The Plant Cell, Vol. 6;1495-1507, October 1994 0 1994 American Society of Plant Physiologists

The AAPTl Gene of Soybean Complements a Cholinephosphotransferase-Deficient Mutant of

Ralph E. Dewey,'" Richard F. Wilson,b William P. Novitzky,b and John H. Goodea a Department of Crop Science, North Carolina State University, Raleigh, North Carolina 27695-7620 b Department of Agriculture, Agricultura1 Research Service, Raleigh, North Carolina 27695-7620

Aminoalcoholphosphotransferases(AAPTases) utilize diacylglycerolsand cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane liplds phosphatidylcholine and phosphatidylethanolamine. A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-l,2-diacylglycerolcholinephosphotrans- ferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPn. The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP4hanolamine:sn- 1,2-diacylglycerol ethanolaminephosphotransferase).Moreover, assays of soybean AAPT1-encoded activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of 14C-CDP-cholineinto phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline. Al- though DNA gel blot analysis suggested that AAPTT-like sequences are represented in soybean as a small multigene family, the same AAPn isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library. Expression assays in yeast using soybean AAPTT cDNAs that differed only in length suggested that sequences in the 5'leader of the transcript were responsiblefor the negative regulation of gene activity in this heterologoussystem. The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPTT reading frame is one model proposed for the observed down-regulation of gene activity.

INTRODUCTION

Phosphatidylcholine (PC) is the major constituent mediated reaction. The resultant products (phosphatidylmethyl- of most eukaryotic cellular membranes (Ansell and Spanner, ethanolamine and phosphatidyldimethylethanolamine) are 1982). lnvestigation of the mechanisms of PC biosynthesisand then rapidly converted to PC by the action of N-methyltrans- its regulation in higher plants has been considered important ferases (Datko and Mudd, 1988a, 1988b; Prud'homme and not only because of its role as a structural component of cellu- Moore, 1992). The specific CDP-aminoalcohol substrate(s) lar membranes but also because of its involvement in the preferentially used in the synthesis of PC can differ signifi- processes that determine the desaturation content of both cantly among plant species. It is not known whether the same membrane lipids and storage triacylglycerols (Ohlrogge et AAPTase is capable of utilizing any of the three CDP-amino- ai., 1991; Somerville and Browse, 1991). One of the essential alcohols to synthesize the correspondingphosphatidyl derivative steps in the predominant pathway of PC biosynthesis in higher or whether distinct are involved. plants, commonly referred to as the nucleotide pathway, in- Adding to the complexity of establishing the precise role and volves the utilization of an aminoalcoholphosphotransferase defining substrates of AAPTases in plants is the current con- (AAPTase) localized in the endoplasmic reticulum to convert fusion regarding whether the same enzyme responsible for sn-l,2-diacylglycerol to PC using a cytidine diphosphate (CDP)- the production of PC can also utilize CDP-ethanolamine as aminoalcohol as the head group donor. Although traditionally a substrate in the synthesis of the closely related membrane this reaction was considered simply in terms of a single lipid phosphatidylethanolamine (PE). Although it is clear that CDP-aminoalcohol substrate (CDP-choline) as the source of separate AAPTases are involved in PC and PE biosynthesis the lipid head group, recent work has revealed a more com- in animais and yeast (Bell and Coleman, 1980; Percy et al., plex picture. In higher plants, it has been shown that 1984), severa1 studies have suggested that the same enzyme CDP-methylethanolamineand CDP-dimethylethanolamineare is utilized in the synthesis of both PC and PE in higher plants also physiologically relevant substrates for the AAPTase- (Macher and Mudd, 1974; Lord, 1975; Sparace et al., 1981; Justin et al., 1985). In yeast, two genetic loci have been identified that encode 1 To whom correspondence should be addressed. AAPTase activities: Cm7, encoding CDPcholine:sn-l,2,-diacyl- 1496 The Plant Cell

glycerol cholinephosphotransferase (EC 2.7.8.2), and fPT7, the yeast strain be mutant at both the CPTl and EPT7 loci to encoding CDP-ethanolamine:sn-1,2-diacylglycerol ethanol- eliminate all endogenous cholinephosphotransferaseactivi- aminephosphotransferase(EC 2.7.8.1). The cholinephospho- ties. Yeast strains HJllO and HJ729 are mutant in CPTl and transferase product of the CPTl gene demonstrates minimal fPT7 gene activities, respectively (Hjelmstad and Bell, 1987, ethanolaminephosphotransferase activity (Hjelmstad and Bell, 1988). Because the strains are of identical mating type (MATa), 1987, 1990). The ethanolaminephosphotransferaseencoded HJ110 was initially crossed to a yeast strain of the opposite by fPT7, however, has been shown to also possess significant mating type (KT1115, MATa), and selected haploids from this cholinephosphotransferase activity (Hjelmstad and Bell, 1988, cross (cpt7, MATa) were crossed to HJ729. A 2:2 segregation 1991). 60th enzymes are localized in the membranes of the (weak signal versus no signal) was observed when haploids endoplasmic reticulum. The two yeast gene products share from this final cross were screened using radiolabeled CDP- 54% amino acid identity and are predicted to be very similar choline in the colony autoradiographic assay (data not shown). in secondary structure and membrane topography. Haploids demonstratinga weak signal were presumedto have By genetic complementation of a yeast mutant lacking retained wild-type ethanolaminephosphotransferaseactivity cholinephosphotransferaseactivity, we isolated a cDNA en- (cpt7, EPTl), and those showing no significant signal were pre- coding a soybean AAPTase based on its ability to utilize sumed to be mutant at both loci (cpt7, ept7). One of the radiolabeled CDP-choline as a substrate in the synthesis of double-mutant haploids was designated RK-ec and selected PC. The predicted amino acid sequence of the soybean gene for use in the complementation experiments. RK-ec provided shows homology similar to both of the yeast AAPTase enzymes: an additional advantage in that it is capable of growing on me- 32.2% identity with the yeast CPTl protein and 33.0% with dia containing galactose as the sole carbon source, a quality the EPTl polypeptide. The identification of a higher plant lacking in both HJllO and HJ729 (R. Dewey, unpublished ob- AAPTase gene will now enable us to study the function, sub- servation). This trait (obtained from the KT1115 parent) permits strate specificities, and regulation of this important enzymatic the screening of RK-ec using yeast expression vectors that reaction. possess galactoseinducible promoters. Two cDNA libraries were constructed in the yeast expression vector pYES2, one using mRNAs isolated from young soybean RESULTS leaves and the other using developing seed tissue 25 days after flowering (DAF). The latter material was chosen because of observations that cholinephosphotransferaseactivity in oil- Complementation of a Yeast Strain Deficient in seeds is significantly induced during seed development, Cholinephosphotransferase and concomitant with triacylglycerol accumulation (Slack et al., Ethanolaminephosphotransferase Activities 1985). The cloning of cDNA inserts adjacent to the galactose- inducible GAL7 promoter of pYES2 facilitates regulated gene Previously, yeast strains deficient in cholinephosphotransfer- expression. The double-mutant yeast strain RK-ec was trans- ase and ethanolaminephosphotransferase activities were formed with the soybean cDNA libraries and plated onto obtained from ethyl methanesulfonate-mutagenized cells, and selective media at a density of -3000 colonies per 90-mm Petri their respective genes were isolated by genetic complemen- dish. To repress expression from the GAL7 promoter, the plat- tation (Hjelmstad and Bell, 1987, 1988, 1990, 1991). Because ing media contained glucose as the sole carbon source. mutations at these loci do not result in an easily discernible Transformed colonies were replica plated onto galactose- phenotype, the use of a colony autoradiographic assay was containing media to induce expression of the transgene. Yeast required for both mutant identification and subsequent com- colonies were subsequently transferred to filter paper and plementation. The assay for cholinephosphotransferase activity subjected to the colony autoradiographic assay using "GCDP- is based on the incorporation of radiolabeled phosphorylcho- choline as the radiolabeled substrate. line from CDP-choline into PC using permeabilized yeast Transformed RK-ec yeast colonies were screened for colonies bound to filter paper. Diacylglycerols endogenous to cholinephosphotransferase activity by colony autoradiography. yeast membranes provide the phosphorylcholine acceptors. Approximately half of the screenings were conducted using Radiolabeled are fixed to the filter paper, and the young leaf cDNA library and half using the 25-DAF de- the unincorporated CDP-choline radiolabel is removed with veloping seed cDNA library. Because the colonies were plated dilute trichloroacetic acid washes. at a high density, the area of the plate corresponding to a puta- The initial goal of this study was to isolate a soybean cDNA tive positive signal was restreaked onto fresh selective media, that encodes an AAPTase responsible for PC biosynthesis by and the colony autoradiographic procedure was repeated. This genetic complementation of a yeast mutant deficient in second screen eliminated the few false positive signals that cholinephosphotransferaseactivity. Because the yeast EPT7 occasionally appeared after the initial assay. After screening gene product displays a cholinephosphotransferase activity more than one million yeast transformants, a colony from an that is -60% as active as its ethanolaminephosphotransfer- experiment using the young leaf library was observed that ase activity (Hjelmstad and Bell, 1988), it was important that maintained a positive signal throughout a second and even The AAPT1 Gene of Soybean 1497

