Chitinase Gene Expression in Responseto Environmental Stresses in Arabidopsis thaliana:Chitinase Inhibitor Allosamidin Enhances Stress Tolerance

Yasuhiro TAKENAKA,1 Sachiko NAKANO,1 Masahiro TAMOI,1

Shohei SAKUDA,2 and Tamo FUKAMIZO1;y

1Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan2Department of Applied Biological Chemistry, The University of Tokyo,1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received November 28, 2008; Accepted January 21, 2009; Online Publication, May 7, 2009

[doi:10.1271/bbb.80837]

The expression levels of three chitinase genes inArabidopsis thaliana, AtChiA (class III), AtChiB (class I),and AtChiV (class IV), were examined under variousstress conditions by semi-quantitative RT-PCR. Undernormal growth conditions, the AtChiB and AtChiV geneswere expressed in most organs of Arabidopsis plantsat all growth stages, whereas the AtChiA gene wasnot expressed at all. The class III AtChiA gene wasexpressed exclusively when the plants were exposed toenvironmental stresses, especially to salt and woundstresses. Treatment of Arabidopsis plants with allosami-din, which inhibits class III chitinases, did not affect thegrowth rate. Surprisingly, however, the plants treatedwith allosamidin were more tolerant of abiotic stresses(cold, freezing, heat, and strong light) than the controlplants. It also appeared that allosamidin enhancesAtChiA and AtChiB expression under heat and stronglight stresses. Allosamidin is likely to enhance abioticstress tolerance, probably through crosstalk between thetwo signaling pathways for biotic and abiotic stressresponses.

Key words: chitinase; Arabidopsis thaliana; stress re-sponse; RT-PCR; allosamidin

Plant chitinases have been characterized with respectto their physiology and molecular structures.1,2) Basedon their deduced amino acid sequences, chitinases arenow classified into several classes (classes I, II, III, IV,V, VI, and VII).3) According to the family classificationproposed by Henrissat and coworkers,4) chitinasesbelonging to classes I, II, and IV are grouped intofamily GH-19, and those belonging to classes III and Vare grouped into family GH-18. Plant chitinases areimplicated in defense responses against pathogen attackson sensitive and unprotected organs. Experimentalevidence for chitinases acting as defense proteins wasobtained using transgenic plants that overexpressedchitinases and exhibited higher resistance to patho-gens.5–7) Recently, however, chitinases have beenrecognized as acting in other plant physiologicalprocesses, such as growth and developmental regula-tion,8–10) programmed cell death,11) symbiosis,12) andtolerance of various environmental stresses.13–16) The

diversity in the physiological functions of chitinasesmight be correlated with structural diversity.Arabidopsis thaliana is frequently used as a model

organism in plant biotechnology and molecular biology,and its genome has been completely sequenced. It has 24open reading frames with chitinase or chitinase-likesequences.17) Although the gene functions have beenanalyzed by gene expression analysis under variousgrowth conditions, their roles in plants are not wellunderstood, and the annotations of most chitinase genesare not complete.18) To utilize fully the function of plantchitinase genes in plant biotechnology, it is highlydesirable to elucidate the physiological functions ofindividual chitinase genes. The enzymological proper-ties of Arabidopsis chitinases are also poorly under-stood. In a previous study on rice chitinases,19) we founda clear difference in the recognition of substrate sugarresidues between a class I chitinase (OsChia1c�ChBD)and a class III chitinase (OsChib1a). The class Ichitinase recognized the three contiguous N-acetylglu-cosamine residues at subsites �2 to +1, whereas theclass III chitinase recognized only one N-acetylglu-cosamine residue at the �1 subsite. (The subsite nomen-clature suggested by Davies et al.20) was employedin this context.) This result suggests that the class Iand class III chitinases function differentially in riceplants. For better understanding of the physiologicalfunction of Arabidopsis chitinases, it is necessary toexamine the mode of gene expression under variousconditions and the substrate specificity of the chitinaseenzymes.On the other hand, allosamidin is well known as an

