Biochemical and molecular characterization of hazelnut ( Corylus avellana ) seed lipoxygenases Angelo Santino 1 , Angelo De Paolis 1 , Antonia Gallo 1, *, Angela Quarta 1 , Rod Casey 2 and Giovanni Mita 1 1 Institute of Sciences of Food Production C.N.R. Section of Lecce, Italy; 2 John Innes Centre, Norwich, UK Plant lipoxygenases (LOXs) are a class of dioxygenases which display diverse functions in several physiological processes such as growth, development and response to biotic and abiotic stresses. Even though LOXs have been characterized from several plant species, the physiological role of seed LOXs is still unclear. With the aim to better clarify the occurrence of LOXs and their influence on hazelnut seed quality, we carried out the biochemical and molecular characterization of the main LOX isoforms expressed during seed development. A genomic clone con- taining a complete LOX gene was isolated and fully char- acterized. The 9887 bp sequence reported contains an open reading frame of 5334 bp encoding a putative polypeptide of 99 kDa. Semiquantitative RT-PCR carried out from RNAs extracted from seeds at different maturation stages showed that LOXs are mainly expressed at early developmental stages. These results were confirmed by LOX activity assays. Biochemical characterization of the reaction products of the hazelnut LOX indicated that it is a 9-LOX. Two cDNAs were isolated by RT-PCR carried out on total RNA from immature hazelnut seeds. Sequence analysis indicated that the two cDNAs are highly homologous (91.9% degree of identity) and one of these corresponded exactly to the genomic clone. The deduced amino acid sequences of the hazelnut LOXs showed that they are closely related to a previously reported almond LOX (79.5% identity) and, to a lesser extent, to some LOXs involved in plant responses to pathogens (cotton and tobacco LOXs, 75.5 and 74.6% identity, respectively). The physiological role of hazelnut LOXs and their role in influencing seed quality are also discussed. Keywords: Corylus avellana; hazelnut; lipoxygenases; seed quality. Lipoxygenases (LOX, EC 1.13.11.12) are a class of widespread dioxygenases that catalyze the addition of oxygen to polyunsaturated fatty acids containing a cis,cis- 1,4-pentadiene structure. The hydroperoxides produced by the LOX reaction are the starting point for a series of other enzymatic reactions which lead to the synthesis of a group of biologically active compounds collectively named oxylipins. In animal cells, the LOX pathway has been extensively studied as it is responsible for the synthesis of leukotrienes and lipoxins belonging to the eicosanoid family which are involved in many physiological processes [1]. In plants the biosynthesis of phytooxylipins is initi- ated by the oxygen insertion at C-9 or C-13 of linoleic (C18 : 2) or linolenic acids (C18 : 3). For this reason plant LOXs are commonly referred as 9-LOXs or 13-LOXs. Both 9- or 13-hydroperoxides are further converted to different compounds through the action of the other enzymes belonging to different branches of the LOX pathway (hydroperoxide lyase, allene oxide synthase, divinyl ether synthase, reductase, peroxygenase). At the end of these enzymatic reactions the molecules synthesized display a wide variety of physiological roles in plant development and response to biotic and abiotic stresses. In this context lipoxygenases, providing the substrates for the activity of all the enzymes located downstream on the pathway, have a primary role in the biosynthesis of phytooxylipins and can affect their availability inside the plant cell. The biological role of specific LOX isoforms has been recently clarified by an antisense approach and their depletion was able to influence plant development or pest/pathogen resistance [2,3]. Transgenic potato plants depleted in the expression of a specific 9-LOX showed an abnormal tuber development [4]. Furthermore Arabidopsis and potato transgenic plants with diminished levels of specific chloroplastic LOXs have reduced levels of wound inducible mRNAs [5,6]. LOXs are widespread in the seeds of many plant species; in some cases such as soybean and other legumes they are abundant proteins and might also function as storage, rather than defence, proteins. This hypothesis is supported Correspondence to A. Santino, ISPA-CNR, via Monteroni, 73100 Lecce, Italy. Fax:/Tel.: + 39 0832 420000, E-mail: angelo.santino@irba.le.cnr.it Abbreviations:9-HODE,(9S,10E,12Z)-9-hydroxy-10,12-octadeca- dienoic acid; 13-HODE, (13S,9Z,11E)-13-hydroxy-9,11-octadeca- dienoic acid; LOX, lipoxygenase; RP-HPLC, reverse-phase HPLC; SP-HPLC, straight-phase HPLC. Enzyme: lipoxygenase (EC 1.13.11.12). Note: a web site is available at http://www.ispa.cnr.it/ Note: The nucleotide sequence reported is in the EMBL database under the accession number AJ417975. *Present address: Institute of Sciences of Food Production C.N.R. Bari, Italy. (Received 18 July 2003, revised 2 September 2003, accepted 12 September 2003) Eur. J. Biochem. 