MXPA99006383A - Polynucleotides encoding choline monooxygenase and plants transformed therewith - Google Patents

Abstract

A full length choline monooxygenase (CMO) cDNA was cloned from spinach and used to transform plants which do not naturally express CMO. A method is presented to improve stress tolerance of crops following engineering of CMO and BADH in plants that lack glycine betaine accumulation. Also provided are fragments useful as probes to isolate other CMO-type genes, and antisense sequences which inhibit the production of CMO. Reduction of glycine betaine as a consequence of antisense expression of CMO in species naturally accumulating glycine betaine improves the transgenic plant's tolerance toward pathogens and pests and/or enhances its nutritional quality.

Description

POLYINUCLEATES THAT CODIFY FOR MONOOXIGENASE HILL, AND PLANTS TRANSFORMED WITH THEMSELVES FIELD OF THE INVENTION The present invention was made with government support under a research project supported by the U.S. Department of Agriculture National Research Initiative Competitive Grants Program, grant No. 95-37100-1596. Mass spectral data were obtained at the Michigan State University-National Institutes of Health (NIH) Mass Spectrometry Facility, which is supported by the NIH, grant RR 00484. The government has certain rights to this invention.

BACKGROUND OF THE INVENTION While the world population is constantly increasing, the need for food to feed this growing population also increases directly. An unfortunate side effect correlated with growing populations is the rapid decrease in land available for crop growing. For example, soil in certain countries in Africa and Asia once marginally capable of sustaining food crops only a few decades ago is now completely arid and infertile, making crop production impossible. Unless drastic measures are taken, this situation is only getting worse with growing populations. In an effort to improve current conditions and to avoid any exacerbation of the problem, much attention has been placed on the ways in which food production can be optimized under sterile and severe conditions. The principle is that the soil currently unable to sustain crops could be used for cultivation. Current research is to consider the defense mechanisms that plants use naturally to survive in environmental conditions under environmental stress, and to determine whether these natural mechanisms can be exploited artificially, for example, through cross-breeding or gene transformation, thus producing more robust. One such defense mechanism that is promising is the accumulation of organic solutes. When bacteria, seaweed and many higher plants are exposed to salinity or drought conditions, they accumulate organic solutes. These solutes include polyols, proline and quaternary ammonium compounds. It is thought that they give the organism tolerance for environmental stress by balancing the osmotic pressures between the outside and the inside of their cells, thus allowing them to maintain their turgidity and growth. Not surprisingly, the biosynthetic pathways for these osmoprotective compounds have become the targets of metabolic engineering to improve the tolerance of an objective species to environmental stress. To date, there have been preliminary studies that have shown that these pathways can be genetically manipulated in higher plants, and that this can improve tolerance to various abiotic stresses (Tarczynski et al., 1993; Kishor et al., 1995; Lilius et al. 1996, Hayashi et al., 1997). There is evidence to suggest that the quaternary ammonium compound glycine betaine may be a more effective osmoprotective than polyols or proline (Mackay et al., 1984; Warr et al., 1988). In addition, glycine betaine is more attractive as a potential target for genetic engineering because, unlike proline or polyols, glycine betaine does not have a subsequent metabolic fate. Thus, in principle, this makes it simpler to genetically engineer the accumulation of glycine betaine because its rate of degradation is not a problem. In plants, such as bacteria, glycine betaine is synthesized by a two-step choline oxidation process. The first step (oxidation of choline to betaine aldehyde) is catalyzed by the enzyme choline monooxygenase (CMO). The second step (oxidation of betaine aldehyde to glycine betaine) is catalyzed by the enzyme betaine aldehyde dehydroxygenase (BADH). Certain upper floors, for example, spinach and sugar beet, accumulate glycine betaine in response to osmotic stress. However, many other species that include tomato, tobacco, potato, legumes, rice and some varieties of corn and sorghum, lack the capacity to synthesize it. The metabolic engineering of glycine betaine synthesis in these crops could therefore improve their tolerance to environmental stress. Although bacterial hill oxidases (Rozwadowski et al., 1991; Hayashi et al., 1997) or dehydrogenases (Lilius et al., 1996) are being explored for this purpose, the use of CMO (in conjunction with BADH) is preferable for the following reason. CMO requires the reduced ferredoxin of the light reactions of photosynthesis to function. Thus, CMO links the synthesis of glycine betaine with the light reactions of photosynthesis. This helps to level the supply of glycine betaine, with the demand for osmotic adjustment and osmoprotection, which rises rapidly after dawn as the water potential and the water content of the leaves under environmental stress due to salts or drought begin to decrease ( Hanson and Hitz, 1982). However, in some circumstances, the synthesis of glycine betaine is an inconvenient event. For example, in the manufacture of sugar beet, glycine betaine is a component of sugar beets that complicates the processing of sugar by inhibiting its crystallization. Therefore, the varieties of sugar beet with reduced levels of glycine betaine or without them in their roots would be convenient. It has been suggested that the accumulation of glycine betaine can make plants susceptible to insect pests (Corcuera, 1993) or microbial pathogens (Pearce et al., 1976). Therefore, under certain circumstances, it may be possible to improve the resistance of the plant to pests or pathogens by blocking the synthesis of glycine betaine. The potential of such application is available for many important crops that naturally accumulate glycine betaine, for example, wheat, barley, corn, sugar cane, sugar beet, spinach, cotton and sunflower. Blocking CMOs in crop species used as animal feed can also improve their nutritional value. Choline is a frequent food supplement for animals, and therefore, cells that contain a higher concentration of choline by virtue of blocking its conversion to glycine betaine, would be convenient. Therefore, a process to genetically alter plants to prevent them from producing glycine betaine would be very beneficial for many agriculturally related industries. To date, the gene encoding the enzyme responsible for the oxidation of betaine aldehyde in glycine betaine, BADH, has been cloned and has been successfully expressed in transformed tobacco. In contrast, the gene coding for CMO is hitherto unknown and, consequently, there have been no means to genetically manipulate plants using the gene for CMO. Since BADH and CMO are required for the production of glycine betaine, the unique transformation of BADH without CMO is useless to increase resistance to environmental stress, since glycine betaine is not produced. Similarly, when it is desired to block the synthesis of glycine betaine, it is more useful to block CMO than BADH. Blocking in the stage catalyzed by BADH can cause the accumulation of betaine aldehyde, which results from the oxidation of choline by CMO. This can inhibit the growth and productivity of the plant, because betaine aldehyde is a toxic metabolite and a structural analog of amino aldehyde intermediates of polyamine catabolism. Due to the above reasons, there is a need for means to isolate a gene that codes for a CMO-like enzyme, and ideally to identify the sequence of a gene that codes for a CMO-like enzyme. As a corollary, there is a need for a purified enzyme similar to the CMO. There is a need for a method to increase or decrease the concentration of glycine betaine in plants. Furthermore, there is a need for a method to genetically engineer organisms to increase their resistance to environmental stress conditions.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to a method for increasing the resistance in plants to severe environmental conditions. A method involving the transformation of plants with a gene encoding a protein having choline monooxygenase activity is specifically exemplified. Although any transformation method can be used, in a preferred embodiment of said method, a host cell of Agrobacterium tumefaciens is transformed with a vector that contains a DNA sequence encoding choline monooxygenase and is cultured; the A. tumefaciens cell is then used to transform a plant cell in accordance with procedures well known in the art. As a specific example to illustrate the teachings herein, it is disclosed that tobacco, which does not express an enzyme of the CMO type, has been successfully transformed by the methods of the present invention, to exhibit CMO activity. Transgenic tobacco that expresses chimeric genes for BADH and CMO synthesizes glycine betaine. This aspect of the invention increases the resistance of the transformed plant to conditions of environmental stress, increasing the concentration of glycine betaine in the tissues. Another aspect of the present invention relates to an isolated DNA sequence that codes for an enzyme that has characteristics of choline monooxygenase. An example of a DNA sequence according to the present invention is shown in Figure 2. Up to now, a sequence coding for CMO has not been obtained. This aspect of the invention provides, in the first instance, sequences that can be used in genetic engineering for the subsequent transformation and expression of proteins that exhibit CMO activity, thus facilitating the biosynthesis of glycine betaine in plant tissues, where previously it was absent . A further aspect of the present invention is directed to a method for isolating DNA sequences for enzymes of the CMO type. Herein, the isolation of a DNA sequence encoding a spinach choline enzyme enzyme (Spinacia oleracea) is described. This sequence provides means to utilize known recombinant DNA techniques to isolate sequences encoding CMO enzymes in other species. For example, the DNA sequence described herein, as well as fragments thereof, can be used as a probe to analyze cDNA libraries of plants known to express a CMO-like enzyme. Such techniques are well known in the art, and are commonly practiced successfully. The nucleotide sequences derived from Figure 2 are yet another aspect of the present invention, as described herein. "Derived from" is used in the present to indicate that it has been taken, obtained, received, tracked, replicated or inherited from a source (chemical and / or biological). A derivative can be produced by chemical or biological manipulation (including, but not limited to, substitution, addition, insertion, suppression, extraction, isolation, mutation and replication) of the original source, by means well known to those skilled in the art. A further aspect of the invention relates to a plant obtained by the methods described herein. This aspect of the invention includes plants transformed with a DNA sequence which hydrides with FIG. 2 under stringent conditions, and which codes for a protein exhibiting CMO activity, as well as fragments of FIG. 2 sufficient to code for CMO activity. . A preferred embodiment is a transgenic plant obtained by the methods described herein, which exhibits CMO activity.

