Note: Descriptions are shown in the official language in which they were submitted.
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1
PLANT GENE EXPRESSION, CONTROLLED BY CONSTITUTIVE
PLANT V-ATPASE PROMOTERS
Plant gene expression under the control of constitutive
plant V-ATPase promoters.
The invention relates to DNA constructs which encompass
a plant V-ATPase promoter which is operatively linked
to a heterologous gene. The invention furthermore
relates to the use of these constructs in the form of
expression cassettes, recombinant vectors and in
transgenic plants, plant cells or protoplasts. In par-
ticular, the invention relates to the promoter of Beta
vulgaris V-ATPase subunit c isoform 2.
Genetic engineering methods allow foreign genes to be
transferred specifically into the genome of a plant.
This process is termed transformation, and the
resulting plants are termed transgenic plants. When
expressing foreign genes in plants, the choice of the
promoter is frequently a critical factor. While it may
be desirable to express a gene only as the response to
a particular abiotic or biotic stimulus or to localized
expression in a specific tissue, other genes should
preferably be expressed constitutively, i.e. in the
entire plant at all times and in all tissues.
Examples for expression which can be induced by a
particular stimulus are the wound induction, which is
described, for example, in WO 93/07279 and, for pota-
toes, in EP 0 375 091 Al, or chemical induction, which
has been described in WO 95/19443 (Ward et al. (1993)
Plant Molecular Biology 22, 361-366), such as, for
example, the induction with salicylate, which is known
from EP 0 337 532 B1, or light induction (Fluhr et al.
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(1986) Science 232, 1106-1112 and WO 91/05054), and
temperature-dependent induction, also described in
EP 0 337 532 B1 and, for tomatoes, in WO 96/12814.
Examples of cell- and tissue-specific expression are
seed-, tuber- and fruit-specific expression (see review
by Edwards and Coruzzi (1990) Annu. Rev. Genet. 24,
275-303 and US 5,753,475).
Phloem-specific expression (SchmUlling et al. (1989)
Plant Cell 1, 665-670), root-nodule-specific expression
(DE 3702497) and meristem-specific expression (Ito et
al. (1994) Plant Molecular Biology 24, 863-878) are
also known.
Promoters which cause constitutive expression of the
genes controlled by them can be employed, for example,
for selecting transformed plant cells (expression of a
selectable marker gene in transgenic plants, generation
of antibiotic-resistant plant cells) or for generating
herbicide-tolerant, insecticide-tolerant and pathogen-
stress-resistant plants, since the products of the
genes controlled by them are present in all parts of
the plant.
Foreign genes of other agronomic, medicinal or other
importance can be expressed in a variety of plants, for
example for generating heterologous recombinant
proteins and for generating plants which contain
mammalian polypeptides. The quantity of the expression
pattern over space and time, of endogenous plant genes,
can also be advantageously altered with the aid of
constitutively active promoters.
The constitutive promoter which is most frequently used
in plant genetics is the viral 35S CaMV promoter
(Franck et al. (1980) Cell 21, 285-294). This promoter
contains different recognition sequences for transcrip-
tional effectors which, in their totality, result in
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constitutive expression of the gene which has been
introduced (Benfey et al. (1989) EMBO J. 8, 2195-2202).
Other constitutive promoters of viral origin are, for
example, the fig-wort mosaic virus promoter, which has
been described in EP 0 426 641, the Australian banana-
infecting badnavirus (WO 99/00492) and the sugar cane
bacilliform virus promoter described in WO 99/09190.
Plant-intrinsic constitutive promoters are, for
example, the maize ALS promoter (WO 96/07746), the rice
actinl promoter (McElroy et al., Plant Cell 2, 163-171,
1990, US 5,641,876), the wheat proline-rich protein
promoter described in WO 91/13991, the raspberry DRU
promoter (WO 97/27307) and the Medicago sativa H3
histone promoter (WO 97/20058).
To express selection genes and resistance genes, it
would be desirable to have available promoters which
show a strong, uniform constitutive activity in, if
possible, all plant tissues or cell types and which,
moreover, show even greater activity, or are not
repressed, under stress conditions. Advantageously,
these promoters should not be derived from plant patho-
gens (as is the 35S CaMV promoter), whose expression
might also differ in different plant tissues.
Regulation of the constitutive 35S promoter by stress
factors has not been described (frequently, elements of
other stress-inducible promoters are coupled to the
CaMV 35S promoter); most of the stress-induced pro-
moters show a virtually undetectable expression under
normal conditions (which is a disadvantage) and are
only strongly induced under the respective inducing
stress conditions. They are therefore unsuitable for
many uses.
WO 94/21793 describes a constitutive promoter which is
modulated by environmental conditions. This is a con-
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stitutive promoter from tobacco which is induced by
heat and hormone shock, by wounding, and by biotic
induction and infection. WO 94/21793 proposes to use
this promoter for selecting crop protection agents or
for protecting the plant from stress situations.
As a further example, EP 0 559 603 describes the con-
stitutive promoter of cauliflower heat-shock protein
hsp80. Parts of this promoter can also lead to consti-
tutive expression in heterologous, non-constitutive
promoters, for example in the case of inducible pro-
moters, promoters which can be regulated by other means
or inactivated promoters. EP 0 559 603 furthermore des-
cribes that genes which mediate resistance to insects,
herbicide resistance genes, antimicrobial genes, anti-
fungal genes, antiviral genes and anti-feedant genes
can be under the control of this promoter. The hsp80
promoter has a very high constitute activity which,
however, is only slightly elevated under stress con-
ditions, which is a disadvantage.
The V-type H+-ATPase (V-ATPase) plays a central role in
the cells of higher plants since it contributes sub-
stantially to establishing the electrochemical H+
gradient on the tonoplast. It accounts for a large pro-
portion (7-35%) of the overall tonoplast protein (Klink
et al. (1990) Bot. Acta 103, 24-31; Fischer-Schliebs et
al. (1997) Biol. Chem. 378, 1131-1139). Moreover,
V-ATPase is also found in the membranes of the Golgi
vesicles. Thus, it is also relatively strongly expres-
sed in cells which do not contain a large central
vacuole. Plant V-ATPases are composed of at least 10
different subunits which, in defined stoichiometry,
form a hollow enzyme of approx. 500 000 kD. In addition
to the vacuole pyrophosphatase (V-PPiase), which is
frequently expressed on the same endomembranes,
V-ATPase plays a central role in processes such as cell
division, cell elongation and metabolite or salt
accumulation in the vacuoles.
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Studies on the expression control of a plurality of
V-ATPase genes in various plants (sugar beet, tobacco,
carrot, maize, Mesembryanthem um crystallinum) have
demonstrated that at least some V-ATPase genes show a
coordinated expression with regard to the transcript
quantities (Rausch et al. (1996) J. Plant Physiol. 148:
425-433; Low et al. (1996) Plant Physiol. 110: 259-265;
Low and Rausch (1996) J. Exp. Bot. 47: 1725-1732;
Kirsch et al. (1996) Plant Mol. Biol. 32: 543-547; Lehr
et al. (1999) Plant Mol. Biol. 39: 463-475).
Summary of the invention
It is an object of the invention to provide a DNA construct with a plant
B. vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No.1] operatively
linked
with a heterologous gene.
It is an object of the invention to provide a polynucleotide comprising the
sequence of the promoter of B. vulgaris V-ATPase subunit c isoform 2 [SEQ ID
No.1 ].
It is an object of the invention to provide a recombinant vector which
comprises a DNA construct of the invention.
It is an object of the invention to provide a microorganism which is
transformed with a recombinant vector of the invention.
It is an object of the invention to provide a transgenic plant cell or
protoplast
whose genome comprises a DNA construct of the invention.
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5a
It is an object of the invention to provide a method for the controlled
expression of a heterologous gene in a plant cell or a protoplast, which
comprises
transforming the cell or the protoplast with a DNA construct of the invention
and
subsequently exposing the transformed cell or the protoplast to such a biotic
or
abiotic stress that the expression of the heterologous gene which has been
transformed by means of the DNA construct is controlled.
It is an object of the invention to provide a method for the controlled
expression of a heterologous gene in a plant, which comprises regenerating
cells or
protoplasts transformed with a DNA construct of the invention to give rise to
a
transgenic plant and subsequently exposing the plant transformed in this way
to
such a biotic or abiotic stress that the expression of the heterologous gene
which
has been transformed by means of the DNA construct is controlled.
It is an object of the invention to provide a method for producing a
recombinant protein in a plant, which comprises:
transforming a plant cell or a protoplast with a DNA construct of the
invention and
subsequently exposing the transformed cell or protoplast to such a biotic or
abiotic
stress that the recombinant protein transformed by means of the DNA construct
is
expressed.
It is an object of the invention to provide a method of producing a
recombinant protein in a plant, which comprises regenerating cells or
protoplasts
transformed with a DNA construct of the invention to give rise to a transgenic
plant
and subsequently exposing the resulting transgenic plant to such a biotic or
abiotic
stress that the recombinant protein transformed by means of the DNA construct
is
expressed.
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5b
It is an object of the invention to provide the use of a DNA construct of the
invention for producing a recombinant protein in a plant cell or a protoplast.
It is an object of the invention to provide the use of a DNA construct of the
invention for producing a recombinant protein in a plant.
It is an object of the invention to provide the use of a DNA construct of the
invention for expressing a gene in a plant under biotic or abiotic stress.
It is an object of the invention to provide the use of a plant V-ATPase
promoter for expressing a gene in a plant under biotic or abiotic stress.
It is an object of the invention to provide the use of a plant B. vulgaris V-
ATPase promoter subunit c isoform 2 [SEQ ID No.1] for expressing a gene in a
plant under biotic or abiotic stress.
It is an object of the invention to provide a plant cell or protoplast which
is
transformed with a DNA construct of the invention and which is resistant to
biotic or
abiotic stress.
It is an object of the invention to provide a plant cell or protoplast of the
invention which is resistant to salt stress.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of the construct pBVA-70(16)/GUS and
pBVA-70(16)/LUC.
Figure 2 shows the construction of the construct pBVA-70/LUC.
Figure 3 Figure 2 shows the construction of the construct pBVA-16/LUC.
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Figure 4 shows the construct pBVA/16-2 promoter deletions.
Figure 5 shows the construct pBVA/16-1 promoter deletions.
Figure 6 shows the comparison of the activity of different promoters under
control
conditions.
Figure 7 shows a Northern Blot analysis of the expression of the C2 isoform in
Beta
Vulgaris.
