MINIREVIEW

J Plant Res (2002) 115:315–320

© The Botanical Society of Japan and Springer-Verlag Tokyo 2002Digital Object Identifier (DOI) 10.1007/s10265-002-0043-9

Oxygen stress and adaptation of a semi-aquatic plant: rice (

Oryza sativa

)

Avijit Das

•

Hirofumi Uchimiya

A. Das

1

•

H. Uchimiya

(

*

)

Laboratory of Cellular Function, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, JapanTel. +81-3-58418466; Fax 81-3-58418466e-mail: uchimiya@iam.u-tokyo.ac.jp

Present address

:

1

Division of Biochemistry, Central Rice Research Institute, Cuttack, Orissa, India

Springer-VerlagTokyoJournal of Plant ResearchJ Plant Res102650918-94401618-0860s10265-002-0043-90043Bot Soc Jpn and Springer-VerlagMinireview

Received: February 8, 2002 / Accepted: May 15, 2002 / Published online: July 18, 2002

Abstract

One of the major abiotic stresses that affects plantgrowth and development is anoxia or hypoxia. Rice is asemi-aquatic plant bestowed with the capability of over-coming oxygen limitation for a considerable period of time.For instance, it can withstand submergence stress eitherby inherent metabolic adaptations (resistant type), or bykeeping its leaves above the water surface by continuouslyelongating the stem (avoiding type). In the former case, aninterplay of several metabolic pathways engaged in anaer-obic fermentation keeps the submerged plant alive for acertain period of time. In the latter type, also known asdeepwater rice, continuous stem elongation brought aboutby a series of reactions in planta enables the shoot to remainabove the water surface and thus maintain respiration andphotosynthesis. However, the earliest event, i.e., sensing theoxygen level that brings about all the changes, has not beenclearly understood. This paper intends to evaluate the met-abolic adaptations of rice plants to oxygen constraints.

Key words

Anaerobic proteins

•

Anoxia

•

Hypoxia

•

Metabolic adaptation

•

Oxygen stress

•

Rice

Introduction

Oxygen is essential for the survival of higher eukaryotes.Plants often encounter hypoxic (a condition of low O

2

) oranoxic (complete deprivation of O

2

) environments whenthey are submerged by heavy rain or an ensuing flood.Because gas diffusion is 10

–4

-fold slower in solution than in

air (Armstrong 1979), the depletion of O

2

is a major featureof flooded sites, which creates hypoxia or anoxia aroundplant tissues such as seeds, or root apices and the stele(Collis and Melville 1974; Kennedy et al. 1992), even if thelatter contain aerenchyma (Armstrong and Beckett 1987;Thomson and Greenway 1991). Moreover, although flood-water O

2

concentration during flash floods is generally high,it may become anoxic in some environments where the O

2

produced during daytime photosynthesis is consumed byrespiration at night. Rice (

Oryza sativa

L.) is a semi-aquaticplant adapted to survive submergence for certain periods oftime. It is grown in a wide range of ecological situationsvarying from irrigated uplands to deep water where waterdepth can sometimes be greater than 1 m during flashfloods. Rice plants have developed various metabolic mech-anisms to deal with such physical problems. Anatomicalchanges maximize the use of available oxygen, and physio-logical adaptations allow metabolic pathways to functionunder limited oxygen conditions. The formation of aeren-chyma is the most important adaptive feature in rice, and isformed in the parenchyma tissues of roots and leaves(Kawai and Uchimiya 2000). The continuum thus formedfacilitates not only O

2

movement down to the roots, butalso the counter flow of volatile products such as methane,which have accumulated in tissues. This pathway may alsobe used to transport some O

2

that is produced by photosyn-thesis when sufficient light penetrates through the water(Vartapetian and Jackson 1997). The metabolic adaptationsin response to flooding are complex (Perata and Alpi 1993)and include such processes as avoidance of self-poisoningand the maintenance of an adequate energy supply (Dhari-wal et al. 1998). Deprivation of oxygen triggers cessation ofthe TCA cycle and the production of ATP is shifted from themitochondrial electron transport chain to substrate-levelphosphorylation of anaerobic glycolysis. This shift results inan alternative carbon flow and limited ATP synthesisthrough alcoholic fermentation. Among the cereals, onlyrice seeds can germinate and undergo coleoptile elongationunder anoxia (Alpi and Beevers 1983; Setter and Ella 1994).Some rice varieties are bestowed with a remarkable capa-bility to elongate their stems upon flooding. Sustained stem