Still further, another aspect of the invention relates to a method for reducing the amount of glycine betaine in plants that normally produce it, and sequences that are antisense to consequences that code for CMO, and which are thus useful in this method. Yet another aspect of the invention is a method for increasing the choline content of a plant cell by inhibiting the conversion of choline to glycine betaine, as well as plant cells and plants affected by this method.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an EPR spectrum of CMO. The CMO spectra reduced by sodium dithionite were obtained at 15K with a microwave energy of 20m W and a modulation amplitude of 10G. The spectrum shown is the average of 16 measurements. Figure 2 shows the nucleotide and deduced amino acid sequence of the pSc clone for cMO for cmo: The amino acid sequence determined for tryptic peptides is underlined; overlaps between peptide sequences are underlined twice. The N-terminus of the processed polypeptide is indicated by an asterisk. The Cys-His pairs conserved in Rieske iron-sulfur proteins are marked with crosses. The stop codon and the putative polyadenylation signal are enclosed in a box. The accession number of the gene bank for the nucleotide sequence shown is U85780. Figure 3 shows the expression of CMO in expanding and expanded spinach leaves. The plants had been irrigated with nutrient solution (0) or, for 10 days before the experiment, with nutrient solution containing 200 mM NaCl (200). (A) RNA biot analysis. The bands contained 5 μg of total RNA. The probe was a 532-bp DNA fragment [positions 660-1191 of pRS3 (fig.2)]. Staining with ethidium bromide showed that all bands contained equivalent amounts of RNA. The densitometry of autoradiographs indicated that salinization increased twice the levels of MMC mRNA in expanding leaves, and 7 times in expanded leaves. (B) Immunoblot Analysis. The bands contained 40 μg of total protein in leaves. Rabbit antibodies against BMD denatured with SDS were used for immunodetection. (C) Extractable activity. The CMO was tested in protein fractions consecutively to polyethylene glycol precipitation as described in Burnet et al. (1995). The bars are averages of three determinations; the standard errors were less than 16% of the average values. Figure 4 shows the nucleotide sequence of the cDNA for choline monooxygenase from sugar beet. Figure 5 shows the deduced amino acid sequence of the sugar beet choline monooxygenase cDNA.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides, in the first instance, a sequence coding for choline monooxygenase or CMO, the enzyme that converts choline to betaine aldehyde, and a novel method for increasing the resistance of plants to conditions under environmental stress. The nucleotide sequence of Figure 2, which represents the gene coding for spinach choline monoxigenase, is illustrative of a sequence encompassed by the present invention. The description of this sequence coding for CMO provides the necessary component to manipulate plants by genetic bioengineering techniques, so that enzymes active in CMO can be produced by plants previously lacking any CMO activity. This CMO coding sequence, as well as the sequences derived therefrom, also provides useful probes for the isolation and determination of gene sequences encoding other related enzymes of other species, by the use of widely known standard techniques. Specifically exemplified herein is a method for producing a plant (previously devoid of CMO enzyme) that expresses a protein exhibiting CMO enzymatic activity. This exemplified method includes transforming an Agrobacterium tumefaciens cell with a vector containing the DNA sequence of Figure 2, culturing the A: tumefaciens cell, and transforming a plant cell with the DNA sequence of Figure 2. Although the transformation of plants is exemplified herein by the Agrobacterium-mediated transformation, other known methods of transformation are encompassed by the present invention. These include, but are not limited to, the following: direct transfer of genes to protoplasts or cells using chemical or physical means such as polyethylene glycol mediated DNA uptake, biolistics, microinjection, electroporation, use of silicon carbide filaments, agroinfection, viral vectors , liposome fusion and liposome injection. Many commercially important crops lack the ability to accumulate glycine betaine, and therefore their tolerance to environmental stress can be improved by genetically engineered CMO and BADH. Crops include, but are not limited to, rice, corn, sorghum, tomato, potato, tobacco, lettuce, oilseed rape, and citrus genotypes. In corn, mutants of individual genes are known that lack glycine betaine accumulation. It was determined that the defect in these mutants lies in the first step of oxidation of choline, that is, that catalyzed by CMO (Rhodes and Rich, 1998; Lerma et al., 1991). In species such as rice, BADH and CMO, they need to be expressed to manipulate the synthesis of glycine betaine by genetic engineering techniques (Rathinasabapathi et al., 1993). However, corn mutants lacking glycine betaine may need to be transformed only with CMO to manipulate the synthesis of glycine betaine by genetic engineering techniques. The transformation of the crops mentioned above, and many other crops of interest, has become customary for the person skilled in the art; and the polynucleotides of interest described herein can be used in the transformation of these species in accordance with known techniques, to exhibit convenient characteristics and otherwise achieve the objectives described herein. Techniques for the transformation of plants are described, for example, in Gartland and Davey (1995), Jones (1995) and Potrykus and Spangenberg (1995). The nucleotide sequences described herein can be modified by any of a variety of mutagenic techniques known in the art. Site-specific mutagenesis is a preferred method, using a series of non-pairing polynucleotide primers to introduce nucleotide sequence variations at any desired locus within a gene or polynucleotide molecule. The exact sequence change produced by said means can be identified by determining the nucleotide sequence in the region affected by the change. A polynucleotide or modified gene can be tested to determine the functional effect of the modification by cloning the gene or polynucleotide into an expression vector, as described herein, or using any other expression system known in the art, and expressing thus the modified gene in a transgenic plant or microorganism.

