Biologia 65/5: 755—759, 2010Section BotanyDOI: 10.2478/s11756-010-0090-y

Review

Role of plant dehydrins in antioxidation mechanisms

Xin Sun1, 2 & Hong-Hui Lin1*1Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan Univer-sity, Chengdu 610064, People’s Republic of China; e-mail: honghuilin@hotmail.com2Agronomy College, Sichuan Agricultural University, Yaan 625014, People’s Republic of China;e-mail: sunxin529@hotmail.com

Abstract: Plant antioxidation system is composed of a series of complex mechanisms, in which many antioxidants includingsome special proteins are involved. Dehydrins are a family of late embryogenesis abundant (LEA) proteins which usuallyaccumulate in plants during late embryogenesis or in response to environmental stresses. They were suggested to be asso-ciated with specific protective functions in plant cells, such as preventing coagulation of macromolecules and maintainingintegrity of crucial cell structures. In recent years, many studies implied that dehydrins also play an antioxidative role toalleviate oxidative damage in stressed plants. They were proposed to scavenge radicals directly and sequester metals whichare sources for radical generation to avoid the production of reactive oxygen species (ROS). In this paper, we will discuss thenovel putative role of dehydrins in plant antioxidation mechanisms and how dehydrins perform their antioxidative activity.

Key words: antioxidation; dehydrin; environmental stresses; metal binding; radical scavenge; reactive oxygen species(ROS).

Introduction

Dehydrins, also known as the group 2 or D-11 fam-ily of late embryogenesis abundant (LEA) proteins, arewidely distributed in the plant kingdom. They are char-acterized by diverse combination of several conserveddomains (Campbell & Close 1997; Allagulova et al.2003). The defining feature of dehydrins is a conserved15-amino acid motif (EKKGIMDKIKEKIPG), namedK segment. It was predicted to form an amphipathic α-helix, which was considered as an important structuralproperty of dehydrins. Many dehydrins contain phos-phorylable serine rich tract, called S segment. Phospho-rylation of S segment was proposed to be related to thenuclear transport of dehydrins. Another conserved se-quence (V/T)DEYGNP, which exists in the N-terminusof many dehydrins, is called Y segment. This segmenthas significant amino acid sequence relatedness to a por-tion of the nucleotide binding site of plant and bacterialchaperones (Close 1996, 1997). According to the num-ber and combination of these three conserved domains,an “YnSnKn” nomenclature scheme within the dehy-drin family has been developed (Close 1996).Dehydrins are typical stress-related proteins and

usually accumulate in plants under environmentalstresses with dehydrative component, such as drought,low temperature, and salinity (Close 1996). Many stud-ies indicated a positive correction between the accumu-lation of dehydrins and stress tolerance of plants. So,

dehydrins were thought to be associated with crucialprotective functions (Allagulova et al. 2003). They weresuggested to play an important role in protecting thefunctions of intracellular molecules and stabilizing cellstructure against dehydration, such as protective activ-ity toward stress-sensitive enzymes, binding and stabi-lizing the plasma membranes (Allagulova et al. 2003).Recently, antioxidative activity has been proposed to bean important function of dehydrins, since the environ-mental conditions which induce the accumulation of de-hydrins also produce reactive oxygen species (ROS) andinduce oxidative stress in plants. ROS, such as super-oxide radical, hydrogen peroxide (H2O2) and hydroxylradical, are toxic to plant cells, especially during stressprocess (Mittler 2002). Therefore, antioxidation mecha-nisms are necessary for plants to eliminate the oxidativedamage caused by excessive ROS. In this paper, we willdiscuss the antioxidative role of dehydrins in plant cells.