316

elongation enables the shoot to remain above the water sur-face and, thus, they are able to survive. Avoidance of com-plete submergence by continuous stem elongation is typicalof deepwater rice, an ecotype that can elongate its stem byup to 25 cm a day to reach a length of about 5 m or more andproduce grains despite several months of deepwater stress(Vergara et al. 1976).

Metabolism under oxygen limitation

Plant metabolism undergoes rapid changes following expo-sure to oxygen stress. Respiration shifts to anaerobic modetogether with an up- and down-regulation of protein syn-thesis. The majority of the up-regulated proteins are directlyor indirectly involved in the processes (e.g., alcoholic fer-mentation) related to plant survival under submergence.

Synthesis of anaerobic proteins

Plants respond to oxygen stress by regulating gene expres-sion at both the transcriptional and translational levels.Such regulation leads to the repression of most aerobic pro-teins and the induction of anaerobic proteins (Sachs et al.1980). When anoxia is imposed, rice tissues synthesize moresoluble proteins. On the other hand, overall synthesis ofglycoproteins is repressed, excepting a few that carry outessential functions such as maintaining membrane integrity(Reggiani et al. 1990). Most of the anaerobic proteins are,however, enzymes involved in carbohydrate metabolism.Six of the inducible proteins have been identified as cytoso-lic enzymes [alcohol dehydrogenase, aldolase, glucose phos-phate isomerase, sucrose synthase, pyruvate decarboxylase(PDC), and glycerol phosphate dehydrogenase] in manycrops including rice (Walker et al. 1987; Kelly 1989; Ricardet al. 1989). These proteins are generally induced severalhours after the imposition of anoxia. Umeda and Uchimiya(1994) identified two categories of genes (types I and II)with respect to the pattern of accumulation of mRNA inresponse to anoxic stress. Transcripts of the type I genes,such as the genes for glucose phosphate isomerase, phos-phofructokinase, glycerol phosphate dehydrogenase, andenolase, reached a maximum level after 24 h of submer-gence. In contrast, transcripts of the type II genes, such asgenes for aldolase and pyruvate kinase, reached a maxi-mum level after 10 h of submergence. Huq and Hodges(1999) identified a gene family, the

aie

(anaerobically induc-ible early) genes, whose mRNA levels peaked after 1.5–3 hof anoxia and continued to remain at a high level even after72 h of anoxia. This family of genes putatively encodes aprotein of about 14 kDa with 127 amino acid residues,which is expected to play an important role in some of theearly events following submergence. Expression of otherenzymes, such as mitochondrial aldehyde dehydrogenase(Nakazono et al. 2000), starch phosphorylase (Das et al.2000), adenylate kinase (Kawai et al. 1997), cytosolic pyru-

vate orthophosphate dikinase (PPDK) (Moons et al. 1998),PDC (Rivoal 1997),

α

-amylase (Perata et al. 1993), andsucrose synthase (Ricard et al. 1991), are also stimulated insubmerged rice plants. Under oxygen limitation, substratelevel phosphorylation in the glycolytic pathway is the onlymeans of ATP production. However, this requires a regularsupply of ADP. Adenylate kinase converts one molecule ofATP and AMP to two molecules of ADP and, thus, it mayplay an important role in maintaining a balance of the com-ponents of the adenine nucleotide pool in the cell underanoxia (Kawai et al. 1992, 1997; Kawai and Uchimiya 1995).PPDK is known as an enzyme that regenerates phosphoe-nol pyruvate (PEP), the primary acceptor of CO

2

in C4 pho-tosynthesis. In seeds, PPDK may be involved in the deliveryof PEP for capturing respiratory CO

2

(Aoyagi and Bassham1989). However, its actual function in anoxic rice plants stillremains to be seen. Reserve foods, such as starch, becomethe main source of energy in submerged plants. Among thecereals, only rice seeds are able to degrade starch duringgermination under anoxia (Atwell and Greenway 1987;Perata et al. 1992) into readily fermentable carbohydratesby a number of hydrolytic enzymes such as

α

-amylase,

α

-glucosidase, and debranching enzyme (Perata et al. 1992;Guglielminetti et al. 1995a, b). This is probably the reasonfor the ability of rice seeds to germinate and elongatecoleoptiles under anoxia (Alpi and Beevers 1983; Setterand Ella 1994).

