Oxygen Deprivation and Flooding HORT 301 – Plant Physiology December 8, 2008 Taiz and Zeiger – Chapter 11 (p. 256-262), Chapter 26 (p. 698-705); Buchanan et al., Chapter 22 and Bailey-Serres and Voesenek (2008) Annu Rev Plant Biol 59:313-339 No or low O2 - insufficient O2 availability causes reduced respiration and inadequate ATP production for optimal growth and development, reduced yield   Anoxia - no O2 Hypoxia - low O2, below atmospheric O2 levels (~21%), diffusion of O2 into actively growing cells from the atmosphere is insufficient, respiration decreases Anaerobic growth - organism growing without O2 Hypoxia and anoxia - associated with water logging or flooding of soils impacting roots

O2 diffusion in air is four orders of magnitude greater than in water Flooding reduces gas exchange that results in reduced O2 and increased CO2 and ethylene Courtesy of Bob Joly Emphasizes the issue of moving oxygen through water, soil solution

Hypoxia and anoxia deleterious effects on plants Flooding of the Midwest (1993) resulted in a 33% reduction in maize yield Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants Without O2, the tricarboxylic acid (TCA)/citric acid cycle and oxidative phosphorylation cannot function as O2 is required for electron transport (terminal electron acceptor), low O2 reduces TCA cycle activity Consequently, NADH cannot be oxidized and NAD+ becomes limiting leading to a cessation of glycolysis

Cells begin fermentative metabolism that oxidizes NADH to NAD+ to generate reducing power for glycolysis, pyruvate to lactate or ethanol, rather than respiration Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants Two (2) molecules of ATP are produced per hexose molecule through fermentation compared to 30 to 32 ATP produced through glycolysis, TCA cycle and oxidative phosphorylation  

Production of lactate in fermentative metabolism causes a decrease in cytosolic pH, which negatively regulates lactate dehydrogenase facilitating ethanol production Critical oxygen pressure (COP) - O2 level that causes a reduction in respiration is about 20% by volume (concentration in air), which is nearly the normal ambient level   The key is for sufficient O2 to reach the actively growing cells in tissues and organ (internal) to support respiration A very low partial pressure of O2 (<1%) in the mitochondrion is sufficient to maintain oxidative phosphorylation, critical enzymes have a low Km for O2 Movement of oxygen from air into internal tissues is restricted because of diffusion through water Only low levels of oxygen reach the mitochondria because of the resistances caused by diffusion through water    

Reduced intracellular pH causes cell death Anaerobic micro-organisms - reduce nitrate (NO3-) to nitrite (NO2-) or to nitrous oxide (N2O) and molecular nitrogen (N2), Fe3+ to Fe2+, and sulfate (SO42-) to hydrogen sulfide (H2S) all of which are toxic to plants   Reduced physiological root function that inhibits shoot growth - e.g. nutrient and water uptake, plants may exhibit wilting, ABA production may induce stomatal closure as a response Reduced intracellular pH causes cell death    

Ethylene is induced by low O2 - production of ethylene leads to epinasty ACC (1-aminocyclopropane-1-carboxylic acid) is produced in roots, transported to shoots in the xylem stream, converted to ethylene causing downward growth of leaves Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants Ethylene causes negative and positive responses to low oxygen Anoxia (because there is no oxygen) blocks ACC oxidase, conversion of ACC to ethylene

Summary of hypoxia and anoxia effects on plants: Low oxygen reduces TCA cycle activity NADH oxidation (to NAD+) is limited ATP production per hexose is reduced from 30/32 to 2 (fermentation cycle to convert NADH to NAD+) Reduced intracellular pH leading to cell death Anaerobic microbes in the soil reduce ions to toxic forms Ethylene production results in epinasty

Adaptive responses to hypoxia and anoxia Low O2 escape syndrome (LOES) – avoidance strategies that mitigate hypoxia: Completion of life cycle between flood periods, seeds are dormant during flooding

