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DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to [email protected]
Project title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE PREDICTION OF
TUBER SUSCEPTIBILITY TO BLACKSPOT BRUISING IN THE FIELD
DEFRA project code
HP0217
Contractor organisation
and location
School of Biological and Biomedical Sciences
University of Durham
Science Laboratories
South Road
Durham
DH1 3LE
UK
Total DEFRA project costs
Project start date
£ 54,783.00
01/03/01
Project end date
28/02/03
Executive summary (maximum 2 sides A4)
A. Introduction
Blackspot bruising is the blue-black tuber discoloration encountered when susceptible potatoes impact on a hard
surface, and is caused by a chemical reaction initiated when tuber cells are physically damaged. This phenomenon greatly
affects the quality of the crop and its value.
A 2-year, BPC/DEFRA funded programme at the University of Durham under the direction of Dr Ron Croy has made
significant advances in the understanding of this important problem facing the potato industry. This knowledge has led to
the development of a detection system for assessing blackspot susceptibility. The detection system will form the basis of a
kit for assessing the crop prior to harvest or during storage and will provide an 'early warning' of a high bruise risk. This
will facilitate implementation of appropriate, gentle harvesting and transport procedures.
When a tuber impacts on a hard surface the tissues are damaged and they respond rapidly by synthesising oxygen free
radicals - a highly active form of oxygen which rapidly reacts with many biological materials including membranes
(lipids), proteins and nucleic acids. The results from this research have confirmed that the level of free radicals produced
indicates how susceptible the tuber is to bruising - a high level indicates a high susceptibility to bruising while a low level
indicates good resistance to bruising. The project has demonstrated the generation of radicals in amounts very closely
correlated with the level of bruise susceptibility in 13 different varieties including Cara, King Edward, Russet Burbank,
Maris Piper and Desiree; as yet no varieties have failed to demonstrate the correlation. As a consequence of the close
relationship between these highly active molecular species and bruise susceptibility it is anticipated that a diagnostic kit
CSG 15 (Rev. 6/02)
1
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
based on a free radical assay will be able to predict bruising status to between 90 and 95% accuracy. This is the first
demonstration of such a clear relationship between a measurable factor and bruise susceptibility.
The method of detection of bruise susceptibility is relatively simple and is based on an assay specific for the
superoxide radical. The level of radical generation is measured using a dye which changes colour when it reacts with the
factor and this colour change is measured to assess the bruise susceptibility. The test is carried out by selecting samples of
the potato crop directly from the field or from storage and exposing these to a standard impact using the impactor
(figure 2). The tuber tissue responds within two hours and then a standard size of core of tissue is excised using the corer
(figure 3), and placed into the dye solution. Following a 20 minute incubation period the solution is mixed with a second
solution which brings about a colour change. The depth of colour of the final solution is measured or compared by eye
with a colour chart and indicates how prone the crop is to bruise damage. The findings from this work have recently been
published in the prestigious journal Plant Physiology (Johnson et al., 2003).
Currently the research is aimed at developing the chemistry and hardware components of the assay to provide a fully
workable and ‘user-friendly’ kit of components for field use by the potato industry.
In the future, investigations will employ cutting-edge biochemical and molecular technologies to identify other factors
present in growing tubers which govern the underlying mechanisms controlling blackspot. This will involve fundamental
studies of the tuber developmental processes which lead to the generation of the factors which pre-determine bruise
susceptibility.
B. Project aims
The overall aim of this project was to exploit the novel observations made by Dr Croy’s group, (and recently reported in
the paper by Johnson, Doherty and Croy, 2003 – Appendix 3) which showed a direct correlation between bruising
susceptibility and free radical generation. These observations were to be adapted into a system for predicting bruise status
in field grown potato crops. The project was directed towards the production of a convenient and easy-to-use bruise
susceptibility diagnostic kit and to test the performance of the detection system under simulated and real field conditions.
C. Project objectives
The objectives of the proposed research were as follows -
i.
Develop the existing assays and investigate new chemical detection systems for free
radicals. The objectives will be to identify assays with the highest sensitivity and
reproducibility with a view to adapting these into a kit format
ii.
Use the selected assay systems to test a range of potatoes grown under controlled field
conditions to simulate bruise susceptible and resistant tuber materials, to confirm the
sensitivity, reproducibility and validity of the assays
iii.
Adapt selected free radical assays into simple prototype test kits
iv.
Design a tuber impactor that will allow a rapid, reproducible impact force on large numbers
of test tubers.
v.
Test prototype kits with a wide range of field-grown tuber materials
vi.
Investigate the possibilities for the future development of an electronic, free radical
biosensor system for diagnosing bruise susceptibility
CSG 15 (Rev. 6/02)
2
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
on
schedule
yes no
date
completed
D. Achievement of project milestones and objectives
Year 1
no.
milestone
due
date
1.
Growth season 1. Production, sampling and storage of defined tuber
material for development and testing (ADAS Gleadthorpe).
10/01

10/01
2.
Design, build and test prototype impactor device
11/01

10/01
3.
Supply of other field grown material from ADAS sites, SBEU and
other sources.
02/02

11/01
4.
Comparison of a range of new detection chemistries with existing
ones. Optimise existing and develop new improved assays for free
radicals.
02/02

01/02
5.
Test a wider range of tuber materials with existing assays – to provide
further proof of concept, detect any deviations from established
correlation.
02/02

04/02
6.
Submit annual report.
02/02

04/02
7.
Submit grower friendly project summary
02/02

04/02
8.
Review of project progress.
02/02

04/02
9.
Modify and refine impactor device as necessary.
08/02

06/02
Year 2
no.
milestone
due
date
10.
Production, sampling and storage of defined replicated tuber material
for kit testing (ADAS Gleadthorpe).
10/02

10/02
11.
Supply of field grown material with documented history from 4 other
ADAS sites; and further material from storage at ADAS sites, SBEU
and other sources.
01/03

12/02
12.
Select most appropriate assay systems and adapt into simple kits.
01/03

02/03
13.
Test prototype kits with test tuber materials; produce data on
reproducibility of tests and establish scale of bruise susceptibility.
01/03

01/03
14.
Investigate future possibilities for biosensor systems to detect free
radicals in tuber tissues.
(Report attached –
Appendix 2).
01/03

02/03
15.
Produce draft instruction manual for test kit. (Document attached –
Appendix 1).
01/03

02/03
CSG 15 (Rev. 6/02)
3
on
schedule
yes
no
date
completed
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
16.
Submit final report. (This document)
01/03

08/03 *
17.
Submit grower friendly project summary - section A of this report.
01/03

02/03
18.
Undertake a minimum of 2 presentations at BPC levy payer meetings
if required. (See Summary of technology transfer and project
deliverables)
02/03

