Role of defense/stress-related marker genes, proteins and secondary metabolites in defining rice...

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Plant Physiology and Biochemistry 44 (2006) 261–273

Review article

Role of defense/stress-related marker genes, proteinsand secondary metabolites in defining rice self-defense mechanisms

Nam-Soo Jwaa, Ganesh Kumar Agrawalb,1, Shigeru Tamogamic, Masami Yonekurad,Oksoo Hane, Hitoshi Iwahashif, Randeep Rakwalb,f,*

aDepartment of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Republic of KoreabResearch Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), GPO Box 8207, Kathmandu, Nepal

c Laboratory of Growth Regulation Chemistry, Department of Biological Production, Akita Prefectural University, Akita 010-0195, JapandFood Function Laboratory, School of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan

* Corresponding autE-mail address: ra

1 Present address:Columbia, 204 Life S

0981-9428/$ - see frondoi:10.1016/j.plaphy.2

eDepartment of Biotechnology, Agricultural Plant Stress Research Center, Biotechnology Research Institute,
College of Agriculture and Life Sciences, Chonnam National University, Buk-gu, Kwangju 500-757, Republic of Korea
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fHuman Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science
and Technology (AIST) WEST, Onogawa 16-1, Tsukuba, Ibaraki 305-8566, Japan
Received 5 October 2005Available online 16 June 2006

Abstract

Rice, a first cereal crop whose draft genome sequence from two subspecies (japonica-type cv. Nipponbare and indica-type 93-11) was avail-able in 2002, along with its almost complete genome sequence in 2005, has drawn the attention of researchers worldwide because of its immenseimpact on human existence. One of the most critical research areas in rice is to discern the self-defense mechanism(s), an innate property of allliving organisms. The last few decades have seen scattered research into rice responses to diverse environmental stimuli and stress factors. Ourunderstanding on rice self-defense mechanism has increased considerably with accelerated research during recent years mainly due to identifica-tion and characterization of several defense/stress-related components, genes, proteins and secondary metabolites. As these identified componentshave been used to study the defense/stress pathways, their compilation in this review will undoubtedly help rice (and others) researchers toeffectively use them as a potential marker for better understanding, and ultimately, in defining rice (and plant) self-defense response pathways.© 2006 Elsevier SAS. All rights reserved.

Keywords: Defense/stress response; Oryza sativa; Pathogenesis-related protein genes; Potential markers; Oxidative stress; Phytoalexins

1. Introduction

The grass family member rice (Oryza sativa L.) along withother important crop plant species, maize, wheat, barley, sor-ghum and sugarcane provide most of the world’s food forhuman consumption and animals. If a single plant specieswere to be voted the most popular by scientists and laymenalike, it would be “rice” [35,135]. Rice is produced all around

[email protected] (R. Rakwal).chemistry Department, University of Missouri-ces Center, Columbia, MO 65211, USA.

atter © 2006 Elsevier SAS. All rights reserved..06.010

the globe from Nepal, which has the highest rice fields in theworld, to Australia, which has the most productive rice lands[Rice Web, http://www.riceweb.org]. Rice (O. sativa ssp.japonica-type cv. Nipponbare and indica-type) is a modelplant among the monocotyledonous cereal crop species basedon the fact that rice i) has a relatively small genome of about440 Mb (the maize genome is 2500 Mb, and that of barley4900 Mb) [20], ii) is suitable for efficient genetic analysisand transformation [69], iii) draft genome sequence of boththe japonica (cv. Nipponbare [58]) and indica (cv. 93-11[169]) subspecies is available [32] along with the completegenome sequence [73], and iv) has high degree of homologywith other cereal genes, where synteny rice and other cereals,especially with maize [32]. Therefore, rice becomes one of the

http://www.riceweb.org












mailto:[email protected]

dx.doi.org/10.1016/j.plaphy.2006.06.010

N.-S. Jwa et al. / Plant Physiology and Biochemistry 44 (2006) 261–273262

most critical plant species to study and improve due to the con-stant threat of global climate change coupled with disastrousyield losses by “pest and pathogen” attack, and a rapid increasein the world’s population growth.

“Stress alleviation or disease control” remains one of themost challenging issues to be addressed, which is especiallytrue for rice considering the largely undefined area of riceself-defense mechanisms. The hypersensitive reaction (HR),one of the most efficient (and visible) parts of the defensemechanisms in nature against invading pathogens [57,99]and/or stresses, is associated with a coordinated and integratedset of metabolic alterations which are instrumental in impedingfurther pathogen ingress or alleviating stress, and includes avariety of novel proteins (genes), and secondary metabolites.Active research on various aspects of defense/stress responsein rice has resulted in the identification of a variety of responsegenes, proteins and antifungal secondary metabolites inducedin response to diverse environmental stresses, including patho-gen attack. These studies have provided novel insight intothese responses and mark a major advance in our understand-ing on the rice self-defense mechanisms. These components arethe responses of integrated signaling networks triggered underunfavorable circumstances, and hence they were assigned as apotential marker(s) based on their responses to a specific sti-muli and/or multiple factors (Fig. 1). As these markers are inti-mately associated with a plant response to its environment,these marker genes, proteins, and secondary metabolites areof particular importance for studying the defense/stress path-

Fig. 1. Environmental factors triggering the rice self-defense mechanisms with a partcomponents are the responses of integrated signaling networks triggered under unfbased on their responses to a specific stimuli and/or multiple factors.

ways. Hence, the purpose of this review is to summarize thepast and present progress made on this important aspect anddiscuss their usefulness in future studies in rice. It is empha-sized here that we mostly deal with published articles in theliterature databases (PubMed and ScienceDirect) and haveincluded the genes, proteins and metabolites that have beenexperimentally demonstrated to be a part of the rice self-defense responses, in the current review. The availability ofthe rice genome sequence no doubt has increased the coverageof the rice genes homologous to known genes, but these remainto be functionally characterized and will be dealt in future stu-dies.