270, 4365–4375 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03831.x by the results on a soybean line lacking the three LOX isoforms which shows similar crop performances in com- parison to control lines [7]. In other cases, LOX genes are expressed only in early developmental stages when the synthesis of storage proteins is not yet initiated. This has been reported for maize L2 LOX [8], almond [9] and peanut [10]. It was reported that peanut LOX gene is also expressed during Aspergillus colonization and consequently a possible defensive role was suggested for this specific isoform [10]. However the physiological role of these LOXs is largely unclear. Because of their role in the generation of pleasant or unpleasant flavours and their involvement in co-oxidation reactions leading to the bleaching of carotenoids and other pigments, LOXs are also of great importance in food science as they can influence the shelf life, the organoleptical and the nutritional characteristics of seeds and other plant products [11]. Hazelnuts are widely used in food industry with the largest part of the world production adsorbed by the pastry industry. Hazelnut seeds contain reasonable levels of linoleic and linolenic acid (about 10 and 0.2%, respectively, of the total fatty acids) and hexanal and nonanal are among the most abundant volatile compounds detected in hazelnut oil [12]. Nevertheless LOX activity was not detected in mature seed [13]. Considering the importance that LOXs could display in influencing the shelf life and the organoleptic features of hazelnut seeds, we carried out a study to characterize hazelnut lipoxygenases at the biochemical and molecular level. Materials and methods Plant material Hazelnut (Corylus avellana L. cv Mortarella) leaves and seeds were harvested at different maturation stages from the experimental field of the Istituto Sperimentale Frutticoltura, Caserta. The leaves and shelled seeds were frozen in liquid nitrogen and stored at )80 °C. PCR amplification of hazelnut genomic probes Two homologous probes (ln32, ln50) were obtained by PCR using hazelnut genomic DNA as template, the primers 5¢-CTATGATTATGATGTCTACAATGATTTG GG-3¢ (ML1) and 5¢-GCAAATTCTTCATCAGTCATC CATGCAGAC-3¢ (ML2) and the following amplification conditions: 94 °C for 5 min (1 cycle); 94 °Cfor1min, 45 °Cfor1min,72°C for 2 min (25 cycles); 72 °Cfor 5 min (1 cycle). Construction and screening of the hazelnut genomic library Hazelnut (cv Mortarella) genomic DNA was extracted from young leaves, and used for the construction of the library as previously described [9]. The library was screened using ln32 and ln50 digoxigenin- labelled probes (Roche). The filters were washed at a final stringency of 0.1· NaCl/Cit, 0.5% SDS, 65 °C. Positive plaques were picked and subjected to two additional screenings at the same conditions. RNA extraction and reverse transcription (RT) PCR analysis Total RNA was isolated from 100 mg of hazelnut seeds using the Rneasy plant mini kit (Quiagen) following the manufacturer’s instructions. RT-PCR was per- formed using 2 lg of total RNA, oligo-(dT) 18 primer and the Superscript II Polymerase (Invitrogen) according to the manufacturer’s instructions. The first strand cDNA was used as template in PCR experiments with oligonucleotides designed on the basis of the genomic sequence. The 5¢ region of the hazelnut LOX cDNA was amplified using the following primers: 5¢-AAGATGAAACGTG AGACGG-3¢ (CA1); 5¢-GATGACATCTCCATGGAA TAC-3¢ (CA2). The 3¢ region of the hazelnut LOX cDNA was amplified using the following primers: 5¢-GTTT GGAAGAGAGATGCTGG-3¢ (CA3); 5¢-AAAGTTTTT AGATTGAGACACTATTGGGAATT-3¢ (CA4). PCR conditions were as follows: 94 °C for 3 min (1 cycle); 94 °C for 45 s, 50 °Cfor45s,72°C for 2 min (30 cycles); 72 °C for 15 min (1 cycle). The amplified fragments were cloned in pCRII-TOPO vector (Invitrogen) and sequenced. For the semiquantitative RT-PCR, the cDNA was amplified with the following oligos: 5¢-GTATTCCATG GAGATGTCATC-3¢ (CA5); 5¢-AAAGTTTTTAGATTG AGACACTATTGGGAATT-3¢ (CA4). Actin cDNA was amplified using the following primers: 5¢-TGGTGTTAT GGTTGGTAT-3¢ (ACTFOR); 5¢-ACCTTCATCTTCAT GCTG-3¢ (ACTREV). Densitometric analysis (using a Bio-Rad Gel Doc2000 and the QUANTITY ONE software) was carried out on the actin amplified fragments to normalize the amount of cDNA to be used in further PCR experiments. Protein extraction and lipoxygenase activity Lipid bodies were first isolated from soluble protein fraction as previously reported [9]. LOX activity was assayed in total protein samples and in micro- somal fraction obtained by centrifugation of total pro- tein samples at 100 000 g for 30 min. The pellet (microsomal fraction) was resuspended in 100 m M sodium carbonate and the pH adjusted to 7.5 before assaying LOX activity. LOX activity was determined polarographically at 25 °C with a Clark O 2 electrode using linoleic acid as substrate. Thirty micromolar stock solution was prepared according to [14]. The reaction mixture (1.0 mL) consisted 100 m M sodium phosphate buffer pH 6 containing 0.3 m M linoleic acid and different amounts of protein samples. Decrease in O 2 concentration was monitored for 3 min and the enzy- matic activity was calculated from the initial rate of O 2 uptake. One unit of LOX activity corresponds to the intake of 1 lmol of O 2 per minute. pH optimum of hazelnut LOX was determined as previously described [9]. LOX activity was also assayed spectrophotometrically monitoring the increase in A 234 of the conjugated-diene structures as previously described [14]. 4366 A. Santino et al. (Eur. J. Biochem. 270) Ó FEBS 2003 SDS/PAGE and Western blot analysis Total proteins (20 lg) extracted from seeds harvested at different developmental stages were subjected to SDS/ PAGE and transferred to nitrocellulose membrane (Amer- sham). Western blot analysis was performed using the ECL protocol (Amersham) and a 1 : 1000 dilution of the pea LOX antibody [15]. Identification of lipoxygenase products Protein samples from different developmental stages were incubated for 1 h in 1 mL of 100 m M sodium phosphate buffer pH 6 containing 0.3 m M linoleic acid. Reaction products were reduced with sodium borohydride, extracted with chloroform/methanol (2 : 1, v/v) and dried. The reaction products were resuspended in methanol/water/ acetic acid (85 : 15 : 0.1, v/v/v) and separated by reverse- phase HPLC (RP-HPLC) using a C18 Ultrasphere column (Beckmann, 0.46 · 25 cm) as already reported [9]. The absorbances at 234 and 210 nm were recorded simulta- neously. The peak adsorbing at 234 nm containing the hydroxy-10,12-octadecadienoic acid (HODE) isomers was collected, dried and resuspended in n-hexane/propan-2-ol/ acetic acid (100 : 2 : 0.1, v/v/v). 9-HODE and 13-HODE were separated by straight-phase HPLC (SP-HPLC) with a Silica Ultrasphere column (Beckmann, 0.46 · 25 cm) as already described [9]. The enantiomer composition of 9-HODE and 13-HODE was carried out by chiral phase- HPLC on a Chiralcel OB column (Daicel Chem. Industries, 0.46 · 25 cm, 5 lm particle size) with a solvent system of hexane/propan-2-ol/acetic acid (100 : 5 : 0.1, v/v/v) and a flow rate of 1 mLÆmin )1 . Authentic standards of 9- and 13-HODE were purchased from ICN. HPLC analyses were carried out using a Beckman System Gold apparatus equipped with a 126 solvent module and a 168 detector. Extraction of genomic DNA and Southern blot analysis Genomic DNA was extracted from young hazelnut leaves using the Plant DNA isolation Kit (Roche). About 10 lg genomic DNA were cleaved with EcoRI, BamHI and HindIII, separated on a 0.8% agarose gel and transferred to Hybond N membrane (Amersham). Hybridizations were carried out at 65 °C overnight using homologous probes digoxigenin labelled according to the manufacturer’s instructions (Roche). Filters were washed at a final stringency of 0.2· NaCl/Cit 65 °C and chemiluminescent detection was carried out using CDP-STAR as substrate according to the manufacturer’s protocol (Roche). Results Lipoxygenase gene isolation and characterization To identify and characterize hazelnut lipoxygenases at the molecular level, we performed PCR experiments using hazelnut genomic DNA as template and the primers ML1 and ML2 bordering a 937-bp conserved region (2103–3039) of the soybean LOX3 gene [16]. An amplification product of about 700 bp was obtained in the above described conditions. The amplified DNA was cloned and sequenced. The results revealed that the amplification product