Section of Lecce, Italy;2John Innes Centre, Norwich, UK Plant lipoxygenases LOXs are a class of dioxygenases which display diverse functions in several physiological processes such as gr
Trang 1Biochemical and molecular characterization of hazelnut
Angelo Santino1, Angelo De Paolis1, Antonia Gallo1,*, Angela Quarta1, Rod Casey2and Giovanni Mita1
1
Institute of Sciences of Food Production C.N.R Section of Lecce, Italy;2John 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)
Trang 2by 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: 94C for 5 min (1 cycle); 94 C for 1 min,
45C for 1 min, 72 C for 2 min (25 cycles); 72 C for
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)18primer 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: 94C for 3 min (1 cycle);
94C for 45 s, 50 C for 45 s, 72 C for 2 min (30 cycles);
72C 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 theQUANTITY 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 mMsodium carbonate and the pH adjusted to 7.5 before assaying LOX activity
LOX activity was determined polarographically at 25C with a Clark O2electrode using linoleic acid as substrate Thirty micromolar stock solution was prepared according
to [14].The reaction mixture (1.0 mL) consisted 100 mM sodium phosphate buffer pH 6 containing 0.3 mMlinoleic acid and different amounts of protein samples.Decrease in
O2 concentration was monitored for 3 min and the enzy-matic activity was calculated from the initial rate of O2 uptake.One unit of LOX activity corresponds to the intake
of 1 lmol of O2per minute.pH optimum of hazelnut LOX was determined as previously described [9].LOX activity was also assayed spectrophotometrically monitoring the increase in A234 of the conjugated-diene structures as previously described [14]
Trang 3SDS/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 mMsodium phosphate
buffer pH 6 containing 0.3 mM 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 65C 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
Trang 4Fig 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.
Trang 5Fig 1 (Continued).
Trang 6two 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) The phylogenetic tree was obtained using the PHYLIP (Phylogeny Inference Package) program.
Fig 3 Analysis of LOX activity and LOX gene expression in hazelnut seedat d ifferent d evelopmental 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)1Æmg)1protein) 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).
Trang 7stage 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
C6 and C9 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 C6and C9aldehydes (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
hazelnut seed were incubated for 30 min in 0 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.
Trang 8and 3¢-end (836 bp) of the isolated CaLOX2 A simple
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).
Trang 9than 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
varies and this is particularly marked in relation to intron I, which can vary in length from 111 to 1245 bp in Arabidopsis LOXgenes, 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:1first 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 CaLOX2in 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:3gene [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 LOXgene expression, enzymatic activity and the synthesis
of C6and C9aldehydes 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.
Trang 10molecules 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.Further investigation,
aiming to evaluate the expression of other genes of the
lipoxygenase pathway involved in oxylipin biosynthesis
(allene oxide synthase, hydroperoxide lyase), will help to
elucidate the role of the LOX pathway in developing seeds
Acknowledgements
We are grateful to Dr Claire Domoney for the gift of pea LOX
antibodies, Dr Pasquale Piccirillo for the hazelnut agronomic aspects,
Dr Danilo Dello Monaco for GC-MS analysis, Professor Liugino
Troisi for chiral phase HPLC analyses and Mr Leone D’Amico for
technical assistance.
This work was supported by Ministero dell’Universita` e della
Ricerca Scientifica e Tecnologica, Cluster C03.
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