thira d colony autoradiographic assay ensuro .T e tha posie tth - Nucleotide Sequence Analysi f pGmaaptso 2 tive signal was attributable to expression of the soybean cDNA and not the result of a genomic reversion of the RK-ec host, The nucleotide sequence of the cDNA insert of pGmaapt2, transformine th g plasmid (designated pGmaapt2 isolates )wa d designated AAPT1, shows i Figurn i singlA . e2 e large open retransformed an d into fresh RK-ec cells shows .A Figurn i e reading frame (ORF) capable of encoding a 389-amino acid 1A, the cholinephosphotransferase-deficient phenotype of long polypeptid identifies ewa d withi cDNAe nth . Data base yeast strain RK-ec was successfully complemented in cells searches comparing the predicted amino acid sequence transformed with pGmaapt2. Furthermore complementee ,th d agains majoe th t r protein data banks revealed extensiv- eho phenotype showed strict inducibilit galactosey yb , confirming molog onl o ydifferent o ytw t sequences CPT1e EPT1d th , an again that the factor responsible for cholinephosphotransfer- gene product yeastf so . Because thes yeaso etw t sequences ase activity is borne on the pGmaapt2 plasmid. share 54% identity at the predicted amino acid level, it is not To verify that the positive autoradiographic signal observed surprising that the AAPT1 gene product would show similarity in the pGmaapt2-transformed RK-ec cells was a result of the to both. Interestingly soybeae th , n polypeptide showed almost synthesis of radiolabeled PC, the filter-bound cells were chlo- equal homology to each of the yeast proteins: 33.0% identity roform-methanol sitextractedn i ue reactioth d an , n products (58.6% similarity) to the yeast EPT1 gene product and 32.2% were characterize thin-layey db r chromatography. Greater than identity (57.0% similarity) to the yeast CPT1 polypeptide. An 98% of the radiolabeled product comigrated with authentic PC alignment of the three amino acid sequences is shown in Fig- (data not shown). ure 3A. Computer analyse yease th f so t CPT EPTd 1an 1 predicted amino acid sequences have previously been use construcdo t t model membrane th r sfo e topograph secondard yan y struc- ture of these proteins (Hjelmstad and Bell, 1990, 1991). Hydropathy profile analysis of the yeast proteins suggested that each possesses seven transmembrane domains. As show hydropathe Figurn th i , e3B y soybeaprofile th f eo n AAPT1 polypeptid vers ei y simila yease th o tt r proteinse th ; polypeptide contains seven segments of sufficient hydropho- bicity and length to span the membrane. In addition to having same th e numbe f predictero d transmembrane domainse th , KT1115 RK-ec: RK-ec: RK-ec:

CAGAAGCCACGCTGTTCTTTCMATCGTAACCGAAGW 41 transferase activities rather than secondary loci that either mk regulatethe activities of the structural genes or act by altering RGATGGTGCGCTGCTGTCGGTGCTTCGCCTGAGTCTCATTCCGTTTGCGCCCACTATCAC 101 mvrccrcfa substrate availability. An additional approach for demonstrating gene function is to express the product in a heterologous AGGTTGTGCTGAGGCTTAACTGGTTCTTG~GCATATTTCAGAGTTTTATCAGAG~CAA161 4 organism that does not possess the activity of interest. E. coli ATGGGTTATATTGGGACTCACGGTGTTGCAGCTCTGCACAGATAT~TATAGTGGAGTA 221 membranes do not contain PC, and the organism completely 1MGKIGTHGVAALHRYKYSGV lacks AAPTase activities. GATCATTCCTATGTGGCCAATGTACTACAACCCTTTTGGAGTCGCTTTGTTAATTTT 281 To facilitate the nucleotide sequencing of the soybean 21DH S YVAKKVLQPFWS RFVN F pGmaapt2 clone, the cDNA insert was initially subcloned into TTCCCCCTATGGATGCCTCCAAACATGATGATAACTCTTATGGGATTTATGTTCTTACTACTG 341 41 F P L W M P P N E; I T L M G F M F L L L E. colivectors pUC119 and pUC120 (see Methods). From the sequence, it was determined that the AAPTl reading frame TCTGCATTGCTTGGTTATATATACTCCCCACAATTGGACACAGCTCCTCCAAGATGGGTT 401 61SALLGK I K S PQLDTAPPRWV in pUC120 (designated pGmaaptl20) was in the sense orien-

CATTTTGCTCATGGACTACTTCTGTTTTTATACCAGACATTTGATGCTGTTGATGGG~G461 tation in relation to the plasmids lacZ promoter and in the 81H FAHG LLL FLYQTFDAVDG K antisense orientation in pUC119 (designated pGmaaptll9). To CAAGCTAGACGAACAAATTCCTCAAGCCCATTGGGGGAGCTCTTTGATCATGGGTGTGAT521 test for the ability of the AAPT7 readingframe to confer choline- 101 QARRTNSSSPLGELFDHGCD phosphotransferaseactivity in E. coli, cells were transformed GCACTTGCTTGCACATTTGAAGCATTGGCTTTTGGGAGCACAGCCATGTGTGGAAGAACT581 with pGmaaptl2O (and pGmaaptll9 as a negative control), 121A L A C T F E A L A F G S T A M C G R T transferred to filter paper, and assayed using colony autoradi- ACTTTCTGGTGGTGGTTAATATCTGCAATTACATTTTATGGTGCAACCTGGGAGCATTAT 641 141T F W W W L I S A I T F Y G A T W E H K ography. As shown in Figure lB, pGmaaptl20-transformed E. coli colonies demonstrated a weak, yet positive signal in TTCACCAATACACTTATACTGCCTGTTATAAATGGGCCTACAGAGGGTCTTATGATAATA 701 161 FTNTLILPVINGPTEGLMII the assay; no signal was detected using the pGmaaptll9 vector.

