Two rice GRAS family genes responsive to N-acetylchitooligosaccharideelicitor are induced by phytoactive gibberellins: evidence for cross-talkbetween elicitor and gibberellin signaling in rice cells

R. Bradley Day1,6,�, Shigeru Tanabe1,�, Masaji Koshioka2, Toshiaki Mitsui3, HironoriItoh4, Miyako Ueguchi-Tanaka4, Makoto Matsuoka4, Hanae Kaku1, Naoto Shibuya5

and Eiichi Minami1,*1Department of Biochemistry, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba 305-8602, Japan (*author for correspondence; e-mail eiminami@nias.affrc.go.jp); 2Department of Genetics andPhysiology, National Institute of Floricultural Science, 2-1, Fujimoto, Tsukuba, Ibaraki 305-8519, Japan;3Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi, 2-8050,Niigata 950-2181, Japan; 4Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan; 5Department of Life Sciences, Faculty of Agriculture, Meiji University, Kawasaki, Kanagawa214-8571, Japan; 6present address: 241 Koshland Hall, Department of Plant and Microbial Biology, Uni-versity of California, Berkeley, CA 94720-3102, USA; �these authors contributed equally to this work

Received 30 October 2003; accepted in revised form 18 February 2004

Key words: N-acetylchitooligosaccharide, gene expression, gibberellin, GRAS family, rice

Abstract

In this study, we present data showing that two members of the GRAS family of genes from rice, CIGR1and CIGR2 (chitin-inducible gibberellin-responsive), inducible by the potent elicitor N-acetylchitooligo-saccharide (GN), are rapidly induced by exogenous gibberellins. The pattern of mRNA accumulation wasdependent on the dose and biological activity of the gibberellins, suggesting that the induction of the genesby gibberellin is mediated by a biological receptor capable of specific recognition and signal transductionupon perception of the phytoactive compounds. Further pharmacological analysis revealed that the CIGR1and CIGR2 mRNA accumulation by treatment with gibberellin is dependent upon protein phosphoryla-tion/dephosphorylation events. In rice calli derived from slender rice 1, a constitutive gibberellin-responsivemutant, or d1, a mutant deficient in the a-subunit of the heterotrimeric G-protein, CIGR1 and CIGR2 wereinduced by a GN elicitor, yet not by gibberellin. Neither gibberellin nor GN showed related activities indefense or development, respectively. These results strongly suggested that the signal transduction cascadefrom gibberellin is independent of that from GN, and further implied that CIGR1 and CIGR2 have dual,distinct roles in defense and development.

Introduction

Gibberellins (GA) are plant hormones that regu-late a variety of growth and developmental pro-cesses such as seed germination, cell elongation, aswell as flower and fruit development. Biochemicaland genetic analyses of the signal transduction ofGA is one of the central interests in plant scienceas is the elucidation of the mechanisms of GAperception and recognition.

Although the receptor for GA has not beencharacterized, components involved in the signal-ing pathway have extensively been studied, with a-amylase as a molecular marker for GA response(Lovegrove and Hooley, 2000). With both phar-macological and genetic approaches, the signalingprocess was shown to involve the heterotrimeric Gprotein complex. A constitutive activator of theheterotrimeric G-protein, mastparan induced theaccumulation of mRNAs for a-amylase in a man-

Plant Molecular Biology 54: 261–272, 2004.� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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ner similar to GA1 in oat aleurone cells (Joneset al., 1998). Similarly, analysis of a rice dwarfmutant, d1, which is deficient in the a-subunit of G-protein (Ga; Ashikari et al., 1999; Fujisawaet al., 1999) revealed the involvement of the Ga inthe induction of a-amylase by GA in a dose-dependent manner. Surprisingly, the dose effect ofGA on the elongation of second leaf sheath in d1was similar to that in wild type (Ueguchi-Tanakaet al., 2000). This selective impairment in the GA-signaling pathway leading to a-amylase inductionin d1 suggests the occurrence of multiple recogni-tion and signaling in GA response. In wheat aleu-rone cells, treatment with okadaic acid, a proteinphosphatase inhibitor, specifically inhibited GA-induced events such as changes in cytoplasmicCa2+ levels and expression of the a-amylase gene(Kuo et al., 1996). These results strongly suggestthat protein phosphorylation is essential elementsrequired for GA action as upstream (e.g., early)events in the GA signaling pathway(s).

