Annals of Botany 86: 929±934, 2000

doi:10.1006/anbo.2000.1255, available online at http://www.idealibrary.com on

Sun¯ower (Helianthus annuus) Leaves Contain Compounds that Reduce Nuclear

Propidium Iodide Fluorescence
H . J A M E S P R I C E , G E O R G E H O D N E T T and J . S P E N C E R J O H N S TO N
Departments of Soil & Crop Sciences and of Entomology, Texas A&M University, College Station, Texas 77843

Received: 25 April 2000 Returned for revision: 6 June 2000 Accepted: 7 July 2000 Published electronically: 30 August 2000

Sun¯ower leaves have unidenti®ed compounds that interfere with propidium iodide (PI) intercalation and/or
¯uorescence. Independently prepared pea leaf nuclei show greater PI ¯uorescence than nuclei from pea leaves
simultaneously processed (co-chopped) with sun¯ower leaves. Di€erences in ¯uorescence persist after mixing the
PI-stained pea and the co-chopped pea/sun¯ower samples, i.e. PI staining protects the nuclei from the e€ects of the
inhibitor. The current results are signi®cant to practical ¯ow cytometric determination of plant nuclear DNA content.
They show: (1) simultaneous processing of nuclear samples from the target and the standard species is necessary to
obtain reliable DNA estimates; (2) a test for the presence of inhibitors should be conducted; and (3) when inhibitors
are present caution should be taken in interpreting di€erences in estimated DNA content. The previously reported
environmentally-induced variation in DNA content in sun¯ower populations is most simply explained by variation in
the amount of environmentally-induced inhibitor that interferes with intercalation and/or ¯uorescence of PI.
Intraspeci®c variation of DNA content for Helianthus annuus needs to be re-evaluated using best practice techniques
comparing physiologically uniform tissues that are free of inhibitors. The best estimate for 2C DNA content of
H. annuus used in this study is 7.3 pg. # 2000 Annals of Botany Company

Key words: Helianthus annuus, DNA content, ¯ow cytometry, propidium iodide, endogenous inhibitors.

