The Structure and Organization of Two Cysteine Endopeptidase

Plant Cell Physiol. 40(4): 462-467 (1999)JSPP © 1999

Short Communication

The Structure and Organization of Two Cysteine Endopeptidase Genes fromRice

Hideki Kato, Ai Shintani and Takao MinamikawaDepartment of Biological Sciences, Tokyo Metropolitan University, Minami-ohsawa, Hachioji, Tokyo, 192-0397 Japan

REP-1 is a major cysteine endopeptidase that digestsseed storage glutelin of rice. A cDNA clone (pRP60) forREP-1 and a cDNA clone (pRP80) for a related enzymewere previously isolated. The expression of both mRNAs isregulated by gibberellin. In this study, we revealed thestructure and organization of Repl and RepA, genes cor-responding to pRP60 and pRP80 mRNAs, respectively.Repl has no introns whereas RepA consists of five exonsand four introns, and both genes exist as one copy gene inthe rice genome. The gibberellin-responsive elements con-served in cereal «-amylase genes are not included in the5-upstream region of Repl or RepA. A molecular phylo-genetic tree of plant cysteine endopeptidases was con-structed, and their relationship was discussed.

Key words: Cysteine endopeptidase — Gibberellic acid —Nucleotide sequences — Oryza sativa — Rice seed.

Cereal grains such as rice, barley and wheat, storemost reserves in the starchy endosperms surrounded byaleurone layers where hydrolases are synthesized. The ex-pression of hydrolases such as a-amylase and proteinase isregulated by gibberellin and ABA (Jacobsen and Beach1985, Koehler and Ho 1990). The regulation of pro-teinase gene expression by plant hormones has not been asextensively studied as the regulation of a-amylase gene ex-pression. In the case of a-amylase gene expression, threeconserved motifs, pyrimidine box, GA3-responsive element(GARE) and box 1 are known to be gibberellin-respon-sive sequences (Huang et al. 1990, Gubler and Jacobsen1992, Rogers and Rogers 1992) and a /rans-element, termedGAmyb, which has an activity to bind GARE was found tobe necessary for the GA3-regulated expression (Gubler etal. 1995).

We previously isolated two cDNA clones, pRP60 andpRP80, from germinated rice seeds (Shintani et al. 1995,Kato and Minamikawa 1996). The pRP60 protein is a

Abbreviation: GARE, GA3-responsive element.The nucleotide sequences reported in this paper have been

submitted to DDBJ, EMBL, Genbank under accession numbersAB004819 and AB004648.

major rice cysteine endopeptidase named REP-1, but therole of pRP80 protein which has features of a cysteineendopeptidase has not been confirmed. Neither mRNAwas detectable in dry seeds, and the levels per seed in-creased sharply upon imbibition, reached peaks at d 6 to 9,and then decreased. When seeds were de-embryonated andincubated, amounts of both mRNAs were reduced to verylow levels, but both mRNAs accumulated at high levels inthe presence of 10~8 to 10~6 M GA3; The addition of ABAor uniconazole, an inhibitor of gibberellin biosynthesis,partly eliminated the effect of GA3 (Shintani et al. 1997).Tnthe present study, we constructed a genomic library fromthe rice plant and obtained two clones corresponding topRP60 and pRP80 mRNAs. Nucleotide sequences of twogenes, named Repl and RepA for pRP60 and pRP80 mR-NAs, respectively, were analyzed with reference to thepresence of the conserved motifs that were reported to beinvolved in the gibberellin-regulated expression of monocota-amylase genes (Gubler and Jacobsen 1992, Itoh et al.

PRP60 pRP80

kbp

23.1-

9.4-

6.6-

2.32.0

Fig. 1 Southern blot analysis of Repl and RepA from rice.Genomic DNA (10 ̂ g) prepared from shoots of rice was digestedwith EcoRl, Pstl and Xbal, and electrophoresed on a 0.1%agarose gel. DNAs were transferred onto a nylon filter and hy-bridized with 32P-labeled pRP60 and pRP80 cDNA inserts.