Figures 8A and 8B show the comparison of the activities of different deleted
promoters under control conditions. The numbers beneath the columns refer to
the
various deletion fragments shown in Figures 4 and 5.
Figures 9A and 9B show the activities of the V-ATPase promoters after exposure
to
125 mM for at least 24 hours in B compared to A (control).
Figure 9C shows a Northern Blot analysis wherein the transcript quantities for
the
V-ATPase genes A, c, and c2 after exposure to salt are elevated compared with
the
control treatment.
Figure 10 shows a Northern Blot analysis for detecting the gene expression of
V-
ATPase and V-PPiase in storage parenchyma cells of sugar beet after mechanical
wounding.
Figure 11 shows a Western Blot analysis with a polyclonal antiserum against
the
K. daigremontiana V-ATPase holoenzyme and shows wound-induced changes in
V-ATPase on the tonoplast in the storage parenchyma of the sugar beet, A)
after
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wounding, subunit c protein quantity increases the enriched tonoplast fraction
as a
function of time, B) in the total microsome fraction, the quantities of the
individual
subunits remain unchanged.
Figure 12 shows wound-induced changes in the H+-pump activity of the V-ATPase
in the microsomal and in the enriched tonoplast fractions, in the presence of
100
M vanadate and 1 mM azide.
Detailed description of the invention
It is an object of the present invention to provide
novel DNA constructs with plant constitutive promoters
which show improved properties over the prior-art plant
or viral promoters and which allow a strong, constitu-
tive gene expression in all plant organs which is
modulated by salt stress and other biotic or abiotic
factors. In particular, the DNA constructs are intended
to be suitable for expressing selection markers and
resistance genes.
We have found that this object is achieved by a DNA
construct in which a plant V-ATPase promoter or its
functional equivalent operatively linked to a hetero-
logous gene is present. According to the invention,
this plant V-ATPase promoter may also be a deleted or
hybrid V-ATPase promoter which remains functionally
active as promoter.
The plant V-ATPase promoter may be derived from either
dicotyledonous or monocotyledonous plants, for example
from sugar beet, tobacco, barley, rice, potatoes,
sunflowers, soya, tomatoes, Canola, wheat, oilseed
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rape, sorghum, carrots, maize, Mesembranthemum
crystallinum or Arabidopsis thaliana.
The plant V-ATPase promoter is preferably the promoter
of the B. vulgaris V-ATPase subunit A [SEQ ID No. 3],
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B. vulgaris V-ATPase subunit c isoform 1 [SEQ ID No. 2]
or B. vulgaris V-ATPase subunit c isoform 2 [SEQ ID
No. 1].
It is a further object of the invention to provide a
novel constitutive plant promoter of the abovementioned
type.
We have achieved this object by a polynucleotide encom-
passing the sequence of the promoter of B. vulgaris
V-ATPase subunit c isoform 2 [SEQ ID No. 1] or a poly-
nucleotide encompassing its functional equivalent.
Expedient embodiments of the invention are characteri-
zed in the subclaims.
Thus, the DNA construct according to the invention can
encompass a second promoter which can be regulated in a
different manner than the first promoter, thus allowing
a more flexible expression control. Moreover, at least
one further pyrimidine stretch can be inserted in the
promoter, and this affects promoter activity.
The DNA construct according to the invention is prefer-
ably an expression cassette. It is further preferred
that the heterologously expressed gene is a selection
marker or a resistance-mediating gene. Examples of such
heterologous genes are insecticidal toxins (such as,
for example, of Bacillus thuringiensis), herbicide
resistance genes, antimicrobial genes, antifungal
genes, antiviral genes and anti-feedant genes. Other
suitable genes are, for example, selectable genes,
reporter genes or killer genes. Examples of selectable
genes are genes for resistance to antibiotics such as
neomycin transferase genes, hygromycin phosphotrans-
ferase genes or phosphinothricin acetyltransferase
genes or, alternatively herbicide resistance genes such
as glufosinate, bromoxynil, sulfonamide or glyphosate
resistance genes (for more information, see Glick and
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Thompson, Methods in Plant Molecular Biology and
Biotechnology, CRC Press London, Chapter 6.7,
pages 71-119). Examples of reporter genes are genes
which code for chloramphenicol acetyltransferase (CAT),
P-glucuronidase (GUS), luciferase (LUC), green fluores-
cent protein (GFP) or thaumatin. Examples of killer
genes are the barnase gene, the TA29 gene or the
Diphtheria toxin gene (for more information, see
"Transgenic Plants", E. Gaulun and A. Breiman, Imperial
College Press, London, 1997).
The invention furthermore provides a recombinant vector
with a DNA construct according to the invention. This
vector may also be a shuttle vector, which facilitates
its handling.
In a further embodiment of the invention, the recombi-
nant vector is an expression vector. Also, transformed
microorganisms are provided together with the recombi-
nant vector, for example a transformed Agrobacterium
tumefaciens.
A preferred embodiment of the invention relates to a
transgenic plant cell or a protoplast whose genome
encompasses a DNA construct according to the invention.
This transgenic plant cell or protoplast can be derived
both from a monocotyledenous and from a dicotyledenous
plant. Preferred cells or protoplasts are those from
sugar beet, tobacco, barley, rice, potatoes, sun-
flowers, soya, tomatoes, Canola, wheat, oilseed rape,
sorghum, carrots, maize, Mesembranthemum crystallinum
or Arabidopsis thaliana.
Especially preferred is a transgenic plant according to
the invention whose genome encompasses a DNA construct
according to the invention. The transgenic plant can be
both a monocotyledenous and a dicotyledenous plant.
Especially preferred are plants such as sugar beet,
tobacco, barley, rice, potatoes, sunflowers, soya,
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tomatoes, Canola, wheat, oilseed rape, sorghum,
carrots, maize, Mesembranthemum crystallinum or
Arabidopsis thaliana.
The invention furthermore provides a process for the
controlled expression of a heterologous gene in a plant
cell or a protoplast which allows a strong, constitu-
tive gene expression in all plant organs which is
modulated by salt stress and other biotic or abiotic
factors. The process according to the invention com-
prises firstly transforming the cell or the protoplast
with a DNA construct according to the invention, for
example an expression cassette, and subsequently
exposing the transformed cell or the protoplast to such
a biotic or abiotic stress that the expression of the
heterologous gene transformed by means of the DNA
construct is controlled efficiently. This stress may
occur in the form of salt stress, for example by NaCl
or KC1, phosphate deficiency, nitrogen deficiency,
sucrose deficiency, wounding, infection, temperature,
drought, herbicides or mechanical stress. According to
the invention, the plant cells or protoplasts used for
the process can be obtained from a monocotyledenous or
dicotyledenous plant, preferably from sugar beet,
tobacco, barley, rice, potatoes, sunflowers, soya,
tomatoes, Canola, wheat, oilseed rape, sorghum,
carrots, maize, Mesembranthemum crystallinum or
Arabidopsis thaliana.
The method according to the invention may also be used
for the controlled expression of a heterologous gene in
a plant. This method comprises regenerating cells or
protoplasts transformed with a DNA construct according
to the invention to give rise to a transgenic plant and
subsequently exposing the transgenic plant to such a
biotic or abiotic stress that the expression of the
heterologous gene transformed by means of the DNA
construct is controlled efficiently. This stress may
occur in the form of salt stress, for example by NaCl
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or KC1, phosphate deficiency, nitrogen deficiency,
sucrose deficiency, wounding, infection, temperature,
drought, herbicides or mechanical stress. The
transgenic plants used for the process can be obtained
from a monocotyledenous or dicotyledenous plant,
preferably from sugar beet, tobacco, barley, rice,
potatoes, sunflowers, soya, tomatoes, Canola, wheat,
oilseed rape, sorghum, carrots, maize, Mesembranthemum
crystallinum or Arabidopsis thaliana.
Furthermore, the invention provides a method which
allows the efficient production of a recombinant
protein in a plant cell or a protoplast. To do this,
the cell or the protoplast is transformed with a DNA
construct according to the invention, and the
transformed cell or the protoplast transformed thus
subsequently exposed to such a biotic or abiotic stress
that the recombinant protein transformed by means of
the DNA construct. is expressed. This stress may occur
in the form of salt stress, for example by NaCl or KC1,
phosphate deficiency, nitrogen deficiency, sucrose
deficiency, wounding, infection, temperature, drought,
herbicides or mechanical stress. The protein produced
in this way can subsequently be isolated from the plant
cell or the protoplast by methods known to those
skilled in the art. The plant cells or protoplasts used
for the process may be derived from a monocotyledenous
or dicotyledenous plant. Especially preferred plant
cells or protoplasts are those from sugar beet,
tobacco, barley, rice, potatoes, sunflowers, soya,
tomatoes, Canola, wheat, oilseed rape, sorghum,
carrots, maize, Mesembranthemum crystallinum or
Arabidopsis thaliana.
Moreover, the invention provides a method which allows
the efficient production of a recombinant protein in a
plant. The method comprises regenerating cells or
protoplasts which have been transformed with a
DNA-construct according to the invention to give rise
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to a transgenic plant and subsequently exposing the
resulting transgenic plant to such a biotic or abiotic
stress that the recombinant protein transformed by
means of the DNA construct is expressed. This stress
may occur in the form of salt stress, for example by
NaCl or KC1, phosphate deficiency, nitrogen deficiency,
sucrose deficiency, wounding, infection, temperature,
drought, herbicides or mechanical stress. The protein
produced in this way can subsequently be isolated from
the plant. The plants used for the process may be
monocotyledenous or dicotyledenous plants. Especially
preferred are plants such as sugar beet, tobacco,
barley, rice, potatoes, sunflowers, soya, tomatoes,
Canola, wheat, oilseed rape, sorghum, carrots, maize,
Mesembranthemum crystallinum or Arabidopsis thaliana.
Moreover, the present invention extends to the use of a
DNA construct according to the invention for producing
a recombinant. protein in a plant, plant cell or a
protoplast. The invention also extends to recombinant
proteins produced by one of the methods according to
the invention.
