Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana

Plant Molecular Biology 30: 647-653, 1996. © 1996 Kluwer Academic Publishers. Printed in Belgium.

Short communication

647

Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana

Takeshi Katagiri x,2, Tsuyoshi Mizoguchi I and Kazuo Shinozaki 1,2,,

1 Laboratory of Plant Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan (*author for correspondence); 2Institute of Biological Sciences, University of Tsukuba, Tennohdai, Tsukuba, lbaraki 305, Japan

Received 15 August 1995; accepted 1 November 1995

Key words: Arabidopsis thaliana, diacylglycerol kinase, EF-hand, cysteine-rich zinc finger, PI turnover, Ca 2 +-binding proteins, signal transduction pathways, plants

Abstract

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of D G K has not been character- ized in plants. We report the cloning of a cDNA, cATDGK1, encoding D G K from Arabidopsis thaliana. The cATDGK1 cDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of D G K as well as phospholipase C suggests the existence of PKC in plants.

In the animal system, extracellular stimuli acti- vate phosphatidylinositol-specific phospholipase C, which hydrolyzes phosphatidylinositol 1,4,5- bisphosphate and generates two secondary mes- sengers, inositol 1,4,5-triphosphate ( IP3) and diacylglycerol (DG). IP 3 induces the release of calcium (Ca 2 +) from intracellular stores, and DG activates protein kinase C (PKC). These initial transmembrane signal-transduction processes constitute the phosphatidylinositol (PI) turnover system. The PI turnover system has been suggested to have important roles in signal transduction pathways in higher plants as well as animals

[9]. The rapid breakdown of inositol phospholipid was observed upon auxin treatment [5]. I P 3 stimulates the release of Ca 2 + from vacuoles [ 1, 20, 27]. In order to study molecular mechanisms of the IP3/Ca 2÷ signal-transduction pathway, we have isolated several genes that are involved in the pathway, such as phospholipase C (PLC) and calcium-dependent protein kinases (CDPK) from Arabidopsis thaliana [8, 30, 31]. At least two genes for PLC and four genes for CDPK have been identified in Arabidopsis [7, 33]. We found that one gene for PLC and two genes for CDPK are induced at the transcriptional level by environ-

The nucleotide sequence data reported will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number D63787.

648

mental stress, such as dehydration, high salinity, and low temperature [8, 31]. In higher plants, Ca 2 + has an important role as a second messenger in various signal-transduction pathways. An increase in cytoplasmic Ca 2+ levels has been reported when plants are exposed to environmen- tal stimuli, such as cold shock, touch, or fungal elicitors [ 11, 12].

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid through an ATP-dependent phosphorylation process of DG, an activator of PKC, which is the first step of the resynthesis of phosphatidylinositols. D G K is thought to regu- late PKC by decreasing the level of DG [10]. In addition, phosphatidic acid may function as a second messenger [15, 16, 26]. The cDNAs of DGK have been isolated from pig, rat, and Droso- phila [6, 13, 24]. The primary structures deduced from the mammalian DGKs contain putative ATP-binding sites, two cysteine-rich zinc finger- like sequences that are similar to those found in PKC, and two EF-hand structures, which are typical structures of Ca 2 +-binding proteins. In Drosophila, retinal degeneration A gene encodes an eye-specific DGK, the absence of which leads to rhabdome degradation due to defective phospholipid turnover [ 13 ].

The DGK enzyme activity in suspension- cultured Catharanthus roseus and Nicotiana tabacum has been reported [9, 32]. However, the existence and the function of D G K in higher plants remain unclear. In this study, we report the molecular cloning o fcDNA that encodes putative DGK in Arabidopsis thaliana, and the structural analysis of the putative D G K protein. To our knowledge, this is the first report describing the D G K gene in higher plants. The putative Arabi- dopsis D G K protein also contains putative ATP- binding sites, two cysteine-rich zinc finger-like sequences, and two EF-hand structures similar to those found in mammalian DGKs.