Increased elongation of stems, petioles and leaves – leaves grow sufficiently to make air contact Thinner leaf blades (cell wall and cuticle thickness) and orientation of chloroplasts to leaf surface – facilitate O2 diffusion into the leaf Bailey-Serres & Voesenek (2008) Annu Rev Plant Biol Figure 1 Blue area (right) indicates the water level, a. (right at top) – aerenchyma in leaf petioles (see a), b. thinner leaf blades – facilitates oxygen diffusion Various species display the low-oxygen escape syndrome (LOES) when submerged. The syndrome includes enhanced elongation of internodes and petioles, the formation of aerenchyma in these organs (air spaces indicated by arrows labeled a), and increased gas exchange with the water layer through reduced leaf thickness and chloroplasts that lie directed toward the epidermis (indicated by arrows labeled b). Photographs are courtesy of Ronald Pierik, Liesje Mommer, MiekeWolters-Arts, and Ankie Ammerlaan

Production of aerenchyma - intracellular spaces formed by programmed cell death (PCD) of cortical cells or separation of cortical cells without cell death In some species aerenchyma formation is constitutive (rice) and in others it is induced (maize) Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants Maize

Low O2 induces ethylene production that leads to aerenchyma formation via PCD, induction of ACC Anoxia results in less production of ethylene and aerenchyma than hypoxia because ACC oxidase is inhibited (lack of O2) Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants Lysigenous aerenchyma – PCD and cell collapse Schizogenous aerenchyma – Cell separation without collapse Signal pathway - low O2 → ethylene → Ca2+, G-proteins, inositol phospholipids, protein kinases & phosphatases → cell wall degrading enzymes (e.g. cellulases, xyloglucan hydrolases, etc.) → PCD → aerenchyma

Adventitious root and lenticel formation - submerged portions of the shoots Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants Adventitious roots – dedifferentation and differentiation of roots, serve root function and closer to oxygen source Lenticel – specialized pore on the epidermis of the stem

Rapid shoot intermodal elongation to access O2 during flooding Deepwater rice is well adapted for growth and development on land where flooding occurs Rapid shoot intermodal elongation to access O2 during flooding Development of adventitious roots post-flood Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants A. Blue line is the water level at different times during the season As flood water recedes the plant lays on the soil Production of adventitious roots, propagation B. Right, elongation after flooding

Responses of different rice varieties to flooding: Intolerant lowland rice - gibberellic acid (GA) induces internodal and leaf elongation (LOES) and carbohydrate consumption through regulation of SUB1C expression Lowland Rice Deepwater Rice Tolerant Intolerant Strategy Quiescence LOES Sub1 haplotype SUB1A-1, SUB1B, SUB1C SUB1B, SUB1C or SUB1A-2, SUB1B, SUSB1C SUB1B, SUB1C or SUB1A-2, SUB1B, SUB1C Carbohydrate consumption Limited by SUB1A-1 High Fermentation capacity Moderate N.D. GA response Inhibited by SUB1A-1 Promoted by SUB1C Figure 2 Rice responds via different strategies to submergence. Flood-tolerant rice varieties invoke a quiescence strategy that is governed by the polygenic Submergence1 (Sub1) locus that encodes two or three ethylene-responsive factor proteins (41, 159). SUB1A is induced by ethylene under submergence and negatively regulates SUB1C mRNA levels. Flood-intolerant varieties avoid submergence via the low oxygen escape syndrome (LOES). To this end SUB1C expression is promoted by gibberellic acid (GA) and is associated with rapid depletion of carbohydrate reserves and enhanced elongation of leaves and internodes. The LOES is unsuccessful when flooding is ephemeral and deep. Deepwater rice varieties survive flooding via a LOES, as long as the rise in depth is sufficiently gradual to allow aerial tissue to escape submergence (61). N.D., not determined. SUB1C promotes depletion of carbohydrate reserves and enhanced elongation of leaves and internodes Bailey-Serres & Voesenek (2008) Annu Rev Plant Biol

SUB1C is an ethylene-response element (ERF) domain interacting transcription factor (ethylene signaling) Stimulates internodal and leaf elongation and rapid depletion of carbohydrate reserves that results in death with prolonged flooding Low O2 → GA → SUB1C → carbohydrate consumption/shoot and leaf elongation