Genetic analysis of slender or dwarf mutantlines, both impaired in the GA-response pathway,have led to the identification of key genes thatregulate GA signaling in a negative manner. InArabidopsis, three such genes, GAI (Peng et al.,1997), RGA (Silverstone et al., 1998) and RGL2(Lee et al., 2002), have been identified and clonedby molecular genetic approaches. Orthologousgenes have been identified in maize (D8; Penget al., 1999), wheat (RHT; Peng et al., 1999), rice(SLR1; Ikeda et al., 2001; also designated as Os-GAI; Ogawa et al., 2000) and barley (SLN1;Chandler et al., 2002). Interestingly, all of theaforementioned gene products have a DELLAmotif at their N-terminal region which has beenshown to be essential for their function in GAsignaling (Dill et al., 2001; Itoh et al., 2002), andare members of the GRAS (GAI, RGA andSCARECROW; Pysh et al., 1999) family of pro-teins, characterized by the presence of a VHIIDmotif (Pysh et al., 1999). In slender rice 1 line (slr1), a constitutive GA response mutant, leakyexpression of a-amylase in the absence of exoge-nous GA was observed (Ikeda et al., 2001), sug-gesting that the expression of a-amylase is alsoregulated by SLR1. However, a detailed mecha-nism of how these regulatory proteins function inGA signaling remains unclear.

The GRAS family, a plant-specific gene family,has best been characterized through the study of

the scarecrow phenotype in Arabidopsis, where theasymmetric divisions of the cortex/endodermalcells initial are disrupted (Di Laurenzio et al.,1996). Subsequently, numerous genes/proteinswithin this family have been identified, such astomato Lateral Suppressor (Ls) (Schumacher et al.,1999), and Arabidopsis PAT1 involved in phyto-chrome A-mediated light signal transduction(Bolle et al., 2000).

In previous reports, we have characterized thebiological action of a potent elicitor, N-acetylchi-tooligosaccharide (GN), in suspension-culturedrice cells. GN induces a set of defense responsessuch as the production of reactive oxygen species(Kuchitsu et al., 1995), ion fluxes (Kuchitsu et al.,1997; Kikuyama et al., 1997), gene expression(Minami et al., 1996; Nishizawa et al., 1999; Takaiet al., 2001), as well as the production of phyto-alexins (Yamada et al., 1993). In this experimentalsystem, we recently isolated two GRAS familygenes responsive to GN and characterized theirexpression patterns (Day et al., 2002, 2003). Ourresults strongly suggest that the two genes areregulated, at least in part, via a high-affinity chitin-binding protein localized in the plasma mem-brane of rice (Shibuya et al., 1996; Ito et al., 1997;Day et al., 2003). Here we present data showingthat these two genes, CIGR1 and CIGR2 (Chitin-Inducible Gibberellin-Responsive), are also res-ponsive to bioactive GA in a dose-dependentmanner in suspension-cultured rice cells. Compar-ison of mRNA accumulation in response to GA3

and N-acetylchitoheptaose (GN7) showed differ-ential effects of inhibitors of protein phosphory-lation and dephosphorylation, further supportingthe hypothesis of multiple signaling mechanismsinvolving CIGR1 and CIGR2 activation.

Materials and methods

Upon request, all novel materials described in thispublication will be made available in a timelymanner for non-commercial research purposes.

Chemicals

Gibberellic acid (GA3) was purchased from WakoChemicals (Osaka, Japan). GA1, GA4, GA13 andGA17 were kindly provided by Dr Richard P.Pharis (University of Calgary, Canada). All gib-

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berellins were dissolved in 50% ethanol at a finalconcentration of 10 mM. N-acetylchitooligosac-charides were prepared as described previously(Yamada et al., 1993). All inhibitors were pur-chased from Wako Chemicals. Inhibitors weredissolved in DMSO, and final concentrations usedwere as follows: K-252a, 10 lM; Lavendustin A,30 lM; Okadaic acid, 1 lM.

Plant materials

Embryogenic calli were induced by incubatinggerminating rice seeds on 1.5% agar containingmodified N-6 medium (Kuchitsu et al., 1993) and2,4-D at 2 mg/l for 10–14 days. Suspension-cul-tured cells of Oryza sativa cv. Nipponbare, its slr1-1 mutant line and d1 (kindly provided by Dr Y.Iwasaki of Fukui Prefectural University) weremaintained in modified N-6 medium and subcul-tured weekly as previously described (Kuchitsuet al., 1993).