The techniques of DNA cloning, isolation, modification, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, as well as various separation techniques, are known and commonly used by those skilled in the art. genetic manipulation technique. Several standard techniques are described in Old and Primrose (1981), Glover (ed.) (1985), Hames and Higgins (eds.) (1985), Sambrook and others (1989), Innis and others (eds.) (1990) and Harwood (1994), all of which are incorporated herein by reference. Abbreviations, when used, are those standard abbreviations in the field, and are commonly used in professional journals such as those cited herein. The fragments of (a) the nucleotide sequence of Figure 2, or (b) the nucleotide sequences derived from Figure 2, including antisense sequences to Figure 2, which are useful in accordance with the teachings herein, they can be produced by the use of restriction enzymes or by digestion limited by ßa / 31 exonuclease. These polynucleotide fragments are then cloned into expression vectors with an appropriate selectable marker, and finally transferred into plant cells according to the methods described above. The plant cells transformed with these fragments are usually grown in calluses, and / or regenerated in plants, which are then tested for the desired characteristics. In this way, fragments of a nucleotide sequence of interest, which are sufficient to confer the desired characteristics, are usually and predictably identified. As is known in the art, non-nucleotide matings at the third base or staggering base at a codon may occur, without causing amino acid substitutions in the final polypeptide sequence thus encoded. Likewise, minor modifications of nucleotides (for example, substitutions, insertions or deletions) in certain regions of a polynucleotide sequence can be tolerated and considered insignificant as long as such modifications result in changes in the sequence of amino acids that do not alter the functionality of the final product. It has been shown that chemically synthesized copies of whole gene sequences, or parts thereof, can replace the corresponding regions in the natural gene without loss of gene function. Homologs of specific DNA sequences can be identified by those skilled in the art using the nucleic acid cross-hybridization test under stringent conditions, as is well known in the art and described in Hames and Higgins (eds.) (1985). . Thus, in this description, it will be understood by those skilled in the art that deliberate variations in homologous sequences may exist or may be deliberately designed. Chemical synthesis of polynucleotides can be achieved manually by using well-established procedures such as those described by Carruthers (1983), or automated chemical synthesis can be carried out using one of several commercially available machines. The present invention also encompasses polynucleotide molecules having sequences that are antisense to polynucleotides that encode CMO enzymatic activity. The expression of an antisense polynucleotide molecule can block the production of the CMO type protein. To be useful in accordance with the teachings herein, it is only necessary that the antisense polynucleotide molecule be of sufficient size to block the production of a protein exhibiting CMO type activity. Such antisense polynucleotides can be constructed by techniques well known in the art, and their utility can be tested by usually determining their ability to block production. of a protein that exhibits CMO type activity in accordance with the teachings herein. The introduction of a chimeric gene for CMO with a sense polynucleotide molecule can also be used to block enzymatic activity by cosuppression. The theory and practice supporting the use of vectors with sense and antisense polynucleotide molecules to obtain transgenic plants with reduced levels of the target enzyme are well known in the art (Green et al., 1986; van der Krol et al., 1988; van der Krol et al., 1990 a and b; Vaucheret et al., 1992). Reducing the level of glycine betaine by blocking BMC can produce agronomically important genotypes in species that naturally accumulate glycine betaine, such as wheat, barley, corn, sugar beet, spinach, cotton and sunflower. The benefits may include greater resistance to pests and pathogens; or industrial processing facility, as in sugar beet, where glycine betaine is an inconvenient by-product that interferes with the crystallization of sugar. Methods for transforming the species mentioned above and many other crops are well known to those skilled in the art: for example, cotton (Umbeck et al., 1987), sunflower (Schoeffl and Baumann, 1985; Alibert et al., 1994), sugar beet (Lindsey and Gallois, 1990; Hall and others, 1996) and corn (Fromm et al., 1986), each of which is incorporated herein by reference. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested by those skilled in the art, and should be included within the spirit and scope of the invention. this application and the scope of the appended claims.