However, the DNA construct according to the invention
may also be used for expressing a gene in a plant under
biotic or abiotic stress. Moreover, the plant V-ATPase
promoter according to the invention can be used for
expressing a gene in a plant under biotic or abiotic
stress. This promoter may also be used in the form of a
deleted or hybrid V-ATPase promoter. Plant V-ATPase
promoters according to the invention from dicoty-
ledenous or monocotyledenous plants may be used. The
use of plant V-ATPase promoters from sugar beet,
tobacco, barley, rice, potatoes, sunflowers, soya,
tomatoes, Canola, wheat, oilseed rape, sorghum,
carrots, maize, Mesembranthemum crystallinum or
Arabidopsis thaliana is preferred. Especially preferred
are the V-ATPase promoters of the Beta vulgaris
V-ATPase subunit A [SEQ ID No. 3], B. vulgaris V-ATPase
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subunit c isoform 1 [SEQ ID No. 2] or B. vulgaris
V-ATPase subunit c isoform 2 [SEQ ID No. 1]. Moreover,
at least one additional pyrimidine stretch may be
inserted into the promoter used.
The invention furthermore relates to a plant cell or
protoplast which has been transformed with a DNA
construct according to the invention and which is
resistant to biotic or abiotic stress. This stress may
occur in the form of salt stress, for example by NaCl
or KC1, phosphate deficiency, nitrogen deficiency,
sucrose deficiency, wounding, infection, temperature,
drought, herbicides or mechanical stress. A transformed
salt-stress-resistant plant cell or protoplast is
preferred.
The invention furthermore extends to a plant trans-
formed with a DNA construct according to the invention
which is resistant to biotic or abiotic stress, prefer-
ably a transformed salt-stress-resistant plant.
A total of three different V-ATPase genes were isolated
from a genomic library of Beta vulgaris. The cDNA
clones and the genomic clones for the subunits A and
cl, which correspond to the peripheral V1 complex and
the membrane-integrated VO complex, have already been
described (Lehr et al., Plant Molecular Biology 39,
463-475 (1999, GenBank/EMBL-Datenbank: X98767, X98851,
Y11038, Y11037).
RNA blot analyses with gene-specific cDNA probes show
expression of the cloned subunits A and cl and of the
isoforms c2, which have been cloned for the purposes of
the present invention, in roots, leaves and in a sugar
beet suspension culture.
Surprisingly, it has emerged that all three promoters
show a very high activity, which, as a rule, exceeds
that of the 35S CaMV promoter.
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It seems that polypyrmidine sequences which are present
in the promoters play an important role in this con-
text. The isoforms A and cl each have two such sequence
stretches while the isoform c2 only has one such
sequence stretch.
It has now surprisingly been found within the context
of the present invention that the plant V-ATPase
promoters are outstandingly suitable for constructing
DNA constructs which can preferably be used as plant
expression cassettes.
These expression cassettes can be used for causing, in
the plant, an expression of heterologous proteins which
is governed by environmental effects. The invention
thus allows the plant or the plant cell to be equipped
with one or even more heterologous genes which are
induced in the plant additionally to its constitutive
expression "when required" by the plant (when the
environment changes).
Luciferase reporter gene studies on the promoter
V-ATPase A and cl from sugar beet and the 35S promoter
were carried out on sugar beet cell cultures under salt
stress (Lehr et al., Plant Mol. Biol. 39, 463-475,
1999). An induction of the promoter activity was detec-
ted for the isoforms A and cl. The 35S CaMV promoter
activity, which was studied for comparison reasons, is
not induced by salt and indeed repressed. Within the
invention, a surprising induction of all three
promoters by 100 mM NaCl or 100 mM KC1 was found, even
though all three promoters differ with regard to their
DNA sequence.
Surprisingly, it has furthermore been found that the
promoters according to the invention show no reduced
promoter activity in the case of phosphate defi-
ciency/nutrient deficiency, which is in contrast to the
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35S CaMV promoter. Thus, the constitutive expression
remains ideally stable.
In contrast, the promoter activity is induced by
wounding, while the 35S CaMV promoter is not induced
under comparable conditions. Also, it has been found
that the promoter activity is affected by sucrose
deficiency; it reduces the activity of cl, but not the
activity of the 35S CaMV promoter.
It has also been found that the promoter activities are
affected by abiotic factors such as high (30-35 C) or
low (2-5 C) temperatures.
In addition, it has been found that various deletions
can be introduced into this promoter, which leads to
this promoter showing either a) an increased activity,
b) an essentially identical activity to that of the
native promoter, c) a reduced activity, d) a higher
inducibility under stress conditions than the native
promoter, or e) a lower inducibility under stress
conditions than the native promoter.
"Operatively linked" means such an arrangement that the
normal function of the components can be carried out.
An encoding sequence which is "operatively linked" with
a control sequence therefore indicates a configuration
in which the encoding sequence can be expressed under
the control of the sequences.
The term "control sequences" describes DNA sequences
which are required for expressing an operatively linked
encoding sequence in a host organism. The control
sequences which are suitable for, for example,
prokaryotes, encompass a promoter, an optional operator
sequence, a ribosome binding site and, possibly, other
sequences which are not well understood as yet.
Eukaryotic cells are known for containing promoters,
polyadenylation signals and enhancers.
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The term "gene" refers to a DNA sequence which encodes
a bioactive polypeptide which can be isolated, or a
precursor. The polypeptide can be encoded by a full-
length sequence or by any part of the encoding sequence
as long as the enzymatic activity is retained.
An aspect of the invention is therefore a DNA construct
with a plant V-ATPase promoter or its functional
equivalent, operatively linked with a heterologous
gene. It is known that minor changes may be present in
the promoter sequence, for example caused by the
degeneration of the genetic code, without substantially
affecting its activity. The present invention therefore
also relates to "functional equivalents" of the plant
V-ATPase promoters which are operatively linked with a
heterologous gene.
The term "functional equivalents" characterizes all DNA
sequences which are complementary to a DNA sequence,
which hybridize with the reference sequence under
stringent conditions and which show an activity which
is similar to that of a plant V-ATPase promoter.
"Stringent hybridization conditions" are to be under-
stood as meaning those conditions under which hybridi-
zation takes place, and remains stable, at 60 C in
2.5 x SSC buffer followed by repeated washing steps at
37 C at a lower buffer concentration.
"Heterologous genes" are DNA sequences which encode
peptides or proteins which are other than the plant
V-ATPase subunits A, ci or c2.
A "deleted or hybrid V-ATPase promoter" is any V-ATPase
promoter which has a deletion or whose make-up is
altered and which still has promoter activity.
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The invention will be described in greater detail
hereinbelow in connection with the drawings.
I. Method of obtaining the genomic library of Beta
vulgaris L.
Generation of a genomic library
A genomic library was established with leaf material of
Beta vulgaris L. type 3A39111 in the "Lambda Fix II/
Xho I partial fill-in" vector by Stratagene.
The use of the "Lambda Fix II/Xho I partial fill-in
kit" allows genomic DNA fragments to be cloned effi-
ciently without the laborious size fractionation via an
agarose gel. To this end, the genomic DNA is subjected
to a partial digest with the restriction endonuclease
Sau3 AI, and the free ends are filled up with Klenow
polymerase, incorporating dGTP and dATP. The resulting
3'-AG-5' overhangs prevent autoligation of the genomic
DNA fragments. Also, the 3'-CT-5' overhangs of the
vector (vector digested with Xho I, partially filled-in
ends with dCTP, dTTP) prevent a religation of the
central vector fragments ("stuffer element"). Amplifi-
cation of wild-type Lambda Fix II phages (containing
the stuffer element) is also prevented by using the P2
lysogenic E. coli strain XLI-Blue MRA(P2).
Thus, the method used allows recombinant genomic clones
in the desired size range to be obtained in very high
yield.
Partial digest of genomic DNA
The optimal enzyme concentration for obtaining frag-
ments in the required size range of 15-23 kb is
determined by a test digest. To do this, individual
reactions together with a DNA marker are fractionated
in a low-concentration agarose gel, and the average
fragment sizes are determined. Since the electro-
phoretic fractionation causes a delay in the migration
behavior in particular in the high-molecular DNA range,
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fragment size cannot be determined directly via the
maximum fluorescence intensity. Rather, Seed et al.
(1982) maintain that fragment size is overestimated by
a factor of 2. To obtain fragments in the desired size
range, only half of the optimal enzyme concentration
determined via gel electrophoresis is therefore
employed for the preparative digest.
Test digest
For the test digest, the restriction enzyme Sau3A I
(10 U/ l, Promega) is diluted in restriction buffer
[1 x buffer B (Promega) supplemented with 0.1 mg/ml
acetylated BSA (Sigma)] to 'concentrations of 0.1 U/[ul,
0.05 U/[l; 0.025 U/ l, 0.01 U/ l], and the enzyme
solutions are stored on ice. 25 g of CsC12-purified
genomic DNA dissolved in TE buffer are resuspended in a
total volume of 1.125 ml of restriction buffer (see
above) and stored in reaction vessels on ice in
225 l-aliquots. The DNA solutions (in each case 5 pg
of DNA) are preheated for approximately 20 minutes in a
waterbath at 37 C, and the individual reactions are
started by adding 25 ill of the restriction enzyme solu-
tion. 25 l of pure buffer solution are added to a
control sample. The digest is carried out for 2 minutes
at 37 C and is stopped by adding 10 l-portions of
0.5 M Nat EDTA. The DNA is then precipitated by adding
1 volume of isopropanol and 0.1 volume of 3 M sodium
acetate (pH 7), washed with 70% ethanol, dried, and
then resuspended in 10 l-portions of TE buffer.
The reaction batches are separated by electrophoresis
overnight in an 0.5% TAE gel (track length 19 cm)
stained with ethidium bromide, overnight at 2 V/cm and
RT. The size marker used in the present experiment was
lambda DNA digested with Hind III, and the 1-kb-DNA
ladder (Gibco, BRL).
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Preparative digest
The conditions for the preparative digest correspond
essentially to those of the test batch. To standardize
the reactions, DNA concentration, overall volume of the
reaction batch, and time, remain unchanged. The enzyme
concentration results from the test digest.
100 g of genomic DNA is digested in aliquots (see
above), 0.11 volume of 10 x STE and 0.3 volume of 1 x
STE are added, and the mixtures are then extracted
twice using 1 volume of phenol/chloroform/isoamyl
alcohol (25:24:11, v/v/v). Precipitation takes place
after addition of 0.5 volume of 7.5 M sodium acetate,
2 volumes of absolute ethanol, 30 minutes at -20 C. The
DNA is washed with 70% ethanol, dried at room tempera-
ture, and resuspended in 200 l of TE buffer while the
pellets are combined. All centrifugation steps are
carried out for 10 minutes at 12,000 g and 4 C. The
digest is stored at -20 C.
