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Blackwell Science, Ltd

Three genes showing distinct regulatory patterns encode the asparagine synthetase of sunflower (Helianthus annuus)

María Begoña Herrera-Rodríguez1, Susana Carrasco-Ballesteros1, José María Maldonado3, Manuel Pineda2, Miguel Aguilar2 and Rafael Pérez-Vicente1

1Departamento de Biología Vegetal, División de Fisiología Vegetal, Universidad de Córdoba, Avda. San Alberto Magno s/n, E−14071 Córdoba, Spain;

2Departamento de Bioquímica y Biología Molecular. Universidad de Córdoba, Campus Rabanales, Edif. C-6, 1a Planta, E−14071 Córdoba, Spain;

3Departamento de Fisiología Vegetal y Ecología, Unidad de Fisiología Vegetal, Facultad de Biología, Universidad de Sevilla, Avda, Reina Mercedes 6,

E−41012 Seville, Spain

Summary

• Asparagine metabolism in sunflower (Helianthus annuus) was investigated bycDNA cloning, sequence characterization and expression analysis of three genesencoding different isoforms of asparagine synthetase (AS, EC 6.3.5.4).• The AS-coding sequences were searched for in leaves, roots and cotyledons byusing a methodology based on the simultaneous amplification of different cDNAs.Three distinct AS-coding genes, HAS1, HAS1.1 and HAS2, were identified.• HAS1 and HAS1.1 are twin genes with closely related sequences that share someregulatory features. By contrast, HAS2 is a singular sequence that encodes an incom-plete AS polypeptide and shows an unusual regulation. The functionality of boththe complete HAS1 and the truncated HAS2 proteins was demonstrated bycomplementation assays. Northern analysis revealed that HAS1, HAS1.1 and HAS2were differentially regulated dependent on the organ, the physiological status, thedevelopmental stage and the light conditions.• Asparagine synthetase from sunflower is encoded by a small gene family whosemembers have achieved a significant degree of specialization to cope with the majorsituations requiring asparagine synthesis.

Key words: asparagine synthetase gene family, differential gene expression,nitrogen metabolism, Helianthus annuus (sunflower).

© New Phytologist (2002) 155: 33–45

Author for correspondence: Rafael Pérez-Vicente Tel: +34 95 7218692 Fax: +34 95 7218939\ Email: bv1pevir@uco.es

Received: 26 November 2001 Accepted: 4 May 2002

Introduction

Asparagine plays a prominent role in nitrogen transport andstorage in plants (Sieciechowicz et al., 1988; Lea et al., 1990).The transfer of the amide group from glutamine (or ammonia)to aspartate, catalysed by asparagine synthetase (AS, EC 6.3.5.4),is the main route for asparagine biosynthesis (Lea et al.,1990). A complete biochemical characterization of plant ASis lacking because of its extreme instability in vitro (Huber &Streeter, 1985; Ta et al., 1989). Therefore, most of the data onplant AS are derived from molecular studies.

Different numbers of genes have been reported to encodeAS in plants. A single gene is found in the majority of species

studied. However, some leguminous plants contain twoclosely related genes that show similar regulatory patterns(Tsai & Coruzzi, 1990; Waterhouse et al., 1996; Hugheset al., 1997; Osuna et al., 2001). An exception is Arabidopsisthaliana, which contains three different AS genes (ASN1,ASN2 and ASN3; Lam et al., 1998). All plant AS genesidentified to date are homologous to asnB, the gene encodingthe glutamine-dependent AS from Escherichia coli (Scofieldet al., 1990).

Data from the characterization of cDNA clones suggestthat plant AS proteins are composed of 579–591 amino acidswith a predicted molecular mass of about 65 kDa. (Shi et al.,1997). Plant AS polypeptides have been divided into group I,

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group II (Lam et al., 1998), type I and type II (Osuna et al.,2001) according to their position in phylogenetic treesderived from sequence comparisons. However, neither thenature of such sequence differences nor their physiologicalsignificance was determined.

Increased levels of asparagine have been observed in carbon-starved tissues from different plant species (Sieciechowiczet al., 1988). Asparagine has a higher nitrogen to carbon(N : C) ratio than glutamine (2 : 4 vs 2 : 5). These datasupport the role of asparagine as the preferred compound fornitrogen transport and storage under conditions of limitedcarbon supply, such as in the absence of light. A typical featureof plant AS genes is an enhanced expression in the darknessthat is repressed by transfer to light. Both phytochrome andthe carbon status of the tissue mediated the light repression ofASN1 from Arabidopsis (Lam et al., 1994).

Nevertheless, asparagine is also required to support thegrowth of plants in the light. It is essential for protein synthe-sis in aerial organs. It is also implicated in photorespiration(Ta et al., 1984; Sieciechowicz et al., 1988), a process that occursin well-illuminated leaves. Moreover, asparagine synthesisin the light has been acknowledged as a mechanism todetoxify high levels of ammonia (Givan, 1979; Sieciechowiczet al., 1988).

Ordinary AS genes that are repressed under illuminationcannot account for a direct supply of the asparagine requiredin the light. An exception to this is Arabidopsis ASN2, a genethat is not repressed but induced by light (Lam et al., 1998).This finding presents Arabidopsis as the only species with ASgenes suitable to satisfy a direct demand of asparagine for bothlight (ASN2) and dark (ASN1) processes (Lam et al., 1994,1998). Another AS gene not repressed by light was alsodetected in Phaseolus vulgaris (PVAS1); however, its expressionis restricted to roots and to cotyledons at specific stages ofgermination, and is undetectable in leaves (Osuna et al., 2001).

While the possession of only light-repressed AS genes is themost common situation in plants, the opposite has also beendocumented. Lotus japonicus contains two AS genes, LJAS1and LJAS2, not repressed by light, but is devoid of ASgenes with enhanced expression in darkness (Waterhouseet al., 1996).