For several decades there has been considerable interest in
the roles of varying DNA content in plant adaptation and
evolution (Bennett and Smith, 1976; Price, 1976, 1988;
Bennett, 1998). Genome size varies by nearly an order of
magnitude among diploid congeneric species. DNA content
positively correlates with physical parameters such as
chromosome size, nuclear volume, and mitotic cycle time
(Price, 1976; Bennett, 1998).
There have been numerous reports of intraspeci®c
variation in genome size in plants (Bennett, 1985; Price,
1988). One notable example is Zea mays, where the observed
37 % variation in DNA content has been correlated with the
number and size of heterochromatic knobs (Laurie and
Bennett, 1985; Rayburn et al., 1985). Another well estab-
lished example is ¯ax, Linum usitatissimum (Durrant, 1962;
Evans, 1968), where increases and decreases in DNA
content over an approximate 15 % range can be environ-
mentally induced by varying amounts of phosphorous and
nitrogen in the fertilizer. In ¯ax, the variation in DNA
content results from di€erential ampli®cation of repetitive
sequences (Cullis, 1985; Cullis and Cleary, 1986).
Most reports of intraspeci®c DNA content variation have
not been independently con®rmed. This has led to ques-
tions concerning the frequency and magnitude of intra-
speci®c DNA content changes (Greilhuber, 1998). Since
there are both technical and biological sources of variation
that can lead to quantitative di€erences in binding and/or
¯uorescence of ¯uorochromes and in Feulgen staining
(Johnston et al., 1996; Greilhuber, 1998) artifactual
di€erences in DNA content may be reported and real
di€erences may go undetected.
0305-7364/00/110929+06 $35.00/00
Sun¯ower, Helianthus annuus, is an example of a species
in which intraspeci®c variation in DNA content determined
by Feulgen microspectrophotometry has been reported by
several di€erent laboratories. Nagl and Capesius (1976),
Olszewska and Osiecka (1983) and Cavillini et al. (1986,
1989) reported up to 60 % variation in DNA content
among H. annuus varieties. Cavillini et al. (1986, 1989) also
reported that the nuclear DNA content was higher in
sun¯ower seedlings grown from achenes from the periphery
of the in¯orescence and progressively decreased in achenes
positioned towards the centre of the head.
Well documented limitations to the acquisition of DNA
contents by Feulgen microspectrophotometry include
problems with standardization, chromatin compaction,
light absorbancy (Price et al., 1980; Price and Johnston,
1996a), and inhibitory e€ects of compounds such as
endogenous tannins (Grelhuber, 1986, 1988). In an attempt
to avoid the limitations of the Feulgen technique, Michael-
son et al. (1991b) initiated a study of DNA content
variation in H. annuus using laser ¯ow cytometry of
propidium iodide (PI) stained nuclei. Flow cytometric
estimates of DNA content variation among leaves and
among plants of the same sun¯ower variety resulted in
extensive variation from approx. 3±8.5 pg (Michaelson
et al., 1991b; Johnston et al., 1996; Price and Johnston,
1996a). This range in DNA content is similar to that
reported among sun¯ower plants from other laboratories
(Arumuganathan and Earle, 1991). Although the cyto-
metric technique of Michaelson et al. (1991b), Johnston
et al. (1996) and Price and Johnston (1996b) utilized a
# 2000 Annals of Botany Company
930 Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves
barley or pea standard, it was independently processed and
stained before being added to the sun¯ower samples.
Johnston et al. (1996) and Price and Johnston (1996b)
reported that PI-based ¯ow cytometrically detected DNA
content variation among sun¯ower plants in growth
chambers and a greenhouse correlated with the quality
and quantity of light under which the plants were grown.
These DNA content di€erences were also apparent from
microspectrophotometric measurements and image analysis
of Feulgen-stained nuclei (Johnston et al., 1996). At that
time it appeared that the DNA content of the sun¯ower
changed during development in response to light quality
and quantity. This hypothesis was further supported by the
measurement of nuclear DNA content of sun¯ower plants
of a natural population where the plants received various
proportions of direct sunlight and irradiance re¯ected from
neighbouring vegetation (Price et al., 1998). However,
factors independent of DNA content could lead to di€er-
ences in the ¯uorescence of PI-stained nuclei. Both
Greilhuber (1998) and Price et al. (1998) suggested that
the observed di€erences in PI ¯uorescence in sun¯ower may
be related to secondary plant products that interfere with
the intercalation and/or ¯uorescence of propidium iodide.
Here we report technical and physiological factors that
quantitatively in¯uence the outcome of ¯ow cytometry
using PI-stained sun¯ower nuclei. It is concluded that the
reported light-induced di€erences in mean PI ¯uorescence
among sun¯ower plants are due to varying levels of
environmentally-induced inhibitor(s) of PI intercalation
and/or ¯uorescence, and do not represent di€erences in
DNA content.
M AT E R I A L S A N D M E T H O D S
Plant materials
The H. annuus plants used in this study were from a wild
population located on the west side of Building 955 on the
Texas A&M campus, College Station, Texas. Pisum sativum
`Minerva Maple' leaves were from plants grown in a growth
chamber adjusted to a 16/8 h, 28/208C day/night cycle.
Laser ¯ow cytometry
Samples of nuclei for ¯ow cytometry were obtained from
leaves. Each sample was chopped with a new razor blade in
a modi®ed Galbraith et al. (1983) bu€er as described by
Price et al. (1998). The bu€er consisted of, per litre, 4.26 g
MgCl2 , 8.84 g sodium citrate, 4.2 g 3-[morpholino]propane
sulfonic acid, 1 ml Triton X-100, 1 mg boiled ribonuclease
A, pH 7.2. The resulting slurry was ®ltered through a 53 mm
nylon ®lter, and propidium iodide (PI) was added to a ®nal
concentration of 50 ppm. The stained samples were stored
in the dark on ice and analysed by ¯ow cytometry after 1 h.
The ¯ow cytometer was a Coulter Epics Elite (Coulter
Electronics, Hialeah FL) equipped with a water-cooled
laser tuned at 514 nm and 500 mW. Fluorescence emission
at 4615 nm was detected with a photomultiplier screened
by a long pass ®lter. O€set and linearity of the ¯ow
cytometer were checked with ¯uorescent check beads
(Coulter Electronics, Hialeah, FL) as recommended by
the manufacturer.
Test for inhibitors
Sun¯ower extracts were tested for unidenti®ed com-
pounds that reduce PI ¯uorescence of pea nuclei as follows.
Nuclei were released from an approximate 40 mm  20 mm
sun¯ower leaf and one-half of a pea leaf that were simul-
taneously processed (co-chopped) and stained with PI
(sample A). Sample B consisted of PI-stained nuclei from
the independently processed and stained other half of the
pea leaf used in sample A. After staining for 1 h, samples A
and B were individually measured for mean PI ¯uorescence,
after which they were mixed and measured up to 17 min, at
60 min, and at 120 min. The experiment was replicated
three times. Reduced ¯uorescence of nuclei from pea leaves
simultaneously processed with sun¯ower leaves, compared
to nuclei from independently processed pea leaves gave
evidence of sun¯ower inhibitors. A second similar exper-
iment was conducted to test the stability of PI ¯uorescence
of prestained pea nuclei after being added to a stained
nuclear sample from simultaneously processed sun¯ower
and pea leaves. Five replicates were measured for PI
¯uorescence during the ®rst 15 min after mixing and
again after 20 h.
Test for concentration e€ects of inhibitors
The e€ects of the concentration of sun¯ower extract on
PI ¯uorescence of pea nuclei were tested. Approximately
13 mm square segments were cut from sun¯ower and pea
leaves and processed separately in 3 ml chopping bu€er.
Four di€erent amounts of the unstained, processed sun-
¯ower sample (adjusted to 2 ml with bu€er) were added to
0.5 ml unstained pea sample to produce a 40-fold range of
sun¯ower/pea extract concentration. PI was added after the
samples were mixed and red ¯uorescence quanti®ed after
1 h. The experiment consisted of three replicates.
The e€ect of bu€er volume on PI ¯uorescence of nuclei from
simultaneously processed sun¯ower and pea leaves
An experiment was designed to test whether or not the
inhibitory e€ect of sun¯ower extracts on pea nuclei PI
¯uorescence could be diluted out. In this experiment,
approx. 13 mm square segments of sun¯ower and pea
leaves were simultaneously processed in 1.5, 15.0 and
30.0 ml bu€er. The samples were stained with PI and the
mean ¯uorescence of the sun¯ower and pea nuclei recorded.
This experiment represented a 20-fold dilution of sample. In
normal practice, 13 mm square leaf segments would
typically be processed in about 1.5±3.0 ml bu€er.
R E S U LT S
Compound(s) in sun¯ower leaves inhibit PI-¯uorescence
Helianthus annuus leaves contain substance(s) that inhibit
PI ¯uorescence of pea nuclei. This is apparent in the ¯ow
 Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves 931
T A B L E 1. Time course of the ratio of the mean (+s.d.) PI
¯uorescence of nuclei (pea 1) of pea leaves simultaneously
B processed and stained with sun¯ower leaves to the mean
50 ¯uorescence of added PI-stained stained nuclei ( pea 2) from
Number of nuclei