10 x STE: 0.1 M NaCl, 10 mM Tris-HC1, pH 8, 1 mM Na2
EDTA
Klenow reaction
50 g of the partially digested genomic DNA in an
overall volume of 350 l of 1 x fill-in buffer (1 x
Klenow buffer: 50 mM Tris-HC1, pH 7.2, 10 MM MgSO4,
0.1 mM DTT, Promega; 0.166 mM dGTP, 0.166 mM dATP) with
15 U Klenow polymerase (5 U/ l, Stratagene). The reac-
tion is stopped by adding 0.5 volume of STE buffer
(0.1 M NaCl, 10 mM Tris-HC1, pH 8, 1 mM Na2 EDTA,
pH 8), and the DNA is then treated with phenol and
chloroform. After 0.5 volume of sodium acetate and
2 volumes of absolute ethanol have been added, the DNA
is precipitated for 30 minutes at -70 C, pelleted for
15 minutes at 12,000 g and 4 C and washed in 70%
ethanol. The dried pellet is resuspended in TE buffer
and stored at -20 C.
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Ligation into the Lambda Fix II/Xho I partial fill-in
vector
In a reaction volume of 10 l, 1 g of lambda DNA
(1 g/ l Lambda Fix II/Xho I partial fill-in vector)
and 0.4 g of genomic DNA fragments in ligase buffer
(10 mM Tris-HC1, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM
rATP, Promega) are resuspended on ice and incubated
overnight at 6-8 C with addition of 3.75 U of T4 DNA
ligase (15 U/ l, Promega). To check the ligation effi-
cacy, 0.3 g of the test insert (50 ng/ l, 12-kb pMF
test insert; BamH I fragment partially filled up with
dATP, dGTP) is ligated with 1 g of lambda DNA under
the abovementioned conditions as a control batch. The
DNA is packaged the following day.
Packaging of the lambda DNA
To generate a representative genomic library, the total
number of independent clones required is calculated
using the formula described by Clarke and Carbon
(1976). Aliquots of the ligation reaction are packaged,
the titer is determined, and this result is used for
calculating the number of independent clones per
packaging reaction. If required, further packaging
reactions are carried out. In total, the calculated
minimum number of independent clones should be reached.
The Gigapack III Gold packaging extract by Stratagene
was used to package the ligation products. A packaging
extract (50 l) is partially defrosted by holding it in
the hand, 1 l of ligation batch are added, and the
mixture is stored on ice. To check the packaging
efficacy, 0.2 g of lambda control DNA (wild type c1857
Sam7, 0.2 g/ l) are packaged in the same manner. The
extract is carefully resuspended using a pipette and
then incubated for 2 hours at room temperature. The
reaction is stopped by adding 500 l of SM buffer
(0.01% gelatin, 50 mM Tris-HC1, pH 7.5, 100 mM NaCl,
8 mM MgSO4) and 20 l of chloroform, and the solution is
mixed by careful shaking. The mixture is then
centrifuged for 1 minute at 16,000 g and 4 C. The
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supernatant is transferred into a reaction vessel and
stored at 4 C until use. The titer plates are estab-
lished the same day.
The following dilution levels of the individual packag-
ing extracts are used:
Packaging extract Dilution level
Ligation of B. vulgaris genomic DNA 1 x 101/1 x 10-1
Ligation of the control insert (pMF) 1 x 10-1/1 x 10-2
Control DNA (wild type c1857 Sam7) 1 x 10-3/1 x 10-4
Amplification of the genomic library
To recover a highly concentrated phage suspension which
can be stored at -80 C as a stable stock culture, the
genomic library of B. vulgaris was amplified. The
minimum number of independent clones required for the
amplification should represent the entire genome
(Clarke and Carbon, 1976).
Amplification takes place in the form of plate lysates
(150 mm 0 LB agar plate). The amount of packaging
extract required depends on the minimum number of
independent clones (see above). 600 l of competent
host cells per plating batch are infected with
50,000 pfu each and incubated for 15 minutes at 37 C
with gentle shaking. 6 ml of top agar is used for
plating. The incubation time is approximately 8 hours
at 37 C. Plaque size should reach a diameter of 1-2 mm.
The plates are then overlaid with 10 ml of SM buffer
and incubated overnight in a refrigerated chamber on a
tumble shaker at the lowest possible speed. The super-
natants are transferred into a sealable centrifuge
vessel, the plates are rinsed with 2 ml of SM buffer,
and the supernatants are combined. After addition of
chloroform (end concentration 5%), the phage suspension
is incubated for 15 minutes at room temperature and
separated from bacterial residues by centrifugation for
10 minutes at 2000 g. The supernatant is transferred
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into a sterile vessel, treated with chloroform (end
concentration 3%), and can now be stored for several
months at 4 C. After the titer of the amplified genomic
library has been determined, aliquots are stored at
-80 C in 7% DMSO (dimethyl sulfoxide) to provide a
stock solution. The following dilution levels are
employed for the titer determination (90 mm 0 LB
plates) : 1 x 10-6, 1 x 10-7, 1 x 10-1.
II. Methods for cloning promoter/reporter gene
constructs for the V-ATPase promoters A, cl and c2,
with the reporter genes luciferase (or P-glucuronidase)
Subcloning genomic clones with promoter regions
A single type of genomic clones was obtained for
subunit A, while two different isoforms were isolated
for subunit c clones. To subject the clone of the
genomic subunit A to sequence analysis, a 4.3 kb EcoRI
fragment (pBVA/70) which hybridizes with subunit A cDNA
was subcloned into the vector pBluescript II SK+. For
subunit c, an 8 kb XbaI/HindIII fragment (pBVA/16-1)
and a 5 kb EcoRV/HindIII fragment were subcloned into
the same vector.
Cloning of promoter/reporter gene constructs
BVA/70 promoter
A 1.3 kb HpaII fragment of the pBVA/70 clone was made
blunt-ended using Klenow polymerase and ligated into
the Smal site of the vector pBluescript in order to
obtain pBVA/70. Restriction with Hindlll and BglII
resulted in a 1.3 kb promoter fragment with its 3' end
in position -28 relative to the transcription start. To
obtain the promoter-GUS fusion, the HindIII/BglII was
ligated with the BamHI/EcoRI GUS cassette of pBI221
(Clontech) and then cloned into the vector pBluescript
in order to obtain pBVA/70p-GUS. To obtain the
promoter-LUC fusion, a PstI/BglII promoter fragment was
ligated upstream of the BamHI/HindIII cassette of
pCaMVLN and cloned into the vector pBluescript.
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BVA/16-1 promoter
The BamHI/EcoRI GUS cassette of plasmid pB1221
(Clontech) was cloned into pBluescript (pBGUS), from
which it was excised using BamHI and Sall. A 1.3 kb
PstI/BglII promoter fragment of pBVA/16-1 with its 3'
end in position -40 relative to the transcription start
was ligated with the GUS cassette and cloned into
PstI/SalI-digested pBluescript in order to obtain
pBVA/16-lp-GUS. The promoter-LUC fusion was cloned as
described for BVA/70 (see above).
The plasmids pFF19G and pCaMVLN were used in each case
as standards for GUS and LUC.
Characterization of the promoter/reporter gene
constructs
To compare the relative promoter activities of subunits
A and cl of the Beta vulgaris V-type H+-ATPase,
fragments of approximately 1.2 kb were isolated from
the region upstream of the encoding region of the
genomic clones pBVA-70 and pBVA-16/1. In this context,
the term promoter characterizes the 5'-regulatory
region and thus not only the promoter region, but also
parts of the 5'-untranslated leader. These fragments
were subsequently ligated into the phagemid vector
pBluescript II SK+ upstream of the corresponding (see
below) reporter gene cassettes. The reporter gene used
was, in addition to (3-glucuronidase structural gene
from Escherichia coli (uid A), the Photinus pyralis
luciferase gene (Jefferson, 1987; de Wet et al., 1987).
The promoter/reporter gene constructs constitute
"transcriptional fusions" since the 3' ends of the
promoter fragments employed are in each case 30-4 bp
upstream of the translation start of the genomic
clones. Figure 1 is a schematic representation of the
individual constructs. The expression vectors are here-
inbelow termed pBVA-70(or 16)/GUS and pBVA-70(or
16)/LUC.
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Characterization of the pBVA-70 promoter fragment
The total size of this fragment is 1.237 kb. It
encompasses the region between positions -1035 to +202
relative to the transcription start (+1) of the genomic
clone pBVA-70, and thus contains, in addition to the
promoter, most of the adjacent leader region (total
length of leader = 230 bp).
Characterization of the pBVA-16/1 promoter fragment
The total size of this fragment is 1.265 kb. It
encompasses the region between positions -1130 to +135
relative to the transcription start (+1) of the genomic
clone pBVA-16/1, and thus contains, in addition to the
promoter sequence, most of the adjacent leader region
(total length of leader = 174 bp).
Figure 1 is a schematic representation of the construct
pBVA-70(16)/GUS and pBVA-70(16)/LUC. Fragments (approx.
1.25 kb) from the region of the 5'-untranslated region
of the genomic clones pBVA-70 and pBVA-16/1 were
ligated into the phagemid vector pBluescript II SK+
upstream of the respective reporter gene cassette. To
prepare the promoter/GUS construct, the BamHI/EcoRI
cassette of vector pBI-221 by Clontech was used. In
addition to the 3-glucuronidase structural gene from
E. coli, it carries the polyadenylation region of the
nopalin synthase gene (Ti plasmid, Agrobacterium
tumefaciens). The promoter/LUC constructs were obtained
using the BamHI/HindIII cassette of the vector pCaMVLN.
It contains the Photinus pyralis luciferase structural
gene and the polyadenylation region of the nopalin
sythase gene. The figures characterize the position of
the cleavage sites of the promoter fragments used
relative to the transcription start (+1) of the genomic
clones.
