J Plant Res (2003) 116:161–164 © The Botanical Society of Japan and Springer-Verlag Tokyo 2003Digital Object Identifier (DOI) 10.1007/s10265-003-0080-z

Springer-VerlagTokyo10265

0918-9440

1618-0860

30669031Journal of Plant Research

J Plant Res008010.1007/s10265-003-0080-z

Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins

SHORT COMMUNICATION

Received: November 5, 2002 / Accepted: December 21, 2002 / Published online: February 18, 2003

Miho Sakai

•

Tomoaki Sakamoto

•

Tamio Saito

•

Makoto Matsuoka

•

Hiroshi Tanaka

•

Masatomo Kobayashi

M. Sakai

1

· H. TanakaNational Institute of Agrobiological Sciences, Tsukuba, Japan

T. SakamotoField Production Science Center, The University of Tokyo, Nishi-Tokyo, Japan

T. Saito · M. Kobayashi (*

)RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, JapanTel.

+

81-298-369048; Fax

+

81-298-369053e-mail: kobayasi@rtc.riken.go.jp

M. MatsuokaBioScience Center, Nagoya University, Nagoya, Japan

Present address:

1

School of Engineering, Nippon Bunri University, Oita, Japan

M. Sakai and T. Sakamoto contributed equally to this work.

Abstract

We have cloned two genes for gibberellin (GA)2-oxidase from rice (

Oryza sativa

L.). Expression of

OsGA2ox2

was not observed. The other gene,

OsGA2ox3

,was expressed in every tissue examined and was enhancedby the application of biologically active GA. RecombinantOsGA2ox3 protein catalyzed the metabolism of GA

1

toGA

8

and GA

20

to GA

29

-catabolite. These results indicatethat

OsGA2ox3

is involved in the homeostatic regulation ofthe endogenous level of biologically active GA in rice.

Key words

Elongation · GA 2-oxidase ·

Oryza sativa

The elongation of rice (

Oryza sativa

L.) is regulated by theendogenous level of a biologically active gibberellin (GA),GA

1

(Kobayashi et al. 1989). It has been proposed thatGA 2-oxidase catalyzes the catabolism of biologically activeGA and its precursors in higher plants (Ross et al. 1995).Recently, genes for GA 2-oxidase have been cloned from

Arabidopsis thaliana

,

Pisum sativum

, and

Phaseoluscoccineus

(Lester et al. 1999; Martin et al. 1999; Thomas etal. 1999). Thomas et al. (1999) reported that the transcriptlevels of two

Arabidopsis

GA 2-oxidase genes,

AtGA2ox1

and

AtGA2ox2

, are reduced in a GA-deficient mutant butare increased after treatment with GA

3

. Based on these

results, they concluded that GA 2-oxidase is involved inmaintenance of the concentration of biologically active GAin plant tissues.

In our previous paper (Sakamoto et al. 2001), wereported the cloning and characterization of a rice GA 2-oxidase gene,

OsGA2ox1

. The gene product of

OsGA2ox1

catalyzed the metabolism of GA

20

to GA

29

and GA

1

to GA

8

(Fig. 1). However, the transcript level of

OsGA2ox1

was notaffected by treatment with GA

3

. This result raised the pos-sibility of the existence of another rice GA 2-oxidase gene,whose expression is affected by biologically active GA. Inthe present paper, we report the cloning and characteriza-tion of two novel rice genes,

OsGA2ox2

and

OsGA2ox3

.Seeds of wild-type rice (

O. sativa

L. cv. “Nipponbare”)were sterilized in 1% NaClO for 1 h and sown on an agarmedium. Seedlings were grown in a growth chamber under

Fig. 1.

Pathways for synthesis and catabolism of gibberellin A

1

(GA

1

)in rice.

Bold arrows

Steps catalyzed by rice GA 2-oxidases. Note thatmetabolism from GA

8

to GA

8

-catabolite has not yet been demon-strated in rice

162

continuous light at 30°C. To investigate the effects of GA

3

and uniconazole on the expression of GA oxidase genes, wetransferred seedlings of wild-type rice to hydroponic culturecontaining 10

m

M GA

3

or 10

m

M uniconazole and grewthem for 3 days.