D
independently processed pea leaves
C
Time after mixing (min) Pea 1/Pea 2*
A
0±2.5 0.70 + 0.05
25
2.5±5.0 0.71 + 0.05
5.0±7.5 0.72 + 0.04
7.5±10.0 0.73 + 0.04
10.0±12.5 0.73 + 0.04
12.5±15.0 0.73 + 0.04
15.0±17.0 0.74 + 0.03
60 0.75 + 0.02
200 400 600 120 0.76 + 0.03
Relative PI fluorescence
* Each ratio is the mean of three replicates. No signi®cant di€erences
F I G . 1. Flow cytogram of ¯uorescence of PI-stained nuclei from (P ˆ 0.05) were observed among the means using a Duncan multiple
simultaneously processed sun¯ower (B) and pea (C) leaves to which PI- range test.
stained nuclei from independently processed pea (D) leaves were added.
PI ¯uorescence was measured 20 h after mixing the samples. Peak (A)
was produced by ¯uorescent calibration beads T A B L E 2. Ratio of mean (+s.d.) PI ¯uorescence of nuclei
( pea 1) of pea leaves simultaneously processed and stained
with sun¯ower leaves to the mean ¯uorescence of added
cytogram shown in Fig. 1. In this cytogram the lowest peak PI-stained nuclei (pea 2) from independently processed
(A) is produced by 0.6 mm 10 % ¯uorescence calibration leaves
beads (Molecular Probes, Eugene, Oregon). Peaks B and C
represent nuclei of sun¯ower and pea leaves that were Time after mixing Pea 1/Pea 2
simultaneously processed prior to PI staining. Peak D is
nuclei from independently processed and PI-stained pea 1.0±15.0 min 0.77 + 0.033
leaves that were added and measured after 20 h. The 20.0 h 0.77 + 0.036
di€erences between the two pea peaks, one inhibited (C)
and the other not inhibited (D) are relatively stable over Each ratio is the mean of ®ve replicates. Measurements were taken at
time. 1.0 to 15 min and at 20 h after mixing.
Table 1 presents a time course of the ratio of the mean
¯uorescence of nuclei of pea leaves, simultaneously E€ect of bu€er volume on PI ¯uorescence of nuclei from
processed and stained with sun¯ower leaves, to the mean simultaneously processed sun¯ower and pea leaves
¯uorescence of added PI-stained nuclei from independently
processed pea leaves. In the 2 h after mixing there was a Testing the e€ect of inhibitor dilution on PI ¯uorescence
progressive, but not signi®cant, increase in the ratio from involved simultaneously processing sun¯ower and pea
0.70 to 0.76 (Table 1). The second experiment showed no leaves in bu€er volumes ranging over a 20-fold range
di€erence in the ratio during the ®rst 15 min after mixing prior to PI staining (Table 4). In this experiment the ratio of
and after 20 h (Table 2). the means of sun¯ower and pea nuclei ¯uorescence
remained similar when leaves were processed in 1.5, 15
and 30 ml bu€er. The PI ¯uorescence of independently
processed pea nuclei decreased with increased volume of
Concentration e€ects of inhibitors chopping bu€er. The estimated DNA content of sun¯ower
The e€ect of concentration of inhibitor(s) was tested by nuclei (7.3±7.4 pg) from leaves processed with pea leaves in
processing same size pea leaf sections in varying concen- this experiment (Table 4) is much higher than the
trations (40-fold range) of bu€er (containing independently 4.5±5.3 pg in the experiment in which sun¯ower and pea
processed sun¯ower leaves) prior to PI staining. The PI leaves were independently processed and then mixed prior
¯uorescence of both sun¯ower and pea nuclei increased to staining (Table 3).
with decreasing concentration of sun¯ower extract
(Table 3). However, the ¯uorescence of pea nuclei in the
DISCUSSION
most dilute solution increased to only 89 % of that of the
control pea nuclei. The mean sun¯ower nuclei ¯uorescence, The current research provides evidence for the presence of
expressed as DNA content, relative to the ¯uorescence of inhibitor(s) of PI intercalation and/or ¯uorescence in
pea nuclei, was similar in the samples containing 0.05, 0.25 sun¯ower leaves. The quantity of inhibitor(s) is apparently
and 0.50 ml of sun¯ower slurry. The sample containing environmentally regulated and the inhibitory e€ect corre-
2 ml sun¯ower slurry had a signi®cantly lower estimated lates with light quantity and quality (Price and Johnston,
DNA content (Table 3). 1996b; Price et al., 1998). The mode of action of the
932 Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves
T A B L E 3. PI ¯uorescence of nuclei from mixtures of independently-chopped sun¯ower and pea leaf samples*