2. Pathogenesis-related protein genes

Plant responses to attack by pathogenic microorganisms arecomplex, and involve the induction of expression of a largearray of genes encoding diverse proteins, many of which arebelieved to have a role in defense/stress. Among these the pro-duction/accumulation of pathogenesis-related (PR) proteins inplants in response to invading pathogen and/or related stresssituations [24,159] is one of the crucial components in theinducible repertoire of the plant’s self-defense mechanism,and used widely as marker genes/proteins to study the plantself-defense mechanism(s). The PR proteins were initially clas-sified into 5 families based on molecular mass, isoelectricpoint, and localization and biological activity [159]. The latestproposition divides the currently known PR proteins into 14

icular focus on endogenous markers involved in defense/stress responses. Theseavorable circumstances, and hence they were assigned as a potential marker(s)





N.-S. Jwa et al. / Plant Physiology and Biochemistry 44 (2006) 261–273 263

families based on sequence or predicted sequence of aminoacids, serological relationships and biological activity [160,161]. In 2001, at the 6th International Workshop on PR pro-teins (Spa, Belgium), 3 new PR families have been added inthis list, namely PR 15 (oxalate oxidase), 16 (OxOLP) and 17(tentative cDNAs-barley NtPRp27).

2.1. OsPR1ab

The PR proteins have been widely studied in dicots, in par-ticular potato, tomato and Arabidopsis thaliana, and focus hasbeen on the PR class 1 (PR1), a dominant group and com-monly used as a marker for systemic acquired resistance(SAR [92]. The widespread occurrence of the PR1-type pro-teins suggests that these proteins share an evolutionary originand possess activity essential to the functioning and survival ofliving organisms. In rice, induction of a basic PR1 (OsPR1b)protein in leaf was first shown against the blast pathogen(Magnaporthe grisea) attack and jasmonic acid (JA [133])treatment by immunoblotting using an antibody raised againstthe major basic PR1 of tomato [145]. The first example of aninducible acidic PR1 (OsPR1a) protein of 17 kDa was reportedin leaf sheaths of 2-week-old rice (cv. Nipponbare) seedlingstreated with JA, using two-dimensional gel electrophoresis (2-DGE) coupled with amino acid sequencing [120]. Later, it wasshown to be induced in JA treated seedling leaves under dark-ness, and after treatment with copper using an antibody raisedagainst the 17 kDa OsPR1a protein [126]. This particular anti-body also cross-reacted with two additional proteins with a ca.molecular mass of 19 and 22 kDa in (and around) lesionsformed after M. grisea inoculation in leaves and leaf sheathsof mature rice plants [126]. These results suggest the presenceof multiple OsPR1a protein isoforms in rice, and their induc-tion depends on the applied stress, tissue used and light signal(s). Simultaneously, our group launched a systematic study on“rice defense/stress response”, and the first rice PR genescloned by our group were the OsPR1a [3] and OsPR1b [6]. Itshould be noted that our group [3,6,10] has consistently used2-week-old rice cv. Nipponbare seedlings leaves as an in vitro-and in vivo-model system to study rice self-defense mechan-isms, unless otherwise stated. These two OsPR1 genes wereshown to be differentially regulated by diverse signals, suchas wounding by cut (hereafter called wounding), global signal-ing molecules, JA, salicylic acid (SA), abscisic acid (ABA),ethylene (using ethylene generator ethephon, ET), hydrogenperoxide (H2O2), potent protein phosphatase (PP) inhibitors,cantharidin (CN) and endothall (EN), and M. grisea infection[10]. From these studies a certain level of complexity involvedin their regulation was suggested. It is important to mentionthat these two studies was the first such demonstration onresponses of these two widely used marker proteins/genes (indicots) against multiple stresses, and which led us to think anduse a diverse array of abiotic and biotic stresses to examine theresponse of a particular gene in detail [10, and referencestherein]. These findings are also in line with the idea thatthere is a substantial network of regulatory interactions andcoordination during plant response to stress among the differ-

ent signaling pathways [53,144]. A comparison of rice OsPR1genes expression with widely studied tobacco PR1 genes man-ifested fundamental and striking differences in their regulation[10]. In light of detailed work on rice OsPR1 genes, a simpli-fied working model was also proposed, where components ofthe signaling pathways as individual entities and their interac-tions up-stream of defense gene(s) activation were shown,which reveals their essential role in defense/stress responsesin rice and use as potent markers.