effective inhibitor of chitinases that belong to familyGH-18.21–23) Class III and class V chitinases are be-lieved to be inhibited by allosamidin,24,25) and inhibitionof the enzymes might affect the physiological state ofthe plants themselves. The effects of allosamidin treat-ment on the states of living organisms, such as fungi,26)

insects,27) and mammals,28) have been examined, and theexperimental data suggest different chitinase functionsin vivo for individual organisms, but no such exper-imental data have been reported for plants. Hence it isunknown how chitinase inhibition by allosamidin affectsthe physiological state of plants. The physiological

y To whom correspondence should be addressed. Tel: +81-742-43-8237; Fax: +81-742-43-8976; E-mail: fukamizo@nara.kindai.ac.jp

Biosci. Biotechnol. Biochem., 73 (5), 1066–1071, 2009

function of Arabidopsis chitinases might be deduced byexamining the effects of allosamidin on chitinase-mediated processes.

In this study, the expression levels of three chitinasesgenes, AtchiA (class III, At5g24090), AtchiB (class I,At3g12500), and AtchiV (class IV, At3g54420), ofwhich the respective transcripts were previously detect-ed in the Arabidopsis plants,29,30) were investigated bysemi-quantitative RT-PCR under various environmentalstresses. The effects of allosamidin on the physiologicalstate of Arabidopsis plants were also examined to gainan insight into the function of chitinases.

Materials and Methods

Materials.Molecular biology reagents and enzymes were purchased

from Takara (Kyoto, Japan). All other chemicals were of an analytical

grade, and were purchased from commercial sources. Allosamidin was

isolated from the mycelium of Streptomyces sp. according to a method

previously reported.21)

Plant materials and stress treatments. Arabidopsis thaliana

ecotype Columbia plants were grown in soil under 16 h light,

25 �C/8 h dark, 22 �C cycles with a light intensity of 100 mmol

photon/m2/s. Two-week-old seedlings were subjected to various

stress conditions. For the wound treatment, leaves were cut into small

pieces with a razor blade and the leaf pieces were kept in water for

24 h. Cold stress was carried out by lowering the temperature to 4 �C

under 100mmol photon/m2/s conditions. Strong light stress was

accomplished by exposure to illumination at 300 mmol photon/m2/s

for 6 h. Drought stress was imposed by withholding water for 7 d.

Salinity stress was imposed by transferring the plants to Hoagland

solution containing 300mM NaCl and growing then for 3 d under

normal conditions. Chemical treatments were imposed by spraying

with 1mg/ml of ethephon or 50mM paraquat, each prepared in 0.05%

(v/v) Tween 20. Ethephon-treated plants were sampled following a

48-h incubation under normal conditions. Paraquat-treated plants were

sampled following 2 h incubation under illumination at 100 mmol

photon/m2/s. The control plants were maintained under normal

conditions and sampled at the same time as the stressed plants. The

plants were collected and frozen in liquid nitrogen and stored at

�80 �C for RNA preparation.

Stress treatment in the presence of allosamidin. Arabidopsis plants

were grown in basic Murashige and Skoog (MS) medium containing

0.8mg/ml (1.3mM) of allosamidin in petri dishes under 16 h light,

25 �C/8 h dark, 22 �C cycles with a light intensity of 100 mmol

photon/m2/s. After 2 weeks, the plates were transferred to cold (4 �C,

1 h), frozen (�20 �C, 1 h), heat (37 �C, 2 h), or strong light (600 mmol

photon/m2/s, 1 h), and were then cultured under normal growth

conditions. For paraquat and NaCl stress, Arabidopsis seeds were

germinated in the MS medium supplemented with 0.8mg/ml

(1.3mM) of allosamidin and 1mM paraquat or 100mM NaCl in a

petri dish under 16 h light, 25 �C/8 h dark, 22 �C cycles with a

light intensity of 100 mmol photon/m2/s. The experiments were

repeated at least 3 times to confirm that the data obtained were fully

reliable.

