structure and expression of two seed-specific cdna clones

Plant CellPhysiol. 37(2): 201-205 (1996)JSPP © 1996

Structure and Expression of Two Seed-Specific cDNA Clones EncodingStearoyl-Acyl Carrier Protein Desaturase from Sesame, Sesamum indicum L.

Yasushi Yukawa1'4, Fumio Takaiwa2, Kazuaki Shoji3, Kyojiro Masuda' and Kyoji Yamada15

1 Department of Biology, Faculty of Science, Toyama University, Gofuku, Toyama, 930 Japan2 National Institute of Agrobiological Resources, Kan-nondai, Tsukuba, 305 Japan3 Biotechnology Section, Toyama Agricultural Research Center, Yoshioka, Toyama, 939 Japan

We have isolated two cDNA clones (CDES01 and 04)encoding stearoyl-acyl carrier protein desaturase (SACPD;EC 1.14.99.6) from immature sesame seeds, and have ana-lyzed accumulation levels of the corresponding mRNAs atdifferent stages and organs in sesame. Clone CDES01 con-tains an open reading frame coding for a 396-amino acidprotein of 45 kDa. CDES04 encodes a partial sequence of141-amino acids. Deduced amino acid sequences of bothclones exhibit a high identity to those of other plantSACPD cDNAs. Northern blots probed with CDES01 andCDES04 indicate that both messages accumulate in a seed-specific manner with a peak at 21 days after anthesis. How-ever, expression patterns also indicate that regulationbetween CDES01 and CDES04 are slightly different. TheCDES01 message accumulates at a low level in young leavesin addition to seeds, whereas accumulation of the RNAtranscript corresponding to CDES04 is restricted to seeds.This observation implies the presence of at least two iso-zymes of SACPD having overlapping but slightly distinctfunctions in sesame.

Key words: cDNA — Fatty acid biosynthesis — Generegulation — Sesame — Stearoyl-acyl carrier protein desat-urase (EC 1.14.99.6).

In higher plants, stearoyl-acyl carrier protein desatura-se (SACPD; EC 1.14.99.6) is a soluble plastid enzyme en-coded in the nuclear genome. SACPD introduces a doublebond at the A9 position of stearoyl-ACP (18:0-ACP) andconverts it into oleoyl-ACP (18:1-ACP). This process is thefirst desaturation step in the pathway of fatty acid biosyn-thesis in plants, and therefore SACPD plays a key rolein determining the ratio of unsaturated to saturated fattyacids in plant membranes and storage oils (McKeon and

Abbreviations: SACPD; stearoyl-acyl carrier protein desatu-rase, ACP; acyl carrier protein desaturase, DAA; days after an-thesis.

The nucleotide sequence data of CDES01 and CDES04 ap-pear in the EMBL, GenBank and DDBJ databases under the acces-sion numbers D422086 and D49833, respectively.4 Present address: Center for Gene Research, Nagoya University,Nagoya, 464-01 Japan.5 To whom correspondence should be addressed.

Stumpf 1982). Previously, SACPD cDNA sequences havebeen obtained from numerous plant species including sever-al oil-bearing crops: Castor (Knutzon et al. 1991), safflower(Thompson et al. 1991), cucumber (Shanklin et al. 1991a),rape (Slocombe et al. 1992), turnip (Knutzon et al. 1992),spinach (Nishida et al. 1992), jojoba (Sato et al. 1992),potato (Talor et al. 1992), flax (Singh et al. 1994) andblack-eyed Susan vine (Cahoon et al. 1994). However, littleis known of the temporal and tissue-specific regulation ofSACPD expression.

Sesame {Sesamum indicum L.) is an important oil-bearing crop. Sesame seeds contain fine quality edible oils,rich in unsaturated fatty acids such as oleic acid andlinoleic acid (85% of the accumulated lipid) (Koh 1987).Thus, sesame is quite adequate for studying on the biosyn-thesis of unsaturated fatty acid. As a first step towardunderstanding the mechanism of regulation of SACPD ex-pression, we isolated two cDNA clones from sesame, de-termined their nucleotide sequences, and examined theirmRNA transcript levels in different organs and at differentdevelopmental stages.