06/03
items attached to this report
* Delay due to requested changes in format and scope of original BPC report. Details subsequently incorporated into the
final DEFRA report
CSG 15 (Rev. 6/02)
4
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Scientific report (maximum 20 sides A4)
Scientific Report
A. Introduction
The following section contains the experimental details and conclusions from the work undertaken during this project
together with relevant results from other associated fundamental investigations contributed by other personnel/project
students in Dr Croy’s group.
B. Materials and methods
Plant materials
Test tubers were grown by ADAS at Gleadthorpe, under conditions designed to produce bruise-susceptible and bruiseresistant crops of the same variety. Initially for the 1st field trial (2001-2) 5 potato varieties exhibiting different degrees of
susceptibility to mechanical damage were studied - Cara, King Edward, Maris Piper, Russet Burbank and Saturna (See
table 1). This selection was later expanded in the 2nd field trial (2002-3) to 8 varieties as indicated in table 1. The figures
refer to the bruise indices for these varieties estimated by our own testing (See later). Tubers were specifically grown and
carefully harvested manually by ADAS to avoid any mechanical stress. Harvested tubers were transported to the ADAS
storage facility at Low Mowthorpe and stored in the dark at 5oC, to inhibit greening and sprouting, until required.
Table 1 Cultivar bruise susceptibilities
Cultivar
Cara
King Edward
Maris Piper
Saturna
Russet Burbank
Bruise Index
Cultivar
1st trial
2nd trial
5.5
4.0
3.1
7.9
9.2
6.2
6.7
7.8
Navan
Cultra
Ambo
Desiree
Pentland Crown
Bruise Index
1st trial
2nd trial
-
6.7
6.5
6.3
7.7
6.2
Bruise susceptibility is on a scale of 0 (highly resistant) to 10 (highly susceptible),
calculated through in-house bruise assessment, comparing bruise to a theoretical
maximum sized bruise. Data shown are the results from materials harvested from 1st
field trials (2001-2) and 2nd field trials (2002-3).
Field Trials - Parameters Used
Three variables were used to generate tuber varieties with a range of susceptibilities. The variables used were – i)
sprouted or unsprouted seed, ii) harvested early or late after ‘burn off’ / ‘die back’ and iii) application of normal levels of
potassium fertilizer (250kg/ha) or no added potassium. In addition to selecting cultivars showing a range of genetic
predispositions to bruising these parameters have been reported to contribute towards bruising. The plots were set up at
ADAS Gleadthorpe which has a sandy soil suitable for ‘minimal’ potassium trials (See example plot layout in figure 1).
Blind Testing
In year 1, samples were assayed for radical assays prior to bruise susceptibility assays being performed. However in year
2, a full ‘blind’ testing regime was adopted, with ADAS allocating a code number to each cultivar prior to collection - to
permit a full analysis to be performed prior to the cultivar identity being revealed.
CSG 15 (Rev. 6/02)
5
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
Project
title
DEFRA
project code
HP0217
figure 1: Diagram of ADAS field plots at Gleadthorpe, 1st field trials
Example of field plot layout used in the first growing season 2001 – 2002. Plot layout was similar in the second growing season
2002-2003 but involved fewer variables and a wider range of varieties (See section B).
CARA
sp 3 bm 3
plot
RUSSET BURBANK
sp 3 bm 4
sp 4 bm 1
2
3
1
S1
6m
sp 4 bm 2
4
PENTLAND CROWN
sp 4 bm 3
5
sp 4 bm 4
6
S2
S1
S2
S1
S2
K2
K2
K2
K2
K2
K2
H1
H1
H1
H1
H1
H1
10m
7
8
9
10
11
12
S1
S2
S1
S2
S1
S2
K1
K1
K1
K1
K1
K1
H2
H2
H2
H2
H2
H2
13
14
15
16
17
18
S1
S2
S1
S2
S1
S2
K2
K2
K2
K2
K2
K2
H2
H2
H2
H2
H2
H2
19
20
21
22
23
24
S1
S2
S1
S2
S1
S2
K1
K1
K1
K1
K1
K1
H1
H1
H1
H1
H1
H1
Treatments
Variety
Nutrition
Seed Condition
Harvest Date
V1
Cara
K1
Zero
S1 Unsprouted
H1 Early
V2
Russet Burbank
K2
Farm Practice 250kg/ha
S2 Sprouted
H2 Late
V3
Pentland Crown
Hardware development
Initial work on bruising employed a simple coach bolt with added weights, dropped from a measured height (usually
30cm) onto the surface of the tuber. The need for a more ‘user-friendly’ device which would deliver a defined and
measurable impact to the surface of a large number of test tubers was recognised early in the potato bruising work.
Preliminary designs were produced as part of the UK and international patents (PCT/GB02/01631, see publication list).
Initial designs described a standardised spring loaded device which drives an impacting head along a barrel aligned with
the area to be impacted and to exert a precise energy of 0.7 joules. Subsequently, an improved design and the first
prototype device were produced in collaboration with the School of Engineering at the University of Durham (figure 2). In
addition a simple device for the rapid sampling of small standard samples (cores) of tuber tissue was required. This was
also produced by the School of Engineering (figure 3).
Tuber mechanical stress (bruise assay)
Tubers were incubated for 48h at 4oC in the dark and then impacted at the stolon end using either i) a falling weight of
240g and a 300mm drop height imparting a standard energy of 0.7J (Croy et al. 1998) OR ii) the prototype impactor set
to deliver the same energy of impact (figure 2). Impacted and control tubers were then incubated at 26 oC to promote
CSG 15 (1/00)
6
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
maximal synthesis of bruise pigments. For bruise index calculations tubers were incubated for 48h then cut in quarters
centred at the impact site. The volume of affected tissue was measured and the intensity of pigmentation estimated on a
scale of 0 (no discoloration) to 3 (deep blue-black coloration). 30 tubers for each of the test samples were used and the
mean bruise index calculated based on bruise extent and intensity. Values were expressed as percentage of a theoretical
maximum bruise volume and intensity.
Assay of superoxide radical generation in tuber tissues
Tuber tissues exposed to the standard mechanical stress were incubated in the dark at 26ºC for various times and then
assayed for superoxide generation. 5mm cubes (125mm3 = 35mg) of tuber tissue were excised from the centre of impact
sites and from distal control sites on the same tubers, washed thoroughly with distilled water, blotted dry and then
incubated at 20ºC for 20 minutes in 200µl 0.12mM XTT in 50mM phosphate buffer, pH 8.2 (Able et al. 1998). The tissue
cube was removed and the assay solution centrifuged (13000g x 5mins). The A 450 of the supernatant was measured and
expressed as μmol superoxide generated per minute using the molar extinction coefficient for the XTT formazan product
of 23,600 M-1 cm-1 (Sutherland and Learmonth, 1997; Sutherland M, personal communication). Superoxide estimations
were carried out in duplicate and all assays were replicated.
Assay of superoxide radical generation in tuber tissues using alternative tetrazolium
derivatives (Dojindo, Japan)
Early work on free radical detection employed a tetrazolium dye – nitroblue tetrazolium (NBT) which reacted with
radicals and the formazan product precipitated out of solution at room temperature. Such a dye substrate is used widely
for histochemical studies to locate radical generation in tissue sections but was of little value for quantitative estimates
due to the insoluble formazan product. A recently developed tetrazolium dye (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)(2H)-tetrazolium-5-carboxanilide (XTT) which reacts with superoxide radicals to produce a soluble formazan product was
initially employed in the development of the assay as indicated below (Able et al., 1998). The soluble product enables an
accurate estimate of superoxide levels by spectrophotometry. Several other available tertrazolium dyes were similarly
tested. More recently a new generation of tetrazolium derivatives (WST’s) have been developed by the Japanese Company
Dojindo. These appear to have some desirable properties and initial studies on small samples provided by contacts at
Dojindo look promising and warrant further investigation.
Estimation of protein modification in tuber tissues
The carbonyl content of oxidatively modified tuber proteins was quantified by the spectrophotometric assay method of
Levine et al (1994). Tubers were exposed to standard mechanical stress and incubated for 48h. Tuber proteins were
extracted from 150mg of control and impacted tubers in 3ml of 50mM phosphate buffer, pH7.4. The carbonyl groups on
extracted proteins were reacted with DNPH and the resulting hydrazone derivatives estimated from the peak absorbance
at 355-390nm using a molar extinction coefficient of 22,000 M -1 cm -1. Protein contents of duplicate samples were
estimated from the A280. Values were expressed as nmol carbonyl mg-1 tuber protein.
Exposure of tubers to inhibitors and free radical scavengers
Tubers were mechanically impacted followed immediately by bisection of the entire tuber through the point of impact.
Each half was then extensively washed three times in distilled water before being gently blotted dry. One half was placed
in 5ml phosphate buffer (50mM pH7.2) and acted as the uninhibited control, the other half was placed in 5ml test
solution (10μM diphenylene iodonium chloride (DPI) or 1μg/ml catalase or 0.5μg/ml superoxide dismutase (SOD) in
50mM phosphate buffer pH7.2) in a petri dish. Each tuber half was then incubated at 27˚C in darkness for the required
time.
Response of tuber cells to extracts from mechanically stressed tissues and cleavage
products of cell wall materials
5mm cubes (125mm3) of tuber tissue were exposed to various tissue extracts and assayed for superoxide generation as
described previously. Extracts were prepared from a 5mm tuber cube excised from the centre of impact sites, 2h and 4h
post-impact, and from non-impacted tubers, washed thoroughly with distilled water, homogenised in 500l 50mM
phosphate buffer (pH7.2) and centrifuged (13000g) for 5 minutes. 200l of each supernatant was added to a fresh,