The involvement of asparagine in translocation of thenitrogen fixed by root nodules (Schubert, 1986) has prompteda more detailed study of its biosynthesis in legumes than innon-legume species such as sunflower, where the role of ASin metabolism is still poorly understood. Previous steps ofnitrogen utilization by sunflower (i.e. the reduction of nitrate toammonia and the incorporation of ammonia into glutamine)have been characterized by our group (de la Haba et al., 1988,1992; Agüera et al., 1990; Maldonado et al., 1990; Montenegroet al., 1998, 1999). We now report on the use of nitrogen onestep further. In this paper, we describe the identification,cDNA characterization and expression analysis of HAS1,HAS1.1 and HAS2, three distinct and differently regulated AS

genes whose existence suggests a rich metabolism of asparag-ine in sunflower.

Materials and Methods

Plant culture conditions and sample collection

Sunflower plants (H. annuus L.) from the isogenic cultivarHA-89 (Semillas Cargill SA, Sevilla, Spain) were grown undera 16-h photoperiod with irradiance of 200 µmol m−2 s−1 PAR(provided by Sylvania cool white F72T12/CW/VHO,160 W fluorescent lamps supplemented with Mazda 60 Wincandescent bulbs) and day/night temperature and relativehumidity regimes of 25°C/19°C and 70%/80%, respectively.Seeds were germinated in plastic trays containing a 1 : 1 (v : v)mixture of perlite and vermiculite. Seedlings were irrigated dailywith a nutrient solution containing 10 mM KNO3 (Hewitt, 1966)and cultured in the trays for up to 25 d. For longer cultureperiods, plants were transferred individually to pots containingComposana substrate (Compo GmbH, Vchte, Germany).

Cotyledons at germination were collected from 3-d-old-seedlings, 7 d before their full expansion. Senescing cotyledons,having lost 40% of their maximum chlorophyll content(0.7 mg g−1 fresh weight), were obtained from 20-d-oldplants, which also provided roots and stems (epicotyls).Immature primary leaves (2–3 cm long) were collected onday 11. Mature, fully expanded primary leaves (9 cm long)were collected on day 20. Dark-adapted leaves were obtainedfrom 20-d-old plants after a 48-h dark treatment. Immatureinflorescences (1.5–2 cm diameter) and flowers wereobtained from 55- and 70-d-old plants, respectively. All plantsamples were frozen by immersion in liquid nitrogen imme-diately after collection and stored at −80°C until use.

Isolation of cDNA clones from three different sunflower AS genes

Single-stranded cDNA with an anchor sequence at the 5′ endwas prepared by priming 5 µg of total RNA with the Qtoligonucleotide (Table 1), as described by Frohman (1994).To amplify the 3′ end of different AS cDNA clones, nesteddegenerate primers AS2 and AS1, designed to fit conservedregions of plant AS, were paired to nested primers QoR andQiR, designed to fit the anchor sequence Qt (Table 1, Fig. 1).Two consecutive nested amplifications were carried out withthe GeneAmp polymerase chain reaction (PCR) Core kit(Perkin-Elmer, Foster City, CA, USA). Each reaction mixcontained: 1 unit of Taq DNA polymerase, 200 µM of each ofthe four dNTPs and 2 mM MgCl2 in 10 mM Tris-HCl/50 mM

KCl, pH 8.3 buffer plus 1 µM of each of the external primersAS2 and QoR and 1 µl of the cDNA solution for the firstPCR reaction, or 1 µM of each of the internal primers AS1 andQiR and 1 µl of a 1 : 19 dilution of the first PCR products forthe second PCR reaction. The PCR reactions were performed

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in a DNA Thermal Cycler 480 (Perkin-Elmer) using thefollowing 40-cycle profile: 60 s at 94°C, 30 s at 60°C and180 s at 72°C. Single-primer PCR reactions were performedas a control to identify non-specific PCR products.

The products of the second PCR (AS1/QiR) were blunt-ended and separated by electrophoresis. The positive fragmentsFas1 and Fas2 were gel-purified and cloned in pBluescriptSK(–) (Stratagene, La Jolla, CA, USA) by standard methods(Sambrook et al., 1989).

Sixty randomly selected Fas1 clones from dark-adaptedleaves and 60 randomly selected Fas2 clones from cotyledonsof germinating seedlings were chosen for restriction analysis.The AS1/QiR inserts from individual Fas1 and Fas2 cloneswere digested with different restriction enzymes. Two differ-ent restriction patterns were observed among the Fas1 clonesdigested with EcoRV, HaeIII, MluI, or MspI, which led to theidentification of a new AS cDNA (Fas1.1).

Partial (3′ end) AS cDNAs Fas 1, Fas 1.1 and Fas2 weresequenced and pairs of nested gene-specific 3′ primers weredesigned to amplify their corresponding 5′ ends. Gene-specific primers 3GS1 and 3GS1n were designed for Fas1,3GS11 and 3GS11n for Fas1.1, and 3GS2 and 3GS2n forFas2 (Table 1). These 3′ primers were located at a distancefrom the beginning of Fas1, Fas1.1 and Fas2 such that the5′ and 3′ ends of the cDNAs would overlap a minimum of130 nucleotides. cDNA fragments of 1570, 1632 and1568 bp, corresponding to the 5′ end of HAS1, HAS1.1and HAS2, were obtained by 5′ rapid amplification ofcDNA ends-polymerase chain reaction (RACE-PCR) byusing the Marathon kit (Clontech, Palo Alto, CA, USA).The sequences of the 5′ and 3′ ends were aligned, allowingthe reconstruction of the full-length cDNAs from HAS1,HAS1.1 and HAS2. In addition, full-length cDNAs fromHAS1 and HAS2 were amplified directly using 5′ and 3′gene-specific primers (GEX5H1 and GEX3H1 for HAS1,and GEX5H2 and GEX3H2 for HAS2; Table 1). Thesefull-length cDNAs were cloned in pBluescript SK(–)forsequencing.