Many studies indicated that plant dehydrinshave antioxidative activity

Results of studies on the subcellular localization sug-gested that dehydrins might have antioxidative activ-ity. In stressed plants, dehydrins are widely distributedin cells and accumulate obviously around the cellularcompartments susceptible to ROS. For example, nu-cleus is an area sensitive to the attack of ROS. Manydehydrins were detected to accumulate in nucleus and

* Corresponding author

c©2010 Institute of Botany, Slovak Academy of Sciences

756 X. Sun & H.-H. Lin

even interact with DNA. This localization of dehydrinsindicates stabilization of chromosome and protectionof the transcriptional machinery (Carjuzaa et al. 2008;Egerton-Warburton et al. 1997; Hara et al. 2009). Somedehydrins also accumulated in the vicinity of plasmamembrane and were suggested to inhibit lipid perox-idation caused by ROS (Danyluk et al. 1998). In thelast a few years, dehydrins have been proved to be dis-tributed in mitochondria and chloroplast (Borovskii etal. 2000; Mueller et al. 2003), which are known to be im-portant ROS-generating places and susceptible to theattack of free radicals (Salin 1991). These results arguedthat the function of dehydrins is associated with ROSor dehydrins may play an antioxidative role in plantsunder environmental stresses.It was demonstrated that a citrus dehydrin Cu-

COR19 showed stronger inhibitory activity against freeradical-inducing peroxidation than that of albumin,glutathione, proline and sucrose, which are known an-tioxidants. Thus, CuCOR19 was proposed to have an-tioxidative activity (Hara et al. 2003). Overexpressionof CuCOR19 also inhibited the lipid peroxidation intransgenic tobacco under cold stress (Hara et al. 2003).Cell fractionation experiment of this transgenic tobaccoshowed that CuCOR19 was predominantly accumu-lated in mitochondria and soluble fraction. Becauselarge numbers of ROS could be generated in mitochon-dria and then leak into cytosol when plants were sub-jected to low temperature, inhibition of lipid peroxi-dation in transgenic tobacco was suggested to be theresult of the antioxidative activity of CuCOR19 accu-mulated in mitochondria and cytosol (Hara et al. 2003).In addition, transgenic tobacco overexpressing rice de-hydrin RAB16A and Brassica juncea dehydrin BjDHNsalso displayed an enhanced stress tolerance with betterantioxidative machinery, as reflected by reduced lipidperoxidation and membrane injury (RoyChoudhury etal. 2007; Xu et al. 2008).Recently, dehydrins were proved to be induced

by ROS. Expression of a bean dehydrin gene PvSR3was up-regulated by H2O2 and heavy metals, whichare known to catalyze the production of toxic ROS.Thus, PvSR3 dehydrin was suggested to protect cellsagainst oxidative damage caused by ROS and metalions (Zhang et al. 2006). Dehydrin expression in plantswas also showed to be enhanced by salicylic acid, a ROS(H2O2) inducer (Li et al. 2004; Zhang et al. 2006). Sal-icylic acid could induce multiple stresses tolerance inplants, but it is also known as an inhibitor of antiox-idant enzyme activities and result in ROS accumula-tion (Vasyukova & Ozeretskovskaya 2007; Yuan & Lin2008). In this case, its effects on the expression of de-hydrins could be proposed to play an important role inplant oxidative tolerance with the reduced antioxidantenzyme activities.Therefore, antioxidative activity would be sug-

gested to be a novel putative function of plant dehy-drins. It is well known that there are two aspects ofantioxidation mechanisms in plant cells: the scavengingof free radicals which already existed, and the avoiding

of ROS production (Mittler 2002). So, the antioxidativeactivity of plant dehydrins can be discussed also fromthese two aspects.

Radical scavenging activity of plant dehydrins

Studies in recent years indicated that dehydrins are an-tioxidative proteins which can directly scavenge freeradicals. For example, the citrus dehydrin CuCOR19has been demonstrated to scavenge hydroxyl radicalsand peroxyl radicals which are toxic to cells. And it wasmore potent than mannitol and approximately equal toBSA, which is known as radical scavenger (Hara et al.2004). This radical scavenging activity was suggested tobe a result of the high content of amino acid residuessusceptible to oxidative modification. It was reportedthat amino acids, such as glycine (Gly), histidine (His),and lysine (Lys), were targets for radical-mediated oxi-dation in proteins (Dean et al. 1997). Analysis of aminoacid composition showed that Gly, His, and Lys aremajor residues in CuCOR19. These three amino acidsaccounted for 15.8%, 12.9%, 12.9%, respectively, and42% together, of all residues. Indeed, oxidative modi-fication of CuCOR19 was detected during the radicalscavenging process (Hara et al. 2004). Thus, CuCOR19was suggested to deplete free radicals through oxidativemodification way.Amino acid composition data showed that content

