Abdelbagi M. Ismail, International Rice Research Institute, The Philippines

The genus Oryza constitutes about 24 species, 20 of which are wild and only O. sativa and O. glaberrima are cultivated. O. sativa is grown worldwide, whereas O. glaberrima is restricted mostly to West Africa. Ecological and geographic distribution of these species is largely determined by temperature and water availability. O. sativa is cultivated on about 144 million ha worldwide, from 50^° N in North China to 35^° S in Australia (New South Wales) and in Argentina. It is also grown from 3 m below sea level in Kerala, India, to as high as 3000 m in Nepal and Bhutan. Two broad categories are generally identified within O. sativa, with some overlaps; japonica varieties are mostly grown in temperate regions, while indica varieties are grown in tropical and subtropical areas. A third category― tropical-japonica ― is mostly grown in the uplands of the tropics and subtropics. Japonica types are known for their better tolerance of low temperatures compared with indica types, and japonica types also have shorter, thicker grains that are softer and stickier when cooked.

Rice is grown on a variety of soils, but the physical ability of the soil to hold water is an important property, so medium- and heavier-textured soils are typically favoured over light-textured sandy soils. It is also grown under variable water regimes and hydrological conditions, from aerobic soils as in uplands, to flooded soils in irrigated and rainfed lowlands, to long-duration flooded conditions in flood-prone areas (Figure 1).

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Figure 1. Rice ecologies and ecosystems based on local hydrology. (Diagram courtesy M. A. Ismail)

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Figure 2. Rice terraces of Madagascar. A typical example where rice is grown under different ecologies, from uplands at the top of the slope, to more favourable rainfed and irrigated areas midway, to flood prone areas at the bottom of the slope. Farmers normally grow different varieties based on adaptation to each condition. (Photograph courtesy A. M. Ismail)

The enormous plasticity in rice to adapt to these diverse ecologies led to the development of substantial numbers of rice cultivars with diverse morphology, phenology and other adaptive and grain characteristics. The Genetic Resource Center of the International Rice Research Institute hosts over 117,000 accessions collected worldwide (http://irri.org/our-work/research/genetic-diversity/international-rice-genebank).

This peculiar diversity within Oryza species made rice one of the most widely grown crops over an extreme range of habitats, and a spectacular model for plant ecophysiological and genetic studies. Various types of models were used to classify rice types based on field ecologies. The most widely used classification distinguishes four broad categories; upland, irrigated lowland, rainfed lowland and flood-prone ecosystems (Maclean et al., 2002). Characters of varieties suitable for each ecology are mostly determined by local hydrology, and in some cases multiple systems co-exist based on the toposequence (Figure 2).

Upland rice is grown in aerobic unbunded soils with topographies ranging from undulating and steep sloping lands with high runoff, to low-laying valleys and well-drained flat lands. Soils vary considerably in texture, fertility and water holding capacity; from poor highly leached soils of West Africa, to fertile soils in Southeast Asia. About 13% of the world rice is grown in uplands, but with low yields of about 1 t ha^-1, and farmers are among the poorest. Upland varieties are mostly short maturing, with deeper roots (drought avoidance) and with higher tolerance of acid soils.

Irrigated ecosystem is the largest rice production system, covering 55% of the world rice area and producing over 75% of world rice grains. Fields have assured water supply and rice is grown in puddled soil in bunded fields with water depths of 2.5-10 cm through most of the season, and with 1-3 crops per year depending on location and farming systems. Dwarf high yielding varieties that are responsive to high use of fertilizers are predominant, and yields are usually high, averaging over 5 t ha^-1.

Rainfed lowlands constitute about one quarter of rice world lands and contribute about 18% of rice production. These areas are generally densely populated with poor communities, and are prone to both drought and submergence because of lack of water control, besides adverse soils, inhibiting adoption of high-yielding varieties and use of high-cost fertilizer inputs. Local landraces with yields of less than 2 t ha^-1 still dominate in most areas; however, new high-yielding varieties tolerant of prevailing abiotic factors are becoming available over recent years and are gradually replacing existing local landraces.

Flood-prone rice ecosystems are subjected to uncontrolled floods, ranging from transient flash-floods causing complete submergence, to longer term floods of 0.5 m to over 4.0 m for most of the season, and sometimes associated with excess salinity, acid sulfates and drought. Over 15 million ha in South and Southeast Asia are annually affected by uncontrolled floods. Yields are low, averaging 1.5 t ha^-1, and yet these areas support over 100 million people. Traditional varieties still dominate because they are better adapted to water fluctuations than modern varieties. Recently, varieties that tolerate complete submergence are becoming available through the incorporation of the SUB1A gene (see also main text). These varieties tolerate 1-2 weeks of complete submergence and considerable yield benefits have been achieved in farmers’ fields, with yield advantages of 1 to over 3 t ha^-1 (Mackill et al., 2012).

The extreme diversity in adaptation to various ecological and hydraulic conditions made rice one of the most widely grown cereal crops worldwide; and an interesting model for crop improvement research. Currently, rice is the most important food crop in developing word and the stable food for over half of the world population.

Further reading on this topic:

Maclean JL, Dawe DC, Hardy B, Hettel GP (2002) Rice Almanac. Los Banos (Philippines): International Rice Research Institute, pp 16-24 http://books.irri.org/0851996361_content.pdf

Mackill DJ, Ismail AM, Singh US, Labios RV, Paris TR (2012) Development and rapid adoption of submergence-tolerant (Sub1) rice varieties. Adv Agron 115: 303-356.

Brian Atwell, Macquarie University, Australia

Flooding is an environmental hazard writ large across the agricultural regions of the planet. Major inundations constrain food production because few of the world’s crop species are hydrophytes (wetland plants). It is essential to discover mechanisms of tolerance that can be used as a basis for breeding and engineering flood tolerance in rice and also other crops.

Rice (Oryza spp.) is notable among the staple crops of the world for its extreme flood tolerance. Thus it has been a natural choice for discovery of flood-tolerance mechanisms. More specifically, the coleoptile of rice is subject in nature to extreme hypoxia or even anoxia and over thousands of years these organs have concentrated gene expression patterns that confer tolerance to O₂ deficits.

The coleoptile, a small sheath that emerges from the seed (caryopsis) as a ‘proto-shoot’, is especially critical in this scenario because it grows preferentially while roots and leaves remain suppressed without O₂ present. Coleoptiles also have special characteristics to make their growth energetically ‘cheap’. They consist of pre-formed cells and can increase in length by 30% each hour (Figure 1 of this Case Study) while the organ as a whole can elongate by 3 cm in a day under water. This illustrates that coleoptiles have evolved to be a perfect emergent organ, providing a conduit from anoxic soil/hypoxic floodwater to the water surface above. Reaching O₂ is essential before energy deficits overwhelm the embryonic meristems. Indeed, this specific role of coleoptiles was called a ‘snorkel’ almost 50 years ago (Kordan, 1974) because it was realised that on reaching a source of O₂, other organs like leaves and roots could grow.

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Figure 1. A time series of rice seedlings growing in hypoxic stagnant solution for 6 days. Note the lack of roots or true leaf. The seedling on the far right has commenced a new phase of development, with the production of a mesocotyl at the base of the coleoptile. This coleoptilar node is the first true shoot meristem. (Photograph courtesy of R. Oldfield and B.J. Atwell).