con- tained two different lipoxygenase sequences showing a high degree of identity but belonging to different genes. The amplified sequences (ln32 and ln50) were used as homolog- ous probes to screen the hazelnut genomic library prepared as described in methods. After three successive screenings we isolated four different genomic clones using the ln32 and one clone with the ln50 probe. In order to identify a genomic clone containing a complete LOX gene, we hybridized the DNA extracted from all the clones with three probes corresponding to the 5¢, central and 3¢ portions of a previously isolated almond lipoxygenase gene [9]. The results indicated that one of the clones identified (ln32/1) possibly contained a complete hazelnut lipoxygenase which was further characterized. Figure 1 shows the 9887 bp characterized sequence and the hazelnut lipoxygenase gene organization. The sequence comprises 4180 bp 5¢-untranslated, an open reading frame of 5334 bp encoding a putative protein with a molecular weight of 99 kDa and a 373-bp 3¢-untranslated region. The gene, named Lox1:Ca:1, contains nine exons and, as already observed for other lipoxygenase genes, a very large first intron of 1871 bp. Sequence analyses revealed that both the homologous probes, ln 32 and ln 50, comprise exon IV and ln 32 probe exactly matches the Lox1:Ca:1 gene from base 6790 to base 7444. FASTA analysis revealed that the hazelnut LOX gene shows the highest degree of identity with a almond LOX gene (79.5% [9]). High identity was also found towards a Gossypium hirsutum lipoxygenase induced by bacteria (75.5% [17]), an elicitor induced tobacco LOX (74.6% [18]); and some potato LOXs (74–72% [19–21]). The dendrogram reported in Fig. 2 confirmed that the described hazelnut LOX is closely related to the almond, Gossypium, tobacco and potato LOXs. In the 5¢ upstream untranslated region, putative TATA and CAAT signals are present at position 4140 ()40 from the ATG) and 4066 ()114), respectively. In the 5¢ upstream untranslated region, we also found four inverted repeats (204–237 bp; 646–715; 1936–61; 2745–2923, bold in Fig. 1); one of which is particularly large (177 bp). LOX expression and enzymatic activity during hazelnut seed development Hazelnut requires more than 6 months to complete seed development as, in Italy, it normally blossoms in December; seeds complete their development in the middle of July and are harvested at the end of August. The maturation stages considered in this work were identified on the basis of seed weight. Stage I (harvested at the end of May 2002) can be considered the beginning of cotyledon development. After this stage, the seeds were collected every 2 weeks until the middle of July (stages II, III and IV). LOX expression was monitored by semiquantitative RT-PCR. Total RNAs extracted from different develop- mental stages were reverse transcribed using an oligo(dT) primer. The obtained cDNAs were used in PCR experi- ments with the LOX primers CA4 and CA5 (matching, respectively, from base 9488 to base 9520 and from base 8366 to base 8387 of the sequence reported in Fig. 1) and Ó FEBS 2003 Characterization of hazelnut seed lipoxygenases (Eur. J. Biochem. 270) 4367 Fig. 1. Organization of the hazelnut lipoxygenase gene lox1:Ca:1. (A) Schematic representation of the hazelnut LOX gene indicating the translation start and the stop codon. The introns and exons are drawn in proportion to their length. (B) Nucleotide and deduced amino acidic sequences of the hazelnut LOX gene. The inverted repeats are in bold and their orientations are indicated by arrows. TATA and CAAT boxes and the initiating ATG are also in bold. 4368 A. Santino et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Fig. 1. (Continued). Ó FEBS 2003 Characterization of hazelnut seed lipoxygenases (Eur. J. Biochem. 270) 4369 two primers designed on the basis of alignment results of several plant actin genes. The LOX amplification product showed the expected size (836 bp) and was further characterized by sequence ana- lysis. The actin amplified fragment was also sequenced. As reported in Fig. 3, hazelnut LOX is mainly expressed during early maturation stages (stages I and II). A faint band is visible in the other maturation stages in overloaded gels. The same RT-PCR experiments, conducted on RNA samples from young leaves, did not reveal any expression of this gene in this organ (data not shown). To verify the expression of the LOX gene corresponding to the ln50 clone, two specific oligos