leaf/internode elongation) Tolerant lowland rice - low O2 causes a quiescent state, respiration is down-regulated and fermentation begins   SUB1A regulates the quiescent strategy by reducing growth (represses SUB1C expression) and respiration Low O2 → ethylene induces SUB1A expression, SUB1A is an ERF domain transcription factor, suppresses the GA response (reduced leaf/internode elongation) Lowland Rice Deepwater Rice Tolerant Intolerant Strategy Quiescence LOES Sub1 haplotype SUB1A-1, SUB1B, SUB1C SUB1B, SUB1C or SUB1A-2, SUB1B, SUSB1C SUB1B, SUB1C or SUB1A-2, SUB1B, SUB1C Carbohydrate consumption Limited by SUB1A-1 High Fermentation capacity Moderate N.D. GA response Inhibited by SUB1A-1 Promoted by SUB1C Figure 2 Rice responds via different strategies to submergence. Flood-tolerant rice varieties invoke a quiescence strategy that is governed by the polygenic Submergence1 (Sub1) locus that encodes two or three ethylene-responsive factor proteins (41, 159). SUB1A is induced by ethylene under submergence and negatively regulates SUB1C mRNA levels. Flood-intolerant varieties avoid submergence via the low oxygen escape syndrome (LOES). To this end SUB1C expression is promoted by gibberellic acid (GA) and is associated with rapid depletion of carbohydrate reserves and enhanced elongation of leaves and internodes. The LOES is unsuccessful when flooding is ephemeral and deep. Deepwater rice varieties survive flooding via a LOES, as long as the rise in depth is sufficiently gradual to allow aerial tissue to escape submergence (61). N.D., not determined. Reduced respiration is an adequate strategy if period of hypoxia and anoxia is short, i.e. flooding is intermittent and temporary Bailey-Serres & Voesenek (2008) Annu Rev Plant Biol

Deepwater rice - LOES, ethylene → GA → internodal and leaf elongation Lowland Rice Deepwater Rice Tolerant Intolerant Strategy Quiescence LOES Sub1 haplotype SUB1A-1, SUB1B, SUB1C SUB1B, SUB1C or SUB1A-2, SUB1B, SUSB1C SUB1B, SUB1C or SUB1A-2, SUB1B, SUB1C Carbohydrate consumption Limited by SUB1A-1 High Fermentation capacity Moderate N.D. GA response Inhibited by SUB1A-1 Promoted by SUB1C Figure 2 Rice responds via different strategies to submergence. Flood-tolerant rice varieties invoke a quiescence strategy that is governed by the polygenic Submergence1 (Sub1) locus that encodes two or three ethylene-responsive factor proteins (41, 159). SUB1A is induced by ethylene under submergence and negatively regulates SUB1C mRNA levels. Flood-intolerant varieties avoid submergence via the low oxygen escape syndrome (LOES). To this end SUB1C expression is promoted by gibberellic acid (GA) and is associated with rapid depletion of carbohydrate reserves and enhanced elongation of leaves and internodes. The LOES is unsuccessful when flooding is ephemeral and deep. Deepwater rice varieties survive flooding via a LOES, as long as the rise in depth is sufficiently gradual to allow aerial tissue to escape submergence (61). N.D., not determined. Bailey-Serres & Voesenek (2008) Annu Rev Plant Biol

Low O2 sensing - hypothetical but the paradigm is: low O2 sensor (receptor) → reactive oxygen species → Ca2+ transients → induction of fermentation enzymes (pyruvate decarboxylates and alcohol & lactate dehydrogenase), ROS scavenging enzymes, hemoglobin-like proteins etc. Low oxygen results in a 70% reduction in protein synthesis (reduces metabolism) and activation of carbohydrate mobilizing enzymes

Fermentation combined with reduced growth is adaptive - production of lactate lowers cytosolic pH, which inhibits lactate dehydrogenase leading to more production of ethanol (relatively nontoxic to the plants) Reduction in cytosolic pH is toxic, which is more acute when growth is rapid Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants

Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants Blue arrows identify reactions that are induced by low O2, including those that are involved in carbohydrate mobilization Figure 3 Metabolic acclimations under O2 deprivation. Plants have multiple routes of sucrose catabolism, ATP production, and NAD+ and NAD(P)+ regeneration. Blue arrows indicate reactions that are promoted during the stress. Metabolites indicated in bold font are major or minor end products of metabolism under hypoxia. Abbreviations are as follows: 2-OGDH, 2-oxyglutarate dehydrogenase; ADH, alcohol dehydrogenase; AlaAT, alanine aminotransferase; ALDH, acetaldehyde dehydrogenase; AspAT, aspartate aminotransferase; CoASH, coenzyme A; CS, citrate synthase; FK, fructokinase; GABA-T, GABA transaminase; GDC, glutamate decarboxylase; GDH, glutamate dehydrogenase; GHBDH, γ-aminobutyrase dehydrogenase; GOGAT, NADPH-dependent glutamine: 2-oxoglutarate aminotransferase; GS, glutamine synthase; HXK, hexokinase; ICDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; NDP kinase, nucleoside diphosphate kinase; NiR, nitrite reductase; NR, nitrate reductase; PCK, phosphenolpyruvate carboxylase kinase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; PEPC, phosphenolpyruvate carboxylase; PFK, ATP-dependent phosphofructokinase; PFP, PPi-dependent phosphofructokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; PK, pyruvate kinase; PPDK, pyruvate Pi dikinase; SDH, succinate dehydrogenase; SSADH, succinate semialdehyde dehydrogenase; Starch Pase, starch phosphorylase; SUS, sucrose synthase; UGPPase, UDP-glucose pyrophosphorylase.