Alcoholic fermentation

When plants are subjected to oxygen deprivation, respira-tion shifts to anaerobic mode. This is reflected by thedecrease in the transcript levels of genes involved in aerobicrespiration (Tsuji et al. 2000). Several reports have revealedthe importance of increased rates of alcoholic fermentationfor energy production needed for plant growth underanoxia (Setter and Ella 1994; Setter et al. 1994; Gibbs et al.2000). There is a good correlation between anoxia toleranceand the rate of ethanol production, as reported by Setteret al. (1994) and Gibbs et al. (2000). The anoxia tolerant cul-tivars, FR13A and Calrose, had higher rates of alcoholicfermentation than the intolerant cultivars, IR 22 and IR42. The importance of alcoholic fermentation in the survivalof rice plants under submergence has also been demon-strated by using the

rad

(reduced ADH activity) mutant(Matsumura et al. 1998).

The two main genes (encoding ADH and PDC) in thealcoholic fermentation pathway are dramatically inducedby anaerobiosis (Umeda and Uchimiya 1994; Sachs et al.1996). Different rice cultivars differ greatly in their rates ofcoleoptile elongation and alcoholic fermentation underanoxia. Tolerant cultivars have a higher rate of coleoptileelongation and alcoholic fermentation (Setter et al. 1994;Gibbs et al. 2000). Tolerant cultivars confer higher activitiesof PDC, ADH, ATP-dependent phosphofructokinase(PFK), and pyrophosphate-dependent PFK (PFP) thansusceptible cultivars. In tolerant cultivars, PFP appears to

317

be the important site of regulation and potential carbonflux. Although this enzyme determines the rate of alcoholicfermentation in tolerant cultivars, in susceptible cultivars,the substrate supply seems to be the limiting step (Gibbs etal. 2000). Anoxia not only increases the level of PFP, butalso its stimulator fructose 2,6-bisphosphate. The potentialadvantage of the use of PFP rather than PFK (6-phosphof-ructo-1-kinase) is that, under anoxia, PFP could result inan increase of up to 50% in ATP yield during glycolysisthrough the use of PPi, a by-product of several biosyntheticreactions, as a phosphate donor (Mertens et al. 1990).

Starch degradation

During submergence, rice plants that have a higher starchreserve survive better than those that have relatively lowerstarch contents (Smith et al. 1987; Sarkar 1998). However,before being utilized as an energy source, starch must firstbe converted to simple sugars. Rice seeds germinatingunder anoxia appear to have a complete set of enzymes:

α

- and

β

-amylase,

α

-glucosidase, debranching enzyme,maltase, and the enzymes needed for the complete degra-dation of starch and its subsequent utilization through gly-colytic flux (Perata et al. 1992; Guglielminetti et al. 1995a,b). Rice has an advantage over other common cereals suchas wheat, barley, oat, and rye in terms of its capacity todegrade starch under anoxia. The inability of other cerealseeds to induce

α

-amylase is because of their inability torespond to gibberellic acid (GA) under conditions of totalO

2

deprivation (Perata et al. 1993). Two phases can be rec-ognized in the metabolism of carbohydrates in rice seedsgerminating under anoxia. The first phase is characterizedby the catabolism of sugars present in the dry seed, and thesecond phase, which starts only after the induction of

α

-amylase, is characterized by increased glucose and sucroseconcentration. The synthesis of sucrose in the germinatingrice seeds under anoxia has been substantiated by thesimultaneous induction of sucrose synthase and glucose-6-phosphate isomerase (Bertani et al. 1981; Ricard et al. 1991;Guglielminetti et al. 1995a). Greatly elevated levels ofsucrose synthase and fructokinase (Guglielminetti 1995a)may be among the adaptations that enable the shoot tissueto import and metabolize sufficient sucrose to supportgrowth, despite the low energy yield of fermentative metab-olism. Growth of rice seedlings under anoxia correlates withthe increased expression of the

