Two thirds of the Earth’s land mass can at least occasionally be flooded (e.g. the monsoon regions of South East Asia or the areas at the lower reaches of the large Siberian rivers). Thus, many plants are exposed to varying degrees of inundation with water. In waterlogged soils, only the roots are affected. However, sometimes even the above-ground organs can be partially or completely submerged. The duration and frequency of flooding range from continuous (for instance, in swamps), to sporadic (for instance, on riverbanks). Flooding can cause severe stress due to inhibition of energy provision, as well as drastic changes in the availability of minerals. Survival under such conditions therefore requires specific adaptations, modifications and acclimations (Bailey-Serres et al. 2012a). Pronounced differences in survival between species represent an important factor determining plant distribution on Earth. In fact, hydrologically defined niches have been suggested to structure plant communities such as those found in meadows (Silvertown et al. 1999). The influence of waterlogging and submergence as one form of extreme water availability has been on the rise over the past 50 years because, as a consequence of anthropogenic influences such as climate and land use change, the incidence of flooding events has increased six- to eightfold on all continents except for Australia (Millennium Ecosystem Assessment, cited in Bailey-Serres et al. (2012a)).

5.1 Conditions of Flooded Soil

Common soils consist of four components: soil particles, water, air and organisms (including plant roots) (Fig. 5.1). Freely draining soils can retain water only in pores with diameters smaller than 10–60 μm. Even at water saturation up to the field capacity, the air-filled pore volume is 10–30% of the total soil volume. However, in partially or permanently waterlogged soils, there are almost no air-filled pores, as the air dissolves in the soil water.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig1_HTML.png — Fig. 5.1
A four-component system: root/soil organism, soil particle, soil water (solution) and soil air

Gas exchange in well-aerated soils occurs mainly through diffusion in the continuum of the air-filled pores. It is accelerated by a number of active processes in the soil and thus becomes a relatively fast process. For example, when oxygen is consumed by the respiratory activity of microorganisms and plant roots, oxygen from the atmosphere flows quickly into the soil, following the concentration gradient. As a result, the partial pressure of O₂ in the soil air, at least in pore-rich soils, remains in the range of 15–20%. Similarly, CO₂ that accumulates in the soil pores quickly leaks out from the soil. The situation is completely different when gas exchange occurs via the water-filled pores of waterlogged soils. Fick’s first law of diffusion describes the amount of gas diffusing per unit of time (i.e. the net gas flux) as being dependent on the diffusion coefficient, D, the size of the exchange area and the concentration gradient. At the same temperature, the diffusion coefficient of oxygen in water is about 10,000 times (exactly 11,300 times) smaller than in air. Furthermore, oxygen has very low solubility in water (0.03 mL O₂ L⁻¹ H₂O). Thus, gas exchange in waterlogged soils is very slow and oxygen becomes one of the limiting factors for growth and the development of plants. Similar considerations apply for CO₂ supply to submerged photosynthetic tissues. Photosynthesis under such conditions can, in addition, be hampered by low availability of light when the floodwater is turbid.

Long-term waterlogged soils have a negative redox potential because of the low oxygen partial pressure—that is, they exhibit reducing properties. Oxygen entering such soils (e.g. through root or earthworm channels) is readily consumed by soil organisms. The dramatic decrease in the redox potential is already observed after only a few days of flooding (Fig. 5.2). Microaerophilic and anaerobic microorganisms start to grow. They mainly utilise the organic matter of the soil as an energy source and require ions as electron acceptors. When nitrate is used as an electron acceptor in a process termed nitrate respiration, it is reduced to nitrite, N₂O and finally N₂ (denitrification). Correspondingly, in sulphate respiration, sulphide is formed from SO₄ ²⁻. These processes reduce the nutrient availability for plants. Similarly, oxidised forms of iron (Fe(III)) and manganese (Mn(IV)) can be reduced to their respective divalent ions. In addition, CO₂ may be used as an electron acceptor, resulting in the production of methane. Table 5.1 shows the sequence of redox reactions occurring in the soil when the redox potential decreases. Such reactions often consume protons—that is, they result in alkalinisation of the soil.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig2_HTML.png — Fig. 5.2
Development of the redox potential of a loamy clay soil as influenced by the water content and the amount of organic matter. (After Amberger (1988))

Table 5.1

Sequence of soil-bound redox reactions (After Marschner (1986))

Redox reaction		Redox potential E (mV) at pH 7
Start of nitrate reduction (denitrification)	${\mathrm{NO}}_3^{-}\to {\mathrm{NO}}_2^{-}$	450–550
Start of manganese reduction	MnO₂ + 4H⁺ + 2e⁻ → Mn²⁺ + 2H₂O	350–450
Absence of free oxygen (due to respiration)	O₂ + 4H⁺ + 4e⁻ → 2H₂O	350
Absence of nitrate	(→ N₂O → N₂)	250
Start of Fe²⁺ formation	Fe(OH)₃ + 3H⁺ +1e⁻ → Fe²⁺ + 3H₂O	150
Complete consumption of Fe³⁺		120
Start of sulphate reduction	${\mathrm{SO}}_4^{2-}+10{\mathrm{H}}^{+}+8{\mathrm{e}}^{-}\to {\mathrm{H}}_2\mathrm{S}+4{\mathrm{H}}_2\mathrm{O}$	−50
Complete consumption of sulphate		−180
Methane formation	CO₂ + 8H⁺ + 8e⁻ → CH₄ + 2H₂O	< −180

The redox potential provides important information about the reactions in the soil, as these reactions take place in the sequence listed (i.e. sulphate is not reduced as long as Fe(III) ions are still present)

Reduced heavy metal ions such as Fe(II) and Mn(II) are more toxic to plants because their availability for uptake is higher relative to the oxidised forms. Thus, the growth of roots not only is inhibited by the lack of oxygen, which is the major stress factor, but can also be affected by toxic ions in the vicinity of the roots (Fig. 5.3). Furthermore, the symbiosis of plant roots with mycorrhizal fungi can be severely compromised in waterlogged soil, thereby further decreasing nutrient acquisition and growth.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig3_HTML.png — Fig. 5.3
Dependence of root growth of the grass *Spartina patens* on the redox potential of the soil. (After DeLaune et al. (1993))

Regarding the relationship between oxygen concentration and metabolism, a situation where biochemical reactions are not limited by partial oxygen pressure is called normoxia. When mitochondrial adenosine triphosphate (ATP) synthesis is affected but not completely inhibited by low O₂, it operates under hypoxia. In the absence of oxygen (anoxia), oxidative phosphorylation in the mitochondria is negligible and ATP synthesis is restricted to substrate phosphorylation in glycolysis. The necessary reoxidation of reduced nicotinamide adenine dinucleotide (NADH) is achieved by fermentative pathways.