Fragments of DNA encoding putative GA 2-oxidasegenes were amplified by PCR using degenerate oligonucle-otides (5

¢

-GGITTYGGIGARCAYACIGAYCCICA-3

¢

and5

¢

-TGIARIVNRTCICCIACRTTIACRAA-3

¢

) as primersand genomic DNA as template. They were cloned intopCR II (Invitrogen, Carlsbad, Calif.), and their sequenceswere determined. A rice genomic library was screenedwith subcloned PCR fragments as previously described(Sakamoto et al. 2001). Nucleotide and amino acidsequences were analyzed by CLUSTALW software (http://www.ddbj.nig.ac.jp/E-mail/homology.html). RNA gel-blotanalysis, determination of GA 2-oxidase activity, and prep-

aration of transgenic plants were performed as describedpreviously (Sakamoto et al. 2001).

Three kinds of DNA fragments were obtained from PCRwith degenerate primers: one corresponded to the previ-ously identified

OsGA2ox1

, and the other two fragmentscorresponded to novel genes, designated

OsGA2ox2

and

OsGA2ox3

. These clones were used to screen the genomiclibrary to obtain full-length genomic clones. After sequenc-ing of genomic clones, the predicted open reading frame(ORF) for

OsGA2ox3

was successfully amplified by RT-PCR, but RT-PCR for

OsGA2ox2

was unsuccessful.The predicted ORF for

OsGA2ox2

contained 1,176 bpencoding a protein of 392 amino acids, and the ORF for

OsGA2ox3

amplified by RT-PCR contained 981 bp encod-ing a protein of 327 amino acids. The amino acids thoughtto associate at the catalytic site and bind with Fe

2+

(Valegardet al. 1998) are conserved in both sequences (Fig. 2A). The

Fig. 2A,B.

Sequence features of rice GA 2-oxidases.

A

Alignment of deduced amino acid sequences of rice GA 2-oxidases.

Dark shading

Identical amino acid residues,

arrowheads

amino acids associated with the proposed catalytic center of 2-oxoglutarate-dependent dioxygenases.

B

Phylogenetic tree of amino acid sequences for GA 20-oxidases, 3-oxidases, and 2-oxidases. The GA 2-oxidase gene family can be classified into two subfamilies:

OsGA2ox3

,

PsGA2ox1

,

PcGA2ox1

, and all of the

Arabidopsis

GA 2-oxidase genes are grouped into the same subfamily, and recombinant proteins encoded by the genes in this subfamily except

AtGA2ox1

are multifunctional.

OsGA2ox1

,

OsGA2ox2

, and

PsGA2ox2

are members of another subfamily, and the products of

OsGA2ox1

and

PsGA2ox2

catalyze single-step oxidation. The figure shows GA 20-oxidases from

Arabidopsis thaliana

(

AtGA20ox1

, X83379;

AtGA20ox2

, X83380;

AtGA20ox3

, X83381) and

Oryza sativa

(

OsGA20ox1

, U50333;

OsGA20ox2

, AB077025), GA 3-oxidases from

A. thaliana

(

AtGA3ox1

, L37126;

AtGA3ox2

, AF070937) and

O. sativa

(

OsGA3ox1

, AB054084;

OsGA3ox2

, AB056519), and GA 2-oxidases from

A. thaliana

(

AtGA2ox1

, AJ132435;

AtGA2ox2

, AJ132436;

AtGA2ox3

, AJ132437),

Phaseolus coccineus

(

PcGA2ox1

, AJ132438),

Pisum sativum

(

PsGA2ox1

, AF100954;

PsGA2ox2

, AF100955), and

O. sativa

(

OsGA2ox1

, AB059416;

OsGA2ox2

, AB092484;

OsGA2ox3

, AB092485)

163

deduced amino acid sequences of the two genes were com-pared with those of other GA oxidase genes (Fig. 2B). Inboth cases, the highest homology was found with GA 2-oxidase genes, indicating that

OsGA2ox2

and

OsGA2ox3

encode GA 2-oxidase.The expression pattern of

OsGA2ox2

and

OsGA2ox3

invarious organs of rice was examined by RNA gel-blot anal-ysis (Fig. 3).

