HCO3(-) in the cytoplasm. pH maintenance would depend on the biochemical and biophysical pH stats (i.e. regulatory systems). Furthermore, there are indications that metabolism is adversely affected when HCO3(-) in the cytoplasm rises above 50 mM, or even lower; succinic dehydrogenase and cytochrome oxidase are inhibited by HCO3(-) as low as 10 mM. Such effects could be mitigated by a decrease in the set point for the pH of the cytoplasm, thus lowering levels of HCO3(-) at the prevailing P(CO2) in the roots. CONCLUSIONS: Measurements are needed on P(CO2) in a range of soil types and in roots of diverse species, during waterlogging and flooding. Species well adapted to high P(CO2) in the root zone, such as rice and other wetland plants, thrive even when P(CO2) is well over 10 kPa; mechanisms of adaptation, or acclimatization, by these species need exploration.

Manipulation of ethanol production in anoxic rice coleoptiles by exogenous glucose determines rates of ion fluxes and provides estimates of energy requirements for cell maintenance during anoxia.

Ethanol production by anoxic, excised, 7-10 mm tips of rice coleoptiles was manipulated using a range of exogenous glucose concentrations. Such a dose-response curve enabled good estimates at which level of ethanol production (and hence by inference ATP production), injury commenced and also allowed assessments of energy requirements for maintenance in anoxia. Rates of net uptake or loss of K+ and P by these excised coleoptile tips were related to rates of ethanol production (r2 of 0.59 and 0.68, respectively). At 72 h anoxia, ATP levels in excised tips were similar at 0, 2.5, and 50 mol m(-3) exogenous glucose, despite large differences in the inferred rates of ATP production. At 96 h anoxia, tips without exogenous glucose had low ATP concentrations; these may be the cause or the consequence of cell injury. In tips without glucose, injury was indicated by losses of K+ and Cl- between 72-96 h anoxia, and during the first hour after re-aeration, while later than 1 h after re-aeration, rates of net uptake were substantially lower than for re-aerated tips previously in anoxia with exogenous glucose. Between 96 h and 124 h anoxia, ion losses from tips without exogenous glucose increased while recovery of net uptake after re-aeration was very sluggish and incomplete. The energy requirement for maintenance of health and survival of anoxic coleoptile tips, expressed on a fresh weight basis, was lower than for three other anoxia-tolerant plant tissues/cells, studied previously. However, the energy requirement on a protein basis was assessed at 1.4 micromol ATP mg(-1) protein h(-1) and this value is 2.6-5.4-fold higher than for the other plant tissues/cells. Yet, this requirement was still only 58-88% of the published values for aerated tissues. The reason for this relatively high ATP requirement per unit protein in anoxic rice coleoptiles remains to be elucidated.

Protein synthesis by rice coleoptiles during prolonged anoxia: implications for glycolysis, growth and energy utilization.

BACKGROUND AND AIMS: Anoxia-tolerant plant tissues synthesize a number of proteins during anoxia, in addition to the 'classical anaerobic proteins' involved in glycolysis and fermentation. The present study used a model system of rice coleoptile tips to elucidate patterns of protein synthesis in this anoxia-tolerant plant tissue. METHODS: Coleoptile tips 7-11 mm long were excised from intact seedlings exposed to anoxia, or excised from hypoxically pre-treated seedlings and then exposed to anoxia for 72 h. Total proteins or 35S-labelled proteins were extracted, separated using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis and analysed using mass spectrometry. KEY RESULTS: The coleoptile tips excised after intact seedlings had been exposed to anoxia for 72 h had a similar proteome to tips that were first excised and then exposed to anoxia. After 72 h anoxia, Bowman-Birk trypsin inhibitors and a glycine-rich RNA-binding protein decreased in abundance, whereas a nucleoside diphosphate kinase and several proteins with unknown functions were strongly enhanced. Using [35S]methionine as label, proteins synthesized at high levels in anoxia, and also in aeration, included a nucleoside diphosphate kinase, a glycine-rich RNA-binding protein, a putative elicitor-inducible protein and a putative actin-depolymerizing factor. Proteins synthesized predominately in anoxia included a pyruvate orthophosphate dikinase (PPDK), alcohol dehydrogenase 1 and 2, fructose 1,6-bisphosphate aldolase and a protein of unknown function. CONCLUSION: The induction of PPDK in anoxic rice coleoptiles might, in combination with pyruvate kinase (PK), enable operation of a 'substrate cycle' producing PPi from ATP. Production of PPi would (a) direct energy to crucial transport processes across the tonoplast (i.e. the H+-PPiase); (b) be required for sucrose hydrolysis via sucrose synthase; and (c) enable acceleration of glycolysis, via pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) acting in parallel with phosphofructokinase (PFK), thus enhancing ATP production in anoxic rice coleoptiles; ATP production would need to be increased if there was a substantial requirement for PPi.