Fast coleoptile growth under water is stimulated by ethylene. The build up of this phytohormone causes coleoptiles of submergence-tolerant genotypes to ‘stretch’ towards to the water surface. Hence, rice coleoptiles constitute an example of the ‘escape’ strategy. As described in Case Study 1, a failure to perceive ethylene in shoots ensures survival in mature plants under long-term floods; the presence of the Sub1A gene induces dormancy and conserves carbohydrates. By contrast with that quiescent strategy, in areas such as river deltas with prolonged deep floods, internode elongation is beneficial for escape and with the discovery of two snorkel genes, has also been shown to be an ethylene-mediated phenomenon.

Mechanisms of anoxia tolerance in plants have been elucidated through studies of coleoptiles. Ethanolic fermentation can accelerate in anoxia because the Pasteur Effect speeds up glycolysis, using carbohydrates from the seed reserves. This provides a modestly better ATP supply than a non-hydrophyte could generate. Indeed, while ethanol was long been thought to be the dominant player in fermentation, other pathways involving haemoglobin and nitric oxide are now being invoked in energy production under anoxia. The realisation that maintenance and even growth in anoxia could be achieved by preserving critical energetically demanding reactions at the cost of non-essential reactions has opened new avenues of research. For example, we now know that protein synthesis becomes a very dominant use of ATP in anoxia while many other energised reactions succumb to energy shortages. Modification of the hierarchy of energy use occurs in animals and could be an evolutionary step unique to hydrophytic plants.

Ultimately, membrane potentials are vital for all living cells. The cost of maintaining potentials has been studied in depth, including under anoxia in coleoptiles. Proton pumping between compartments is vital and in anoxia, ATP-driven proton pumps become disabled. This induces a fall in the vital cytosolic pH but this fall is modest in rice whereas acidification can be lethal in intolerant species. Most remarkably, pyrophosphate is an alternative energy source to ATP for pumping protons in plants. This relatively ‘cheap’ energy source appears to energise proton pumping into the cell vacuole by a unique protein that is expressed most strongly in anoxia-tolerant cultivars of rice and is inducible within < 2 h of imposition of anoxia. Genetically knocking out the gene for this tonoplastic proton pump increases sensitivity to anoxia. Thus, genes encoding a pyrophosphate-driven pump at the tonoplast have been identified as targets for improving survival during flooding and have also recently been implicated in regulation of Na⁺ compartmentation crucial also to salinity tolerance.

This short account of some of the applications of rice coleoptiles to improve our understanding of stress tolerance illustrates the power of an ideal model. That one simple, undifferentiated organ could instruct us about hormone physiology, energy metabolism and membrane integrity is truly remarkable. This does not preclude the need for studies in more organisms, including wild plants or animals, but does reinforce the importance of model systems

Further reading:

Kordan HA (1974) The rice shoot in relation to oxygen supply and root growth in seedlings germinating under water. New Phytol 73: 695–697

Edwards JM, Roberts TH, Atwell BJ (2012) Quantifying ATP turnover in anoxic coleoptiles of rice (Oryza sativa) demonstrates preferential allocation of energy to protein synthesis. J Exp Bot 63: 4389-4402

When plants are completely submerged and in darkness, so that no photosynthesis occurs, O₂ can become exhausted by respiration resulting in tissue anoxia, especially in tissues/organs buried in anoxic soil. Prolonged anoxia is tolerated by rhizomes, tubers and some shoot organs of wetland species, and by germinating seeds of rice and some paddy weeds (e.g. barnyard grass). More commonly, anoxia can occur in portions of the plant body (e.g. roots), or parts of tissues within roots. ‘Anoxic cores’, coexistence of an anoxic stele and aerobic cortex, were demonstrated for maize roots in hypoxic conditions, using O₂-microelectrodes (Figure 18.5) and biochemical indicators of fermentative metabolism (Thomson and Greenway 1991).

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Figure 18.5. O₂ concentration (mM) measured across a maize seedling root by using an O₂-microelectrode. The profile was taken radially through differentiated tissues 75 mm behind the apex of a 135 mm long root. O₂ concentration in the bathing medium was about 0.05 mM (hypoxia), so that the cortex received O₂ whereas the stele had an ‘anoxic core’. The abrupt gradient in O₂ status results from the lower porosity and higher metabolic demand in steal tissue as compared with the cortex. (Profile reproduced from Gibbs et al., 1988 with courtesy of W. Armstrong).

Roots in drained soil respire by catabolising carbohydrates in the tricarboxylic acid (TCA) cycle, with the ‘reducing power’ produced used in the electron transport chain (ETC) with O₂ as the terminal electron acceptor. Energy in the form of ATP is generated, predominantly through oxidative phosphorylation in mitochondria (Chapter 2.4). However, in waterlogged soils O₂ is scarce or even absent and therefore respiration is inhibited. Carbohydrates are then broken down via fermentative pathways to yield at least some ATP, produced during substrate-level phosphorylation in glycolysis (Figure 18.6). Conversion of the pyruvate to an end-product, such as ethanol, is essential to remove this metabolite as well as to recycle the NADH to NAD⁺, so that the pathway can continue to flow. Breakdown of carbohydrates to ethanol and CO₂ is the principal fermentative pathway in plants. Some lactate and alanine are also produced, but in contrast to fermentation leading to lactate and alanine, ethanolic fermentation can be sustained over days in anoxic tissues, end-product feedback or even toxicity being minimised by leakage of ethanol and CO₂ to the environment.

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Figure 18.6. Scheme denoting the important metabolic reactions during anaerobic carbohydrate catabolism. Anoxia prevents pyruvate from entering the TCA cycle because O₂ is unavailable as a terminal electron acceptor. Carbon is diverted to fermentative end-products, allowing oxidation of NADH and sustained catabolism of carbohydrates. Key enzymes are: 1. ATP-dependent phosphofructokinase; 2, PPi-dependent phosphofructokinase (PFK); 3, lactate dehydrogenase; 4, pyruvate decarboxylase (PDC); 5, alcohol dehydrogenase (ADH); 6, glutamate-pyruvate transaminase; 7, pyruvate dehydrogenase. The enzyme that catalyses oxidation of NADH as pyruvate is converted to alanine has not been identified. Note that some reactions are reversible (two-way arrows).

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Figure 18.7. Curves showing pH optima of enzymes at the branch point for carbon flow to aerobic and anaerobic pathways. These in vitro determinations from extracts of rice coleoptiles indicate how cytoplasmic pH controls carbon flow. In aerobic conditions, pyruvate dehydrogenase (PDH) catalyses entry of pyruvate to the TCA cycle when pH is above 7. Anoxia results in a decline in cytoplasmic pH to below 7, causing PDH activity to give way to pyruvate decarboxylase (PDC) and fermentation to commence (i.e. PDC becomes engaged at pH below 7, whereas PDH ceases to function). In addition, PDC extracted from coleoptiles of rice seedlings previously exposed to anoxia is in a more active state, enhancing pyruvate consumption for ethanol production. Based on Morrell et al. (1989).