2.2. OsPR2 (endo-β-glucanases)

The family of PR2 proteins, β-1, 3-glucanases (glucan endo-1,3-β-glucosidases, E.C. 3.2.1.39), are able to catalyze endo-type hydrolytic cleavage of the 1,3-β-D-glucosidic linkages inβ-1,3-glucans. β-1,3-glucanases, abundant, highly regulatedenzymes widely distributed in seed-plant species, exist as mul-tiple structural isoforms that differ in size, isoelectric points,primary structure, cellular localization and pattern of regula-tion. The tobacco β-1,3-glucanases, which form a multigenefamily, are classified into three structural classes (class I–III)[90]. Interestingly, a pronounced synergistic effect in reducingthe susceptibility of plants to infection by certain fungi wasobserved in plants expressing β-1,3-glucanases transgenesalone or in combination with chitinase transgenes [100,172].Rice β-1,3-glucanase gene, Gns1 (GenBank X58877) was thefirst to be cloned and characterized [147]. Its expression wasshown to induce at much higher level in shoots upon wound-ing, and treatment with ethylene, cytokinin, SA, and fungalelicitors derived from the pathogen Sclerotium oryzae or fromthe non-pathogen Saccharomyces cerevisiae. Later, 13 new riceβ-glucanase-encoding genes, and together with other monocotβ-glucanases, were classified into four subfamilies (A–D)based on their structure and function [136]. Our characteriza-tion of the OsGns1 [subfamily B] transcript in leaves revealedits potent up-regulation by JA, CN and EN, further suggestinga role for OsGns1 in defense/stress response [Agrawal et al.,unpublished data]. Moreover, a JA-inducible protein in maturerice (cv. Hitomebore) leaves, showing 88% homology with β-glucanase precursor was identified using 2-DGE, which pro-vided first functional evidence for induced accumulation of aGns-like protein in rice [123]. Further functional evidence onOsGns1 comes from its overexpression, under the control ofthe CaMV (Caulimosaic virus) 35S promoter in rice, whichimparted resistance against M. grisea, accompanied by HRand increased PR protein expression [116].

2.3. Chitinases

Outstanding among the PR proteins are the chitinases (EC3.2.1.14), that catalyze the hydrolysis of chitin, a linear homo-polymer of β-1,4-linked N-acetylglucosamine residues [40].Chitinases, belonging to the PR3, 4, 8 and 11 families, havebeen grouped into six classes (class I–VI) based on the primarystructure [40,89,112]. A major natural role for chitinase indefense has been proposed mainly due to their ability to inhibitthe growth of many fungi in vitro, and accumulate around fun-


gal hyphae material in planta [25,26,31,40]. In rice only fewchitinase genes have been identified and characterized amongthis large chitinase family. For example, rice class I chitinases(Cht-1-3) were shown to be induced in the leaves by wound-ing, a fungal elicitor, ethylene, and SA [113–115,167,171].The functional significance of chitinase genes comes fromrice plants overexpressing these genes, imparting resistance tofungal attack [93,172]. Class I chitinases are known to be regu-lated in response to various stresses [119]. On the other hand, asingle rice class II acidic chitinase, Rcht2 (cv. Cheongcheong-byeo) is known, and whose transcriptional activation with bothglycol chitin and fungal elicitor was shown [80]. The transcriptof a basic chitinase gene, designated RC24, was observed inwounded leaf, and rapidly shown to accumulate within 1 hafter fungal elicitor treatment of suspension-cultured cells[167]. An independent group used genetic manipulation ofrice OsPR3 chitinase cDNA (RC7), isolated from Rhizoctoniasolania infected rice plants, into indica rice cultivars IR72,IR64, IR68899B, MH63, and Chinsurah Boro II to controlrice sheath blight disease [42]. Recently, a novel OsPR4(accession number AY050642) single copy gene was identified[14]. Its transcript differentially accumulated in abundancewith time during a compatible and incompatible host-pathogen interaction. Interestingly, wounding did not induceits weak constitutive expression, whereas JA, ABA CN, ENand okadaic acid (OA), but not SA, ET and H2O2, stronglyup-regulated its transcript. These studies suggest that there arechitinases belonging to different families and classes in rice,and the discovery of novel chitinases (especially of family 8and 11) will further shed light on our understanding howthese genes function and are regulated by environmental fac-tors.

2.4. OsPR5 (thaumatin-like proteins)

The PR5 family comprises proteins displaying a certaindegree of homology over their amino acid sequences to thesweet tasting thaumatins from Thaumatococcus daniellii [51]and also includes proteins termed osmotins [148,149] and per-matins [162]. PR5s accumulate in plants in response to stressconditions, e.g. high salt concentrations, wounding or pathogenattack. In vitro bioassays have shown their antifungal activity[66,162]. Constitutively, they are thought to play a significantrole in protecting seeds against fungal attack during storage orgermination [41,66]. The presence of a low molecular weight(19 kDa) OsPR5 protein was first reported in leaves of maturerice (cv. Hitomebore) plants treated with JA, heavy metal cop-per, and short wave UV-irradiation, using 2-DGE [123].Furthermore, initial study on the overexpression of OsPR5gene conferred increased resistance to sheath blight disease[43]. Later, using a specific anti-OsPR5 polyclonal antibody,its induction was demonstrated in JA and copper treated leaves,leaf sheaths and roots of 2-week-old rice seedlings and inmature plants after M. grisea infection [120,126]. A pathogeninducible rice OsPR5 cDNA (pPIR2, EMBL accession no.X68197) having the same N-terminal amino acid sequence asthe OsPR5 protein [123] was cloned and shown to be induced

after infection with Pseudomonas syringae pv. syringae [131].Its detailed characterization revealed its weak constitutive nat-ure in seedling leaves, which is induced by wounding, JA, SA,ET, a cytokinin, kinetin (KN), but not ABA, 3-indolacetic acid(IAA), and gibberellin (GA3), and PP inhibitors, CN and EN[121]. As our Southern analysis data indicates the presence ofan OsPR5 multigene family in the rice genome (Rakwal et al.,unpublished data), it is believed that further identification andcharacterization of novel rice PR5 genes will provide addi-tional information on their role/function in rice plant defense.