Rice plants were grown in a growth chamberunder 14 h light (28 �C) and 10 h dark (24 �C) for10 days.

RNA gel blot analysis

Total RNAs were isolated from cultured cells orleaves by the SDS-phenol method (Watanabe andPrice, 1982). Total RNA (10 lg) was denatured byglyoxal, fractionated by electrophoresis on a 1.4%agarose gel and transferred onto a Biodyne nylonmembrane (Pall BioSupport, East Hills, NY).Hybridizations were performed at 42 �C, over-night, with a 32P-labeled full-length cDNA probewith a specific activity of ca. 109 cpm/lg DNA.Membranes were washed as follows: 0.2 · SSCwith 0.1% SDS at room temperature for 15 min,followed by two washes at 65 �C with the samewash buffer. Membranes were exposed to X-rayfilm (X-Omat AR, Eastman Kodak, Rochester,NY) and developed according to the supplier’sspecifications. In some experiments, membraneswere exposed to a BAS-III phosphor-imagingplate (Fuji Photo Film, Tokyo, Japan) and readwith a Fuji-X BAS 2000 plate reader (Fuji PhotoFilm) for quantitative analysis. As a loading con-trol of RNA, 25S rDNA (pRR217; Takaiwa et al.,1984) was used as a probe.

a-Amylase induction in half seeds of rice

Embryoless half seeds and induction of a-amylasewere performed as previously described (Kashemet al., 1998). Rice seeds (cv. Nipponbare) werehulled and mechanically separated into two halves.Both the embryoless and embryo containing half-seeds were sterilized in 1% v/v hypochlorite for 15and 2 min, respectively, and rinsed thoroughlywith sterile distilled H2O (dH2O). Scutellar tissueswere dissected from the embryo containing half-seeds and soaked in sterile dH2O for 24 h at 30 �C,in the dark. Twenty half-seeds were incubated inmicro-titer plates (15 mm) containing 0.75 ml ofinduction media containing GN4 or GN8 at vari-ous concentrations, GA3 (0.5 lM, positive con-trol) or sterile dH2O for 4 days.

a-Amylase activity was measured as previouslydescribed (Mitsui et al., 1993). The enzyme reac-tion was carried out in 0.4 ml of assay mixture(25 mM sodium acetate pH 5.3, 0.25 mM CaCl2,2.5 mM EGTA, 0.17% w/v starch) at 37 �C for20 min. Reactions were stopped by the addition of0.5 ml of freshly prepared iodine reagent consist-ing of 0.05 M HCl, 0.12% w/v KI and 0.012% w/vI2. Samples were extracted with 0.1% w/v TritonX-100 and centrifuged at 15 000 · g for 20 min,and a-amylase activities were measured as previ-ously described (Okamoto and Akazawa, 1978).

ROS production assay

An 80 mg portion of cells were incubated in 1 mlof fresh medium at 28 �C for 2 h in a shaker, andtreated with GN7 (1 lM) or GA3 (50 lM) for 1 h.Medium (50 ll portions) was assayed for theaccumulation of H2O2 by the luminol method(Schwacke and Hager, 1992).

Elongation assay of the second leaf sheath

The elongation assay of the second leaf sheath ofrice seedlings was carried out as described byMurakami (1968) with slight modification. Riceseeds (cv. Tanginbouzu) were sterilized and im-bibed in sterile water at 30 �C for 24 h followed bytreatment with 7 lM uniconazole for 24 h. Onemicrolitre of GA3 (0.1 lM) or GN7 (0.5 lM) dis-solved in 50% acetone was applied to germinatedseeds. Plants were further incubated for 4 days at30 �C under illumination and the length of second

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leaf sheath was measured. As a negative control,50% acetone was used. Ten seedlings were used foreach assay.

In situ hybridization

Tissue embedding, sectioning and in situ hybrid-izations were performed as previously described(Kouchi and Hata, 1993). Tissue sections werefixed in 4% paraformaldehyde, 0.25% glutaralde-hyde, 50 mM NaPO4 pH 7.4 for 12 h, dehydratedby passing through a series of ethanol/t-butanol at30 �C and embedded in molten (ca. 65 �C)Paraplast Plus Tissue Embedding Medium (Ox-ford Labware, St. Louis, MO).