Amy3

subfamily genesencoding

α

-amylase in the embryo (Hwang et al. 1999). Thetwo

α

-amylase isozymes, which are found in anoxic riceseedlings, but not in aerobic seedlings, may be encoded bythe

Amy3

family of genes. Under aerobic conditions, thealeurone is the primary site of

α

-amylase production,whereas the scutellum tissue is the major producer of Amy3isozyme under anoxia (Hwang et al. 1999). Amylase induc-tion in submerged rice (deepwater rice) has previously beendescribed by Kende and his associates (Raskin and Kende1984a; Smith et al. 1987). These authors have suggested thatenhanced

α

-amylase activities are probably responsible for

the mobilization of carbohydrates, which are needed tosupport internode elongation during submergence of deep-water rice. Even when 3-week-old rice plants were sub-jected to submergence, the activity of starch phosphorylase,an enzyme reported to be involved in the synthesis as wellas the degradation of starch, increased in both submer-gence-tolerant and susceptible cultivars. However, theincrease was much higher in the tolerant cultivars than thesusceptible cultivars (Das et al. 2000).

Role of hormones

Rice plants can overcome submergence stress temporarilyeither by inherent defense mechanisms (tolerant type),or by continuously elongating the stem so that the shootalways remains above the water level (avoidance type). Thelatter type is also known as deepwater rice. The elongationof the stem by deepwater rice with increasing water level ismediated by ethylene. Ethylene accumulates as a result ofphysical entrapment because of its low rate of diffusion inwater. In addition, the sub-atmospheric oxygen level alsoleads to an increase in de novo ethylene synthesis (approx-imately eightfold) through the activation of 1-aminocyclo-propane-1-carboxylic acid (ACC) synthase (ACS; Metrauxand Kende 1983; Cohen and Kende 1987). ACS is encodedby a multigene family that consists of at least five membersin rice (Zarembinski and Theologis 1993, 1997; Van DerStraeten et al. 2001).

Os-ACS1

and, very recently,

Os-ACS5

are the only rice

ACS

genes that have been implicated insubmergence response thus far (Zarembinski and Theologis1997; Van Der Straeten et al. 2001). Partial submergenceinduces the expression of

OS-ACS1

, but suppresses the

OS-ACS2

gene. The induction of

OS-ACS1

occurs within 12 h ofpartial submergence under low O

2

concentrations (2.5% orless). The

OS-ACS1

mRNA accumulation parallels the ACCaccumulation in the intercalary meristem (IM) zone abovethe second node of the rice plant. In contrast,

OS-ACS-5

isinduced by short-term submergence (1 h). A strong accu-mulation of

OS-ACS-5

mRNA was also observed after2 days of submergence and the accumulation was muchhigher than

OS-ACS1

mRNA. It has been suggested that inseedlings

OS-ACS5

may contribute to an initial growth-promoting increase in ethylene synthesis. Thus, accumula-tion of ethylene by limited gas diffusion and an enhancedethylene production may lead to the burst of growth in theearly phase. Although the growth response of seedlings andadult plants of lowland and deepwater rice cultivars is quitesimilar with short-term submergence, a marked differencearises with the long-term submergence of seedlings. In con-trast to the lowland cultivar, deepwater rice has a boost of

OS-ACS5

and

OS-ACS1

mRNA accumulation, which is cor-related with sustained growth. Ethylene may take a role ininternode elongation of deepwater rice (Metraux andKende 1983; Raskin and Kende 1984a) by increasing theresponsiveness for and the levels of GA (Raskin and Kende1984a, b; Hoffman-Benning and Kende 1992; Sauter andKende 1992) and by decreasing ABA contents in the IM

318

zone (Hoffman-Benning and Kende 1992; Azuma et al.1995). Because the effects of ABA are antagonistic to thoseof GA (Hoffman-Benning and Kende 1992), the effectiveGA concentration increases more than fourfold. Theincrease in GA level, coupled with the increase in respon-siveness, is believed to induce cell division followed by cellelongation in the IM zone (Sauter and Kende 1992;Lorbiecke and Sauter 1998).