Performance of seminal and nodal roots of wheat in stagnant solution: K+ and P uptake and effects of increasing O2 partial pressures around the shoot on nodal root elongation.

Roots of intact wheat plants were grown for 7-12 d in stagnant nutrient solution, containing 0.1% agar, to mimic the lack of convection in waterlogged soil. Net K+ and P uptakes by seminal and nodal roots were measured separately using a split root system. For seminal roots in stagnant solution, net uptakes as a percentage of aerated roots were between 0% and 16% for P, while K+ ranged between 15% uptake and 54% loss. For the more waterlogging-tolerant nodal roots, net uptakes in stagnant nutrient solution, as a percentage of aerated roots, were 31-73% for P and 69-115% for K+. Elongation rates of nodal roots in stagnant nutrient were about 35-43% of those for roots in aerated solution. This partial inhibition occurred in these nodal roots despite their 15% porosity (v/v). Elevation of O2 partial pressures around the shoots to 40 kPa and then to 80 kPa substantially accelerated nodal root elongation in stagnant solution, demonstrating that most of the inhibition seen with ambient O2 around the shoots was associated with a restricted O2 supply to these nodal roots. Thus, in wheat nodal roots, with a partial pressure of 20 kPa O2 around the shoots, O2 diffusion from the shoots did not completely relieve the restrictions on elongation resulting from stagnancy in the nutrient solution. These results contrast with those in the literature for rice, in which roots function efficiently in stagnant solutions (0.1% agar). So, when wheat roots are aerenchymatous there are still restrictions to O2 diffusion in the gas space continuum between the atmosphere and the functional tissues of the roots. This poor acclimation must have been due to inefficiency of the aerenchymatous axes, which may include persistence of anoxic steles, and/or restricted O2 diffusion in other parts of the gas space continuum, in either the shoots and shoot-root junction or in the root tip.

Anoxia tolerance in rice seedlings: exogenous glucose improves growth of an anoxia-'intolerant', but not of a 'tolerant' genotype.

This study demonstrated that, in rice seedlings, genotypic difference in tolerance to anoxia only occurred when anoxia was imposed at imbibition, but not at 3 d after imbibition. When seeds were imbibed and grown in anoxia, IR22 (anoxia-'intolerant') grew much slower and had lower soluble sugar concentrations in coleoptiles and seeds than Amaroo (anoxia-'tolerant'), while Calrose was intermediate. After 3 d in anoxia, the sugar concentrations in embryos and endosperms of anoxic seedlings were nearly 4-fold lower in IR22 than in Amaroo. Sugar deficit in the embryo of IR22 is presumably due to the limitation of sugar mobilization rather than the capacity of transport as shown by similar sugar accumulation ratios of 1.8 between embryo and endosperm in IR22 and Amaroo at 3 d in anoxia. With 20 mol m-3 exogenous glucose, coleoptile extension and fresh weight increments in anoxic seedlings of IR22 were much closer to those in the two other genotypes, nevertheless protein concentration remained lowest on a fresh weight basis in the coleoptiles of IR22; indicating that protein synthesis has a lower priority for energy apportionment during anoxia than processes crucial to coleoptile extension. In contrast to these responses to anoxia imposed at imbibition, IR22 had nearly the same high tolerance to anoxia as Calrose and Amaroo, when anoxia was imposed on seedlings subsequent to 48 h aeration followed by 16 h hypoxic pretreatment. In fact, coleoptiles of anoxic IR22 had higher sugar concentrations and grew faster than Calrose, and exogenous glucose had no effect on the coleoptile extension of IR22. Excised coleoptile tips of IR22 and Amaroo with exogenous glucose had similar rates of ethanol production and were equally tolerant to anoxia. In conclusion, much of the anoxia 'intolerance' of IR22 when germinated in anoxia could be attributed to limited substrate availability to the embryo and coleoptile, presumably due to slow starch hydrolysis in the endosperm.

Review: Mechanisms of anoxia tolerance in plants. I. Growth, survival and anaerobic catabolism.

