Fig. 2. Structures of the two antisense lipoxygenases LOX constructs; A the construct p2ALX contained a 1.2 Kb antisense sequence of TomloxA under the control of the 2A11 fruit specific promoter; B the pPGLX construct contained a 396 bp antisense
sequence of TomloxA under the control of the ripening specific polygalacturonase PG promoter. Abbreviations: NPT II, kanamycin selectable marker; B, BamHI; C, ClaI; K, KpnI; X, XhoI; S, SpeI.
the antisense constructs were designed to target this particular region of the LOX mRNA. We
report on the generation of transgenic antisense LOX tomato fruit and their analysis in terms of
mRNA expression studies, LOX enzyme activity and aroma volatile analysis.
2. Materials and methods
2
.
1
. Screening of a cDNA library Screening of an early ripening tomato cDNA
library Picton et al., 1993, using a potato LOX cDNA lox
1:
St
:1
Casey, 1995, resulted in the identification
of three
previously published
tomato LOX clones. PTL1 was homologous to TomloxA Ferrie et al., 1994; PTL2 was identical
to TomloxB Ferrie et al., 1994 and a clone designated U13681 Kausch and Handa, 1995,
but PTL2 lacked 300 nucleotides from the 5 end; LOX3 which was not full length at 1.2 Kb was
homologous to the 3 region of TomloxC Heitz et al., 1997. PTL1, PTL2 and LOX3 will be referred
to henceforth as TomloxA, TomloxB and Tom- loxC respectively.
2
.
2
. Construction of antisense LOX transformation constructs using the
2
A
11
fruit specific promoter and the polygalacturonase
promoter All molecular cloning procedures were carried
out using standard methods Sambrook et al., 1989. The 2A11 promoter described by Van
Haaren and Houck 1991, 1993, was obtained via polymerase chain reaction PCR supplied by Dr
Sumant Chengappa, Unilever Research, Colworth House. The construct pA2LX was assembled to
generate an antisense transgene to down-regulate the endogenous TomloxA and TomloxB mRNA
Fig. 2A. A partial length 3 sequence of the cDNA of TomloxA, in the antisense orientation,
was assembled by ligating a 1.2 Kb KpnIClaI fragment of TomloxA in front of the 2A11 pro-
moter at the KpnIClaI site contained in the pBluescript derived vector pBS2A11. The pres-
ence and orientation of the TomloxA insert was confirmed by restriction enzyme analysis and
PCR, using primers specific to the highly con- served region of the LOX sequence and the T7
promoter of pBluescript. These approaches confi- rmed that the LOX sequence was in the antisense
orientation with respect to the 2A11 promoter. No terminator was incorporated into this con-
struct. This expression cassette containing the 2A11 promoter and LOX insert was then released
by a KpnIBamHI digest to give a 5.2 Kb frag- ment. This fragment was then ligated into the
binary transformation vector BIN19 Bevan, 1984 at the KpnIBamHI site to give p2ALX.
Colonies were checked for the presence of the expression cassette by blotting onto Hybond-N +
membranes and probed using the original Tom- loxA KpnIClaI insert as a probe. Further confir-
mation of the presence and orientation of the 2A11 promoter and the LOX antisense insert was
achieved by restriction enzyme analysis and plas- mid Southern analysis.
The second construct, pPGLX, designed to down regulate endogenous LOX mRNA expres-
sion utilised the 4.8 Kb polygalacturonase PG promoter Nicholass et al., 1995, along with a
partial 3 antisense orientated sequence of Tom- loxA and the 1.8 Kb PG terminator Fig. 2B.
The PG promoter and terminator had previously been assembled into the binary vector pBIN19
Supplied by Dr Colin Bird and Dr Rachael Drake, Zeneca Plant Science and was designated
pRD12. A 396 bp XhoISpeI fragment of Tom- loxA was assembled in the antisense orientation in
front of the PG promoter at the XhoISpeI site of the pRD12 vector. Clones containing the insert
were identified by restriction enzyme analysis, fol- lowed by plasmid Southern analysis and also by
PCR using primers specific to the highly con- served LOX region and the PG promoter.
2
.
3
. Plant transformation For plant transformations the binary vectors
were introduced into competent cells of Agrobac- terium tumefaciens strain LBA4404 Bevan, 1984.