Physiologically, primary and secondary hypoxia (or anaerobiosis) should be differentiated. In primary hypoxia, germination of a plant already takes place in an oxygen-deficient environment, which does not change during the whole lifetime of the plant. This applies, for example, to obligate marsh plants. Secondary hypoxia occurs when plants that normally grow in well-aerated soils are temporarily flooded. This hypoxia gradually develops, allowing plants to acclimate. Accordingly, one has to differentiate constitutively expressed mechanisms to survive prolonged inundation from those induced by flooding events. A second distinction should be made between responses to waterlogging (which affects only root respiration) and submergence (which in addition affects photosynthesis and respiration in the shoot).

5.2 Hypoxia-Induced Damage: Energy Metabolism of Plants Under Oxygen Deficiency

Root cells need to produce ATP via respiration in order to support transport processes and biosyntheses—for example, the uptake of nutrient anions from the soil solution against a negative membrane potential or the activation of sugars to build cell wall polysaccharides, respectively. The daily oxygen demands of soils during the growth period of plants are in the range of 10–20 L/m², depending on the density of the roots and the activity of soil microbes. There is a direct positive correlation between O₂ partial pressure and root growth. The minimum oxygen partial pressure in the soil for the growth of flooding-sensitive plants is 2–3% (about 5 kPa). Inhibition of growth under hypoxic conditions is a multifactorial phenomenon, which is basically caused by the very low efficiency of the energy metabolism. During inhibition of mitochondrial respiration, many heterotrophic organisms and plant tissues are able to switch to fermentative metabolism, which can be regarded as an acclimative response to oxygen deficiency. This type of metabolism, however, requires increased throughput of energy carriers such as glucose because of the much lower energy yield (2 moles of ATP per mole of glucose via glycolysis, compared with 34–36 moles of ATP per mole of glucose via oxidative phosphorylation). Under these conditions, reserve material is quickly consumed. Despite stimulation of glycolysis and fermentation—the so-called Pasteur effect—the energy charge of cells remains low, so during extended periods of hypoxia, or even short-term anoxia, values below 0.5 result (Fig. 5.4). These values are too low for anabolic metabolism (i.e. for growth). Inhibition of phloem transport and phloem unloading is another consequence of the low energy charge of the plant tissues. Thus, cells are depleted not only because of the faster turnover of storage material but also because of a drop in supply of photosynthates.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig4_HTML.png — Fig. 5.4
Energy charge (EC) of lettuce seeds a or rice grains b during germination in air or under nitrogen. The energy charge of cells is usually determined by the degree of phosphorylation of the adenylate system. The following formula is applied: EC = ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP]). By definition, the maximum EC equals 1. Since ADP possesses only one energy-rich phosphate bond, its concentration has to be multiplied by the factor 0.5. A cell supplied with sufficient oxygen has an EC between 0.8 and 0.95. Under anaerobic conditions the energy charge may drop to 0.2. (After Pradet et al. (1985))

In addition to the energy deficit caused by hypoxia and anaerobiosis, fermentation leads to the accumulation of toxic metabolic products. The first product is lactate, produced by lactate dehydrogenase (LDH), which is rapidly activated upon O₂ deficiency. A rise in lactate concentrations causes acidification of the cytosol, with potentially detrimental effects on metabolism. The next acclimative response, therefore, is inactivation of LDH by acidic pH (Sect. 5.4.2). As the pH optimum of LDH is in the neutral range, it inhibits itself upon acidification of the cytosol. Pyruvate decarboxylase is less susceptible to acidity and therefore takes over, producing acetaldehyde, which is converted to ethanol by alcohol dehydrogenase. Higher concentrations of this poisonous compound destroy the selective permeability of membranes and prevent formation of proton gradients and, in turn, the gain of energy. On the other hand, ethanol easily permeates through cellular membranes and cell walls and thus only rarely reaches damaging concentrations of 50–100 nM in the cell. This limits toxicity but results in a loss of reduced carbon. Acetaldehyde, the biochemical precursor of ethanol, is much more toxic than ethanol but is usually reduced immediately. It accumulates only when alcohol dehydrogenase is nonfunctional or switched off by mutation or regulation, respectively.

Fine root systems and root meristems are particularly sensitive to oxygen deficiency. In species not tolerant of flooding, those parts of the root system used for water and ion uptake die off at oxygen partial pressures below 0.5–5 kPa and, as a consequence, the plant becomes stressed as if exposed to drought even though it is standing in water. This is indicated by stomatal closure. The rates of photosynthesis and growth decrease. Finally, the plants become stunted, while their leaves show strong epinasty (downward bending of the leaves and petioles because of increased relative growth of the upper side; this response is hypothesised to limit transpirational water loss because exposure to light is reduced). Such phenomena are often observed in indoor plants that are watered too much. Hypoxia in the water-saturated soil leads to death of the root system and withering of the shoot. In fact, most plants are more sensitive to flooding than to desiccation.

5.3 Natural Variation in the Ability to Endure Inundation by Water

Strong variation exists in the ability to withstand conditions of low oxygen availability. Many terrestrial plant species, including nearly all crops, are sensitive and do not survive longer than a few days of waterlogging. At the other extreme are plants adapted to life in wetlands (swamps, marshes, bogs, etc.), such as Iris pseudacorus, Typha latifolia, Phragmites australis and Rorippa sylvestris. They can tolerate submergence for months (Bailey-Serres et al. 2012b) and thus are adapted to primary hypoxia. Many other species show moderate tolerance of secondary hypoxia that reflects the hydrological signature of their natural environments—that is, variation in flooding tolerance determines, to a large extent, the distribution of plant species in the many areas worldwide that can be exposed to flooding. This can easily be seen in riparian vegetation, which shows pronounced zonation attributable to variation in flooding tolerance. A well-documented case is represented by the genus Rumex. It comprises species that are more hypoxia tolerant (e.g. R. maritimus and R. palustris) and thrive in zones prone to extended flooding—for instance, in the Rhine Valley (Fig. 5.5, Table 5.2)—as well as more hypoxia-sensitive species (e.g. R. acetosa and R. acetosella), which are found in zones with less frequent and lower-amplitude flooding events. Similarly, certain tree species (e.g. of the genera Alnus, Populus, Salix and Quercus) dominate along rivers and in alluvial forests.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig5_HTML.png — Fig. 5.5
Water level changes of the Rhine river near Nijmegen (the Netherlands) over 2 years, and vertical zonation of species differing in submergence tolerance. (Redrawn after Blom et al. (1993))