Isolation and sequencing of a cDNA that encodes DGKfrom Arabidopsis thaliana

To understand signal transduction-pathways in plants, we isolated the cDNAs that encode pro-

tein kinases, G-proteins, and transcription fac- tors in plants. Following amplification using the polymerase chain reaction (PCR), we isolated a DNA sequence (clone c7) that contained a partial gene sequence for D G K from A. thaliana (Fig. 1). The inserted clone c7 DNA was used as a probe to screen an Arabidopsis cDNA library for cloning cDNAs that encode DGK homologues. Four positive clones were obtained from a total of 9 x 105 plaques. The cDNA inserts were sub- cloned into the pSKII - vector. Partial-sequence analysis revealed that all of the cloned DNA inserts originated from the same gene. We sequenced the largest insert and named it cATDGK1. Figure 1 shows the nucleotide sequence of the cDNA and the corresponding deduced amino acid sequence, cATDGK1 contains a 2184 bp open reading frame. There is an in- frame stop codon (TAA) 75 bp upstream of the putative initiation codon at nucleotide 90. The cDNA putatively encodes a protein of 728 amino acids with a molecular mass of 79.4 kDa.

Primary structure of the putative A TDGK1 protein

The putative catalytic domain of ATDGK1 has significant homology to those of rat 88K DGK (41~o identity) [6], pig 80K DGK~ (40~o identity) [24] and Drosophila DGK (37~o identity) [13] (Fig. 2A). Like animal DGKs, ATDGK1 contains two cysteine-rich, zinc finger-like sequences at residues 95-137 and 169-212 (Fig. 2B). Such sequences have been reported to exist in PKC and DNA-binding proteins that function in transcriptional regulation. It has been suggested that D G K and PKC possess similar regulatory mechanisms, because the cysteine-rich, zinc finger-like sequences of DGK are very similar to those of PKC. These sequences in PKC as phorbol ester or phospholipid binding sites [3, 18]. This function raises possibility that ATDGK1 activity is also regulated by phospholipids.

ATDGK1 contains two EF-hand motif sequences, typical structures of Ca 2 + -binding proteins, between the zinc finger-like sequences and

649

C CTGAC CAATACAATAATCTT TTTG GGGC TTCTTATTGGCT C C~ATTYCAGGGAT~TT CACACTTGTAGTAGAGTTG CAACATAGACAAATC-GACGACGA 100

M D D D 4

TGGAGAATT ~TGTTCTT TCCTAGCT GGAC CAGCAAGAATC CTATTGACACGGT TGAATCTC GTGGT ~AATGT T CTCTTGTTTTGTTGC TGCC CTT 200

G E L G M F F P S W T S K N P I D T V E S R G L M F S C F V A A L 37

GTTC-GTATATTGAC CAT C GC C TACAC TC-C TTTT CAATGGCGAAGAAATATTAATq'gAAGTT GGAC GAAAGCCATAGC CAGGTC_%AAGAAAAAT C CAAAGG 300

