Plant Molecular Biology 24: 965-967, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium.

Short communication

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Nucleotide sequence of a rice eDNA similar to a maize NADP-dependent malic enzyme

Takaomi Fushimi 1,4,, Masaaki Umeda 1, Tetsuo Shimazaki2, Atsushi Kato 2, Kinya Toriyama 3 and Hirofumi Uchimiya 1 I Instituw of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan," 2Department of Botany, Hokkaido University, Sapporo 060, Japan; 3Faculty of Agriculture, Tohoku University, Sendai 98i, Japan," 4 Shiraoka Research Station of Biological Science, Nissan Chemical Industries, Ltd., Minamisaitama Saitama 349-02, Japan (*author for correspondence)

Received 20 July 1993; accepted in revised form 16 December 1993

Key words." rice (Oriza sativa L.), cDNA and NADP-malic enzyme

Abstract

We have isolated a rice cDNA clone that is homologous to the gene for the maize NADP-dependent malic enzyme (EC 1.1.1.40; NADP-ME). The deduced amino acid sequence coded for by the cDNA indicates a high level of homology to chloroplast type NADP-ME, including a transit peptide with pronounced hydrophobic properties at the amino terminus. Northern blot analysis indicates that the expression of this gene is regulated by external stress such as submergence.

NADP-dependent malic enzyme (NADP-ME) catalyzes the decarboxylation of malate with the production of pyruvate. In plants, the cytosolic form regulates intercellular pH in conjunction with phosphoenolpyruvate carboxylase [1], and the chloroplast form releases the CO2 for carbon fixation of the photosynthetic pathway in C4 plants [2]. So far, the chloroplast type of NADP-ME cDNA had been identified in C4 plants such as maize [2] and Flaveria [ 3 ], whereas the cytosolic type had been identified in C3 plants such as poplar [4] and bean [5]. The chloroplast type NADP-ME from maize and Flaveria con- tains a transit peptide, but cytosolic type from

poplar and bean do not. Although the mitochon- drial type NADP-ME has been identified in mammalian tissue [6], it has not been identified yet in plants.

Previously, we have identified numerous cod- ing sequences by random sequencing of cDNA clones prepared from rice suspension cultured cells [7]. We found one clone (S108) showing high level of homology with maize NADP-ME gene when comparing partial sequences of cDNA clones to those in the GenBank database. Using S 108 a cDNA possessing partial coding region, we isolated a full-length cDNA from the cDNA library of rice anthers at the uninucleate

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number D16499.

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R MLSARAAATAAAAA-ASP LWKRGE GG S SGS GS GC T SCREVRRRAAVRVRTPVP RRVEAVAME SAAETEKKEEVAAAGGGVEDMATEEVPVTPWAF SVAS G 99 M ***TRT**V**S*SP***-**L--**•*EG*AS*DG**TY*NTLRR*AAPAKV*ALPPRRvDAv*MVSNA*TETEKEQ••AA••*S**L**M***T***** 97 P MESTLKEMRDG* SVLDMDPKSTVGGGVEDVYG*DR* * *DQ* * * * *TI ** ** * 52

R YTLLRDPHHNKGLAFsEKERDAHYL~GLLPPAVVSQDLQVKKIMHNL~QYsvPLQRYMAMMDLQERNERLFYKLLIDNVEELLP~~QKYGS 199 • ******************** **************************** ***************************************************** M G Q ..... ::: :::::::::,::::::::::::::,,::::::::::::::::: 1 9 7

P *******************************•••**•**•**•*•*****••***•*•***•*•******************************************* 152

R I FRQPQGLYVS LKDKGKVLDVLRNWPERNI QVIVVTD G E R ~ CQGMGI PVGKL SLYTALGGVRP *ACLP I T I DVGTNNEQLLNDEFY I GLRQRR 299

P **KR*****I***E ........ K***QKS .......... ***************************** ............ V ............ K ........... 252

R A~GKEYHELMEEFMSAvKQIYG~KVLIQFEDFANHNAFDLLAKYSK~HL~!IQ~SVv~GLLs~VVGGT~HTYLFL~A~EAGT~ZLIAL 399 M *************************************************************************************************************** 397

R EI SKQTKAP ••••RKKVWLLDSKGLIVNSRKE•LQAFKKPWAH•*•PVTTLLDA•QS•KPTvLIG••G•GKTF•KEvI•AMAsFNERPV•FsLANPTS•S 499 M **************************************************************************************************** 497 P ************************************************************************************************* 452

R E CTAEEAYNWSQGRAVFASGSPFD PVEYNGKI HVP GQSNNAY I FP GF GLGWI SGAVRVHEDMLLAAS E TLAD QATQENFEKGS I FP PF TNI RKI SARIA 599 M *************************************************************************************************** 597 P ************************************************************************************************* 552

R RIAATVAAKAYELGLATRLPQP RD LEKYAE S CMY TPVYRS YR 638 M ****************************************** 636 P ****************************************** 591

Fig. 1. Alignment of deduced amino acid sequences coded for by the rice cDNA (R), maize cDNA (M), and poplar cDNA (P). Each amino acid is represented by the standard single-letter code. Identical amino acid residues are indicated by an asterisk. The shaded boxes indicate regions that are highly homologous in the NADP and NAD-dependent malic enzymes.

microspore stage. [8]. By plaque hybridization, using the cDNA of S108 as a probe, 15 positive clones were isolated. Of these, nine clones pos- sessed inserts longer than 2.3 kb. Each cDNA fragment was subcloned into the Eco RI site of pBluescript for the determination of nucleotide sequence.

The entire nucleotide and deduced amino acid sequence of the longest cDNA clone (rice NADP-ME gene) contained 2430 bp. The first ATG start codon is located at position 194, whereas the TAA stop codon is located at posi- tion 2108. The sequence therefore contains a 193 bp 5'-non-coding region, a 245 bp 3'-non-coding region and part of a poly(A) tail (a stretch of about 78 adenine residues). Comparison of the nucleotide sequence with that of maize shows 80~o homology.

Figure 1 shows comparison of deduced amino acid sequences of rice NADP-ME from several plants. The deduced amino acid sequence of rice NADP-ME shows 81 ~o and 78~ homology with that of maize [2] and poplar [4], respectively. Amino acid residues which have been identified as comprising the NADP binding region were

Fig. 2. Northern blot and hybridization analysis. Total RNA (10 ;~g) isolated from 7-day-old seedlings was hybridized with malic enzyme cDNA. A. Autoradiogram of northern analysis. B. Quantitative analysis of 2.3 kb transcript using a Fujix BAS2000 Bio-Imaging Analyzer (Fuji Photo Film Co.). Un- treated control was represented as 100%. Lane 1, untreated control; lane 2, treated with 20% sucrose for 24 h; lane 3, treated with 0.5% NaC1 for 24 h; lane 4, treated with low temperature (10 °C) for 24 h; lane 5, treated with submer- gence stress for 24 h.

identical in all plant NADP-MEs. Moreover, a highly hydrophobic amino terminus consisting of about 20 amino acids are apparent. It is suggested that in C3 plants NADP + -linked malic enzyme is confined to the plastids [9]. Thus the rice NADP-ME identified here may be transported from cytosol into chloroplasts. Further studies are required to confirm this speculation.

Northern blot analysis of mRNA from rice plants shows that a 2.3 kb mRNA hybridizes to the rice NADP-ME cDNA probe. The cDNA clone identified in this report therefore consisted of a full-length coding sequence (Fig. 2). The ex- pression of rice NADP-ME gene is activated about three-fold by submerging the plants in water, suggesting that the rice NADP-ME gene is activated by anaerobic stress.

Acknowledgements

This research was supported by Grants-in-Aid for Scientific Research, Ministry of Education, Culture and Science, Japan and a grant from the Rockefeller Foundation.

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