Martínez-Izquierdo, J A

Menossi, M

Puigdomènech, P

BioTechniques

English

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Improved	analysis	of	promoter	activity	in
biolistically	transformed	plant	cells.
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Financial support from the European
Commission (Marie Curie Grant No. ERB-
4001-GT-97-0136) is gratefully acknowl-
edged. Address correspondence to Franck
Atienzar, Department of Biological Sciences
and Plymouth Environmental ResearchCen-
ter (PERC), University of Plymouth, Drake
Circus,Plymouth,PL48AA,England,UK.In-
ternet: fatienzar@plymouth.ac.uk
Received 28 June 1999; accepted 19
October 1999.
F. Atienzar, A. Evenden,
A. Jha, D. Savva1 and
M. Depledge
University of Plymouth
Plymouth, England, UK
1The University of Reading
Reading, England, UK
Improved Analysis of
Promoter Activity in
Biolistically Transformed
Plant Cells
BioTechniques 28:54-58 (January 2000)
A common way to analyze gene ex-
pression involves the use of transgenic
plants. Transient assays that use particle
bombardment (9) are a powerful alter-
native to rapidly evaluate gene expres-
sion in intact plant tissues. If the partic-
ular system in use has been validated by
another approach, the results provide a
meaningful indicator of the expression
of stable genes in plants. Even for veri-
fied transient assay systems, a critical
problem often associated with particle
bombardment is that of high data dis-
persion. On the other hand, the choice
of the assay (histochemical or fluoro-
metric) used to quantify promoter activ-
ity in gus-based constructs could lead to
misinterpretation of the data because of
the different sensitivities for GUS de-
tection among assays. The aim of this
work was to find a way to decrease data
dispersion and to evaluate the consis-
tency of the two assays of GUS activity
used to determine the promoter activity
in transient assays.
To address the data dispersion prob-
lem, we used the C1 and B-Peru genes
from maize as internal controls. These
genes code for transcriptional factors
that activate the anthocyanin biosyn-
thetic pathway (1,10). The level of an-
thocyanin was used as a visual marker
to select only samples with high levels
of transformed cells. A major factor
causing data dispersion is the unequal
dispersion of particles among different
bombardments and among distinct
samples in the same bombardment. In-
ternal controls coding for other reporter
enzymes under the control of constitu-
tive promoters are generally used to
normalize the data dispersion. Howev-
er, when using internal controls, it is de-
sirable to identify efficiently transfected
samples before undertaking the labori-
ous process of protein extraction and
quantification of the promoter activities
of the test and control constructs.
In these experiments, we evaluated
the co-bombardment of the test DNA
construct together with constructs con-
taining the C1 and B-Peru genes (1,10).
After bombarding tissues from maize,
wheat, petunia, pea and white clover
with these two genes under the control
of the cauliflower mosaic virus (CaMV)
35S promoter, the transformed cells be-
come colored as a result of anthocyanin
accumulation and can be seen with the
naked eye (1,2,7,10,11,13).
The constructs p35SC1, pB-Peru
(anthocyanin accumulation) and pAH-
18 (luciferase production) were used as
internal controls, and p699 was used as
a test construct to direct GUS produc-
tion. GUS activity is expressed as pmol
4-methylumbelliferone (4-MU) min-1
mg-1. Both p35SC1 and pB-Peru con-
tain the CaMV 35S promoter and the
first intron of maize Adh1, fused to the
C1 and B-Peru gene coding regions, re-
spectively (kindly provided by S. Wess-
ler). The pAH18 contains the maize
ubiquitin promoter and its first intron
fused to the firefly luciferase coding re-
gion (5). The p699 is a deletion of the
Hrgp promoter obtained from an AC-
1503 maize genomic clone (EMBL Ac-
cession No. AJ131535) with the 3′ end
at +16 and the 5′ end at -699 (number-
ing relative to the ATG start codon),
fused in frame to a gusA/3′NOS frag-
ment (12). The maize Hrgp gene codes
for a hydroxyproline-rich glycoprotein
that is present in cell walls (15), and the
promoter shows high activity in young
shoots and immature embryos (12).
When intact maize tissues with ir-
regular shapes were bombarded, there
was a high level of data dispersion,
even among samples in the same petri