Generation of a BVA/16-2 promoter/LUC construct
A further reporter gene employed for preparing a
construct was the Photinus pyralis luciferase gene (de
Wet et al. 1987), EMBL Acc. No. M15077. To prepare a
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promoter/luciferase construct, the BamHi/Hindill cas-
sette is excised from vector pCLN (pCaMVLN; Callis et
al. 1987). It contains the Photinus pyralis luciferase
structural gene (LUC) with the polyadenylation region
of the nopalin synthase gene. This cassette is cloned
into the vector pBluescript II SK (Stratagene) which
has been cleaved with BamHi/Hindlll. The promoter/
leader fragment of the gene of subunit c (isoform 2) of
the Beta vulgaris V-type H+-ATPase is excised from the
construct pBVA/16-2 GUS (see above) using BamHI. The
vector pBluescript II SK, into which the luciferase
cassette has been cloned, is also opened up using
BamHI, and the promoter with sticky ends thus cloned
upstream of the luciferase. The position of the
promoter fragment in the construct in the correct
orientation (5'-3') upstream of the luciferase gene is
checked by a variety of restriction digests (for
example BamHI, SalI or XmnI). The transitions from the
MCS of the vector to the promoter and from the other
side of the MCS to the luciferase are checked by
sequencing with the vector primers "M13 Forward" and
"M 13 Reverse". The promoter/leader LUC construct is
termed pBVA/16-2 LUC.
III. Methods for measuring the promoter activities in
Beta cell cultures
Ballistic transformation
The ballistic transformation method (particle gum
bombardment) was employed for the transient expression
of promoter gene constructs in Beta vulgaris cell
suspension cultures. This is a direct gene transfer
method, whereby the DNA reaches the plant cell owing to
the fact that the cell wall is destroyed mechanically
(Sanford et al. (1987)).
A Biolistic PDS-1000/He particle delivery system,
BIORAD was used.
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During the experiment, the gas pressure in the pressure
chamber rises and leads to the bursting disks being
destroyed. The number of bursting disks used governs
the pressure generated in the pressure chamber. As a
consequence, the stream of helium gas escapes in a
shocklike manner from the pressure chamber into the
previously generated vacuum chamber. The escaping
stream of gas accelerates the particle-coated macro-
carrier, which is slowed down after only a few
centimeters by means of a retaining net. The DNA-loaded
microcarriers (here: tungsten particles), in contrast,
pass through the interstices of the net and rupture the
cell walls when they hit the plant material.
Microcarrier preparation
In a reaction vessel, 1 ml of 70% ethanol is added to
30 mg of tungsten particles (microcarrier), and the
mixture is vortexed for 20 seconds. A subsequent
incubation for 10 minutes allows the particles to
settle, and they are then sedimented for 30 seconds at
1300 g (unbraked) at room temperature. After the
supernatant has been removed, the particles are washed
with 500 ml of sterile double-distilled H2O as follows:
vortex for 10 seconds, leave to stand for 10 minutes,
unbraked centrifugation for 30 seconds at 1300 g. The
sedimented particles are then treated with 500 l of
50% glycerol and mixed by vortexing. They are stored at
-20 C.
Loading of the microcarriers with DNA
The plasmid DNA required for the transformation should
be free from impurities (protein, RNA) and should
predominantly be present in supercoiled form. To
isolate the DNA, commercially available plasmid kits
are employed. To load the particles (microcarriers), it
is recommended to assume a minimum number of 10-15
bombardments. The microcarriers rapidly settle out in
the suspension during preparation, and small volumes
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make resuspending difficult. The volumes which follow
are in each case per bombardment.
All steps with the exception of the centrifugation
steps are carried out in a clean bench. The particle
suspension is mixed thoroughly on a Vortex mixer, and
9- l-batches of the solution are subsequently trans-
ferred into a reaction vessel. 1 l of plasmid DNA
(1 g/ l in TE buffer) is added with constant vortex-
ing. The mixture is stored for 15 minutes in an ice
bath. Then, 9 l of 2.5 M of CaCl2 (sterile), 3.6 l of
1.2 mM spermidine (filter-sterilized) and 18.2 l of
absolute ethanol are subsequently added in succession,
with gentle vortexing. After incubation for 10 minutes
in an ice bath, the particles are sedimented for
5 seconds at 180 g at room temperature, and the super-
natant is then removed. 6.4 l of absolute ethanol are
added, and the particles are carefully resuspended by
"finger vortexing" and stored in the ice bath until
further use.
Preparation of the cell material
For the gene transfer, the cell suspension culture used
(here: 3-4 day-old Beta vulgaris culture) is trans-
ferred to agar plates. To improve transformation
efficiency, the osmotic value of the medium is
increased by adding mannitol and sorbitol, and the
cells are preincubated on this medium for approx.
4 hours. The effect of the osmotic treatment on the
transformation efficiency of the plant cells can
probably be attributed to the fact that plasmolysis
occurs (Vain et al., 1993). It is possible that this
prevents the cytoplasm from leaking out after the
particle has entered the cell (Armaleo et al., 1990;
Sanford et al., 1992).
All steps, with the exception of the centrifuging
steps, are carried out in the clean bench. To this end,
10 ml-disposable pipettes, tweezers, filter paper disks
(45 mm 0), Buchner funnels and a wash flask are
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prepared. The materials with the exception of the
disposable pipettes and the tweezers are autoclaved
beforehand. Petri dishes (50 mm 0) provided with
Gamborg B5 medium (0.9% agar supplemented with 0.5 g/l
casein hydrolysate, 125 mm sorbitol, 125 mM mannitol,
after Ingersoll et al. 1996) are also used.
The Buchner funnel is provided with a filter paper
disk. Then, 3.3 ml of suspension culture, corresponding
to approx. 1 ml of cell packed volume (see below) are
distributed uniformly on the filter surface using a
disposable pipette. The excess medium is removed via
the wash flask by briefly applying a vacuum (approx.
2 seconds). The filter is transferred to a Petri dish
containing nutrient medium, and the cells prepared in
this way are stored in the clean bench at room
temperature for 3-4 hours until the gene transfer takes
place.
CPV determination
At the beginning of the experiment, the cell packed
volume (CPV) of the cell suspension culture is deter-
mined. To this end, 10 ml of the suspension are removed
under sterile conditions and sedimented for 5 minutes
at 1130 g (swing-bucket rotor) in a 15 ml graduated
centrifuge tube. The cell packed volume is determined
with reference to the graduation.
Transformation
All steps with the exception of the ultrasonic treat-
ment of the particle suspension (see below) are carried
out in the clean bench. Beforehand, the inside of the
particle gun and all metal and plastic supports are
cleaned with absolute ethanol. Equally, the retaining
nets and the bursting disks are treated with ethanol
and dried in the clean bench. The macrocarriers (M-20,
BIORAD) are sterilized by briefly immersing them in
absolute ethanol and transferring them into a
desiccator.
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27
The tungsten particle suspension is sonicated for
2 seconds at the maximum setting (Sonopuls HD 60,
Bandelin), and 5- l-portions of the suspension (approx.
422 g particles, 0.78 g DNA) are carefully pipetted
onto the macrocarrier surface. To prevent settling out,
the particles are kept in solution between the
pipetting steps by repeatedly finger-vortexing the
reaction vessel. Then, a vacuum is applied to the
desiccator for 20 minutes. Before the bombardment, the
bursting disks (3 disks), the particle-loaded macro-
carriers and the retaining net are fixed in accordance
with the instructions. Position 3 is chosen for the
Petri dish support charged with the suspension culture.
Then, the chamber is evacuated down to a vacuum of
27 inches (Hg), and the helium gas is passed into the
pressure chamber. At a pressure of 1200 psi, the
bursting disks rupture. The Petri dishes are then
sealed under sterile conditions (Parafilm), and the
cultures are incubated for 2 days at 24 C in permanent
darkness.
Histochemical detection of 0-glucuronidase activity
The transient expression of the P-glucuronidase struc-
tural gene can be detected by a histochemical method.
To this end, after transformation has been carried out
and an incubation time of 24 hours has elapsed, paper
filters of suspension cultures are transferred into a
Petri dish (50 mm 0) and overlaid with 1 ml of reaction
buffer (see below). Incubation is carried out overnight
at 37 C in an incubator. The number of zones which are
stained blue is determined at 40 x magnification using
a stereomicroscope.
To prepare the reaction buffer, 70 mg of X-Gluc
(5-bromo-4-chloro-3-indolyl-p-D-glucuronic acid,
Duchefa) are predissolved in 500 l of dimethyl sul-
foxide and the solution is resuspended in 100 ml of a
buffer solution (115 mM NaH2PO4 x 2 H20/Na2HPO4 x 2 H2O
pH 7, 10 mM Na2-EDTA, 0.5 mM K3 [Fe(CN)6], 0.5 mM K4
* Trademarks
CA 02379498 2009-06-23
28
[Fe(CN)6]). Aliquots of the reaction buffer thus
obtained can be stored over several weeks at -20 C.
Methods for quantifying the 0-glucuronidase and
luciferase activities
To quantify the enzyme activity of the reporter gene
products used, detection methods were used which rely
on bioluminescence and chemiluminescence. To determine
the enzyme activity, the proteins are extracted before-
hand. The "light quantity" generated in the subsequent
enzymatic reactions is quantified in a luminescence
spectrometer (Berthold, Lumat 9501). The measurement
value obtained represents the light quantity integrated
over a period of time. The measurement value is
indicated as light units measured, "LU".
Preparation of the protein extracts
The proteins are extracted using the "GUS-Light Assay"
(Tropix) and the "Luciferase Assay System" (Promega).
To prepare the protein extracts, the suspension
cultures are removed from the paper filters using a
spatula, the fresh weight is determined, and the cells
are then disrupted using a pestle and mortar with
addition of liquid nitrogen. Extraction buffer
(500 gl/g FW) is pipetted to the frozen material, and
the extract is defrosted and then thoroughly
homogenized. Using cut-off pipette tips, the suspension
is transferred into a reaction vessel on ice and
centrifuged for 1 minute at 4 C and 16,000 g. The
supernatants are kept on ice until the activity is
determined.
Extraction buffer: ("GUS-Light Assay")
The buffer solution (GUS lysis solution) is supplemen-
ted with Q-mercaptoethanol prior to use (final concen-
tration: 50 mm sodium phosphate buffer pH 7, 10 mM
Na2-EDTA, 0.1% SDS, 0.1% Triton* 10 mM (3-mercapto-
ethanol). The solution is stored at room temperature
until use.
* Trademark
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Extraction buffer: (Luciferase Assay System)
The buffer (cell culture lysis reagent), which has a
concentration of 5 x, is diluted with sterile double-
distilled H2O at a ratio of 1:5 (final concentration:
25 mM Tris-HC1 pH 7.8 with H3PO4, 2 mM CDTA, 2 mM DTT,
10% glycerol, 1% Triton) and stored at room temperature
until use.
