AU780117B2 - Plant gene expression, controlled by constitutive plant V-ATpase promoters - Google Patents

Description

NAE19991074PC 1 as orginally filed Plant gene expression under the control of 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 particular, 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 potatoes, 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 BI, or light induction (Fluhr et al.

NAE19991074PC 2 (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 (Schmilling 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 pathogenstress-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 transcriptional effectors which, in their totality, result in NAE19991074PC IC 3 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 bananainfecting 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 pathogens (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 promoters 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- NAE19991074PC 4 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 constitutive promoter of cauliflower heat-shock protein Parts of this promoter can also lead to constitutive expression in heterologous, non-constitutive promoters, for example in the case of inducible promoters, promoters which can be regulated by other means or inactivated promoters. EP 0 559 603 furthermore describes that genes which mediate resistance to insects, herbicide resistance genes, antimicrobial genes, antifungal genes, antiviral genes and anti-feedant genes can be under the control of this promoter. The promoter has a very high constitute activity which, however, is only slightly elevated under stress conditions, which is a disadvantage.

The V-type H+-ATPase (V-ATPase) plays a central role in the cells of higher plants since it contributes substantially to establishing the electrochemical H gradient on the tonoplast. It accounts for a large proportion 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 expressed in cells which do not contain a large central vacuole. Plant V-ATPases are composed of at least 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.

Studies on the expression control of a plurality of V-ATPase genes in various plants (sugar beet, tobacco, carrot, maize, Mesembryanthemum 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; L8w et al. (1996) Plant Physiol. 110: 259-265; L6w 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).

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, constitutive 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.

In a first aspect of the invention there is provided a DNA construct in which 15 a plant V-ATPase promoter or its functional equivalent operatively linked to a heterologous 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. In particular, there is provided a DNA construct including a nucleotide sequence encoding a plant B. vulgaris V-ATPase promoter 20 subunit c isoform 2 [SEQ ID No. operatively linked with a heterologous gene.

Still further, there is provided a DNA construct including a nucleotide sequence of a plant B. vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No. 1] wherein said nucleotide sequence contains a deletion or is a hybrid of the B. vulgaris V-ATPase promoter subunit c isoform 2 sequence [SEQ ID No. 1] and the sequences of other homologous V-ATPase promoters.

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 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. 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 desired that the invention provide a novel constitutive plant promoter of the abovementioned type.

In a further aspect of the invention there is provided a polynucleotide encompassing the sequence of the promoter of B. vulgaris V-ATPase subunit c isoform 2 [SEQ ID No. This polynucleotide may contain a deletion or be a hybrid of the V-ATPase subunit c isoform 2 and the sequences of other homologous V-ATPase promoters.

Expedient embodiments of the invention are characterized 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 15 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 preferably an expression cassette. It is further preferred that the heterologously expressed gene is a 20 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 phosphotransferase 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 NAE19991074PC 7 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 fluorescent 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 recombinant vector is an expression vector. Also, transformed microorganisms are provided together with the recombinant 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, sunflowers, 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, NAE19991074PC 8 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, constitutive gene expression in all plant organs which is modulated by salt stress and other biotic or abiotic factors. The process according to the invention comprises 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 NAE19991074PC 9 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 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 NaCI or KCI, 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 i: 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 15 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 20 form of a deleted or hybrid V-ATPase promoter. Plant V-ATPase promoters according to the invention from dicotyledenous 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 is the V-ATPase promoter of the Beta vulgaris V-ATPase subunit c isoform 2 [SEQ ID No. 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 NaCI or KCI, 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.

11 The invention furthermore extends to a plant transformed with a DNA construct according to the invention which is resistant to biotic or abiotic stress, preferably 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 membraneintegrated 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 15 activity, which, as a rule, exceeds that of the 35S CaMV promoter.

oO* *e •go

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*ego ooa *oo• *•oo *oo *o* NAE19991074PC 12 It seems that polypyrmidine sequences which are present in the promoters play an important role in this context. 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 detected 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 KCl 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 deficiency/nutrient deficiency, which is in contrast to the NAE19991074PC 13 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 0 C) or low (2-5 0 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, poiyadenylation signals and enhancers.