Table 5.2

Effect of flooding on biomass production and on nutrient content of the leaves of two flooding-sensitive representatives and one flooding-tolerant representative of the genus Rumex (After Laan et al. (1989))

Species/conditions for growth	Dry weight of shoot (g)	Nutrient content of leaves (µmol g⁻¹ dry weight)
Species/conditions for growth	Dry weight of shoot (g)	Nitrogen	Phosphorus	Sodium	Calcium	Magnesium
R. thyrsiflorus
Dry site	15.2 ± 1.4	1878 ± 45	143 ± 10	1238 ± 37	323 ± 11	435 ± 22
Flooded site	7.2 ± 1.4	1006 ± 64	44 ± 2	355 ± 15	190 ± 2	218 ± 3
R. crispus
Dry site	13.6 ± 1.4	1372 ± 54	90 ± 5	857 ± 39	532 ±27	311 ± 7
Flooded site	12.7 ± 2.5	702 ± 35	58 ± 10	347 ± 23	315 ± 13	160 ± 8
R. maritimus
Dry site	24.8 ± 2.6	1018 ± 91	59 ± 2	478 ± 11	615 ± 19	401 ± 4
Flooded site	25.4 ± 3.9	1052 ± 52	59 ± 2	272 ± 10	761 ± 40	398 ± 11

The data are average values of five identical experiments ± standard deviations

In contrast to most cultivated plants, rice is flooding tolerant, and lowland rice varieties are normally grown in paddy fields (i.e. parcels of land covered with water 5–50 cm deep). Still, considerable intraspecific variation exists. Several low-yielding landraces are able to withstand particularly severe floods. Others can be directly planted as seeds into shallow paddies and develop despite low oxygen availability (Bailey-Serres and Voesenek 2008) (Sect. 5.6).

A wide range of mechanisms explains the distribution of plants in flood-prone environments. Some species (e.g. Chenopodium rubrum) are able to circumvent the adverse effects of hypoxia by completing their life cycle between floods—which in many habitats occur with certain regularity—and by enduring flooding events as dormant life stages (Blom and Voesenek 1996). Most other plants show morphological, anatomical, developmental and metabolic characteristics that help them to avoid or truly tolerate oxygen deprivation (Voesenek and Bailey-Serres 2015) (Fig. 5.6).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig6_HTML.png — Fig. 5.6
Morphological and developmental adaptations or acclimative modifications that help flooding-tolerant plant species avoid anoxic conditions (for details, Sect. 5.4.1)

Hypoxia survival traits represent a very good example of how plant stress endurance strategies have in recent years been elucidated from the organismic level to the molecular level, and from model plants to species initially studied purely ecophysiologically. This has been possible thanks to the fruitful collaboration of ecologists with molecular biologists. The current level of understanding has implications for agriculture too, as mechanistic insights can now be used in breeding programmes aimed at developing plant varieties better adapted to conditions that are becoming more prevalent because of climate change (Xu et al. 2006) (Box 5.1).

5.4 Adaptations to Flooding-Prone Habitats

5.4.1 Anatomical–Morphological Adaptations and Modifications

An obvious way to alleviate the consequences of inundation with water is the formation of structures that supply oxygen to the roots or, in the case of submergence, to the shoot as well. A hallmark of helophytes (marsh plants) are large intercellular channels extending from the shoot and leaves into the roots. Such gas-filled tissues (termed aerenchyma) maintain a sufficiently high oxygen concentration in the roots.

In plants adapted to conditions of primary hypoxia, the formation of aerenchymas is a constitutive trait. Many other hypoxia-tolerant species (both monocots and dicots) are able to develop aerenchymas in roots and the basal part of the shoot in response to flooding. Documented examples include maize (Fig. 5.7), the coastal grass Spartina patens or sunflower (Drew et al. 2000). Formation of aerenchyma not only guarantees the aeration of tissues but also reduces the number of oxygen-consuming cells in those tissues.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig7_HTML.png — Fig. 5.7
Aerenchyma formation in a maize root: a normoxia; b hypoxia; c hypoxia + neutral red (disintegrating cells are coloured purple by neutral red); d hypoxia + Evans blue (Evans blue accumulates in dying cells). (After He et al. (1996) and Drew et al. (2000))

While, during development, aerenchyma arise by separation of cells at the middle lamella (schizogeny), inducible aerenchyma formation requires programmed cell death (PCD) and disintegration of cells (lysigeny). PCD does not take place in differentiated older cells. Rather, an aerenchyma is initiated near the elongation zone of the organ. The competence to produce aerenchyma has been directly linked to flooding survival. A large-scale investigation of wetland, non-wetland and intermediate species found a clear positive correlation between flooding survival and root porosity (Justin and Armstrong 1987). Such correlations apply also to petioles when partial or complete submergence is considered (Mommer et al. 2006), showing that the formation of longitudinally interconnected pathways for gas flow extends from leaves to root tips.

Another way to enhance oxygen supply is initiation of adventitious roots with a well-developed aerenchyma. Some plants (e.g. maize, ash, willow, Forsythia and Rumex palustris) are able, within a few days, to produce them from basal shoot parts or the lower nodes (Fig. 5.8). These roots do not penetrate as deeply into the soil as the primary root system does into a well-aerated substrate. Formation of adventitious roots involves programmed cell death too. The epidermal cell layer covering adventitious root primordia has to be weakened to allow emergence of the adventitious roots. The mechanical force exerted by the growth stimulation of the primordial cells is sufficient to trigger programmed cell death (Steffens et al. 2012).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig8_HTML.png — Fig. 5.8
Formation of adventitious roots in the flooding-tolerant *Rumex palustris* upon flooding of the root bed. The newly formed roots appear white as a consequence of the air-filled spaces in the aerenchyma and are thus clearly distinguished from roots grown under aerobic conditions, which senesce under prolonged hypoxia (Laan et al. 1991)

Oxygen reaching root cells via aerenchyma should be available for respiration and not diffuse out. Radial loss of oxygen from the interior of the root to the surrounding anaerobic soil is often reduced by the formation of a diffusion barrier. This is found, for instance, in deepwater rice, which produces a suberin-impregnated exodermis. Many wetland species show suberised and lignified secondary cell walls very close (within a few millimetres) to the root tip. If such a diffusion barrier tissue is missing, oxygen leaks out of the aerenchyma to the surrounding soil, where the heavy metal ions in the immediate proximity of the roots are oxidised, forming rusty spots and root channels in pseudogley (or stagnosol), the main soil type of wetlands. The oxidation detoxifies the metals for soil organisms—an effect that can also be beneficial for the roots themselves.