Fig. 1 Polymerase chain reaction (PCR) amplification of sunflower asparagine synthetase (AS)-coding cDNAs. (a) Schematic model of the AS cDNA with indication of the position of primers AS1, AS2, QoR and QiR. The 5′ and 3′ untranslated sequences are represented by lines. (b) Samples of cDNA from leaves (L), roots (R), cotyledons of germinating seeds (CG), senescing cotyledons (SC), and leaves of plants adapted to darkness (DL) were amplified with primers AS2 and QoR, and then reamplified with primers AS1 and QiR (lanes marked with a C). Single-primer PCR reactions with AS1 or QiR were performed to detect nonspecific products (lanes AS1 or QiR). Reactions that did not render detectable fragments are not shown. Bands corresponding to AS-coding cDNA fragments are marked with an asterisk. The PCR products were separated in a high-resolution agarose gel and stained with ethidium bromide. Five microlitres from each amplification reaction (total 50 µl) were loaded per lane. Gel image is shown in negative. Position and length of DNA size markers are indicated.

Name Sequence

Qt CCA GTG AGC AGA GTG ACG AGG ACT CGA GCT CAA GC(T)17QoR CCA GTG AGC AGA GTG ACG AGG AQiR AGT GAC GAG GAC TCG AGC TCA AGAS1 GGT GTT GGC TAT RGI TTG ATH GAY GAS2 CTC TAC AGG CAG AAA GAR CAR TTY AGY GA3GS1 GAC GGT CAA CTT AGC CGA ATT CTG A3GS1n AGG GAA AAG CCG CTC AAA GAT CAT T3GS11 TGC AGT GCT GCA AGC CAC G3GS11n GCT TGC TCC ACC CGG GAC A3GS2 AAC CGT TAA CCT TGC AGC ATT CTT G3GS2n TCT TGG GGA AGA ACT TTT CAA AGA TTT TGGEX5H1 CTA GTC TAG ACA TGT GTG GAA TAC TTG CTG TCT TGG GTGEX3H1 ACC CAA GCT TCG CTA GCC CTG AAT CAC GAG TTGEX5H2 CCG GAA TTC TAA TGT GTG GAA TAC TCG CCG TTGEX3H2 ACC GCT CGA GAT TCA CTT GGC GTC TTC GTAlab1 CTA AAA AAC AAA TTT ATT TAA AAG Clab11 AGG TGT AAT CAA TCG TCT GAAlab2 AAT AAG AGA GGT ATT TTG GAA TTT G

Table 1 Primers used for cDNA synthesis, amplification and labelling

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Sequencing and sequence analysis

The cDNA inserts in pBluescript SK(–) were sequenced byusing the AmpliTaq DyeDeoxy Terminator Cycle Sequencingkit (Perkin-Elmer). Two to three independent clones of eachcDNA type were sequenced to avoid PCR-induced mutations.Computer applications used for sequence analysis were thoseincluded in the Wisconsin software package (version 9.1-UNIX, 1997) from Genetics Computer Group (Madison, WI,USA) and the Lasergene software package (version 3.0) fromDNASTAR (Madison, WI, USA). Multiple sequence align-ments were edited and displayed with the Multiple SequenceAlignment Editor & Shading Utility from Genedoc (v. 2.4).

Complementation of an Escherichia coli asparagine auxotroph

HAS1 and HAS2 coding regions were obtained by directamplification of cDNA with 5′ and 3′ gene-specific primers(GEX5H1, GEX5H2, GEX3H1 and GEX3H2), and clonedin pGEX-KG (Guan & Dixon, 1991) to obtain pHAS1ex andpHAS2ex constructs.

The E. coli asparagine auxotroph strain ER (asnA, asnB, thi-1, relA, spoT1) from the Genetic Stock Center (New Haven,CI, USA) was transformed with the constructs pHAS1ex,pHAS2ex or with the empty vector pGEX-KG, as a control.The ER cells carrying pHAS1ex, pHAS2ex or pGEX-KGand XL1-Blue wild-type cells (Stratagene) were grown at28°C, in M9 minimal medium (Sambrook et al., 1989)without asparagine. pHAS1ex and pHAS2ex expression wasinduced by adding 1 mM isopropyl β-D-thiogalactoside(IPTG) to the medium. M9 medium was supplemented withampicillin (100 µg ml−1) when needed. Bacterial growth wasdetermined by measuring the absorbance of the cultures at550 nm.

Analyses of DNA and RNA

Total RNA was isolated from different organs by selectiveprecipitation with LiCl, according to the method of Manning(1991). Genomic DNA was obtained from the LiClsupernatant by precipitation with ethanol.

Gene-specific probes for HAS1, HAS1.1 and HAS2 wereprepared from their 3′ untranslated regions. Fragments con-taining the 3′ untranslated regions were excised from Fas1,Fas1.1 and Fas2 cDNAs with EcoRV, HpaI and BbsI, respec-tively. Single-stranded probes were then prepared by PCRwith α32P-dATP, as described by Konat et al. (1994). Primersused for PCR labelling were lab1, lab11 and lab2 (Table 1) forHAS1, HAS1.1 and HAS2, respectively. The HAS1-specificprobe encompassed 1765–1941 nt of the cDNA sequence,the HAS1.1-specific probe encompassed 1817–2017 nt of thecDNA sequence and the HAS2-specific probe encompassed1696–1819 nt of the cDNA sequence.

An AS conserved probe was obtained from a 1 : 1 mix offragments from the coding region of HAS1 (nt 1393–1764)and HAS2 (nt 1391–1695) that were labelled with α32P-dCTP by random priming with the oligolabellling kit fromPharmacia (Uppsala, Sweden).

Seven micrograms of DNA were digested with XbaI,EcoRV or HindIII, electrophoresed in agarose gels and blottedonto a nylon filter for Southern analysis. Medium stringencyhybridization was performed at 65°C, overnight, as describedpreviously (Sambrook et al., 1989) and filters were washedthree times (15 min each) in 1× standard saline citrate (SSC),0.1% (w : v) sodium dodecyl sulphate (SDS) at 65°C.

Samples of total RNA from different organs were separatedby denaturing electrophoresis in formaldehyde agarose gelsand blotted onto nylon filters according to standard proce-dures (Sambrook et al., 1989). Hybridization was performedat 42°C, overnight, in solutions containing 50% formamideand the corresponding gene-specific probe. After hybridiza-tion, filters were washed twice for 15 min in 0.2× SSC, 0.1%(w : v) SDS at 65°C. Results of the Southern and Northernanalyses were revealed by autoradiography after exposing X-ray films (Kodak X-Omat AR) to the filters for 6 d and 5 d at−80°C, respectively.