NAE19991074PC 14 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 fulllength 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 understood as meaning those conditions under which hybridization takes place, and remains stable, at 60 0 C in x SSC buffer followed by repeated washing steps at 37 0 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, cl 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.

NAE19991074PC 15 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 efficiently 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 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"). Amplification 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 fragments 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 electrophoretic fractionation causes a delay in the migration behavior in particular in the high-molecular DNA range, NAE19991074PC 16 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 U/pl, 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/l, 0.05 U/pl, 0.025 U/pl, 0.01 U/pl], and the enzyme solutions are stored on ice. 25 of CsCl 2 -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 pl-aliquots. The DNA solutions (in each case 5 pg of DNA) are preheated for approximately 20 minutes in a waterbath at 37 0 C, and the individual reactions are started by adding 25 p. of the restriction enzyme solution. 25 ptl of pure buffer solution are added to a control sample. The digest is carried out for 2 minutes at 37 0 C and is stopped by adding 10 .1-portions of M Na 2 EDTA. The DNA is then precipitated by adding 1 volume of isopropanol and 0.1 volume of 3 M sodium acetate (pH washed with 70% ethanol, dried, and then resuspended in 10 pl-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).

NAE19991074PC 17 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, 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 temperature, and resuspended in 200 il 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 0

C.

10 x STE: 0.1 M NaCl, 10 mM Tris-HCl, pH 8, 1 mM Na 2

EDTA

Klenow reaction pg of the partially digested genomic DNA in an overall volume of 350 p1 of 1 x fill-in buffer (1 x Klenow buffer: 50 mM Tris-HCl, pH 7.2, 10 mM MgSO 4 0.1 mM DTT, Promega; 0.166 mM dGTP, 0.166 mM dATP) with U Klenow polymerase (5 U/il, Stratagene). The reaction is stopped by adding 0.5 volume of STE buffer (0.1 M NaCl, 10 mM Tris-HCl, pH 8, 1 mM Na 2

EDTA,

pH 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 ethanol. The dried pellet is resuspended in TE buffer and stored at -200C.

NAE19991074PC 18 Ligation into the Lambda Fix II/Xho I partial fill-in vector In a reaction volume of 10 p1, 1 Rg of lambda DNA (1 Rg/il Lambda Fix II/Xho I partial fill-in vector) and 0.4 [tg of genomic DNA fragments in ligase buffer mM Tris-HCl, pH 7.8, 10 mM MgC1 2 10 mM DTT, 1 mM rATP, Promega) are resuspended on ice and incubated overnight at 6-8 0 C with addition of 3.75 U of T4 DNA ligase (15 U/il, Promega). To check the ligation efficacy, 0.3 [g of the test insert (50 ng/pl, 12-kb pMF test insert; BamH I fragment partially filled up with dATP, dGTP) is ligated with 1 [pg 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 pl) is partially defrosted by holding it in the hand, 1 1l of ligation batch are added, and the mixture is stored on ice. To check the packaging efficacy, 0.2 pg of lambda control DNA (wild type c1857 Sam7, 0.2 Rg/pl) 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 tl of SM buffer (0.01% gelatin, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 8 mM MgSO 4 and 20 1l of chloroform, and the solution is mixed by careful shaking. The mixture is then centrifuged for 1 minute at 16,000 g and 4 0 C. The NAE19991074PC 19 supernatant is transferred into a reaction vessel and stored at 4 0 C until use. The titer plates are established the same day.