Total RNA extraction and gene expression analysis by RT-PCR.

Total RNA was isolated using Sepasol-RNA I super (Nakarai tesque,

Kyoto, Japan) from various organs (root, rosette, stem, leaf, flower, and

silique) of wild-type plants, and mRNA was prepared using an RNeasy

Plant Mini Kit (Qiagen, Valencia, CA). To eliminate any DNA

contamination, 50mg of total RNA was treated with DNase I (Takara).

Total RNA was converted into cDNA using ReverTra Ace (Toyobo,

Osaka, Japan) with oligo (dT)20 primer. To detect the mRNAs of

AtchiA, AtchiB, and AtchiV, we used the oligonucleotide primers listed

in Table 1. PCR amplification was performed in 100ml reaction

mixtures containing 1xPCR buffer supplied by the manufacturer

(Takara), 2.5mM MgCl2, 200mM dNTPs, 2.5 units of recombinant Taq

DNA polymerase (Takara), each of the primers at 1.0mM, and template

cDNA. Actin transcript was used as the constitutive control. PCR

amplification was performed for 20–26 cycles of 95 �C for 60 s, 55 �C

for 60 s, and 72 �C for 60 s, followed by 72 �C for 10min. The PCR

products were analyzed on 1% agarose gels. Equal loading of each

amplified gene sequence was determined with the control Actin8 PCR

product. To confirm the reliability of the experimental data, the

quantitative RT-PCR experiments were repeated 3 times.

Results

Expression levels of AtchiA (class III), AtchiB(class I), and AtchiV (class IV) as determined bysemi-quantitative RT-PCRUnder normal growth conditions, we monitored the

expression levels of individual chitinase genes inindividual organs at various growth stages of theArabidopsis plants. The results are shown in Fig. 1.AtchiB and AtchiV were expressed in most organs testedduring all growth stages, but the expression of thesegenes was up- or down-regulated according to thegrowth stage in individual organs. Specifically forAtChiB, expression was strongly enhanced in the rootsat germination and in individual organs during thesilique ripening stage. AtChiV expression appeared to besuppressed in the later growth stages. On the other hand,the AtChiA transcript was not detected in any organsat any growth stage. The three chitinase genes aredifferentially regulated in Arabidopsis plants.

Effects of environmental stresses on chitinase geneexpressionArabidopsis plants were exposed to various environ-

mental stresses, and the chitinase gene transcripts weredetermined by semi-quantitative RT-PCR to determinetheir stress responses. The expression profiles areshown in Fig. 2. Interestingly, the AtChiA transcript,which was not detected under normal conditions,clearly appeared in response to most environmentalstresses. Expression of the AtChiA gene was mostintensive under salt stress in roots and wound stress inleaves. For the AtChiB gene, expression was up-regulated in shoots by cold and drought stress, andalso by ethephon and paraquat treatment, but was notsignificantly affected in the roots. Up-regulation of theAtChiB gene was also observed under wound stress inleaves. The effects of environmental stresses on AtChiVgene expression were much smaller than those on theAtChiA and AtChiB genes, except that wound stressbrought about down-regulation of AtChiV expression.It should be noted that in Arabidopsis leaves treatedwith wound stress, the expression levels of AtChiA andAtChiB were up-regulated but that of AtChiV wasdown-regulated.