of Gly, His, and Lys in dehydrins, not only citrus Cu-COR19, are always high. Therefore, the amino acid-responsible radical scavenging activity should not beexclusive to individual types of dehydrins, but can besuggested to be a common feature of plant dehydrins.For example, Arabidopsis possesses ten genes for de-hydrins and dehydrin-like proteins. The Gly, His, andLys in Arabidopsis dehydrins account on average for12.4%, 6.9%, and 13.5%, respectively, of all amino acidresidues (Hara et al. 2004). The situation in barley, awidely used material in research of dehydrins, is similarto that of in Arabidopsis. A statistical analysis of thetotal collection of genomic clones brings the estimatedsize of barley dehydrin gene family to thirteen, whichwere known as Dhn 1 to 13 (Choi et al. 1999). The pro-portions of Gly, His, and Lys in the proteins encodedby these thirteen genes (DHN 1 to 13) are describedin Table 1. And recent studies also suggested that thebarley dehydrins might be oxidatively modified by freeradicals under stress conditions (Sun et al. 2006, 2009).Because of the wide distribution in plant cells, dehy-drins are supposed to be a family of radical scavengingproteins.

Avoiding of ROS production by plant dehydrins

In recent years, dehydrins were proposed to preventROS production by sequestering metals which aresources for radical generation. Metal-binding activityhas been reported to be shared by many dehydrins be-long to different types (Table 2). For example, wheatdehydrin WCOR410 was suggested to be an ion-binding

Dehydrins in antioxidation mechanisms 757

Table 1. Proportions of amino acid residues susceptible to oxidative modification and the number of His-His pairs or His-X3–His motifsin barley dehydrins.

Protein name Dehydrin type % Gly % His % Lys Number of His-His pairs Number of His-X3–His

DHN1 YSK2 21.6 5.8 10.1 0 0DHN2 YSK2 20.3 5.6 9.8 0 0DHN3 YSK2 25.2 7.7 7.7 0 0DHN4 YSK2 30.4 7.3 4.5 0 0DHN5 K9 24.5 9.4 7.8 0 0DHN6 Y2SK3 32.5 9.2 3.8 1 11DHN7 YSK2 27.6 6.1 7.2 0 0DHN8 SK3 7.5 3.9 14.9 1 0DHN9 YSK2 22.6 5.5 8.9 0 0DHN10 YSK3 22.7 5.8 6.4 0 0DHN11 Y2SK2 18.5 4.7 6.9 1 1DHN12 YSK2 23.4 4.3 7.1 0 0DHN13 KS 13.1 10.3 28.0 0 0

Table 2. Proportion of His and the number of His-His pairs or His-X3–His motifs in several metal-binding dehydrins.

Protein name Dehydrin type Species % His Number of His-His pairs Number of His-X3–His

CuCOR15 K2S Citrus unshiu Marcov. 13.9 4 5ITP KS Ricinus communis L. 19.0 0 3ERD14 SK2 Arabidopsis thaliana 3.2 0 0

ERD10/LTI29 SK3 Arabidopsis thaliana 4.2 0 0COR47 SK3 Arabidopsis thaliana 4.9 2 0LTI30 K6 Arabidopsis thaliana 13.5 11 3RAB18 Y2SK2 Arabidopsis thaliana 4.3 3 0WCOR410 SK3 Triticum aestivum L. 4.5 1 0

protein in the vicinity of plasma membrane (Danyluk etal. 1998). A 32-kDa soy dehydrin could be captured bya Cu2+-charged chelating column (Herzer et al. 2003).An iron transport dehydrin ITP in castor bean prefer-entially bound to Fe3+ and also complexes Cu2+, Zn2+,Mn2+ (Kruger et al. 2002). Four Arabidopsis dehydrins(RAB18, COR47, LTI29 and LTI30) could interact withCu2+ and Ni2+ . LTI30 could also bind Co2+ and Zn2+

(Svensson et al. 2000). In addition, Arabidopsis dehy-drins COR47, ERD10, and ERD14 had the ability tointeract with Fe2+, Mn2+, and Mg2+ (Alsheikh et al.2005). These three dehydrins and a celery dehydrinVCaB45 also had a phosphorylation-dependent Ca2+