EXAMPLE 1 Isolation and analysis of proteins The enzymatically active CMO was purified, as described, from leaves of spinach plants (Spinacia oleracea L. cultivated variety Savoy, hybrid 612) that had been cultured and salinated with NaCl at 200 mM (Burnet et al., 1995). Non-heme iron was determined colorimetrically (Atkin et al., 1973) and acid-labile sulfur (Beinert, 1983). The protein was tested by the bicinchoninic acid method (Smith et al., 1985).

EXAMPLE 2 EPR spectroscopy Purified CMO samples were adjusted to pH 10 with glycine-KOH, and reduced by the addition of 1 mg of sodium dithionite per 300 μl. They were analyzed using a Bruker ECS 106 EPR X-band spectrometer with ER 4116 DM resonator and an Oxford liquid helium cryostat. The temperature was controlled by an intelligent Oxford controller, and monitored with a 3 mm thermocouple under the sample tube with liquid nitrogen as a reference. The frequency of the microwaves was sampled by a 5340A frequency counter from Hewlett-Packard. Data manipulations were carried out using the IgorPro 2.04 program (Wavemetrics, Lake Oswego, OR).

EXAMPLE 3 Determination of peptide microsequences Purified CMO was subjected to SDS-PAGE, and the Mf 5,000 band was subjected to blotting with polyvinylidene difluoride membrane. Tryptic peptides were obtained by the method of Fernandez et al. (1994), and purified by inverted phase HPLC using an Aquapore RP-300 column (C-8, 2.1 x 220 mm) developed with a gradient of trifluoroacetic acid-aceton. trilo. The isolated peptides were subjected to sequence analysis in an ABI protein / peptide sequence determiner model 476A (Perkin-Eimer ABD). The N-terminal sequence of the intact protein was determined in a sample further purified by inverted phase HPLC.