Submerged plants additionally show typical leaf modifications that enhance photosynthesis and gas exchange. Leaves of Rumex palustris developing under water have a greater specific leaf area and a thinner cuticle, and the chloroplasts are oriented towards the leaf surface (Voesenek et al. 2006). Complete submergence of plants abolishes access to atmospheric O₂. Supply of O₂ then is largely dependent on photosynthesis, which explains why light availability can support submergence survival. Some plants, however, show a pronounced “snorkel response” under these conditions—that is, they massively elongate internodes and petioles to escape the hypoxic environment. This reaction has been termed the low-oxygen escape syndrome (LOES). Applying the definitions used throughout this book (Chap. 2), it would be more appropriately termed an avoidance syndrome, because the consequences (hypoxia) of a stress (submergence) are mitigated by expanding parts of the plant so they reach the surface and have access to oxygen. LOES has been particularly well studied in deepwater rice, where the submerged shoots elongate by up to 25 cm/day (Fig. 5.9). Alternatively, hyponastic growth of leaves (i.e. a change in the orientation of petioles to vertical) can elevate them above the water surface (Voesenek et al. 2006). Such a response is well documented for R. palustris (Fig. 5.10).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig9_HTML.jpg — Fig. 5.9
Contrasting flooding survival strategies of rice. Among flooding-tolerant plant species, a continuum of survival strategies can be observed. The extremes of this continuum are represented here by rice accessions. *Top:* Low-oxygen escape syndrome (LOES); deepwater rice can cope with a slow progressive and long-lasting flood by rapid elongation of internodes. *Bottom:* Low-oxygen quiescence syndrome (LOQS); some rice accessions endure deep but transient flash floods through a strong reduction of growth and metabolic adjustments that maintain cell viability under anoxic conditions. LOES is controlled by *SNORKEL* genes (*SK1* and *SK2*); LOQS is controlled by the *SUB1A* locus. Interestingly, both *SK1/2* and *SUB1A* encode ethylene response factors (ERFs). These ERFs trigger contrasting responses in the respective rice accessions induced by the same signal transduction pathway (Fig. 5.14). (Modified from Bailey-Serres et al. (2012a))

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig10_HTML.jpg — Fig. 5.10
*Rumex palustris* shows hyponastic growth upon submergence. Leaves of a submerged plant (*right*) re-orient into a more vertical position. Strong petiole growth then moves the leaves towards the water surface. On the *left*, a plant that was not submerged is shown for comparison (Voesenek et al. 2006)

The strategy to escape low oxygen availability by stimulated growth involves substantial metabolic costs. For instance, cell wall material has to be synthesised, which requires carbohydrates and ATP. Especially given the metabolic constraints that submerged plants are subjected to, these investments can be fatal when the atmosphere is not reached. Indeed, it has been found that the escape is associated with a particular type of flooding—namely, prolonged but rather shallow floods that can be outgrown by internode elongation or hyponastic growth. In the Rhine Valley, elongating species are exclusively found in areas with slow drainage (Voesenek et al. 2004) (Figs. 5.5 and 5.17).

When floods are more transient or deep, escape via accelerated growth as an inducible avoidance strategy is not viable. Instead, species and genotypes exposed to submergence caused by these types of floods display an alternative strategy, the low-oxygen quiescence syndrome (LOQS). They tolerate hypoxic conditions in an energy-saving mode—that is, they restrict growth until the water recedes to a level where above-ground tissues are in direct contact with the atmosphere again. This has been documented best for a limited number of rice accessions (Fig. 5.9) but can also be observed in many other species successfully colonising flooding-prone habitats—for example, other Rumex species such as R. acetosa (Fig. 5.17). LOES and LOQS can be regarded as the extremes of a continuum of survival strategies employed by flooding-tolerant plant species (Voesenek and Bailey-Serres 2015).

5.4.2 Biochemical Modifications

Reoxidation of NADH to NAD⁺ in fermentative reactions is an absolute requirement to sustain energy provision by glycolysis in low-O₂ conditions, since without NAD⁺ as a substrate, glycolytic reactions cannot take place (Sect. 5.2). Correspondingly, up-regulation of enzymes such as lactate dehydrogenase and alcohol dehydrogenase belongs to the first acclimative responses elucidated molecularly. In flooding-tolerant plants, hypoxia—which under natural flooding stress conditions precedes anoxia—triggers increases in the levels of “anaerobic polypeptides” (ANPs). This process has been intensively studied in maize roots. Besides fermentative proteins, glycolytic enzymes such as aldolase, enolase and glyceraldehyde-3-phosphate dehydrogenase have been predominantly identified (Drew 1997). Higher concentrations of these proteins are a combined result of stronger gene expression and preferential translation. The promoters of the anaerobic genes share a consensus sequence, the so-called anaerobic response element (ARE) (Christopher and Good 1996). The presence of this cis-element allows coordinated regulation. In addition, it has been well documented, at least for alcohol dehydrogenase (ADH) messenger RNA (mRNA), that under hypoxia stress it is more efficiently translated than mRNAs of housekeeping genes. While overall protein synthesis is strongly reduced under low-O₂ conditions in spite of continuous gene expression, ADH protein is synthesised at high rates. This is dependent on specific sequence motifs in the 5′ and 3′ untranslated regions of the ADH mRNA.

Inability to mount these responses is detrimental. Mutant plants with compromised fermentative and glycolytic activities are unable to acclimate to decreasing O₂ availability and thus die more rapidly when exposed to anoxia (Bailey-Serres and Voesenek 2008). This has been demonstrated for maize as an example of a flooding-tolerant species. Plants lacking ADH1 are more flooding sensitive than near-isogenic lines with functional ADH1.