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Structures of two rice cysteine endopeptidase genes 463

1995, Rogers and Rogers 1992). We compare the structureand organization of cereal and legume cysteine endopepti-dases and discuss their relationship.

Southern blot analysis of Repl and RepA genes—Rice seeds {Oryza sativa L. cv. Koshihikari) were pur-chased from Shibata-shi Agricultural Cooperative Associ-

ation, and were allowed to germinate for 9 d at 27 °C indarkness. Rice seedlings were ground in liquid N2, andnuclear DNA was extracted according to Rogers andBendich (1985). Nuclear DNA (10 ̂ g) was digested by res-triction enzymes, and electrophoresed on a 0.7% agarosegel. After electrophoresis, DNA was transferred to a nylon

AGCGCGAACCTAGAACAATGAGTTTTAAAA

-600 ACTTTTTAATCGCTGTCCTAACGTATCAACCGCGCAGGCGGATCAATCAC

-300 AACTTCCCAGGATTCAATCACGACGCCATTCTACGTGAATTTTCCCGCCG

301

601

901

1201

1501

AAATCTGTCATTTAAGTCAATATAAAAATTGAATATTTTTAATAACTTACAACTCGGTGGCAGCCCCTCGAGAGCAAAGCCAACCCAGTAAGTCATSMSGGCAACTCACCGGACACCATCAAATCCCAG

TGGAGAAGATACCTTGAGAAATATGAATAGATAAAAATAATTTACGAGTATCTCTGCGGCGCCTCCACGAAGCCATTCCTCGAGCATCCA&A&AACCGATGTAGCCCGCCCGCACGCATC

CCGTGAAAACTCCCTCCGTGTAAATCATATTTGTCTTGGAAGTTAAAGTGGAGGGAGTTCTGCCTCATCCGCCATTAACACGTCACGTTAAAATTGCACGATCCGTATGCTAACGTAGTTTCTGTTACGTCTCCCTCC

AACTGGGCTGTACTCGTAAAGAATAACTATAAAAATAACATTGTGTTTTAAAGAGCTAGCCGCAACCAATTACCGTCCTAAGCCCGACCACACCTAAGCTTTGAATCTGGCAGCCGTCCACCCCATTTAACTTCTCCTTC

TGAGACTDAGGAAAATCGTTCAAGTTGTTGTACATATATCGAGACCGTGTCGATCAACCGTCCTACACGCGCCAAGCTACCCCAGTGATAACCTACACTACCAGAATCTTTCCCAACTCCATCCGTCTCGCTCCTCATCG

CCCTCACCCTCACTGCTACT

GGGGAGGGTTG R VGGCGGTGGAGA V EGCTGTGGGATL W DGAAGCACCGCK H RGCGCGGCGGCR G GGTTCCGCGCCF R A

GCCGCCGCTCP P L

GCGCCGAAAGR R KGTTCTCCACGF S TGCTGTCGGAGL S EGCTCATGGAGL M ECTACCCGTACY P Y

GGTGGTGATCV V ICGTCGCCAACV A NCTCCGACGGCS D GCGGCTACGGCG Y G

AGCCTGGGGCA W GCTGCGGCATCC G I

AAGGCGCGCCR R AACGTGCCCGCAAATGGTGCTGAGGCGATCATGCTAAATAAATGTGGTGTT

CCAATTCCAAGCGATCGATC

ATTAGCAGCTI S S WCTGTGCGCCGL C A ACTGTACGAGCL Y E RCGGTTCGGCGR F G ACGGGGCTACCR G Y RACGTTCGCCGT F A GCCGGGGTTCAP G F MGGCGCGGTCAG A V TGTGGTGTCCGV V S VCAGGAGCTGAQ E L IAACGCGTTCGN A F ECGCGCCGCCAR A A NGACGGGCACC0 G H QCAGCCCGTCTQ P V SGTCTTCGCCGV F A GGAGACCAACGE T N DGAGGGCGGCTE G G YGCCATGGAAGA M E ACTTGGCGCCAL G A KCTACGAGCCTATTATTGTTGGGGAAAAGAAGTTTTAGATGCTTCGTGGCA