Anaerobic signal transduction

A very important aspect of anaerobic metabolism in organ-isms is the sensing of oxygen level at the cell surface. In N

2

-fixing bacteria, this is carried out by a two-component reg-ulatory cascade, which consists of a membrane-bound haemprotein (FixL) and a cytoplasmic protein (FixJ; Gilles-Gonzalez et al. 1991). However, not much is known aboutanaerobic signal transduction in plants. As in other cases, inplants the signal is thought to be transduced inside the cellthrough the production of a second messenger. Duringanoxia, transcription of genes encoding anaerobic proteinssuch as ADH, sucrose synthase requires Ca

2+

mobilizationfrom intracellular stores to cytoplasm (Subbaiah et al.1994). Moreover, the Ca

2+

/calmodulin (CaM) complex hasbeen shown to control anaerobic protein degradation andsolute release (Aurisano et al. 1995). Evidence for theinvolvement of Ca

2+

as a physiological transducer of low O

2

concentration in rice plants has come from the study of Tsujiet al. (2000), who reported that Ca

2+

indeed acts as a phys-iological transducer for the expression of alternate oxidase1a (

AOX1a

) gene, but not for the expression of genesinvolved in the cytochrome respiratory pathway. Anothertarget of anaerobic signal transduction is CaM-dependentglutamic acid decarboxylase (GAD). This enzyme convertsglutamic acid to

γ

-amino butyric acid (GABA), which accu-mulates at high levels in rice seedlings in response to anoxia(Reggiani et al. 1988). Anaerobic GABA accumulation inrice roots is Ca

2+

dependent. The level of GABA is alsoincreased by stimulation of GTP-binding proteins byGTP

γ

S or aluminum fluoride (AlF

4–

) in rice roots, whereasthe opposite is observed by inhibiting G-proteins with GDPor GDP

β

S (Aurisano et al. 1996). In higher plants, a possi-ble target for activated GTP-binding proteins is phospholi-pase C (PLC). PLC hydrolyzes phosphatidyl inositol4,5-bisphosphate and two second messengers, inositol1,4,5-triphosphate (IP

3

, an inducer of Ca

2+

release) and 1,2-diacylglycerol (an activator of protein kinase C), are gener-ated. Pretreatment of rice roots for 2 h in aerobic conditionswith inhibitors of PLC inhibited the accumulation ofGABA, and increased the loss of K

+

in the medium during3 h of anoxia. A similar treatment of rice roots also abol-ished the anaerobic increase in the concentration of thePLC product IP3. Moreover, the stimulation of the anaer-obic signal transduction pathway with AlF

4–

was attenuatedby PLC inhibitors. These studies have emphasized the roleof PLC in anaerobic signal transduction in rice plants(Reggiani and Laoreti 2000).

Post-submergence oxidative injury

When floodwater recedes, the plants that have been underwater start experiencing higher O

2

levels on returning to anormal atmosphere. Re-exposure to air results in oxidativeinjuries caused by reactive O

2

species such as O

2–

, H

2

O

2

, andOH

–

(Crawford et al. 1992; Blokhina et al. 2001). Thesereactive species damage cellular and organelle membranesby oxidizing unsaturated fatty acids of the membrane-lipidbilayer. This leads to leakage of cellular contents and affectsrespiratory activity in mitochondria and the carbon-fixingability of chloroplasts (Scandalios 1993). Plants, thus, musthave some active oxygen-scavenging systems as a defensemechanism against these reactive oxygen species in order tosurvive injury after submergence. Antioxidative enzymes,such as catalase (CAT), superoxide dismutase (SOD),ascorbate peroxidase (APX), monodehydroascorbatereductase (MDAR), dehydroascorbate reductase (DHAR),and glutathione reductase (GR), are involved in the detox-ification of these reactive species in plants (Asada andTakahashi 1987). Post-anoxic (hypoxic) injury, which is oxi-dative damage after transfer to normoxia (atmospheric O

2

level), has been reported in rice seedlings (Ushimaru et al.1994). The antioxidative system is immature in rice seed-lings germinated under water, but is rapidly (within 24 h)synthesized after exposure of the seedlings to air. Activitiesof the active O