EXAMPLE 4 Cloning of cDNA Total RNA was extracted from salinized spinach leaves, as described (Hall et al., 1978), except that a step was added to precipitate carbohydrates with BaCI2 at 75 mM. Poly (A) + RNA was isolated using Sephadex Poly (U) (Hondred et al., 1987), and used to construct a cDNA library (9x106 pfu) in? UniZap XR (Stratagene). A 532 bp DNA fragment was obtained by (RT) -PCR for reverse transcription with primers based on CMO peptides; the initiators (+) and (-) were respectively: 5'-CCIGA (A / G) CA (A / G) AA (T / C) (T / C) TNGA (C / T) CCIAA (A / G) G-3 'and 5'-CCATCAT (A / G) TT (C / T) TC (C / T) TC (T / G / A) AT (A / G) TA (A / G) TA (A / G) TC-3 '. The RT-PCR reaction (100 μl) contained 3 ng of the first strand cDNA, 40 pmol of each primer, 20 μM of each of the four dNTPs, KCl at 50 mM, MgC.2 at 1.5 mM, gelatin a 0.001% (w / v) and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer) in Tris-HCl at 10 mM, pH 8.3. 40 cycles of 0.5 minutes each were carried out at 94 ° C and 30 ° C, and 1 minute at 50 ° C. The 532-bp fragment was isolated from low melting point agarose (Sambrook et al., 1989), and labeled with [α-32P] dCTP (> 3x109 cpm / μg) by the random primer method. Analysis of the library and excision in vivo were in accordance with the supplier's instructions. Analysis of the amplified library ("2x105 plates") with this probe yielded 18 positive clones, of which the largest was 1 189 bp. The unamplified library ("2x10 5 plates") was then analyzed with an EcoRI fragment of 223 bp from the 5 'region of this clone. Of eight positive clones, the sequence of two having the longest insertions (pRS3 and pRS5) in both strands was determined using the chain-terminated fluorescent dideoxynucleotide method (Prober et al., 1987). They were identical, except that pRS5 lacked 150 bp at the 5 'end and had a base change in the 3' non-coding region. The sequences were analyzed with the GCG Wisconsin sequence analysis package.

EXAMPLE 5 Biot analysis of DNA and RNA Genomic DNA from leaves was prepared, as described (Sambrook et al., 1989). Total RNA was isolated from control and saiinized leaves (Puissant and Houdebine 1990), denatured and subjected to electrophoresis in formaldehyde / 1.2% agarose gels (Sambrook et al., 1989). The RNA was quantified by the orcinol method (Dawson et al., 1986). Blotting and hybridizations were carried out using standard protocols (Sambrook et al., 1989). The molecular size markers were an RNA ladder (0.24-9.5 kb, GibcoBRL) for RNA blots, and DNA fragments? digested with Hind \\\ for DNA blots.

EXAMPLE 6 Production of antibodies and immunoblot analysis Rabbit antibodies against CMO were purified by SDS-PAGE and by inverted-phase HPLC, respectively. To determine the effect of salinization on CMO expression, proteins from spinach leaves were precipitated with PEG 8000 (Burnet et al., 1995), separated by SDS-PAGE, and transferred to nitrocellulose (Tokuhisa et al., 1985). . Mr pre-tinted markers (BioRad) were run simultaneously. The blots were treated with a probe with a dilution of 1: 500 of rabbit serum (Tokuhisa et al., 1985).

EXAMPLE 7 Evidence of a f2Fe-2S1 center of the Rieske type We searched for definitive evidence for a Fe-S center using EPR spectroscopy and chemical tests of acid-labile sulfur and non-heme iron. In EPR studies, after reduction by sodium dithionite, a rhombic spectrum with apparent g values of 2,008, 1,915 and 1,736 was observed (Figure 1). The maximum value of narrow and low field and the maximum value of wide and high field resemble those reported for certain types of groups [2Fe-2S] (Johnson, 1994). The gpr0m (x + gy + g? / 3) of this spectrum was 1.89, similar to that of many groups [2Fe-2S] of Rieske type bound with ligand at 2 His-, 2 Cys (Mason et al., 1992) . In contrast, [2Fe-2S] groups linked with ligand by 4 Cys residues typically have a gprom = 1-94 (33). The spectrum for CMO reached a maximum intensity at 15 K, a little lower than what is typical of the Rieske type groups, but still within the expected scale. Consistent with these results, Fe and S analyzes indicated that CMO contains approximately 2Fe and 2S per subunit (Table 1).

EXAMPLE 8 Cloning of cDNA The amino acid sequences were obtained for the N-terminus of CMO, and for 12 tryptic peptides. Two internal sequences were used to design primers for RT-PCR, which gave a DNA of 532 bp. Analysis of a library with this fragment produced several truncated CMC cDNA molecules; the 5 'region of the longest of these cDNA molecules was then used as a probe to isolate a full-length cDNA (Figure 2). This cDNA (1622 bp) had 5 'and 3' non-coding regions of 56 and 246 bp, respectively. A putative polyadenylation signal (AAATAAT) preceded the poly (A) sequence in 58 bp. The open reading frame (1320 bp) encoded 440 amino acids that included a 60-residue transit peptide. As the open reading frame begins with two adjacent ATG codons, it may be the beginning of the translation. However, the sequences flanking the second ATG codon link the consensus motif of translation initiation in plants (Joshi, 1987). The coding region included all the amino acid sequences determined for purified CMO. The size and composition of the deduced transit peptide were typical for a peptide targeting the chloroplast stroma (Cline et al., 1996), consistent with the location of CMO in the stroma (Brouquisse et al., 1989). The predicted Mr marker of the processed polypeptide was 42,884. In view of the fact that this value differs from that obtained by MALDI-MS in less than the experimental error of the method, the polypeptide for CMO is subject to very few modifications of post-translation, if any.

EXAMPLE 9 Comparisons of the primary structure No sequence in the public database had close general homology with CMO, so that no oxygenase of this type is known to date. The iron-sulfur proteins of the Rieske type share a consensus sequence Cys-X-His (15 to 17 amino acids) Cys-X-X-His, where X = any amino acid. This motif, which is considered to be involved in the union of the group [2Fe-2S] (Mason et al., 1992), was conserved in CMO (Figure 2). This finding strongly supports the chemical and EPR data that indicate that CMO has a center [2Fe-2S]. In addition to this conserved motif, some local homologies were found with several bacterial oxygenates, particularly members of the benzene dioxygenase enzyme family (Harayama et al., 1992). We also found weak local homology with bacterial alkyl group hydrolases and with iron-sulfur proteins of the Rieske type of mitochondria and chloroplasts. Representative data for each of these three families are shown in Table 2. The presence of the [2Fe-2S] group of the Rieske type, and the amino acid sequence of the protein, place the CMO in a new class of oxygenases of plants.