OsGA2ox3

transcripts were abundant in stems,flowers, and roots but were less abundant in vegetativeshoot apices, leaf blades, and leaf sheaths. On the otherhand,

OsGA2ox2

transcripts were not detected in anytissues by RNA gel-blot analysis. The expression of

OsGA2ox2

was also not detected by RT-PCR (data notshown).

For the functional analysis of

OsGA2ox3

, recombinantprotein was prepared and incubated with deuterium-labeled GA

20

, GA

29

, and GA

1

as described previously(Sakamoto et al. 2001). Full-scan gas chromatography-massspectrometry (GC-MS) analysis revealed that GA

1

was con-verted to the corresponding 2

b

-hydroxylated product, GA

8

(Table 1). GA

8

-catabolite was not detected. GA

20

was, how-ever, metabolized to GA

29

-catabolite. When GA

29

was usedas a substrate, it was also metabolized to GA

29

-catabolite.These results indicate that OsGA2ox3 catalyzes two steps

of oxidation, namely GA

20

to GA

29

and GA

29

to GA

29

-catabolite (Fig. 1).

To examine the activity of the

OsGA2ox3

gene productin vivo, full-length

OsGA2ox3

cDNA was fused to the riceactin promoter and introduced into wild-type rice byAgrobacterium-mediated gene transfer. All of the trans-genic plants from 27 independent lines showed anextremely dwarfed phenotype (Fig. 4). Their leaf bladeswere dark green, and were shorter and wider than those ofwild-type plants. All of these phenotypes are typical of GA-deficient dwarf rice. Whereas wild-type plants floweredabout 3 months after sowing, these plants did not flowereven 4 months after sowing.

The effects of GA3 and uniconazole, an inhibitor of GAbiosynthesis (Izumi et al. 1985), on the levels of OsGA2ox2and OsGA2ox3 transcripts were examined (Fig. 5A).OsGA2ox3 transcript levels were low in wild-type plantsand plants treated with uniconazole but increased uponGA3 application (Fig. 5A). OsGA2ox2 transcripts werenot detected even after treatment with GA3. Further ex-periments revealed that the OsGA2ox3 transcript level

Table 1. Identification of products from incubation of OsGA2ox3 with C19-giberellins (GA)

aProducts were identified by gas chromatography-mass spectrometry on the basis of Kovats retention indices (KRI) and full-scan mass spectraof the methyl ester trimethylsilyl ether derivatives

Substrate Producta KRI Characteristic ions, m/z (% relative abundance)

[2H2]GA1 [2H2]GA8 2,821 596 (100), 581 (7), 537 (8), 450 (23), 209 (61)[2H2]GA20 [2H2]GA29-catabolite 2,688 448 (100), 419 (52), 389 (35), 329 (14), 240 (23)[2H2]GA29 [2H2]GA29-catabolite 2,687 448 (100), 419 (68), 389 (31), 329 (15), 240 (23)

Fig. 3. Expression of OsGA2ox2 and OsGA2ox3 in various organs ofwild-type rice. Northern blots of total RNA (10 mg) from vegetativeshoot apices (Sa), stems (St), leaf blades (Lb), leaf sheaths (Ls), flowers(Fl) and roots (Rt) were hybridized to 32P-labeled cDNA forOsGA2ox2 (upper panel) and OsGA2ox3 (middle panel). Lower panelEthidium bromide-stained agarose gel image Fig. 4. Transgenic rice plant over-expressing OsGA2ox3 cDNA

164

increased within 1 h after the application of GA3, but 6 hafter the application transcript levels were even lower thanthe initial level (Fig. 5B).

We have isolated two novel genes, OsGA2ox2 andOsGA2ox3, from rice. We confirmed that OsGA2ox3encodes an active GA 2-oxidase that inactivates GA1 andits immediate precursor, GA20 (Fig. 1). Therefore, there areat least two active GA 2-oxidase genes in rice: OsGA2ox1and OsGA2ox3. In contrast to OsGA2ox1, the gene productof OsGA2ox3 catalyzed the two-step oxidation of GA20; theexpression of OsGA2ox3 was observed in every tissueexamined and was regulated by biologically active GA in afeed-forward manner. The effect of GA3 application on theexpression of OsGA2ox3 was transient (Fig. 5B), suggestingthat regulation of the mRNA level of this gene must beimportant for the maintenance of the elongation rate. Thefeed-forward regulation of a GA 2-oxidase gene was firstreported for AtGA2ox1 and AtGA2ox2 (Thomas et al.1999); this is the second such report.