Materials and Methods

Plant material—Sesame (Sesamum indicum L. strain 4294)was grown in a field at Toyama University. Seedlings were incubat-ed on vermiculite under a 16 h light/8 h dark cycle at 28°C for twoweeks.

Slot blot analysis—Total RNA was prepared from immaturesesame seed at different stages (27, 39, 45, 49, 60, and 70 days afteranthesis; DAA) with the extraction reagent, ISOGEN (Nippon-gene, Japan). Ten fig, of each RNA sample was blotted on Hybond-N + membranes (Amersham, U.K.) using a filtration apparatus,Bio-Dot SF (BioRad, U.S.A.). Hybridization was performedunder standard conditions using digoxigenin-labeled (BochringerMannheim, Germany) safflower SACPD RNA (Thompson et al.1991) as a probe. Signal detection was carried out by chemilumi-nescence and exposure to X-ray film (New RX, Fuji, Japan).

Construction and screening of a cDNA library—MessengerRNA was purified from immature sesame seeds (27 DAA) using theFast Track mRNA isolation kit (Invitrogen, U.S.A.). Complimen-tary DNA was synthesized with the Time Saver cDNA synthesiskit (Pharmacia, Sweden) and then ligated into AgtlO arms (Strata-gene, U.S.A.). The resulting recombinant plaques were trans-ferred to Hybond-N+ and probed with a 420-base pair fragmentof the safflower SACPD gene (Thompson et al. 1991). Positiveclones were subcloned into pBluescriptll KS+ (Stratagene) for se-quencing.

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202 Y. Yukawa et al.

Sequencing—The nucleotide sequences were determined bythe dideoxy chain termination method with the UcoBEST sequenc-ing kit (Takara, Japan) and [a-"P]dATP (ICN, U.S.A.). DNA se-quences were analyzed using Genetyx Mac software (SoftwareDevelopment, Japan).

Northern blot analysis—Sesame total RNA was extractedfrom immature seeds (7-35 DAA), leaves, stems and roots of seed-lings with ISOGEN. Total RNA (10 ̂ g) was fractionated in a1.5% agarose gel containing 0.66 M formaldehyde, and then trans-ferred to Hybond-N+. The membranes were hybridized witheither a 1,200-base pair fragment of cDNA clone CDES01 en-coding the entire length of SACPD or a 440-base pair fragment ofcDNA clone CDES04 encoding the partial segment of SACPD.Hybridization with the 32P-labeled probe was carried out understringent conditions (5xSSC, lOxDenhardt's soltion, 10 mMNa2PO4 pH 6.5, 0.5% SDS, 50% formamide, 5% sodium dextransulfate, 0.1 mg ml"1 denatured salmon sperm DNA at 65°C for16 h), with final wash conditions of 0.2 x SSC, 0.1% SDS at 65°C.The hybridized blots were exposed to a sheet of Hypernlm-/miax(Amersham) for two weeks at room temperature.

Results and Discussion

Isolation and structure of cDNA clones for sesameSACPD—Slot blot analysis indicated that SACPD mes-sage was accumulated at highest levels early in the develop-ing phase of the sesame seed (data not shown). There-fore, a cDNA library was constructed from immature seedat 27 DAA. Positive clones were detected at a rate of one in100,000 recombinants. We isolated in total eleven cloneswhich were subsequently divided into two groups basedon their nucleotide sequences. A representative clone wasselected from each group and designated CDES01 andCDES04, respectively. CDES01 contained an open readingframe encoding a 396 amino acid protein of about 45 kDa,which was large enough for SACPD (Fig. 1). The deducedamino acid sequence of the sesame SACPD showed identi-

Table 1 Comparison of putative SACPD amino acid se-quences between sesame and those from other plants

Plant species Identity"%

Ricinus communisCucumis sativusCarthamus tinctoriusBrassica rapaBrassica napusSpinacia oleraceaSolanum commersoniiThunbergia alata (pTAD2)Solanum tuberosumSimmondsia chinensisLinum usitatissimum

89.483.883.382.982.581.780.178.976.876.673.6

° Based on a precursor amino acid sequence deduced from cDNAclone, CDES01.