Table 1. Oligonucleotide Primers Used in RT-PCR Amplification

Gene Primer sequenceProduct size

(bp)

AtChiA-N 50-CATCAAGGTTATGTTGTCTC-30673

AtChiA-C 50-GATACACATACAATGGCCAT-30

AtChiB-N 50-GGATCTATATATATCTTTCATGCC-301036

AtChiB-C 50-CTCTCGTACTAAATAGCAGCCT-30

AtChiV-N 50-CAACATCAAAATGTTGACTCCC-30843

AtChiV-C 50-GATGTTTTTGTTAGCAAGTGAGG-30

Plant Chitinases and Environmental Stresses 1067

Effects of allosamidin on plant growth and chitinasegene expression

As described above, the AtChiA gene encodingclass III chitinase is responsive specifically to environ-mental stresses. Hence we focused our attention on theclass III chitinase inhibitor allosamidin and its effect onthe stress response. The effects of allosamidin were firstexamined under normal growth conditions. After culti-vation of Arabidopsis plants for 30 d in MS mediumcontaining the inhibitor (0.8mg/ml, 1.3mM), the phe-notype was compared with that of the control plants. Asshown in Fig. 3, allosamidin did not affect the growthrate or morphology of the Arabidopsis plants. Theeffects of allosamidin on the levels of chitinase geneexpression were also examined by semi-quantitative RT-PCR. Even when the highest concentration of theinhibitor (0.8mg/ml, 1.3mM) was added to the MSmedium, the AtChiA transcript did not appear at all(Fig. 4a). The expression levels of the AtChiB andAtChiV genes were up-regulated only slightly upon theaddition of allosamidin.

Effects of allosamidin on environmental stress toleranceNext, the stress tolerance of the Arabidopsis plants

was examined in the presence of allosamidin. Aftercultivation for 10 d in MS medium containing 0.8mg/ml(1.3mM) of allosamidin, the phenotypes of the Arabi-dopsis plants were compared with those of the controlplants under various stress conditions. The results are

shown in Fig. 5. Surprisingly, although the controlplants were partly damaged under cold (4 �C for 10 d)and freezing conditions (�20 �C for 1 h), the allosami-din-treated plants were tolerant of these stresses. Similarenhancement of stress tolerance was observed for heatstress (37 �C for 2 h) and strong light stress (600 mmol/m2/s for 1 h) conditions. Under the paraquat and NaCltreatments, the stress tolerance was not enhanced byadding allosamidin (data not shown).

Chitinase gene expression in allosamidin-treatedplants under environmental stressesAliquots containing 5 mg of total RNA extracted from

Arabidopsis plants that had been exposed to one oranother of the environmental stresses were used for RT-PCR. The results are shown in Fig. 4b. AtChiA expres-sion was clearly enhanced by allosamidin treatment understrong light stress conditions, and slightly enhanced underheat stress conditions. As in the case of the controlexperiment (Fig. 4a), enhancement of AtChiB expressionby allosamidin was similarly observed under strong lightand heat stress conditions. The effect of allosamidin onAtChiV expression was not clear in this case.

Discussion

It is recognized that some plant chitinases areresponsive to various environmental stresses. Antifreezeactivity has been found in class I and class II chitinases

Germination Rossetegrowth

Inflorescence emergence

Flowering Silique ripening Senescence

AtchiB

Roo

t

Shoo

t

Ros

sete

leaf

Stem

Infl

ores

cenc

e

Siliq

ue

Roo

t

Ros

sete

leaf

Roo

t

Stem

Roo

t

Infl

ores

cenc

eSt

em

Roo

t

Cau

line

leaf

Siliq

ue

Infl

ores

cenc

eSt

em

Roo

t

Ros

sete

leaf

Ros

sete

leaf

Ros

sete

leaf

Cau

line

leaf

AtchiV

actin

AtchiA

Fig. 1. Expression of the AtchiA, AtchiB, and AtchiV Genes in Different Plant Tissues.Semi-quantitative reverse transcription-PCR was performed using specific primers for Atchi genes and Actin8 on total RNA from various

organs. PCR amplification was performed with 20–26 cycles of 95 �C for 60 s, 55 �C for 60 s, and 72 �C for 60 s, followed by 72 �C for 10min.Aliquots of the products were analyzed on 1% agarose gel.