Ethylene down-regulates ABA levels by reducing NCED expression Ethylene - low O2 induces ethylene biosynthetic genes and is the initial signal leading to shoot and leaf elongation Ethylene down-regulates ABA levels by reducing NCED expression Ethylene promotes GA function because ABA activates gibberellin oxidase (GA degrading enzyme) gene expression, i.e. ABA reduces GA levels Bailey-Serres & Voesenek (2008) Annu Rev Plant Biol GA activates genes that regulate , cell cycle and cell expansion and shoot and leaf elongation and starch breakdown Figure 4 Schematic model of the plant processes, hormones, and genes involved in submergence-induced shoot elongation (blue signifies upregulated genes and red signifies downregulated genes). Gene abbreviations are as follows: CYC2Os, cyclin; CDC2Os, cyclin-dependent kinase; OsACO and RpACO, ACC oxidase; OsACS and RpACS, ACC synthase; OsDD, differentially displayed (61); OsAMY, amylase (41); OsEXP, RdEXP, and RpEXP, expansins; OsGRF, growth-regulating factor (22); OsRPA, replication protein A1; OsSBF, sodium/bile acid symporter family (108); OsSUB1, submergence1; OsTMK, transmembrane protein kinase (133); OsUSP, universal stress protein (117); RpERS1, ethylene receptor (155); RpNCED, 9-cis-epoxycarotenoid dioxygenase; RpGA3ox, gibberellin 3-oxidase (8); OsXTR, xyloglucan endotransglucosylase-related (27); OsABA8ox, ABA 8-hydroxylase (110). Os indicates Oryza sativa, Rd indicates Regnellidium diphyllum, and Rp indicates Rumex palustris.

Low O2 regulates gene expression at the transcriptional and post-transcriptional levels, some transcripts are more efficiently translated

Hypoxia acclimates plants to tolerate anoxia Hypoxia facilitates ethanol and lactate (lowers pH) diffusion

Bailey-Serres & Voesenek (2008) Annu Rev Plant Biol Figure 4 Schematic model of the plant processes, hormones, and genes involved in submergence-induced shoot elongation (blue signifies upregulated genes and red signifies downregulated genes). Gene abbreviations are as follows: CYC2Os, cyclin; CDC2Os, cyclin-dependent kinase; OsACO and RpACO, ACC oxidase; OsACS and RpACS, ACC synthase; OsDD, differentially displayed (61); OsAMY, amylase (41); OsEXP, RdEXP, and RpEXP, expansins; OsGRF, growth-regulating factor (22); OsRPA, replication protein A1; OsSBF, sodium/bile acid symporter family (108); OsSUB1, submergence1; OsTMK, transmembrane protein kinase (133); OsUSP, universal stress protein (117); RpERS1, ethylene receptor (155); RpNCED, 9-cis-epoxycarotenoid dioxygenase; RpGA3ox, gibberellin 3-oxidase (8); OsXTR, xyloglucan endotransglucosylase-related (27); OsABA8ox, ABA 8-hydroxylase (110). Os indicates Oryza sativa, Rd indicates Regnellidium diphyllum, and Rp indicates Rumex palustris.