Determination of (3-glucuronidase activity
Detection reaction:
gusA gene - (3-glucuronidase
3-glucuronidase
adamantyl-1,2-dioxethane-arylglucuronide
methyl-3-oxybenzoic acid (glucurone) + light (470 nm) +
adamantyl group
To detect the P-glucuronidase activity, the GUS-Light
Kit (Tropix) was used. The manufacturer's instructions
were followed for this.
Protein extract, reaction buffer and "accelerator" are
pre-warmed to room temperature prior to use. After
20 l of protein extract have been transferred into a
test tube (10 ml), the reactions are started by the
staggered (20 seconds) addition of reaction buffer (in
each case 180 l). The batches are incubated for 1 hour
at room temperature in permanent darkness. Then, in
each case 300 l of accelerator solution are pipetted
to the reactions (staggered, see above). Immediately
after the solution has been added, the individual
samples are measured in a luminescence spectrometer
(Lumat 9501, Berthold) with a delay of 5 seconds over a
period of, again, 5 seconds.
Negative control
The absolute values determined by the luminescence
spectrometer cannot be attributed directly to the
activity of a foreign gene. To determine the background
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value, which, in the case of 3-glucuronidase, may pos-
sibly also be attributed to a comparable, endogenous
activity, untransformed cell suspension cultures are
studied by means of the above-described detection
methods. The measurement values of this negative
control (BW, blank value; see Tables la and lb) addi-
tionally also contain the background values caused by
buffer substances. To calculate the promoter activi-
ties, the blank value is subtracted from the measure-
ment values of the transformed culture.
Reaction buffer ("GUS Reaction Buffer")
Prior to use, the substrate (glucurone, chemilumines-
cent substrate) is diluted 1:100 in "GUS Reaction
Buffer" (not complemented: 0.1 M sodium phosphate
buffer, pH 7, 10 mM Na2-EDTA).
Luciferase activity determination
Detection reaction:-
luc gene - luciferase (Photinus pyralis)
luciferase + Mg2+
ATP + Luciferin + 02 AMP + oxyluciferin + PPi +
light (560 nm)
To determine the luciferase activity, the Luciferase
Assay System (Promega) was used. The ratio of protein
extract to reaction buffer was varied.
Both protein extract and reaction buffer (see below)
are prewarmed to room temperature. After in each case
20 l (100 l) of protein extract have been transferred
into a 10 ml test tube, 100 l (50 l) of reaction
buffer are added to the sample and the batch is then
measured immediately in the luminescence spectrometer
(Lumat 9501, Berthold) over a period of 10 seconds.
According to what has been said for determining the
(3-glucuronidase activity, a blank value is determined
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which is used for correcting the measurement values of
transformed cultures.
Reaction buffer ("Luciferase Assay Reagent")
To prepare the reaction buffer, the substrate
(Luciferase Assay Substrate) is dissolved in the
"Luciferase Assay Buffer" (final concentration: 470 M
luciferin, 270 M coenzyme A (lithium salt), 530 M
ATP, 20 mM trizin, 1.07 mM (MgCO3)4Mg(OH)2 x 5H20,
2.67 mM MgSO4, 0.1 mM Na2-EDTA, 33.3 mM DTT, pH 7.8).
The reaction buffer is divided into aliquots and stored
at -70 C.
Determination of the relative promoter activity
To quantify the promoter activity in promoter/ reporter
gene constructs, the transient expression of the repor-
ter genes is measured by means of the above-described
enzymatic detection methods. However, when using direct
gene transfer to determine promoter activity, the
variation, between the individual transformations, of
the measurement values obtained is frequently substan-
tial. In the case of ballistic transformation, the
efficiency of the gene transfer is affected on the one
hand by the physiological state of the transformed
tissue after the treatment. On the other hand, physical
factors are also of importance, for example differences
in the loading of the microcarriers or in the
distribution of the particle suspension on the
macrocarrier. The measurement values must therefore be
supported by a calibration of the measurement system.
Theory of calibration
Test plasmid
Test promoter: To quantify the promoter activity, the
transient expression of the reporter gene is determined
by determining the enzymatic activity of its gene
product. However, the promoter activity of the test
promoter cannot be shown as an absolute value of the
enzymatic activity determination. Quantification
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requires a comparison with the constitutive promoter
(standard promoter).
Standard promoter: In a parallel set-up, the expression
of the reporter gene under a constitute promoter (here:
CaMV 35S promoter) is studied. The promoter activity of
the test promoter is indicated as the ratio activitytest
promoter/ actiVitystandard promoter, the activity of the
standard promoter equalling 1. Using expression of the
reporter gene under a standard promoter as the
reference base allows, inter alia, a comparison of the
results from repeated experiments.
Calibration plasmid (internal standard)
To record the transformation efficiency of individual
bombardments (transformations), the microcarriers are
loaded simultaneously with a test plasmid and a
calibration plasmid. The constructs used (test plasmid,
calibration plasmid) carry different reporter genes,
thus allowing their expression data (enzyme activities)
to be recorded in parallel.
The structural gene of the calibration plasmid is under
the control of a constitutive promoter (here: CaMV
d35S, strength 35S promoter). The measurement values
(enzyme activities) for the calibration plasmid reflect
the transformation efficiencies of individual
bombardments. All measurement values obtained for the
internal standard (calibration plasmid) in one
experiment are considered together and divided by the
maximum value obtained in the experiment. Thus, the
highest measurement value leads to a transformation
efficiency of 1 (corresponds to 100%). The
transformation efficiency (from 0-1) is indicated as
relative light units "rLU". The absolute measurement
values obtained in the study for the test plasmid (test
promoter, standard promoter) are corrected taking into
consideration the transformation efficiency of the
bombardment. To this end, the activity determined for
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the test plasmid is divided by the transformation
efficiency. The corrected values are termed relative
activities. The ratio of test plasmid to calibration
plasmid (7:3, w/w) is retained in all experiments.
Copy number
Identical quantities of test plasmid DNA are employed
in the transformation (here approx. 0.7 g per bombard-
ment). When comparing the activities of test promoters
and standard promoter (see above), plasmid size, i.e.
the number of molecules (copy factor) per pg of test
plasmid DNA must be taken into consideration. The
number of standard promoter molecules (test plasmid)
employed per bombardment is taken as the reference
value and made equal to 1. The measurement values for
the activity of the test promoters are corrected taking
into consideration the number of molecules (per
bombardment).
Calculation of the relative promoter activity
The example which follows shows an example of indi-
vidual calculation steps to clarify this further.
The data refer to a study on the quantification of the
relative promoter activity of the A-subunit promoter
(70 kD) of the Beta vulgaris V-type H+-ATPase (test
promoter, plasmid: pBVA-70/LUC). The CaMV 35S promoter
(plasmid: PCaMVLN) was used as reference (standard
promoter). Both test plasmids carry the luciferase
structural gene (LUC). To record the transformation
efficiency, the calibration plasmid pFF19G was used in
the cotransformation. It carries the 3-glucuronidase
structural gene (GUS) under the control of the enhanced
35S CaMV promoter.
The following combinations of test plasmid x calibra-
tion plasmid were employed:
1. Test promoter: 70 kD UE-ATPase (pBVA-70/LUC) x
CaMV d35S (pFF19G)
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2. Standard promoter: CamV 35S (PcAMVLN) x CaMV d35S
(pFF19G)
Plate LUC LUC GUS GUS Effi- Relative Mean
acti- activity acti- activity ciency LUC
vity (-BV) vity (-BV) activity
LU LU LU LU rLU rLU rLU
1 8070 7967 28734 22286 0.65 12257
2 10977 10874 40238 33790 0.99 10984
13 13834 13731 40640 34192 1 13731 12324
Table la: Calculation of the relative luciferase
activity for the promoter construct of the 70 kD
subunit of Beta vulgaris V-type H+-ATPase (70 kD UE
ATPase/LUC x CaMV d35S/GUS)
Background activity: The absolute values obtained from
the determination of the enzyme activity (LUC and a-GUS
activity) of transformed cells were corrected by taking
into consideration the measurement values of the nega-
tive control (absolute value - blank value (BV)). The
blank value for the LUC activity was 103 LU. The blank
value for the GUS activity was 6448 LU. The LU (light
units) indicated refer to the quantity of protein
extract employed (here: 20 l). Transformation
efficiency: the highest measurement value (GUS acti-
vity) for the internal standard CaMV d35S/GUS, and thus
the highest transformation efficiency, was obtained in
plate 3. The values for the LUC activities of the
remaining transformation batches were corrected by
taking into consideration the respective transformation
efficiency.
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Plate LUC LUC GUS GUS Effi- Corrected Mean
acti- activity acti- activity ciency LUC
vity (-BV) vity (-BV) activity
LU LU LU LU rLU rLU rLU
1 4518 4415 13756 7308 0.21 21024
2 13572 13469 20566 14118 0.41 32851
13 11256 11153 17051 10603 0.31 35977 29951
Table lb: Calculation of the relative luciferase
activity for the CaMV 35S promoter (35S CamV/LUC x CaMV
d35S/GUS); see legend to Table la
To calculate the activity of the test promoter, the
mean of the relative luciferase activity (see Table la)
is corrected by taking into consideration the molecule
number (copy factor) (see below). The relative strength
of the test promoter is now expressed as the ratio
between activity of the test promoter and activity of
the standard promoter. In the present example, a value
of 0.49 results for the relative promoter activity of
the subunit A promoter of the Beta vulgaris V-type
H-ATPase .
Promoter LUC Copy Corrected Promoter
activity factor LUC activity strength
(rLU) (rLU)
70 kD subunit 12324 1.2 14789 0.49
ATPase
35S CaMV 29951 1 29951 1
IV. Construction of the plasmid constructs for the
ballistic transformation
Figure 2 shows the construction of the construct
pBVA-70/LUC. To obtain the luciferase construct
pBVA-70/LUC, the 5'-regulatory region (1236 bp) of the
gene for subunit A of the Beta vulgaris V-type
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H+-ATPase was liberated from plasmid pBVA-70 by means
of a PstI/BglII digest. The genomic fragment was subse-
quently cloned into the vector pBluescript SKII+
(Stratagene) upstream of the BamHI/HindIII cassette of
vector pCaMVLN via the PstI/HindIII cleavage sites. The
cassette contains the luciferase structural gene and
also the polyadenylation region of nopalin synthase
gene. Vector regions (U) are emphasized particularly in
the BVA-70 promoter/LUC/CaMV/terminator cassette.