2.5. Proteinase inhibitors

Proteinase inhibitors (PIs), identified before the discovery ofPR proteins, belongs to the PR6 family, and are widely distrib-uted in the plant kingdom. They are of special interest becausethey appear to be an essential part of a plant’s natural defenseagainst pathogens, including a role in development (for review,see [65,87,137,138]). Among the PIs, serine-PIs have been stu-died in detail, where two non-homologous PIs, inhibitor I andII, are by far the best examples [137]. Two other known sub-classes are the Bowman–Birk- (BB) and Kunitz-type inhibi-tors, which were isolated in 1946 from soybean, and sincethen their homologues have been characterized from otherplants. Although found in many plant species, the cysteine-,aspartyl-, and metallo-PI classes have not been analyzed exten-sively [65,138]. Two cysteine-PIs (also called “cystatins”), ory-zacystatin I/II have been characterized from rice seeds [1,84].Two studies reflect the importance of PIs in rice plant defense.The introduction of dicot PI genes (cowpea trypsin-PI andpotato PI-II) into rice caused significant resistance againstinsect pests [49,166], and transfer of a corn cystatin gene intothe rice genome showed that crude extracts of transgenic plantsefficiently inhibited cysteine proteinase activity in the midgetsof the rice pest Sitophilus zeamais [75]. Recently, we reportedtwo novel PI genes, OsBBPI and OsPIN from rice leaves [12,122]. OsBBPI, part of a multigene family in the rice genome,was shown to be transcriptionally up-regulated in seedlingleaves in response to cut, JA, CN and EN, in a light/dark-,time- and dose-dependent manner. On the other hand, OsPIN,isolated from a differentially expressed cDNA library of therice leaves infected with M. grisea, was specifically inducedin abundance with time in a compatible but not incompatiblehost-pathogen interaction. Considering its response only topathogen, it is believed that OsPIN can be used as a markerfor studying the signaling pathway triggered uponrice–M. grisea interaction.

2.6. OsPR10

The PR10s, a ubiquitous class of intracellular (in contrast tothe extracellular nature of most PR proteins) defense-relatedproteins, were first described in cultured parsley cells upon eli-citor treatment [151]. Although PR10s are similar to the PR1sin respect to molecular mass and acidic nature, they are notstructurally related to the classical PR1 protein markers oftobacco [159–161]. PR10s have been attributed a


“ribonuclease-like function” due to sequence homology withginseng ribonucleases [111,160]. Interestingly, PR10s alsoshare amino acid sequence similarity to pollen allergens oftrees [30] and the major food allergen of celery [29], suggest-ing their diverse functions. The first rice PR10 gene was iden-tified after treatment with probenazole (PBZ), and hencetermed PBZ1 [108]. Later, using 2-DGE, we identified threeOsPR10 proteins, OsPR10a-c, ranging in size from 18–20kDa, from stressed rice plants [63,123,124,128]. OsPR10a issimilar to the previously cloned PBZ1, and was characterizedin detail at transcript level [7,124]. OsPR10a, which did notexpress constitutively in leaves, was unresponsive to wound-ing, but potently up-regulated by JA, ET, KN, CN and EN[124]. Moreover, in the same study, a good correlation betweenOsPR10a gene expression and its protein accumulation wasestablished using 2-DGE. The same year, an independentgroup took a genomic approach and cloned three rice PR10genes, named RPR10a-c [106], where RPR10a was 99% iden-tical to the OsPR10a gene. The RPR10a and b genes were alsofound to be up-regulated by M. grisea infection, whereRPR10a is expressed most strongly in a localized fashion[106]. This result complements previous data on OsPR10sinvolvement in defense. Indeed in pea, a relationship betweenthe expression of a PR10 protein and disease resistance hasbeen demonstrated [134]. The wide spread occurrence and con-servation of the PR10 proteins within the plant kingdom,including the monocot sorghum [97] and the dicot cotton[105] that were different from the characterized OsPR10 pro-teins, led to the identification of JA-inducible PR10 gene,termed JIOsPR10, a homolog of sorghum PR10 gene [78].The expression of JIOsPR10 was not constitutive and respon-sive to wounding, and it was up-regulated by JA, SA, H2O2,CN, EN, and M. grisea infection, but not by ET and ABA. Asthe OsPR10a-c proteins were recently detected from leaves ofrice seedlings exposed to notorious environmental pollutantsozone [O3, 15] and sulfur dioxide [SO2, 129], and after treat-ment with a fungal elicitor chitosan [16], they can serve asglobal marker for defense/stress responses.