washed 5mm test cube from a non-impacted tuber and incubated at 20C for 2h. The tuber tissue was then removed,
washed and immediately assayed for superoxide generation as described earlier.
Tissue cores were also tested for superoxide generation following exposure to exogenously generated fragments from cell
walls or pectin extract. 50l of 1% (w/v) aqueous suspension of purified tuber cell walls (see below) or 50l of 5% (w/v)
citrus peel pectin in 200l 50mM sodium phosphate buffer pH 7.8 were treated either with 5units of polygalacturonase,
CSG 15 (1/00)
7
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
5l pectinase (Pectinex 3X L, Novozyme/Sigma-Aldrich, UK) or a superoxide-generation solution. Superoxide generation
was initiated by addition of 50l 2.8M phenazine methosulphate to 25l 196M NADH, 0.2M EDTA in 50mM sodium
phosphate buffer pH7.8; superoxide generation lasted around 20 minutes. Mixtures were incubated for 30 minutes at
room temperature before centrifugation (15000g, 10minutes, 16c) and retention of the supernatants.
Cell walls were prepared from tubers of the varieties Cara and Russet Burbank (RB). 100g of tuber cortex (stolon
end) was grated and extensively homogenised in 100ml of 2mM dithiothreitol, using a mortar and pestle. The
homogenate was filtered through muslin and the cell walls, retained in the muslin, recovered and re-homogenised until
most of the starch grains were removed. The cell walls were then purified from residual starch by centrifugation onto a
cushion of 100% Percoll (Pharmacia Life Sciences) at 2000g, 5min, 10 oC. The starch grains passed through the Percoll
cushion and the cell walls collected at the interface. These were judged pure by fluorescence microscopy using calcofluor
stain and by the absence of starch grains.
C. Results
Hardware development
The figures shown below illustrate the first prototype of the impactor (figure 2) and the corer (figure 3). The impactor is
largely made from machined aluminium and stainless steel with a precision steel spring driving a steel impact piston
calculated to give an energy of impact of 0.7J. The corer design was based on a simple cork borer with a steel blade
inserted centrally within the tube. In use the sharpened end of the tube is driven into the tuber to a fixed depth and is then
rotated. The inserted blade causes the tuber core to shear off at the base and the tissue sample is retained within the corer
when it is withdrawn. The tissue sample is then ejected, using a separate plunger, into the test solution. This action
effectively produces a core of the required dimensions divided into two halves. An increased surface area of cells for
testing makes the assay more rapid / sensitive. Other results have confirmed that the assay determines radical generation
in the outer cell layers only not the whole volume of tissue ie. radical levels are proportional to the surface area rather than
the volume. Experience in using the first prototypes has led to design changes and improvements as indicated in figures 4
and 5 showing diagrams of second prototype devices – engineering drawings for these are held by Dr Croy.
Plant materials
Of the variables used in year 1 field trial to alter the bruise susceptibility of individual cultivars, only the application of
potassium fertilizer had any significant effect – growth of the selected cultivars on the Gleadthorpe site without added
potassium fertilizer gave tubers displaying higher levels of bruise susceptibility; application of potassium fertilizer caused
a marked decrease in bruise susceptibility in all 5 cultivars tested (See interim report for 2002). As a result in the second
field trials ± potassium fertilizer was the only parameter used to provide materials with a range of susceptibilities. The
number of cultivars grown for testing was increased to 8 (table 1).
Tuber mechanical stress
In optimising the assay conditions it was essential to check the variation in response between susceptible and resistant
varieties to a range of different impact energies. This would be important for fixing the standard impact for the test kit.
Tissue explants excised from impact sites on tubers were shown to respond to mechanical stress by generating superoxide
radicals. When the energy of impact on tuber tissues from a mechanically sensitive line (cv. Russet Burbank) was varied
the resulting maximum level of superoxide production also varied (figure 6). An impact energy as low as 0.1J was
sufficient to initiate detectable levels of radical generation and this increased linearly with increasing energies up to 0.7J.
Above 0.7J did not result in a further linear increase but rather reached a plateau at maximal superoxide production. In
contrast, a more mechanically resistant line (cv. Cara) was comparatively insensitive to the energy of impact. All energies
tested induced only a small, constant superoxide production above background level, amounting to about 10% of the
maximal response of the susceptible line. Thus 0.7J was selected as the standard energy of impact for all subsequent
experiments. It was clear from these results that the level of superoxide generation is dependent upon the
magnitude of the impact energy.
CSG 15 (1/00)
8
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Hardware - first prototypes
figure 2: First prototype impactor– used to deliver a standard impact to the test potato tuber
figure 3: First prototype corer – used to excise a standard tissue sample from test tubers. Figures
show the corer in use.
CSG 15 (1/00)
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Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
Designs for second prototypes of hardware devices
figure 4: Impactor –prototype 2
Anodised Barrel
plastic
handle
figure 5: Corer –prototype 2
core release
ergonomic grip
core depth control
CSG 15 (1/00)
10
DEFRA
project code
HP0217
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Figure 6
Tuber mechanical stress - differential response of bruise
susceptible and resistant tuber varieties to variable mechanical impact as
indicated by assays for radical generation. Superoxide generation by tuber cells
exposed to increasing mechanical stress. Tubers of two different potato lines with
differing mechanical susceptibilities, were exposed to impact energies ranging
between 0.1 and 1.4 joules and the resulting generation of superoxide above
background levels measured as described in the Materials and Methods susceptible cultivar Russet Burbank () and resistant cultivar Cara (). The data
plotted are the results of duplicate estimates (two samples from the same tuber)
from two independent experiments. Error bars are SD, where not visible are
hidden by symbols.
Assay of superoxide radical generation in tuber tissues
Tissue explants excised from impact sites on tubers were shown to respond to mechanical stress by generating superoxide
radicals (figure 7). The magnitude of superoxide generation was found to be highly variable between different genetic lines
(Data not presented here; see Johnson et al. 2003 for further details). Tissues from those varieties highly susceptible to
mechanical stress (cv Russet Burbank and Saturna) generated much higher levels of superoxide compared to the more
resistant varieties (cv King Edward and Maris Piper). The kinetics of radical synthesis were non-linear with time. Radical
generation above background levels was first detectable in the most susceptible lines within about an hour post-impact.
Intriguingly, in all the varieties tested, superoxide production occurred in a distinctive and reproducible two-phase
pattern. In the first phase superoxide generation reached a peak 1-2 hours after impact. This was followed by a second
phase with maximal generation 4-5 hours after impact. The response of cv. Saturna (figure 7) was typical of the biphasic
responses by susceptible lines and invariably the second peak was of greater intensity than the first. Both peaks were
reduced in magnitude with increasing varietal resistance to mechanical stress while in the most resistant varieties the first
peak was barely discernable above background, control levels. No significant increase in superoxide generation was seen
in control tissues sampled distal to the point of impact suggesting that the oxidative burst was specifically a localised
response to mechanical stress. When the maximal levels of superoxide generation were quantitatively compared (figure
8) to the bruise indices in the different genetic lines a high degree of correlation was observed (Pearson correlation
coefficient R2 = 0.9386) indicating that the two events are closely related and potentially are causally linked.
CSG 15 (1/00)
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Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
Figure 7 Superoxide radical generation
in tuber tissues - 1. The biphasic mode of
radical generation in a mechanically susceptible
variety of potato following a mechanical impact.
Peaks of superoxide generation are evident at
approximately 2h and 4h post-impact. The
results for cv Saturna are shown here.
DEFRA
project code
HP0217
Figure 8 Superoxide radical generation in
tuber tissues – 2. Correlation graph showing
the
direct
relationship
between
bruise
susceptibility of tuber samples with the ability to
generate free radicals. The Pearson R2 value of
0.9386 (radical generation) indicates a strong
correlation with response to mechanical stress.
Assay of superoxide radical generation in tuber tissues using alternative tetrazolium
derivatives (Dojindo, Japan)
A range of alternative tetrazolium dyes have been tested for their ability to detect superoxide radicals (Table 2). Using a
system in which superoxide generation was accurately quantified it was possible to assess the efficiency with which
tetrazolium dyes could detect the radicals.
Table 2: Performance of alternative tetrazolium derivatives
Tetrazolium
% efficiency of superoxide
detection
Tetrazolium Red
MTS
MTT
NBT
XTT
28
30
34
55
94