The following dilution levels of the individual packaging extracts are used: Packaging extract Dilution level Ligation of B. vulgaris genomic DNA 1 x 100/1 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 0 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 Rl 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 0 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 supernatants 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 the phage suspension is incubated for 15 minutes at room temperature and separated from bacterial residues by centrifugation for minutes at 2000 g. The supernatant is transferred NAE19991074PC 20 into a sterile vessel, treated with chloroform (end concentration and can now be stored for several months at 4 0 C. After the titer of the amplified genomic library has been determined, aliquots are stored at -80 0 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- 8 II. Methods for cloning promoter/reporter gene constructs for the V-ATPase promoters A, cl and c2, with the reporter genes luciferase (or 0-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 promoter A 1.3 kb HpaII fragment of the pBVA/70 clone was made blunt-ended using Klenow polymerase and ligated into the SmaI site of the vector pBluescript in order to obtain pBVA/70. Restriction with HindIII 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.

NAE19991074PC 21 BVA/16-1 promoter The BamHI/EcoRI GUS cassette of plasmid pBI221 (Clontech) was cloned into pBluescript (pBGUS), from which it was excised using BamHI and SalI. 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 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 p-glucuronidase structural gene from Escherichia coli (uid 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 hereinbelow termed pBVA-70(or 16)/GUS and 16)/LUC.

NAE19991074PC 22 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 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 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 P-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 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 NAE19991074PC 23 promoter/luciferase construct, the BamHI/HindIII cassette 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/HindIII. 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 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.

NAE19991074PC 24 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 macrocarrier, 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 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 H 2 0 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 pl of glycerol and mixed by vortexing. They are stored at 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 NAE19991074PC 25 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 transferred into a reaction vessel. 1 il of plasmid DNA (1 p.g/pil in TE buffer) is added with constant vortexing. The mixture is stored for 15 minutes in an ice bath. Then, 9 il of 2.5 M of CaCl 2 (sterile), 3.6 il of 1.2 mM spermidine (filter-sterilized) and 18.2 jil 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 supernatant is then removed. 6.4 Rl 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 transferred 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, ml-disposable pipettes, tweezers, filter paper disks mm Bichner funnels and a wash flask are NAE19991074PC 26 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 agar supplemented with 0.5 g/l casein hydrolysate, 125 mM sorbitol, 125 mM mannitol, after Ingersoll et al. 1996) are also used.

The Biichner 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 determined. 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 treatment 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 BIORAD) are sterilized by briefly immersing them in absolute ethanol and transferring them into a desiccator.

NAE19991074PC 27 The tungsten particle suspension is sonicated for 2 seconds at the maximum setting (Sonopuls HD Bandelin), and 5-Rl-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 macrocarriers 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 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 0 C in permanent darkness.

Histochemical detection of P-glucuronidase activity The transient expression of the P-glucuronidase structural 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 0 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 pl of dimethyl sulfoxide and the solution is resuspended in 100 ml of a buffer solution (115 mM NaH 2

PO

4 x 2 H 2 0/Na 2

HPO

4 x 2 H 2 0 pH 7, 10 mM Na 2 -EDTA, 0.5 mM K3 [Fe(CN) 6 0.5 mM K4 NAE19991074PC 28 [Fe(CN) 6 Aliquots of the reaction buffer thus obtained can be stored over several weeks at -20 0

C.

Methods for quantifying the P-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 beforehand. 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 [tl/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 supplemented with -mercaptoethanol prior to use (final concentration: 50 mM sodium phosphate buffer pH 7, 10 mM Na 2 -EDTA, 0.1% SDS, 0.1% Triton, 10 mM p-mercaptoethanol). The solution is stored at room temperature until use.

NAE19991074PC 29 Extraction buffer: (Luciferase Assay System) The buffer (cell culture lysis reagent), which has a concentration of 5 x, is diluted with sterile doubledistilled H 2 0 at a ratio of 1:5 (final concentration: 25 mM Tris-HCl pH 7.8 with H 3 P0 4 2 mM CDTA, 2 mM DTT, glycerol, 1% Triton) and stored at room temperature until use.

Determination of P-glucuronidase activity Detection reaction: gusA gene P-glucuronidase P-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 p.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 p1). The batches are incubated for 1 hour at room temperature in permanent darkness. Then, in each case 300 [pl 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 NAE19991074PC 30 value, which, in the case of p-glucuronidase, may possibly 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 Ib) additionally also contain the background values caused by buffer substances. To calculate the promoter activities, the blank value is subtracted from the measurement values of the transformed culture.