2.7. Thionins (PR13)

Thionins, present in monocot and dicot species, are of par-ticular interest because of their antimicrobial activity in plants.These proteins are present in cell walls, vacuoles, and proteinbodies, and have been isolated as the 5 kDa mature (butsynthesized as much larger precursors) proteins. They havebeen grouped into four major classes (I–IV) based on the num-ber of cysteine residues and the pattern of disulfide bridges.The well studied barley and Arabidopsis thionins form asmall gene family, and are possibly involved in defense,where the Arabidopsis Thi2.1 gene is a known marker gene forJA-inducible defense/stress responses (reviewed in [23]).Recently, a genetic manipulation of oat thionin into rice wasshown to result in resistance to infection by two major seed-transmitted soil borne bacteria [74]. Authors used oat thioningene for understanding the role of thionin in rice, reasoning,“the expression of endogenous thionin genes alone is not

enough to prevent bacterial infection in rice plants”. However,they did not provide any evidence in concluding the abovestatement, though they argued “seedlings of japonica rice cul-tivars (Nipponbare and Chiyohonami) are known to be vulner-able to seed-transmitted bacteria such as Burkholderia plantariiand glumae, indicating that expression of rice thionin genesincluding OsThi1, did not protect against the infection”. Oneshould be cautious in interpreting this result without havingany additional data, such as gain- or loss-of function of ricethionins. Our group also identified a rice thionin-like genethat did not express constitutively in leaf and leaf sheath, andwas not induced by major signaling molecules JA and SA(each at 0.1 mM concentrations, Agrawal et al., unpublisheddata). Though initial study indicates OsThi1 induction by JA(albeit at high concentration of 1 mM [74]) in leaf sheath at24 h after treatment, a detailed characterization is needed toknow whether or not JA specifically induces OsThi1 beforeits use as a JA-inducible defense/stress marker in rice, likethat in Arabidopsis.

2.8. OsGST1/2

The glutathione S-transferases (GSTs, EC 2.5.1.18) aredimeric, multifunctional proteins found as isozymes in mam-mals [101,102], insects [39], and plants [48,104,107]. Amajor function of these enzymes is to detoxify a variety ofhydrophobic, electrophilic compounds by catalyzing their con-jugation with glutathione [102]. In rice, a GST-like cDNA(OsGST1) was cloned and suggested to have evolved to servea specialized function in cold protection [21,48]. Recently, weidentified a second rice GST gene, OsGST2 (EMBL accessionnumber AJ486976), which might exist as a single copy gene inthe rice genome [4]. As M. grisea infection specifically andpotently elicited the accumulation of OsGST2 mRNA in leavesin an incompatible versus compatible interaction, it can be con-sidered as a PR-like gene. Among numerous GSTs, a singlepathogen-induced wheat gene that encodes a glutathione S-transferase is known among monocots [50]. However, it shouldbe noted that the induction of GST rapidly and systemically bypathogen-derived signals has been previously reported in aclassical paper on Arabidopsis–Pseudomonas syringae pv.tomato strain DC3000 [18], indicating that the GST familymembers are pathogen or pathogen-derived signal induciblegenes. Characterization of OsGST1 and OsGST2 has suggestedtheir role in rice defense/stress response pathway(s), and hencesuitability as specific markers for cold and pathogen, respec-tively.

2.9. Lipid transfer proteins

Identification of proteins that facilitate transport of lipids invitro from one membrane to another led to the discovery ofplant lipid transfer proteins (LTP), which are encoded bysmall multigene families. Many studies have demonstratedthat LTPs are present in most species and that they areexpressed in a variety of tissues and physiological conditions[33,55,79]. Several Ltp genes have been shown to be respon-


sive to environmental changes such as salt, drought, abscisicacid, cold, and pathogen infection. Rice also has a “smallgene family” for LTP, and at least nine genes are known,belonging to classes I–V in group I [163,164]. Like otherplant LTPs, preliminary studies have indicated that rice LTPgenes are also responsive to stresses, such as SA, ABA, salt(NaCl), mannitol, and P. syringae [22,54,164], and thereforeare the emerging markers in rice defense/stress responses. Itis emphasized that although the currently characterized LTPgenes suggest a small family in the rice genome, there areabout 60 hits in the genome database search, indicating thatthis (small gene family) may not be the case. Therefore theever increasing genome annotations will help in identifyingnovel gene members, which then must be characterized indivi-dually to ascertain their organization and function in the ricegenome.

3. Oxidative stress related protein genes

Prior (and/or in parallel) to the transcriptional activation ofthe PR genes, is the generation of reactive oxygen species(ROS). ROS include the superoxide anion (O2

–), H2O2, andthe hydroxyl radical (OH–), constituting the oxidative burst,and have been established as a characteristic feature of theHR (reviewed in [27,59,86,99]). Among these, H2O2 (a stableand less reactive ROS) from the oxidative burst plays a centralrole in the expression of HR. To escape from these damagingoxidative injuries, plants have developed enzymatic systemsfor scavenging these highly reactive forms of oxygen (O2).Superoxide dismutase (SOD, EC 1.15.1.1) catalyzes the con-version of O2

– to O2 and H2O2 [28]. Catalase (CAT, EC1.11.1.6) and/or ascorbate peroxidase (APX, EC 1.11.1.11) inturn convert H2O2 to water and O2 [19,142]. Besides these, theextracellular class III peroxidases (POX, EC 1.11.1.7) catalyzethe oxireduction between H2O2 and several different reduc-tants, and their activity has also been correlated with plantdefense against pathogens [37,56]. Another class of less stu-died antioxidant enzymes is the phospholipid hydroperoxideglutathione peroxidase (PHGPX), which is considered themain line of enzymatic defense against oxidative biomembranedestruction [11,157]. In rice, the enzymatic activity of most ofthese enzymes has been measured against stresses, such aschilling, drought, salt, and heat stress [139–141,155,156,158].Although changes in these enzyme activities can itself serve asa marker, it should be kept in mind that these enzymes usuallyhave multiple isoforms, and thus reflect changes in total cellu-lar enzymatic activity. Nevertheless, recent studies on genesencoding these enzymes at transcriptional level further demon-strate the usefulness of individual ROS scavenging genes asmarkers for investigating the defense/stress pathways.