In conclusion, our results suggest that OsGA2ox3 isresponsible for homeostatic regulation of the concentrationof biologically active GA in rice, whereas OsGA2ox1 mayhave a specific role such as regulation of apical meristemdevelopment (Sakamoto et al. 2001). It is noteworthy thatthere are two rice GA 3-oxidase genes (OsGA3ox1 andOsGA3ox2), and that the expression of OsGA3ox2 is underfeedback regulation by the level of biologically active GAwhereas that of OsGA3ox1 is not (Itoh et al. 2001). Thus,the concentration of GA1 may be maintained at the correctlevel through homeostatic regulation of the expression ofOsGA3ox2 (synthesis) and OsGA2ox3 (catabolism). Thismechanism must be important in rice for regulating verticalelongation.

Acknowledgements This work was supported in part by a grant fromthe Program for Promotion of Basic Research Activities for InnovativeBiosciences (to H.T. and M.M.).

References

Itoh H, Ueguchi-Tanaka M, Sentoku N, Kitano H, Matsuoka M,Kobayashi M (2001) Cloning and functional analysis of two gibber-ellin 3b-hydroxylase genes that are differently expressed during thegrowth of rice. Proc Natl Acad Sci USA 98:8909–8914

Izumi K, Kamiya Y, Sakurai A, Oshio H, Takahashi N (1985)Studies of sites of action of a new plant growth retardant (E)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol(S3307) and comparative effects of its stereoisomers in a cell-freesystem from Cucurbita maxima. Plant Cell Physiol 26:821–827

Kobayashi M, Sakurai A, Saka H, Takahashi N (1989) Quantitativeanalysis of endogenous gibberellins in normal and dwarf cultivars ofrice. Plant Cell Physiol 30:963–969

Lester DR, Ross JJ, Smith JJ, Elliott RC, Reid JB (1999) Gib-berellin 2-oxidation and the SLN gene of Pisum sativum. Plant J19:65–73

Martin DN, Proebsting WM, Hedden P (1999) The SLENDERgene of pea encodes a gibberellin 2-oxidase. Plant Physiol 121:775–781

Ross JJ, Reid JB, Swain SM, Hasan O, Poole AT, Hedden P, Willis CL(1995) Genetic regulation of gibberellin deactivation in Pisum. PlantJ 7:513–523

Sakamoto T, Kobayashi M, Itoh H, Tagiri A, Kayano T, Tanaka H,Iwahori S, Matsuoka M (2001) Expression of a gibberellin 2-oxidasegene around the shoot apex is related to phase transition in rice.Plant Physiol 125:1508–1516

Thomas SG, Phillips AL, Hedden P (1999) Molecular cloning andfunctional expression of gibberellin 2-oxidases, multifunctionalenzymes involved in gibberellin deactivation. Proc Natl Acad SciUSA 96:4698–4703

Valegard, K, van Scheltinga ACT, Lloyd MD, Hara T, Ramaswamy S,Perrakis A, Thompson A, Lee HJ, Baldwin JE, Schofield CJ, HajduJ, Andersson I (1998) Structure of a cephalosporin synthase. Nature394:805–809

Fig. 5A,B. Effects of GA and uniconazole on the transcript abundanceof OsGA2ox2 and OsGA2ox3. A Upper panel Northern blots of totalRNA (10 mg) from wild-type seedlings treated with 10 mM GA3 (G) or10 mM uniconazole (U), or untreated (C) were hybridized to 32P-labeled cDNA for OsGA2ox2, OsGA2ox3, and OsGA20ox2. Theexpression pattern of OsGA20ox2 indicates the effectiveness of treat-ments. Lower panel Ethidium bromide-stained agarose gel images. BChange in the transcript abundance of OsGA2ox3 after the applicationof 10 mM GA3. Upper panel Total RNA (10 mg) was prepared 0, 1, 3,6, 12, and 24 h after the application and used for blotting. Lower panelEthidium bromide-stained agarose gel image