Anoxia can be one consequence of waterlogging and submergence of plants. Anoxia in plant tissues reduces the rate of energy production by 65-97% compared with the rate in air. Thus, adaptation to anoxia always includes coping with an energy crisis. Tolerance to anoxia is relevant to wetland species, rice cultivation and transient waterlogging of other agricultural and horticultural crops. This perspective, in two parts, examines mechanisms of anoxia tolerance in plants. Part 1 covers anoxia tolerance in terms of growth and survival, the interaction of anoxia tolerance with other environmental factors, and the development of anoxic cores within plant tissues. Equally importantly, Part 1 also examines anaerobic carbohydrate catabolism (principally ethanolic fermentation in plants) and its regulation. We put forward two modes of anoxia tolerance, one based on reduced rates of anaerobic carbohydrate catabolism and the other on accelerated rates (Pasteur effect). Further, Part 1 examines mechanisms of post-anoxic injury. In Part 2 (Greenway and Gibbs, manuscript in preparation) we consider flow of the limited amount of energy produced under anoxia to processes essential for cell survival. We show that acclimation to anoxia in plants involves integration of a set of sophisticated characteristics, as a consequence of which the habitat within the anoxic cell is a very different world to that of the aerobic cell.

Review: Mechanisms of anoxia tolerance in plants. I. Growth, survival and anaerobic catabolism.

Anoxia can be one consequence of waterlogging and submergence of plants. Anoxia in plant tissues reduces the rate of energy production by 65-97% compared with the rate in air. Thus, adaptation to anoxia always includes coping with an energy crisis. Tolerance to anoxia is relevant to wetland species, rice cultivation and transient waterlogging of other agricultural and horticultural crops. This perspective, in two parts, examines mechanisms of anoxia tolerance in plants. Part 1 covers anoxia tolerance in terms of growth and survival, the interaction of anoxia tolerance with other environmental factors, and the development of anoxic cores within plant tissues. Equally importantly, Part 1 also examines anaerobic carbohydrate catabolism (principally ethanolic fermentation in plants) and its regulation. We put forward two modes of anoxia tolerance, one based on reduced rates of anaerobic carbohydrate catabolism and the other on accelerated rates (Pasteur effect). Further, Part 1 examines mechanisms of post-anoxic injury. In Part 2 (Greenway and Gibbs, manuscript in preparation) we consider flow of the limited amount of energy produced under anoxia to processes essential for cell survival. We show that acclimation to anoxia in plants involves integration of a set of sophisticated characteristics, as a consequence of which the habitat within the anoxic cell is a very different world to that of the aerobic cell.

Review: Mechanisms of anoxia tolerance in plants. II. Energy requirements for maintenance and energy distribution to essential processes.

Anoxia in plant tissues results in an energy crisis (Gibbs and Greenway 2003). How anoxia-tolerant tissues cope with such an energy crisis is relevant not only to anoxia tolerance, but also to adverse conditions in air that cause an energy crisis.To survive an energy crisis, plant cells need to reduce their energy requirements for maintenance, and also direct the limited amount of energy produced during anaerobic catabolism to the energy-consuming processes that are critical to survival.We postulate that during anoxia, reductions in ion fluxes and protein turnover achieve economies in energy consumption. Processes receiving energy from the limited supply available under anoxia include synthesis of anaerobic proteins and energy-dependent substrate transport. Energy would also be required for maintenance of membrane integrity and for regulation of cytoplasmic pH (pHcyt). We suggest that a moderate decrease in the set point of pHcyt, from approximately 7.5 to approximately 7.0 is an acclimation to the energy crisis in anoxia-tolerant tissues. This decrease in the set point of pHcyt would favour metabolism of acclimative value, such as reduction in protein synthesis and stimulation of ethanolic fermentation. During anoxia lasting several days, a proportion of the scarce energy produced may need to be spent to mitigate the acidifying effect on pHcyt arising from fluxes of undissociated organic acids across the tonoplast as a consequence of high concentrations of organic acids in the vacuole. Increases in vacuolar pH (pHvac), with concomitant decreases in the vacuolar concentrations of undissociated acids, would mitigate such an 'acid load' on the cytoplasm. We present evidence that a preferential engagement of V-PPiases, over that of V-ATPases, may direct energy flow at the tonoplast to maintain pHcyt.We conclude that the likely causes of death under anoxia are firstly, a decrease in pHcyt below 7.0. Cytoplasmic acidosis occurs in several anoxia-intolerant tissues and may contribute to their death. Such adverse decreases in pHcyt can be mitigated by the biochemical pH stat. Secondly, deterioration in membrane selectivity culminating in loss of membrane integrity would be fatal. We suggest these two causes are not mutually exclusive but may act in concert.