TACATCTGTCACTTTTTCACTGCTATCGTGGGTGCTGAGTGGTGGGTTCAGC~TTTGGA161 Furthermore, the cholinephosphotransferaseactivity was in- 181K I C H F F T A I V G A E W W V Q Q F G ducible by isopropyl P-o-thiogalactopyranoside, as would be AAGTCTTTGCCATTCTTRAATTGGCTTCCTTATCTTGGGGGAATCCCGACATTCAAAGCT 821 expected from a gene expressed from the lacZpromoter.Thin- 201K S L P F L N W L P K L G G I P T F K A layer chromatography analysis of the radiolabeled products ATATTGTGTTTAATGATAGCTTTTGGTGTTACACCAACAGTCACATGC~TGTTAGT~T 881 confirmed that the radiolabel was incorporated into PC (data 2211 L C L M I A F G V T P T V T C N V S N not shown). The ability of the soybean cDNA to direct synthe- GTTTACAAGGTTGTGAAGGGRAAGAATGGAAGCATGCATGCCACTTGCATTGGC~TGCTTTAC941 sis of PC in E. coli using 14C-CDP-cholineprovided compelling 241 VYKVVKGKNGSMPLALAMLY evidence that theAAPT7 gene encodes a structural AAPTase CCATTTGTTGTACTTGTGGGAGGAGTGCTTGTGTGGGATTATTTGTCACCATCAGACATC1001 261P F V V L V G G V L V W D K L S P S D I enzyme.

ATGGGGRAATATCCACATTTAGTTGTTATAGGACTTACTTTCGGATATCTTGTG1061 281M G K Y P H L V V I G T G L T F G Y L V

GGGAGGATGATTTTGGCACACTTATGTGATGAACCTAAGGGTCTGAAAACTGGGATGTGC1121 lnhibition of Soybean AAPTT-Mediated 301G R M I L A H L C D E P K G L K T G M C Cholinephosphotransferase Activity by ATGTCCCTCATGTTTCTCCCATTGGCCATTGCCAATGTACTTGCATCCAGGCTAAATGAT1181 CDP-Ethanolamine 321 MSLMFLPLAIANVLASRLND GGGGTTCCTTTAGTAGATGAGAGACTAGTTCTTCTTGGTTACTGTGCATTTTCAGTGACA1241 Severa1 studies have presented evidence suggesting that 341G V P L V D E R L V L L G Y C A F S V T cholinephosphotransferase and ethanolaminephosphotrans- CTATACTTGCATTTTGCCACATCAGTCATTCATTCATG~TTACCAATGCCCTGGG~TATAT1301 361L K L H F A T S V I H E I T N A L G I K ferase activities in higher plants may be catalyzed by a single enzyme (Macher and Mudd, 1974; Lord, 1975; Sparace TGTTTCAGGATAACTAGGAAGGAAGCTTGAATCCACCCAAGATTTCCTTTAGCTTGCAAG1361 381C F R I T R K E A et al., 1981; Justin et al., 1985). Among the data supporting

TCGAGCAAAATATTGGCAAGGGTCAGTACTAATTTTGCTTTATTTGACT~CATGGATCT1421 this hypothesis are observations that CDP-choline and CDP- TGTAATTATATATTGATTCTTTATCCTTTTTTTTTTAAGTTCTTAAGTTTTATATGTGAC1481 ethanolamine are competitive inhibitors of ethanolaminephos- CAGGTCTGTTGAAATTTACATCGTTGATTATGATCACAATATAAGTCATACAACGTTTTA1541 TCTTTT(61 As1 1608 photransferase and cholinephosphotransferase activities, Figure 2. Nucleotide and Predicted Amino Acid Sequences of a Soy- respectively. By the expression of pGmaapt2 in the heterologous bean AAPTl cDNA. yeast system, we have demonstrated that the AAPWencoded polypeptide possesses significant cholinephosphotransferase The sequence presented corresponds to the longest AAPT7 cDNA activity (Figure 1). The most direct approach to establishing characterized in this study (frorn pBSaaptl.6). An arrow indicates the the degree to which the AAPT7 gene product may also pos- beginning of the pGrnaapt2 cDNA that was identified by complementa- tion of yeast Strain RK-ec. The nucleotide sequence of the uORF motif sess ethanolaminephosphotransferaseactivity would be to located in the 5’flanking region of the transcript is underlined (predicted assay its ability to use radiolabeled CDP-ethanolaminein syn- arnino acid sequence is in lowercase letters). The GenBank accession thesizing labeled PE. Because radiolabeled CDP-ethanolamine number of the AAPT7 cDNA sequence is U12735. is currently unavailable commercially, we tested the ability of unlabeled CDP-ethanolamineto inhibit the cholinephospho- transferase activity of the AAPTl enzyme as a preliminary step AAPT1e Th Gen f Soybeaeo n 1499

A B

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6.6 — — 1.6kb

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2.3 — 2.0 — CPT1 201 F I A V O ...TIWHTKBP V AOFSBODFVF DVEHvHLUYA AAPT 4 HFF18 1 | BAE WWVQOFOKSB PFLNHLPYLG GIPUFKAILC a 3 EPT1 187 L I L SKQ ...VIWHTY™ FTITVGDKVI DVDDLDIVFS § a

8 CPTF.CTGALIF23 1 N IVTAHTy R YYESOSTKSV A TPSKTAENIS AAPT 4 HMIAgfflvTP22 1 T VTCNVSG VVKGKNGgup LALAMLYPFV 65°C 50°C 4 J.AV^BLVMEPT22 I N ALSAKF YY.RNSlHsA NNITOIEQDS I CPT1 277 K A| AAPT1 264 V L EPT1 262 . A I K| I

CPT1 317 F V NFPFLIPTIQ nVLYAFMVYV l BloFigurGe t . Analysee4 f Soybeaso n Usin AAPT1n ga Probe. AAPT1 298 YL GMCQSLMFLP ^AIANV | FT 1 EPT30 1 NAPELIPSCQ IVLYKICLR (A) Genomic DMA gel blot of AAPT1 in soybean. Each lane represents 6 CPTHDYQKGSIV35 1 S T L A I HGI 10 ng of EcoRI-digested genomic soybean DMA hybridized to the AAPT1 AAPT1 338 HNDGVPLVDE VTLYLHHAT F V I K N ES I G W 0 EPT34 I LGVH probe under condition higf so h (50°C(65°Cw lo r )o hybridization strin-

CPT1 395 gencies. The arrow indicates a hybridizing band that is visualized only AAPT1 378 under low-stringency conditions. Molecular length markers are given EPT1 379 lefe kilobasesan i tth t . (B) RNA gel blot of AAPT1 transcripts in different tissues of soybean. B Ten micrograms of poly(A)+ RNA was used for each lane. The molec- Soybean AAPT1 ular length of the hybridizing transcript was estimated using RNA size standards (dat t shown)devF ano DA . seed5 .2 , developing seed5 s2 DAFmature Th . e 1.6-kb AAPT1 transcrip indicateds i t .

toward addressin questioe gth f whetheno soybeae rth - nen zyme can catalyze both of the AAPTase reactions. Microsomal membrane preparation f yeasso t strain RK-ec transformed with pGmaapt2 were assaye their dfo r abilito yt synthesize radiolabeled PC using 14C-CDP-choline and en- dogenous diacylglycerol substratess sa . Increasing amounts of nonradiolabeled CDP-choline or CDP-ethanolamine were added to compare the relative effects of each compound in reducing the incorporation of radiolabeled CDP-choline into Yeast EPT1 PC. These result showe sar Figurn i expected s A . e5 , reduc- specifie th g cin activit reactioe th f yo n (i.e., increasing quantities of unlabeled CDP-choline) resulted in a continual reduction in the synthesis of radiolabeled PC. Interestingly, a very similar patter observes nwa d when nonradiolabeled CDP-ethanolamine was assaye useth dn i .