3.1. OsCATC

Plant catalases are thought to play an important role in anti-oxidant defenses, in response to environmental and physiologi-cal signals [143]. Among the monocots, CAT genes have beenidentified and characterized from barley [150], maize [61], and

rice [8,70]. In rice, three CAT genes (OsCATA-C) are known.The OsCATA gene did not respond to any stress agents, exceptslightly to methyl viologen (a free radical generator) [70].Characterization of the OsCATC gene against signaling mole-cules and PP inhibitors revealed an active down-regulation ofits expression, which was influenced by light signals [8]. Basedon this result, it was proposed that for activation of defense/stress response pathways, a certain threshold level of the intra-cellular signal, H2O2, might be necessary early in the inductionof these pathways, and which (H2O2) if scavenged by the H2O2

oxireductase enzymes (including the most potent, catalase)may halt this response(s). Indeed, using a catalase-deficientmutant, it has been shown that normal levels of catalase arerequired for proper growth/development and survival of aplant [2].

3.2. OsPOX

Peroxidases occur in numerous isoforms and have beenimplicated in a wide range of physiological processes, suchas auxin metabolism, ethylene biosynthesis, lignin formation,respiration, light mediated processes, growth and senescence(for review, see [37]). In rice, at least 42 different POX ESTs(established sequence tags) are known [168]. Initially, 5 POXgenes, POXgP2.3, gPOX8.1, gPOX22.3, POXgX9, andPOX5.1 were characterized [36,37]. Recently, expression pro-files of 21 POX genes were examined using 5- and 16-day-oldcv. Nipponbare plants, which revealed that POX genes havediverse biological functions during growth and in response toenvironmental stresses [72]. Most recently, a rice peroxidase(OsPOX) gene, identified from screening a JA-treated riceseedling leaf cDNA library, whose nucleotide sequence wasessentially similar to the previously cloned pathogen inducibleOSPER/POX22.3, was characterized in detail [9]. Its transcrip-tional profiles revealed a high responsiveness to wounding inleaves. Moreover, JA, SA, ET, KN, CN and EN were found topotently up-regulate its expression, whereas, ABA caused adramatic down-regulation of its transcript. A completeresponse profile of individual POX genes against environmen-tal cues will help in selecting a particular POX gene as a spe-cific marker.

3.3. OsAPX

Physiological, biochemical and molecular approaches usedto study the function of APX indicate that APX isozymes arecritical components that prevent oxidative stress in photosyn-thetic organisms. Additionally, the response of APX expres-sion to some stress conditions and pathogen attack indicatethe importance of APX activity in controlling the H2O2 con-centration in intracellular signaling [146]. In spite of itsimmense importance in plant protection against oxidativedamage, only few reports on it are available to date in rice.In one such report, the effect of chilling stress was investigatedin temperature sensitive and tolerant cultivars of rice, and itwas concluded that chilling injury is closely linked with APXactivity [140]. In their recent paper, changes in APX activity


and transcript of cytosolic APX gene in rice plants exposed tohigh temperature and subsequent chilling were examined[141]. It was reported that high temperature stress results inincreased APX activity and mRNA level. These studies indi-cate a role for APX in heat-treatment-mediated protection ofrice seedlings against chilling injury. Another group alsofound increase in abundance of the APX protein in both thetolerant rice cultivar Pokkali and the sensitive IR29 upon saltstress, by using 2-DGE [139]. Other stresses, such as O3 andSO2 were also shown to cause marked increase in the cytosolicAPX protein, using 2-DGE coupled with amino acid sequen-cing and immunoblotting [15,129]. Furthermore, a detailedstudy APX genes (OsAPX1 and OsAPX2) expression at tran-script level has also revealed its up-regulation by diverse envir-onmental cues, including M. grisea infection [5].

3.4. OsPHGPX

OsPHGPX is the only PHGPX family member gene isolatedfrom rice [91] and characterized in some detail by our group[11]. Its potent up-regulation by a variety of defense/stress-related stimuli, including blast pathogen (M. grisea) attackhas only now demonstrated its usefulness as a marker.

3.5. OsATX

The ATX1 (antioxidant) gene first identified from Sacchar-omyces cerevisiae affords protection against oxidative damage(ATX1 [94]). ATX homologs have been identified from humans,mouse, and in Arabidopsis, and shown to have dual roles underoxidative stress and in copper trafficking [64,71,81,95]. Anortholog gene from rice, OsATX, existing as a single copygene in the rice genome, was cloned from the JA-treated riceseedling leaf cDNA library [13]. Its expression analysisrevealed responsiveness to cut, over its weak constitutiveexpression in the healthy leaves. OsATX was further shown tobe potently up-regulated by a variety of signaling molecules,like JA and H2O2, but not ET, PP inhibitors, CN, EN and OA,and M. grisea infection. As expected from the presence of ametal-binding domain in the OsATX protein, copper, a heavymetal, also induced its expression.

4. Secondary metabolite pathway genes linkingto phytoalexin production

Other than the production of PRs during the defense/stressresponse(s), plants accumulate a variety of low molecular massnatural products, known as secondary metabolites. Secondarymetabolites, derived from the isoprenoid, phenylpropanoid,alkaloid or fatty acid/polyketide pathways, are distinct fromthe components of intermediary (primary) metabolism in thatthey are generally non-essential for the basic metabolic pro-cesses of the plant [45]. The antimicrobial secondary metabo-lites (“phytoalexins”) contribute to the self-defense systems inplants, due to their high antifungal activity and accumulationaround infection sites soon after pathogen attack [46,60,62,85,103,118]. Virtually, all the enzymes of flavonoid and isoflavo-

noid biosynthesis, the early steps of mono-, sesqui- andditerpene-biosynthesis, leading to phytoalexin productionhave now been isolated and functionally characterized [45].Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), which cata-lyzes the deamination of L-phenylalanine to trans-cinnamic, isthe first step in the biosynthesis of a large class of plant naturalproducts based on the phenylpropane-skeleton, including ligninmonomers as well certain classes of phytoalexins [46,76].Chalcone synthase (CHS; EC 2.3.1.74) catalyses the first com-mitted step in the biosynthesis of flavonoids, i.e. condensationof a hydroxycinnamoyl-CoA starter ester and three malonyl-CoA molecules to form a chalcone, constituting an importantstep in the synthesis of secondary metabolites. The CHS isconsidered a key enzyme, as its action is obligatory for thebiosynthesis of all flavonoids and anthocyanins in plants [68,165]. In rice, only PAL and CHS have been studied to someextent, and which has been discussed below.

4.1. OsPAL and OsCHS

While PAL genes belonging to a small multigene familyhave been well studied in dicots, little is known about PALexpression/regulation in monocots [170]. Available data inrice also suggest the presence of a small gene family, encodingPAL, and to date three OsPAL genes have been isolated fromrice, namely GP-1, GP-28, and ZB8 [109,110,170]. The GP-1expression was enhanced during greening [109]. The ZB8 tran-script was shown to accumulate to a high level in stems, ascompared to low levels in roots and leaves, and upon wound-ing in leaf tissues, and in suspension cell cultures uponM. grisea fungal elicitor [170]. Its induction in cell cultureswas reported to be too rapid and transient, reaching the max-imum level within 2 h as compared to the transcript maximaseen at 24 h in wounded leaf. A separate study also implicatedthe ZB8 gene in rice defense response based on its up-regulation by SA and Pseudomonas syringae elicitor [22].Recently, our group reported that CN-induced accumulationof phytoalexin sakuranetin was preceded by an increase inPAL activity [128].

On the other hand, CHS-like genes in plants have evolvedinto a supergene family whose members have both differentregulation and capacity to code for different but related enzy-matic activities [67]. The molecular analysis of CHS genes in awide variety of plant species (both monocot and dicot) hasrevealed that they occur in variable copy number from 1 to10 ([130] and references therein). Moreover, the CHS geneexpression has been examined in plants against a wide rangeof abiotic and biotic stresses [38,52,88,98]. In rice, a CHS(OsCHS) gene was isolated from a leaf cDNA library of anindica cv. Purpleputtu and mapped to the centromeric regionof chromosome 11 [130]. The presence of a CHS protein(immunological similar to that of maize) in rice seedlings andits development stage-specific expression has been demon-strated by western analysis. However, identification of othernovel rice PAL and CHS genes and their in-depth characteriza-tion against environmental cues remains to be done.


4.2. Phytoalexins

Leaves of rice plants produce a wide variety of both pre-formed and induced antifungal compounds. Rice leaf phytoa-lexins comprise both flavonoids (sakuranetin and naringenin)and diterpenenoid (momilactones A and B, oryzalexins A–Fand S, and phytocassanes A–D) constituents [60]. Sakuranetinand momilactone A are among the most potent (momilactoneA being more antifungal) phytoalexins induced in rice plantsunder a variety of abiotic and biotic (including M. grisea infec-tion) stimuli [82,83,125,127–129,132,152–154]. Note thatsakuranetin is now known to exist constitutively as a phytoan-ticipin (pre-formed infectional inhibitors) in the leaf glands ofblackcurrant [45]. Moreover, an independent study conductedon rice genotypes of different susceptibility to Pyricularia ory-zae (M. grisea) using short wave UV-irradiation as an elicitor,revealed high quantities of sakuranetin, momilactone A, andoryzalexin S in the resistant cultivars than in the susceptiblecultivars [44]. On the other hand, the amount of phytocassanesin rice (cv. Jukkoku) leaves infected with M. grisea, incompa-tible race 031, were also shown to be higher than those in riceleaves infected with compatible race 007, indicating that phy-tocassanes are synthesized in association with defense reac-tions in rice plants [82]. Though several phytoalexins havebeen identified to date, only sakuranetin and momilactone Ahave been most widely used by independent research groupsas a marker for defense/stress responses and disease resistance[44,83,117,129].