Results

Cloning and sequence analysis of three AS-coding cDNAs from sunflower

An intensive search for AS-coding sequences was performedby amplifying cDNA samples from leaves, roots, senescingcotyledons and cotyledons from germinating seeds, as wellas from leaves from plants adapted to darkness. Degenerateprimers against conserved regions were used to amplifysequences from different AS-coding genes. The PCR strategyand results are shown in Fig. 1.

Two positive fragments named Fas1 (582 bp) and Fas2(462 bp) were simultaneously amplified from senescingcotyledons (Fig. 1). The analysis of their sequences showed thatFas1 and Fas2 corresponded to the 3′ ends of two differentAS-coding genes that were named HAS1 and HAS2, respec-tively (Fig. 2). Fragments with the same sequence as Fas2 werealso amplified from cotyledons of germinating seeds, leaves,dark-adapted leaves and roots. Fragments with the samesequence as Fas1 were amplified only from root and dark-adapted leaves, in addition to senescing cotyledons (Fig. 1).

Under the PCR conditions employed, the amplification ofa single band does not imply the amplification of a singlecDNA. Several cDNAs for AS with a similar size but differentsequences can be simultaneously amplified and groupedtogether in a single band. To assess this possibility, thesequence homogeneity of 60 independent clones of Fas1obtained from dark-adapted leaves was checked by restrictionanalysis. The detection of Fas1 clones showing different

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restriction patterns with the same enzymes allowed identi-fication of a third AS-coding sequence named Fas1.1 (622 bp)that had been amplified simultaneously with Fas1 cDNA fromdark-adapted leaves, and was electrophoretically indistin-guishable from it. The sequence of Fas 1.1 derived from the3′ end of a new AS-coding gene named HAS1.1 (Fig. 2). Morethan 60 independent Fas2 clones were also checked with 20restriction enzymes and no sequence heterogeneity was detected.

The sequences of the Fas1, Fas 1.1 and Fas2 partial cDNAswere used to design gene-specific primers to amplify thecorresponding 5′ ends of each cDNA by 5′ RACE-PCR.Fragments encompassing the 5′ ends were amplified andsequenced, allowing the reconstruction of three full-lengthcDNAs whose properties are summarized in Table 2.

A final PCR reaction was performed with gene-specificprimers located at the ends of each reconstructed cDNA that

allowed the direct amplification of full-length cDNAs.Sequence comparison between the reconstructed and thedirectly amplified full-length cDNAs allowed us to discard thepossibility that reconstructed cDNAs could be hybrids of 5′and 3′ ends from different genes.

In the 3′ untranslated regions of HAS1, HAS1.1 and HAS2,several cis-acting elements that may control message termina-tion and processing or stability were identified. Both T-richfar upstream elements (FUEs) and near upstream AATAAAelements (NUEs) involved in polyadenylation (Hunt, 1994;Li & Hunt, 1997) were found (Fig. 2).

The deduced sequences of HAS1, HAS1.1 and HAS2polypeptides were aligned with AS amino acid sequences from14 plant species plus the E. coli glutamine-dependent AS asnB(Fig. 3; to save space, only part of the alignment is displayed)and the dendrogram in Fig. 4 was constructed. The three

Fig. 2 Alignment of the 3′ ends of the asparagine synthetase (AS)-coding genes HAS1, HAS1.1 and HAS2. Sequences shown were obtained from the cDNA fragments Fas1, Fas1.1 and Fas2, respectively. Arrowhead indicates the beginning of the 3′ variable region. Stop codons are blocked in black. A putative ancestral stop codon is boxed. Putative polyadenylation signals are underlined with a single line (far upstream element) or appear in bold type (near upstream element).

Table 2 Summary of sequence properties of the asparagine synthetase (AS) gene family from sunflower

Gene name

GenBank accession no.

cDNA length (bp)

Polypeptide length (aa)

Molecular mass (kDa)

Isoelectric point

C-terminal variable region

Gln-binding domain

HAS1 AF190728 1971 591 66.4 6.24 Yes YesHAS1.1 AF263432 2035 589 66.2 6.44 Yes YesHAS2 AF190729 1850 558 63.2 6.11 No Yes

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Fig. 3 Sequence alignment of the polypeptides encoded by HAS1, HAS 1.1 and HAS2 with others deduced asparagine synthetases (AS). The AS polypeptides shown are sunflower HAS1, HAS1.1 and HAS2, Arabidopsis ASN1, ASN2 and ASN3, Sandersonia AS (SAND), rice AS (ORY) and the glutamine-dependent AS from Escherichia coli (asnB). Identical amino acids are indicated by dots; dashes denote gaps introduced to maximize the homology of the compared sequences. Essential residues from the glutamine-binding domain are marked with an open inverted triangle. Residues proposed to facilitate the binding of aspartate are marked with a dot. An inverted black triangle marks the residues proposed to be responsible for the anchoring of the AMP moiety. The pyrophosphate-binding region is marked with asterisks. Unique positions specific for the class II AS from Fig. 4 are in bold type and underlined. The arrowhead indicates the beginning of the C-terminal variable region.

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sunflower AS exhibited a high overall sequence identity withthe rest of the plant AS examined. The minimum sequencehomology value found for the comparison of sunflowerHAS1.1 to maize (Zea mays) AS corresponded to 77% ofsequence identity at the amino acid level.