Lowland Rice Deepwater Rice Tolerant Intolerant Strategy Quiescence LOES Sub1 haplotype SUB1A-1, SUB1B, SUB1C SUB1B, SUB1C or SUB1A-2, SUB1B, SUSB1C SUB1B, SUB1C or SUB1A-2, SUB1B, SUB1C Carbohydrate consumption Limited by SUB1A-1 High Fermentation capacity Moderate N.D. GA response Inhibited by SUB1A-1 Promoted by SUB1C Figure 2 Rice responds via different strategies to submergence. Flood-tolerant rice varieties invoke a quiescence strategy that is governed by the polygenic Submergence1 (Sub1) locus that encodes two or three ethylene-responsive factor proteins (41, 159). SUB1A is induced by ethylene under submergence and negatively regulates SUB1C mRNA levels. Flood-intolerant varieties avoid submergence via the low oxygen escape syndrome (LOES). To this end SUB1C expression is promoted by gibberellic acid (GA) and is associated with rapid depletion of carbohydrate reserves and enhanced elongation of leaves and internodes. The LOES is unsuccessful when flooding is ephemeral and deep. Deepwater rice varieties survive flooding via a LOES, as long as the rise in depth is sufficiently gradual to allow aerial tissue to escape submergence (61). N.D., not determined.

Figure 1 Various species display the low-oxygen escape syndrome (LOES) when submerged. The syndrome includes enhanced elongation of internodes and petioles, the formation of aerenchyma in these organs (air spaces indicated by arrows labeled a), and increased gas exchange with the water layer through reduced leaf thickness and chloroplasts that lie directed toward the epidermis (indicated by arrows labeled b). Photographs are courtesy of Ronald Pierik, Liesje Mommer, MiekeWolters-Arts, and Ankie Ammerlaan.

Figure 3 Metabolic acclimations under O2 deprivation. Plants have multiple routes of sucrose catabolism, ATP production, and NAD+ and NAD(P)+ regeneration. Blue arrows indicate reactions that are promoted during the stress. Metabolites indicated in bold font are major or minor end products of metabolism under hypoxia. Abbreviations are as follows: 2-OGDH, 2-oxyglutarate dehydrogenase; ADH, alcohol dehydrogenase; AlaAT, alanine aminotransferase; ALDH, acetaldehyde dehydrogenase; AspAT, aspartate aminotransferase; CoASH, coenzyme A; CS, citrate synthase; FK, fructokinase; GABA-T, GABA transaminase; GDC, glutamate decarboxylase; GDH, glutamate dehydrogenase; GHBDH, γ-aminobutyrase dehydrogenase; GOGAT, NADPH-dependent glutamine: 2-oxoglutarate aminotransferase; GS, glutamine synthase; HXK, hexokinase; ICDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; NDP kinase, nucleoside diphosphate kinase; NiR, nitrite reductase; NR, nitrate reductase; PCK, phosphenolpyruvate carboxylase kinase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; PEPC, phosphenolpyruvate carboxylase; PFK, ATP-dependent phosphofructokinase; PFP, PPi-dependent phosphofructokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; PK, pyruvate kinase; PPDK, pyruvate Pi dikinase; SDH, succinate dehydrogenase; SSADH, succinate semialdehyde dehydrogenase; Starch Pase, starch phosphorylase; SUS, sucrose synthase; UGPPase, UDP-glucose pyrophosphorylase.

Figure 4 Schematic model of the plant processes, hormones, and genes involved in submergence-induced shoot elongation (blue signifies upregulated genes and red signifies downregulated genes). Gene abbreviations are as follows: CYC2Os, cyclin; CDC2Os, cyclin-dependent kinase; OsACO and RpACO, ACC oxidase; OsACS and RpACS, ACC synthase; OsDD, differentially displayed (61); OsAMY, amylase (41); OsEXP, RdEXP, and RpEXP, expansins; OsGRF, growth-regulating factor (22); OsRPA, replication protein A1; OsSBF, sodium/bile acid symporter family (108); OsSUB1, submergence1; OsTMK, transmembrane protein kinase (133); OsUSP, universal stress protein (117); RpERS1, ethylene receptor (155); RpNCED, 9-cis-epoxycarotenoid dioxygenase; RpGA3ox, gibberellin 3-oxidase (8); OsXTR, xyloglucan endotransglucosylase-related (27); OsABA8ox, ABA 8-hydroxylase (110). Os indicates Oryza sativa, Rd indicates Regnellidium diphyllum, and Rp indicates Rumex palustris.

Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants

Buchanan et al. (2000) (Biochemistry & Molecular Biology of Plants

Courtesy of Bob Joly Courtesy of Bob Joly

Summary of hypoxia and anoxia effects on plants through time Courtesy of Bob Joly