Mean PI ¯uorescence
Sun¯ower DNA
Composition{ Sun¯ower Pea Sun¯ower/pea ratio content ( pg){ Duncan's grouping}

2 ml sun¯ower 176.7 + 25.5 372.5 + 17.7 0.47 4.5 A

0.5 ml pea
0.5 ml sun¯ower 272.7 + 4.7 490.5 + 13.6 0.56 5.3 B
0.5 ml bu€er
0.5 ml pea
0.25 ml sun¯ower 296.7 + 3.7 538.5 + 12.5 0.55 5.3 B
1.75 ml bu€er
0.05 ml sun¯ower 332.3 + 4.1 615.4 + 14.3 0.54 5.2 B
1.95 ml bu€er
0.5 ml pea
0 ml sun¯ower 689.4 + 8.5
2.00 ml bu€er
0.5 ml pea

* All values are the means of three replicates +s.d.

{ Slurries containing the pea and sun¯ower nuclei were mixed before the propidium iodide was added.
{ The mean DNA content of the sun¯ower was calculated by the formula: DNA content ˆ (mean ¯uorescence sun¯ower/mean ¯uorescence
pea)(mean DNA content of the pea standard). The 2C DNA content of pea is 9.56 pg (Johnston et al., 1999).
} Multiple range test groupings (P ˆ 0.05). Means with the same letter are not signi®cantly di€erent.