dish. In a representative experiment
(Figure 1), young seedlings were bom-
Benchmarks
54 BioTechniques Vol. 28, No. 1 (2000)
barded with pB-Peru and p35SC1, to-
gether with p699 and pAH18 con-
structs. The number of bronze spots
representing the anthocyanin genes ex-
pression in each seedling was scored
under a stereomicroscope 24 h after
bombardment. GUS activity in each
seedling was assayed fluorometrically
and varied from zero to approximately
300 pmol of 4-MU min-1mg-1. There
was a positive correlation between the
GUS activity of each seedling and the
number of bronze spots (Figure 1). The
mean GUS (± SEM) of all the samples
shown in Figure 1 was 85 ± 22 pmol 4
MU/min/mg protein: the SEM corre-
sponded to 26% of the mean.
After eliminating samples with less
than 150 bronze spots that produce low
GUS activity, the mean GUS activity in-
creased to 124 ± 23, with the SEM rep-
resenting 18% of the mean. To further
reduce data variability, the luciferase ac-
tivity directed by pAH18 in each
seedling also was assayed. To normalize
the activity driven by the p699 construct
in each seeding, the GUS activity (pmol
4-MU min-1 mg-1) was divided by the
luciferase  activity (mV per 10). Using
this second internal control, the SEM as
a percentage of the mean was further re-
duced from 18% to 10%. This approach
gave also excellent results when imma-
ture embryos, endosperms, young
leaves and BMS suspension cell cul-
tures were transformed by particle bom-
bardment (data not shown).
Another possible problem associat-
ed with transient expression assays that
use particle bombardment is the
method for quantifying GUS activity.
The quantification of promoter activity
by histochemical detection of GUS ac-
Vol. 28, No. 1 (2000) BioTechniques 55
Figure 1. Correlation between anthocyanin ac-
cumulation and GUS activity in young maize
seedlings. Maize seedlings were bombarded with
p699, pAH18 and pB-Peru and p35SC1 plas-
mids. Particle preparation and DNA coating were
carried out (12) using 8 µL of DNA-GUS con-
struct (0.5 µg/µL in TE buffer, pH 8.0) and 15 µL
of a control mixture containing 25 ng of pB-Peru,
25 ng of p35SC1 and 1 µg of pAH18 in each
preparation. Bombardment was done with a
PDS1000/He device (Bio-Rad Laboratories, Her-
cules, CA, USA) using settings already described
(12). Nine young seedlings (three days after ger-
mination) of the maize inbred line W64A were
distributed in petri dishes containing MS media
(MS salts and vitamins, 30g sucrose/L, 2.4g Gel-
rite/L, pH 5.8). The number of bronze spots in the
shoot of each seedling was scored 24 h after bom-
bardment. A protein extract from each shoot was
subsequently obtained by homogenization in 150
µL of luciferase lysis buffer (luciferase assay sys-
tem, Promega, Madison, WI, USA). The GUS
activity, directed by p699, was quantified fluoro-
metrically with a TK 1000 fluorometer (Amer-
sham Pharmacia Biotech, Piscataway, NJ, USA).
Points represent the GUS activity (pmol 4-MU
min-1 mg-1) versus the number of bronze spots
scored in each seedling.
Figure 2. Comparison of histochemical and
fluorometric assays for the quantification of
GUS activity. Maize embryos (14 days after pol-
lination) were flattened onto MS media (9 per
dish) and bombarded with p35SI, p1076 and
p159 in co-transformation with pAH18, as de-
scribed in Figure 1. The histochemical assay of
GUS activity using X-Gluc as substrate was per-
formed as described (12) and the blue spots in
each embryo were counted under a stereomicro-
scope. Fluorometric measurements of GUS activ-
ity were performed as before by using the protein
extract prepared from the 9 embryos of each dish.
Luciferase activity was quantified in a 1250 Lu-
minometer (BioOrbit, Turku, Finland) by mixing
20 µL of protein extract with 100 µL of reaction
buffer (luciferase assay system; Promega) and
taking the measurments after 10 s. To normalize
promoter activity, the GUS value detected in each
sample was divided by the luciferase value ob-
tained in the same sample. Black bars indicate the
average number of blue spots seen following the
histochemical assay (n=58) and the white bars
represent the mean GUS/luciferase activities
based on the fluorometric assay (n=4). In both
cases, the activity of p35SI was considered as
100%. The error bars represent the SEM.
tivity, followed by counting the number
of blue spots produced, is widely used
because of its simplicity (4,6,14). How-