Carbon flow from pyruvate to ethanol (with CO₂ also produced) occurs via the fermentative enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) (Figure 18.6). This flow is probably regulated by the activity of PDC which catalyses the first step of ethanolic fermentation. In wheat roots, for example, the PDC in vitro activity approximates the measured in vivo rate of ethanol production. Increases in the amounts of PDC and ADH proteins have been observed in a range of plant genotypes and tissues in response to O₂ deprivation. Indeed, these enzymes form part of a suite of ‘anaerobic proteins’, enzymes synthesised during anoxia. In addition to increased protein abundance, post-translational regulation of PDC activity is also exerted by changes in cytoplasmic pH, which decreases from around 7.5 in aerobic cells to around 6.8–7.2 in anoxic cells. Below pH 7.2, the activity of PDC reaches its optimum. For example, PDC extracted from anoxic rice coleoptiles becomes very active as pH drops below 7 according to the broad pH response curve in Figure 18.7. Following a return to aerobic conditions, cytoplasmic pH increases back to its normal level, the activity of PDC decreases, and carbon then flows again via pyruvate dehydrogenase (PDH) to the TCA cycle, rather than via PDC for fermentation to ethanol.

During anoxia, normal protein synthesis is replaced by the selective transcription and translation of a set of proteins called ‘anaerobic proteins’. In maize roots, there are 20–22 of these proteins which include fermentative enzymes (e.g. PDC and ADH), enzymes involved in anaerobic carbohydrate catabolism (e.g. sucrose synthase and enzymes responsible for the reversible breakdown of sucrose) and several glycolytic enzymes (e.g. aldolase). Other ‘anaerobic proteins’ of maize include superoxide dismutase (SOD), responsible for scavenging O₂-free radicals. ‘Anaerobic proteins’ are also formed in rice embryos, with a suit of proteins similar to those described for maize but also others - one very interesting additional ‘anaerobic protein’ in rice is the tonoplast H⁺-pyrophosphatase (Carystinos et al. 1995). By maintaining an ‘energised’ tonoplast capable of ion and solute transport, this enzyme might help stabilise cytoplasmic pH. Use of pyrophosphate (PPi) as an energy source reduces the dependence of tonoplast ion transport on ATP regeneration to drive the H⁺-ATPase. Studies using knockout mutants in rice have further demonstrated the importance of the H⁺-pyrophosphatase for anoxia tolerance (see Case Study 2).

Root tissues can acclimate to low O₂ with improved anoxia tolerance, if exposed to hypoxia (low, but not zero O₂) prior to the onset of anoxia. As examples, roots of maize and wheat survive anoxia more than three times longer if exposed first to hypoxia rather than abrupt transfer from aerated solution into anoxia (Table 18.4). The elimination of ADH activity reduced the survival of maize Adh^-

Mutants to almost zero following anoxic shock but allowed recovery following hypoxic pre-treatment (Table 18.4)

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Metabolic acclimation set in train by hypoxia included changes in gene expression and therefore the protein complement (‘proteome’) in cells. Hypoxic pretreatment raised activities of the fermentative enzymes PDC and ADH, and resulted in a faster rate of ethanolic fermentation during the subsequent anoxia. How plants sense and initiate signal cascades to invoke these metabolic acclimations is a current topic of debate and could involve sensing of changes in cellular energy charge, cytosolic pH, and/or possibly oxygen (see Case Study 18.3) or other possibilities.

The changes in fermentative enzymes, together with observations that exogenous sugars prolong tissue survival during anoxia, point to carbohydrate catabolism as an important factor in tolerance to anoxia. Even with fermentation operational, however, the anoxic root cells still face an ‘energy crisis’, as the ATP generated via fermentation is often insufficient even for cell maintenance in some species. Compared with respiration, fermentation produces 85–95% less ATP per hexose unit consumed. So, although such anaerobic energy generation is vital, a rapid rate of fermentation alone does not endow anoxia tolerance. Pea root tips, for example, ferment 45% faster than maize root tips, but survive less than half as long in anoxia. Greenway and Gibbs (2003) have highlighted that in addition to fermentation, other more subtle aspects of energy consumption must also be involved in anoxia tolerance, such as a reduction of energy requirements for cell maintenance and the redirection of energy flow to essential cellular processes, including maintenance of membrane integrity, regulation of cytoplasmic pH, and synthesis of appropriate ‘anaerobic proteins’.

The key to anoxia tolerance therefore lies in integration of energy production via anaerobic carbohydrate catabolism and energy consumption in reactions essential for survival. Accumulating evidence suggests two modes of tolerance based on slow and rapid rates of fermentation (Greenway and Gibbs, 2003). As one example of the ‘slow fermentation mode’, lettuce seeds appear to survive anoxia by slowing carbohydrate catabolism in anoxia to less than 35% of the rate in air. After 14 d without O₂, lettuce seeds germinate normally (Raymond and Pradet, 1980). Other plant tissues which survive but do not grow in anoxia, produce an initial burst of fermentative activity over 6–24 h before settling to slower fermentation rates. This two-phase pattern presumably provides the higher ATP required as cells acclimate to anoxia, but then the lower rates of fermentation would conserve carbohydrates for long-term survival. To be of adaptive value, this conservation of substrates through slower catabolism must be compatible with the smaller ATP yield available for cell maintenance. Calculations show that, for example, non-growing beetroot tissue in anoxia used 10- to 25-fold less ATP for cell maintenance than aerobic tissues (Zhang and Greenway, 1994).

The coleoptile of rice provides an example of the ‘fast fermentation mode’, this organ grows in anoxia (a second example is the stem of Potamogeton spp.). Fast fermentation is sustained by accelerated glycolysis, a phenomenon known as the ‘Pasteur Effect’. However, even in rice, glycolytic rate is only about twice as fast in anoxia as in air (Table 18.5).

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The glycolytic enzyme ATP-dependent phosphofructokinase (PFK), might in addition to PDC, contribute to control of glycolysis (Figure 18.6) and thus fermentation in the coleoptile of rice. Starch breakdown and sugar transport from the endosperm to coleoptile of rice seedlings in anoxia fuels the ethanolic fermentation. For plants without such starch reserves, however, low carbohydrate levels would limit the rate of anaerobic carbohydrate catabolism in tissues that experience anoxia.

LACJ (Rens) Voesenek, Utrecht University, The Netherlands

Plant life relies on light-energy driven fixation of CO₂ into carbohydrates through photosynthesis. These carbohydrates are subsequently used to construct various plant structures and fuel energy production through respiration in non-photosynthetic tissues and even in photosynthetic cells during dark periods. Respiration requires a sufficient supply of O₂. Cells in various organs of submerged terrestrial plants typically suffer from severe shortage of O₂ and thus energy deficits. The extremely slow diffusion rate of O₂ through water to plant organs and cells means that O₂ supply becomes limiting for respiration. Low O₂ conditions during flooding most frequently occur in root tissues as these are surrounded by a soil environment characterized by very low O₂ levels and because of the absence of photosynthetic capacity to generate O₂. Upon low O₂ conditions metabolism shifts from efficient mitochondrial ATP production (O₂ dependent) to inefficient anaerobic substrate-level production of ATP (glycolysis linked to fermentation), as long as sugar substrates are available. Further adjustments involve restrictions in ATP consumption and translational activities so that general protein turnover slows to save energy.