were designed and used in RT-PCR experiments. In our experimental conditions we were not able to observe any expression of this gene in seed or in leaves (not shown). We carried out Western blot analysis on total protein samples from hazelnut seeds at different developmental stages using pea LOX antibodies [15]. As reported in Fig. 3, a polypeptide in the molecular mass range expected for LOXs (99 kDa) was detected in protein samples from stage I and II seeds. To analyze LOX activity throughout seed development we set up a procedure aimed to reduce the interference of endogenous lipids on enzymatic assay. For this reason the lipid bodies fraction was first removed and LOX activity was assayed in total protein samples or in the microsomal fraction. The enzymatic activity was preliminarily analyzed at different pH values (pH 5–9) and we found that the hazelnut LOX has a pH optimum of about 6. In these experimental conditions, LOX activity was detected in all the developmental stages considered with a maximum in Fig. 2. Phylogenetic tree of hazelnut LOXs. The amino acid sequences of hazelnut lipoxy- genases were compared with other plant LOXs (accession numbers are in parentheses). Thephylogenetictreewasobtainedusing the PHYLIP (Phylogeny Inference Package) program. Fig. 3. Analysis of LOX activity and LOX gene expression in hazelnut seed at different developmental stages. Hazelnut seeds, collected at different maturation stages, were weighed (A) and used to extract total proteins for LOX immunodetection and LOX activity quantification. Twenty micrograms of total proteins were separated by SDS/PAGE, transferred to nitrocellulose and LOX was immunodetected with a pea LOX antibody (B). LOX activity was assayed measuring the con- sumption of oxygen (nmolÆmin )1 Æmg )1 protein) with a Clark electrode using linoleic acid as a substrate (C). cDNAs were generated using an oligo(dT) 18 primer and total RNA extracted from the different mat- uration stages. PCR experiments were carried out with LOX specific primers (D) or with actin specific primers as internal control (E). 4370 A. Santino et al. (Eur. J. Biochem. 270) Ó FEBS 2003 stage I. After this stage it declined throughout seed development and was negligible in mature seed (Fig. 3). We also tested the presence of LOX activity in either the microsomal or the lipid bodies fractions (data not shown). In both these fractions, low levels of LOX activity were recorded but at present it is unclear if the same LOX isoforms found in the soluble fraction are responsible for this activity or if it derives from other isoforms. Plant LOXs are usually classified as 9-LOXs and 13-LOXs on the basis of their product specificity. In order to better characterize the hazelnut LOX we analyzed by RP- HPLC the reaction products using linoleic acid as substrate. The reaction products were reduced with sodium boro- hydride and separated by RP-HPLC. The peak adsorbing at 234 nm containing the hydroperoxides was collected and subjected to SP-HPLC to separate 13- and 9-HODE. The retention times of the reaction products of the hazelnut LOX were compared with the reaction products of soybean LOX type I and authentic standards of 9- and 13-HODE. As shown in Fig. 4, 9-HODE is the main product of the hazelnut LOX reaction (about 70% of total hydroper- oxides). We carried out chiral-phase HPLC on both 9- and 13-HODE to separate R and S enantiomers. As reported in Fig. 4 the products of hazelnut LOX are predominantly in the S configuration indicating that these compounds derive from the activity of a specific lipoxygenase. Similar results were obtained using linolenic acid as substrate (data not shown). From these results we can conclude that a 9-LOX is largely responsible for the LOX activity in immature hazelnut seed. In order to correlate LOX activity and the production of C 6 and C 9 aldehydes deriving from the activity of other enzymes of the LOX pathway, we carried out a gas- chromatographic analysis of the head space volatiles compounds present in the hazelnut seeds at different developmental stages. Both C 6 and C 9 aldehydes (and the corresponding alcohol) were detected in all the samples and, similarly to the pattern of LOX expression and activity, their levels were higher in stage I seed and rapidly decline during maturation (data not shown). LOX cDNA isolation and characterization To better characterize LOX mRNAs expressed in hazelnut seeds, we performed RT-PCR experiments using RNA extracted from hazelnut