that fatty
acid hydroxyperoxides
and their
metabolites play some role in flower senescence and the activity of lipoxygenase, the key enzyme
of hydroxyperoxidation of fatty acids, is elevated during senescence of carnation flowers [22].
Methyl jasmonate, a product of the octadecanoid pathway from the fatty acid hydroperoxide, has
been shown to promote senescence of petunia and dendrobium flowers [29]. Therefore, we decided to
investigate whether a lipoxygenase was involved in the senescence of rose flowers.
In this study, we have isolated a cDNA encod- ing a lipoxygenase isoform and found that this
cDNA is expressed in rose petals in response to senescence.
2. Materials and methods
2
.
1
. Plant materials Petals of Rosa hybrida cv. Kardinal grown in a
green house in Victoria, Australia were harvested from flowers at different developmental stages
defined as follows: stage 1, closed and pigmenting buds; stage 2, buds still closed and heavily pig-
mented; stage 3, flowers whose outer petals were just opening. Flower stems were harvested at stage
3 and the bottom half of the cut stems were kept in distilled water at room temperature; stage 4,
flowers 2 days after harvest; stage 5, flowers 4 days after harvest; stage 6, flowers 6 days after harvest;
stage 7, flowers 8 days after harvest. Each of the floral organs as well as young red leaves and
mature green leaves were obtained from flower stems on the day of harvest.
2
.
2
. Ethylene treatment For the treatment of rose flowers with ethylene,
excised stems with flowers were kept for 24, 48 or 72 h in an 850 ml chamber with a solution of
0.25M Tris – HCl pH 8.0 and 0.4 Ethrel May and Baker Rural Pty., Sydney, NSW, Australia;
the concentration of ethephon, a precursor to ethylene, is 480 gl in Ethrel. Under these condi-
tions, the concentration of ethylene in the chamber was calculated to be 650 ppm, as described previ-
ously [30]. As a control, excised flower stems were incubated under the same conditions in the ab-
sence of Ethrel.
2
.
3
. Isolation and molecular analysis of rose lipoxygenase cDNAs
Total RNA was prepared by differential precipi- tation using 2-butoxyethanol [31]. A cDNA library
was constructed from polyA
+
RNAs isolated from petals at stage 5 see above as described previ-
ously [28]. DNA fragments from two tomato lipoxygenase
cDNAs, tomloxA and tomloxB [19], were ob- tained by PCR using oligonucleotide primers cor-
responding to their 5 and 3 sequences, using a tomato fruit cDNA library breaker stage, Clon-
tech, Inc. Palo Alto, California, USA as a tem- plate. The amplified fragments were labeled with
digoxigenin-dUTP DIG-dUTP using a DIG la- beling system Boehringer, Heidelberg, Germany
and used to screen the rose petal cDNA library. The screening procedures were essentially the same
as described previously [32] except for the strin- gency of screening conditions; the hybridization
solution contained 30 formamide, and washing was performed at 55°C with 5 × SSC 0.75 M
NaCl, 0.75 M sodium citrate containing 1 SDS. The sequences of the cDNAs were determined and
analyzed as described previously [32]. The possible localization of the rose lipoxygenase was predicted
by a PSORT protein sorting analysis program National Institute for Basic Biology, Okazaki,
Japan, E-mail: psortnibb. ac. jp.
2
.
4
. Expression of a rose lipoxygenase cDNA in E.coli
The expression vector pTrc99A Pharmacia Bio- tech, Uppsala, Sweden, was used to express a rose
lipoxygenase cDNA Rlox1 in E. coli strain JM109. The 5-half portion of the Rlox1 cDNA
146 bp in length was amplified by PCR so that a NcoI site was created just before the putative
initiation codon in the Rlox1 cDNA. The am- plified fragment and the remaining 3-half portion
of the Rlox1 cDNA were digested with NcoI and P6uI, or P6uI and KpnI, respectively, and inserted
together into pTrc99A predigested with NcoI and KpnI to produce an expression construct, pTrcR-
lox1. These procedures caused a mutation in the second amino acid residue; leucine was replaced
by valine in the expressed Rlox1 product. Trans- formed E. coli cells were grown in LB bacto-
tryptonl, 0.5 bacto-yeast extract, 1 NaCl, pH
7.0 broth containing 50 mgl ampicillin at 37°C. The expression of Rlox1 cDNA was induced by
addition of 2 mM isopropyl b-
D
-thio-galactoside final concentration, after which the cells were
grown for 4 h and used for purification of the recombinant Rlox1 protein.