As a consequence XTT was determined to be the only tetrazolium dye worthy of further investigation. Subsequently a new
range of Water Soluble Tetrazolium dyes (WST) became available. Two of the new WST compounds were kindly supplied
by the manufacturer – Dojindo. Preliminary tests indicated these to have 85-90% efficiency of detection. However, this
slight decrease in sensitivity was compensated by a considerable improvement in chemical stability. These new
derivatives warrant further investigation.
Response of tuber cells to extracts from mechanically stressed tissues and cleavage
products of cell wall materials
A key finding of this work was that superoxide generation, identical to that seen in mechanically impacted tissue, could be
induced in non-impacted tissues by exposure to cell wall extracts from impacted tissue. Thus, in this way, a purified cell
wall extract from impacted Russet Burbank tubers could induce a full oxidative burst in non-impacted Maris Piper tubers
(a resistant variety)(figure 9). This is a key result as it indicates that the differential oxidative responses observed between
resistant and susceptible varieties is due to a difference in perception of the mechanical impact and not a difference in
capacity to generate radicals.
CSG 15 (1/00)
12
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
B
4
3
2
1
6
-1
5
4
-1
5
-1
-1
Superoxide generation
6
(nmol g min )
Superoxide generation
A
(nmol g min )
Project
title
0
3
2
1
0
0
1
2
3
4
0
Time (hours)
1
2
3
4
Time (hours)
Figure 9 - Response of tuber cells to extracts from mechanically stressed tissues and
cleavage products of cell wall materials. Elicitation of superoxide generation by cell-free extracts
from impacted tuber tissues. A) exposure of a non-impacted bruise resistant cultivar (cv. Cara ) and a
non-impacted bruise susceptible cultivar (cv. Russet Burbank) to cell free extracts of the impacted
bruise susceptible cultivar showing the generation of a single burst of superoxide generation after 1-2 h.
B) Conversely where the two cultivars are exposed to a crude extract prepared from a bruise resistant
cultivar (cv. Cara ) no comparable superoxide generation results. Abbreviations used are RB – cv.
Russet Burbank, CA – cv. Cara. Data are the mean values for two replicates.
Exposure of tubers to
inhibitors and free radical
scavengers
80
60
40
Superoxide (NI)
DPI+Superoxide
Catalase
SOD
0
DPI
20
Control
Percentage of Control
100
Treatment
Figure 10 Exposure of tubers to inhibitors and free radical
scavengers. Histogram showing the effects of inhibitors of oxidative
reactions on the generation of oxidatively modified proteins in
mechanically impacted tubers of cv. Russet Burbank. Inhibitors used
were diphenylene iodonium chloride (DPI), superoxide dismutase
(SOD), or catalase. Confirmation of alterations to AOS generation
were by analysing secondary carbonyl accumulation in each of the
three treatments (data not shown). The results were expressed as
percentage of mean control value and data plotted are duplicates (two
samples from the same tuber) from four independent experiments.
Error bars are SD, where not visible are hidden by symbols.
CSG 15 (1/00)
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To identify specifically which of the
family of oxygen free radicals were
implicated in pigment synthesis a range
of experiments were undertaken using
free radical scavengers and enzyme
inhibitors.
The use of diphenylene
iodonium chloride (DPI) indicated that
by inhibiting NADPH Oxidase – one of
the key enzymes proposed to be involved
in superoxide generation, a high level of
inhibition of pigment synthesis was
achieved. Superoxide dismutase (SOD)
and Catalase (Cat) are both free radical
scavenging enzymes – SOD removes
superoxide radicals, and Cat removes
hydrogen peroxide. Interestingly, even
though these compounds are closely
related chemically, only alteration in
superoxide levels (as indicated through
SOD activity) affected pigment synthesis
significantly (figure 10), implicating
superoxide and not hydrogen peroxide in
the pigment synthesis process.
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Estimation of protein modification in tuber tissues
As well as direct effects upon the cell components, oxygen free radicals, especially superoxide and peroxyl radicals
(derivatives from superoxide) have a deleterious effect upon proteins. Typically amino acids are modified through
exposure to superoxide, with lysine and histidine residues being particularly susceptible. The modifications are
characterised by the carbonylation of amino acid residues. Such secondary carbonyl modifications can be detected by
derivitisation with dinitrophenylhydrazine (DNPH) in concentrated triflouroacetic acid. Initial studies with antibodies to
the DNPH moiety and electrophoretic analyses were successful in showing substantial differences between impacted and
non-impacted tissues, however a switch to a spectrophotometric method permitted quantification of oxidative damage.
When level of oxidative damage was plotted against bruise susceptibility of a particular variety an extremely tight
correlation was observed (R2=0.945), which is the highest correlation of any factor with blackspot susceptibility at the
time of writing (figure 11).
15
4
10
3
2
5
1
0
2.5
5.0
Protein carbonyl content
(nmol. mg-1. protein)
Superoxide generation
(nmol. g-1. min-1)
5
0
10.0
7.5
Bruise index
Figure 11 - Relationship between levels of tuber protein oxidative
modification (carbonyl content) () and tuber susceptibility to mechanical
stress (bruise indices). The Pearson R2 value of 0.9448 for the protein carbonyl
content indicates a strong correlation with reponse to mechanical stress. Shown
alongside for comparison are the data for superoxide radical generation () reproduced
from figure 6. The cultivars used to establish these correlations were Russet Burbank ;
Saturna ; Cara ; King Edward and Maris Piper. Data plotted are the means of three
replicates.
D. Discussion of project findings
We describe here the novel detection of superoxide radicals generated in potato tuber cells in response to
mechanical stress and particularly differences in response between mechanically resistant and susceptible varieties.
Detection is based on immersion of tissue explants from test tubers directly into a tetrazolium dye solution which
sequesters the radicals immediately they are synthesized. As a result the assay provides a measure of generation potential
rather than levels of accumulation. Due to the high reactivity of the free radicals and the low diffusion rate in the tuber
apoplast we believe that the observed results represent the responses of only a few cell layers near the surface of the tissue
cube. Assays in which the explants were bisected showed that radical production approximates more to the surface area
rather than the volume of tissue.
We have used this assay to follow in detail the time course and factors affecting the level of radical generation in
potato tuber cells following impact. Cells in tuber tissues exposed to mechanical stress respond with a rapid production of
superoxide radicals. Susceptible potato varieties such as cv. Russet Burbank display a large burst of radical synthesis
while more resistant varieties such as cv. Cara show relatively low levels of radical production. Depending on the variety
used the response is proportional to the level of stress (impact energy) imparted up to a maximum level (figure 6). This
CSG 15 (1/00)
14
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
indicates that the perception of the stress and the signal transduction to the superoxide synthesis complex are
quantitatively linked but there is an upper limit defined by other factors such as cell wall strength.
The level of oxidative burst is genetically highly variable since different potato varieties show diverse responses
and quantities of superoxide generated (figure 8). We have followed superoxide generation over several hours following
exposure to a standard impact. The phenomenon was most evident in Russet Burbank and Saturna, cultivars which also
exhibit the highest susceptibility to mechanical stress i.e. display the highest bruise indices (table 1, figure 7, Johnson et
al. 2003). A novel feature of the superoxide synthesis was the biphasic pattern of generation with peak levels detectable at
1-2 hours and 4-5 hours post-impact (figure 7). This pattern is reminiscent of the response reported by Baker and Orlandi
(1995) in which cultured plant cells treated with a pathogen-derived elicitor displayed two peaks of hydrogen peroxide
generation over a period of 6 hours. Phase 1 was suggested to be a non-specific biological response to stress while phase 2
was determined to be a specific interaction between the pathogen hrp complex and the plant receptors leading to
hypersensitive cell death. In the present tuber system we see an equivalent initial peak which probably corresponds to a
response initiated by the shock wave passing through the cells causing a perturbation of the cell organisation and ion
balance or activation of a membrane-associated 'stretch receptor' as proposed by Cazalé et al. (1999). The second peak is
potentially of much greater interest. The observation that a second burst arises in mechanically stressed tissues in the
absence of pathogens or pathogen-derived elicitors suggests activation via a cell receptor which further induces or
amplifies the cells responses to the mechanical stimuli. Other studies have demonstrated AOS production in response to
mechanical stress. Yahraus et al. (1995) exerted mechanical stress in soybean cell suspension cultures by hypo-osmotic
media or by exerting physical pressure on cells under a microscope slide and were able to demonstrate peroxide
production histochemically after a few minutes. Legendre et al. (1993) and Cazalé et al. (1998, 1999) used physical mixing
of cell suspensions to impose mechanical stress and showed an oxidative burst of peroxide synthesis. However these