(B) Hydropathy profiles of the predicted amino acid sequences of the Figure 3. Comparison of AAPTases from Soybean and Yeast. soybean and yeast AAPTases. Hydropathic values were calculated and (A) Alignment of the amino acid sequences derived from the yeast CPT1, plotted accordin methoe th go t Kytf Doolittldd o ean e (1982) usin winga - EPT1, soybead an n AAPT1 cDNAs. Identical residue soybeae th r sfo n dow residues0 siz2 f e o sevee Th . n candidate membrane-spanning AAPT1 protein and the yeast CPT1 protein or the soybean AAPT1 pro- domains for the yeast EPT1 polypeptide proposed by Hjelmstad and yease th teid t EPTnan 1 polypeptid showe blacear a n no k background. Bell (1991) are indicated as M1 to M7. 1500 The Plant Cell

RK-ec:pGmaaptS When the same assay was used to measure inhibition of the cholinephosphotransferase activity mediated by the yeast 30) 1 CPT7 gene, a much different result was obtained. Yeast strain HJ729 is mutant at the EPT7 locus but contains a wild-type CPT7 gene (Hjelmstad and Bell, 1988). Total cholinephospho- transferase activity (nanomoles of 1%-PC per milligram of protein per hour) was approximately four times greater in the RK-ec cells transformed with the soybean gene than was ob- served using HJ729 (Figure 5). This is most likely a result of the fact that the soybean sequence was expressed from a multiple copy plasmid vector (and a different promoter) in con- O 100 200 300 400 500 600 trast to the single chromosomal copy of CPT7 in HJ729. Similar pM unlabeled CDP-Amlnoalcohol to the RK-ec:pGmaaptP results, increasing concentrations of nonradiolabeled CDP-choline decreased the production of ra- diolabeled PC. The addition of CDP-ethanolamine to the HJ 729 reaction, however, failed to inhibit synthesis of radiolabeled 10 PC in yeast strain HJ729 (Figure 5). This result demonstrated that the cholinephosphotransferaseactivity encoded by the yeast CPT7 gene differs radically from the cholinephospho- transferase activity of soybean AAPT7 with regard to the ability of CDP-ethanolamine to inhibit the respective reactions. Yeast strain HJ110 is mutant at the CPT7 locus, but contains a wild-type EPT7 gene (Hjelmstad and Bell, 1987). Although EPT7 encodes an enzyme that is believed to function primarily - as an ethanolaminephosphotransferase,significant choline- 01.1. I 'I.'.'.' O 100 200 300 400 500 600 phosphotransferase activity has also been attributed to this gene product. The rate of 14C-PCsynthesis measured using WMunlabeled CDP-Aminoalcohol HJ110 microsomal membranes was approximately fourfold less than that observed from HJ729 and 15 times less than the HJ 110 RKec:pGmaapt2 membrane preparations. Similar to the results I obtained from the CDP-aminoalcohol inhibition profiles of the soybean AAPT7 gene product, both CDP-choline and CDP- ethanolamine were effective in reducing incorporation of 14C-CDP-cholineinto PC in assays conducted using HJ110 membranes. In contrast to the soybean AAPTGencoded en- zyme, however, the assays for cholinephosphotransferase activity mediated by EPT7 appeared to be inhibited to a greater extent by CDP-ethanolamine than CDP-choline; at any given inhibitor concentration, the rate of 14C-PCsynthesis was two- to threefold less in assays using CDP-ethanolamine as the o i00 200 300 400 500 600 competitive inhibitor than was observed using CDP-choline $4 unlabeled CDP-Amlnoalcohol (Figure 5). Figure 5. Effect of CDP-Choline and CDP-Ethanolamineon the Incor- poration of I4C-CDP-Cholineinto Phospholipid by Soybean and Yeast AAPTases. Microsomal membrane fractions were isolated from the following yeast Cloning of AAPTl cDNAs from a Developing Seed strains: RK-ec expressing pGmaapt2 (AAPT1, cpt7, epfl), HJ729 (CpT1, cDNA Library ept7), and HJllO (cpf7,EPT7). Cholinephosphotransferase assays were conducted for each membrane preparation in the presence of increas- Current models of triacylglycerol biosynthesis in oilseeds predict ing concentrations of unlabeled CDP-choline (U). or unlabeled a specialized role for the enzyme cholinephosphotransferase CDP-ethanolamine (O). Enzyme activity was measured as nanomoles during seed development by facilitating the temporary mobili- of radiolabeled PC ("PC) produced per milligram of membrane protein zation of fatty acids to PC, thereby allowing the polyunsaturation per hour. of the acyl chains by the 18:l and 18:2 desaturases (Ohlrogge et al., 1991; Somerville and Browse, 1991). Because the soy- bean AAPT7 cDNA was capable of expressing high levels of cholinephosphotransferaseactivity in yeast, we considered it The AAPT7 Gene of Soybean 1501

likely that either the AAPT7 gene or a gene closely related to Evidence of Post-TranscriptionalRegulation of AAPTT it is responsible for the cholinephosphotransferase activity ob- served during this important stage of development. DNA gel In addition to providing evidence that the AAPTl polypeptides blot assays suggested that AAPT7-like sequences may exist produced in the leaf and developing seeds are likely to be the as a small multigene family (Figure 4A); therefore, it is possi- same, analysis of the seed-specific cDNAs also provided in- ble that a different isoform of the gene is expressed during teresting information regarding the 5'noncoding sequence of storage oil biosynthesis than that used for the production of theAAPT7 mRNA. Four of the five seed-specific clones exam- membrane lipids in young leaves (the tissue from which the ined had cDNA inserts that were somewhat longer than that pGmaapt2 cDNA was derived). originally characterized from pGmaapt2 (1.44 kb for the leaf To determine whether a different isoform of AAPT7 is ex- clone versus -1.6 kb for the longest cDNA from the develop- pressed during seed development, a hZAPll cDNA library ing seed cDNA library). Moreover, the RNA gel blot assays generated from 25-DAF developing soybean seed mRNAs was shown in Figure 48 revealed a mature AAPT7 transcript of screened using the pGmaapt2 cDNA insert as a hybridization 4.6 kb in all tissues examined. probe. Hybridizationswere conducted at low stringency in case The cDNA insert of pGmaapt2 contains 44 bp of 5'noncod- significant divergence had occurred between the leaf- and ing sequence upstream of the Met initiation codon (Figure 2). seed-specific members of the gene family (and because the The authenticity of this proposed initiator start site is supported DNA gel blot data indicated that an additional band could be by the following observations: (1) there is an in-framestop codon visualized at low-stringency conditions). AAPT7 hybridizing (TGA) 33 bp upstream of the putative translational start site plaques were fairly abundant in the library, occurring at a fre- and (2) the proposed AAPT7 Met initiation codon aligns per- quency of approximately one in every 7000 plaques screened fectly with the yeast EPT7 start codon (Figure 3). Sequence (data not shown). Eight positively hybridizing plaques were pu- analysis of pBSaaptl.6 revealed that the full-length mRNA tran- rified to homogeneity, and their corresponding plasmids were script contains an additional short ORF (11 codons in length) excised. Although the cDNA inserts varied somewhat in total 124 bp upstream of the AAPT7-encoding reading frame (Fig- length (ranging from 1.2 to 1.6 kb), a detailed restriction en- ure 2). Three of the five characterized AAPT7 cDNAs from the zyme analysis revealed no differences in the restriction profiles seed-specific library contain 5' leader sequences that possess of the various clones for any enzyme tested, and neither were this small reading frame, one terminated within it, and the fi- differences in hybridization intensities observed using the nal cDNA terminated within the AAPT7 reading frame (data AAPT7 cDNA probe in DNA gel blot assays (data not shown). not shown). To further characterize the seed-specificAAPT7 cDNAs, each In eukaryotes, translational initiationtypically proceeds from clone was subjected to limited nucleotide sequence analysis. the first AUG codon encountered by the ribosome. For the great More than 350 bases of nucleotide sequence information were majority of eukaryotic genes, the first AUG codon of the ma- obtained from both the 3'and the 5'ends for each clone (data ture mRNA represents the beginning of the reading frame of not shown). Three of the eight individuals were assumed to' the encoded protein product. Severa1 examples have been be duplicate copies of other of the cDNAs (resulting from the documented, however, where one or more short upstream amplification of the cDNA library) as judged by perfect iden- ORFs (uORFs) will precede the larger, primary reading frame tity in both size and sequence. For each of the five unique of the transcript (for review, see Kozak, 1989). In systems where cDNAs, the length of the poly(A)+ tails varied, and some mi- their effect on gene expression has been examined, these nor variation in the fidelity of where the polyadenylationinitiated uORFs typically result in a decrease in translational efficiency was observed. At the 5'end of the cDNAs, the only variations of the principal ORF. detected were in total length. Otherwise, the nucleotide se- To determine whether sequences present in the 5'flanking quences of each clone were identical not only to each other region play a role in the post-transcriptionalregulation of AAPT7, but also to the original AAPT7 cDNA isolated from the young the insert from pBSaaptl.6 was cloned (in the sense orienta- leaf-specific library. tion with respect to the GAL7 promoter) into the yeast The complete nucleotide sequence of the longest AAPT7 expression vector pYES2. Mutant yeast strain RK-ec was trans- clone isolated from the developing seed library, designated formed with this plasmid, designated pGmaaptl.6, and assayed pBSaaptl.6, was determined and is shown in Figure 2. The for cholinephosphotransferase activity using the colony autora- AAPT7 cDNA sequence from pBSaaptl.6 differed from the diographic procedure. As shown in Figure 6, a great decrease pGmaapt2 insert only in the lengths of the 5'leader sequence in cholinephosphotransferaseactivity was seen in RK-ec cells and poly(A)+ tail. The inability to detect nucleotide sequence transformed with pGmaaptl.6 when compared with the origi- differences between pGmaapt2 and pBSaaptl.6 and in more nal pGmaapt2 vector that lacks the extended 5'flanking region. than 700 bases of nucleotide sequence (5' and 3' end infor- To provide an estimate of the decrease in enzyme activity at- mation combined) among each of four additional independent tributable to the 5' flanking region of clone pGmaaptl.6, the cDNAs suggests that the sameAAPT7 isoform recovered from filter-bound yeast colonies from Figure 6 were chloroform- a young leaf-specific cDNA library is likely to be the predomi- methanol extracted, and the %-PC was analyzed by thin-layer nant, if not sole, AAPT7 gene that is expressed during seed chromatography after each sample was standardized accord- development. ing to total lipid content (data not shown). Quantitation of the 1502 The Plant Cell