Reliable sensing of O₂ levels would allow rapid acclimation to declining O₂ in flooded plants. It was shown recently that O₂ sensing is achieved by a mechanism in which the N-end rule pathway of protein degradation serves to regulate the low O₂ response in plants (Gibbs et al. 2011; Licausi et al. 2011). Up to now the unraveling of the O₂ sensing machinery was one of the biggest challenges in flooding research. The identification of such a mechanism sheds light on the earliest step in the signaling pathway leading to low O₂ acclimation (Figure 1).

Many genes that are typically associated with low O₂ conditions in plants are regulated by transcription factors belonging to the so-called group VII Ethylene Response Factors (ERFs). Typical for these ERFs in Arabidopsis (5 members) is that they possess a specific N-terminal motif (N-degron). Due to this motif these proteins are post-translationally modified in an O₂-dependent manner via the N-end rule pathway of protein degradation. The N-terminal of Arabidopsis group VII ERFs is composed of a methionine followed by a cysteine as the second residue. The constitutive activity of methionine amino peptidase cleaves these ERF proteins between the methionine and the cysteine, yielding an N-terminal exposed cysteine. In this conformation the cysteine can be oxidized in an O₂-dependent manner. Under normoxic conditions, cysteine is oxidized and an arginine residue is added to the cysteine, catalyzed by an arginyl tRNA transferase (ATE). In this form the ERF protein can be recognized by the E3 ligase PROTEOLYSIS 6 (PRT6), leading to ubiquitination and 26S proteosome-mediated degradation. However, when O₂ is limited (hypoxia or anoxia) as in many organs of flooded plants, degradation of ERFs is inhibited as a consequence of a lack of cysteine oxidation. Under these conditions stable ERFs can function as transcription factors and drive transcription of genes needed in plants to survive in low-O₂ environments. As soon as the plant is re-oxygenated (e.g. upon withdrawal of flood water) the ERFs are again destabilized and the transcription of hypoxia-induced genes is halted.

At least one Arabidopsis ERF, RAP2.12, is sequestered at the plasma membrane, mediated by an interaction with the membrane-bound Acyl CoA binding proteins 1 and 2 (ACBP1/2). This sequestration of RAP2.12 is functional to prevent degradation by the N-end rule pathway under normoxic conditions. Via docking to ACBP1/2, high levels of RAP2.12 can be maintained even under normoxic conditions, without the risk of being degraded. Upon hypoxia RAP2.12 translocates rapidly to the nucleus to switch on acclimative pathways for low O₂ conditions (Figure 1).

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Figure 1. A model describing O₂-sensing in plants. Under normoxic conditions the ERF protein RAP2.12 has a protected plasma membrane localization due to its interaction with Acyl CoA binding proteins (ACBPs). Upon hypoxia RAP2.12 and ACBP dissociate and RAP2.12 moves to the nucleus where it induces transcription of adaptive hypoxia-response genes. Upon re-oxygenation RAP2.12 is rapidly degraded via the N-end rule pathway to down regulate the hypoxia response (Licausi et al. 2011).

Further reading:

Bailey-Serres J, Fukao T, Gibbs DJ et al. (2012) Making sense of low oxygen sensing. Trends Plant Sci 17: 129-138

Gibbs DJ, Lee SC, Isa NM et al. (2011) Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature 479: 415-418

Licausi F, Kosmacz M, Weits DA et al. (2011) Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature 479: 419-422

Hypoxic conditions can favour generation of reactive oxygen species (ROS). The major sources of ROS generation are ETC in mitochondria and oxidase activities (Blokhina et al., 2003). Various ROS species may be produced; among the major ones are superoxide radical (O₂^•-), hydroxyl radical (OH^•), and hydrogen peroxide (H₂O₂). Although H₂O₂ is less reactive than the two other ROS, in the presence of reduced transition metals such as Fe²⁺ (abundant in waterlogged soils), the formation of OH^• can occur in the Fenton reaction. These ROS can damage plant cells by causing lipid peroxidation in membranes, DNA damage, protein denaturation, carbohydrate oxidation, pigment breakdown and an impairment of enzymatic activity (Noctor and Foyer, 1998).

The extent of the ROS-induced damage to cells depends on duration and severity of stress. Short-term O₂ deprivation results in a limited accumulation of ROS and lipid peroxidation. In the short-term, the rate of ROS formation and the degree of lipid peroxidation can be regulated by constitutive endogenous antioxidants (Blokhina et al. 2003). In addition, hypoxia induces increased activities of antioxidant systems. Prolonged deprivation of O₂, however, can diminish or even abolish synthesis, transport and turnover of antioxidants. As a consequence of the depleted antioxidants and associated enzymes, cells are unable to cope with the ROS and lipid peroxidation can become severe, particularly during re-oxygenation (see also section 18.6). In addition to causing non-specific increases in membrane permeability resulting from lipid peroxidation, both H₂O₂ and OH^• have also been shown to directly control activity of Ca²⁺- and K⁺-permeable plasma membrane ion channels (Demidchik et al. 2007, 2010). Perturbations in intracellular ionic homeostasis may initiate programmed cell death (Demidchik et al. 2010).

Plant nutrient acquisition is dramatically reduced in sensitive species when in waterlogged soil. Upon waterlogging, root growth can be immediately arrested whereas shoots can continue to grow. The resulting increased shoot:root ratio causes an imbalance between shoot nutrient demands and supply by roots. Nutrient ion uptake be roots is also greatly reduced on a per root weight basis (Elzenga and van Veen 2010; Colmer and Greenway, 2011), primarily as a result of reduced O₂ availability inhibiting respiration. Ion uptake by roots consumes energy. The plasma membrane proton pump (H⁺-ATPase) requires ATP and the proton motive force generated is used to drive symporter-mediated ion uptake. Indeed, all anions (e.g. NO₃^-) enter root cells via H⁺-anion symporters. Furthermore, the H⁺-ATPase maintains the negative membrane potential, essential to creating electrochemical gradients allowing channel-mediated uptake of cations (e.g. K⁺ uptake). Absence of O₂ inhibited respiration and lowered H⁺-ATPase pumping, causing a substantial membrane depolarization, making such cation uptake via channels thermodynamically impossible (Pang and Shabala 2010). Not only is K⁺ uptake significantly reduced, but roots can also loose substantial amounts of K⁺ through depolarization-activated channels. It is not surprising, therefore, that waterlogged plants often exhibit acute K⁺ deficiency. The organic acids present in waterlogged soils, from anaerobic microbial metabolism, can also lead to membrane depolarisation of root cells and reduced ion uptake.

The diminished capacity for ion transport, together with initial ‘dilution’ of shoot nutrient concentrations by continued shoot growth relative to roots, explains a range of nutrient deficiencies observed in leaves of intolerant plants under waterlogged conditions. Waterlogging tolerant species with adequate O₂ supply to roots via large volumes of aerenchyma, can sustain root respiration and therefore plasmamembrane H⁺-ATPase functioning for nutrient uptake, as well as having adequate O₂ and energy for deeper root penetration. Efficient internal aeration of roots, together with a barrier to ROL in basal zones, also enables an aerobic rhizosphere at the root tips and regions of dense laterals, altering the rhizosphere (e.g. diminished soil toxins), presumably also with benefits for nutrient uptake by the roots.