immature seeds. Primers were synthesized on the basis of the sequence of the isolated genomic clone. Attempts to amplify a complete LOX cDNA in a one-step PCR amplification were unsuccessful, so we used two pairs of oligonucleotides to obtain separately the 5¢ and 3¢ regions. The amplified fragments were cloned and sequenced. The 5¢ (1757 nt) and the 3¢ region (1487 nt) overlapped for 672 nt and were considered different portions of the same gene. The 2572 bp cDNA (CaLOX1) corresponded exactly to the deduced cDNA from the genomic clone Lox1:Ca1, but missed the first 51 bp at the 5¢ end as the sense primer was designed considering the ATG located at position 4235 of the genomic clone as the translation start. The characterization of other clones revealed the presence of another cDNA (CaLOX2) sharing high identity, 91.9%, with the CaLOX1. The comparison of the deduced amino acidic sequences of CaLOX1 and CaLOX2 indicated that the two proteins differ in 68 positions. Considering the high degree of identity of the two cDNAs, the expression results obtained by semiquantitative RT-PCR reported in Fig. 3 have to be considered as the sum of the expression of both CaLOX1 and CaLOX2. The sequence of 10 independent clones deriving from RT-PCR experiments, however, revealed that nine corresponded to CaLOX2 and one to CaLOX1, suggesting that CaLOX2 is the predominant LOX cDNA in hazelnut seeds. After comparison with other plant LOXs (almond, cotton, Arabidopsis, tomato, potato and soybean LOX1) we verified the presence of all of the most conserved regions (Fig. 5). Among these are the conserved domains involved in substrate binding (AWRTDEEFGREMLAG, positions 373–387) and in oxygen binding (ASALHAAVNFGQY, positions 723–735). The highly conserved C-terminal motif GIPNSVSI is also present. The histidine residues (His535, His540, His727), asparagine (Asn731) and isoleucine (Ile873) essential for active site iron binding are present at the expected positions. The TV motif, thought to be characteristic of 9-LOXs [22], is present in both the two identified cDNAs (at positions 591–592 of Lox1:Ca:1). Southern analysis Southern blot analysis of hazelnut DNA was carried out by probing two different regions spanning the 5¢- (1757 bp) Fig. 4. HPLC analysis of hydroxyl-fatty acids produced by the hazelnut LOX. Two hundred micrograms of total proteins from immature hazelnutseedwereincubatedfor30minin0.1 M sodium phosphate buffer pH 6 containing 0.3 m M linoleic acid. Reaction products were first separated by RP-HPLC and then by SP-HPLC as described in Materials and methods. Inset shows the retention time of the isomers produced by soybean LOX1 compared with authentic standards of 9- and 13-hydroxy linoleic acid. Boxes: chiral phase HPLC showing the positional isomers composition of 13- and 9-HPOD. Ó FEBS 2003 Characterization of hazelnut seed lipoxygenases (Eur. J. Biochem. 270) 4371 and 3¢-end (836 bp) of the isolated CaLOX2.Asimple hybridization pattern was obtained with both the probes. Figure 6 shows the hybridization signals obtained with the 836 bp fragment corresponding to the 3¢ portion of the cDNA. On the basis of restriction analysis of the genomic clone, a 1897-bp fragment was expected in the EcoRI digestion; a fragment larger than 1627 bp fragment was expected with the HindIII digestion and a fragment larger Fig. 5. Comparison of the cDNA-deduced protein sequences of hazelnut LOXs. Sequences were aligned using the CLUSTALW 1.7 program. Hazelnut CaLOX1 refers to the putative protein from the genomic clone Lox1:Ca:1 (accession no. AJ417975), hazelnut CaLOX2 is deduced from the cDNA clone (CaLOX2) described in the text. The other aligned plant LOXs were as follows: almond (accession no. AJ404331), cotton (accession number AF361893), Arabidopsis lox1 (accession no. JQ2267), tomato loxA (accession number P38415), tomato loxB (accession number P38416), potato lox1 (accession no. U60200) and soybean lox1 (accession no. J02795). 4372 A. Santino et al. (Eur. J. Biochem. 