studies did not follow the longer time course described in the present study and only measured hydrogen peroxide levels
rather than superoxide radicals.
The observation that those varieties exhibiting a large oxidative burst were also the ones showing high mechanical
susceptibility as judged by high levels of melanin synthesis, posed the question as to the relationship between these two
processes. The correlation data presented in figure 8 indicates that there is a direct and tight quantitative relationship
between the degree of mechanical susceptibility of a variety and the level of superoxide generated by its cells on impact.
Experiments designed to remove superoxide radicals or inhibit superoxide generation also demonstrated that this radical
was directly required for generation of melanin in response to mechanical stress (figure 10). Use of nitroblue tetrazolium
as a histochemical stain for superoxide radicals indicated that the oxidative burst was spatially restricted to the impact
zone and coincident with the region of melanin pigment synthesis (data not presented). Melanin and related complexes
are known to be free radical scavengers and melanin radicals have been demonstrated in animal pigments under
conditions which elevate free radicals (Qu et al. 2000), so it seems plausible that radical production and melanin
synthesis may be causally related. Furthermore, work by Valverde et al. 1996 in mouse melanoma cells has provided
direct evidence that PPO, the key enzyme involved in melanin pigment synthesis, may preferentially utilise superoxide
radicals as a co-substrate rather than any other form of molecular oxygen. In this way a high level of superoxide radicals
generated in a susceptible variety would provide an excess of a preferred co-substrate for PPO. One of the consequences of
radical production is the oxidative modification of proteins in which carbonyl groups are introduced into protein side
groups through modification of lysine, proline and arginine residues (Stadtman 1993). Such protein modifications are
well documented in animal systems but the present report is the first demonstration of the effect occurring as an outcome
of mechanical stress in plants. Since oxidative modification of tuber proteins arises through interaction with the oxygen
free radicals it is not unexpected that levels of protein modification produced a similar direct relationship with the bruise
indices (Figure 11). This result also provides independent corroboration of the levels of radical production.
A large oxidative burst was inducible in both susceptible and resistant varieties by exposure of non-impacted
tuber cells to cell-free extracts from an impacted susceptible variety (figure 9). In contrast, an extract from an impacted
resistant variety was unable to induce superoxide generation in non-impacted cells of either a resistant or a susceptible
variety. These results implicate a factor produced endogenously in impacted susceptible tuber cells but which is absent
from impacted resistant cells. Since the factor can elicit a large oxidative burst in both susceptible and resistant varieties
this indicates that both are equally receptive to its presence and respond to the same extent. However the resistant variety
is incapable or does not produce the factor on impact. Exposure to the factor initiated a single burst of superoxide
generation of comparable magnitude to the second peak observed in the biphasic response to impact. This suggests that
the second phase arises by the production of the fragment in vivo.
We extended these experiments further in an effort to try to elucidate the nature of this factor (Johnson et al.,
2003). Pectinase and superoxide radicals produce fragments from both varieties which can elicit a superoxide burst.
Significantly, fragments from the resistant variety, Cara, were able to induce a large oxidative burst in Cara tissues. This
confirms that Cara can, but does not, produce the fragment in response to impact and precludes the inability to produce
the fragment as suggested earlier (figure 9). Evidence from 'cross activation' experiments indicated that the fragments
generated in susceptible and resistant varieties are most probably the same since Russet Burbank generated fragments
elicit a response in Cara and vice versa. Clearly the factor is associated with cell walls. Treatment of purified cell walls by
pectinase or superoxide action produced fragments causing elevated superoxide synthesis in non-impacted tissues of both
varieties.
CSG 15 (1/00)
15
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
The conclusion from the complex set of results shown in figure 6G of Johnson et al., (2003) suggest that the most
likely identity of the factor is a pectin-derived fragment (results not presented here). As suggested by other workers, active
pectic fragments can be generated in the absence of enzymatic action by free radical oxidative scission. Fry, (1998)
suggested that scission of plant cell wall polysaccharides by hydroxyl radicals could yield biologically active fragments.
These may act as ligands in the activation of a plasma membrane receptor which initiates a signalling cascade leading to a
large oxidative burst. Our results support this contention and furthermore provide an explanation for the observed
biphasic superoxide synthesis. Since superoxide radicals rapidly dismutate to other AOS including hydroxyl radicals the
model proposed here could provide an in vivo example of the process of radical scission described by Fry and others
(Fry,1998: Fry et al. 2001: Miller and Fry, 2001).
E. Conclusions and practical recommendations (See also section H)
The biphasic superoxide generation described here provides evidence of two possible receptor functions - a
mechano-receptor which detects the transient shock-wave due to mechanical impact and induces an initial burst of
superoxide synthesis into the apoplast of the tuber tissue. As a result of radical oxidative scission of cell wall pectin,
biologically active pectic fragments are produced. Secondly, an elicitor-type receptor detects these active fragments and is
activated leading to initiation of a signalling cascade which causes the second, larger burst of superoxide synthesis. The
second phase of superoxide synthesis represents a significant amplification of the original response and suggests that in
other stress situations a similar amplification may operate. Whether the elicitor-type receptor proposed here is unique or
is the same as pathogen-type receptors remains to be elucidated.
The genetic variation observed between mechanically susceptible and resistant potato varieties appears to be
largely based on the ability or inability of tuber cells to initiate the first synthesis of superoxide. It is clear from the
present work that there are no compositional differences between the cell wall polysaccharides which precludes the
generation of the active fragments. Nor is there significant variation in the level of receptor which initiates superoxide
generation. Importantly this does not preclude variation in cell wall protein or polysaccharide composition which may
influence the physical strength of the cell walls and determine its level of mechanical resistance to impact. The
amplification of the initial superoxide burst in susceptible potato varieties leads not only to the elevated synthesis of
melanin pigmentation but also to the manifestation of other deleterious effects including protein modification, membrane
disruption and cell death (Croy et al. 1998, Laerke et al. 2000, Partington et al. 1999). The genetic basis of the variation in
initial response remains to be elucidated but would ideally provide the basis for a fundamental investigation of gene
expression during tuber growth and development which correlates with the manifestation of mechanical susceptibility or
resistance.
The results from this research programme have confirmed the concept that varieties generate levels of superoxide
radicals directly proportional to their susceptibility to bruising. Thus resistant varieties such as Cara and Maris piper
show little or no free radical generation while susceptible varieties such as Russet Burbank and Saturna generate levels of
radicals directly proportional to the level of susceptibility (See figure 11). The relationship holds for every variety and field
conditions tested over at least 3 growing seasons. The correlation was even maintained in crops which suffered a large
scab infection during the 2002 – 2003 growing season leading to badly blemished skin. This was a problem since several
of the varieties selected specifically to provide a particular bruise index were also varieties highly susceptible to scab under
the soil conditions. The disease is characterised by a surface blemishes of altered, spongier texture which does not
transmit the impact energy as effectively as in normal tuber tissue. In such material care was required to select areas of
the tuber skin devoid of scab for impacting. The worst scab-affected varieties could not be impacted due to the extensive
surface damage. The whole of the tuber material from the 2nd field trial was adversely affected by the disease and other
factors, such that the bruise indices were increased (table 1). Despite these problems the cultivars which could be tested
with the radical generation assay still adhered to the correlation showing accurate predictability of their actual bruise
status (table 3).
The aim of the project has been achieved and we are in a good position to continue work on the hardware and the
chemistry needed for the commercial development of a field kit for the rapid determination of bruise susceptibility. The
programme has also produced some interesting and novel observations which with further fundamental investigation
could provide improved diagnostic tools in the future.
CSG 15 (1/00)
16
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Table 3: Results of ‘Blind’ testing of tuber materials from 2nd field trials (2002-3)
Rank
Variety
K+
1
Cara
2
Navan
3
Maris
Piper
Pentland
Crown
Russet
Burbank