Reaction buffer ("GUS Reaction Buffer") Prior to use, the substrate (glucurone, chemiluminescent substrate) is diluted 1:100 in "GUS Reaction Buffer" (not complemented: 0.1 M sodium phosphate buffer, pH 7, 10 mM Na 2

-EDTA).

Luciferase activity determination Detection reaction: luc gene luciferase (Photinus pyralis) luciferase Mg 2 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 il (100 il) of protein extract have been transferred into a 10 ml test tube, 100 pl (50 pl) 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 P-glucuronidase activity, a blank value is determined NAE19991074PC E- 31 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 pM luciferin, 270 pM coenzyme A (lithium salt), 530 pM ATP, 20 mM trizin, 1.07 mM (MgCO 3 )4Mg(OH)2 x 5H 2 0, 2.67 mM MgSO 4 0.1 mM Na 2 -EDTA, 33.3 mM DTT, pH 7.8).

The reaction buffer is divided into aliquots and stored at -70 0

C.

Determination of the relative promoter activity To quantify the promoter activity in promoter/reporter gene constructs, the transient expression of the reporter 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 substantial. 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 NAE19991074PC 32 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 NAE19991074PC 33 the test plasmid is divided by the transformation efficiency. The corrected values are termed relative activities. The ratio of test plasmid to calibration plasmid w/w) is retained in all experiments.

Copy number Identical quantities of test plasmid DNA are employed in the transformation (here approx. 0.7 Rg per bombardment). 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 individual 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 -glucuronidase structural gene (GUS) under the control of the enhanced CaMV promoter.

The following combinations of test plasmid x calibration plasmid were employed: 1. Test promoter: 70 kD UE-ATPase (pBVA-70/LUC) x CaMV d35S (pFF19G) NAE19991074PC 34 2. Standard promoter: CamV 35S (PcAMVLN) x CaMV (pFF19G) Plate LUC LUC GUS GUS Effi- Relative Mean acti- activity acti- activity ciency LUC vity vity activity LU LU LU LU rLU rLU rLU 1 8070 7967 28734 22286 0.65 12257 2 10977 10874 40238 33790 0.99 10984 3 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 Background activity: The absolute values obtained from the determination of the enzyme activity (LUC and P-GUS activity) of transformed cells were corrected by taking into consideration the measurement values of the negative control (absolute value blank value 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 il). Transformation efficiency: the highest measurement value (GUS activity) 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.

NAE19991074PC 35 Plate LUC LUC GUS GUS Effi- Corrected Mean acti- activity acti- activity ciency LUC vity vity activity LU LU LU LU rLU rLU rLU 1 4518 4415 13756 7308 0.21 21024 2 13572 13469 20566 14118 0.41 32851 3 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) kD subunit 12324 1.2 14789 0.49 ATPase CaMV 29951 1 29951 1 IV. Construction of the plasmid constructs ballistic transformation for the Figure 2 shows the construction of the construct To obtain the luciferase construct the 5'-regulatory region (1236 bp) of the gene for subunit A of the Beta vulgaris V-type NAE19991074PC 36 H -ATPase was liberated from plasmid pBVA-70 by means of a PstI/BglII digest. The genomic fragment was subsequently 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 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/HindIII 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 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 The individual deletion clones are shown schematically underneath, and the number of the deletion clone is given. The number on the left-hand NAE19991074PC 37 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 NAE19991074PC 38 (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 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 NAE19991074PC 39 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 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 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 -1035 Table 2: Deletion constructs of 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 NAE19991074PC 40 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 561355 14.06 4.64 2. pBVA/16-2 LUC pFF19G 120958 3.03 1 3. pCLN pFF19G 39912 1 4. pBVA/70 LUC pFF19G 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 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.