T A B L E 4. Mean PI ¯uorescence of nuclei from pea and (Bharathan et al., 1994) have no measurable e€ect on
sun¯ower leaves simultaneously processed in di€erent reducing the inhibition. Greilhuber (1998) showed that
volumes of bu€er* tannins interfere with Feulgen staining. It is possible that
they could inhibit PI ¯uorescence as well. However, we have
Mean PI ¯uorescence Sun¯ower shown that soluble tannins isolated from sun¯ower leaves
Sun¯ower/ DNA content increase the variance of the peaks but do not inhibit PI
Bu€er volume Sun¯ower Pea pea ratio ( pg){ ¯uorescence of pea nuclei (Price, Hodnett and Johnston,
unpubl. res.). Therefore, tannins are apparently not among
Pea ‡ sun¯ower
1.5 ml 414 + 10 534 + 20 0.78 7.4 the compounds that interfere with PI ¯uorescence and/or
15 ml 407 + 37 532 + 45 0.77 7.3 intercalation.
30 ml 461 + 28 601 + 32 0.77 7.3 The presence of inhibitors compromises the reliability of
estimated DNA content, particularly if detection of small
Pea controls
1.5 ml 800 + 18 di€erences in DNA content is desired. Combined proces-
15 ml 719 + 31 sing of the target and standard species reduces potential
30 ml 707 + 20 variability and improves standardization (DolezÏel, 1991;
Baranyi and Greilhuber, 1995). This procedure may
* All values are means of ®ve replicates +s.d. minimize but not entirely eliminate the e€ect of inhibitors
{ The mean 2C DNA content of the sun¯ower was calculated by the on estimated DNA content. The practice of measuring
formula: DNA content ˆ (mean ¯uorescence sun¯ower/mean ¯uor-
escence pea)(mean DNA content of the pea standard). The 2C DNA mixed, independently-processed and stained nuclei from
content of pea is 9.56pg (Johnston et al., 1999). standard and target species, although not uncommon in the
literature (O'Brien et al., 1996; Price and Johnston, 1996b;
Rayburn et al., 1997; and others), may result in greatly
inhibitor(s) is unknown, but appears to involve either an exaggerated di€erences in ¯uorochrome ¯uorescence that
intercalation into the DNA of the nuclei or interaction with may not be due to di€erences in DNA amount. This is
the proteins of chromatin that inhibits the access of PI to apparent when comparing the large di€erences between
DNA. It is also possible that the inhibitor acts directly on estimated sun¯ower DNA content in Table 3 (4.5±5.3 pg)
the PI molecule and interferes with its ¯uorescence. This and Table 4 (7.3±7.4 pg). The values in Table 3 involved
hypothesis would require that intercalated PI be less mixing independently processed pea and sun¯ower samples
sensitive to the inhibitor than free PI. before staining with PI. The sun¯ower DNA contents of
The chemical nature of the inhibitor(s) of PI ¯uorescence Table 4 were calculated from nuclei of simultaneously
is not known but probably involves one or more of the processed and stained sun¯ower and pea leaves. Our best
numerous secondary metabolites produced by plants. Our estimate for the 2C nuclear DNA content of H. annuus used
preliminary experiments indicate that antioxidants such as in the current study is 7.3 pg.
polyvinylpyrrolidine, ascorbate, 2-mercaptoethanol, and Environmentally-induced DNA content di€erences such
dithiothreitol that are sometimes used in chopping bu€ers as those reported by Price and Johnston (1996b) and
 Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves
Price et al. (1998) are most simply explained as due to
environmentally-induced variation in the amount of
inhibitors that interfere with the intercalation and/or
¯uorescence of PI. Due to e€ects of endogenous inhibitors,
intraspeci®c variation in DNA content of H. annuus needs
to be re-evaluated by comparing physiologically uniform
tissues that are free of inhibitors and using best practice
¯ow cytometry procedures.
Flow cytometry of PI-stained nuclei is generally con-
sidered to be a reliable method for estimating plant DNA
content (Michaelson, 1991a; Price and Johnston, 1996a;
DolezÏel et al., 1998). However, the best practice techniques
for ¯ow cytometry are still being developed. For example,
Johnston et al. (1999) emphasized the importance of using
internationally accepted plant species as standards in the
determination of plant DNA contents. The results of the
current study are important to best practice ¯ow cytometry.
First, it is apparent that the simultaneous processing of
tissue of the target and standard species is absolutely
necessary to obtain reliable DNA content estimates. The
current study also establishes that compounds in sun¯ower
leaves greatly inhibit the PI ¯uorescence of the standard pea
nuclei. The proportion of plant species with natural inhibi-
tors of PI intercalation or ¯uorescence remains to be deter-
mined. A preliminary study detected inhibitors in four out
of ten randomly chosen angiosperm species (Bennett, pers.
comm.). It is likely that naturally occurring inhibitors that
decrease ¯uorochrome ¯uorescence of plant nuclei are com-
mon. Therefore, a test for the presence of inhibitors should
be used in all ¯ow cytometry studies. This can be done
simply by comparing the mean PI ¯uorescence of the
standard species nuclei, from samples containing simul-
taneously processed target and standard tissues, with the
mean ¯uorescence of standard nuclei from tissues that have
been processed in the absence of target species tissue. A
lower mean ¯uorescence of the standard nuclei in samples
of simultaneously processed standard and target tissues
indicates the presence of at least one inhibitor. When
inhibitors are present, additional caution should be taken in
interpreting di€erences in DNA content among plant
samples.
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