270) Ó FEBS 2003 than 3387 bp was expected with the BamHI digestion. The most intense signals visible in Fig. 6 correspond to these fragments, while the faint hybridization signals could indicate the presence of other LOX genes. Discussion PCR experiments carried out on genomic DNA resulted in the amplification of two different LOX sequences (ln32 and ln50). A genomic clone was shown to contain a complete LOX gene (Lox1:Ca:1). RT-PCR experiments, performed with RNA from immature seeds, revealed the presence of two different LOX cDNAs, one of which corresponded exactly to the characterized Lox1:Ca:1 gene. The second clone shared a high degree of similarity (91.9% identity at the amino acid level) and is encoded by a different LOX gene. Sequence results and Southern blot analysis collec- tively identified the presence of three different types of LOX gene in the hazelnut genome; it is likely that more will be detected with time. Most plant species have multiple LOX genes in several classes, including tomato, potato, pea, soybean and Arabidopsis [23,24]. Although the numbers and relative positions of introns within plant LOX genes are fairly constant, their length variesandthisisparticularlymarkedinrelationtointronI, which can vary in length from 111 to 1245 bp in Arabidopsis LOX genes, be as large as 1201 bp in almond seed LOX [9] or even 2542 bp in the Glycine max L-4 gene (see [25]). The lox1:Ca:1 first intron is 1871 bp. There is some evidence to suggest that those LOX genes expressed predominantly in vegetative tissues have parti- cularly large first introns (see [25] for references). In this regard, the lox1:Ca:1 gene is interesting because it is expressed only at early stages of embryo development and not during the mid-to-late maturation phases of seed development, in a fashion reminiscent of the pea C2 LOX, the RNA for which is detected only at very early stages of seed development, and which is the only LOX detected in pea leaves [25]. Considering the high level of expression of CaLOX2 in hazelnut seeds, it would be of interest to isolate and characterize the corresponding genomic clone in order to determine if it has a similar genomic organization to Lox1:Ca:1. Hazelnut CaLOX1 and CaLOX2 seem not to be expressed in leaves. At present the physiological role of hazelnut seed LOXs is unclear. LOX gene expression, the enzymatic activity, and the accumulation of LOX protein in developing seeds could be associated with the intense mitotic activity and the consequent rapid remodelling of cell membranes occurring in developing seed. FASTA analysis suggests that, despite the clear evidence for expression in early seed development, the lox1:Ca:1 gene is more similar to some vegetatively/tuber-expressed LOX genes than those expressed in seeds. Possibly the conditions in the very early stages of seed development, when growth is mitotic rather than through differentiation and cell expan- sion, promote the expression of Lox1:Ca:1. The 5¢-non- coding promoter sequence of lox1:Ca:1 contains a number of putative transcription factor binding motifs, and also inverted repeats that could form stem loop structures reminiscent of those proposed for the promoter of the pea lox1:Ps:3 gene [26]. The functional significance of any such elements is unknown and requires further analysis. The hazelnut lipoxygenase shows dual product specificity, forming a mixture of 9- and 13-hydroperoxides in an approximate 2 : 1 ratio. This is not uncommon; pea seed LOX-3, for instance, also produces 9- and 13-hydroper- oxides from linoleic acid in a 2 : 1 ratio. Pea seed LOX-3 additionally produces keto-fatty acids, which are thought to arise from the dismutation of a prematurely released fatty acid peroxyl radical [27]. The hazelnut lipoxygenase does not, however, form such products, indicating that there is no particular link between dual product specificity and keto- fatty acid production (see also [23]). There are a number of similarities between the hazelnut and almond seed lipoxygenases [9]. Their amino acid sequences are 80% identical; they have a similar pH optimum; and they both produce 9-hydroperoxides from linoleic acid, although the LOX activity (in terms of specific activity) is much higher in almond. The almond LOX, however, produces exclusively 9-hydroperoxides, whereas the hazelnut LOX forms a mixture of 9- and 13-products. LOX gene expression, enzymatic activity and the synthesis of C 6 and C 9 aldehydes all have a similar trend throughout hazelnut seed development, being fairly limited to the early developmental stages. It is likely that the synthesis of these Fig. 6. Southern hybridization of hazelnut genomic DNA with a digo- xigenin-labelled LOX probe. Genomic DNA (10 lg) was digested with HindIII (H); BamHI (B) and EcoRI (E) and hybridized with an 836-bp fragment from the 3-end of the hazelnut CaLOX2. DNA size stand- ards in kb are shown to the left. Ó FEBS 2003 Characterization of hazelnut seed lipoxygenases (Eur. J. Biochem. 