An immediate threat of lactate fermentation is a potentially damaging drop in pH (cytosolic acidosis). Therefore, an essential component of metabolic responses to hypoxia and anoxia is the switch to ethanol fermentation. This is brought about by a characteristic of pyruvate decarboxylase—namely, the marked increase in activity at a pH below the usual physiological value. As a consequence, cytosolic acidification due to lactate synthesis switches on ethanol fermentation. Conversely, lactate fermentation is reduced because lactate dehydrogenase becomes progressively less active with the lowering of cytosolic pH. This type of regulation is sometimes referred to as pH-stat (Fig. 5.11). In addition, pyruvate decarboxylase gene expression is induced. An alternative way of counteracting acidification is the activation of lactate efflux in hypoxic root cells. This has been documented in maize.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig11_HTML.png — Fig. 5.11
Metabolic adjustments under hypoxic conditions. When respiration is decreased because of reduced oxygen availability, metabolic changes occur that maximise substrate-level adenosine triphosphate (ATP) production and counteract negative consequences of this shift. The processes that are shown have been reported in flooding-tolerant plant species. Please note that they do not necessarily occur all or with the same rates in every species. Sucrose metabolism may be stimulated to meet an increase in carbon demand. Flux through glycolysis is enhanced to at least partially compensate for the much lower ATP yield compared to respiration. Lactate and ethanol fermentation provide nicotinamide adenine dinucleotide (NAD⁺) to maintain glycolytic activity. Alanine production reduces the carbon loss of ethanol fermentation. The γ-aminobutyric acid (GABA) shunt consumes protons and thus stabilises cytosolic pH (which can be lowered by lactate fermentation). *Yellow boxes* summarise prominent metabolic adjustments; *red lines* indicate pathways enhanced during hypoxia; *grey dashed lines* indicate reactions that are inhibited during hypoxia. Metabolites that accumulate under these conditions are shown in *red*; metabolites that decrease in abundance are shown in *blue*; important enzymes are shown in *green boxes*. *ADH* alcohol dehydrogenase, *GAD* glutamic acid decarboxylase, *GDH* glutamate dehydrogenase, *INV* invertase, *LDH* lactate dehydrogenase, *MDH* malate dehydrogenase, *PDC* pyruvate decarboxylase, *SCS* succinyl CoA ligase, *SUS* sucrose synthase. (Modified from Bailey-Serres et al. (2012a))

Several additional metabolic responses that support survival of flooding conditions have been discovered in subsets of hypoxia-tolerant plant species (Bailey-Serres et al. 2012a). For instance, more biochemical modifications are known that help prevent some of the negative consequences of fermentation. The loss of carbon due to ethanol formation and its diffusion out of cells is reduced when pyruvate is converted to alanine instead. The amino group is provided by glutamate, which is converted to α-ketoglutarate. Metabolism of α-ketoglutarate to succinate in the citric acid cycle provides extra ATP. In addition to lactate and ethanol, rice seedlings and sweet flag (Acorus calamus) rhizomes mainly produce basic amino acids (asparagine, arginine and γ-aminobutyric acid (GABA)) as fermentation end products. Synthesis of GABA from glutamate releases CO₂ and consumes protons, thereby reducing the risk of cytosolic acidosis (Fig. 5.11).

Reliance solely on glycolysis and the ensuing energy crisis necessitate a suite of modifications to optimise ATP production and to minimise energy consumption. Contributing to the latter is the aforementioned down-regulation of housekeeping protein synthesis. An example of ATP-saving metabolism is a switch in sucrose mobilisation from the invertase pathway to sucrose synthase, which reduces the amount of ATP needed to channel sucrose into glycolysis from 2 moles of ATP per mole of sucrose to 1 mole of pyrophosphate per mole of sucrose (Fig. 5.11).

Because of the low ATP gain of glycolysis in comparison with mitochondrial respiration, energy provision under O₂ deficiency is dependent on the rapid mobilisation of starch and other reserves. However, in accordance with the contrasting escape and quiescence strategies, flooded plants differ in the rate of starch mobilisation. Two different metabolic modes of anoxia tolerance can be distinguished, one based on high rates of anaerobic carbohydrate metabolism to supply ATP (energy consumption), and one based on reduced rates of anaerobic carbohydrate metabolism (energy saving) allowing a low rate of energy provision to be sustained for extended periods (Gibbs and Greenway 2003). Over time, many O₂-deprived cells move from the first to the second strategy.

Differences in the ability to grow in conditions of primary hypoxia are already apparent at the germination stage and are associated with starch metabolism. Neither wheat nor barley seeds are able to germinate under anaerobic conditions, but rice can because of differences in starch mobilisation (Fig. 5.4). Dry cereal seeds contain reserve carbohydrates, mainly in the form of starch. In order to germinate, they require catabolising enzymes: α- and β-amylase, amylopectin-debranching enzymes and α-glucosidases (maltase, diastase). In the rice grain, starch debranching enzymes and α-glucosidases are present as inactive precursors, which are activated during germination, even without oxygen. Upon germination in the absence of oxygen, α- and β-amylases are synthesised de novo (Guglielminetti et al. 1995). This happens during the first 2 days of germination when the soluble carbohydrates already present serve as the energy source. After this, starch-catabolising enzymes become active, starch is hydrolytically degraded and the degradation products become available for further metabolism, predominantly as glucose-6-P and fructose-6-P.

An additional aspect of metabolism under hypoxic conditions is post-anoxic stress. Tissues tolerating hypoxic stress are often damaged by subsequent aeration because the sudden availability of oxygen triggers reactive oxygen species (ROS) production (Chap. 2, Sect. 2.2). Cells that are metabolically acclimated to hypoxic or anoxic conditions have a comparatively negative redox potential—that is, high electron pressure from a high NADH to NAD⁺ ratio. In the presence of O₂ this leads to oxygen reduction and to the formation of ROS. During the hypoxic phase, activities of enzymes detoxifying ROS are decreased and the pools of scavenger metabolites are reduced, so the tissue is not capable of coping with increased oxidative stress. However, some plants (e.g. Iris pseudacorus, an ornamental aquatic plant native to Europe, western Asia and northern Africa but invasive in the USA) are known to tolerate post-anoxic stress well, owing to the up-regulation of enzymes such as superoxide dismutase in response to hypoxia.

5.5 Sensing of Flooding and Ensuing Signal Transduction

Elongation growth, programmed cell death and metabolic adjustments all represent acclimations and modifications that are activated upon waterlogging and/or submergence, and often depend on changes in gene expression. This clearly implies the existence of sensing and signalling mechanisms. The gaseous hormone ethylene plays a central role as a response mediator under O₂ deficiency. The key modifications—aerenchyma formation, adventitious root emergence, hyponastic growth and stem elongation—are all controlled by ethylene as the trigger. Ethylene is constitutively produced in all cells of a plant. Upon flooding, ethylene immediately (within 1 h) accumulates in and around roots and submerged shoots because of the strongly reduced gas exchange under water. Thus, it represents an early and very reliable indicator of flooding. As detailed in Sect. 5.6, ethylene is employed for controlling contrasting strategies via the regulation of differential gene expression through ethylene response factors (ERFs).