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Figure 18.9. Transverse sections of (a) adventitious root of rice showing lysigenous aerenchyma, and (b) lateral root of Rumex hydrolapathum showing schizogenous aerenchyma. (a) taken 50 mm behind the root apex, (b) taken 5 mm behind the root apex. (Micrographs courtesy of W. Armstrong).

The amount of aerenchyma within roots determines the capacity for internal O₂ transport. Aerenchyma formation is constitutive in roots of many wetland species, although the amount is often further enhanced when soils are waterlogged (Justin and Armstrong, 1987). Many non-wetland species can also form root aerenchyma, but its development can take a couple of days following the onset of waterlogging. Roots of non-wetland species typically, however, form less aerenchyma than wetland species, and some dryland species cannot form aerenchyma. Nevertheless, even in relatively intolerant species such as wheat, newly-formed adventitious roots develop aerenchyma, and these new roots are important since the bulk of the seminal roots die.

Aerenchyma can form in the cortex of roots by two main, distinct, developmental processes: (i) lysigeny - the collapse of files of cortical cells to leave behind gas-filled voids (Figure 18.9a), or (ii) schizogeny - cell separation in a radial direction, so that large gas-filled channels form between cells (Figure 18.9b). Lysigenous aerenchyma results from selective programmed cell death in the cortex and this type occurs in many monocots, including important crops (e.g., barley, wheat, maize, rice). Schizogenous aerenchyma is formed by separation of cells (without death) and is found in some wetland species and particularly dicots (e.g., Rumex spp.). The ‘honeycomb-type’ schizogenous aerenchyma shown in Figure 18.9b for Rumex hydrolapathum forms due to cells being forced apart owing to oblique divisions by some of the cortical cells in radial rows.

The involvement of ethylene in formation of lysigenous aerenchyma has been studied in maize roots, and although major gaps exist in understanding of lysigenous aerenchyma, even less is known of the regulation of schizogenous aerenchyma. Evidence for the involvement of ethylene signalling in lysigenous aerenchyma formation is that inhibitors of ethylene action (e.g., silver ions) or of ethylene synthesis (e.g., aminoethoxyvinylglycine, AVG) block aerenchyma formation in hypoxic roots. Hypoxia enhances the activity of an enzyme involved in ethylene biosynthesis (1-aminocyclopropan-1-carboxylic acid (ACC synthase), ACC concentration increased in hypoxic roots, and ethylene synthesis was stimulated. The involvement of ethylene signalling in aerenchyma formation was also supported by experiments in which exogenously supplied ethylene induced aerenchyma formation in aerated roots.

Aerenchyma formation can be quantified by taking root cross-sections and measuring areas of gas-filled spaces relative to the total cross-sectional area. Many studies have quantified root porosity, the gas-filled volume per unit of root volume, and porosity includes the total gas volume in the roots (i.e. large aerenchyma channels plus the smaller intercellular spaces). Porosity in roots of plants grown in waterlogged soil varied from below 1% in some non-wetland species to as much as 53% in one wetland species, demonstrating the wide variation amongst species in their capacities for internal aeration of their roots when in waterlogged soil (Justin and Armstrong, 1987).

The importance of high porosity for root growth in waterlogged soil was demonstrated in a study of 91 species differing in aerenchyma volumes; species with roots of < 5% porosity penetrated only 30-95 mm, whereas those with > 35% porosity grew 150-345 mm into a waterlogged potting mix (Justin and Armstrong, 1987). Mathematical modelling has also highlighted the importance of root porosity for internal O₂ diffusion and therefore root growth in anaerobic substrates (Armstrong, 1979). O₂ supply via aerenchyma determines the respiratory activity and therefore energy status in roots, as demonstrated by measurements of adenylate energy charge (AEC) in root tips of maize seedling roots in an O₂-free medium (Drew et al., 1985). For roots reliant on an internal O₂ supply, the AEC was ~ 0.7 for tips of roots with aerenchyma (porosity ~13%), compared with ~ 0.4 in those without aerenchyma (porosity ~4%). Thus, O₂ supplied via the aerenchyma enables respiration and the ATP produced is essential for the survival, functioning and growth of roots in waterlogged soil.

Figure_18.10.jpg

Figure 18.10. Profiles of O₂ with distance behind the apex of the main axis of an intact adventitious root of Phragmites australis, when in an O₂-free medium with shoots in air. O₂ within the root cortex increases at positions closer towards the root-rhizome junction, consistent with higher concentrations towards the source of O₂ diffusing down the root. By contrast, O₂ on the exterior of the root surface is highest near the tip, and very low in the basal part of the root. A barrier to radial O₂ loss largely prevents O₂ movement to the rhizosphere in these basal positions (see also description in the text). Jackson and Armstrong (1999).

In addition to large volumes of aerenchyma (i.e. high porosity), roots of many wetland plants also possess a barrier to radial O₂ loss (ROL) in the root exterior (Armstrong, 1979; Colmer, 2003). The ROL barrier diminishes losses of O₂ to the rhizosphere and thus enhances longitudinal diffusion towards the root apex. Loss of O₂ from roots to waterlogged soil can be substantial, owing to the steep concentration gradient from root-to-soil. Many, but not all, wetland species can restrict O₂ losses from the basal parts of their roots. An example of a functional barrier to ROL is shown in Figure 18.10 for a root of common reed (Phragmites australis). When in a low O₂ medium, the concentration of O₂ at the root surface was relatively high near the tip, but it was extremely low at 30 mm and further behind the apex. This pattern of low surface O₂ in basal regions, despite the internal O₂ being higher towards the root base (closer to the O₂ source), indicates a high resistance to ROL across the outer cell layers in sub-apical positions. This resistance to radial O₂ diffusion results from suberin depositions in the hypodermis/exodermis. A ‘tight’ barrier to ROL develops constitutively in the basal zones of adventitious roots of many wetland species, but was induced by growth in stagnant deoxygenated medium in several others, including rice (Colmer, 2003). 

Even for roots with a ‘tight’ barrier to ROL, losses of O₂ are substantial near the root tip, and also from laterals. ROL around the root tip protects this sensitive growing point from reduced toxins, as these would be re-oxidised as the tip advances into the otherwise anaerobic soil (e.g. Fe²⁺ oxidised to insoluble Fe₂O₃). Thus, ROL may ‘protect’ the apex against reduced toxins, with the barrier to ROL loss in mature zones not only restricting exit of O₂ but also excluding reduced phytotoxins in the soil.

Pressurised through-flows of gas greatly increase the rate of O₂ transport along rhizomes of several emergent and floating-leaved wetland species, compared to that achieved by diffusion alone. These through-flows increase the concentration of O₂ in rhizomes above those if only diffusion occurred, and the higher rhizome O₂ increases diffusion into roots arising from the rhizomes. Through-flows occur when pressure gradients are established along the aerenchymatous pathway with a low-resistance exit from the plant to the atmosphere (Beckett et al. 1988). Flows can be substantial, for example in the yellow waterlily (Nuphar luteum) gas flow rates within aerenchymatous petioles were described by Dacey (1980) as ‘internal winds’. Through-flows can result in an increase of two orders of magnitude in the effective length of aeration in culms and rhizomes above that possible via diffusion (Armstrong et al. 1991), enabling some wetland plants to inhabit areas with permanent deep waters and for rhizome growth deep into waterlogged soils. It is important to note that even in species with through-flows along rhizomes, O₂ movement into and along roots occurs via diffusion (the roots are a dead-end side-path so through flows cannot occur without an ‘exit’).