AtchiA

AtchiB

AtchiV

Actin

Col

d

Con

trol

Eth

epho

n

Dro

ught

Hig

h lig

ht

Salt

Wou

nd

Par

aqua

t

R S Lr Ls Lr Ls

Con

trol

R S R S R S R S R S R S

Fig. 2. Effects of Stress Treatment on Expression Levels of AtchiA, AtchiB, and AtchiV Genes.The RT-PCR conditions were the same as in Fig. 1. The stress conditions are described in ‘‘Materials and Methods.’’ R, root; S, shoot;

Lr, rossete leaves; and Ls, stem leaves.

1068 Y. TAKENAKA et al.

from winter rye (Secale cereale).31–34) These classes ofchitinases were also induced in salt-adapted cells oftobacco (Nicotiana tabacum)14) and winged bean (Pso-phocarpus tetragonolobus).16) However, chitinases thatpossess such a function have been characterized onlypartly in the well-studied plant Arabidopsis thaliana.Recently, in the Arabidopsis hot2 mutant, which lacksthermotolerance, a mutation of AtCTL1 that encodes fora class II chitinase was found to be responsible for itsabnormal phenotypes.35) The AtCTL1 gene appeared tobe essential for tolerance to salt and drought stresses. Allof the chitinase genes that have been identified as stress-responsive genes thus far belong to the GH-19 family(class I and class II). In this study, we found that theAtChiB chitinase gene, which belongs to the class Isubfamily, was up-regulated by cold stress and ethe-phone treatment in shoots and wound stress in leaves.This is consistent with the data previously reported forother class I plant chitinases.

In contrast, we found that the AtChiA gene, whichencodes a class III chitinase (family GH-18) was ex-pressed exclusively in response to various environ-

mental stresses. This is the first report of a family GH-18chitinase gene that is specifically responsive to environ-mental stresses. A specificity analysis of rice chitinasesrevealed that the class III chitinase recognized only oneN-acetylglucosamine residue at the catalytic site (subsite�1).19) Such a specificity in a class III chitinase suggeststhat the chitinase hydrolyzes not only the chitin polysac-charide but also a complex carbohydrate containing atleast one N-acetylglucosamine residue. The gene prod-uct AtChiA, which belongs to the class III subfamily,might have specificity similar to that of rice class IIIchitinase. Thus the target of AtChiA might be a complexcarbohydrate containing an N-acetylglucosamine resi-due. Carbohydrate processing by AtChiA might partic-ipate in the stress responses in an unidentified manner.Differential display experiments using wild-type andtransgenic Arabidopsis plants that express antisense

Cold(4°°C)

Frozen(-20°C)

Heat(2 h)

Strong light(1 h)

actin

AtchiV

AtchiB

AtchiA

A

A, +Allosamidin (1.3 mM)C, control

Control

a

C A C A C A C A C

b

Fig. 4. Effects of Allosamidin (0.8mg/ml, 1.3mM) on the Expression of the AtchiA, AtchiB, and AtchiV Genes in Leaves.a, Under normal conditions. b, Under various stress conditions. The RT-PCR conditions were the same as in Fig. 1.

Cold (4°°C)

C, ControlA, +Allosamidin (1.3 mM)

C C

C C

Frozen (-20°C, 1 h)

Heat (37°C, 2 h) High light(600 mol/m2/s, 1 h)

A A

AA

µ

Fig. 5. Effects of Allosamidin Treatment on Growth under VariousStress Conditions.

Arabidopsis plants were grown on basic Murashige and Skoog(MS) medium containing 0.8mg/ml (1.3mM) of allosamidin in petridishes under 16 h light, 25 �C/8 h dark, 22 �C cycles with a lightintensity of 100mmol photon/m2/s. After 2 weeks, plates wereexposed to various abiotic stresses. The stress conditions aredescribed in the text.