5.5.1 Ethylene Signal Transduction

The simple alkene ethylene regulates a multitude of developmental processes in plants, including seed germination, leaf abscission and fruit ripening, as well as many responses to abiotic and biotic stresses. The ethylene signalling pathway has been elucidated in Arabidopsis thaliana (Fig. 5.12). In dark-grown A. thaliana seedlings, ethylene induces the so-called triple response: an increased apical hook of the cotyledons, thickening of the hypocotyl instead of extension growth, and reduced root elongation. The triple response is easy to score and allows the isolation of mutants showing either ethylene insensitivity (etr, ein) or constitutive responses in the absence of ethylene (ctr). Molecular analysis of these mutants has defined the core pathway of ethylene signalling, which has since been found to be highly conserved in the plant kingdom.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig12_HTML.jpg — Fig. 5.12
Ethylene signal transduction. According to the current model of ethylene signal transduction in *Arabidopsis thaliana*, the five ethylene receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) (shown as *green structures*; two receptors are shown exemplarily) reside in the membrane of the endoplasmic reticulum as homodimers. Copper (shown as *red circles*) serves as a cofactor for ethylene binding. The ethylene receptors are negative regulators. In the absence of a critical ethylene concentration (*left side*), the receptors activate the kinase CTR1 (shown in *yellow*), which suppresses the response. The positive regulator EIN2 (shown in *blue*) is inactivated by phosphorylation through CTR1 and is tagged for degradation in the 26S proteasome by the F-box proteins ETP1 and ETP2. Two other F-box proteins, EBF1 and EBF2, mediate degradation of the transcription factors EIN3 and EIL1 (shown in *red*) in the nucleus. No transcription of the ethylene response genes occurs. When the ethylene concentration rises above a critical threshold—for example, because of strongly reduced diffusion out of plant tissues into flooded soil—the receptors bind the hormone and become inactivated. This switches off CTR1 and prevents the phosphorylation of EIN2. The C-terminal end of EIN2 moves to the nucleus after cleavage, stabilises the transcription factors EIN3/EIL1 and induces degradation of EBF1/2. The transcription factors dimerise and bind to *cis* elements in the promoters of ethylene response genes such as *ERF1*, thereby activating their expression. ERF1 and other products of early genes then activate expression of hundreds of additional ethylene response genes. Their combined activities bring about acclimative changes in morphology and metabolism. (Modified from Merchante et al. (2013))

The ethylene signal is perceived by ethylene receptors. They share sequence similarity with the bacterial two-component histidine kinases, which indicates their evolutionary origin. Interaction with the extremely simple ligand C₂H₄ requires Cu as a cofactor. Apparently, all terrestrial plants, including mosses, possess several receptors that can homodimerise and form higher order complexes. A. thaliana has five ethylene receptors, with ETR1 being the most studied. Ethylene receptors are negative regulators that suppress responses in the absence of the signal. They do this by activating another negative regulator, the serine/threonine kinase CTR1, which inactivates the next downstream component, EIN2, through phosphorylation of its C-terminus (Fig. 5.12).

Ethylene receptors reside in the membrane of the endoplasmic reticulum. This is possible because ethylene freely diffuses through aqueous and lipid phases. Upon binding of the ligand, the receptors become inactivated and switch off CTR1. The positive regulator EIN2 is thus no longer phosphorylated, which triggers cleavage of the C-terminus of EIN2, its movement into the nucleus and the triggering of the transcriptional cascade constituting the ethylene response. The EIN2 C-terminus stabilises the transcription factors EIN3 and EIL1, which in the absence of ethylene are tagged for proteasomal degradation by EBF1 and EBF2. EIN3/EIL1 dimerise and activate transcription of genes encoding transcription factors such as the ERFs (e.g. SUB1A in rice; Fig. 5.14 and Box 5.1), which then activate hundreds of other ethylene-responsive genes.

This linear core pathway is modulated by various additional mechanisms. For instance, Cu supply to the ethylene receptors is dependent on the Cu-ATPase RAN1. More recently discovered regulatory components promote, for example, the transition of the ethylene receptors from the inactive to the active state (RTE1), or they influence the stability of EIN2 (ETPs) (Merchante et al. 2013).

5.5.2 Oxygen Sensing

A second indicator of flooding is, of course, the O₂ status. However, while ethylene concentrations rapidly increase in all organs upon submergence, the situation is more complex for O₂. In contrast to root cells, which can become anoxic quite rapidly, shoot O₂ levels can show a pronounced diurnal pattern with comparatively high concentrations during the light period because of photosynthetic O₂ generation (Voesenek and Sasidharan 2013). The existence of a direct oxygen-sensing mechanism in plants has long been debated. The alternative scenario postulated an indirect sensing of O₂ levels through the perception of, for instance, the energy charge of cells or the cytosolic pH. However, an oxygen sensor was finally discovered in A. thaliana (Licausi et al. 2011; Gibbs et al. 2011). It regulates ethylene response factors, which are important for low-oxygen survival. In this way the two important indicators of flooding stress are integrated. It is important to note, however, that acclimations and modifications differ with respect to the relative roles that ethylene and low O₂ play in activating them. Overall, oxygen sensing is mainly important for metabolic changes, while ethylene signalling has a broader role and is essential for most responses upon flooding, including morphological modifications.

The sensor uses the oxygen dependence of the amino (N)-end rule pathway for targeted proteolysis of proteins that carry a cysteine at the N-terminus right after the first amino acid, methionine (Fig. 5.13). Following cleavage of the methionine, the cysteine can be oxidised enzymatically. This enables arginylation, that is, the addition of an arginine residue, which tags the proteins for proteasomal degradation. Thus, under normoxic conditions in a cell, these proteins are destabilised. Among the proteins with a methionine–cysteine combination at the N-terminus in A. thaliana are the class VII ERFs, which mediate various hypoxia responses (Sect. 5.6). Thus, they are rapidly degraded when O₂ for the cysteine oxidation is available and cannot activate hypoxia responses. In contrast, under low-oxygen conditions, this oxidation of the cysteine no longer occurs, resulting in greater stability of the response factors and the activation of metabolic and developmental changes supporting survival of flooding or submergence.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig13_HTML.jpg — Fig. 5.13
The oxygen sensor in *Arabidopsis thaliana*. Under hypoxic conditions, ethylene signalling activates transcription of group VII ethylene response factors (ERFs; Fig. 5.12). These ERFs (HRE1 and HRE2 are shown as examples) all share the N-terminal sequence Met-Cys (MC) and are thus substrates of the N-end rule pathway. Methionine (M) is cleaved by a methionine aminopeptidase (MAP). In the presence of O₂ (normoxic conditions) or NO, the cysteine is oxidised. After addition of an arginine (R) by arginyl transfer RNA (tRNA) transferase (ATE), the protein is recognised by the E3 ligase PRT6, which tags the arginylated ERF for degradation in the 26S proteasome by adding several ubiquitins (Ub). Thus, the ERFs cannot activate expression of ethylene response genes. However, when the cellular O₂ concentration drops, the cysteine oxidation eventually cannot occur. ERFs are not degraded and now activate the hypoxia responses. (Modified from Bailey-Serres et al. (2012a))