Hybridizations were performed with eithersense or antisense digoxigenin-11-UTP-labeledprobes of the 3¢-untranslated region (1853–2086)of CIGR1 cDNA synthesized with the digoxigeninnucleic acid detection kit (Boehringer Mannheim,Mannheim, Germany), according to the manu-facturer’s specifications. Slides were air-dried,mounted, and photographed by light microscopy(Olympus Vanox-T AH-2 FL compound micro-scope, Olympus Optical, Tokyo, Japan). Com-posite images were assembled with AdobePhotoshop Software 5.0LE (Adobe Systems, SanJose, CA).

Results

CIGR1 and CIGR2 respond to exogenous GA

Both CIGR1 and CIGR2 were identified by DNAmicroarray analysis as genes responsive to GN7

that belong to the GRAS family (Day et al., 2002,2003). Based on the responsiveness of OsGAI(SLR1) to exogenous GA (Ogawa et al., 2000), wesought to determine the relationship between GAperception and CIGR1 and CIGR2 mRNA accu-mulation. GA3 induced a rapid and transientaccumulation of mRNAs for both genes in sus-pension-cultured cells of rice (Oryza sativa cv.Nipponbare) in media depleted of 2,4-D withpeaks at 10 min upon the addition of GA3 (Fig-ure 1). Accumulation of the mRNAs for thesegenes was detected in rice leaves sprayed with GA3

(Figure 1). On the contrary, EL3, a rapidlyresponsive gene to GN (Minami et al., 1996), didnot respond to GA3 (Figure 1), as well as other

elicitor-responsive genes such as phenylalanineammonia-lyase (PAL) or chitinases (Day andMinami, data not shown).

Induction of CIGR1 and CIGR2 is specific to theperception of phytoactive gibberellins

Because CIGR1 and CIGR2 are responsive to GN,it was likely that the mRNAs accumulated as aresult of a stress response that was imposed bytreatment with GA3. GA shows much variabilityin the extent of activity according to the chemicalstructure (Crozier et al., 1970). Therefore, as a firststep to elucidate the biological significance ofCIGR1 and CIGR2 in the GA response, it isessential to determine whether or not the accu-mulation of mRNAs for CIGR1 and CIGR2 canbe correlated with the bioactivities of GA. Ricesuspension-cultured cells were treated for 10 minwith active GA (e.g., GA1, GA3, GA4) as well asinactive GA (e.g., GA13 and GA17). As shown inFigure 2, induction of CIGR1 and CIGR2 wasobserved only upon application of phytoactiveGA, suggesting that the accumulation of bothmRNAs is dependent on the specific activity ofGA alone, and therefore not related to the induc-tion of a non-specific stress response. Further-more, these results clearly demonstrate thatCIGR1 and CIGR2 are excellent markers of GA-signal transduction in suspension-cultured cells ofrice.

Figure 1. Accumulation of mRNAs for CIGR1 and CIGR2 in

suspension-cultured cells and seedlings of rice in response to

GA3. Suspension cells: suspension-cultured cells were treated

with 50 lM GA3 before (+2,4-D) or after ()2,4-D) washing

with media depleted of 2,4-D and re-suspended in the same

medium for 2 h. Leaves: rice seedlings were sprayed with 50 lMGA3. Total RNAs were isolated and subjected to northern blot

hybridization with CIGR1, CIGR2 and EL3 as probes.

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Dose effect of GA on the accumulation of CIGR1and CIGR2 mRNAs

Because of its biochemical and physiologicalhomogeneity, suspension-cultured cells are anexcellent system for studying cell signaling in vivo.As such, we further analyzed the mode of induc-tion of CIGR1 and CIGR2 by using suspension-cultured rice cells.

Figure 3 shows the dose-dependent effect ofGA3 on the accumulation of CIGR1 and CIGR2mRNAs. Suspension cells were treated withincreasing concentrations of GA3 for 10 min andexamined for the accumulation of CIGR1 andCIGR2 mRNAs by northern blot hybridization.Both mRNAs accumulated in a dose-dependentmanner by the application of exogenous GA3. Amarked accumulation of the mRNAs was shownwhen cells were treated with 10)6 M GA3, andstrongly induced at 10)5 M or higher (Figure 3).