The importance of through-flows for growing in deep water is especially visible in lakeshore vegetation. On lakeshores, wetland plants such as Typha spp. and common reed (Phragmites australis) that have flows along rhizomes and grow more deeply than morphologically similar plants that rely solely on diffusive movement of O₂ (Vretare Strand 2002). The deeper the water, the more advantageous it is for a plant to transport gases by pressurised flow rather than diffusion. Emergent plants with efficient through-flows can readily grow in water up to 3 m depth (Sorrell and Hawes 2010), and some floating-leaved plants such as sacred lotus (Nelumbo nucifera) are found in up to 5 m water depth.

Rates of through-flow are determined by the pressure gradient and the resistance to flow along the aeration system. The pressure gradient can result from: (i) pressurisation of gas in live shoots due to gradients in water vapour concentration between the interior and exterior of an enclosed space with the surface of the enclosure containing micro-pores (e.g., several wetland species, Brix et al. 1992; Armstrong et al. 1996), or (ii) venturi-induced suction caused by wind blowing over the open-ends of tall, broken culms so that gas is sucked out, and air enters via shorter culms exposed to lower wind speeds (only documented so far in common reed, Phragmites australis, Armstrong et al. 1996). The processes for pressurisation in leaves, and for venturi-induced suction, have been evaluated using physical and mathematical models.

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Figure 18.11. Mangroves are trees or large shrubs which grow within the intertidal zone. Mangroves have specialised structures for root aeration, such as (A) pneumatophores (vertical ‘air roots’) or (B) ‘knee roots’ (also called ‘prop roots’). The surfaces of these roots have lenticels (C; surface of knee roots) which are pores that allow gas exchange between the atmosphere and the internal tissues. At high tide these root structures are submerged whereas at low tide these parts of the roots have direct contact with air and oxygen can then enter via the lenticels. Photographs taken in north-western Australia by Ole Pedersen.

Waterlogging tolerant species tend to develop larger adventitious root systems than intolerant species, and these roots contain aerenchyma. The initiation and outgrowth of adventitious roots has been studied in wetland species such as rice and Rumex palustris; accumulation of ethylene appears to be the primary signal for this response, and auxin and H₂O₂ are also involved in the downstream signalling cascade (Visser et al. 1996; Steffens and Sauter 2009).

Newly-formed adventitious roots with aerenchyma can grow into anoxic waterlogged soil, but in many cases adventitious roots also grow close to the soil surface, or when floods submerge a significant portion of the stem they emerge into the water column. Surface roots are common for species with low amounts of aerenchyma – the superficial root system is therefore restricted to the surface oxidised layer of the soil. Such plants often develop a ‘sprawling’ growth form in shallow water with many adventitious roots in the water column, barely entering the soil surface, and taking up nutrients from the surface water. These plants are most common in eutrophic, relatively still water, as they rely on high nutrient concentrations in water given that they cannot exploit soil nutrients. The shallow root system means such plants are vulnerable to uprooting. Aquatic roots that grow into the floodwater are exposed to light and can form chloroplasts, with photosynthesis resulting in high endogenous O₂ levels during the daytime (e.g. Rich et al. 2012).

Pneumatophores and ‘knee roots’ are specific features of mangroves (Figure 18.11), and are the point of entry for the atmospheric O₂ that is transported to roots. The surfaces contain lenticels, pores which allow gas exchange between the atmosphere and these woody organs. Lenticels are also common on stems and the number and size increase in response to flooding on the trunks of flood-tolerant trees. Their openings become ‘hypertrophied’, i.e. impregnated with hydrophobic compounds, to prevent water infiltrating the aerenchyma when submerged by high tides or rising floodwaters.

Grey mangroves (Avicennia spp.) have long, horizontal roots (‘cable roots’) close to the soil surface, from which arise hundreds of ca. 1 cm thick, 30 cm long vertical aerial roots termed pneumatophores. O₂ enters the pneumatophores and diffuses via aerenchyma to the underground roots. Even the pneumatophores, however, become temporarily submerged and thus cut off from the atmosphere at high tide. Early researchers suggested that aeration via the pneumatophores might involve pressurised gas flows, but this effect is negligible, and most O₂ transport in mangroves is by diffusion (Beckett et al. 1988). The red mangrove (Rhizophora spp.) lacks pneumatophores, instead having aerial ‘knee’ or ‘prop’ roots that elevate the trunk above the water surface, and with hypertrophied lenticels serving as entry ports for O₂ which then diffuses to underground roots.

Even with O₂ transport in aerenchyma, trees in wetlands are unable to grow roots as deeply into soil as terrestrial trees, often leaving them vulnerable to toppling. Many flood-tolerant trees in freshwater swamps therefore feature extensions to their lower trunks (buttresses and knees) that provide mechanical stabilisation, and with lenticels present these enable entry of O₂ into the root aerenchyma.

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Figure 18.15. Leaf gas films on common reed (Phragmites australis) during complete submergence. Gas films form on hydrophobic leaf surfaces. Leaf gas films enhance gas-exchange with the surrounding floodwater: CO₂ entry for photosynthesis (in light) and O₂ entry for internal aeration for respiration (in dark), of submerged plants. (Photograph courtesy of Ole Pedersen).

Photosynthesis in completely submerged wetland plants is severely impeded by low light, and the slow diffusion of CO₂ across the aqueous diffusive boundary layer (DBL) adjacent to leaves. Aquatic and amphibious plants have evolved a number of leaf traits to reduce the total resistance to CO₂ uptake, including: (i) dissected leaves, (ii) undulating leaf edges to increase turbulence across the leaf, (iii) thin leaves, (iv) reduced cuticle, and (v) chloroplasts in epidermal cells. The first three traits (i, ii & iii) all serve to erode/reduce the DBL and thus decrease the distance of molecular diffusion (i.e. decreased total external resistance to CO₂ uptake), and (iv & v) reduce the resistance within the tissue for the diffusion pathway to chloroplasts. Likewise, many submerged terrestrial wetland plants also display some acclimation to inundation such as thinner leaves, reduced cuticle and chloroplast orientation towards the source of CO₂. Amphibious plants are positioned between the truly aquatic plants and the terrestrial wetland plants and display a suite of leaf acclimation traits that allows efficient gas exchange by leaves in air, as well as those formed under water.

Rates of net photosynthesis by submerged leaves are typically much lower than rates achieved in air, even for acclimated leaves. Underwater net photosynthesis by submerged terrestrial plants is generally lower than the rates achieved by aquatic plants. However, this is only true when photosynthetic rates are expressed on a per unit area basis, the units commonly used in terrestrial plant physiology. When net photosynthesis is expressed on a per unit dry mass basis, rates in aquatic plants > amphibious plants > terrestrial plants and this order reflects the higher carbon-return per unit of dry mass investment by the aquatic leaf types, as compared with terrestrial leaf types, when submerged.

Gas films on leaves of submerged terrestrial wetland plants have also been shown to facilitate underwater photosynthesis. Gas films form on hydrophobic leaf surfaces of many wetland plants when submerged (Figure 18.15); e.g., species of Phragmites, Typha, Spartina, Carex, Phalaris and Oryza (including cultivated rice), and the gas film forms a large gas-water interface that facilitates gas exchange with the surrounding water.