ties of 89.4% and 73.6% against castor bean (Knutzon etal. 1991) and flax (Singh et al. 1994) SACPDs, respective-ly (Table 1). The deduced amino acid sequence of sesameSACPD was considerably conserved in comparison withother SACPDs, confirming that it is an essential enzyme inhigher plants. As is the case of castor bean and cucumber(Shanklin et al. 1991a), no homology was shown betweensesame SACPD and the stearoyl-CoA desaturases previ-ously described from animals (Thiede et al. 1986), yeast(Stukey et al. 1990) and cyanobacteria (Sakamoto et al.1994). It is suggested that CDES01 contains a 33 amino acidleader sequence which may function as a transit peptide,and the putative mature peptide comprises 363 residues. Totake this leader peptide into account, the identities withSACPDs from other plants are higher than in the wholeprecursor sequence; from 11.5% to 91.2%.

It is likely that clone CDES04 contained a cDNA in-sert encoding another SACPD (Fig. 2). Although CDES04is not a full-length cDNA clone, the deduced amino acidsequence of the partial segment has identities of 78.7% to89.4% with SACPDs from other plants (data not shown).In addition, since CDES04 has a 85.1% identity toCDES01, it is probable that CDES01 and CDES04, al-though showing reatively high homology to palmitoyl-ACP desaturase as previously reported by Cahoon et al.(1992), are isozymes of sesame SACPD. To assess the copynumber of SACPD gene in sesame, we carried out southernblotting analysis of genomic DNA using CDES01 cDNA asa probe. The resulting banding patterns were consistentwith the presumption of the existence of at least two genesin S. indicum (data not shown). To date, SACPD isozymeshave been reported only for rape seed (Slocombe et al.1994) and black-eyed Susan vine (Cahoon et al. 1994).

Temporal accumulation of SACPD messages—The temporal gene regulation of two SACPD isozymes(CDES01 and CDES04) was analyzed in the developingseed of sesame. We obtained preliminary data by RNAslot blot analysis using RNA samples from developingseeds between 28 and 70 DAA and a gene fragment ofsafflower SACPD as a probe. The level of accumulatedtranscripts peaked at 28 DAA, and then leveled off (datanot shown). Consequently, we conducted further analysisof mRNA accumulation in developing seeds between 7 and35 DAA by northern hybridization (Fig. 3A). Results in-dicated that CDES01 transcription was already induced at7 DAA, peaking at 21 DAA, and reducing rapidly after 35DAA. This result is in accordance with SACPD function infatty acid unsaturation since this process precedes lipid syn-thesis. By contrast, although mRNA of CDES04 exhibiteda similar peak in accumulation at 21 DAA, it was lessaccumulated than CDES01 in the early developing phase(7-14 DAA). Therefore, it is implied that CDES01 andCDES04 may have slightly different functions.

Organ specific accumulation of two SACPD tran-


Stearoyl-ACP desaturase cDNA of sesame 203

CCCTGTCCCACCTCTCTCTCTCTCTTCTCACTCTGTCTCTTACATAGCTTGCGACTCTCTCTTTTGTGGAGGAAACAAAA 80CCTGAAAAAGAATACAAGAACAGCAAATAGAGGATGGCCTTGAAGCTGAATGCCATCAATTTTCAATCCCCGAAATGCCC 160

1 M A L K L N A j I N F Q S P K C PTTCGTTTGCTCTTCCACCGGTTGCCAGCGTTAGATCTCCTAAGTTCTTCATGGCTTCCACTCTTCGCTCTGGTTCTAAGG 240

17 S F A L P P V A S V R S P K F F M A S T L R S G S K EAGGTCGAGACGGTCAAGAGGCCTTTTAATCCTCCCCGAGAGGTTCATGTTCAAGTGACACACTCTATGCCACCACAAAAG 320

44 V E T V K R P F N P P R E V H V O V T H S M P P O KATTGAGATCTTCAAAGCTCTGGAAGACTGGGCTGACAATAATATACTTGTTCACCTTAAGCCTGTTGAGAAATGTTGGCA 400

70 I E I F K A L E D W A D N N I L V H L K P V E K C W OACCTCAGGATTTCCTGCCTGATCCATCTTCTGACGGATTCGATGATCAGGTCAAGGAATTGAGGGAGAGAGCCAAGGAGA 480

97 P O D F L P D P S S D G F D D O V K E L R E R A K E ITTCCAGATGATTATTTTGTTGTTTTAGTCGGTGATATGATCACAGAAGAAGCCCTTCCAACATATCAAACAATGCTTAAT 560