NAE19991074PC 41 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 pg 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 cl(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 NAE19991074PC 42 described for cl (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 pg 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 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 NAE19991074PC 43 Promoter activities of various 5'-deletions of the V-ATPase A promoter in comparison to the CaMV 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 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.

NAE19991074PC 44 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 deletions of the V-ATPase cl promoter and of the CaMV promoter during the grown of a Beta cell culture.

The activities were determined in Beta suspension cells 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) d0/164(-863) 75141 51857 106816 165252 75170 59147 132474 219219 63916 44955 108072 157218 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 Effect of NaCi (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 cl 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.

NAE19991074PC 45 Promoter Control LUC activity +125 mM NaCl +125 mM KC1 32527(100) 8384(26) 4711(14) d164(cl) 67841(100) 51380(76) 22379(33) 20924(100) 5036(24) n.d.

d4/46(A) 23165(100) 9886(43) n.d.

Table 7: Effect of NaCI or KCl 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 NaCI or KCl stress of the cells than the 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, cl, c2 and 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 cl promoter (d164 in Beta cell cultures 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.

NAE19991074PC 46 LUC activity Promoter Control Phosphate 33084(100) 6842(20) d164(cl) 97417(100) 32897(34) 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 cl promoter (d164 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, cl, c2 and 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 in Beta cell cultures 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.

NAE19991074PC 47 LUC activity Promoter Control Sucrose 33084(100) 31740(95) d164(cl) 97417(100) 71017(73) 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 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 promoter.

XII. Coordinated wound induction of the V-ATPase genes A, cl, 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 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 NAE19991074PC 48 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 NAE19991074PC 49 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.

NAE19991074PC 50 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.

MAE19991074PC 51 SEQUENCE LISTING <1 1O> BASF AG <120> Plant gene expression under the control of constitutive plant V-ATPase promoters 130> BASF NAE 1074/99 <140> <141> <160> 3 <170> Patentln Ver. 2.1 <210> 1 <21 1 >2090 <212> DNA <213> Beta vulgaris <220> <223> Promoter subunit c isoform 2 <220> <221 promoter <222> (1 923) <400> 1 gatatcacac attcgtccat cgacgatttg cgaactttca aataggtacg taattcttt aatcttcaaa gttatttcac attagtcgac tattatgtag ttgaaaaaat ggagataata 120 ggaattagtt gaaaagggtg tttatataat tagacttaaa tttgattcat tttcatatat 180 ctgaaaacaa ggtatgtatg aaatttgatt catttatgac actgatgaaa aagttaacga 240 MAE19991074PC -5 52 tttagttctt ttttttaaaa ttccaatata aatttttgcc caaaactttt gcaaaatatc 300 catgttcgga aataaatttt gaaaaacaaa acaatatraa acttgc gaac-aacttt 360 tacaaaaatc cattttcaga aaaaaaaatt tacattaact tgcgaaatca aattgtgtat 420 gaaaaattta saattcctt tcacctataa ttgaaactca aagtgttaaa atttagaaaa 480 ggagaaaaat aaaaaatgac catttcatgc gaaatcaaat tgtgtatgaa aaacttaaaa .540 tittatitta aatataattg aaattcaaag tgttaaaatt tagaaaagga gaaaaattaa 600 aatgaccatt tcattcaaaa tcagattgtg tatgaaaaat ttaaattttt atttcaaata 660 taattgaaac tcaaagtgtg aacatttaga aaaggagaaa aattaaaatg atgaaaattt 720 gtaaaacatc aatttgtgaa atcagaattt agaagttaga caaggaaaaa aaactgaatt 780 gtcttatact tttcggttac aattttggga tcataaagaa attactgaaa tccatatcaa 840 aaactattat aaattacaaa aatgaataaa accraaaaa gaagaacatg acgatatttc 900 gtaaagaaca tcatactgat tataaaagaa catgcgcata ttagaattga gaaacaaaaa 960 actattcaaa atcacaaaaa tggataacaa catacaaaga acatgaaaga atcttattca 1020 caaaatggag gtgaacttaa atactaacft gcattttcag tttatttact acttagtatt 1080 aggcctaaaa atatcatcgc acgcatcgcg tgcacaaaag actagtgtta agtatcacaa 1140 gtcacaaact cacaactgat tttcatftag gctccatttg gtagggcgta aaacgttttc 1200 ccggaar-act atttttctcc atttttagtt ttacattgtt tggttgacaa aagagtgtaa 1260 aaccgttttc ccttggggta aaattactct tccaatgatc gaaaaccatt ttcctttcaa 1320 aatgaaggga aaactgtttt ccttatctct cttgttacac ttttctcact acctcettac 1380 tttccctttt attttacttt catttcatca tfttctftgc atgaaaccaa acaacggaaa 1440 actaatttg gaattgtgtt ttccattgta aattgttttc catgaaaatc attttacact 1500 gaaaatgttu tacaccctac caaacagagc cttcgtgtgc catgaatgca tgaccgattt 1560 caaattcgaa actggtgttt accgtttcct aattggtttc gagttcacaa ccaagtattc 1620 -TEactatgttg ctcacectaa agatgaatat ggtaaaacct tgaggtgggc tttggctaaa 1680 aaaagtccca ccaagcccca attctaggct cccaaaacca cgaaatttcc tggtactatt 1740 ccaaaacaaa aacaercacc tcctgatcaa accagaaaaa ataaaarata tttttgtttt 1800 cteccaattt ttcattttaa ttatcacgg gaaagtacac aataattcaa tcaagggtaa 1860 aaaaaataaa ataaaaagaa aagataagta taaacaaaag aaatttgtct tctctacatc 1920 ctcatatttc atccacgctc tcttcttcct ctcctctcat ctcctttttc aattcccaga 1980 tcggatcaag caattcatcg aacaccttcc gatcatcacc atcaaaaaaa atgtcaacag 2040 tctttaacgg cgatgaaacg gcgccgttct tcggctttct gggtgctgct 2090 MAE19991074PC 53 <210> 2 <211> 1153 <212> DNA <213> Beta vulgaris <220> <223> Promoter subunit c isoform 1 <300> <302> cDNA and genomic cloning of sugar beet V-type H+-ATPase subunit A and c isoforms: evidence for coordinate expression during plant development and coordinate induction in response to high salinity <303> Plant Mol. Biol.