Figure 3 shows the construction of construct
pBVA-16/LUC. To obtain the luciferase construct
pBVA-16/LUC, the PstI/BglII fragment of the genomic
subclone pBVA-16/1 (which was described in Figure 2)
was cloned into the vector pBluescript II SK+
(Stratagene) upstream of the BamHi/Hindlil cassette of
the vector pCaMVLN via the cleavage sites PstI/HindIII.
See legend to Figure 2.
V. Representation of the cloned 5'-deletions for the
V-ATPase promoters A, cl and c2
Promoter deletions of the BVA/16-2 promoter/LUC
construct
The starting construct for the promoter deletions is
the promoter/leader luciferase construct pBVA/16-2 LUC.
This construct contains the overall promoter region
with 1923 bp and additionally 87 bp of the leader
(-1923 to +87 = 2010 bp). The ATPase promoter is to be
deleted with the aid of the "Exo Mung Bean Deletion
Kit" (Stratagene, 1997), starting from the 5'-end.
Figure 4 shows the BVA/16-2 promoter deletions. The top
row of the figure shows the promoter region of the
original genomic clone pBVA/16-2 with the leader up to
the translation start (ATG). The transcription start is
shown as +1. The individual deletion clones are shown
schematically underneath, and the number of, the
deletion clone is given. The number on the left-hand
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side shows the length of the remaining promoter, and
the number on the right-hand side at the end of each
row shows the total length of the deleted promoter and
the leader portion (in each case +87 bp) in the
promoter/ luciferase construct.
To prevent digestion of the vector, too, a restriction
cleavage site for an enzyme in the MCS on the left of
the vector is selected which cleaves neither in the
promoter nor in the reporter gene. A restriction enzyme
with a 3'-overhang requires no fill-in reaction. In the
case of a restriction enzyme with a 5'-overhang, for
example NotI, the sticky end is filled up after the
digest with a-thio dNTPs with the aid of Klenow
polymerase. First, the 5'-end of the promoter is
shortened by 1121 bp with the aid of an Spel digest,
starting at the 5-end. In the promoter/leader fragment
of the construct, 812 bp of the promoter plus 87 bp of
the leader = 889 bp remain. The Spel digest results in
a 5'-restriction overhang. The enzyme exonuclease III
digests 5'-restriction overhangs which have not been
filled up with a-thio-dNTPs. After the construct has
been digested with NotI, subsequently filled up with
a-thio-dNTPs and then digested with Spel, the
5'-deletion of the promoter with exonuclease III can be
started (initially, 10 deletion points are chosen). The
vector end of the construct remains unaltered. After
the exonuclease III has been digested, the remaining
overhangs are digested with mung bean nuclease. This
gives rise to two smooth ends which can be ligated and
subsequently transformed into E. coli competent cells.
After plasmid isolations and subsequent test digests
with SacI/XbaI, the selected plasmids are subjected to
incipient sequencing (TOPLAB) with the T3 vector primer
(sense primer). Starting from the MCS on the left-hand
side, the overhang of the vector pBluescript II SK into
the 5'-deleted promoter is sequenced. Selected deletion
zones are employed in the ballistic transformation
using the particle gun: No. 4 with 600 bp promoter
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(687 bp promoter + leader), No. 9 with 541 bp promoter
(628 bp promoter + leader), No. 17 with 364 bp promoter
(451 bp promoter + leader), No. 20 with 294 bp promoter
(381 bp promoter + leader), No. 19 with 223 bp promoter
(310 bp promoter + leader) and No. 22 with 180 bp
promoter (267 bp promoter + leader).
Promoter deletions of the BVA/16-1 promoter/LUC
construct
In the subsequent studies, the promoter of isoform 2 of
the subunit c of B. vulgaris V-type H-ATPase
(BVA/16-2) will be compared with the promoter of the
other isoform known to date (isoform 1 subunit c of
Beta vulgaris V-type H+-ATPase, BVA/16-1). Thus, the
promoter/leader luciferase construct pBVA/16-1 LUC
(Lehr et al. 1999) is also subjected to 5'-promoter
deletions (see above). This promoter (BVA/16-1) was
cloned into vector pBluescript II SK+ upstream of the
BamHI/HindIII cassette of the vector pCLN (with the
luciferase structural gene) as a PstI/BglII fragment.
The original construct contained the entire promoter
region with 1126 bp and additional 131 bp of the leader
(-1126 to +131 = 1257 bp). Finally, the deletion clones
are also subjected to incipient sequencing. A clone in
which deletions are carried out as far as into the
leader is generated to act as a control for the
promoter deletion clones. This clone therefore only
contains 51 bp (+81 to +131) of the leader upstream of
the luciferase. Initially, selected deletion clones are
employed in the ballistic transformation with the
particle gun: No. 164 with 863 bp promoter (994 bp
promoter + leader), No. 1 with 662 bp promoter (793 bp
promoter + leader), No. 34 with 361 bp promoter (492 bp
promoter + leader), No. 55 with 110 bp promoter (241 bp
promoter + leader) and the control No. 93 with 51 bp of
the leader without promoter.
Figure 5 shows the BVA/16-1 promoter deletions. The top
row shows the promoter region of the original genomic
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clone pBVA/16-1 with the leader up to the translation
start (ATG). The transcription start is shown as +1.
The individual deletion clones are shown schematically
underneath, and the number of the deletion clone is
given. The number on the left-hand side shows the
length of the remaining promoter, and the number on the
right-hand side at the end of each row shows the total
length of the deleted promoter and the leader portion
(in each case +131 bp) in the promoter/ reporter gene
construct.
Deletions for the V-ATPase A promoter
The original construct pBVA/70-LUC contained 1035 bp
5'-upstream sequence based on the transcription start.
The following deletions were generated analogously to
the procedure for cl and c2:
Deletion Position based on the
transcription start
d 15/47 -30
d 15/ -108
d 15/49 -164
d 13/4 -270
d 9/180 -356
s 6/80 -494
d 4/47 -591
d 4/46 -682
7-5LUC -1035
(=pBVA-70-LUC)
Table 2: Deletion constructs of pBVA/70-LUC
VI. Comparison of the promoter activities of A, cl, c2
and CaMV35S under standard conditions
Figure 6 shows the comparison of the activity of
different promoters under control conditions. In this
experiment with the transient gene expression by means
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of the ballistic transformation, the activities of the
individual promoters are determined indirectly by the
luciferase activity. The three V-type H-ATPase
promoters BVA/16-1, BVA/16-2 and BVA/70 are compared
with the CaMV 35S promoter. The end values of the
luciferase activities are corrected with regard to each
other by a cotransformation with the construct pFF19G.
The diagram shows the corrected end values with the
standard deviations.
Luciferase
activities:
corrected end
values [RLU]
1. pBVA/16-1 LUC pFF19G (1:1) 561355 14.06 4.64
2. pBVA/16-2 LUC pFF19G (1:1) 120958 3.03 1
3. pCLN pFF19G (1:1) 39912 1
4. pBVA/70 LUC pFF19G (1:1) 134384 3.36
Table 3: End values together with promoter strengths
(based on pCLN or 16-2)
in this experiment under control conditions, the
V-ATPase BVA/16-1 promoter is approximately 5 times
more active than the BVA/16-2 promoter. The promoters
BVA/16-2 and BVA/70 show comparable activities (the
activity of BVA/70 is slightly greater than that of
BVA/16-2). In comparison with the CaMV 35S promoter in
the construct pCLN, the V-ATPase promoters BVA/16-2 and
BVA/70 are approximately 3 times more active, and the
strongest of these three ATPase promoters, BVA/16-1,
shows an activity which is 14 times higher than that of
pCLN.
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Detection of the constitutive activity of the V-ATPase
promoters for the isoforms cl and c2 in Beta vulgaris
leaves
The activities of the promoter/reporter gene constructs
for c2 (full-length promoter) and cl (5'-deletion d164)
were determined by particle bombardment on fully
expanded leaves of plants which have been raised in the
field. To this end, in each case 2 leaf disks (0 5 cm)
one on top of the other in Petri dishes on moist paper
filters were bombarded with the particle gun (1
bursting disk, 900 psi). Bombardment with 0.5 ug of
plasmid (V-ATPase promoter/pFF19G=7.3). After the
bombardment, the leaf disks were incubated for 24 hours
in the light while floating on water in Petri dishes.
Promoter Experiment No. LUC activity
CaMV 35S 1 2348
2 829
3 832
cl (d164) 565
527
534
c2 1691
1404
669
Table 4: Detection of the constitutive activity of the
V-ATPase promoters for the isoforms cl and c2 in Beta
vulgaris leaves
The results in Table 4 demonstrate that the promoters
c2 and c1(d164) show activities which are comparable
with CaMV 35S, even in fully expanded leaves. In
parallel, a Northern blot analysis revealed that, as
has already been shown for cl, the c2 isoform is also
expressed constitutively in the root, in young leaves
and in old leaves, as expected. As has already been
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described for ci (Lehr et al. 1999), salt stress of the
plants results in an increased amount of transcript.
Northern blot analysis on the expression of the c2
isoform in Beta vulgaris
Figure 7 shows a Northern blot analysis on the
expression of the c2 isoform in Beta vulgaris. In each
case, 10 Ng of total RNA were applied and hybridized
with a gene-specific probe from the 3-UTR region. In
each case two samples from control plants (left,
center) and one sample from salt-treated plants
(100 mM, 48 hours; right) were applied.
VII. Relative activities of 5'-deletions of the
promoters A, cl and c2 in comparison to CaMV35S
Figure 8 shows the comparison of the activities of
different deleted promoters under control conditions.
In this experiment with the transient gene expression
by means of the ballistic transformation, the
activities of the individual promoters are determined
indirectly by the luciferase activity. The deleted
V-type H+-ATPase promoters BVA/16-1 and BVA/16-2 are
compared with the CaMV 35S promoter. The end values of
the luciferase activities are corrected with regard to
each other by a cotransformation with the construct
pFF19G. The diagram shows the corrected end values with
the standard deviations. The numbers beneath the
columns refer to the various deletion fragments shown
in Figures 4 and 5.