It is likely that the stomata remain open underneath the gas film. The gas films enable leaves of such terrestrial wetland plants to photosynthesize under water, albeit at rates much reduced when compared with in air, but without further acclimation and this strategy may therefore be particularly advantageous under short floods; as examples, frequent tidal submergence or short duration flash floods such as in some rice-growing areas and natural wetlands where water recedes after a week or two. The improved O₂ and sugar status of submerged rice owing to the beneficial effects of leaf gas films would enhance survival during complete submergence.

Ole Pedersen, University of Copenhagen, Denmark

Development of microelectrodes robust enough for use in field conditions has enabled in situ measurements of O₂ dynamics in submerged plants (Figure 1). Eco-physiological research on submerged plants has been a challenge; infrared gas analyzers do not work under water! Field studies (in situ) have revealed how fluctuating light, diurnal changes in temperature and water column O₂ concentrations all influence internal aeration of submerged aquatic plants. In the case of seagrasses, application of sulphide microelectrodes has also added to the growing evidence of sulphide poisoning as a likely cause of extensive die-backs.

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Figure 1. Diver operating O₂ and H₂S microelectrodes (left) in a Thallasia seagrass meadow in the Caribbean. Development of in situ equipment has enabled measurements of internal aeration under challenging field conditions in aquatic systems (right). (Photographs courtesy of Malene Hedegård Petersen (left) and Ole Pedersen (right).

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Figure 2. Oxygen dynamics in the rhizome of the seagrass Zostera marina during a diurnal cycle. O₂ in the rhizome closely follows incoming light during daytime, whereas during the night internal aeration relies on supply of O₂ from the water column. The dependence on water column pO₂ during the night for internal aeration becomes particularly clear when the tide carries water across the seagrass meadow with lower pO₂ as the internal pO₂ of the rhizomes immediately declines (see arrows). During the day, it is the other way around as fluctuations in incoming light is immediately reflected in rhizome pO₂ as daytime rhizome pO₂ follows underwater photosynthesis. Data from Greve et al. (2003).

Seagrasses are flowering plants with roots, rhizomes, sometimes stems, and almost always strap shaped leaves to reduce pressure drag and thus the uprooting forces created by wave action. Seagrasses are key marine ecosystem engineers and habitat for various marine animals, but seagrasses are under world-wide threat from human activities (eutrophication, dredging and other physical disturbances). Eutrophication impacts directly on seagrasses by decreasing the available light (stimulates growth of epiphytes and planktonic algae) but also indirectly by stimulating the decomposition of organic matter in the sediment (mineralization of algae and seagrass litter is often limited by N and P) and thus increases the O₂ demand of the sediment. In marine sediments, sulphate reduction by microorganisms can be substantial under anoxic conditions, producing sulphide, a potent phytotoxin with toxicity and mode of action similar to that of cyanide. Sulphide exists in three different chemical forms in water (H₂S, HS^- and S^2-) with the gaseous H₂S dominating at low pH and S^2- at high pH. Gaseous H₂S can enter the root and rhizome aerenchyma and move via diffusion to other parts of the seagrass, such as to leaf meristems that are relatively sensitive to sulphide. The resulting sulphide poisoning is a major cause of the worldwide die back of seagrasses observed in temperate as well as in tropical seagrass meadows.

Mechanistic field studies employing in situ microelectrodes have improved our understanding of internal aeration and sulphide intrusion in natural seagrass meadows. During the day, internal aeration of roots and rhizomes relies on O₂ production in underwater photosynthesis, as does radial O₂ loss (ROL) from roots to the sediments. There is a strong relationship between incoming light and O₂ partial pressure in roots (Figure 2). Clouds immediately lead to a decline in root O₂, whereas during periods of high sunlight root O₂ was highest. During the night time, however, internal aeration relies on a steady flux of O₂ from the water and into the leaves, and via the aerenchyma, further down into the roots and rhizomes.

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Figure 3. Oxygen dynamics in the rhizome of the seagrass Zostera marina during the day (A) and during the night (B). During the day, underwater photosynthesis produces O₂ in the leaves and rhizome pO₂ is thus a function of incoming light as O₂ easily diffuses from the chloroplast and into the root via the porous tissues. In contrast, during the night, the water column is the only source of O₂ for the plant and O₂ diffuses from the water and into the leaves and further down into the belowground tissues. Consequently, rhizome pO₂ is strongly correlated to water column pO₂ during darkness. Re-analyzed data from Greve et al. (2003).

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Figure 4. Lobelia dortmanna and other isoetids in a Norwegian oligotrophic lobelia lake. The water of such lakes contains only little dissolved CO₂ but L. dortmanna can take up CO₂ from the sediment where the concentration is typically 100‑fold higher. As a consequence, L. dortmanna has no barrier to radial O₂ loss (ROL) in its roots and almost all O₂ produced in underwater photosynthesis is lost to the sediment via the roots. (Photograph courtesy of Ole Pedersen).

Critically low water column O₂ can occur during nights in areas with still, warm waters, resulting from net system respiration faster than inwards movement of O₂ into the stagnant waters. Under such night time conditions, roots can experience anoxia. Cessation of ROL to the sediments means there is no longer chemical oxidation of H₂S to SO₄^2- in the rhizosphere, so that gaseous H₂S enters the aerenchyma and spreads via gas phase diffusion to all parts of the seagrass. The very metabolically active leaf meristems are thought to be particularly sensitive to sulphide poisoning. It is thought provoking that during most sudden die backs of seagrasses, the shoots are found drifting in the water with apparently healthy leaves but detached from the vertical stem exactly where the basal leaf meristems is located.

In contrast to coastal marine habitats of seagrasses, lobelia lakes are highly transparent oligotrophic, low alkaline lakes of the northern hemisphere. The vegetation consist of several evergreen species that are morphologically strikingly similar: short stiff leaves arranged in a rosette and with unbranched roots that can make up more than 50% of the biomass. As a whole, the type of vegetation is referred to as isoetids from the genus Isoetes that occurs in most lobelia lakes. Although not present in all lobelia lakes, Lobelia dortmanna is key species (Figure 4). Isoetids take up CO₂ from the sediment via the roots and some are even CAM plants, although conservation of water is probably the least of all concerns for these plants. CO₂ concentrations are highest at night, so CAM enables storage of malate for subsequent decarboxylation providing CO₂ for photosynthesis the next day.

Many sandy sediments in shallow lobelia lakes are permanently oxic. Oxic sediments are a consequence of inherently low mineralization rates as the oligotrophic conditions lead to very low production of organic matter that is subsequently decomposed in the sediment. Nevertheless, the CO₂ concentration in these sediments can be 100-fold higher than in the water above and L. dortmanna, along with the other isoetids, tap into this rich source of CO₂ with their large root systems. The large gradient in partial pressure of CO₂ between sendiment and photosynthetic leaves drives a flux of CO₂ from the sediment, into the root aerenchyma (radial CO₂ uptake) and then upwards into the porous leaves. Interestingly, the leaves are covered with a relatively thick cuticle to prevent loss of CO₂ to the surrounding water, and as a result up to 100% of the O₂ produced in underwater photosynthesis is lost via ROL from the roots (Figure 5).