EXAMPLE 10 Induction of CMO by salinity In non-salinized plants, CMO mRNA levels were low in expanding and expanded leaves. Salinization increased these levels, especially in expanded leaves (Figure 3A). The CMO levels were similar to these changes (Figure 3B). The increase in extractable CMO activity in salinized plants observed previously (Brouquisse et al., 1989) can therefore be attributed to an increase in gene expression for CMO. The magnitudes of the increases in the amounts of protein and mRNA for CMO induced by salts, are comparable to those reported for BADH (Rhodes et al., 1993).

EXAMPLE 11 Analysis of genomic DNA After digestion of spinach genomic DNA with HindW ?, EcoRV or EcoRI, the biot analysis revealed individual bands of approximately 18, 9 and 3.7 kb, respectively (not shown). This is consistent with the existence of a gene for CMO that contains a large intron. The reconstruction experiments also suggested a single copy of BMC per haploid genome (not shown).

EXAMPLE 12 Transformation of tobacco The pGACMOl expression vector was constructed in plants in the following manner: an S / pal-EcoRVADN fragment containing the entire cDNA coding sequence for CMO was subcloned into the Hpa site of pGA643 (An et al., 1988) . The construct contained the following sequences between the 35S promoter of pGA643 and the cDNA: 15 bp of the SmaI to EcoRI vector sequence of pBluescript, 9 bp of sequences added to the cDNA for CMO during cDNA cloning (CGGCACGAG), and 21 bp of the sequence of the multiple cloning region of pGA643. In this expression vector, the insertion sequence was flanked by the 35S promoter of the cauliflower mosaic virus at the 5 'end and the 7' 3 'untranslated region of the Agrobacterium transcript (158 bp) at the 3' end. After the introduction of pGACMOl vector in LBA 4404 of Agrobacterium tumefaciens, tobacco was transformed by the leaf disc method (Ebert et al., 1988). Transformants were selected for resistance to kanamycin sulfate (100 mg / l). The presence of ANDc for CMO in the transgenic plants was verified using the polymerase chain reaction to amplify a cDNA fragment for CMO from genomic DNA isolated from leaves.

EXAMPLE 13 Expression of CMO in transgenic tobacco Total RNA was isolated from tobacco leaves of wild type Wisconsin 38, from vector control alone, and from fourteen plants positive for CMO cDNA by PCR analysis. These RNA samples (10 to 30 μg per band) were separated on an agarose gel in formaldehyde, and subjected to blotting. RNA blots were probed with cDNA for spinach CMO (Accl-EcoRV fragment). Vector control alone and wild-type tobacco had no signal, but ten plants of transgenic tobacco for CMO had an individual band that corresponded in intensity to approximately 50 to 100% of that observed in salinated spinach plants. The size of the message expressed in the transgenic tobacco was as expected (from approximately 1.8 to 1.9 kbp) (data not shown). Protein extracts were obtained from fully expanded young tobacco leaves (wild type tobacco, vector control and transgenic expressing mRNA for CMO). Partially purified protein fractions (PEG precipitation and molecular size exclusion chromatography) were used in a protein biot, and were treated with probe with rabbit antibody produced against spinach-purified CMO. In these biot, controls (vector control and wild type) lacked a positive signal, and samples from CMO positive plants had a band in the expected subunit size (approximately 45 kDa). The intensity of the CMO band was comparable to about 2% of the salinized spinach (data not shown). Transgenic tobacco expressing cDNA for CMO has only BMC but lacks the spinach betaine aldehyde dehydrogenase (BADH) enzyme. However, wild-type tobacco has a weak but detectable BADH activity (Rathinasabapathi et al., 1994). Therefore, it was expected that tobacco transformed only with spinach CMO synthesized glycine betaine. Glycine betaine levels were measured in vector controls alone, wild-type tobacco and transgenic tobacco positive for CMO, using mass spectrometry with fast atom bombardment (Rhodes and Hanson, 1993, incorporated herein by reference). The wild-type tobacco or the vector-transformed tobacco only contained small amounts of glycine betaine (ie, approximately 75 nmol per dry weight in grams). Transgenic tobacco expressing CMO had glycine betaine levels 2 to 4 times higher, indicating that the spinach CMO is functionally expressed in transgenic tobacco plants (Table 3). The synthesis of glycine betaine in the transgenic tobacco transformed with cDNA for spinach CMO, indicates that the cDNA of spinach for CMO introduced in the tobacco, is sufficient to express a functional CMO enzyme. In addition, the constitutive expression of the spinach CMO in tobacco had no detrimental effect on the growth and development of the plants.

EXAMPLE 14 Test of transgenic tobacco for tolerance to environmental stress Transgenic tobacco plants constitutively expressing CMO of spinach, wild type and vector control were grown only in large pots under greenhouse conditions that promote real evaporation demands. Plants of approximately 6 weeks of age were salinised at various levels by gradual increments of salinity (ratio of Na7Ca2 + of 5.7: 1) to the final level of 250 mM, and developed for several weeks before harvest. The tolerance of these plants to environmental stress was evaluated by measuring growth (changes in fresh weight) and water status (solute potential, data not shown) using standard techniques known in the art. The results shown in table 4 based on the fresh weight of shoots during harvest, indicate that two (235-3 and 231-23) of three plants expressing spinach CMO had significantly increased their shoot biomass over low controls control environmental stress and salinity. These results show that the transgene conferred an advantage for growth over the control in conditions under environmental stress.