Distinct effects of inhibitors of protein phosphorylation on the accumulation of CIGR1 and CIGR2mRNAs by GN7 and GA3

Both CIGR1 and CIGR2 are responsive to GNwith a strict requirement for its chemical structure(Day et al., 2003), suggesting that the signal istransmitted via a specific receptor, likely to be ahigh-affinity chitin-binding protein localized to theplasma membrane of rice cells (Shibuya et al.,1996; Ito et al., 1997). In previous studies, wedemonstrated that protein phosphorylation/dephosphorylation plays an essential role in thesignal transduction leading from GN perception togene activation (He et al., 1998; Nishizawa et al.,1999; Takai et al., 2001). To further characterizethe signaling pathway from GA and GN, weemployed a pharmacological approach usinginhibitors of protein phosphorylation/dephospho-rylation.

As shown in Figure 4, pretreatment of sus-pension-cultured rice cells with K-252a, an inhib-itor of protein kinases possessing a broad-basedspecific activity which includes serine/threoninekinases and tyrosine kinases, abolished the accu-mulation of mRNAs for CIGR1 and CIGR2 in-duced by GN7 (Figure 4A) and, similarly, by GA3

(Figure 4D). Pretreatment with lavendustin A, aninhibitor of receptor tyrosine kinases in mamma-lian cells (Hu and Fan, 1995), partially inhibited

Figure 2. Correlation of the accumulation of mRNAs for

CIGR1 and CIGR2 with the biological activities of GA. Sus-

pension-cultured rice cells pre-incubated in the absence of 2,4-D

were treated with GA1, GA3, GA4 (active GAs), GA13, and

GA17 (less active) at 50 lM for 10 min, and total RNAs were

analyzed by northern blot hybridization.

Figure 3. Dose effect of GA3 on the accumulation of mRNAs

for CIGR1 and CIGR2. Suspension-cultured rice cells were

treated with GA3 at the concentrations indicated for 10 min

and total RNAs were analyzed by northern blot hybridization.

Figure 4. Effects of inhibitors of protein phosphorylation on

the accumulation of mRNAs for CIGR1 and CIGR2 in re-

sponse to GN7 and GA3. Suspension-cultured rice cells were

treated with K-252a (A and D), lavendustin A (B and E), and

okadaic acid (C and F) followed by treatment with GN7 (A–C)

or GA3 (D–F) for the time indicated. Total RNAs were ana-

lyzed by northern blot hybridization.

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the GN-induced expression of CIGR1 and CIGR2(Figure 4B) and strongly inhibited the GA-dependent induction (Figure 4E), suggesting theinvolvement of protein phosphorylation events,likely tyrosine phosphorylation, in the GA sig-naling pathway(s) leading to CIGR1 and CIGR2expression.

In wheat aleurone, okadaic acid, a potentinhibitor of type 1 and 2A protein phosphatases(Cohen et al., 1990), specifically inhibited GA-in-duced events such as an elevation in the level ofcytosolic Ca2+, the induction of a-amylase andprogrammed cell death (Kuo et al., 1996). Okadaicacid did not show clear inhibition of the GN-stimulated accumulation of mRNAs for CIGR1and CIGR2 (compare Figure 4C and A), whereasit strongly inhibited mRNA accumulation by GA3

(Figure 4F). These results suggest that CIGR1 andCIGR2 are activated by GA3 via a distinct sig-naling pathway from that of GN7. In theseexperiments, we did not find a significant effect of

the depletion of 2,4-D on the mRNA accumula-tion induced by GN7 (data not shown).

Neither GN nor GA has effects on the cellularevents specifically induced by one another. Theobservation that the GN7-inducible nature ofCIGR1 and CIGR2 are responsive to GA3 led us tospeculate that GN7 has GA-like activities inaddition to its elicitor activity, and/or vice versa.To test this hypothesis, we compared the activityof GN7 and GA3 for the activation of a-amylase inrice seeds, the elongation of the second leaf sheathas well as for the production of reactive oxygenspecies (ROS).