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Figure 5. Oxygen dynamics in leaves and roots of Lobelia dortmanna and the surrounding water column and sediment in Lake Värsjö, Sweden. In light, underwater photosynthesis drives leaf pO₂ up above 30 kPa and the steep gradient to roots results in a substantial O₂ flux into roots. The roots have no barrier to ROL and thus the majority of O₂ produced in photosynthesis is lost to the sediment resulting in large diurnal fluctuation in sediment pO₂; in fact, the sediment remains permanently oxic. Data modified from Sand-Jensen et al. 2005.

In conclusion, the isoetids tested so far do not form a barrier to ROL in their roots and isoetids are thus restricted to sediments with very low O₂ demand; any root barrier would also restrict CO₂ uptake. In contrast, the few species of seagrasses studied all show a strong barrier to ROL and in the marine H₂S rich environment the barrier would also reduce the inward flux of gaseous H₂S.

References

Greve TM, Borum J, Pedersen O (2003) Meristematic oxygen variability in eelgrass (Zostera marina). Limnol Oceanogr 48: 210-216

Sand-Jensen K, Pedersen O, Binzer T, Borum J (2005) Contrasting oxygen dynamics in the freshwater isoetid Lobelia dortmanna and the marine seagrass Zostera marina. Ann Bot 96: 613-623

Pedersen O, Colmer TD, Sand-Jensen  K (2013) Underwater photosynthesis of submerged plants – recent advances and methods. Front Plant Sci 4 DOI: 10.3389/fpls.2013.0014

During daytime, underwater photosynthesis not only provides sugars but it also produces O₂ which results in significant aeration of belowground tissues. In light, the partial pressure gradient of O₂ from shoot to root is huge (high O₂ in the surrounding water, zero O₂ in the anoxic soil) and can thus drive a substantial flux of O₂ from shoot to root in well-developed aerenchyma as this pathway poses relatively little resistance to diffusion. In fact, O₂ in the roots of rice displays a normal saturation curve relationship when plotted against light available to the shoots, a pattern also found in truly aquatic plants (see Case Study 4).

At night, the floodwater surrounding the shoot is the main source of O₂ for internal aeration also of belowground tissues. The O₂ “stored” in aerenchymous tissues cannot support night time respiration of belowground tissues as the O₂ in the aerenchyma relatively quickly equilibrates with the environment (soil in the case of roots and water column in the case of shoot). However, with ample O₂ in the floodwater a large partial pressure gradient exists for O₂ movement from the floodwater into the leaves and further into the roots (both respiring and thus consuming O₂). As a result, the relationship between floodwater O₂ and root pO₂ in darkness is often linear (see Case Study 4). The floodwater O₂ threshold concentration required for O₂ to enter and reach the root extremities is determined by the total resistance to molecular diffusion into (DBL, surface gas films, stomatal resistance, cuticular resistance) and through the plant body (tissue porosity, diffusion distance) plus loss of O₂ along the route (respiration and any ROL). As with underwater photosynthesis, leaf gas films reduce the resistance to gas exchange between floodwater and leaves, so enhancing O₂ entry at night-time. Plants with gas films have been shown to maintain better internal aeration as compared to plants with the gas films experimentally removed.

As detailed in Section 18.5, complete submergence restricts light intensity and gas exchange, slows O₂ and CO₂ exchange between shoot tissue and floodwater. Reduced photosynthetic activity, together with excessive growth during submergence, result in severe carbohydrate starvation and consequently, death and disintegration of most tissues when flooding persists for longer duration.

Visual symptoms of stress generally start developing soon after desubmergence, with sensitive genotypes showing leaf senescence and decay, followed by mortality within a few days after desubmergence.

Excessive growth during submergence is common, and due to accumulation of the phytohormone ethylene. Submerged plants tend to elongate excessively, an “elongation escape” adaption that allows their leaves to maintain contact with air until the floodwaters are too deep. This elongation capacity is mediated through ethylene which suppresses ABA synthesis but enhances synthesis and sensitivity to GA, resulting in leaf and internode elongation (Das et al. 2005). Ethylene accumulation also triggers chlorophyll degradation and leaf senescence (Ella et al. 2003b), rendering leaves less fit for photosynthesis both underwater and upon resumption of contact with air after desubmergence.

The sudden aeration and exposure to high illumination upon desubmergence causes oxidative stress resulting from ROS generated in leaves that have limited capacity for photosynthesis following submergence (Ella et al. 2003a).

Recovery after submergence therefore, depends on maintenance of carbohydrate reserves, during and shortly after flooding (Das et al., 2005), and the maintenance of a functional photosynthetic system. In rice, as explained in Section 18.5, tolerance of submergence is conferred by an ethylene response-like transcription factor SUB1A. Induced by ethylene that accumulates within plants during submergence, SUB1A disrupts the elongation escape strategy typical of most lowland rice varieties through suppressing GA-promoted elongation, and also slows ethylene-induced leaf senescence (Bailey-Serres et al. 2008). Survival and recovery are enhanced in two ways: (i) less energy is consumed on elongation growth and carbohydrates are conserved, and (ii) leaf senescence is prevented. Thus, plants continue photosynthesis while underwater, and can resume optimal rates of carbon fixation upon re-exposure to air and high illumination and so minimise ROS damage after desubmergence. The sudden exposure to high O₂ and high light increases the generation of ROS. The ability to recover quickly and produce new tillers following desubmergence is important because only these new tillers will become effective in contributing to grain yield.

Re-oxygenation injury is well-documented for both animal and plant tissues. A highly reduced intracellular environment (including transition metal ions) and low energy supply, such as occurs during soil waterlogging, are the factors which favour ROS generation. Free radicals are formed soon after O₂ re-enters, in a so-called oxidative burst. At the same time, activity of most plant antioxidant systems is compromised due to metabolic perturbations caused by the previous period of anoxia or severe hypoxia (Blokhina et al. 2003). Production of ROS upon reaeration might impede ion uptake by roots; H₂O₂ causes membrane depolarization and K⁺ efflux (Chen et al. 2007). Recovery of nutrient uptake upon re-aeration following anoxia in roots of wheat showed a short time lag (~ 4 h) before net K⁺ uptake accelerated. The time lags could have been associated with repair of general metabolism (e.g. lag in recovery of mitochondria), repair of membranes and membrane transporters, or the prevention of damage from ROS.

Recovery of root growth upon drainage is important for crops in rain-fed agriculture, as deep roots will be required to obtain sufficient water from the soil later in the season (e.g. wheat in Mediterranean climates). Seminal roots of wheat cease growing soon after waterlogging, and if waterlogging exceeds several days these roots show little capacity for re-growth upon drainage and soil aeration. Apices of the main axes of the seminal roots typically die, with any regrowth from laterals. Adventitious roots, by contrast, are able to grow in waterlogged soils and retain the potential of the main axes to elongate upon soil aeration. Resumption of growth of adventitious roots following drainage can be fast, so that these roots continue in their importance for future shoot growth. Nevertheless, the adventitious root system could not compensate for the severe inhibition of the seminal roots of wheat. For waterlogging-sensitive crops, even short periods of transient waterlogging can have longer-term adverse effects. In wheat, for example, 3 d waterlogging severely retarded development even in the longer term after drainage (Malik et al. 2002), highlighting the need for improved waterlogging tolerance in our crops.