X

X

X

X

X
4
5
Predicted
Actual
susceptibility susceptibility
5.18
5.20
7.40
7.29
5.17
5.55
7.60
7.83
5.44
5.79
7.51
7.66
5.32
6.12
6.50
6.20
7.22
7.37
8.66
8.28
∆
-0.02
+0.11
-0.38
-0.23
-0.35
-0.15
-0.80
+0.30
-0.15
+0.38
%
accuracy
99
98
93
97
94
98
87
95
98
95
F. References
Able AJ, Guest DI, Sutherland, MW (1998) Use of a new tetrazolium-based assay to study the production of
superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of Phytophthora parasitica var
nicotianae. Plant Physiol 117: 491-499
Adams S, Green P, Claxton R, Simcox S, Williams MV, Walsh K, Leeuwenburgh C (2001) Reactive
carbonyl formation by oxidative and non-oxidative pathways. Frontiers Biosci 6: a17-24
Anonymous (1994) Factsheet 1. Potato Marketing Board, Cowley, UK
Antosiewicz DM, Polisensky DH, Braam J (1995) Cellular-localization of the Ca2+ binding TCH3 protein of
arabidopsis. Plant Journal 8: 623-636
Bachem CWB, Speckmann G-J, van der Linde PCG, Verheggen FTM, Hunt MD, Steffens JC, Zabeau M
(1994) Antisense expression of polyphenol oxidase genes inhibits enzymatic browning in potato tubers. Bio-Tech 12:
1101-1105
Baker GJ, Orlandi EW (1995) Active oxygen in plant pathogenesis. Annu Rev Phytopathol 33: 299-321
Bolwell GP, Butt VS, Davies DR, Zimmerlin A (1995) The origin of the oxidative burst in plants. Free Rad Res
23: 517-532
Bolwell GP, Wojtaszek P (1997) Mechanisms for the generation of reactive oxygen species in plant defence – a
broad perspective. Physiol Mol Plant Pathol 51: 347-366
Braam J, Davis RW (1990) Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes
in Arabidopsis. Cell 60: 357-364
Brisson LF, Tenhaken R, Lamb C (1994) Function of oxidative cross-linking of cell wall structural proteins in
plant disease resistance. Plant Cell 6: 1703-1712
British Potato Council (2000) in The British Seed Potato Variety Handbook pp172-174, British Potato Council.
Cazalé AC, Rouet-Mayer MA, Heberle-Bors H, Barbier-Brygoo, Mathieu Y, Laurière C (1998) Oxidative
burst and hypoosmotic stress in tobacco cell suspensions Plant Physiol 116: 659-669
Cazalé AC, Droillard MJ, Wilson C, Heberle-Bors E, Barbier-Brygoo H, Laurière C (1999) MAP kinase
activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response? Plant J 19: 297307
CSG 15 (1/00)
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Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Coetzer C, Corsini D, Love S, Pavek J, Tumer N (2001) Control of enzymatic browning in potato (Solanum
tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase. J Agric Food Chem 49 652-657
Corsini DL, Pavek JJ, Dean B (1992) Differences in free and protein-bound tyrosine among potato genotypes and
the relationship to internal blackspot resistance. Am Pot J 69: 423-435
Croy RRD, Baxter R, Deakin W, Edwards R, Gatehouse JA, Gates P, Harris N, Hole C, Johnson SM,
Raemaekers R (1998) Blackspot bruising in potatoes: structural and molecular approaches to the identification of
factors associated with tuber bruising susceptibility. Asp App Biol 52: 207-214
Dixon TJ (1992) in Potato Varieties, published by Potato Marketing Board and National Institute of Agricultural
Botany, Wooster P (Ed), (ISBN 0903623307), Performance Characters, 156-163
Doke N (1995) NADPH-dependent O2- generation in membrane fractions isolated from wounded potato tubers
inoculated with Phytophthora infestans. Physiol Plant Pathol 27: 311-322
Elliott KA, Shirsat A (1998) Promoter regions of the extA extensin gene from Brassica napus control activation in
response to wounding and tensile stress. Plant Molecular Biology 37: 675–687
Friedman M (1997) Chemistry, biochemistry, and dietary role of potato polyphenols. A review. J Agric Food Chem
45: 1523-1540
Fry SC (1998) Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals. Biochem J
332:443-453
Fry SC, Dumville, JC, Miller, JG (2001) Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro
and in the cell walls of ripening pear fruit. Biochem J 357, 729-737
Hudson DE (1975) The relationship of cell size, intercellular space, and specific gravity to bruise depth in potatoes.
Am Pot J 52: 9-14
Jaffe MJ (1973) Thigmomorphogenesis: the response of plant growth and development to mechanical stimulation.
Planta 114: 143-157
Jaffe MJ, Forbes, S (1993) Thigmomorphogenesis: the effect of mechanical perturbation on plants. Plant Growth
Regul 12: 313-324
Jaffe MJ, Huberman M, Johnson J, Telewski FW (1985) Thigmomorpho-genesis: the induction of callose
formation and ethylene evolution by mechanical perturbation in bean stems. Physiol Plant 64: 271–279
Kleinschmidt G, Thornton M (1991) Bulletin 725, University of Idaho Cooperative Extension System, Moscow,
USA
Krohn BM, Hollier AA, Darchuk S, Stark DM (1998) Improving potato varieties through biotechnology. Asp
App Biol 52: 239-254
Laerke, PE, Brierley, ER, Cobb, AH (2000) Impact-induced blackspots and membrane deterioration in potato
(Solanum tuberosum L) tubers. J.Sci.Food Agric. 80: 1332-1338
Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol
48: 251-275
Legendre L, Rueter S, Heinstein PF, Low PS (1993) Characterisation of the oligogalacturonide-induced
oxidative burst in cultured soybean (Glycine max) cell. Plant Physiol 102: 233-240
Levine RL, Williams JA, Stadtman ER, Shacter E (1994) Carbonyl assays for determination of oxidatively
modified proteins. Methods Enzymol 233: 346-357
Low PS, Merida JR (1996) The oxidative burst in plant defense: Function and signal transduction. Physiol Plant
96: 533-542
Mehdy MC, Sharma YK, Sathasivan K, Bays NW (1996) The role of activated oxygen species in plant disease
resistance. Physiol Plant 98: 365-374
CSG 15 (1/00)
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Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Miller, JG, Fry, SC (2001) Characteristics of xyloglucan after attack by hydroxyl radicals. Carbohydrate Res 332,
389-403
Partington, JC, Smith, C, Bolwell, GP (1999) Changes in the location of polyphenol oxidase in potato (Solanum
tuberosum L.) tuber during cell death in response to impact injury: comparison with wound tissue. Planta 207: 449460
O’Leary AG, Iritani WM (1969) Potato bruise detection. Am Pot J 46: 352-35Qu X, Kirschenbaum LJ, Borish
ET (2000) Hydroxyterephthalate as a fluorescent probe for hydroxyl radicals: application to hair melanin. Photochem
Photobiol 71: 307-313Rastovski AA van es. (1987) Storage of potatoes: Post-harvest behaviour, store, design,
storage practice, handling. Pudoc, Wageningen, Netherlands pp163-170