Embryo-less half seeds were incubated in thepresence of GA3 (0.5 lM), GN4 (0.1 and 1.0 lM)or GN8 (0.1 and 1.0 lM) for 4 days, and theactivities of a-amylase were measured. NeitherGN4 nor GN8 induced the activation of a-amylase,nor inhibited the activation of a-amylase by GA3

(Figure 5A). GN7 did not induce the elongation ofthe second leaf sheath (Figure 5B). Similarly, GA3

Figure 5. Effects of GN7 and GA3 on the induction of a-amylase, elongation of second leaf sheath and ROS production. (A). Twenty

embryo-less half seeds of rice were incubated for 4 days in culture medium containing GN4, GN8 (1, 0.1 lM) or GA3 (0.5 lM) as

indicated and the activity of a-amylase was assayed as described in Materials and methods. The average and standard deviation of four

independent experiments are shown. (B). Germinated rice seeds were treated with GA3 or GN7 and incubated for 4 days. Elongation of

second leaf sheath in response to GA3 or GN7 was carried out as described in Materials and methods. (C). Suspension-cultured rice

cells (cv. Nipponbare) were treated with GN7 (1 lM) or GA3 (50 lM) for 1 h, and H2O2 in the medium was assayed as described in

Materials and methods.

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did not induce the production of ROS in suspen-sion-cultured cells (Figure 5C). These results fur-ther support our hypothesis that the signalingpathway(s) upon GA perception is independentfrom that of GN, as strengthened by the differ-ential effects of inhibitors of protein phosphory-lation/dephosphorylation as shown in Figure 4.

GA3-stimulated expression of CIGR1 and CIGR2is dependent on D1 and SLR1

In the rice d1 line, the induction of GA-responsivegenes in the aleurone layer was significantly im-paired, whereas the dose effect of GA on theelongation of second leaf sheath in d1 was notdistinguishable from that in wild type (Ueguchi-Tanaka et al., 2000). It was, therefore, interestingto examine the role of Ga in the signal transduc-tion in non-differentiated calli with CIGR1 andCIGR2 as molecular markers for GA and GNaction. Using embryogenic calli derived from theseedlings of d1, we sought to monitor the induc-tion of CIGR1 and CIGR2 by exogenously appliedGA3 and GN7. As shown in Figure 6A, additionof GA3 at 50 lM failed to induce mRNA accu-mulation of either transcript, whereas the response

of both CIGR1 and CIGR2 genes to GN7 was notsignificantly impaired in d1, consistent with ourprevious observation that Ga is not essentiallyinvolved in GN signaling (Tsukadaet al., 2002), although the induced level within thetime investigated was lower compared with that inwild-type rice cells.

It is thought that GAI/RGA/RGL2/RHT/SLR1/D8/SLN1 may in fact be the masterswitches negatively controlling GA signaling. Arice mutant, slender rice 1-1 (slr1-1), which carriesa single base deletion in the nuclear localizationsignal (NLS) of the SLR1 protein resulting in theformation of a premature protein, shows a con-stitutive GA-response phenotype (Ikeda et al.,2001). Therefore, it was expected that the level ofmRNAs for CIGR1 and CIGR2 in slr1-1 would beconstitutively high, being insensitive to exogenousGA. To test this hypothesis, we induced anembryogenic calli from slr1-1 homozygous lineand monitored the response of CIGR1 and CIGR2to the addition of exogenous GA3. The mutationdid not significantly impair the accumulation ofmRNAs for CIGR1 and CIGR2 in response toGN7, however, GA3 failed to induce the mRNAaccumulation for both transcripts in slr 1-1 back-ground (Figure 6B).

In situ hybridization analysis of CIGR1

The apparently positive action of SLR1 in theinduction of two GA-responsive genes was foundin suspension-cultured cells of rice, which arelikely comprised of non-differentiated cells.Therefore, it was necessary to see if SLR1 plays thesame role in the response of the genes to GA bycomparing wild-type rice plant with slr1-1 line. Toaddress this question, mode of expression ofCIGR1 in leaves was examined by in situ hybrid-ization experiment. Expression of CIGR1 wasspecifically detected in a subset of alternating epi-dermal dumbbell-shaped cells classified as phyto-liths, among which are cork cells and ordinarysilicified cells (collectively referred to in rice as‘short cells’; Hoshikawa, 1989) of untreated wild-type rice cv. Nipponbare, and was induced bytreatment with GN7 and GA3 (Figure 7A). In slr1-1 line, the signal was detected in the same cells ofuntreated plants and significantly enhanced whentreated with GN7 but not by GA3 (Figure 7B).

Figure 6. Dependency on D1 and SLR1 of the accumulation of

mRNAs for CIGR1 and CIGR2 in response to GN7 and GA3.