Prud'homm Moored ean , 1992). These product subsee sar - quentl actioye converteth A/-methyltransferasesy f nb o C P do t . preferree Th d biosynthesiroutC P f eo s also appeare b o st very different among the various plant species examined. In soybean leaves and cell cultures, it appears that CDP-methyl- ethanolamine is the primary substrate used by the AAPTase reaction that leads to the production of PC. In contrast, the RK-ec: RK-ec: principal physiological substrat CDP-choline likels ei b o yt e pGmaapt2 pGmaaptl.6 in Lemna and castor bean endosperm, and all three CDP- Figur . Colone6 y Autoradiographic Assay Using AAPT1 cDNAs Con- aminoalcohols (methylethanolamine, dimethylethanolamine, taining 5' Leader Sequences That Differ in Length. cholined an ) appea involvee b biosynthesio e rt th dn i n i C P f so AAFT1 cDNAs cloned intpYESe oth 2 expression vector were transformed carrot (Datk Muddd oan , 1988b; Prud'homm Moored ean , 1992). into strain RK-e plated can d onto inducing media (containing galactose). The evidence presente thidn i s study clearly demonstrates pGmaapt originae th 2s i l AAPT1 construct identifie abilits it y comdo yb t - that the AAPT1 gene product of soybean possesses significant plemen mutane th t t yeast phenotype. pGmaapt possesse6 l. longese sth t cholinephosphotransferase activity . Wha (Figure5) d t an s1 AAPT1 cDNA characterize thidn i s study. Length difference' 5 e th n si remains to be determined is the degree to which the enzyme flanking regions of the two cDNAs are shown in Figure 2. may also utilize CDP-ethanolamine in catalyzing the synthesis CDP-methylethanolamind an , oPE f CDP-dimethylethd ean - anolamine in producing PC via phosphatidylmethylethanolamine radiolabeled products was conducted using an imaging scan- phosphatidyldimethylethanolamind an e intermediatesa s A . ner and demonstrated that RK-ec cells transformed with preliminary investigation to address these questions, the syn- pGmaaptl .6 were ~20-fold less effective in synthesizing radio- thesi f 14so C-P C using 14C-CDP-cholin measures ewa n i d labele thaC dP n cells transformed with pGmaapt2. microsomal membranes of yeast cells expressing the soybean gene in the presence of increasing amounts of nonradioac- tive CDP-choline or CDP-ethanolamine. The rate of reduction DISCUSSION synthesin i radiolabelef so observeC dP loweriny db spee gth - cific activity of the radiolabeled substrate (i.e., increasing nonradiolabeled CDP-choline concentration vers ywa ) simi- Current models describing PC and PE biosynthesis suggest lar to that observed when nonradiolabeled CDP-ethanolamine tha role AAPTasef tth e o thisn i s proces diffey sma r somewhat was adde reactioe th d(Figuro t x thinn mi I s. erespect5) e ,th between plan animad an t fungar o l l systems animaln I . d san soybean enzyme differed dramatically fro yease mth t choline- yeast, CDP-choline and CDP-ethanolamine are believed to be phosphotransferase encode CPT1e th y d b gen e that failedo t primare th y substrate separato tw r sfo e AAPTases, choline- inhibitee b evet da n very high concentration CDP-ethanolf so - phosphotransferase and ethanolaminephosphotransferase, amine inhibitioe Th . n profilsoybeae th f eo n AAPT1-encoded respectively (Bell and Coleman, 1980; Percy et al., 1984; enzyme more closely reflecte dyease thath f to EPT1 gene prod- Hjelmsta Belld dan , 1991) highen I . r plants, however quese ,th - uctn enzyma , e s thabeeha t n show o possest n s both tion of whether a single or separate enzyme(s) catalyzes the cholinephosphotransferas ethanolaminephosphotransd ean - respective reactions remains unanswered. Several studies in ferase activities. Consistent wit presumes hit d primary function plants have presented compelling evidence suggesting that of catalyzin ethanolaminephosphotransferase gth e reaction, same th e enzyme catalyzes both reactions (Mache Muddd ran , however, CDP-ethanolamine appeare more b do et effective than 1974; Lord, 1975; Sparace et al., 1981; Justin et al., 1985). The CDP-cholin competitive th en i e inhibitio cholinephose th f no - strongest data supporting this hypothesis are observations that photransferase activity of the EPT1 gene product (Figure 5). CDP-choline and CDP-ethanolamine are competitive inhibitors One model tha compatibls i t e wit above hth e observations of ethanolaminephosphotransferas cholinephosphotransd an e - is that the soybean AAPT1 gene product displays similar ferase activities, respectively failure th find eo t d,an inhibitors substrate binding affinities to both CDP-choline and CDP- that can differentiate the two activities. Reports suggesting ethanolamine and is capable of synthesizing either PC or PE, that separate enzymes catalyz respective eth e reactione sar depending upon substrate availability. A more direct enzymo- based largely on molecular species labeling studies (Dykes logical characterization, however, will be necessary to determine et al., 1976; Harwood, 1976). relative th e degre whiceo t >AAP7"7-encodee hth d enzymy ema The issue is further complicated by observations that CDP- catalyz onlt ethanolaminephosphotransferase no y th e reaction cholin CDP-ethanolamind ean exclusive th t no e esubstratear s using CDP-ethanolamine, but also its ability to utilize CDP- AAPTase foth r e reaction highen si r plants. Current models methylethanolamine and CDP-dimethylethanolamine as sub- predict that CDP-methylethanolamine and CDP-dimethyleth- strate helso t p establis rols h it catalyzin en i g foue eacth rf ho anolamine can also be utilized by AAPTases to produce physiologically relevant AAPTase-mediated reactions involved phosphatidylmethylethanolamin phosphatidyldimethyld ean - highen i C P r synthesid plantse th an n i E P . f Witso isolae hth - ethanolamine, respectively (Datko and Mudd, 1988a, 19885; tion of the soybean AAPT1 cDNA and the ability to express The AAPT7 Gene of Soybean 1503