V G I L T I A Y T A F Q W R R N I N L S W T K A I A R S K K N P K A 71

CACGACATAAGGTTCCTGTTGCCC CACATAGCTGGGAACTC GAC C C TATAGCT CGTGCC=AAAAAC TTGAACTGTTGT GTTTG CTTGAAGTCCATGTC GCC 400

R H K V P V A P H S W E L D P I A R A K N L N C C V C L K S M S P 104

ATCTCAGGCAATTGTAGCq~f CAGAA GTTT ~TC CACAGGT GCACAATCTGTGGAGCAGCAGCACATTTTAAC TGTTC TTCAAGTGCGC CAAAAGAT TGC 500

S Q A I V A S E S F F H R C T I C G A A A H F N C S S S A P K D C 137

AAATGTGTC TCCATGGTTGGATTTGAGCATGTGGTGCACCAGTGGGCAGTGCGGTGGACAGAAGGGGCTGATCAGAC TGATGACT C CTCGTTTTGTAGCT 600

K C V S M V G F E H V V H Q W A V R W T E G A D Q T D D S S F C S Y 171

ACTGTGACGAGTCATGTAGTAGCTCC TTT CTI"GGGGGTTCT C CrATATGGTGC TGCTTGTGGTGT CAACGTCTTGTGCACGTTGAC TGTCACAGTAACAT 700

C D E S C S S S F L G G S P I W C C L W C Q R L V H V D C H S N M 204

GTCAAATGA.%ACTGGTGACATTTGTGATT TAGGC CCGCTTAGAAGGTTAATAT TGTGC CCACTCTACGTTAAGGAATTGACGCGGAATC C CT C T GGAGGA 800

S N E T G D I C D L G P L R R L I L C P L Y V K E L T R N P S G G 237

TTTCTGAGCTCAATCACTCATGGTGCTAACGAACTTGCATCTACCGCCCTTGC CAGTATCAGGATTCAAA AAA TACAAGCAAACTAATGAAACTT 900

F L S S I T H G A N E L A S T A L A S I R I Q S K K Y K Q T N E T S 271

CAC-CTGACACTGGTAATAGTGGTAGCAATTGCGATGAATCCACAGAAAGCACAGCTGATACAGGTCCAACTGTTAATC-GCGCTCATGCCGTACTGGAAAA 1000

A D T G N S G S N C D E S T E S T A D T G P T V N G A H A V L E N 304

TT CTATCAGCGTTATGAATGGGGAT TCTT CTAACGGGGACAGTGACAGCAAT~GCTC-GAAAAGAAGCCGAGTGTTAAAAGAACCGGGT C TTTT GGT 1100

S I S V M N G D S S N G D S D S N G K L E K K P S V K R T G S F G 337

CAGAAGGAATAT CATGCACTAAGGT CGAAACTTAAGTATC=AACTAGCTGATTT GCCTT CAGATGCAAGAC CGTTGTT GGTTT~'fATTAACAAAAAGAGTG 1200

Q K E Y H A L R S K L K Y E L A D L P S D A R P L L V F I N K K S G 371

GTGCTCAAC GAGGTGATTCCC TTC GT CAAC GTC TTCATCI~ CATCTAAAT CCTGTGCAGGTATI~TGAATTGAGTTCAGTGCAGGGACCAGAAGTGGGACT 1300

A Q R G D S L R Q R L H L H L N P V Q V F E L S S V Q G P E V G L 404

T TTTC TCTTCAGGAAGGTTC C TCAC TTTAG~ETTCTTGTTTGTGGTGGAGATGGCAC C GCI~GTTGGGTA~'rAGATGCCATAGAGAAACAGAATTI-f ATT 1400

F L F R K V P H F R V L V C G G D G T A G W V L D A I E K Q N F I 437

TCTCCTCCT GCAGTTGCTATAC TGC CTC-C TGGAACCGGGAATGAT CTATC CCGAGTATTAAATrG GGGTGGTGGTTTC43~TTCTGTTGAGAGACAAGGAG 1500

S P P A V A I L P A G T G N D L S R V L N W G G G L G S V E R Q G G 471

GCTTGTCGACAGTATTA~CATAGAGCATGCTGCAGTCACTGTCCTTGAT~GTTGGAAAGTATCGATTCTGAATCAACAAGGAAAGCAACTCCAGCC 1600

L S T V L Q N I E H A A V T V L D R W K V S I L N Q Q G K Q L Q P 504

GCCAAAATATATGACCAATTATATAGGGGT TGGGTGTGATG C TAAGGTTGCCC TTGAGAT TCACAATCTACGC-GAGGAGAAT CCAG~3AGATTTTATAGC 1700

P K Y M T N Y I G V G C D A K V A L E I H N L R E E N P E R F Y S 537

CAGTTTATGAACAAAGT C CTCTATGC CAGAGAAGGTGCAAGGAGTATAATGGACAGAACATTCGAAGATTTC C CTTGGCAAGTTCGAGTTGAGGTGGATG 1800

Q F M N K V L Y A R E G A R S I M D R T F E D F P W Q V R V E V D G 571

GTGTTGACATCGAGGTTC CTGAGGATGCGGAAGGAATACTGGTr GCAAACATTGGAAGTTACATGGGAGGGGTGGATTTATGGCAGAATGAAGA AAC 1900

V D I E V P E D A E G I L V A N I G S Y M G G V D L W Q N E D E T 604