Suspension-cultured cells of rice mutant lines, d1 (A) and slr1-1

(B), were treated with GN7 (1 lM) or GA3 (50 lM) and incu-

bated for the time indicated. Total RNAs were analyzed by

northern blot hybridization.

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Discussion

The GA-signal transduction pathway

In this study, we used suspension-cultured ricecells derived from germinating seedlings to moni-tor the expression patterns of two GN- and GA-responsive cDNAs, CIGR1 and CIGR2 (Figure 1).Rice suspension cells are known to constitutivelyexpress a-amylase genes, which are transcription-ally regulated by sucrose and ABA (Yu et al.,1991; Kashem et al., 1998) but not by GA (Yuet al., 1991). The observation that both CIGR1and CIGR2 responded to exogenous GA inaccordance with the biological activity in suspen-sion-cultured rice cells (Figure 2) shows that sus-pension-cultured cells, at least in part, retain themachinery necessary for the recognition of GA.Failure to detect the induction of a-amylase by GAindicates CIGR1 and CIGR2 function in a differentpathway from that leading to the expression of a-amylase. The expression of CIGR1 and CIGR2 inresponse to GA3 was completely inhibited by aprotein kinase inhibitor, K-252a (Figure 4). This isin contrast to the expression of a-amylase in wheataleurone, which was not inhibited by K-252a (Kuoet al., 1996), suggesting that the molecularmachinery for the GA-induced expression ofCIGR1 and CIGR2 is distinct from that leading toa-amylase expression.

Lavendustin A, an inhibitor of receptor-tyro-sine kinase in mammalian cells, and okadaic acid

strongly inhibited the expression of CIGR1 andCIGR2 in response to GA3 (Figure 4). Tyrosinekinases are known to play important roles in signaltransduction in non-plant organisms, but not inhigher plants except for limited number of exam-ples such as MAP kinase (Mizoguchi et al., 1997).However, reports on tyrosine kinase activity (Aliet al., 1994) and tyrosine phosphatase (Xu et al.,1998) strongly indicate that phosphotyrosine actsas a signal molecule in higher plants. It would benoteworthy that, by analogy to STAT (signaltransducers and activators of transcription) pro-teins in non-plant organisms, which play impor-tant roles in receptor-mediated signaling (Darnel,1997), the GRAS family of proteins, to whichCIGR1 and CIGR2 belong, are speculated tocarry a tyrosine residue in a corresponding posi-tion to that of STAT proteins that is phosphory-lated by receptor tyrosine kinases (Richards et al.,2000). Inhibition of gene expression by okadaicacid is commonly observed in the GA responses,i.e. the expression of a-amylase, elevation ofcytoplasmic Ca2+ level, and programmed celldeath in wheat aleurone (Kuo et al., 1996). Thesepharmacological results strongly indicate thatprotein phosphorylation/dephosphorylation playscrucial role in the early step of GA-signal cascade.

K-252a strongly inhibited GN-induced defenseresponses such as gene expression and ion fluxes insuspension-cultured rice cells (He et al., 1998;Takai et al., 2001). In this study, the application ofK-252a inhibited the expression of CIGR1 and

Figure 7. In situ hybridization of CIGR1 in wild type and slr1-1 of rice cv. Nipponbare. (A). Ten-day old rice seedlings (cv. Nip-

ponbare) were sprayed with solution of N-acetylchitoheptaose (1 lM, GN7) or GA3 (50 lM) and leaves were harvested at 0, 30 min,

3 h and 6 h and subjected to in situ hybridization with CIGR1 as a probe. (B). Ten-day old seedlings of slr1-1 were processed as in (A).

A bar indicates 50 lm.

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CIGR2 in response to GN7 (Figure 4), consistentwith the previous results. Conversely, lavendustinA and okadaic acid exhibited a slight and noinhibition to the induction of CIGR1 and CIGR2by GN7, respectively. These results clearly indicatethat the signal transduction pathway from GA3 isdistinct from GN7.