2

-scavenging enzymes and the levels of anti-oxidants (oxidized ascorbic acid, reduced glutathione)increase during the first 24 h after de-submergence(Ushimaru et al. 1992, 1999) accompanied by the develop-ment of the electron transport system in mitochondria(Shibasaka and Tsuji 1988). Moreover, the activities ofCAT, DHAR, and GR exceed the control level (Ushimaruet al. 1992). These observations imply that the antioxidativesystem, in addition to the oxygen-utilizing system, is regu-lated in response to changes in oxygen tension. Hypoxictreatment of aerobic seedlings decreases the activities ofSOD, MDAR, and DHAR to some degree. On the otherhand, the activities of APX and GR show no marked changeafter 24 h of submergence. In contrast, catalase activity isincreased by the shift to anoxia (Ushimaru et al. 1999). Theincrease in catalase activity may reflect its necessity duringsubmergence. It has been reported that rice plants that ger-minate under water can utilize a small amount of molecularoxygen, which may be used for respiration in mitochondriaand

β

-oxidation in glyoxisomes (Ushimaru et al. 1992). Thelatter process produces H

2

O

2

and the former generates O

2–

,which is also converted to H

2

O

2

by the action of SOD. It islikely that a small amount of H

2

O

2

may be generated underhypoxic conditions. Thus, the increased catalase activitymight contribute to the conversion of H

2

O

2

generated in thecells to O

2

because the seedlings that possess low alcoholdehydrogenase activity seem to be unable to survive by fer-mentation alone (Ushimaru et al. 1999). In another study,Sarkar and Das (2000) observed that submergence, in gen-eral, reduced the activities of anti-oxidative enzymes such asSOD, CAT, peroxidase (PER), and ascorbic acid oxidase(AAO). The activities of SOD, AAO, CAT, and PER

319

increased from submerged to air-adapted conditions; how-ever, although the activities of CAT and PER reached thesame level as that of the air-grown seedlings, the activities ofSOD and AAO still remained lower than that of the aerobicseedlings. The tolerant cultivar, FR13A, had higher activi-ties of CAT and PER and this was attributed to the higherroot activities, as measured by

α

-napthylamine oxidation, ofthe tolerant cultivar and hence it was able to utilize more O

2

compared with the susceptible cultivar IR42 (Sarkar andBera 1997). Both ascorbic acid and aldehyde contentsincrease under submergence, but decrease on re-exposureto air. Aldehydes, such as acetaldehyde, are highly toxic tothe cell and are released at higher rates under submergedconditions and continue to be released, albeit at slowerrates, even when re-exposed to air. The susceptible cultivar,IR42, releases more aldehydes under all conditions (Sarkarand Das 2000). Lipid peroxidation has been reported to be1.2- to 1.4-fold higher in IR42 than FR13A. The activities ofenzymes such as APX (except for the first 24 h after de-submergence), SOD, and GR, and the level of oxidizedascorbic acid measured after de-submergence, are higher inFR13A than in IR42 (Ito et al. 1999).

It is highly conceivable that during evolution, rice plantsdeveloped composite adaptive mechanisms for respondingto anoxia, resulting in suitable functional and morphologi-cal phenotypes. The high tolerance of rice plants to anoxiaappears to be composed of a cross talk of genetically pro-grammed elements. Understanding these genetic and bio-chemical programs will pave the way for the developmentof cultivars with superior agronomic traits. The survival ofrice plants under sustained submergence is modulated atthe level of the signaling hormones (e.g., ethylene) and thehormones that control the elongation response. The rela-tionship between enhanced ethylene level and the stimula-tion of aerenchyma formation associated with programmedcell death (Kawai et al. 1998; Kawai and Uchimiya 2000;Matsukura et al. 2000) still remains to be investigated.Moreover, abiotic stresses, such as salt, have been reportedto adversely affect cell division in rice roots (Samarajeewaet al. 1999). However, how and to what extent oxygen stressaffects this process still remains to be seen.

Acknowledgment

This research was supported by Research for theFuture from the Japan Society for the Promotion of Science.

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