HAS1, HAS1.1 and HAS2 polypeptides contained theproposed glutamine-binding site that is shared by all NH2-terminal nucleophilic (Ntn) amidotransferases (Zalkin &Smith, 1998), a family of enzymes, represented by theglutamine phosphoribosyl pyrophosphate amidotransferase(GPRPPA; EC 2.4.2.14), that uses glutamine as amide donor.All the essential residues of the glutamine-binding siteCys1–Arg26–Gly27–Gly32–Arg73–Pro86–Asn101–Gly102–Asp127

(GPRPPA numbering; Zalkin & Smith, 1998), were conservedin the sunflower polypeptides (Fig. 3). Essential residuesfor the binding of other substrates were also conserved inHAS1, HAS1.1 and HAS2. Threonine residues 317 and 318,and arginine residue 320 (HAS1 numbering) were proved totake part in aspartate-binding in E. coli asnB (Boehlein et al.,1997a). Cysteine 524 (HAS1 numbering) was also suggestedto facilitate the reaction between aspartate and the enzyme-bound ATP in asnB (Boehlein et al., 1997b). The amino acidsoccurring between serine residues 234 and 239 (HAS1numbering) are a conserved motif in enzymes that hydrolyseATP to AMP and pyrophosphate, such as GMP synthase(Mäntsälä & Zalkin, 1992) or argininosuccinate synthe-tase (Ratner, 1973; Surh et al., 1988), and constitute apyrophosphate-binding element (Richards & Schuster, 1998).This segment was conserved in the sunflower AS polypeptides(Fig. 3). Finally, four amino acid residues (Leu233, Val269

Ser343 and Gly344; HAS1 numbering) have been recognized as

the points of anchoring of the AMP moiety in asnB (Larsenet al., 1999). Those residues were conserved in the sunflowerpolypeptides, although valine 269 has been replaced by anisoleucine in HAS2 (Fig. 3).

All the essential residues cited above were also conserved inthe AS from A. thaliana, rice and Sandersonia aurantiaca(Fig. 3), as well as in the rest of the plant AS listed in Fig. 4(data not shown), with the following exceptions: threonine317 from the aspartate-binding site has been replaced byserine in the pea (Pisum sativum) AS2 polypeptide, cysteine524 by lysine in the AS from Sandersonia , valine 269 fromthe AMP-binding site is replaced by isoleucine in ASN2 andASN3 from Arabidopsis and in the polypeptides from rice andmaize, and serine 343 by cysteine in Phaseolus PVAS1.

Against the background of a high overall homology, sev-eral unique features differentiated the sunflower AS fromeach other. HAS1 and HAS 1.1 that encoded two extremelyclosely related polypeptides (92% sequence identity), differedfrom one another because of the 3′ variable region (Lam et al.,1994), a poorly conserved stretch encoding the last 35amino acids from the C-terminal end that is specific forplant AS (Figs 2 and 3). On the other side, HAS2 divergedfrom HAS1 and HAS1.1 both in size and sequence(Table 2; Fig. 3). The main difference between HAS2 andthe other sunflower AS was the absence of the C-terminalvariable region due to an early stop codon in the sequenceof HAS2 (Figs 2 and 3). A single-nucleotide deletion mayexplain the origin of this stop codon that produces thetruncated HAS2 polypeptide (Fig. 2). A putative ancestralstop codon is found 74 nucleotides downstream from theactual stop codon (Fig. 2). The translation of the mRNA

Fig. 4 Dendrogram of relationships among different asparagine synthetase (AS) polypeptides. The dendrogram was obtained after a multiple sequence comparison. The sequences compared were from Pisum sativum 1 (AS1; X52179) and 2 (AS2; X52180), Lotus japonicus 1 (LJAS1; X89409) and 2 (LJAS2; X89410), Glycine max 1 (SAS1; U77679) and 2 (SAS2; U77678), Arabidopsis thaliana 1 (ASN1; L29083), 2 (ASN2; AF095453) and 3 (ASN3; AF095452), Helianthus annuus 1 (HAS1; AF190728), 1.1 (HAS1.1; AF263432) and 2 (HAS2; AF190729), Phaseolus vulgaris 1 (PVAS1; AJ33522) and 2 (PVAS2; AJ009952), Vicia faba (VFAS1; Z72354), Medicago sativa (U89923), Brassica oleracea (X84448), Elaeagnus umbellata (AS; AF061740), Triphysaria versicolor (AS; AF014055), Asparagus officinalis (X67958), Sandersonia aurantiaca (SAND1; AF005724), Oryza sativa (D83378), Zea mays (X82849) and Escherichia coli (asnB; J05554).

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up to this putative ancestral stop codon would give a poly-peptide containing the C-terminal variable region. Thisfinding strongly suggests that HAS2 has derived, by a single-nucleotide deletion, from a longer sequence that possessedthe C-terminal variable region.

HAS2 also differed from HAS1 and HAS1.1 by about 100divergent residues scattered along its entire length (Fig. 3). Itshares a greater sequence identity with Arabidopsis ASN2(91%) and ASN3 (90%) and with rice AS (88%) than withthe other AS from sunflower (84%).

The dendrogram derived from the comparison of differentplant AS polypeptides showed two major dendritic groups(Fig. 4). A large dendritic group (class I) containing most ofthe AS compared, including HAS1 and HAS1.1, and a smalldendritic group (class II) formed by HAS2 plus the AS fromrice, maize and Arabidopsis (ASN2 and ASN3) (Fig. 4).Unique sequence features specific to class II sequences lay inseven positions: Lys131, Phe157, Ala163, Ser165, Leu187, Thr264

and Ile267 (HAS2 numbering, Fig. 3). The residues in thosepositions were not repeated in the other plant AS. Residuesthat occupy the corresponding positions in the rest of theplant AS were also significantly conserved.

Existence of additional AS-coding genes in sunflower

The existence of additional AS genes was investigated bySouthern analysis of the sunflower genome. A probe forAS-conserved regions was prepared as a tool for detectingfragments from any AS-coding gene present in the sunflowergenome. This probe was obtained from conserved sequencesof both HAS1 and HAS2, and was hybridized to total digestedDNA under medium stringency conditions in order tobroaden its hybridization spectrum. Gene-specific probes forHAS1, HAS1.1 and HAS2 were prepared from unique regionsof their 3′ untranslated sequences.