This hypothesis is further supported by theobservation that the expression of CIGR1 andCIGR2 induced by GN7 is not significantly im-paired in d1 or slr1-1 line. In this study, we foundthat the expression of CIGR1 and CIGR2 by GA3

are dependent on D1 and SLR1 (Figure 6). In thealeurone layer of d1, expression of a-amylase andCa2+-ATPase were insensitive to GA3, whereas, inthe elongation assay of the second leaf sheath, thesensitivity to GA3 in the d1 line was indistin-guishable from that of wild-type rice plants (Ue-guchi-Tanaka et al., 2000). In this context, theGA-signaling mechanism leading to the expressionof CIGR1 and CIGR2 is similar to that of theexpression of a-amylase in the aleurone layer. Bycrossing the mutants, D1 was shown to be epistaticto SLR1 (Ueguchi-Tanaka et al., 2000), and SLR1protein shown to disappear in the nucleus upontreatment with GA (Itoh et al., 2002) as also ob-served for Arabidopsis RGA (Silverstone et al.,2001) and barley SLN1 (Gubler et al., 2002). In thestudies using rice and barley aleurone, the level ofSLR1 or SLN1 appear to have a reverse correla-tion with regard to the expression of the a-amylasegene (Ikeda et al., 2001) or its activator, GAMYB(Gubler et al., 2002), respectively. Recalling thatthe mRNA expression patterns of a-amylase andGAMYB are both negatively regulated by SLR1 orSLN1, we found it interesting that CIGR1 andCIGR2 appear to be positively regulated by SLR1both in suspension-cultured cells (Figure 6) and inplanta (Figure 7). Taken together with the obser-vation that SLR1 (OsGAI) has an apparentactivity/function as a transcriptional activator(Ogawa et al., 2000), it is tempting to speculatethat SLR1 acts as a transcriptional activator ofCIGR1 and CIGR2 before degradation in responseto GA.

GA and stress response

To the best of our knowledge, there is no evidenceindicating that GA is directly involved in the stressresponse(s) of plants. Some reports suggesting the

role of GA in the stress response indicate thecomplexity of GA signaling, yet do not specificallyaddress the role, or mechanism, of GA in suchresponses. In rice for example, treatment of plantswith NaCl induces a gene, salT, in leaves, and thisinduction is enhanced by treatment with GA3.However, GA3 alone is not effective in inducingsalT expression (Garcia et al., 1998). In deepwaterrice, GA-responsive b-expansin genes are also in-duced by wounding (Lee and Kende, 2001).

In the cereal aleurone layer, GA triggers pro-grammed cell death. It was recently shown inbarley that GA significantly enhances the sensi-tivity of aleurone cells to ROS (Bethke and Jones,2001), which is hypothesized to be a causal signalleading to cell death initiated during the hyper-sensitive response in plant defense reactions (Jabset al., 1996). Conversely, reports are availableshowing that GA acts as an antagonist to stressresponse. For example, expression of tomatoGAD1, which shows striking similarity to a pro-teinase inhibitor, pinII, was induced by woundingand reduced by GA3 (Jacobsen and Olzewski,1996). In the present study, we failed to detectactivities of ROS production by exogenously ap-plied GA (Figure 5), suggesting that CIGR1 andCIGR2 have distinct roles in GA3 response andGN7 response (e.g., growth/development versusdefense), despite that CIGR1 was induced exactlyin the same cell type by GN7 and GA3 (Figure 7).Similarly, some classes of PR genes are known tobe regulated developmentally (Grunner and Pfitz-ner, 1994; Warner et al., 1994; Cheong et al.,2000), and while we can make no assumptions asof yet that CIGR1 and CIGR2 are developmentallycontrolled, the classification of CIGR1 andCIGR2 as members of the GRAS family of pro-teins, which do, to a large extent, function in adevelopmental capacity, is prima fascia evidencefor such a role. As a whole, GRAS proteins arelikely to have post-translational modifications(Richards et al., 2000), by analogy with the puta-tive orthologues in non-plant cells, STAT, and assuch, phosphorylation and possible dimerizationmay play significant roles in the signaling mecha-nisms involving these proteins. Further work isrequired to understand the biological significanceof crossing of GA and GN signal at CIGR1 andCIGR2, and functional analyses of these two genescould be a clue to reveal the relationship betweendevelopment and defense in higher plants.

269

Acknowledgements

We wish to thank Dr Russell Jones of Universityof California, Berkeley for invaluable discussions,and Dr Hidemi Kitano of Nagoya Univ. forprovidingus with slr1-1 seeds. Special thanks to DrNoel Day for critical reading of the manuscript.This work was supported by a MAFF grant (MA-2115, IP-4003) and the Program for Promotion ofBasic Research Activities for Innovative Bio-sciences (Tokyo, Japan) to E.M., and an NSF-STA Postdoctoral Fellowship to R.B.D.

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