EXAMPLE 15 Isolation of cDNA for CMO from sugar beet, and its use in the transformation of beet Total RNA was extracted from salinized leaves of sugar beet, as described (Hall et al., 1978), except that a step was added to precipitate carbohydrates with BaCI2 at 75 mM. Poly (A) + RNA was isolated using Sephadex Poly (U) (Hondred et al., 1987), and was used to construct a cDNA library in? UniZap XR (Stratagene). A spinach cDNA fragment was isolated including most of the coding region from low melting point agarose (Sambrook et al., 1989), and was labeled with [oc-2 PjdCTP (> 3x109 cpm / μa /) using the random initiator method. After analyzing the library, the sequence of the clones having the longest insertions in both chains was determined using the chain-terminated fluorescent dideoxynucleotide method (Prober et al., 1987). The sequences were analyzed with the GCG Wisconsin sequence analysis package. Two clones thus isolated represented a 1751 bp total length cDNA encoding a 446 amino acid polypeptide, and have a 3 'untranslated region of 377 bp. The deduced amino acid sequence of beet CMO comprised a transit peptide and a mature peptide of 381 residues that was 84% identical (97% similar) to that of spinach, and that showed the same consensus motive for coordinating a group [2Fe-2S ] of the Rieske type. As in the spinach CMO, a mononuclear Fe binding motif was also present (Jiang et al., 1996). The accession number of the gene bank for the mixed nucleotide sequence of beet BMC is AF 023132 (Figure 4). CDNA for sugar beet CMO in pBluescript is subcloned into a binary expression vector such as pGA643 (An et al., 1988) in antisense orientation. The binary vector is then mobilized in LBA4404 of Agrobacterium tumefaciens by triparental crossing (An et al., 1988). The transformed bacterium Agrobacterium is selected for resistance to streptomycin, kanamycin and tetracycline. The Kwerta sugar beet variety is propagated in vitro as shoots cultures in MS medium (Murashige &Skoog, 1962), supplemented with 30 g / l of sucrose and 0.25 mg / l of BAP (Lindsey and Gallois, 1990) under light keep going. For transformation, explants of the stem base (approximately 1 cm x 1 cm x 2 cm) are derived from axenic shoot cultures. The explants are incubated in a suspension of Agrobacterium (2.5-5x108 cells / ml) for 24 hours, and then cultured in selective medium (MS medium supplemented with 1 mg / l of BAP, 200 mg / l of carbenicillin, 100 mg / l of kanamycin and glycine betaine at 5 mM). The tissue sections are transferred to fresh selective medium every 15 days. The putative transgenic shoots identified by resistance to kanamycin are then transferred to rooting medium (MS medium supplemented with 5 mg / l NAA).

CMC cRNA levels are compared in vector controls alone and antisense CMO transformants by biot analysis of plant cRNAs under control conditions and under salt environmental stress (NaCl at 300 mM). The activity of CMO in leaf extracts is measured using the radiometric test described above (Burnet et al., 1995, incorporated herein by reference). The CMO protein in transgenic plants is analyzed by immunoblotting using antibodies specific for CMO. The levels of glycine betaine in various tissues of vector controls alone and antisense CMO transgenic beet are determined using mass spectrometry with rapid atom bombardment (Rhodes and Hanson, 1993). Beet antisense CMO transformants containing low levels of glycine betaine are compared to vector control plants only for salinity tolerance, growth characteristics and sugar level for root storage in plants under controlled conditions. The differential damage of these transformants by diseases and pests of specific insects is evaluated in a field test of multiple localities. The plants are grown under a standard pest management protocol. The severity of the incidence of pests and diseases on transgenic plants is evaluated at four different locations using a scale of 1 to 10 calculated for each pest or disease.

TABLE 1 Content of non-heme iron and acid-labile sulfur of CMO Analito nmoles mi "1 CMO subunit 57 (0.3) Fe 100 (5.0) 99 (6.0) Enzymatically active CMO was prepared as described (Burnet and others, 1995). The inverted phase HPLC elution profile (absorbance at 280 nm) of the preparation indicated that CMO represented 51% of the total protein. This value, together with a Mr of 43,026, was used to calculate the molar concentration of the CMO subunit. The values are means and standard error (in parentheses) for 3 or 4 determinations.

TABLE 2 Homology of amino acid sequence between CMO and other proteins with iron-sulfur centers of the Rieske type Species similarity Region size% compared sequence (residues) identity Fe-S mitochondrial protein Type Rieske Zea mays 72 29.2 36.1 Naphthalene Pseudomonas dioxygenase putida 195 29.2 43.0 Vanilato demethylase Pseudomonas sp. 58 31.0 43.0 The data shown is for members of representative families. The regions of homology that were compared included the conserved [2Fe-2S] group binding motif. Local homologies were first identified using BLASTP with non-redundant sequences in the NCBI database, and individual entries were compared using FASTA.