Hybridization of the conserved probe to DNA digestedwith either XbaI, EcoRV or HindIII resulted in a multiplebanding pattern, as expected from the occurrence of severalAS genes in the sunflower genome (Fig. 5). All the bandsobtained with the conserved probe could be identified,although with varying intensities, in the banding patterns

obtained with the gene-specific probes. This is generallyaccepted as an indication of the absence of additional genes tothe ones the gene-specific probes were made for. However,under the medium stringency conditions used, multiplebands were also obtained with the probe for HAS1.1. Astrongly hybridizing band plus two to four weakly hybridizingbands were obtained for each hybridization with the HAS1.1-specific probe, indicating the existence of fragments homo-logous to the 3′ untranslated sequences of HAS1.1. (Fig. 5).Those weakly hybridizing bands were not detected underhigh stringency conditions (results not shown). The fact thatthose bands were detected by the conserved probe (Fig. 5,XbaI digestion) indicates that they also contain AS conservedregions. This strongly suggests the existence of additional ASgenes, closely related to HAS1.1, in the sunflower genome. Avery low expression level of those genes, below the PCRdetection limit, may explain the failure of our approach toamplify them.

Complementation of an asparagine auxotroph mutant strain of E. coli

To test the functionality of the cloned sequences, the regionsencoding the proteins HAS1 and HAS2 were inserted in-frame in the pGEX-KG expression vector (Guan & Dixon,1991). The new constructs (pHAS1ex and pHAS2ex) weretransformed and expressed into the E. coli auxotroph ERstrain (asnA, asnB, thi-1, relA, spoT1) lacking AS activity(Felton et al., 1980). As expected, growth of E. coli ERtransformed with the empty vector was very poor whencultured in a medium without asparagine (Fig. 6). Highergrowth levels were obtained in the same medium when the ERstrain was transformed with either pHAS1ex or pHAS2ex(40% and 75% of maximum growth level, respectively).However, none of the transformants, carrying eitherpHAS1ex or pHAS2ex, achieved the growth of the wild-type E. coli used as a positive control. This is, nevertheless,the expected result since transformation with a singleheterologous AS gene (pHAS1ex or pHAS2ex) only partlyovercomes the lack of two homologous AS genes (asnA andasnB) from E. coli.

Fig. 5 Southern analysis of sunflower DNA. Seven micrograms of total DNA were digested with (a) XbaI, (b) EcoRV or (c) HindIII, separated by agarose gel electrophoresis and blotted onto a nylon filter. Four identical replica filters were hybridized with gene-specific probes for HAS1 (1), HAS1.1 (1.1) HAS2 (2) and with a conserved probe (C). Hybridization was performed under medium stringency conditions to allow the conserved probe to bind sequences from different AS genes. The position and length of DNA size markers are indicated.

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Patterns of expression of HAS1, HAS1.1 and HAS2 in sunflower

The abundance of transcripts from each one of the three ASgenes from sunflower was determined in a variety of organsunder different physiological conditions and developmentalstages. Selected organs included immature and mature leaves,leaves of dark-adapted plants, roots, stems (epicotyls), cotyledonsfrom germinating seedlings, senescing cotyledons, immatureinflorescences and flowers.

According to the abundance of its transcripts, the expressionof HAS1.1 in plants cultured under standard photoperiodicconditions was restricted to roots. However, HAS1 was

also expressed in senescing cotyledons, immature inflores-cences and flowers. Transcripts from HAS2 were detected inall organs tested, being especially abundant in cotyledonsduring germination and in immature leaves.

Darkness greatly increased the level of HAS1 and HAS1.1transcripts in leaves, but not that of HAS2 (Fig. 7).

Differences in transcript accumulation also depended onthe developmental stage. HAS1 was expressed both in imma-ture inflorescences and in flowers, although a greater accumu-lation of transcripts was found in the mature organ. Bycontrast, HAS2 transcripts were much more abundant both inthe immature inflorescences and immature leaves than inmature leaves and flowers (Fig. 7). Transcripts from HAS1.1could not be detected in those organs.

Discussion

Identification of three genes encoding functional asparagine synthetases in sunflower

Most plant species have been found to contain a single ASgene. Model plant A. thaliana was first reported to contain asingle AS gene that was isolated by heterologous hybridization(ASN1; Lam et al., 1994). A second approach based on thecomplementation of a yeast asparagine auxotroph revealedtwo additional genes for AS in this crucifer (ASN2 and ASN3,Lam et al., 1998). The fact that a plant with a small genomesuch as Arabidopsis contained three genes for AS led usto expect multiple AS genes in sunflower. Our PCR-basedapproach permitted us to examine cDNA sequences fromseveral organs in different physiological situations, and led tothe identification of three different AS genes: HAS1, HAS1.1and HAS2. Subsequent Southern analysis (Fig. 5) provideddata indicating the existence of additional HAS1.1-relatedgenes in the sunflower genome.

Multigene families are a common feature of plant genomesand have been reported for other enzymes of nitrogen

Fig. 6 Complementation of an Escherichia coli asparagine auxotrophic strain (ER). Mutant strain ER was transformed with the constructs pHAS1ex, pHAS2ex, or with the empty vector pGEX-KG, as a control. Bacterial strains were inoculated to 0.1 unit of absorbance at 550 nm, at zero time, in minimal media with (filled bars) or without (open bars) 1 mM isopropyl β-D-thiogalactoside (IPTG). Bacterial growth was determined 24 h after inoculation by measuring the absorbance at 550 nm of the culture. Growth percentages represent the mean of three independent experiments with differences below 10% among them. Maximal growth levels obtained by the wild-type E. coli XL1-Blue strain in minimal medium corresponded to 1.40 (+ IPTG) and 1.58 (– IPTG) absorbance units.

Fig. 7 Relative levels of HAS1, HAS1.1 and HAS2 mRNA in organs under different physiological and developmental conditions. Twenty micrograms of total RNA from roots (R), epicotyls (E), cotyledons from germinating seedlings (CG), senescing cotyledons (SC), immature leaves (IL), fully expanded mature leaves (ML), immature inflorescences (II), flowers (F) and mature leaves from plants adapted to darkness for 48 h (DL) were separated by electrophoresis, stained with ethidium bromide (bottom) and blotted onto a nylon filter. The filter was hybridized sequentially to probes specific for HAS1, HAS1.1 and HAS2. The old probe was stripped off the filter before each new hybridization. Results were revealed by autoradiography after 5 d of exposure. Hybridization bands correspond to an mRNA of about 2 kb.