270) 4373 molecules is a consequence of the LOX and hydroperoxide lyase activities. Further studies are in progress to clarify the role of these enzymes in the production of volatile compounds, which are important constituents of hazelnut flavour and aroma. The hazelnut and almond LOXs are closely related to some plant LOXs involved in plant response to biotic stresses. Among these, the highest identity was found towards a cotton LOX, induced in the hypersensitive reaction towards Xanthomonas campestris. Jalloud and coworkers [17] showed that this cotton LOX is a 9-LOX and (9S)-hydroperoxides are largely predominant during the hypersensitive response of cotton cells. A high degree of identity (74.6%) was also found towards the tobacco 9-LOX specifically induced by Phytophthora parasitica [18]. The importance of this 9-LOX in the resistance to this pathogen was further confirmed in transgenic tobacco plants, in which the activity of this specific LOX was down-regulated by an antisense strategy. In contrast to wild type plants, transgenic plants became susceptible to Phytophthora parasitica [28]. Despite these clear indications on the importance of 9-LOXs in plant defence, the nature and the biological activities of oxylipins produced from 9-hydroperoxides are still largely unclear. Recently Go ¨ bel et al. [29] reported that cultured potato cells treated with a Phytophthora infestans elicitor accumulated high levels of 9-LOX and 13-LOX. However, oxylipin profiling revealed that the main oxylipins detected in treated potato cells were derived from the 9-hydroperoxides. Among these are colneleic and colnelenic acids which are produced by the activity of the divinyl ether synthase [30,31] and may act as antifungal molecules inhibiting the germination of the fungal spores [32]. Moreover some other oxylipins deriving from 9-hydro- peroxides such as 10-oxo-11,15-phytodienoic acid (10-OPDA), have been shown to display a regulatory function in response to biotic stresses as well as in plant developmental processes [33]. This could suggest an import- ant role for 9-LOX metabolism. The importance of oxylipins derived from 9-LOX metabolism in seed develop- ment has not yet been demonstrated. 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Biochem. 270) Ó FEBS 2003 [...]...Ó FEBS 2003 Characterization of hazelnut seed lipoxygenases (Eur J Biochem 270) 4375 22 Hornung, E., Walther, M., Kuhn, H & Feussner, I (1999) ¨ Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis Proc Natl Acad Sci USA 96, 4192–4197 23 Casey, R & Hughes, R.K (2003) Recombinant lipoxygenases and oxylipin metabolism in relation... Hardy, D., West, S.I., Fairhurst, S.A & Casey, R (1998) Characterization of authentic recombinant pea -seed lipoxygenases with distinct properties and reaction mechanisms Biochem J 333, 33–43 ´ ´ ´ 28 Rance, I., Fournier, J & Esquerre-Tugaye, M.T (1998) The incompatible interaction between Phytophthora parasitica var 29 30 31 32 33 nicotianae race 0 and tobacco is suppressed in transgenic plants expressing... preferential stimulation of the 9-lipoxygenase pathway in elicitor-treated potato cells J Biol Chem 276, 6267– 6273 Stumpe, M., Kandzia, R., Gobel, C., Roshal, S & Feussner, I ¨ (2001) A pathogen induced divinyl ether synthase (CYP74D) from elicitor-treated potato suspension cells FEBS Lett 507, 371–376 Itoh, A & Howe, G.A (2001) Molecular cloning of a divinyl ether synthase: identification of a CYP74 cytochrome... press 24 Casey, R., West, S.I., Hardy, D., Robinson, D.S., Wu, Z & Hughes, R.K (1999) New frontiers in food enzymology: recombinant lipoxygenases Trends Food Sci Technol 10, 297–302 25 Knox, M., Forster, C., Domoney, C & Casey, R (1994) Structure of the Pisum sativum seed lipoxygenase gene lox1:Ps:3 Biochim Biophys Acta 1214, 341–343 26 Domoney, C., Firmin, J.L., Sidebottom, C., Ealing, P.M., Slabas,... fatty acid synthesis in late blight-diseased potato leaves Plant Cell 11, 485–493 Itoh, A., Schilmiller, A.L., McCaig, B.C & Howe, G.A (2002) Identification of a jasmonate-regulated allene oxide synthase that metabolizes 9-hydroperoxides of linoleic and linolenic acids J Biol Chem 277, 46051–46058 . shelf life and the organoleptic features of hazelnut seeds, we carried out a study to characterize hazelnut lipoxygenases at the biochemical and molecular level. Materials. pastry industry. Hazelnut seeds contain reasonable levels of linoleic and linolenic acid (about 10 and 0.2%, respectively, of the total fatty acids) and hexanal and