its product in yeast, we are now in a position to conduct inves- Evidence of potential negative post-transcriptional regula- tigations to definitively establish substrate specificities and tion of the soybean AAPT7 gene may have parallels with two more precisely determine its role in phospholipid biosynthesis. other genes in the glycerolipid biosynthetic pathway of plants. In addition, to help determine whether other AAPT7-like genes Hannapel and Ohlrogge (1988)demonstrated that the acyl car- exist and encode AAPTases with different substrate specifici- rier protein (ACP)/mRNA ratio in developing soybean seeds ties, the AAPT7 sequence could be useful as a hybridization was significantly less than that observed for lectin, causing probe in the screening of cDNA libraries. them to suggest that reduced translational efficiency may con- The scanning model is widely accepted as the mechanism stitute one aspect of ACP regulation. Long runs of CT repeats by which translation of eukaryotic mRNAs is initiated (for re- and/or the conservation of a 7-bp GC-rich motif in the 5‘ non- view, see Kozak, 1991). Simply stated, eukaryotic ribosomes coding region of the mRNA was considered to be responsible initially bind to the 5’end of an mRNA and initiate translation for reducing translational efficiency of the ACP gene (Ohlrogge at the first AUG codon in a “good” context. Although many of et al., 1991). Recently, evidence suggesting the negative post- the nucleotides surrounding the initiationAUG codon are capa- transcriptional regulation of the 18:l desaturase of Arabidopsis ble of affecting the translational efficiency of a given mRNA, was also presented (Okuley et al., 1994). A T-DNA insertion generally positions -3 and +4 (relative to the A of the AUG mutation in the 5’flanking region of the 18:l desaturase resulted codon) have the greatest influence. The presence of a purine in a nearly 90% reduction of its steady state mRNA levels. This residue at position -3 appears to be the single most impor- low leve1of transcript, however, was sufficient to confer greater tant feature that is required for optimal translation of a message than 50% of the 18:l desaturation attributable to the wild-type (Kozak, 1986). In the absence of a purine at -3, however, a gene. One possible explanation of these results was that G residue at position +4 becomes essential for efficient trans- sequences in the wild-type 5‘ flanking region of the 18:l de- lation, and the effects of other positions are more pronounced saturase mRNA may confer negative translational regulation. (Kozak, 1987). The negative post-transcriptional regulation observed when Severa1examples have been described in which one or more a “full-length” soybean AAPT7 cDNA was expressed in yeast small uORFs precede the primary reading frame of an mRNA. also provides a likely explanation regarding the great number When the translational efficiencies of these mRNAs were of colonies (more than one million) that were necessary to tested, it was found that a small peptide is produced from the screen before we were successful in isolating the gene using uORF and that translation of the larger downstream reading genetic complementation. When the pGmaapt2 insert was frame is greatly reduced (Khalili et al., 1987;Werner et al., 1987; used as a hybridization probe in screening the developing seed Damiani and Wessler, 1993;Han et al., 1993). The fact that cDNA library, it was revealed that cDNAs corresponding to any translational initiation is allowed to proceed from a down- AAPT7 were represented in the library at a frequency of ap- stream ORF is believed to be a result of a small percentage proximately one in 7000 transformants. By comparing the of reinitiation of the ribosomal complex, allowing those ribo- intensity of hybridization signals of AAPT7 transcripts in de- somes to “scan” to the next AUG (Kozak, 1989). The proposed veloping seed versus young leaf tissue using RNA gel blot initiator AUG codon of the uORF in the 5’ leader sequence assays (Figure 48),one would also predict the representation of the AAPT7 transcript contains an A residue at position -3 of AAPT7 cDNAs to be somewhat similar in a young leaf cDNA (Figure 2) and would appear to be in a good context for trans- library. Given the fact that a cDNA possessing a long 5’flank- lational initiation. Furthermore, the third codon of the uORF ing region produced a signal that was barely detectable using also specifies an AUG codon and therefore may act as an ad- this assay (Figure S), it is unlikely that such a cDNA would ditional site of translational initiation. have been identified in the original screening. If the uORF in Expression in yeast of the longest soybean AAPT7 cDNA the 5‘ leader sequence proves to be responsible for the ob- characterized in this study demonstrated that sequences in served repression of gene activity, it is possible that the only the 5‘flanking region of the full-length transcript have a nega- cDNAs in the expression libraries that could have been effec- tive effect on gene expression in this heterologousenvironment tively isolated by this technique were ones derived from mRNAs (Figure 6).Although it cannot be concluded from these data long enough so that they contained the entireAAPT7 reading that the uORF was responsible for this phenomenon, similari- frame, yet not so long as to also possess the uORF (in addi- ties between the organization of the AAPT7 transcript and tion to being in correct orientation with respect to the vector‘s genes that have been found to be negatively regulated via short GAL7 promoter). Because of the increasing number of genes 5‘ proximal ORFs suggest that this motif is a likely candidate involved in glycerolipid biosynthesisthat are appearing to dem- as the agent that mediates the observed repression in gene onstrate negative post-transcriptional regulation (AAPT7, ACP, activity. Alternatively, strong secondary structure in the 5’flank- and the 18:l desaturase), future attempts to isolate genes of ing region of some eukaryotic mRNAs has been proposed as this pathway by genetic complementation should be conducted a mechanism of negative translational regulation (Kozak, 1991). with the understanding that such a form of gene regulation Computer analysis of the predicted optimal RNA secondary could present significant obstacles when using this strategy. structure of the AAPT7 mRNA, however, failed to detect any The role of AAPTases in mediating the synthesis of PC is contiguous stretches of sequence complementarity longer than of particular interest during seed development. The majority 6 bp in the 5‘flanking region of the transcript (data not shown). of polyunsaturated lipid biosynthesis in the developing seed 1504 The Plant Cell occurs via 18:l and 18:2 desaturases located on the endoplas- necessarily increasing steady state accumulation of the AAPT7 mic reticulum (Ohlrogge et al., 1991; Somerville and Browse, mRNA. 1991). The substrate for these desaturase enzymes has been In conclusion, the cloning and characterizationof theAAPT7 shown to be PC (Slack et ai., 1979; Stymne and Appelqvist, cDNA of soybean has provided valuable information concern- 1980; Browse and Slack, 1981). The fatty acids of PC that have ing the structure and regulation of a gene encoding an essential become polyunsaturated may subsequently be made avail- step in the glycerolipid biosynthetic pathway. In addition to able for incorporation into storage triacylglycerols by two providing a tool that will facilitate the study of substrate speci- mechanisms: (1) reentry into the acyl-coenzyme A pool by spe- ficities and the kinetics of a higher plant AAPTase, the soybean cific acyltransferases that exchange acyl groups at position AAPT7 clone will also provide an opportunity to study the ef- sn-2 of PC with those of acyl-coenzymeA (Stymne et al., 1983; fects of manipulating AAFTase activity in transgenic plants by Griffiths et ai., 1988) or (2) reentry into the diacylglycerol pool by either overexpression or antisense-mediated inhibition of gene the reversible reaction of the cholinephosphotransferaseen- activity. Such studies will help further elucidate the role of phos- zyme (Slack et al., 1983; Stymne and Stobart, 1984). Consistent pholipid composition and function during plant growth and with this model is the observation that cholinephosphotrans- development. ferase activity increases dramatically during seed development in safflower, concomitant with increases in PC and triacylgly- cerol accumulation (Slack et al., 1985). Two observations METHODS suggest that the AAPT7 product is the predominant (and possibly the only) AAF'Tase responsible for the cholinephos- photransferaseactivity in developing soybean seeds: (1) AAPT7 Yeast Strains and Growth Conditions cDNAs were well represented within the 25-DAF developing Haploid yeast (Saccharomycescerevisiae) strains HJllO (MATa, cpt7, seed cDNA library and (2) even under low-stringency hybrid- gal-, his3-A 7, leu2-3, leu2-772, ura3-52, trp-289) and HJ729 (MATa, ization conditions, noAAPT7-like sequences different from our ept7, gal-, his3-A 7,leu2-3, leu2-772, ura3-52, trp-289) were obtained original AAPT7 cDNA probe were recovered from the seed- from R.M. Bell (Duke University Medical Center, Durham, NC). Strain specific cDNA library. KT1115 (MATa, leu2-3,leu2-772, ura3-52) was obtained from K. Tatchell RNA gel blot analysis demonstrated that the AAPT7 gene (North Carolina State University, Raleigh, NC). Yeast was cultured in is expressed in each tissue examined (Figure 48). Because either YPD (1% yeast extract, 2% peptone, 2% dextrose) or synthetic of the role of AAFTases in the synthesis of membrane phos- minimal media (0.67% yeast nitrogen base without amino acids, 2% pholipids in virtually all cell types, this result was expected. dextrose) supplemented with amino acids at 20 pglmL. lnduction of expression from the GAL7 promoter of pYES2 was conducted by cul- It is interesting, however, that the greatest accumulation of ture of the appropriate transformed yeast strain in YP-Gal media (1% steady state transcript was observed in the root and stem tis- yeast extract, 2% peptone, and 2% galactose). sues. One possible explanation for the greater abundance of Haploid strain RH-6D (MATa, cpn, his3-A 7,leu2-3,leo2-772, "-52) AAPT7 mRNAs in root than in leaf tissue may be the fact that was recovered from a cross between HJllO and KT1115. Haploid strain the major membrane lipids in leaves are the galactolipids of RK-ec (MATa, cpn, epn, his3-A 7,leu2-3,leu2-772, ura3-52)was selected the , whereas in nonphotosynthetic tissues PC and from a cross between RH-6D and HJ729. All yeast manipulations and PE are the predominant cellular lipids. Although there are crosses were conducted according to standard protocols (Guthrie and studies that report increases in cholinephosphotransferase ac- Fink, 1991). tivity during seed development (Slacket al., 1985), it is difficult to discuss them in the context of our RNA gel blot data because Construction of Soybean cDNA Libraries they did not include comparison of the cholinephosphotrans- ferase activity in developing seeds with the activity found in Size-selectedcDNA (>500 bp) was synthesized from mRNA isolated other plant tissues such as roots or leaves. Moreover, although from young soybean (Glycinemaxcv Dare) leaves and developing soy- 25 DAF represents a developmental stage during which triacyl- bean seeds 25 days after flowering (DAF) using moloney murine glycerol accumulation in soybeans is expected to be maximal leukemia virus reverse transcriptase as described by the manufac- (Wilson, 1987), RNA gel blot analyses using mRNA isolated turer (Gibco BRL). cDNA libraries corresponding to each of the two from developing seed tissue at numerous time points would developmental stages were constructed in the yeast expression vec- be required to determine whether this stage represents the tor pYES2 (Invitrogen, San Diego, CA). pYES2 is designed to utilize period of maximal AAPT7 transcript accumulation. Finally, any the high-efficiency inverted BstXl cloning strategy of Aruffo and Seed attempts to correlateAAFW mRNA accumulation with enzyme (1987). BstXl adapters were ligated to the soybean cDNA and cloned into BstXI-digestedpYES2 vectors as outlined by Becker et al. (1991). activity must be interpreted in the context that post-transcrip- Ligation reactions were phenol-chloroformextracted, precipitated, and tional control may also play an important role in gene regulation. transformed into electrocompetent Escherichia coli ToplOF' cells ac- Should AAPT7 in soybean show the same negative post-tran- cording to the manufacturer's protocol (Invitrogen). Transformed cells scriptional control of gene expression that was observed in were plated at a density of ~2 x 106 colonies per 150-mm Petri dish. the heterologous yeast system, it would be possible for en- Colonies were scraped directly from the surface of the Petri dishes, zyme activity to increase in a given tissue that was capable and plasmids were recovered by alkaline lysis and CsCl gradient cen- of overcoming the mechanism of negative control without trifugation as described previously (Sambrook et al., 1989). For each The AAPT7 Gene of Soybean 1505