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metabolism (i.e. glutamine synthetase, EC 6.3.2.1) Thisenzyme is encoded by four, five and six isogenes in Arabidopsis,sunflower and maize, respectively (Peterman & Goodman,1991; Li et al., 1993; Montenegro et al., 1998). The identifica-tion of three genes for AS in sunflower and in Arabidopsis stronglysuggests that multiple AS genes occur in most species of plants.

All three AS cDNAs from sunflower contained an openreading frame encoding an AS polypeptide very similar topreviously isolated plant AS in length, sequence (Fig. 3) andother molecular parameters (Table 2) (Tsai & Coruzzi, 1990;Lam et al., 1994; Chevalier et al., 1996; Waterhouse et al.,1996; Hughes et al., 1997; Shi et al., 1997; Lam et al., 1998;Osuna et al., 1999, 2001). Moreover, polypeptides coded byHAS1 and HAS2 have proved their functionality by comple-menting an asparagine auxotroph strain of E. coli (Fig. 6).

Sequence characterization of HAS1, HAS1.1 and HAS2

The biochemical characterization of plant AS is very poor. Inaddition to an extreme in vitro instability, the presence ofendogenous inhibitors and contaminating asparaginase inplant extracts have made AS activity extremely difficult toassay (Streeter, 1977; Joy et al., 1983; Huber & Streeter, 1985;Ta et al., 1989). In the absence of direct kinetic data, thepossession of the glutamine-binding triad consisting of threeconserved residues (Cys1, Asp29 and His101; Mei & Zalkin,1989), has been the main criterion to identify a plant AS asa glutamine-utilizing amidotransferase (Lam et al., 1994;Chevalier et al., 1996; Waterhouse et al., 1996; Eason &King, 1997; Hughes et al., 1997; Shi et al., 1997; Lam et al.,1998; Eason et al., 2000). However, further evidence hasdemonstrated that His101 and Asp29 are too distant fromCys1 to participate in catalysis (Smith et al., 1994; Zalkin &Smith, 1998), thus challenging the reliability of the triad as acriterion to identify glutamine-utilizing enzymes.

On the basis of X-ray structural models and mutagenesisexperiments, nine invariant residues have been identified asthe distinctive fingerprints of the glutamine-binding domainof Ntn amidotransferases (Zalkin & Smith, 1998). Thoseinvariant residues were conserved in the sequence of allsunflower AS (Cys1–Arg30–Gly31–Gly36–Arg49–Pro61–Asn74–Gly75–Asp99, HAS1 numbering; Fig. 3), as well as in the restof plant AS (Figs 3 and 4). This finding allows the classifi-cation of all the AS examined in this paper as genuineglutamine-utilizing Ntn-amidotransferases, including AS2from Pisum sativum, where the triad essential residues are notconserved (Tsai & Coruzzi, 1990; Lam et al., 1994). Datafrom direct kinetic studies, when available, will confirm this.

HAS1 and HAS1.1 are twin genes encoding polypeptidesof nearly the same length (591 aa and 589 aa, respectively)that share a high sequence identity (92%). Nonetheless, ahigher number of amino acid residues in common with therest of the plant AS examined (result not shown) suggests thatHAS1 is evolutionarily older than HAS1.1.

The identification of highly homologous, almost redun-dant, AS genes has been the rule in plants containing two ASgenes, such as Pisum sativum (AS1 and AS2 ; Tsai & Coruzzi,1990), Lotus japonicus (LJAS1 and LJAS2 ; Waterhouse et al.,1996), Glycine max (SAS1 and SAS2 ; Hughes et al., 1997)and Phaseolus vulgaris (PVAS1 and PVAS2, Osuna et al., 1999,2001).

The third AS gene found in sunflower, HAS2, encodes asingular polypeptide that has greater similarity to other plantAS than to HAS1 and HAS1.1. In fact, HAS2, together withArabidopsis ASN2 and ASN3 and rice and maize AS, is classi-fied in a separate dendritic group (Fig. 4).

The division of plant AS into two main groups, one con-sisting of the AS from rice and maize (the monocot group)and another including the rest of the plant AS available tothe authors (the dicot group), was first reported by Shi et al.(1997). The inclusion of ASN2 and ASN3 in the monocotgroup by Lam et al. (1998) demonstrated that the separationof this group was not due to properties exclusive to monocotAS. Groups were named as group II, containing the AS fromrice and maize plus ASN2 and ASN3, and group I, containing10 AS from plants of the Liliaceae, Brassicaceae and Fabaceae(Lam et al., 1998). We have extended the comparison byincluding representatives from distantly related families suchas Asteraceae (sunflower), Elaeagnaceae (Elaeagnus), andOrobanchaceae (Triphysaria), and confirmed the existence oftwo major classes of plant AS by identifying and cloning newmembers from both classes.

Yet another division into type I and type II AS has beenestablished to differentiate plant AS (Osuna et al., 2001). Ourextended sequence comparison confirms this division andshows that both types I and II are subgroups that involve,exclusively, closely related legume AS, all of which areincluded in the higher group I (Fig. 4). Since some confusionmay arise from the similarity of the names used for the differ-ent kinds of plant AS (type I and II, group I and II), we sug-gest a change in the nomenclature to class I and II for thoseAS belonging to the two major groups, and type a and b forthe legume-specific types of AS.

Despite constituting a separate group, class II AS did notdiffer significantly from class I AS in terms of primary struc-ture. In fact, the number of residues specific to class II AS issmall (Lys131; Phe157; Ala163; Ser165; Leu187; Thr264 and Ile2,HAS2 numbering). The similarity between class I and class IIAS is significant enough to propose that their genes havederived from the duplication of a common ancestor. Thisshould have happened before the separation of plants intomonocot and dicot species, since both classes of AS have beenisolated from both kinds of plants (Fig. 4). More recent dupli-cations of class I genes might have originated the two closelyrelated types of legumes AS, as well as the twin genes HAS1and HAS1.1 from sunflower.