Control Allosamidin (1.3 m

Rossetegrowth

Control Allosamidin (1.3 mM

Siliqueripening

)+

+ M)

Fig. 3. Effects of Allosamidin Treatment on Growth under NormalConditions.Arabidopsis plants were grown for 30 d in basic Murashige and

Skoog (MS) medium containing 0.8mg/ml (1.3mM) of allosamidinin petri dishes under 16 h light, 25 �C/8 h dark, 22 �C cycles with alight intensity of 100 mmol photon/m2/s.

Plant Chitinases and Environmental Stresses 1069

RNA to the AtChiA gene suggested that knockdown ofAtChiA expression down-regulated the gene responsiblefor glycolipid synthesis (Tamoi et al., unpublished).Carbohydrate processing by AtChiA might lead to thegeneration or degradation of a glycolipid that acts as asignal molecule in the induction of proteins functioningin the abiotic stress tolerance.

Allosamidin is a potent inhibitor of family GH-18chitinases. The allosamizoline moiety of this compoundmimics the oxazoline intermediate proposed for thereaction catalyzed by family GH-18 chitinases, andstrongly binds to the catalytic center of the en-zymes.36,37) To control the biological processes mediatedby the chitinases, allosamidin has been used for insectcontrol with partial success.27,38,39) On the other hand, inStreptomyces sp. AJ9463, allosamidin has been foundto enhance chitinase production and cell growth.40)

Similar allosamidin effects were observed in otherStreptomyces strains.41) Suzuki et al.42) proposed thatthe enhancement of chitinase production is not derivedfrom the inhibitory action of allosamidin, but fromsignaling through allosamidin binding to the two-component regulatory system. The effects of allosamidinon the state of living organisms appear to be morecomplicated than expected.

Gerhardt et al.30) examined the role of an Arabidopsischitinase gene that is identical to AtChiV from theviewpoint of host defense mechanisms against patho-gens. AtChiV transcript accumulated very rapidly in theleaves after inoculation with Xanthomonas campestris,suggesting the involvement of AtChiV in the initialevents of the hypersensitive reaction. In the presentstudy, abiotic stresses did not significantly affect AtChiVgene expression. The AtChiV gene is probably respon-sible for host defense against pathogens, but dispensablefor abiotic stress tolerance. On the other hand, Yasudaet al.43) recently reported antagonism between thesignaling pathways for biotic and abiotic stresses inArabidopsis plants. Enhancement of the pathway forenvironmental (abiotic) stress signals suppresses theinduction of systemic acquired resistance, that is, thepathogenic (biotic) stress response. Activation of thepathogenic response conversely suppresses the environ-mental stress response. With such antagonistic crosstalkbetween the signaling pathways, plants effectivelyrespond to various stress types by using the minimumenergy to survive in biologically or climatically rigorousenvironments. As shown in Fig. 2, wound stress appliedto Arabidopsis leaves up-regulated the expression levelsof AtChiA and AtChiB, but down-regulated AtChiVexpression. This is consistent with the idea of antago-nistic crosstalk proposed by Yasuda et al.43)

Allosamidin possibly inhibits the class III or class Vchitinases that are responsible for the initial events ofhypertensive reactions, suppressing the signaling path-way for pathogenic stress. Through the antagonisticcrosstalk proposed by Yasuda et al.,43) the suppressionof the pathogenic signaling pathway by allosamidininhibition might enhance the environmental stressresponse. This is a possible explanation of the enhance-ment of stress tolerance by allosamidin that is shown inFig. 5. In fact, allosamidin up-regulated the expressionof the AtChiA and AtChiB genes but down-regulatedthe AtChiV gene under strong light stress conditions

(Fig. 4b). Under cold and freezing conditions, chitinasegene expression was not clearly observed. Other genefactors might be involved in tolerance to such stresses.Further experiments are needed to explain completelythe enhancement of stress tolerance by allosamidin.

Acknowledgment

This work was supported by the ‘‘Academic Frontier’’Project for Private Universities: a matching fund subsidyfrom MEXT, 2004–2008.

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