library, we obtained >5 x 107 primary transformants per pg of input and pUC120 (Vieira and Messing, 1987), designated pGmaaptll9 and cDNA. As judged by the frequency of plasmidscontaining inserts (ob- pGmaaptl20, respectively. This cloning strategy placed the inserts in tained from 40 randomly selected colonies), we estimated that >90% the respective vectors in opposite orientation in relation to the univer- of the clones from the developing seed cDNA library and >75% of sal primer attachment site. To facilitate sequencing of the entire cDNA, the young leaf cDNA library plasmids contained soybean cDNA inserts. a nested set of deletions was generated using the Erase-A-Base kit For the isolation of soybean aminoalcoholphosphotransferase according to the manufacturer's protocol (Promega). The complete (AAPTase) cDNAs expressed during seed development using the leaf- cDNA sequence was determined in both orientations using Sequenase specific AAPT7 cDNA as a hybridization probe, an additional cDNA Version 2.0 (U.S. Biochemical Corp.). library was constructed in LZAPII. cDNAs from developing soybean Computer data base searches and hydropathy plots of the soybean seeds 25 DAF were ligated to EcoRl adapters and cloned into the AAPT7 cDNA and predicted amino acid sequences were conducted EcoRCdigested,phosphatase-treated arms of UAPII according to the using the MACVECTOR software package (IBI, North Haven, CT). Pair- manufacturer's instructions (Stratagene). wise comparisonsto establish percent identity between the soybean AAPT7-encodedpolypeptide and the yeast CPT7 and EPT7 gene prod- ucts were calculated using the GAP program of the University of Screening of Yeast Straln RK-ec nansformed with a Soybean Wisconsin (Madison) Genetics ComputingGroup (UWGCG) software cDNA Library Uslng Colony Autoradiography package. The multiple sequence alignment of the three protein se- quences (Figure 3A) was deduced using the UWGCG PILEUP program The soybean cDNA librariesconstructed in the pYES2 expressionvector (gap weight = 3.0, gap length weight = 0.1). were transformed into strain RK-ec according to the protocol of Gietz et al. (1992). Transformants were plated at a density of m3000 colo- nies per 90-mm Petri dish on selective media(synthetic minimal media DNA and mRNA Gel Elot Analyses supplemented with amino acids, minus uracil). After growth for 3 days at 3OoC, colonies were replica plated onto YP-Gal media and grown Total genomic DNA from young soybean leaves was isolatedas previ- for an additional 24 hr at 3OoC to induce expression of the transgene. ously described (Murray and Thompson, 1980). Ten micrograms of To screen for mutant complementation, a diacylglycerolcholinephos- soybean DNA was digested with EcoRl and separated by electropho- photransferase colony autoradiographic assay was conducted resis on a 1% agarose gel. Total cellular RNA was isolated from various essentiallyas described by Hjelmstad and Bell(1987). Briefly, the trans- soybean tissues as outlined by Grimes et al. (1992). Poly(A)+ RNA was formed, induced yeast colonies were transferred to Whatman No. 42 recovered using oligo(dT) columns as outlined by the manufacturer filter paper (90-mm circles) and frozen at -80% for a minimum of 2 hr. (Gibco BRL) and separated electrophoretically on a 1.2% formalde- Yeast colonies were permeabilized by drying in a direct stream of air hyde gel (Sambrook et al., 1989). from an electric fan for 30 min at room temperature. Individual filters Nucleic acids were transferred to nylon membranes (Gibco BRL) were incubatedin Petri dishes in 1.2 mL of a reaction solution contain- according to Sambrook et al. (1989). Both RNA and DNAgel blots were ing 50 mM S(Kmorpho1ino)propanesulfonic acid (Mops)-NaOH, pH 7.5, hybridized overnight to 32P-labeledAAPT7 cDNA in 6 x SSPE (1 x 20 mM MgCI2, 1.5 mg/mL BSA ( free), 1 mM dithiothreitol, SSPE is 0.15 M NaCI, 10 mM sodium phosphate, 1 mM EDTA, pH 7.4), and 5 pM .14C-cytidinediphosphate (CDP)-choline (58 mCi/mmol; 10 x Denhardt's solution (1 x Denhardt's solution is 0.02% Ficoll, Amersham Corp.). After a 1-hr incubation at 3OoC, filters were trans- 0.02% PVP, 0.02% BSA), 1% (w/v) SDS, and 100 pg/mL of denatured ferred to a new Petri dish containing 1 mL of cold (4OC) 10% herring sperm DNA at 65OC. Blots were washed twice at room temper- trichloroacetic acid to stop the reaction and fix the labeled phosphatidyl- ature in 2 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate), choline (PC) to the filter paper. After a 30-min incubation at 4OC, the 0.1% (w/v) SDS for 15 min, once at 65OC in 1 x SSC, 0.1% SDS for filters were washed six times with 25 mL of cold (4OC) 2% trichloro- 15 min, and once at 65OC in 0.2 x SSC, 0.1% SDS for 15 min. All acetic acid using a Buchner funnel. Air-dried filters were sprayed with 32P-labeledDNA probes were generated using random primers ac- three light coats of a fluorographic enhancer (EN3HANCE; Du cording to the method of Feinberg and Vogelstein (1983). To ensure to Kodak XAR-5 film for I Pont-New England Nuclear) and exposed that each lane of the RNA gel blots contained similar amounts of un- to 5 days. Colony autoradiographic assays for cholinephosphotrans- degraded RNA, filters that had been hybridized to an AAPT7 probe ferase activity in coli (strain MV1190) were conducted exactly as E. were subsequentlystripped and rehybridized with a 32P-labeled25s describedfor yeast cells with the exception that the filter-boundE. coli ribosomal RNA gene from pea (data not shown). colonies were initially permeabilizedusing a 10 mg/mL lysozyme, 10 Low-stringency hybridizationswere conducted in the same hy- mM EDTA solution as described by Raetz (1975). bridization solution described above using an incubation temperature Radioactive products were extracted by soaking the filter papers of 50%. Filters for the low-stringency hybridizationswere washed twice in 10 mL of 1:l chloroform-methanol overnight at 4OC. After the addi- using 2 x SSC, 0.1% (w/v) SDS at room temperature for 15 min and tion of 6.25 mL of 1 N HCI, the chloroformphase was recovered, dried once with a 1 x SSC, 0.1% SDS solution at 5OoC for 15 min. to completion, redissolved in 50 pL of 2:l chloroform-methanol, and analyzed by thin-layer chromatography on Silica Gel 60 plates (Analtech, Newark, DE) using a developing solvent of 25:15:4:2 chlo- lsolation of AAPn cDNAs from a Developing Seed Soybean roform-methanol-water-acetic acid. Comigration of radiolabeled cDNA Llbrary products with authentic PC run as a standard was verified using a Bio- scan System 400 lmaging Scanner (Bioscan Inc., Washington, DC). The developing seed soybean cDNA library in hZAPll was plated at a density oí 4 x 104 plaque-formingunits per 150-mm Petri dish ac- Sequence Analysis cording to the procedures outlined by Stratagene. Plaques were transferred to nylon membranes (Gibco BRL) and hybridized to To determine the nucleotide sequence of the soybean cDNA from plas- 32P-labeledinsert DNA from pGmaapt2 using the conditions of low- mid pGmaapt2, the insert was initiallysubcloned intovectors pUCll9 hybridization stringency described above. Plaques showing positive 1506 The Plant Cell