<304> 1999 <305> 39 <306> 463-475 <400> 2 gggtgggggt gaagtggggg ctgcaggggt gggtgggggg ggctggtggc aggtattggt agtggtgcta ggtggtgatt atggaggaag aagggaaata gaagtggtta ac-accgaaaa 120 gtgagattaa gttttatctt gcaaaaataa tttatttttt ggtttgtttt cacaaagaat 180 gctttattaa ggtttttttt tgcaaaaaac tgaactttta aaaggttttt tttgataaat 240 ttcctaata ttaataaatg aaaatttttc taacatggga gatagcctgg acctataact 300 agttgttata acttataaga agagaattt gggcaaCtat gCggaaaata ggggaaattt 360 tggaaaaaat aagtaaaaga ttttaggtgt aaacttgcft gttataagtt ataaacttga 420 atttgttcta ttgatagtgt tggttaataa tgtgacaact aaatcgaaca taaaggttta 480 ggacccttte gacatcattg agagttagag gtgggcatga gccgggccag ttcgcgtgct 540 ggaacggcca gttcgegggc tggtacggcc aggcecgagc ccgatcaagg cacgacccgg 600 cttgttgtcc gctacgtgga ccgtgggccc atcacggtca aaaaaaaata tttaggctc 660 gcaccagcac gagcccgcgt aggccagccc gcgggctgaa tgttcctttc ctttgttt 720 tagcctaaaa ctgtcataaa tacaaaaaga tascataaaa eccggctag gccctcaata 780 ggcacgagcc cgtggaaaaa atccgaaaat tccaagccta gctagttact cacaagctca 840 NAE19991074PC -554 gacccgattc gtcatttc caaacccgtg ggccgagccg gtgaacaggc tcggcccggc 900 tcatgtccag gtgacaggtc tactgagagt tatgagttgt ttccccatgg gctacttgac 960 tacaaacctt ccatagttcc atgacacttc catctgagcc catgagataa aattaaggaa 1020 tacaaacaca aaactcaaca accaacccga aatatcgatc ggacttgtta gcacgtgtta 1080 tgttgggtcc catccaagaa actttctctc tcctcctcct tcataaaaaa accttctcac 1140 tgatcccatc cag 1153 <210O> 3 <21 1> 1073 <212> DNA 213> Beta vulgaris <220> <223> Promoter subunit A <300> <302> cDNA and genomic cloning of sugar beet V-type H+-ATPase subunit A and c isoforms: evidence for coordinate expression during plant development and coordinate induction in response to high salinity <303> Plant Mol. Biol.