-binding activity (Alsheikh et al. 2005; Heyen et al.2002).Catalytic metals, such as Fe and Cu, mainly exist

as metal-protein forms in plants under normal growthconditions. However, they can be released as free ionswhen plants are exposed to environmental stresses.These free ions are required to produce highly toxicradicals via the Haber-Weiss or the Fenton reactions(Jiang 1999; Mittler 2002). So sequestering of free metalions could avoid the formation of toxic ROS and alle-viate the oxidative damage suffered by stressed plants.Antioxidative proteins, such as metallothionein, cerulo-plasmin, and serum albumin, were indicated to controlthe generation of radicals by chelating metals (Kanget al. 2001; Soriani et al. 1994; Vasak & Hasler 2000).Therefore, plant dehydrins could also analogously pre-vent the production of ROS by sequestering metal ions,especially the catalytic metals.

Since no metal-binding motif was found, metal-binding activity of dehydrins was suggested to be at-tributed to the high content of His, which was in-dicated to be involved in metal-protein interactions(Battaglia et al. 2008; Svensson et al. 2000). In gen-eral, His residues are rare in proteins, with the propor-tion being 2% of amino acid content (Ueda et al. 2003).However, plant dehydrins contain a much higher pro-portion of His. For example, the range of His contentin ten Arabidopsis dehydrins and dehydrin-like proteinswas 3.2% to 13.5% (Hara et al. 2005). The proportionof His in several metal-binding dehydrins is describedin Table 2. It is also noticed that many dehydrins con-tain His-His pairs and His-X3-His motifs (Table 1, 2,and others not shown; X3 represents other three aminoacid residues). The His-His pairs were reported to ab-sorb metals more strongly than single His residues (Po-rath 1992; Svensson et al. 2000). And the His-X3-Hismotifs also showed strong metal-binding activity by lo-cating the two His close together on the surface of aself-forming helix (Suzuki et al. 1998). A recent studyalso indicated that a His-rich sequence in citrus dehy-drin CuCOR15, HKGEHHSGDHH, showed a similarbinding activity to CuCOR15 itself, and was suggestedto be a core sequence to bind metals. This sequencewas suggested to form a loop structure similar to Znfinger motif and provide the two His-His pairs to con-nect together in order to bind more metals (Hara etal. 2005). In addition, dehydrins are known as Gly-rich proteins, and the flexibility imbued by a high Glycontent was suggested to allow multiple His residues

758 X. Sun & H.-H. Lin

to make contact with metal ions (Tunnacliffe & Wise2007).Because of the high content of His and the strong

metal-binding domains like His-His pairs and His-X3-His motifs, plant dehydrins can be suggested to be ion-sequestering antioxidative proteins which can preventthe metal-required production of radicals.

Concluding remarks and future perspectives

From the above, a role of dehydrins in plant antioxi-dation could be proposed since the ability to scavengefree radicals directly and prevent the production of ROSby binding metals. The antioxidative activity of dehy-drins is mainly due to their amino acid composition.Content of amino acid residues which are susceptibleto oxidative modification and involved in metal-proteininteractions are higher in dehydrins than in general. En-vironmental stresses such as drought, cold, and salin-ity always cause oxidative damage to plants. Therefore,the accumulation of antioxidative dehydrins is possiblycrucial for plants to adapt to a wide range of stresses.However, such conclusion is mainly speculated on theresults from experiments in vitro and direct evidencesare still limited to clarify the antioxidative role of de-hydrins under physiological conditions. It is noticeablethat activity of dehydrins may vary under situations invivo and in vitro. For example, the dehydrin ITP in cas-tor bean could interact with Fe3+ plus Cu2+, Zn2+, andMn2+ in vitro, but only bound Fe3+ in vivo (Kruger etal. 2002). Therefore, further investigation is necessaryto understand the detail role of dehydrins in plant an-tioxidation mechanism.

Acknowledgements

This work was supported by the grants from the Na-tional Natural Science Foundation of China (30670166),the National Key Basic Research “973” Program of China(2009CB118500) and the Program for New Century Excel-lent Talents in University (NCET-05-0786).

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Received June 29, 2009Accepted January 12, 2010