hybridization signals were purified to homogeneity and their corre- REFERENCES sponding plasmids excised as described in the ZAPII cloning kit (Stratagene). Nucleotidesequence information for 350 to 400 bp was obtained from each end of the selected clones using the universal and Ansell, G.B., and Spanner, S. (1982). Phosphatidylserine, phospha- reverse primers with the Sequenase Version 2.0 sequencing kit. tidylethanolamine and phosphatidylcholine.In New Comprehensive Biochemistry, Vol. 4, A. Neuberger and L.L.M. van Deenen, eds (New York: Elsevier), pp. 1-50. Chollnephosphotransferase Assays of Yeast Microsomal Aruffo, A., and Seed, B. (1987). Molecular cloning of a CD28 cDNA Membranes by a high-efficiencyCOS cell expression system. Proc. Natl. Acad. Sci. USA 84, 8573-8577. Yeast colonies were inoculated into mL of YPD (HJ110 and HJ729) 200 Becker, D.M., Fikes, J.D., and Guarente, L. (1991). A cDNA encod- or YP-Gal (RK-ec:pGmaapt2) and grown to an AGO0of 1.0 to 2.0. Cells ing a human CCAAT-binding protein cloned by functional were harvested by centrifugation at lOOOg for 10 min and washed once complementation in yeast. Proc. Natl. Acad. Sci. USA88,1968-1972. with 100 mL of water and once with 25 mL of 20% glycerol, 50 mM Mops-NaOH, pH 7.5, 1 mM EDTA (GME buffer). The final pellet was Bell, R.M., and Coleman, R.A. (1980). Enzymes of glycerolipid syn- resuspended in 1 mL of GME buffer, and the cells were disrupted using thesis in eukaryotes. Annu. Rev. Biochem. 49, 459-487. a mini-beadbeaterasdescribed by Tillman and Bell(1986). The resulting Bradford, M. (1976). A rapid and sensitive method for the quantitation homogenatewas centrifuged for 15 min at 14,0009 to remove unbroken of microgram quantities of protein utilizing the principle of protein- cells. GME buffer was added to the supernatant to a final volume of dye binding. Anal. Biochem. 72, 248-254. 12 mL and centrifuged for 1.5 hr at 100,OOOg. The pellet was Browse, J.A., and Slack, C.R. 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Transla- North Carolina State University, Raleigh) for yeast strain KT1115 and tional repression of br7A expression prevents premature development advice in the techniques of yeast molecular genetics. We thank Cathy in Aspergillus. EMBO J. 12, 2449-2457. Varner for assistance in generating the RK-ec yeast mutant. Hannapel, D.J., and Ohlrogge, J.B. (1988). Regulation of acyl car- rier protein messenger RNA levels during seed and leaf development. Received June 7, 1994; accepted August 9, 1994. Plant Physiol. 86, 1174-1178. The AAPTl Gene of Soybean 1507

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