ever, theoretical considerations indicate
that the fluorometric assay of GUS ac-
tivity may be better for this purpose.
The number of blue spots in immature
embryos that were bombarded with
constructs containing GUS fused to
three Hrgp promoter deletions was
found to be similar, although the inten-
sity of the spots differed (data not
shown). These variations probably re-
flect distinct levels of GUS activity-
driven promoters, which differed in
strength. In this case, counting the spots
would erroneously estimate the activity
of promoters that varied in strength.
To test this hypothesis, immature
embryos were bombarded with p35SI
and p1076 and p159 constructs, and two
methods for detecting GUS activities
were compared. The p35SI construct
contains a 450 bp 35S promoter fused to
the first intron of the Adh1-S gene (3), to
a gusA coding region and to nopaline
synthase terminator (3’NOS) from
pBi121 (8). The constructs p1076 and
p159 are deletions of the HRGP pro-
moter fused to the gusA gene, with the
5′ end at -1076 and -159, respectively.
The two assays detected that GUS
activity varied with the promoter, al-
though there were discrepancies in the
activity of a given promoter (Figure 2).
Thus, the activity of p1076 was 70%
that of p35SI when the blue spot count-
ing method was used, but only 40%
when the fluorometric assay was used.
The p159 construct showed even greater
discrepancies (40% of the p35SI con-
struct value in the blue spot counting
method and only 6% in the fluorometric
assay). An essential assumption of the
blue spot counting method is that the
stronger the promoter, the higher the
probability that one transformed cell
will produce a sufficient amount of re-
action product to give a detectable blue
spot. However, our results indicate that
with this method, there is a clear overes-
timation of the activity of weak promot-
ers. The GUS fluorometric assay was
more accurate in detecting differences
in the activities directed by distinct con-
structions and provided a more faithful
determination of promoter activity. This
is probably because the histochemical
spot-counting method does not allow
for the distinction between light and
dark blue spots. Thus, once a cell (or a
group of cells) reaches the threshold for
color detection by the naked eye, it will
be considered a unit in the same manner
as dark spots with higher GUS activity.
In summary, our approach using two
internal control systems greatly de-
creases data variability. It also decreas-
es the amount of time and money nec-
essary to prepare protein extracts and
assay GUS and luciferase activities
from non-transformed and poorly
Benchmarks
transformed samples. This system will
probably be useful in other species that
respond to transformation with the C1
and B-Peru genes. Our results also
show that the histochemical spot-count-
ing method for the quantification of
promoter-driven GUS activity may un-
derstimate the activity of strong pro-
moters. Irrespective of the species be-
ing studied, the fluorometric assay is
preferred for this reason when quanti-
fying promoter-driven GUS activity.
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The authors thank Dr. P. Fontanet for in-
valuable help with maize plant materials,
Dr. R. Raz for providing the AC1503 maize
Hrgp genomic clone and Drs. S. Wessler
and V. Walbot for providing plasmid con-
structs. This research was supported by the
Dirección General de Política Científica
(Grant No. PB93-0043) and Plan Nacional
de Investigación Científica y Técnica
(Grant No. BIO 94-0734). M.M. was the re-
cipient of a predoctoral fellowship from
CNPq/RHAE, Brazil. Address correspon-
dence to Dr. Marcelo Menossi, Centro de
Biologia Molecular e Engenharia Genética,
Departamento de Genética e Evolução, C.P.
6109, Universidade Estadual de Campinas,
CEP 13083-970, Campinas, SP, Brazil. In-
tenet: menossi@ unicamp.br
Received 2 March 1999; accepted 4
October 1999.
Marcelo Menossi1,2, Pere
Puigdomènech1 and José A.
Martínez-Izquierdo1
1Centro de Investigación y
Desarrollo, CSIC
Barcelona, Spain
2Universidade Estadual de 
Campinas
Campinas, SP, Brazil
Benchmarks
Vol. 28, No. 1 (2000)


Improved Analysis Of Promoter Activity In Biolistically Transformed Plant Cells.

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Improved Analysis Of Promoter Activity In Biolistically Transformed Plant Cells.

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