5.6 Regulation of Avoidance and Tolerance Strategies

The metabolic and morphological plasticity of flooding-tolerant plants enables a wide range of survival strategies, of which escape and quiescence can be regarded as the extreme poles. Molecular dissection of these contrasting strategies in rice has revealed that they are regulated by very similar factors. This provides direct insights into how, during evolution, successful adaptation to a variety of habitats can arise within a species or a genus. Several low-yielding rice landraces cultivated in flood-prone areas carry loci that are responsible for the rapid shoot elongation trait (SNORKEL1 and SNORKEL2 (SK1 and SK2) (Hattori et al. 2009)) or the energy-saving mode (SUBMERGENCE TOLERANCE1A (SUB1A) (Xu et al. 2006)) (Fig. 5.9). Encoded by these loci are group VII ethylene response factors—that is, members of the family that in A. thaliana has been shown to be regulated by the oxygen-sensing pathway (Fig. 5.13). Thus, the integration of the ethylene signal and the O₂ sensing is utilised to trigger very different responses to oxygen deprivation through variation in the targets of ethylene and low-oxygen signalling. The underlying circuitry and its recruitment for contrasting strategies exemplify the central role of differential growth control for the adaptation to stress conditions (Chap. 2, Sect. 2.3).

Shoot growth is dependent on the plant growth hormone gibberellic acid (GA). One of its classic activities is stimulation of internode elongation. Accordingly, the SNORKEL genes stimulate the GA response (Fig. 5.14). In addition, ethylene triggers the decrease in the endogenous abscisic acid (ABA) level and thus in turn increases the effectiveness of GA. Part of the GA response is the induction of expansins—small proteins that are involved in the loosening of hydrogen bonds between cellulose and hemicelluloses in the cell wall. This is a prerequisite for cell expansion. In the shoots of deepwater rice, submerged internodes accumulate considerably more expansins than those in air. The proteins occur in the intercalary meristem and in the adjacent extension zone but not in the differentiation zone (Cho and Kende 1997). Auxin also plays a role in shoot expansion: it stimulates acidification of the cell wall by activating the ATP-dependent proton pumps in the plasma membrane.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig14_HTML.png — Fig. 5.14
Contrasting submergence survival strategies are mediated by ethylene response factors in rice accessions. Upon submergence of aerial organs, ethylene concentrations rise, adenosine triphosphate (ATP) becomes depleted and sucrose is rapidly consumed. These events trigger contrasting strategies in different rice genotypes: either promotion of elongation growth in deepwater rice (escape) or repression of growth in accessions tolerant of deep submergence (quiescence). The escape strategy is controlled by the ethylene response factors SNORKEL1 (SK1) and SNORKEL2 (SK2). They stimulate gibberellic acid (GA)–dependent elongation of internodes. Among the activated proteins are expansins. GA also induces another ethylene response factor, SUB1C, which activates starch mobilisation and the provision of ATP through anaerobic metabolism (Fig. 5.11; *ADH* alcohol dehydrogenase, *PDC* pyruvate decarboxylase, *SUS* sucrose synthase). Depletion of ATP and sucrose pools trigger these metabolic processes through kinases such as CIPK15 and SnRK1A. By contrast, in rice genotypes carrying the *SUB1A* gene, ethylene leads to an increased accumulation of the GA response inhibitors SLENDER RICE 1 (SLR1) and SLENDER RICE-LIKE 1 (SLRL1). Furthermore, the consumption of sucrose and starch, as well as the synthesis of ethylene, are inhibited. (Modified from Bailey-Serres and Voesenek (2010))

Conversely, when the SUB1A gene is present in rice, the GA response is inhibited through the activation of negative regulators. SLENDER RICE 1 and a related gene in rice encode DELLA proteins, which repress the induction of GA response genes such as expansins. The energy conservation is further supported by reducing the mobilisation of carbohydrate reserves—for instance, through the inhibition of sucrose synthase activity. SUB1A also supports recovery from post-anoxic shock upon subsidence of floodwater by up-regulating antioxidant defences. The function of SUB1A as a major regulator of flooding responses has enabled successful breeding approaches (Box 5.1).

Box 5.1: Molecular Breeding for Enhanced Flooding Tolerance

Molecular elucidation of stress tolerance mechanisms and adaptation greatly facilitates the breeding of elite cultivars, using the available diversity. The SUB1A gene represents an impressive example of the potential of this approach. Some rice cultivars are highly tolerant of prolonged and complete submergence. This ability is linked to a major quantitative trait locus (QTL), SUBMERGENCE TOLERANCE1. It has been molecularly identified and found to carry three genes (SUB1A, SUB1B and SUB1C) (Fig. 5.15) encoding ethylene response factors (Xu et al. 2006). While SUB1B and SUB1C are ubiquitously present in rice cultivars, there is variation for SUB1A. First, it is present only in a subset of rice cultivars. Second, two different alleles have been found to be associated with submergence intolerance (SUB1A-2) or submergence tolerance (SUB1A-1). Using marker-assisted breeding, SUB1 has been introgressed into several flooding-sensitive elite rice cultivars. In all cases examined, it was found that varieties can be generated that show substantial increases in flooding tolerance yet retain yield potential and other desired agronomic traits (Xu et al. 2006; Septiningsih et al. 2009) (Fig. 5.16).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig15_HTML.jpg — Fig. 5.15
Structure of the *SUB1* locus in rice. *SUB1* is a major quantitative trait locus explaining some of the natural variation in submergence tolerance among rice accessions. The SUB1 locus comprises a set of genes encoding ethylene response factors. They activate contrasting submergence survival strategies upon a rise in ethylene concentrations (Fig. 5.14) (Fukao et al. 2009)

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig16_HTML.jpg — Fig. 5.16
Generation of more flooding-tolerant varieties by SUB1 introgression. The SUB1 haplotype from a flooding-tolerant variety (R49830, *right*) was introduced into an intolerant variety (Swarma, *left*) by marker-assisted selection, and confers submergence tolerance. In the *centre*, two individual plants selected from backcrosses that carried the *Sub1* haplotype with the least IR49830 background are shown. Fourteen-day-old seedlings were submerged for 14 days and photographed 14 days after de-submergence (Xu et al. 2006)

Variation at the SUB1 locus and the involvement in contrasting strategies (Fig. 5.9) suggest that duplication and divergence of group VII ERFs underlie distinctions in flooding responses in rice, as well as in many other flooding-tolerant species (Bailey-Serres and Voesenek 2010).