TABLE 3 Glycine betaine genotype (nmol "1 x g dry weight) Wisconsin 38 of wild type 62 ± 10 Control of vector pGA 88 ± 10 231 - 23 CMO, transgenic 1 169 ± 28 * 235 - 3 CMO, transgenic 2 270 ± 51 * 231-14 CMO, transgenic 3 293 ± 68 * Glycine betaine levels measured in fully expanded young leaves of Wisconsin 38 tobacco, vector control and three transgenic tobacco plants expressing spinach CMO. The transgenic plants were primary transformants. The replicas were generated by micropropagation. All the plants were grown under conditions without identical environmental stress. The values are means and standard errors for three independent samples. * = significantly different from the controls at p = 0.05.

TABLE 4 Genotype Fresh weight of the rods (g per plant) Salinized Control Vector control pGA 379a ± 41 195c ± 15 Wisconsin 38 384a ± 21 193c ± 22 231-23 CMO, transgenic 1 459b ± 27 235d ± 28 235-3 CMO, transgenic 2 478b ± 42 256d ± 12 231-14 CMO, transgenic 3 418b ± 41 222c ± 40 Growth of Wisconsin 38 wild-type tobacco plants, vector control and three transgenic tobacco plants expressing spinach CMO, under control conditions and environmental stress by salinity. The control plants were irrigated with nutrient solution, and the plants submitted under environmental stress by salinity with nutrient solution containing salts increasing in steps of 50mM every three days up to 150mM, concentration at which the treated plants were developed during three weeks. The transgenic plants were primary transformants. The replicas were generated by micropropagation. The final fresh weight of the rods is reported. The values are means and standard errors for four plants (Nuccio, M., Russell, B., North, K., Rathinasabapathi, B and A. D. Hanson, unpublished results). The means a - d followed by different letters are statistically significant at p = 0.05.

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Claims (46)

NOVELTY OF THE INVENTION CLAIMS

1. - A substantially purified polynucleotide molecule useful as a probe, characterized in that said polynucleotide molecule hybridizes to the DNA sequence of Figure 2 under astringent conditions.

2. The polynucleotide molecule according to claim 1, further characterized in that it encodes a protein that exhibits CMO activity.

3. The polynucleotide according to claim 2, characterized in that it has the nucleotide sequence of Figure 2.

4.- An expression vector comprising the polynucleotide molecule according to claim 1.

5.- A procedure for increase the resistance in plants to environmental stress conditions, characterized in that it comprises incorporating the polynucleotide molecule according to claim 2, in the genome of said plant under conditions whereby said polynucleotide is expressed, whereby said plant produces glycine betaine .

6. A plant produced by the method according to claim 5, or descendants of said plant.

7. The method according to claim 5, characterized in that said plant is tobacco.

8. - The plant according to claim 6, characterized in that said plant is tobacco.

9. The method according to claim 5, further characterized in that said plant is wheat.

10. The plant according to claim 6, further characterized in that said plant is wheat.

11. The method according to claim 5, further characterized in that said plant is barley.

12. The plant according to claim 6, further characterized in that said plant is barley.

13. The method according to claim 5, further characterized in that said plant is corn.

14. The plant according to claim 6, further characterized in that said plant is corn.

15. The method according to claim 5, further characterized in that said plant is sugar cane.

16. The plant according to claim 6, further characterized in that said plant is sugar cane.

17. The method according to claim 5, further characterized in that said plant is sugar beet.

18. The plant according to claim 6, further characterized in that said plant is sugar beet.

19. - The method according to claim 5, further characterized in that said plant is spinach.

20. The plant according to claim 6, further characterized in that said plant is spinach.

21. The method according to claim 5, further characterized in that said plant is cotton.

22. The plant according to claim 6, further characterized in that said plant is cotton.

23. The method according to claim 5, further characterized in that said plant is sunflower.

24. The plant according to claim 6, further characterized in that said plant is sunflower.

25. The method according to claim 5, further characterized in that said plant is rice.

26. The plant according to claim 6, further characterized in that said plant is rice.

27. The method according to claim 5, further characterized in that said plant is sorghum.

28.- The plant according to claim 6, further characterized because said plant is sorghum.

29. The method according to claim 5, further characterized in that said plant is tomato.

30. - The plant according to claim 6, further characterized in that said plant is tomato.

31. The method according to claim 5, further characterized in that said plant is potato.

32. The plant according to claim 6, further characterized because said plant is potato. 33.- The method according to claim 5, further characterized because said plant is lettuce. 34. The plant according to claim 6, further characterized in that said plant is lettuce. 35. The method according to claim 5, further characterized in that said plant is oilseed rape. 36.- The plant according to claim 6, further characterized in that said plant is oilseed rape. 37. The method according to claim 5, further characterized in that said plant is a citrus genotype. 38.- The plant according to claim 6, further characterized in that said plant is a citrus genotype. 39.- The seed of the plant according to claim 6. 40.- A method for decreasing the production of glycine betaine in a plant, characterized in that it comprises incorporating the polynucleotide molecule according to claim 2 in antisense form, the genome of said plant under conditions whereby said antisense polynucleotide is expressed, with which said plant does not produce glycine betaine or produces a smaller amount thereof. 41.- The plant produced by the method according to claim 10, or descendants of said plant. 42.- The seed of the plant according to claim 41. The method according to claim 40, further characterized in that said plant is sugar beet, 44.- The plant according to claim 41, further characterized because that plant is sugar beet. 45.- The substantially purified polynucleotide molecule useful as a probe, further characterized in that said polynucleotide molecule hybridizes to the DNA sequence of Figure 4 under astringent conditions. 46. The polynucleotide molecule according to claim 45, further characterized in that it encodes a protein that exhibits CMO activity.