124 P D D Y F V V L V G D M I T E E A L P T Y O T M L NACCTTAGACGGTGTGCGGGATGAAACGGGGGCCAGCCCAACTTCTTGGGCTATTTGGACAAGGGCATGGACTGCTGAAGA 640

150 T L D G V R D E T G A S P T S W A I W T R A W T A E EAAATAGGCATGGGGACCTTCTAAATAAATATCTCTATCTCTCTGGACGAGTAGATATGAGACAAATTGAGAAGACTATCC 720

1 7 7 N R H G O L L N K Y L Y L S G R V D M R Q I E K T I 0AGTATCTGATAGGATCAGGAATGGATCCACGGACAGAAAACAGCCCATATCTTGGATTTATCTATACATCCTTCCAAGAA 800

204 Y L I G S G H D P R T E N S P Y L G F I Y T S F O EAGGGCTACTTTCATCTCCCATGGCAACACTGCAAGACTTGCAAGGGAACATGGGGACTTGAAGCTGGCCCAAATCTGCGG 880

230 R A T F I S H G N T A R L A R E H G D L K L A O I C GCACAATTGCCGCAGATGAGAAGCGTCATGAAACTGCATACACCAAGATAGTGGAAAAGCTATTTGAGATTGACCCCAACG 960

2 5 7 T I A A D E K R H E T A Y T K I V E K L F E I D P N DACACTGTTCTTGCTTTTGCTGACATGATGAGGAAGAAGATCTCCATGCCAGCCCACTTGATGTATGATGGCCGTGACGAT 1040

284 T V L A F A D M M R K K I S M P A H L M Y D G R D DAACCTCTTCGACCACTTCTCATCTGTTGCTCAGCGGCTTGGCGTCTATACGGCCAAAGACTATGCCGACATCCTAGAACA 1120

310 N L F D H F S S V A O R L G V Y T A K D Y A D I L E HCTTGGTCGCCAGATGGAAAGTGGCAAATTTAACAGGACTATCTGCTGATGGCAGAAAAGCCCAAGACTATGTCTGTGGGC 1200

337 L V A R W K V A N L T G L S A D G R K A O D Y V C G LTGCCCCCAAGAATCAGGAGGTTAGAGGAGAGAGCTCAAGGGCGGGCCAAGCAAGCACCAAAGATCCCATTTAGCTGGATA 1280

364 P P R I R R L E E R A Q G R A K Q A P K I P F S W ICATGATCGAGAGGTGCAACTCTGAGTTCAGTTCGGATGAGTTTCTTGTGCTCCATCGATGTAACTATTGGTAAGAAAAGA 1360

390 H D R E V Q L *AATTGCATTGTAGCTTGTTTGCACCACCGATTTATTGTTCCTTCAGAAGGATTATGTCGGGAGTGAAAGAGACTGTAGAT 1440AGTGGTTTGTTATCTTTTCGAAGTCTTTTAGATCTTCATATAGGGGCAGCTCTAGTTGTTACGTTGTTCTAATGTGGGCT 1520GGTATGATTTCTTGTCTAGCTATTGTGACTGAAACTGCCAAATATCGTAAGGGAATTTTGGT 1582

Fig. 1 Nucleotide and deduced amino acid sequence of sesame SACPD cDNA, CDES01. Stop codon is denoted by an asterisk. Anarrow head indicates a putative cleavage site of transit peptide.

scripts—We further investigated possible differences in two mRNAs are seed specific, however slight differences inorgan specific accumulation between mRNA correspond- the expression patterns between them were detectable. Aing to CDES01 and CDES04 (Fig. 3B). Total RNA was pre- weak signal was obtained in leaf using CDES01, but notpared from leaf, stem, root and immature seed (21 DAA). for CDES04, as a probe. We can presume from this resultWith both SACPDs, strong signals appeared in seeds. This that the two SACPDs of sesame have not similar spe-result is consistent with SACPD's role in the production of cificities and may have somewhat different roles in lipid bio-unsaturated fatty acids which in sesame seeds comprise synthesis. The fact that CDES01 can crosshybridize with85% of the total fatty acid. For the most part, both the CDES04 depending on hybridization conditions (data not