The differential developmental and metabolic response to ethylene is not unique for rice but conserved among flooding-tolerant plants. Species of the genus Rumex, as well as other plant species in the Rhine Valley floodplain, display varying degrees of flooding tolerance, and their growth behaviour can be placed at different positions along the continuum between escape and quiescence (Fig. 5.5). R. palustris tries to reach the atmosphere by hyponastic leaf growth (Fig. 5.10); the closely related species R. acetosa does not. Ethylene invariably accumulates in submerged tissues, yet only R. palustris shows activation of cell expansion. These differences can also be seen when more species are tested. Ethylene treatment stimulates growth in only a subset of the species (Fig. 5.17). All of them—for example, Ranunculus sceleratus and Rumex maritimus (Fig. 5.5)—belong to the same niches in the Rhine Valley. These niches are defined mostly by gradients in flooding duration and the speed of soil drying after a flooding event. The escape strategy is associated with long-lasting floods and slow drainage.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_5_Chapter/72100_2_En_5_Fig17_HTML.png — Fig. 5.17
Differential responses to ethylene in herbaceous species from floodplains of the Rhine River. For 11 species belonging to different niche classes, ethylene-induced shoot elongation (5-day treatment with air containing 10 μL/L ethylene) was determined relative to the mean elongation under control conditions (treatment with air) (standardised at 1). *Asterisks* indicate significant differences (p < 0.05) between ethylene and control treatments. The niche classes are characterised by differences in the duration of flooding and in the speed of run-off and drying after a flooding event. The elongating species are found in sites with long-lasting and slowly receding floods. Abbreviated species names: Acm, *Achillea millefolium*; Ags, *Agrostis stolonifera*; Chr, *Chenopodium rubrum*; Pll, *Plantago lanceolata*; Por, *Potentilla reptans*; Ras, *Ranunculus sceleratus*; Rua, *Rumex acetosa*; Ruc, *Rumex crispus*; Rum, *Rumex maritimus*; Rup, *Rumex palustris*; Trr, *Trifolium repens*. (Modified from Voesenek et al. (2004))

According to genome-wide transcriptome analyses of the two model Rumex species R. palustris and R. acetosa, using RNA sequencing, the differential behaviour can be associated with the activation of different processes similar to what has been found in rice (van Veen et al. 2013). Submerged R. acetosa undergoes metabolic reprogramming consistent with quiescence, while R. palustris activates photomorphogenesis and shade avoidance pathways to support the growth response (i.e. escape).

5.7 Summary

Many plants are exposed to varying degrees of inundation with water. Both waterlogging and submergence cause a lack of oxygen because the diffusion of oxygen in water is about 10,000 times slower than in air. Normoxia, hypoxia and anoxia refer to conditions of sufficient oxygen supply, reduced oxygen supply affecting mitochondrial respiration, and lack of oxygen, respectively. Hypoxia and anoxia produce negative redox potentials of the soils. Under these conditions, oxidised heavy metal ions become reduced and in this form can be toxic, thereby adding to the stress caused by inundation.
Low oxygen partial pressure affects the energy metabolism of plant organs. Adenosine triphosphate (ATP) synthesis is restricted to substrate phosphorylation in glycolysis. The necessary reoxidation of reduced nicotinamide adenine dinucleotide (NADH) is achieved by fermentative pathways but results in accumulation of the potentially toxic products lactate and ethanol. Thus, prolonged oxygen deficiency results in the death of root tissue in non-adapted plants. Adapted plants are capable of metabolic adjustments that limit the damage caused by fermentation.
Strong variation exists in the ability to withstand conditions of low oxygen availability. Many plant species have evolved the ability to tolerate primary hypoxia—for example, helophytes (swamp-inhabiting plants) are able to thrive in continuously oxygen-limited conditions. Other species withstand secondary hypoxia caused by flooding and can therefore colonise flooding-prone habitats.
A hallmark of helophytes are aerenchyma—large intercellular channels extending from the shoot and leaves into the roots. Such gas-filled tissues supply roots and rhizomes with oxygen. The formation of suberin-impregnated diffusion barriers in the root limits oxygen loss to the surrounding soil or water.
Secondary hypoxia triggers morphological, anatomical, developmental and metabolic changes that help the plant avoid or truly tolerate oxygen deprivation. The gaseous phytohormone ethylene plays a key role in activating these processes. Its concentration rises rapidly in roots and submerged shoots upon flooding, because of the strongly reduced gas exchange under water.
The inducible formation of aerenchyma and adventitious roots provides better root aeration. Both processes require programmed cell death.
The extremes of a continuum of survival strategies employed by submergence-tolerant plant species are the low-oxygen escape syndrome (LOES) and the low-oxygen quiescence syndrome (LOQS). LOES refers to a “snorkel response”—the stimulation of rapid shoot elongation or hyponastic growth of leaves in order to reach the water surface. LOQS, on the other hand, summarises mechanisms that suppress growth and restrict energy consumption to survive until a flood recedes. Both extremes can be observed within one species (e.g. rice) or between closely related species (e.g. in the genus Rumex).
Growth stimulation in LOES is dependent on another phytohormone, gibberellic acid. In LOQS the gibberellic acid response is inhibited and the metabolism of carbohydrate reserves is suppressed.
Ethylene controls these contrasting strategies via the regulation of differential gene expression through ethylene response factors (ERFs). The presence of particular sets of ERF genes in the genome determines the survival strategy. Therefore, transfer of alleles via marker-assisted breeding can confer increases in flooding tolerance.
Ethylene signal transduction proceeds via the inactivation of ethylene receptors, which act as negative regulators of signalling.
Ethylene signalling is integrated with a second mode of perception, the sensing of intracellular oxygen levels. Oxygen sensing depends on the N-end rule pathway, an O₂-dependent protein degradation pathway that results in the rapid degradation of ERFs and other proteins when the oxygen supply is sufficient.

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