TCCCTCCCAAGGTTGCAAAG

GGAGGGTTCTR V L

CGATACCGTTI P F

GGTGGCAGGAW Q E

CGTTCAAGGAF K D

GCCTCCGGCTL R L

GGTCCCACGCS H A

TGTACGAGGGY E G

CCGGCGTCAAG V K

TGGAGGGCATE G I

TCGACTGCGAD C D

AGTACATCAAY I K

ACGGAACGTGG T C

AGAACGTGCCN V P

CCGTCGCCATV A I

GCGACTGCGGD C G

ACGGCACGGAG T E

ACATCCGGATI R H

CCTCCTACCCS Y P

AGGAAACCCAE T Q

GGATCAACGGCTGCTCTTTGTTACCTGAAATGAGGTGAGGCCGGGAAATA

GAACTAGCTGTTAATTAATT

TGCGGTGGTGA V VCGACGAGAGGD E R

GCACCACCACH H H

CAACGTGAGGN V R

CAACCGCTTCN R F

CAACGACCTCN D L

CGTCCGCGACV R DGGACCAGGGCD Q G

CAACGCGATCN A I

CACGGCGGACT A D

GCACAGCGGCH S G

CGACGCCGTCD A V

GGCCAACAGCA N SCGACGCCGGCD A G

CACCGACCTCT D L

GTACTGGATCY W I

GCAGCGCGACQ R D

CGTCAAGTTCV K FGTGATCGAGC*

CTCTGGTCGCTTAATTGCCTTCGTCTAAGTTCGATCTATCTATATAAGAA

ATCACGACGAAGTGGCGATC

GCTGCTTTGAA A L MGATTTGGAGTD L E SGTGCCGCGCCV P R HTACATCCACGY ~I I EGGCGACATGGG D M GCGCCGCGACGR R D GCTCCCCCGCGL P R AAAGTGCGGCAK C G SCGGACGGGGCR T G RAACAGCGGCTN S G CGGCATCACCAG I T TCGCGCGCGGCR A R RGAGGCCGCGCE A A LGACCAGTCCTD Q S FGACCACGGCGD H G VGTCAAGAACTV K N STCCGGCTACGS G Y DTCGCCAAATCS P N RCGGCGCCATC

GTCTCCTCCTTGTGTTTATGTACTAGTGTTATGTAATGTAGGTTGTTGTT

GCAGCTAGTCGATCGATCAT

MTGGCCATGGCA M A

CCGACGAGGCD E A

ACCACGGCGAH G E

AGCACAACAAH N K

GCAGAGAGGAR E E

GCCTCGCCGCL A A

CCGTCGACTGV D W

GCTGCTGGGCC W A

GGCTGGTGTCL V S

GCCAGGGCGGQ G G

CCGAGTCCGCE S A

GCGCGCCGCTA P L

TCGCCAAGGCA K A

TCCAGTTCTAQ F Y

TCGCGGTGGTA V V

CGTGGGGCACW G T

ACGGCGGCCTG G L

GTGTCACGCCV T P

TCCGCCGTAC

CCAGCTTGCATAATTCGTGTATTTCGTGTGCTGAACTTATATGACTAA^G

TTATGCCAAATATCTCAATTTACGACTCGT

TTTCATCCAAGCTTCAGTTTGGGATTCGCA

AAAAGACTAATTAGCTTGTCACCAAAATCG

GGTTATGCCATCGCTGGGCGAAGACGAGAG

AATTTCATCCTTCCTATGACCTTTGCAACC

AAAAAAAAGTTTATGTGAGTGGAGAG

Fig. 2 Nucleotide sequence of Repl, a gene for REP-1, and deduced amino acids. Serial deletions of pgRP60 were prepared with aKilo-sequence Deletion Kit (Takara Shuzo). Deletions were sequenced by an automated DNA sequencer (Applied Biosystems Model373A) with a dideoxy terminator cycle sequencing kit (Applied Biosystems). The numbering is based on the putative transcription startsite. The putative TATA box is underlined. The translational region is boxed. The stop codon is indicated by an asterisk. Nucleo-tide sequences that resemble the conserved sequences, GARE, box 1 and pyrimidine box, were double-underlined (cf. Fig. 5).


464 Structures of two rice cysteine endopeptidase genes

filter, and the fixation was performed under ultravioletlight. Hybridization was first conducted with pRP80cDNA used as a probe (Sambrook et al. 1989). After ex-posure, the nylon filter was washed completely with boiled0.1% SDS solution until it showed no radioactivity. ThenpRP60 cDNA was used as a probe. Neither cDNA cross-hybridized to each other under high stringency conditions(Shintani et al. 1997).

When genomic DNA was digested with EcoRl, Pstl orXbal, each of which digests neither pRP60 nor pRP80cDNA, a single band was probed with the radiolabeledcDNA insert (Fig. 1). These indicate that both Repl andRep A exist as single genes in the rice genome, whereas

other gibberellin-inducible cereal hydrolases such as ct-am-ylase (Huang et al. 1990, Huttly et al. 1988, Khursheed andRogers 1988) and EP-B (Mikkonen et al. 1996), a majorendopeptidase from barley, are encoded by multigenefamilies. Repl and RepA existing as single genes may be ofadvantage to investigate the mechanism of gibberellin-response expression of genes in cereal aleurone layers.

Nucleotide sequences of Repl and RepA genes—Nu-clear DNA (10 /ig) was partially digested by Mbol, and10 to 20 kbp DNA was fractionated by sucrose densitygradient centrifugation. The partially digested DNAs wereinserted into the BamHl cloning site of AEMBL-3 clon-ing vector (Stratagene). The in vitro packaging was con-

ATCTGATCAA TCCCCCAGCA AGAAAACCAA CCAATCATGT AACATGCACA AGGTAGCCTCJTTTCTTCTC CCCAAAACTT ATCCGGCCAT GCATATGATC ATGATCGCCA ACCGCCAAAACCATCTCCCA CCTCTCCCGC CATCTATCTC TGACCTCTCC TCTCCACATG CTCATCGATC

_AATGGCGTTT TGCCGCCCCA CGCTACAACT CTTATCATAT CTTCOCCXAJ ATATATCGCTIGCTCATTGAA[cpTGATATAT

CTGCCGCTTTAATTAGTTTGTTTCAGCTTCCCTTCAAGTTCCAG

AGGTCAACGGCCTTCCCTTTTAACTTCCCGTCACTGCTCATTTCGTTTCA

ACTTCGCATT

^TGAATAAAG TCGGTGTGCT ATAGCTACCA TTCCCCCGGA CCTGTGTGTTTTGTAATTTTTTTTTCTTTT

TCCAAAAAGT

TTTTTTTACGTCATTTCAGC

ATCTTTCTAGTCACTCAACT

AGTTGTAATCGAATAAAGTC

CGAAATGCTGH L

CGTTCACGGA

r T ECGGCCGGCAGR P A AGTACATCCACY I II

AGTTCCGCCGr a n

TTCCGGTACGT B Y GGGATCAAGGCD Q G

AGGTGCTTCCR C F LGAGCGACCTCS D L

CATCAGGAGGS G G

GAGGCGAACAE A H nCACCTACGCGT Y A

GCGGAGACGAG D D

CAATGCdGTAQ C C

CAAATGCAGTTAACAGGATTTTAAATTTAGTTTTTTTTCA

GTTTATGGCACACTGAACCCAAAGTGGCAC

TGGTCGCGGCV A A

TCGTCGGAGGS S E ECGTTGGCAACV G H

GGAGGGGCGGR G G

GGCTCCACGGG S R ACGAGGACAACE D N

GGCGGCGGTGA A V

AGAGCCTGCGS L R

GACGACGGCGD O G ECCGGCCGTTCR P F

CGCGCCACCAR H H

CTGCCGCCCGL P P A

GCGTTGGCTGA L A AGGCGCTGTACA L Y

AGGCTCGCCGA R R

AGGCTGGCGCR L A LCCGCAGCCTGR S L

CGGTCGACTG

ACJCTAGCTTGCGGCGGCGGC

A A AGAGCGGTGGCE R W RGCGGTTCAACR F NTGAACAAGTT

N K FAGCGGCGGCCS G G RGCGCGAGCGCR E R

GGCGCTGTCG.TTTTCTGCTC

GTATCTGGGTTATCAATAGA

TATTTTAGGTAGCGCCGGCGA P A

GGAGCCGGTAS R Y

GTGTTCGTGGV F V ECGCCGACATGA D M

GCGGCGGCGAG G E

GGCGCCGTCAG A V T

GAAAACCGTCAGGGCTATCCR A I PCACCGTGTCGT V S

AGAACGCGAGN A R

ACGACGGACGT T D EGGGGGGCTCCG G S

CCGGCATCAAG I K

AAAAAGCGTG AACATTTTTT TCTCGTTCTA AAAAGCATAT AACATTTCTA TGGAAAAAGC

CAAAAAAACATAAAATTTCGTCAAAAACTCAAATGGTTTTAAAGATCTTAACATGTCATAAAAGTTAAAT

TATGGATAGAGTTTCCGAAAAAAATATATTTTACTGTTGGTATTTACTTCGACACATGAGGTCCCATTTA

TATAGTACAATTTTGGAATCTAAATTCAGTCTACACTTTTGAAAATAATTAGCCACATGTCTTCGTGCAC

ATTATGCAAACCTGTTAACATAGATTTTTATTCTCAGAATAACTATTTGCCATATAXAACGTGTGTGCTA

ATCATTAGATCGAAATTTTTAATAATTTCTATTTTATCXACACTCTTACGAACTGACAAAGTCACTCGGC

TATTACGGCAGGATTTTTTTTGCATAAGTGTPPftTftTAATAGTGGCGATATCCTTAATGGTCTTATTGGT

CATTATTTTCCTAATTATTCAAAATACGATTAAGCATATAAAGGATTTGCCCACATCTCAGTGATTTTAA

GbGAGCTGCTI S C WGGAGCAGGAGE Q ETCAAGAGGAA| K R N

GGGCGTTCTCA F S

CTGGTGGACTL V D CCGGAGGGATCG G I

GACGGTGGCGT V A

GCGACACCGGD T G

ACCACCGAGTT T E S

GCGGTGGAGGA V E GTGATAACCAGD N Q

CCAACTACCCN Y P

GAGTGAACAAV N K

GGCTGCGACGG C D GATATAGAGCGY R A

GATCAAGACGI K T

GCGGGCTCATG L M

GAGCAGGGGAE Q G R

GGGAGGTTGGG R L VGGATTACGCGD Y A

GGTGTAACAAC N K

TGACGCTGTCT L S