204 Y. Yukawa et al.

CDES01 241:RLAREHGDLKLAQICKCDES04 1 : K

:AADEKRHETAYTKIVEKLFEIDFNDI"VLAF&DMMRKKISMPAITLAADEKRHETAYTKIVEKLFEIDFDGRVLAUDMMRKKVSMPA

CDES01 301:HLMYDGRCELMYDGRCDNLMYDGRHEN

LFDHFSSVAQRLGVYTAKDYADILEHLVARVICDES04 46:HLMYDGRdENLFDHFSlAh/A0RLGVYTAKDYADILBFLVARWEMEKLTGLSlGE

CVARWKMANLl:lVARWEMEKL1

NLTGLSADGRKAQDYVIRKAODYV

CDES01 361:CDES04 106:

:GLPPRIRRLEERA|QGfcAKQA|PKIpFSW]lH[)p\fliIGLPPRIRRLEERAIHARAKOAISPVPFSWIYGREV

<IPF'VRf

Fig. 2 Alignment of deduced amino acid sequences of two cDNA clones, CDESOl and CDES04.CDES04 clone is not a full-length cDNA.

30045

360105

396141

Identical residues are boxed. The

shown) does not exclude the possibility that, in sesame,there are two different SACPDs which may have specializ-ed functions in different plastid types, and/or may playdifferent roles such as for the synthesis of neutral lipids orphospholipids of varying compositions.

The message corresponding to CDESOl in sesameleaves is not as low as compared to the cases of rape seed(Slocombe et al. 1992) or castor bean (Shanklin et al.1991b). This result is probably related to the fact thatsesame synthesizes not insignificant amounts of lipids inother organs besides seeds such as in leaves and stems. Ac-cording to recent investigations, in Brassica SACPD is ex-pressed in oil bearing tissue or rapidly growing tissue suchas in the anther or meristem (Slocombe et al. 1994). Ourresults with CDESOl are in general agreement with those

CDESOl1 2 3 4 5

CDES041 2 3 4 5

IB CDESOl

1 2 3 4 5 6CDES04

1 2 3 4 5 6

••Fig. 3 Northern blot analysis of total RNA from each stage dur-ing embryo development (A) or from each different organ (B) inS. indicum. Total RNA (10|/g) from each tissue was analyzedusing the desaturase cDNA clones, CDESOl and CDES04 as pro-bes. Hybridization was performed by stringent conditions as de-scribed in the materials and methods. (A) lane 1, 7 DAA; lane 2,14 DAA; lane 3, 21 DAA; lane 4, 28 DAA; lane 5, 35 DAA. (B)lane 1, immature seed (before 14 DAA); lane 2, immature seed (21DAA); lane 3, root; lane 4, stem; lane 5, young leaf; lane 6,mature leaf.

reports. We think that SACPD regulation as exemplifiedwith CDESOl is related to the ability of individual organsof particular plants for lipid synthesis.

From our results and other reports, SACPD gene isregulated temporally, tissue specifically (Slocombe et al.1991, 1994), and is growth-temperature dependent (Chees-brough 1990). These results suggest that the promoter ofsesame SACPD may be useful for the purpose of geneticengineering. SACPD is a key enzyme that determinesthe proportion of lipid saturation. Consequently, theexpression and activity of SACPD also plays a promi-nent role in determining storage oil quality and chilling tol-erance. Recently, in turnip, a case of antisense inhibition ofSACPD gene was reported (Knutzon et al. 1992). We hopeto use the SACPD promoter for sense and antisense trans-genic experiments using both homologous and hetero-logous genes. In particular, we are aiming for the genera-tion of new sesame strains that produce well-balanced fattyacids. We are now studying other desaturases and thegenomic gene structure of SACPD in attempt to identifythe mechanisms governing their regulation. We are also de-veloping methodologies for the production of transgenicsesame, to investigate further the mechanisms of fatty acidbiosynthesis and the regulation of SACPD and other genesin sesame.

We thank Miss Naomi Yoshida for technical assistance andDr. Jon Y. Suzuki for critical reading of the manuscript. This workwas supported by a grant from the Japanese Ministry of Agricul-ture, Forestry and Fisheries.

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(Received October 17, 1995; Accepted January 5, 1996)


structure and expression of two seed-specific cdna clones

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