ATATGAAAACTTTGATC CGCAATCGATC-CACGACAAGATAGTTGAAGTTGTGAGTATATCTGGGACATGGCACCTTGGGAAACTT CAGGT CGGGTTG T CT 2000

Y E N F D P Q S M H D K I V E V V S I S G T W H L G K L Q V G L S 637

CGTGCGAGAAC-GCTAGCT CAAGGGT CAGCAGTCAAGATACAACTTTGTGC GCCTTrACCAGTGCAAATT GATGGAGAGCC TTGGAATCAGCAAC CAT G TA 2100

R A R R L A Q G S A V K I Q L C A P L P V Q I D G E P W N Q Q P C T 671

C CTTAACCATATCG CAC CATGGCCAC-GCT TTCAT GCTAAAGAGAGC CGCGGAAGAGC CACTGGGT CACGCAGCAGCTATAATCACAGATGTTCTAGAGAA 2200

L T I S H H G Q A F M L K R A A E E P L G H A A A I I T D V L E N 704

TG CAGAAAC CAATCAAGTGATAAAC GCTT CTCAGAAACGAAC GCTGCTTCAAGAAATGC-CTCTAC GGC TAAC TTAAAACAG GAAAAGA 2300

A E T N Q V I N A S Q K R T L L Q E M A L R L T * 728

CAGAAGACAAAAC C TTC C CTC TTCAACAAC CAATCAATCATCACTGGTTAGTTACAAATATTTACAAATGCGTGTATT~-fACATATCTT GTAC CTCTGCC 2400 TAATGAAGGGAATGTTAAAAAGTC T TGAAGATC GAAAT CCGC TGATTGACAAAG CGGGTTCTGTG GAAGTAGGAAAAAGGAAC CAAAAATTGC=AATCT 2500 GG T TCCTAGAT TITTGGCTGTTCTCA TAGTGAATGAAAGCAAAACGTGT TTAAT T A A ~ 2580

Fig. 1. Nucleotide and deduced amino acid sequence o fcATDGK1. The sequences corresponding to the probe (the inserted DNA of the clone c7) that was used to screen the library is underlined. Oligonucleotide sequences (primer h 5 ' -G A A T T CA T GGAG- TA(TC)(AT)IIGA(TC)TG(TC)GG-3' and primer 2: 5 ' -GAATTCCATGTAIGTI(GC)(AT)IGTICCIACGAAIGT-3 ' ) were used as primers for the isolation of PCR-amplified DNA fragments that contained the partial gene sequence for D G K from Arabidopsis. PCR amplification, cloning, and sequencing were done as described previously [ 14].

650 ATDGKI RAT88KDGK

PIGDGY-IX

DrosophilaDGK

ATDGKI RAT88KDGK

PIGDGIq~

DrosophilaDGK

ATDGKI RAT88KDGK

PIGDGI~IX

Drosoph~aDGK

ATDGKI RAT88KDGK

PIGDGI{~

DrosophilaDGK

ATDGKI RAT88KDGK

PIGDGF4x

DrosophilaDGK

ATD(~[I RAT88KDGK

PIGDGF4x

DrosophilaDGK

ATDGKI RAT88KDGK

PIGDGK~

DrosophilaDGK

ATDGKI RAT88KDGK

PIGDGI¢~

DrosophilaDGK

KRTGSFGQKEYHALRSKLK'YELADLPS-DARPLLVFINKKSGAQRGDSLR 379 ---SDS*AAAKGE*VM .... QYKII**PGTH****LV*P***GRQ*ERIL 449 ...... S**TTDD*NLSTSEA*RID*VSNTH*****V*P***GKQ*ERVL 393 ...... R**GKEEKKEPRAFIVKPI**PEVI*VI****P***GNQ*HK*L 827

QRLI4-LHLNPVQVFELSSVQGPEVGLFLFRKVPHFRVLVCGGDGTAGWVLD 429 ****************-****************************** 498 --*************-******************************** 442 ****************-******************************* 876

AIE--KQNFISPPAVAILPAGTGNDLSRVLNWGGGLGSVERQGGLSTVLQ 477 ***--********************************* .... *S*TKI*K 542 ***--******************************** .... QN*GKI*K 486 VLDQIQPPLQPA***GV**L******A*A*G****YTD .... EPIGKI*R 922

NIEHAAVTVLDRWICqSILNQQGKQLQ ......... PPKYMTNYIGVGCDA 518 E**QSPL*M****YLEVMPREEVENGD ...... QV*YNI*N**FSI*V** 586 DL*ASK**HM***S*EVIP**TEEKSD ...... PV*FQIIN**FSI*V** 530 E*GMSQC*LM***R*KVTPNDDVTDDHVDRSKPNV*LNVIN**FSF*V** 972

KVALEIHNLREENPERFYSQFMNKVLYAREGARSIMDRTFEDFPWQVRVE 568 SI*HRF*VM**KH**K*N*RMK**LW*FEF*TSETFAA*CKKLHDHIEL* 636 SI*HRF*IM**KY**K*N*RMK**LW*FEFATSESIFS*CKKLEESLT** 580 HI***F*EA**AH****N*RLR**MY*GQM*GKDLIL*QYRNLSQW*TL* 1022