<304> 1999 <305> 39 <306> 463-475 <400> 3 gaattctctg ctctggattc gacatcaaaa tttccaccgg gttactattc aaacagactg ctcaaatctc atttcttctc ttcgttctat ttcgacggtg cctcgctcta tctactggtc 120 tgtatgccag attatgagtc taacaggaaa tcttcactgg tgttttctgc ttaaagtaaa 180 ttgtcaaaaa gttcaaaagg cacaaaatgt ggctaagcta tgtgctcaaa tttgttgttt 240 actacttgat ttgttttctt tctctttatg cctgttttt ccccctttgt aaaaaaaaga 300 tacaagtata gatgaaggga ttctttatat tggccccatg tgttcgttag ataattacat 360 MAE19991074PC -5 55 tgtacoctca attcctcatc fttcttgaag ttctacgtag taccattgtg gttgcataag 420 caaatgataa tcatactttc atatcttagt taatgtacat cgtcacttgt gcttaacatg 480 tcataaacta atttccttgg tttaatactg gttacattaa ctaaatcttt tattcttaaa 540 tatttaagaa gtgtgcagta aattaagttt cttccaaatc ctcaataaga cattatactt 600 agaaaacttc tataaagtta cttatcaact tacagataac gaccaaagaa tcatcaccaa 660 aacagttatc gaacc-acata gaagctgcat agcttttgaa aaaggtgaag gtacaatata 720 aatctccaac aaatatagtg tatctactcc caaaagctat cctagtaata tccctatctc 780 aaaaacatat cttttatcaa ctttttccca acacaaactc aattgttaaa aactacaagg 840 aaacgttgtt taaccaatca ctatatttac attaaccata tttttaattt agttaaacct 900 ctcaagtctc actacattct taaaaaaagt tggagataga gtcatcataa ttcatagaga 960 aggaattgac aacatctaat aagaacgaat tacgaacgtg gcaaaatcac agacgaaaca 1020 tagcaaaact taaccctgca aatctcaatc agatttaaat aatccttttg ctg 1073

Claims (30)

1. A DNA construct including a nucleotide sequence encoding a plant B. vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No. operatively linked with a heterologous gene.

2. A DNA construct including a nucleotide sequence of a plant B. vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No. 1] wherein said nucleotide sequence contains a deletion or is a hybrid of the B. vulgaris V-ATPase promoter subunitc isoform 2 sequence [SEQ ID No. 1] and the sequences of other homologous V-ATPase promoters.

3. The DNA construct as claimed in claim 1 or 2 which further includes a second promoter which can be regulated in a different manner to that of the first promoter. *e

4. The DNA construct as claimed in any one of claims 1 to 3, wherein at least one further pyrimidine stretch is inserted in the plant promoter. S 15

5. The DNA construct as claimed in any one of claims 1 to 4, wherein the heterologous gene is a selection marker or a resistance-mediating gene or a gene of other medicinal, agronomical or other interest.

6. A polynucleotide including the sequence of a promoter of B. vulgaris V-ATPase subunit c isoform 2 [SEQ ID No. 1].

7. A polynucleotide including a nucleotide sequence of the plant B. vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No. 1] wherein said nucleotide sequence contains a deletion or is a hybrid of the sequence of the B. vulgaris V-ATPase subunit c isoform 2 promoter [SEQ ID No. 1] and the sequence of a different homologous V-ATPase promoter.