5. Concluding remarks

This is a first review on rice self-defense mechanisms deal-ing specifically with its components (genes-proteins-secondarymetabolites) that have a potential significance as markers forunderstanding the complex and sophisticated plant innateimmune response. A schematic illustration depicted in Fig. 1

Table 1Genes discussed in the present study and their behavior under diverse environment

Gene/protein Up-regulation/down-regulationOsPR1a Wounding, JA, SA and diverse eOsPR1b Wounding, JA, SA and diverse eOsPR2 Wounding, ethylene, cytokinin, SOsGns1 JA, CN and ENChitinases Wounding, JA, SA, fungal elicitoOsPR5 Wounding, JA, SA, copper, UV-CProteinase Inhibitors Wounding, JA, CN, EN and pathOsPR10s JA, SA, PBZ, ET, KN, CN, EN,Thionins JA (?)OsGST1/2 Cold and pathogenLipid Transfer proteins SA, ABA, salt, mannitol and pathOsCATC Multiple stresses [8]OsPOX Wounding, JA, SA, pathogen, ETOsAPX Wounding, JA, SA, high temperaOsPHGPX Wounding, JA, SA, pathogen andOsATX Wounding, JA, H2O2 and copperOsPAL Wounding, SA, fungal elicitor anOsCHS Wounding and radiation*

Down-regulation is indicated by italics; asterisks denote unpublished results [Rakwdin; EN, endothall; ABA, abscisic acid; OA, okadaic acid; ET, ethephon; KN, kdioxide.

incorporates the components of the rice self-defense mechan-isms outlined in this review. The environmental factors triggerdownstream-interlinked signaling pathways leading to modula-tion of a variety of defense/stress associated response “markergenes/proteins/phytoalexins”, categorized into three individualbut overlapping groups (circled), whose combined effect deter-mines the fate of overall response against biotic and abioticstresses. This valuable information will certainly be helpful torice researchers in using at least these available endogenousrice markers, reversing the trend of using heterologous probesfrom dicot species. Moreover, as most of these markers areinduced by critical signals (JA and SA) modulating the plantimmune responses, and pathogens (see also [96]), they have afunctional significance in rice self-defense mechanisms. Thegenes discussed in the present study and those responding todiverse abiotic and biotic factors, including to the most impor-tant of chemical signals, JA and SA, are provided in Table 1for clarity and as a reference for the readers.

Furthermore, this report reflects on the “availability oflimited- and lack of specific markers associated with a particu-lar signaling pathway” in rice. It is believed that the release ofthe complete genome sequence [73] will be a great resource foridentifying each and every component of the defense/stressresponse pathways, and their subsequent characterization willprovide us with novel markers. Moreover, rice mutants, such aslesion mimic mutants [77], with enhanced resistance or sus-ceptibility against environmental stresses, including pathogen,and those impaired in signaling pathways leading to defense/stress responses will further help in assigning individual mar-kers to specific diverse environmental stimuli. It is emphasizedthat the annotation of large number of genes remains an issue,and which needs to be resolved in the coming few years for abetter utilization of the genome database. Two recent databasearticles in the literature provide i) an almost up-to-date refer-ence of most, if not all, papers describing the gene expression

al stresses

nvironmental stress signals [10]nvironmental stress signals [10]A and fungal elicitor

r, ethylene, chitin, ABA, CN, EN and OA, pathogen, ET and KNogenpathogen, H2O2, O3, SO2 and chitosan

ogen

, KN, CN, EN and ABAture, salt, O3, SO2 and diverse environmental stress signalsdiverse environmental stress signals [11]

d radiation*

al et al.]. Abbreviations: JA, jasmonic acid; SA, salicylic acid; CN, canthari-inetin; PBZ, probenazole; H2O2, hydrogen peroxide; O3, ozone; SO2, sulfur

Fig. 2. The era of functional genomics, where “omics” technologies play an important role in defining the plant phenotype. Multiple approaches, such as genetic(reverse and forward) and transgenic, will be needed to firmly define the function of a gene responsible for a particular phenotype. Genomics, transcriptomics,proteomics, and metabolomics, will be needed to reveal the function of each and every gene in the genome and their networks, ultimately leading to ourunderstanding of the whole plant.


in plants and rice in particular (The DRASTIC database [34])and ii) information on a large set of rice defense genes [47].These studies provide important and detailed information at thedatabase level, and will complement our present review.

Achievement of these goals will ultimately help in definingthe rice self-defense mechanisms. Functional genomics istherefore the next frontier, and will require systematic andmulti-parallel high-throughput approaches. This goal willinevitably lead to an unprecedented pace in the analysis anddeduction of gene function in a short time, at the transcrip-tomics, proteomics, and metabolomics levels, which are theintegral pillars of functional genomics (Fig. 2). Proteomics isone such hot research field which is gaining momentum inplant (including rice) science as the next step towards definingthe “functional genes” and will be instrumental in identifyingnew biomarker proteins in not only plant growth and develop-ment but also under abnormal or unfavorable environmentalconditions (for review, see [17]). Collective information fromthese pillars will answer the most fundamental question—howdo biological systems work and interact in the environment?

Acknowledgements

The study has been supported by institutional funds fromHSS, AIST, Japan. This research was also supported by theCrop Functional Genomics Center of the 21st Century FrontierResearch Program (Grant No. CG2260), and a grant from the

Plant Signaling Network Research Center (The Korea Scienceand Engineering Foundation). We are indebted to Dr. Vishwa-nath Prasad Agrawal (RLABB) for his continuous encourage-ment and advice. We also thank Ms. Junko Shibato (HSS,AIST) for her help with the references and preparation of fig-ures/table. Finally we thank the anonymous referees whosecomments helped improve the manuscript.

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Role of defense/stress-related marker genes, proteins and secondary metabolites in defining rice...

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