TTCCAGTTCA

F o r i_GGCAAA^GTAA K

ATTGCACGGTAATAACTTAAGATGCATGGTTTATGTTGCA

GAAATCTCATTTTTTGCGCATAATAAACAC

GAAAAATTATAAGCGTTCGGTTGTTTTAGG

ATTTATGTTAAGGAAACATAACTAAAATGA

ACTATACTAAAATGTTTATCAGTTAGTCTG

AAAAAATCAAATGAAAAAAGAATACAAACT

TGTTGAATTTATCTGCTCCGGAAAATTTGC

AOGT

GAAGAAAAAAAGTAAAGAAACATCTGGTGT

AAAGCAGTCGK A V ACAACATGACCGCGTGTGTCT

QGCAAGCTCT

JA s sCAAACCAGCC

N Q P

CACGATGTAAH D V TTGTGGCTGTT

CGATTGATGGI D G

GCAGTAGAGG

CTACGAGGACY E D

CTAGCGGCCAS G Q

GTTCCTGCCAV P A NAGATTTCCAGD F Q

ATGACGAGTCD E S

TTCTATTCGGF Y S E

GGCTCTACAGA L 0

AACAACCTAGAATTATACTC

CTACACGATAAAAGATACTA

TGTCTGAAATATACCATCAT

TAATCAACAGATATATATCA

TTCAACACATCTTGGGAATA

GATCTACACAATCAAACTAG

AGETA

TATTCAGTGTGGTGTCTTCA

CCGGAGAATGG E C

ATCGTCAAGAI V K NTGGGCTGTGTG L C

TTAAAGACGAK D E

AGTAGTGCTATCAGAAGTTA

CGGCACGGATG T D

ACTCGTGGGGS W G

GGCATCGCGAG I A MGATGTGAGCC

H *GCTAAGCTTTGGTAAAATAA

CTCGACCATG GTGTTGCCGCL D II G V A ACGAGGATTGG GGCGAGAGAGE D W G E R G

TGGAGGCTTC TTACCCTGTCZ A S Y P V

TGTGATCGAT GGAGGGATAT

CGTTGTCAACATTCAAAGAG

TGGATTTAATGAGCATGACT

TGTCGGTTACV G Y

GGTACATCAGY I R

AAATCAGGAGK S G AGATATGGATA

CTTCTACTGTAACATAATGT

GGTATAACTCG I T RAATGCAGCGTH Q R

CACGCAATGCn N A

TGATCGATGC

TCACGGTTGTGCTCAACTGG

GAGACGGCACD G T

GGTGTCTCGTG V S STGCTGCAAGTA A S

TTTATGTGTG

AATCGACATTTTTGACACCACCTATATAAAACGGTCAAACCCTGTAGAATTAATGCGCCGAGGCAGGCCTCTATATAATCCATGCATTCCTTTTTTTTCATGATGCTTTC

ACTGGGAAAATTGACTTTTTAATATATTTAATGTACTAAAGTGCCGGGGGCCTGAGAAGACTTAAAGCTTTTCTTCCTCCCTCTCTTCTTGCTATGATTGATTACCCAAA

TATTTTTTGCAGTACATGTTACAATGAATCAAGTCAACGGAGGGGAGGAGTACATTTTGCCACATACGAACTGAGCCGTAACGATTTTATTGGCGGATCTATATATGTAT

AGACAAGGTGTGACCGTTCGAAATAATAGATGTCAAACATGGCAAGATGGACAGGAGGAGGATGTACCTTCACAATTCATTTTTATTTTTATGTATACACATTGCAACTT

CACCTATCTTTCTTATTCAAAAAAGAATTATTCAAAACGGCCATCCATCTCACTTAAGGCCTCAGGTGGCACGATATTTCAGGCCTTAATCTTGATGGCATTCCAATCAC

TTCAACGGGTAAACTTTTGTATAATTACTTAGGGAGTATAGCGAAGACCAGTCCTACCTGATCTTAAGTTTTCGGTGGATAGTAGTGAAATGTTGTCCTCCTAGATATAG

ACTGTACGCCATTGCTIJAAA

JG V F TCAAGTACTGGK Y WCAGACTCGAA

D S HAACAGAGTGGM R V VCTTCGCTGAA

AGGTGTCAAA

ACTCCCTCCGGAAATATGTAAAATTTTTTTTTTTTGGCACATCTTCTTAGTGATACTACTATCTGCTTGGTTTTCTACAGGCACCATGTGTCATCCCTTG

AATTTGCTCATTTCAAAATGAAATTATAGGAATTAGACGACATCGGTCCTGTAGACGACACCATTTTCCAGAAGATGGCTTTATTAGATCCATATAATCTTTCTTAAATG

Fig. 3Fig. 2.

Nucleotide sequence of RepA, a gene for pRP80 mRNA, and deduced amino acids. Figure details are as given in the legend to



ducted according to the manufacturer's manual and 1 x107 recombinant phages were obtained. The genomic li-brary was primarily screened with pRP60 and pRP80cDNA inserts as probes, and four clones were obtained.The second screening was performed with each of thecDNAs as a probe, and three clones hybridized with pRP60cDNA and one clone with pRP80 cDNA. The longest cloneof the three which hybridized to pRP60 was 18 kbp, andthe clone which hybridized to pRP80 was 15 kbp. Theseclones were named AgRP60 and AgRP80. The fragments of2.5 kbp between two EcoRl sites of AgRP60 and 3.5 kbpbetween Sad and Xbal sites of AgRP80 were subclonedinto pUC118, and named pgRP60 and pgRP80, respec-tively. Nucleotide sequencing of pgRP60 and pgRP80 rev-ealed structures of Rep 1 and RepA, respectively (Fig. 2, 3).

Structures of Repl and RepA genes and phylogenet-ic relationship of plant cysteine endopeptidases—TotalRNA fraction was prepared from d-6 aleurone layers andendosperms according to Karrer et al. (1991) with minormodifications. mRNA (20//g) was purified with OligotexdT 30 Super (Takara Shuzo) and used for the determi-nation of transcription start sites of Repl and RepA. Twospecific primers for pRP60 and pRP80 were designed; 5'-TCC CCA TGA TCG ATC GAT CGC CAC T-3' (position102 to 125 in pgRP60; Fig. 2) and 5-CCC AGA TAC CGACAG CGC CAA CAC ACA GGT-3' (position 56 to 80 inpgRP80; Fig. 3). The first strand cDNA was synthesizedand radiolabeled with 32P by a first strand cDNA synthe-sis kit (Takara Shuzo). The sequencing reaction of pgRP60and pgRP80 was performed by a BcaBEST dideoxy se-quencing kit (Takara Shuzo) using two specific primers.C and G nucleotides 119 and 245 bp upstream from thetranslation start sites were determined as major transcrip-tion start sites of Repl and RepA, respectively (Fig. 2, 3).Comparison of the nucleotide sequences of pgRP60 andpgRP80 with the corresponding cDNAs showed that Replhas no introns whereas RepA consists of five exons andfour introns (Fig. 2, 3). As discussed above, Repl exist asone copy gene in the rice genome, indicating that Repl isnot a pseudogene. Barley EP-B genes, EPB1 and EPB2,were reported to have no introns (Mikkonen et al. 1996),while intronless genes are rare among functional eukary-otic genes. Thus, we postulate that Repl and EP-B genesgenerated from a common ancestor via retroviral dupli-cation. The structure and organization of RepA are simi-lar not only to that of the gene for barley cysteine endo-peptidase, EP-A, but also to those of the genes for legumecysteine endopeptidases, SH-EP and EP-C1 (Fig.4A). Thelength and position of the 2nd to 5th exons of RepA cor-respond to those of the 1st to 4th exons of these endopep-tidase genes.

In the phylogenetic tree of plant cysteine endopepti-dases, REP-1 and EP-B are classified into one group, andthree legume cysteine endopeptidases, EP-C1, SH-EP and

B100,

ss95

89

EP-C1

SH-EP

proteinase A

100 L

- EP-A

pRP80 protein

EP-B

REP-1oryzain u

- RD21

• papam

0.05100L

Fig. 4 Phylogenetic relationship of REP-1 and related cysteineendopeptidases. (A) Gene structures were drawn diagrammati-cally with shaded squares (exons) and lines (introns). The fol-lowing sequences were used: SH-EP gene, Akasofu et al. (1990);EP-C1 and EP-A genes, (X63102 and Z97021). (B) The phyloge-netic tree constructed by using the neighbor-joining method(Saitou and Nei 1987). Numbers at branches indicate bootstrapvalues (Felsenstein 1985). The following sequences are used: EP-Cl , Tanaka et al. (1991); SH-EP, Akasofu et al. (1989); pRP80,Shintani et al. (1995); EP-B, Koehler and Ho (1990); REP-1, Katoand Minamikawa (1996); aleurain, Rogers et al. (1985); oryzain a,Watanabe et al. (1991); proteinase A, (Z34895); EP-A (Z97023);papain, (M152O3); RD21, (D13043); and maize-cp (X99936).

proteinase A, united into another group (Fig.4B). Anoth-er group of the pRP80 protein and EP-A, with genestructures resembling those of legume cysteine endopepti-dases, are located near these two groups. This suggests thatthe cereal EP-A and pRP80 genes and the legume SH-EP,EP-C1 and proteinase A genes derived from a commonancestor, and that the REP-1 and EP-B genes were thengenerated from the ancestor of the EP-A and pRP80 genes.

Possible regulatory elements for gibberellin responsein Repl and RepA genes—In the case of cereal a-amylasegenes, three ris-elements, box 1, pyrimidine box andGARE, which are required for the gibberellin-inducedgene expression, are clustered and located around the— 200 bp upstream region from the transcription start site(Huang et al. 1990). Because the levels of REP-1 andpRP80 mRNAs in germinated rice seeds were regulatedby gibberellin and ABA (Shintani et al. 1997), we searchedfor possible gibberellin-responsive elements in Repl and


466 Structures of two rice cysteine endopeptidase genes

GenePutative gibberellin-responsive elements

GARE Box1 Pyrimidinebox

,RAmy1A TAACAAA TATCCAT CCTTTT

Rep1 GAACAAA CATCCAC ACTTTT

RepA TAACAAG TATCCAT CCTTTT

B PG B

415

-I—nb—£

RAmylA

. Repl200 bp

B G. RepA

Fig. 5 A comparison of gibberellin-responsive elements in Repl and RepA. RAmylA represents a typical a-amylase gene (Huang etal. 1990). (A) Typical nucleotide sequences of gibberellin-responsive elements of a-amylase genes are shown in the line of RAmylA.Nucleotide sequences that resembled gibberellin-responsive elements are presented in the lines of Repl and RepA. (B) Structures of threegenes are illustrated diagrammatically. Relative positions of consensus and related sequences are indicated by capital letters; P, py-rimidine box; G, GARE; B, box 1; and + 1 , putative transcription start sites.

RepA, but found no typical sequences in them except for a

pyrimidine box in the 5'-untranscribed region of RepA

(Fig. 5). Sequences analogous to GARE and box 1 were

dispersedly found in the 5'-upstream and transcribed

regions of Repl. In RepA, GARE- and box 1-like se-

quences were present in the 2nd intron far from the TATA

box. These findings are in conflict with a previous report

that GARE and box 1 act cooperatively when they are

located at correct positions (Gubler and Jacobsen 1992).

Thus, GARE- and box 1-like sequences may not be re-

sponsible for the gibberellin-regulated expression of Repl

and RepA. Previously, we reported that the level of pRP80

mRNA in d-5 germinated seeds was markedly lowered by

exogenously supplied ABA or uniconazole, whereas the

levels of REP-1 and its mRNA, pRP60 mRNA, in d-5

seeds were only slightly affected (Shintani et al. 1997),

which indicates that the regulation mechanism for the ex-

pression of pRP60 mRNA may be different from that for

pRP80 mRNA.

This work was supported in part by a grant-in-aid (no.09640776) from the Ministry of Education, Science, and Cultureof Japan.

References

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Nucleotide sequence of cDNA for sulfhydryl-endopeptidase (SH-EP)from cotyledons of germinating Vigna mungo seeds. Nucl. Acids Res.17: 6733.

Akasofu, H., Yamauchi, D. and Minamikawa, T. (1990) Nucleotide se-quence of the gene for the Vigna mungo sulfhydryl-endopeptidase(SH-EP). Nucl. Acids Res. 18: 1892.

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(Received November 9, 1998; Accepted February 15, 1999)


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The Structure and Organization of Two Cysteine Endopeptidase