8. A recombinant vector which includes a DNA construct as claimed in any one of claims 1 to 57

9. The recombinant vector as claimed in claim 8, which is an expression vector.

A microorganism which is transformed with a recombinant vector as claimed in claim 8 or 9.

11. A transgenic plant cell or protoplast whose genome includes a DNA construct as claimed in any one of claims 1 to

12. A transgenic plant whose genome includes a DNA construct as claimed in any one of claims 1 to

13. A method for the controlled expression of a heterologous gene in a plant S 10 cell or a protoplast, which includes a) transforming the cell or the protoplast with a DNA construct as claimed in any one of claims 1 to 5 and b) subsequently exposing the transformed cell or the protoplast to a biotic or abiotic stress wherein the expression of the heterologous gene which has been transformed by means of the DNA construct is controlled.

14. A method for the controlled expression of a heterologous gene in a plant, which includes a) regenerating plant cells or protoplasts transformed with a DNA construct as claimed in any one of claims 1 to 5 to produce a transgenic plant and b) subsequently exposing the transgenic plant to a biotic or abiotic stress wherein the expression of the heterologous gene which has been transformed by means of the DNA construct is controlled.

15. A method for producing a recombinant protein, which includes a) transforming a plant cell or a protoplast with a DNA construct as claimed in any one of claims 1 to 5 and 58 b) subsequently exposing the transformed cell or protoplast to a biotic or abiotic stress wherein the recombinant protein transformed by means of the DNA construct is expressed.

16. A method of producing a recombinant protein in a plant, which includes a) regenerating plant cells or protoplasts transformed with a DNA construct as claimed in any one of claims 1 to 5 to produce a transgenic plant and b) subsequently exposing the transgenic plant to a biotic or abiotic stress wherein the recombinant protein transformed by means of the DNA construct is expressed.

17. A recombinant protein obtained by a method as claimed in claim 15 or 16.

18. The use of a DNA construct as claimed in any one of claims 1 to 5 for producing a recombinant protein in a plant cell or a protoplast.

19. The use of a DNA construct as claimed in any one of claims 1 to 5 for producing a recombinant protein in a plant. S**

20. The use of a DNA construct as claimed in any one of claims 1 to 5 for expressing a gene in a plant under biotic or abiotic stress.

21. The use of a nucleotide sequence encoding 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.

22. The use of a nucleotide sequence encoding a plant B. vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No. 1] wherein said sequence contains a deletion or is a hybrid of the B. vulgaris V-ATPase promoter subunit c isoform 2 sequence [SEQ ID No. 1] and the sequences of the other homologous V-ATPase promoters for expressing a gene in a plant under biotic or abiotic stress.

23. The use as claimed in claim 21 or 22, wherein at least one further pyrimidine stretch is inserted into the plant promoter.

24. A plant cell or protoplast which is transformed with a DNA construct as claimed in any one of claims 1 to 5 and which is resistant to biotic or abiotic stress.

A plant cell or protoplast as claimed in claim 24 which is resistant to salt stress.

26. A plant which is transformed with a DNA construct as claimed in any one of claims 1 to 5 and which is resistant to biotic or abiotic stress. 10

27. A plant as claimed in claim 26 which is resistant to salt stress.

28. A DNA construct including the B. vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No. 1] as hereinbefore described with reference to the Examples.

29. A recombinant vector which includes a DNA construct including the B. 15 vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No. 1] as hereinbefore described with reference to the Examples.

30. A transgenic plant cell, protoplast or plant whose genome includes a DNA construct including the B. vulgaris V-ATPase promoter subunit c isoform 2 [SEQ ID No. 1] as hereinbefore described with reference to the Examples. DATED this 5th day of January 2005 BASF PLANT SCIENCE GMBH WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA P20893AU00 CJH/ALJ/MEH