Sieczka JB, Thornton RE (1993) Commercial Potato Production In North America, Potato Association of America,
Orono, Maine U.S.A.
Shirsat AH, Bell A, Spence J, Harris N (1996) The Brassica napus extA extensin gene is expressed in regions of
the plant subject to tensile stresses. Planta 199: 618–624 (1996).
Skrobacki A, Halderson JL, Pavek JJ, Corsini DL (1989) Determining potato tuber resistance to impact
damage. Am Pot J 66: 401-415
Stadtman ER (1993) Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metalcatalysed reactions. Annu Rev Biochem 62: 797-821
Stevens LH, Davelaar E (1996) Isolation and characterization of blackspot pigments from potato tubers.
Phytochemistry 42: 941-947
Stevens LH, Davelaar E (1997) Biochemical potential of potato tubers to synthesize blackspot pigments in relation
to their actual blackspot susceptibility. J Agric Food Chem 45: 4221-4226
Sutherland MW, Learmonth BA (1997) The tetrazolium dyes MTS and XTT provide new quantitative assays for
superoxide and superoxide dismutase. Free Rad Res 27: 283-289
Tenhaken R, Levine A, Brisson LF, Dixon RA, Lamb C (1995) Function of the oxidative burst in hypersensitive
disease resistance. Proc Natl Acad Sci USA 92: 4158-4163
Valverde P, Manning P, McNeil CJ, Thody AJ (1996) Activation of tyrosinase reduces the cytotoxic effects of the
superoxide anion in B16 mouse melnoma cells. Pigment Cell Res 9: 77-84
Wojtaszek P (1997) Oxidative burst: an early plant response to pathogen infection. Biochem J 322: 681-692
Yahraus T, Chandra S, Legendre L, Low PS (1995) Evidence for a mechanically induced oxidative burst. Plant
Physiol 109: 1259-1
G. Summary of technology transfer activities and project deliverables
CSG 15 (1/00)
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A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
Project
title
DEFRA
project code
HP0217
Details of the scientific papers, publications, exhibitions and conferences at which results of the
work from this programme have been presented
i.
Johnson, SM, Doherty, SJ and Croy, RRD (2003) Biphasic superoxide generation in potato tubers:
a self-amplifying response to stress. Plant Physiology (2003) 131, 1440–1449.
ii.
Johnson, SM and Croy, RRD (2001) A novel superoxide radical response to mechanical stress in
potato. Poster presented to Society for Free Radical Research – Oxidative Stress in Plants
conference – Nice, November 2001.
iii.
Hammond, T ‘Strategy for Success’ (2002), exhibition attended by the Prime Minister and the
Minister of Science and Technology, Lord Sainsbury and held at Samsung Exhibition Centre, 26th
July 2002.
iv.
Venture Capitalists Exhibition (2002) - North-East, entrepreneurs/investors – Newcastle-uponTyne, May 2002
v.
Eyewitness, August 2002 ‘Beating Bruising’
vi.
Johnson, SM and Croy, RRD (2002) A novel, fast and accurate test for blackspot susceptibility.
Poster presented at The Potato Storage Event 2002 – Sutton Bridge, Lincolnshire, May 2002
vii.
As a result of appearing at ‘The Potato Storage Event 2002’ the project was featured in issues of the
Crops Magazine, Potato Review, Farmers Weekly
viii.
Eyewitness, October 2001 ‘Bruising Research'
ix.
International Patent PCT/GB02/01631 - ‘Method and Apparatus for Detecting Response to Damage
and Diagnostic Method Therefor’
x.
UK Patent 0209229.4 - ‘Method and Apparatus for Identifying Gene Expression Relating to
Response to Damage and Diagnostic Method Therefor’
H. Project conclusions and priority areas for continued research
i.
Potato tuber tissues respond to mechanical damage by generating free radicals into the
extracellular spaces.
ii.
Free radical generation can be assayed in mechanically damaged tubers by excising a sample core of
the tissue and immersing it in a solution of a tetrazolium dye which sequesters the radicals
producing a colour change which is directly proportional to the amount of radicals generated.
iii.
Radical assays of mechanically damaged tubers of different potato varieties indicate differential
levels of radical generation.
iv.
Comparison of bruise susceptibilities of varieties with their differential generation of radicals has
revealed a high level of correlation. The correlation is the highest of any previously proposed
factors and gives >90% accuracy in predicting the bruise status of potato tubers.
v.
Optimisation of the radical assay could provide an accuarate field diagnostic kit for
vi.
The molecular basis for the correlation is unknown although subsequent evidence indicates that
superoxide radicals may be the preferred substrate for the enzyme polyphenol oxidase (PPO) which
plays a key role in the oxidative reactions leading to the conversion of monophenols, such as
tyrosine, to the bruise pigment melanin. PPO is widespread in plant tissues and is an important
factor in food quality due to the ‘browning’ reaction in cut or damaged tissues. Investigation of the
relationship could provide key information on the mechanism of action of this important enzyme
and prevention of tissue discolouration.
CSG 15 (1/00)
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Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
vii.
The differential basis of the generation of free radicals which directly affects the ability of tuber
tissues to synthesise the bruise pigments is similarly unknown. This is a key question for two
reasons: firstly elucidation of the mechanism whereby the tuber cells perceive and respond to
mechanical stress would indicate which factor(s) predispose the tissues to greater or lesser
susceptibility to mechanical damage – little is known about this aspect in plant tissues. Secondly it
should be possible to devise an early diagnostic test which would indicate susceptibility to
mechanical stress while the crop is developing in the field and even in tissues other than the tubers
themselves (eg. leaves).
viii.
A major priority for future research will be the development of the hardware and chemistry to
exploit the novel findings described in this report.
CSG 15 (1/00)
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A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
Project
title
DEFRA
project code
HP0217
Appendix 1
Draft Instruction Manual for Blackspot Protect
INSTRUCTION
MANUAL
(draft)
Enhancing quality through Science
Introduction
Blackspot Protect is a highly sensitive diagnostic system for the rapid and accurate assessment of
susceptibility to Blackspot Bruising in your potato crop. It is ideally suited for diagnostic tests on both preharvest and stored tubers.
Components