The transformation of tomato cotyledons Lycop- ersicon esculentum cv Ailsa Craig involved a stan-
dard transformation protocol Bird et al., 1988. Successfully transformed plant material with roots
was regenerated from callus grown on 100 mgml kanamycin and then transferred to the glasshouse.
These plants were later potted up into compost Levington M2 and grown under identical
glasshouse conditions used for non-transformed control plants.
2
.
4
. RNA isolation and Northern analysis Tomato fruit pericarp tissue was harvested
from the combined pericarp of two to three fruit, frozen in liquid nitrogen and then stored at −
80°C until required. Total RNA was extracted from approximately 10 g of the pericarp tissue.
The extraction, quantification, blotting, hybridisa- tion and probe synthesis protocols were as de-
scribed by Griffiths et al. 1999. Hybridisation was at 42°C in 50 vv deionised formamide,
1 wv SDS, 1 M NaCl, 10 wv dextran sulphate and 100 mgml salmon sperm DNA.
In addition to using autoradiography to analyse the
signal intensity,
the membranes
were quantified directly using a Packard InstantIm-
ager™ 2024 radioanalytical imaging detector and the data analysed using InstantImager™ software.
2
.
5
. DNA isolation and Southern analysis Genomic DNA was extracted according to the
protocol of Bernatzky and Tanksley 1986, ex- cept that frozen leaf samples 2 – 3 g were initially
ground in liquid nitrogen using a pestle and mor- tar, followed by the addition of extraction buffer.
The isolated DNA was digested with the appro- priate restriction enzymes and fractionated on a
1.0 agarose gel and blotted onto Gene Screen Plus membranes Gene Screen, Du Pont. Prehy-
bridisation and hybridisation conditions were car- ried
out at
65°C as
described in
the manufacturer’s instructions Gene Screen, Du
Pont.
2
.
6
. Extraction and assaying of LOX acti6ity Fruit samples were harvested at various stages
of development from breaker to 7 days post- breaker. Pericarp tissue was diced and frozen
immediately in liquid nitrogen before storage at −
80°C until required. For the LOX assay 12 g of pericarp tissue was thawed and extracted using a
pestle and mortar in 12 ml of 0.1 M phosphate buffer pH 6.5, including 0.1 wv Triton X-
100 and 1 mM EDTA. LOX enzyme activity measurements were made using a Clark-type oxy-
gen electrode, where oxygen consumption was measured in the presence of the substrate linoleic
acid as previously described Smith et al., 1997.
2
.
7
. Volatile sampling
2
.
7
.
1
. Headspace sampling Individual tomatoes 28 – 66 g were placed in a
plastic stomacher bag and an internal standard 0.025 mg of 2-octanone in 100 ml water was
added. Tomatoes were macerated for 1 min by the action of paddles in the stomacher machine Se-
ward M50-110, London, UK. After maceration, headspace gas 160 ml was collected onto a
Tenax trap Unijector, SGE, Milton Keynes, UK for 1 min using a vacuum pump Charles Austin,
B100 SEC. The trap was then removed and the volatiles analysed by GC-MS as described below.
2
.
7
.
2
. Chromatographic conditions Tenax traps were desorbed in a headspace injec-
tor Unijector; SGE connected to a Hewlett Packard
5890 Series
II gas
chromatograph column head pressure 18 psi, helium carrier gas.
The volatiles were desorbed from the traps 3 min, 240°C and cryofocused onto a 400 mm region of
the column BP-1, 25 m × 0.22 mm ID, 1 mm film thickness; SGE. After desorption, the column
was held at 40°C 2 min, then temperature pro- grammed from 40 to 106°C at 4°Cmin and from
106 to 145°C at 15°Cmin. Compounds were de- tected using an MD 800 mass spectrometer oper-
ating in the mz range 30 – 150 Fisons Scientific, Manchester, UK.
2
.
7
.
3
. Data analysis The relative amounts of compounds were deter-
mined by measuring the peak areas of characteris- tic ions for the volatiles of interest hexanal mz
92; Z-3-hexenal mz 98; E-2-hexenal mz 83; 2-octanone mz 58. Peak areas for each sample
were corrected by reference to the internal stan- dard and expressed as peak area of volatile per 50
g fresh weight.
3. Results