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Promoter activities of various 5'-deletions of the
V-ATPase A promoter in comparison to the CaMV 35S
promoter
Promoter LUC activity
CaMV 35S 48766
70-5LUC(-1035) 93459
(full-length promoter)
d4/46 (-682) 90716
d4/47(-591) 89906
d6/80 (-494) 62572
d9/180(-356) 56545
d13/14(-270) 77619
d15/49(-164) 43623
d15/7(-108) 43046
d15/479(-30) 44448
Table 5: Comparison of the activities of various
deleted promoters of the V-ATPase promoter of subunit A
under control conditions
Table 5 shows the comparison of the activities of
different deleted promoters of the V-ATPase promoter of
subunit A under control conditions. In this experiment
with the transient gene expression by means of the
ballistic transformation, the activities of the
individual promoters are determined indirectly by the
luciferase activity. The deleted V-type H+-ATPase
promoters of BVA/70 are compared with the CaMV 35S
promoter. The end values of the luciferase activities
are corrected with regard to each other by a
cotransformation with the construct pFF19G. The diagram
shows the corrected end values. The numbers of the
promoters indicate the various deletion fragments given
in Table 3.
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VIII. Promoter activities of various 5'-deletions of
the V-ATPase cl promoter and of the CaMV 35S promoter
during the grown of a Beta cell culture
Table 6 shows the Promoter activities of various 5'-
deletions of the V-ATPase cl promoter and of the CaMV
35S promoter during the grown of a Beta cell culture.
The activities were determined in Beta suspension cells
1.5, 3.5, 5.5 and 7.5 days after they were transferred
to fresh medium.
Days after Promoter activities (LUC)
transfer CaMV 35S d5/55(-110) d3/34(-361) dO/164(-863)
1.5 75141 51857 106816 165252
3.5 75170 59147 132474 219219
5.5 63916 44955 108072 157218
7.5 18192 16954 31877 51930
Table 6: Promoter activities of various 5'-deletions of
the V-ATPase cl promoter and of the CaMV 35S promoter
during the growth of a Beta cell culture
IX. Effect of NaCl(KC1) stress on the promoter
activities of A, cl, c2 and CaMV35S
Effect of NaCl (KC1 stress (125 mM/48 hours) on the
activities of a 5'-deletion of the A promoter (d4/46 [-
682bp]) and a 5'-deletion of the ci promoter
(d164[-863]) in Beta cell cultures
1.5 days after the last transfer, the cells were
suction-filtered on filter paper disks and incubated on
Petri dishes with control medium or with addition of
125 mM NaCl (KC1). After the bombardment, they were
incubated for a further 24 hours. Then, the LUC
activities were determined.
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Promoter Control LUC activity
+125 mM NaCl +125 mM KC1
CaMV35S 32527(100) 8384(26) 4711(14)
d164(cl) 67841(100) 51380(76) 22379(33)
CaMV35S 20924(100) 5036(24) n.d.
d4/46(A) 23165(100) 9886(43) n.d.
Table 7: Effect of NaCl or KC1 stress on the activities
of the V-ATPase promoters
The results demonstrate that the activities of the
V-ATPase promoters are considerably less affected by
NaCl or KC1 stress of the cells than the CaMV35S
promoter. Further information on this can be found in
Lehr et al. (1999).
Figure 9 demonstrates that the comparable effect is
also observed for the c2 promoter. The data shown are
the activities after exposure to 125 mM NaCl for at
least 24 hours. Also, Figure 9, at the bottom, shows in
the Northern blot analysis that the transcript
quantities for the V-APTase genes A, cl and c2 after
exposure to salt are all elevated compared with the
control treatment.
X. Effect of phosphate deficiency on the promoter
activities of A, ci, c2 and CaMV35S
Effect of phosphate deficiency (48 hours) on the
activities of a 5'-deletion of the A promoter (d4/46
[-682bp]) and a 5'-deletion of the ci promoter (d164
[-863]) in Beta cell cultures
1.5 days after the last transfer, the cells were
suction-filtered onto filter paper disks and these were
incubated on Petri dishes for 48 hours either in
control medium or in phosphate-free medium. After the
bombardment, they were incubated for a further
24 hours. Then, the LUC activities were determined.
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LUC activity
Promoter Control (+P) - Phosphate
CaMV35S 33084(100) 6842(20)
d164(cl) 97417(100) 32897(34)
CaMV35S 20924(100) 2678(13)
d4/46(A) 23165(100) 7406(32)
Table 8: Effect of phosphate deficiency (48 hours) on
the activities of a 5'-deletion of the A promoter
(d4/46 [-682bp]) and a 5'-deletion of the ci promoter
(d164 [-863]) in Beta cell cultures
The results demonstrate that the activities of the
V-ATPase promoters are markedly less affected by
phosphate deficiency of the cells than the activity of
the CaMV35S promoter.
XI. Effect of sucrose deficiency on the promoter
activities of A, ci, c2 and CaMV35S
Effect of sucrose deficiency (48 hours) on the
activities of a 5'-deletion of the A promoter (d4/46
[-682b ]) and a 5'-deletion of the ci promoter (d164
[-863]) in Beta cell cultures
1.5 days after the last transfer, the cells were
suction-filtered onto filter paper disks and these were
incubated on Petri dishes for 48 hours either in
control medium or in sucrose-free medium. After the
bombardment, they were incubated for a further
24 hours. Then, the LUC activities were determined.
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LUC activity
Promoter Control (+S) - Sucrose
CaMV35S 33084(100) 31740(95)
d164(cl) 97417(100) 71017(73)
CaMV35S 20924(100) 6933(33)
d4/46(A) 23165(100) 6780(29)
Table 9: Effect of sucrose deficiency (48 hours) on the
activities of a 5'-deletion of the A promoter (d4/46
[-682bp]) and a 5'-deletion of the cl promoter (d164
[-863]) in Beta cell cultures
The results demonstrate that sucrose deficiency of
cells affects the activities of the V-ATPase promoters
to a similar extent as the activity of the CaMV35S
promoter.
XII. Coordinated wound induction of the V-ATPase genes
A, c1, C2 and E in storage tissue of Beta beet
Expression of V-ATPase and V-PPiase genes after
wounding
To detect the steady-state transcript levels of the
V-PPiase and various V-ATPase subunits from the head,
stalk and membrane-integral region, samples were taken
at various points in time after wounding. After the RNA
was isolated, a Northern blot was carried out in which
15 pg of RNA were applied per point in time. The same
blot was developed repeatedly in succession with
different homologous biotin-labeled probes. To detect
the transcripts of the two isoforms of proteolipid c,
the gene-specific cl probe was stripped from the
membrane after detection before hybridization with the
gene-specific c2 probe took place. As can be seen from
Figure 10, the cl probe was not stripped completely;
the bands of the slightly larger transcript of the
isoform cl can still be recognized weakly above the
signals of isoform c2, which, however, demonstrates
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that the mRNAs of the isoforms were indeed detected
specifically. Since the transcripts of the A subunit
and of the V-PPiase also migrate over a similar
distance in the gel, the membrane was again stripped
between the detection of the mRNA for the A subunit and
for the V-PPiase.
The storage root of sugar beet is a tissue which
requires a great deal of energy supply to the tonoplast
in order to maintain its sucrose storage capacity.
Figure 10 shows that indeed both proton pumps in the
vacuoles show a significant basal expression, as can be
expected after staining with neutral red.
Figure 10 shows the Northern blot analysis for
detecting the gene expression of V-ATPase and V-PPiase
in storage parenchyma cells of sugar beet after
mechanical wounding. In each case 15 pg of RNA were
applied.
Wound-induced changes in V-ATPase at the protein level
A Western blot analysis is chosen to test if the
elevated transcription levels of a large number of
V-ATPase genes which are observed after wounding in
Northern blots are also reflected in elevated protein
quantities. Membrane proteins and the enriched
tonoplast fraction as well as the total microsome
fraction, all of which had been isolated 0, 10 and
72 hours after wounding, were fractionated by
electrophoresis in a 13% PAA gel and blotted onto a
PVDF membrane. The membrane was subsequently developed
with the antiserum against the K. daigremontiana
V-ATPase holoenzyme. It emerged that no significant
quantitative change in the V-ATPase subunits is
observed in the total microsome fraction (Figure 11B).
In the enriched tonoplast fraction, too, the protein
quantities of the subunits clearly remain constant
after wounding, with one exception: subunit c, whose
isoforms had also shown the strongest induction at the
CA 02379498 2002-02-18
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mRNA level, was markedly increased after wounding. This
induction was equally strong in the enriched tonoplast
fractions of two beets which were isolated
independently of each other (Figure 11A).
Figure 11 shows the Western blot analysis with a
polyclonal antiserum against the K. daigremontiana
V-ATPase holoenzyme and shows wound-induced changes in
V-ATPase on the tonoplast in the storage parenchyma of
the sugar beet. A. After wounding, the subunit
c protein quantity increases in the enriched tonoplast
fraction as a function of time. B. In the total
microsome fraction, the quantities of the individual
subunits remain unchanged. In each case 5 pg of protein
of the isolated membranes of two sugar beet were
applied, beet 1: tracks 1, 3 and 5; beet 2: tracks 2, 4
and 6.
Reduced V-ATPase H+-pump activity after wounding
As has been described in the above chapters, an
induction at the mRNA level was found for several
V-ATPase genes, and an elevated subunit c protein level
on the tonoplast was found by Western blot analysis,
after wounding. This gave rise to the question of
whether these changes would also lead to an elevated
proton pump performance of the V-ATPase. To study this,
the H+-pump activity of isolated membrane vesicles of
the microsomal and of the enriched tonoplast fraction
in the presence of the inhibitors vanadate for
P-ATPases and azide for F-ATPases was measured by means
of the fluorescent dye acridine orange. Instead of
sucrose, the pump medium contained 250 mM sorbitol to
exclude effects caused by the H+ sucrose antiporter.
As shown in Figure 12, the H+-pump activity of the
V-ATPase in the microsomal fractions is equally high
before and after wounding. In contrast, 3 days after
wounding, the proton pump activity in the enriched
tonoplast fraction is increased by a factor of 3.3.
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Accordingly, the protein levels of proteolipid c on the
tonoplast (Figure 12), which are elevated after
wounding, are accompanied by a significantly elevated
H+-pump activity of the V-ATPase localized therein.
Figure 12 shows wound-induced changes in the H+-pump
activity of the V-ATPase in the microsomal and in the
enriched tonoplast fraction in the presence of 100 pM
vanadate and 1 mM azide. While the initial rate of the
decrease in fluorescene ()F540) of acridine orange after
addition of 1 mM MgATP to vesicles of the microsomal
fraction (40 pg of protein) is equally high before and
after wounding, the enriched tonoplast fraction (10 pg
of protein) shows an increase by a factor of 3.3. The
data shown are the means of in each case 3 individual
measurements.