2
.
5
. Partial purification of the recombinant lipoxygenase protein
The following purification procedures were per- formed at 4°C. The E.coli cells expressing the
recombinant Rlox1 protein 64 g, wet wt were harvested by centrifugation and were ground with
128 g of levigated alminum oxide in a mortar chilled on ice. The homogenate was then sus-
pended in 0.01M potassium phosphate buffer, pH 7.2, containing 0.25 M sucrose buffer A followed
by centrifugation at 8000g for 15 min. The super- natant was dialyzed against buffer A, and applied
to a column of Sepabeads FPDA13 400 ml, Mit- subishi Chemical Industries, Tokyo equilibrated
with buffer A. The lipoxygenase activity was eluted by a linear gradient of 0 – 1.0 M NaCl in
buffer A 1.01 each. The active fractions were dialyzed extensively against buffer A followed by
Fast Protein Liqid Chromatography FPLC with a HiPrep-Q 1610 column Pharmacia equili-
brated with buffer A. After extensive washing of the column with buffer A, lipoxygenase activity
was eluted with a linear gradient of 0 – 1.0 M NaCl in Buffer A. The active fractions were dialyzed
against buffer A and rechromatographed under the same FPLC conditions as described above
except that a linear gradient of 0 – 0.5 M NaCl was used. The active fractions were concentrated using
an Amicon 8200 ultrafiltration unit with a PM 10 membrane. The concentrate was subject to gel
filtration chromatography on Ultrogel AcA44 Pharmacia, 2 × 64 cm equilibrated with buffer A
containing 0.2 M NaCl. The active fractions pu- rity was 20 as judged by SDS-PAGE were used
for further characterization.
2
.
6
. Assay of lipoxygenase acti6ity
2
.
6
.
1
. Method I Lipoxygenase activity was assayed by a conju-
gated diene method [33]. The reaction mixture 1.0 ml consisted of 0.1 M sodium acetate buffer, pH
5.0, 0.0025 Tween 20, and 0.2 mM linoleic acid final concentrations. After incubation of the mix-
ture at 37°C, the reaction was started by adding enzyme up to 30 ml and the increase in ab-
sorbance at 234 nm was monitored with a Shi- madzu UV-160 spectrophotometer kinetic mode
with a temperature-controlled cell positioner CPS- 240A. The extinction coefficient for the conjugated
diene of 25 000M
− 1
cm
− 1
[2] was used for the unit calculation.
2
.
6
.
2
. Method II Formation of lipid hydroperoxides was moni-
tored by a visible-spectrophotomeric method as described previously [34].
2
.
7
. Characterization of enzymatic acti6ity For pH – activity profiles, lipoxygenase activity
was assayed using method I except that the reac- tion mixture contained 100 mM of one of the
following buffers: pH 3.0 – 6.0, sodium acetate; pH 6.0 – 8.0, potassium phosphate; pH 8.0 – 9.0, Tris-
HCl; and pH 8.0 – 10.0, sodium borate.
For temperature – activity profiles, lipoxygenase activity was assayed at various temperatures indi-
cated in Fig. 2B, essentially using assay method I. For pH stability, the enzyme was incubated at
4°C in the following buffers final concentration, 50 mM: pH 3.0 – 6.0, sodium acetate; pH 6.0 – 8.0,
potassium phosphate, pH 8.0 – 9.0, Tris – HCl; and pH 8.0 – 10.0, sodium borate. After incubation for
24 h or 3 weeks, 60 ml of each enzyme solution was withdraw and assayed for remaining activity by
using method I.
2
.
8
. Northern blot analysis The procedure for Northern blot analysis is
essentially the same as described previously [28]. EcoRI-digested Rlox1 cDNA fragment, 0.25 kb,
was used as a probe for Northern blot analysis. This fragment contained all of the 3-noncoding
region of Rlox1 together with a 24 bp 3-terminal sequence from the coding region.
3. Results and discussion