Blackspot Protect impactor
Blackspot Protect corer
Blackspot Protect colorimeter (exclusive to Blackspot Protect Plus)
indelible marker pen
10 x filtered syringe barrels, containing detection solution A
covered syringe holder
reaction tube, containing detection solution B
colour chart for Blackspot susceptibility
reaction cuvettes (exclusive to Blackspot Protect Plus)
Safety Precautions




the Blackspot Protect Section Corer contains sharp blades – handle with care.
the Blackspot Protect impactor should only be used for impacting potato tubers
solution A – avoid any contact with skin or eyes - in the event of contact wash with plenty of water
solution B – is corrosive, avoid any contact with skin or eyes - in the event of
contact wash with plenty of water and seek medical advice as soon as possible
Method
The method of detection of bruise susceptibility is quite simple. When a tuber impacts on a hard surface the tissues are damaged and
respond by generating a highly active factor. The level of this factor produced indicates how susceptible the tuber is to bruising - a
high level of factor indicates a high susceptibility to bruising while a low level of factor indicates resistance to bruising. We measure
the level using a dye which changes colour when it reacts with the factor and this colour change is measured to assess the bruise
CSG 15 (1/00)
22
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
susceptibility. The test is carried out by selecting samples of the potato crop directly from the field or from storage and exposing
these to a measured impact using the Impactor. The tuber tissue responds within two hours and then a standard sample of tissue
(core) is excised using the Corer, and placed into the dye solution. Following a 20 minute incubation period the solution is mixed
with a second solution which brings about a colour change. The depth of colour of the final solution is measured or compared by eye
with a colour chart and the corresponding bruise susceptibility indicated.
Test procedure: The detailed steps are as follows:
1. Select 10 tubers to be tested sampled at random from different parts of field. For pre-harvest testing after haulm
destruction, carefully hand harvest the tubers avoiding any impacts. Avoid any damaged or diseased tubers.
!! For a more representative assessment of the bruise susceptibility of the crop it is best to sample tubers from
plants growing at least 10 metres apart !!
2. Remove any surface soil from the stolon end of each tuber - it is acceptable to wash the tubers gently prior to testing.
3. Using the Blackspot Protect impactor, impose a single impact onto a smooth area of the stolon end of the tuber ( See
diagram below).
!! Do not impact directly onto the stolon !!
4. Mark the site of impact using the indelible marker pen.
5. After 2 hours use the Blackspot Protect Corer to take a sample of the impacted tuber.
6. Place each section in a filtered syringe barrel containing solution A.
7. Shake gently for 10 seconds and store in the dark in the covered holder.
8. After 20 minutes inject the contents of the syringe barrels into the attached reaction tube containing solution B - shake
briefly to mix.
9. Compare the colour of the solution against the colour chart provided to give an estimate of the bruise susceptibility of
your tested tubers – the darker the colour the more susceptible the crop.
10. For Blackspot Protect Plus users only – after step 8 the coloured solution can be read directly in the Blackspot Protect
colorimeter. The digital display will give a measure of the blackspot bruise susceptibility of the tested tuber. Calculate
the average value for the tuber samples tested and then consult the table of bruise values to estimate the bruise
susceptibility of the crop.
CSG 15 (1/00)
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Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
A) Identify the stolon end of the tuber
stolon end of tuber
B) Impacting a
sample tuber at the
stolon end but avoid
impacting at the
stolon itself.
C) Remove a small sample
of tuber tissue using the
corer
---- End ----
CSG 15 (1/00)
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Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Appendix 2
Prospects for the development of an electronic biosensor system
to detect free radicals in tuber tissues.
1. Biosensors are electronic devices comprising of a 'solid-state' detection system, usually a membrane or
conducting metal film, with specific detector molecules immobilised on the surface. When target
molecules are present in solution or gas surrounding the biosensor they interact with the detector
molecules causing a change in the electrical properties of the detection system. These changes are
amplified electronically and provide a measure of the concentration of target molecules in test solutions.
2. Biosensors have been developed for a range of substances and are widely used in situations where
continuous monitoring of the level of a target molecule is required. Applications include monitoring
pollutants in effluent treatment and rivers, and monitoring of levels of drugs or metabolites within body
fluids. In the latter situation miniaturisation of the design has provided micro-biosensors.
3. Construction of biosensors for the detection of free radicals has been achieved based on detector molecules
such as cytochrome C, superoxide dismutase. Currently these biosensors are under development for
medical diagnostic application where elevation of free radicals is diagnostic of trauma such as heart attack
and chromic disease.
4. All of these systems rely on the sensor being immersed in the solution in which the target radicals are
generated. Thus the free radicals will have ready access, via diffusion in free solution, to the detector
molecules and an equilibrium will be established between bound radical and free radical in solution. This
will change depending on changing levels of the radical in solution.
5. With regard to the detection of elevated free radical generation in potato tuber tissues. A major problem in
detecting free radicals is their high activity - they will react with almost any other molecule they make
contact with. As a result the half life of a free radical, particularly in a biological environment, is very short.
Detection of free radicals using chemical methods is successful because the tissue samples are bathed in the
detection solution and as soon as a radical molecule is generated it is sequestered by the detecting
chemicals.
6. Since free radicals are very reactive it is possible that the spatial separation between the site of generation
of the radical (cell surface) and the biosensor may be too great for accurate detection - the radicals react
with other chemicals before they can contact the biosensor surface. Furthermore it is questionable whether
the structure of the tissue into which the biosensor is inserted is sufficiently homogeneous to establish the
equilibrium situation required for accurate detection.
7. An alternative approach for consideration which gets around the potential problems described in 6. might
be the use of biosensors designed to detect the immediate products of radical dismutation such as
peroxides. Such chemicals although highly reactive are much more stable than free radicals and therefore
could survive long enough to be detected by the sensor.
Conclusions: The advantages of a detection system that relies entirely on solid state detection system,
does not require any 'wet chemistry', and which might be more rapid and accurate make the proposition of a
free radical sensor suited for bruise susceptibility detection an exciting prospect for future investigation.
The reservations discussed above can only be resolved by experimentation. It is proposed that discussions
with biosensor experts at the Universities of Durham, Newcastle and Glasgow, during the course of the
DEFRA - LINK programme would enable such experimentation and provide a measure of the feasibility of
the future development of such a detection system.
CSG 15 (1/00)
25
Project
title
A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE
PREDICTION OF TUBER SUSCEPTIBILITY TO
BLACKSPOT BRUISING IN THE FIELD
DEFRA
project code
HP0217
Appendix 3
Biphasic superoxide generation in potato tubers: a
self-amplifying response to stress.
Johnson, SM1, Doherty, SJ2 and Croy, RRD1
Crop Protection Group, School of Biological and Biomedical Sciences, University of Durham, South Road,
Durham DH1 3LE, United Kingdom
2 Avecia Life Science Molecules, Belasis Avenue,
Billingham, Teesside TS25 1TN, UK.
Plant Physiology (2003) 131, 1440–1449.
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