VDGVDIEVPEDA---EGILVANIGSYMGGVDLWQNEDETYENFDP . . . . . 610 C***EV--DLSNIFL***AIL**P*MY**TN**GETKKNRAVIRESR--- 681 IC*KPL--DLSNLSL***A*L**P*TH**SN**GDTKRPHGDIHGINQAL 628 C**Q*FTGKLRDAGCHAV*FL**P**G**THP*NDS .............. 1058

........... c ..... QSMHDKIVEWSISGTWHLGKLQVGLSRA-RRL 642

.... KSVTDPKELKCCV*DLS*QLL***GLE*AMEM*QIYT**KS*G*** 727 GAMAKVITDPDILKTCVPDLS**RL***GLE*AIEM*QIYTK*KN*GH** 678 ............ FGASKP*ID*GLM***GL-T*YQLPM**A*MH--GTCI 1093

AQGSAVKIQLCAPLPVQIDGEPWNQQPCTLTISHHGQAFML **********--***************************

*KC*EITFHTTKT**M*******M*T**TIK*T*RN*MP** C*CRKAR*ITKRT**M*V***ACRVK*SVIE*ELLNK*L**

683 768 719

1134

ATDGKI 95 ~CV~KSMSPSQAIVASESFFH~~SSAPKD---~ 137 DGK~ 218 ~FI~SSIG ..... LGKQGL--~C~TV~DQ~IiA-LP----~ 252 PKCI 50 ~S~TDF IW ...... GFGKQGF~V~C~P~EFVTFS - - - -~ 86 Pxc2 11s ~ps~.r~Y ...... G~,i~~~q~vlmrPsr~---~ iSl

1 , 9 .12 DGK(~ 281 ~3R~KK-IRIYHSLVGL .... ~F~C~LEI~DI~2~PAMGH .... ~ 318 PKCI 50 ~SH~PDFIWGFGKQGFQ ..... - ~ Q V ~ ~ E F V T F .... ~ 86 PKC2 1 1 5 ~ D ~ G S L L Y G L I H Q G I ~ . . . . . . ~ D T ~ D M ~ Q ~ V I N V P S . . . . L ~ 1 5 1

X Y Z-Y-X -Z X Y Z-Y-X -Z

DG~: o~ 122 ID~r,Ds~-33- p~f~vsu~l ATCAM 3 21 ~DGD~ITTI~-25- ~JA~IDFI:~

ATDGK1

DGK~x

728 a.a.

N ~ c 734 a.a.

[ ] E-F hand

• zinc finger-like domain

• DG kinase catalytic domain

Drosophila DGK r~ [ ~ C

1454 a.a.

651

the putative D G K catalytic domain (Fig. 2C, D), while mammalian D G K s contain two EF-hand motifs in their N terminals. The Ca 2+-binding proteins calmodulin and calpain have four EF- hand Ca 2 + -binding loops that function as Ca 2 + acceptors [2, 29]. Pig D G K ~ also has such motif sequences and pig DGK~ proteins expressed in Escherichia coli and COS-7 cells are shown not only to bind Ca 2+ but also to be activated by Ca 2+. Pig DGK~, calmodulin, and other small EF-hand proteins are known to undergo confor- mational changes upon Ca 2+ binding; these changes can be detected as mobility shifts in al- kaline urea-PAGE (polyacrylamide gel electro- phoresis) analysis [22]. In a preliminary experi- ment, however, the Ca 2 + binding activity of the ATDGK1 protein expressed in bacteria was not detected through sodium dodecyl sulfate (SDS)- PAGE with or without the presence of Ca 2÷. Based on its structure, it is likely that ATDGK1 protein is regulated by Ca 2 +, but it is still un- known whether the EF-hand motifs of ATDGK1 function as Ca 2 + acceptors.

IP 3 stimulates Ca 2 ÷ release from microsomal vesicles [4, 21] and intact vacuoles in plants [1 ]. D G K may be regulated by Ca 2+, because it contains two EF-hand motif sequences. Based on structural analyses, A T D G K 1 may have a role in a mechanisms that links Ca 2 + signaling and the PI turnover system in Arabidopsis.

Fig. 3. Southern blot analysis of the genomic sequence that corresponds to cATDGK1. Genomic DNA was digested with BamHI, EcoRI and HindIII, separated on 0.7% agarose gels and transferred to nylon membranes. Filters were hybridized with a [32p]-labeled fragment of the cATDGK1 cDNA at 42 °C and washed in either 0.5 x SSC/0.5% SDS at 50 °C (low stringency) or 0.1 × S SC/0.1% SDS at 65 ° C (high stringency). High and Low represent high- and low-stringency hy- bridization conditions, respectively. The sizes of the DNA markers are indicated in kb.