A unique feature of the polypeptide encoded by HAS2 isthe lack of the C-terminal variable region, a poorly conserved

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sequence stretch for which no essential roles have beenreported. Despite that, it is maintained in most of the plantAS, suggesting some unknown function. Among the otherplant AS compared in this paper (Fig. 4), only Sandersonia AS(Eason & King, 1997) lacks the C-terminal variable region(Fig. 3).

Plant AS seem to have evolved from bacterial AS, assuggested by their high degree of sequence identity. Sincebacterial AS possess no C-terminal variable region, this regionmight have been acquired by plant AS, most probably byextending the coding region into part of the 3′ untranslatedsequences.

Except for the case of sunflower, no direct evidence hasbeen presented to discard the functionality of this region. Inthis case, the ability of the truncated HAS2 polypeptide tocomplement an asparagine auxotroph strain of E. coli clearlyindicates the absence of essential catalytic functions in thispart of the protein. However, a role of the C-terminal variableregion in promoting interactions with other polypeptides orcell components cannot be excluded.

Differential regulation of HAS1, HAS1.1 and HAS2

In addition to a different primary structure, HAS1, HAS1.1and HAS2 genes showed distinct patterns of regulationaccording to the organ, the physiological status, thedevelopmental stage and the light conditions.

The expression of HAS1 and HAS1.1 under standard lightconditions was confined to a few organs. However, their tran-scripts accumulated to high levels in leaves of plants adaptedto darkness (Fig. 7).

Limitation of the expression to particular organs andrepression by light are the two most frequent traits of plant ASgenes. Negative effects of light on the expression of AS geneshave been reported in P. sativum, Nicotiana tabacum,Nicotiana plumbaginifolia (Tsai & Coruzzi, 1991), Z. mays(Chevalier et al., 1996), Medicago sativa (Shi et al., 1997),Arabidopsis (Lam et al., 1998) and P. vulgaris (Osuna et al.,2001).

Light repression of HAS1 and HAS1.1, as well as most ofplant AS genes, is consistent with the role of asparagine innitrogen transport and storage when carbon supply is limited(Sieciechowicz et al., 1988). However, this pattern of expres-sion does not explain how asparagine is supplied for proteinsynthesis in well-illuminated leaves and for photorespiration(Ta et al., 1984; Sieciechowicz et al., 1988).

HAS2 did not follow the typical expression pattern of aplant AS since its transcripts were widely distributed in avariety of organs and its expression was not repressed by light(Fig. 7). This unusual pattern strongly suggests that theisozyme encoded by HAS2 is the direct supplier of asparaginein well-illuminated tissues.

Differences in the regulation by light between class I and IIAS has been demonstrated in Arabidopsis (Lam et al., 1998),

the other species where both classes of AS genes have beenisolated. Light repressed the expression of class I ASN1 butinduced the expression of class II ASN2. This resembles thesituation found in sunflower except that class II HAS2 is unaf-fected rather than induced by light. The gene encoding AS inrice is another non-light-repressible class II gene (Nakanoet al., 2000). The finding of non-light-repressible class IIgenes in distantly related species such as Arabidopsis, rice andsunflower suggests a wide distribution of this type of gene,thus encouraging a deeper search for class II genes in specieswhere class I genes have already been found. For that purpose,a nonclassical, high-resolution sequence searching methodo-logy such as ours is recommended.

Not all class II genes lack repression by light, as deducedfrom the absence of AS transcripts in illuminated tissue ofmaize (Chevalier et al., 1996). Similarly, although most of theclass I genes are repressible by light, there are some exceptionssuch as Lotus LJAS1 and LJAS2 (Waterhouse et al., 1996) andPhaseolus PVAS1 (Osuna et al., 2001), showing that expres-sion in the light is a valuable trait that has been positivelyselected in the evolution of both classes of AS genes.

The members of the AS gene family from sunflower exhibita considerable degree of specialization that is not limited tospecific response to light. HAS1 and HAS2 were also develop-mentally regulated (Fig. 7). The fact that the level of HAS2transcripts was always much higher in young than in maturetissues from both flowers and leaves indicates a role of HAS2in the construction of these organs rather than in its mainte-nance. HAS1 appears to have a function complementary tothat of HAS2 in floral tissue, since its transcripts accumulatein mature flowers rather than in immature inflorescences(Fig. 7).

Moreover, synthesis of asparagine required for different sit-uations involving nitrogen mobilization, such as germinationor senescence, is assisted by different AS genes in sunflower.According to the data of transcript accumulation, HAS2 isexpected to play a major role in the synthesis of asparagineduring germination, with no significant collaboration fromHAS1 and HAS1.1. Conversely, the asparagine formed duringcotyledon senescence is expected to be produced primarily bythe activity of HAS1 and HAS2, with little contribution fromHAS1.1 (Fig. 7).

The expression of sunflower AS genes have been measuredin terms of transcript abundance, which may not correlatewith the actual levels of AS protein. However, changes in ASmRNA abundance are physiologically relevant in Arabidopsis,as they parallel the level of free asparagine in the plants (Lamet al., 1998).

Class I AS genes have been reported to be expressed in peacotyledons during germination (Tsai & Coruzzi, 1990), inasparagus (Asparagus officinalis) spears during harvest-inducedsenescence (Davies & King, 1993) and in senescing flowersfrom Sandersonia (Eason et al., 2000). However, data on theactivity of class I and II AS genes in response to various

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situations of nitrogen mobilization, or to different develop-mental stages to compare with the data obtained from sun-flower have not yet been presented.

The finding of a small AS gene family, whose members aredifferentially regulated, supports the existence of a complexand finely regulated asparagine metabolism in sunflower.Major aspects of this metabolism, such as asparagine synthesisin germination and senescence, in young and mature tissueand in light and dark conditions can be accounted for by theAS genes described here. However, a more precise under-standing of the in vivo role of each sunflower AS will requirethe determination of the relative levels of all AS polypeptidesand their location in the tissue; this work is in progress.

Acknowledgements

This work was funded by a grant from Dirección General deEnseñanza Superior e Investigación Científica (DGESIC &BXX 2000–0289) and by Plan Andaluz de Investigación(PAI, group CV-0159), Spain.

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