Southern and northern blot analyses

The number of D G K genes in an Arabidopsis genome was estimated by Southern blot analysis (Fig. 3). Nuclear D N A from Arabidopsis plants

was digested with BamHI, EcoRI, and HindlII, electrophoresed on agarose gels, blotted onto nylon membranes, and hybridized under both high- and low-stringency conditions using cATDGK1 as a probe. Under both high- and

Fig. 2. Comparison ofamino acid sequences amongATDGK1 and animal DGKs" Asterisks indicate identical amin° acids" Dashes indicate gaps introduced to maximize alignment. A. Amino acid sequences of D G K catalytic domains. Proteins used for comparison were rat 88K DGK, pig 80K DGKct, and Drosophila DGK. Asterisks indicate conserved amino acids. B. Amino acid sequences of systeine-rich, zinc finger-like regions of ATDGK1, DGKct, and PKC. Rat PKC~ was studied as a representative of the PKC family [ 17]. Putative zinc binding amino acids were boxed. C. Comparison of amino acid sequences of Ca 2 + -binding loops in EF-hand motifs among ATDGK1, DGKct, and Arabidopsis calmodulin (ATCAM-3) [19]. Identical amino acids are boxed. Numbers in parentheses denote the residues number of the first amino acid of reach protein. Numbers between amino-acid sequences represent amino-acid residues between the Ca 2 + -binding loops. X, Y, Z, -X, -Y and -Z denote the calcium-coordinating positions [29]. D. Predicted structures of ATDGK1, DGK~, and Drosophila D G K proteins. Putative catalytic domains, cysteine- rich, zinc finger-like regions, and EF-hand motifs are indicated.

652

Fig. 4. Northern analysis of the expression of ATDGK1 mRNA in Arabidopsis organs. For the detection of ATDGK1 mRNA, 40 mg RNA prepared from roots, shoots, leaves, siliques, and flowers was electrophoresed on 1% agarose gels that containing formaldehyde [25]. The RNA was then blotted onto a nylon membrane and hybridized with [ 3 2 p ] _ l a b e l e d

cATDGK1.

low-stringency conditions, one hybridized band was detected in each restriction digest. These re- sults suggest that A TDGK1 is a single gene in the Arabidopsis genome.

Organ-specific expression of ATDGK1 was examined by northern blot analysis (Fig. 4). ATDGK1 mRNA was detected in roots, shoots, and leaves, whereas it was undetected in flowers and siliques.

Expression of the A TDGK1 protein in E. coli and COS-7 cells

To examine whether ATDGK 1 protein had D G K activity, and whether its activity can be regulated by Ca 2 + and phospholipids, the protein encoded by A TDGK1 cDNA was expressed in E. coli cells as a product of fusion with glutathione S- transferase using a pGEX expression vector [28]. D G K activity of the fusion protein was measured by the deoxycholate method [23], but no in- creased activity was detected when compared with the negative control. Since the DGK activity of pig DGK~ was detected using the transient expression system of COS-7 cells, we then tried detecting the D G K activity of the ATDGK 1 protein using the transient expression system of COS-7 cells. No D G K activity was detected by this experimental method, however (T. Katagiri,

F. Sakane, H. Kanoh and K. Shinozaki, unpub- lished data). This lack of the detectable activity may mean that the expression level of the ATDGK1 protein may be too low to detect, or that protein modification, such as phosphorylation or glycosylation, may be required for the full activation of ATDGK1 in plants.

Analysis of transgenic plants, in which the ADTGK1 gene is overexpressed or downregu- lated, can further the investigation into the function of ATDGK1 in Arabidopsis. This analysis may also provide us with some information on the function of D G K in the PI turnover system and the C a 2 + signal-transduction pathway in plants. Although the existence of PKC in plants has not been reported, the finding of D G K and PLC genes from Arabidopsis strongly suggests the existence of PKC in higher plants.

Acknowledgements

We thank for Professor H. Kanoh and Dr F. Sa- kane of the Department of Biochemistry, Sap- poro Medical College, for the expression of the ATDGK1 protein in COS-7 cells and for helpful discussion. This work was supported in part by the Special Coordination Fund of the Science and Technology Agency of the Japanese Govern- ment, and a grant-in-aid from the Ministry of Education, Science and Culture of Japan, to K.S. T.M. was supported by and a fellowship from the Science and Technology Agency of Japan.

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Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana