US 5,344,923 AGrant
Nucleotide Sequence Encoding for Bifunctional Enzyme for Proline Production
Issue Date:1994-09-06
•2 Claims
•8 Drawing Sheets
Abstract
The invention is directed to the isolation and identification of a mothbean (Vigna aconitifolia) cDNA clone encoding a bifunctional enzyme, delta.sup.1 -pyrroline-5-carboxylate synthetase, hereinafter P5CS, with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in plant proline production, also called biosynthesis.
Metadata
Assignee
- The Ohio State University Research Foundation
Inventors
- Desh P. S. Verma
- Chien-an A. Hu
- Ashton J. Delauney
Application Information
Application Number:US 9536957
Filing Date:1992-09-29
Priority Date:1992-09-29
Art Unit:182
Classifications
IPC:
C07H 1700C12N 1500
Field of Search:
80053643520523.2;23.6;23.4172.3
Patent Drawings (8 sheets)
Description
TECHNICAL FIELD
The invention relates to a novel bifunctional enzyme which catalyzes the first two steps in proline production in plants leading to osmoregulation in the plants.
BACKGROUND ART
Many plants synthesize and accumulate proline in response to osmotic stress. Drought and high salinity are the most important environmental factors that cause osmotic stress and impact negatively on plant growth and crop productivity. The role of proline biosynthesis in osmoregulation in bacteria is well established in, for example, Csonka, Microbiol. Rev. 53, 1989, pp. 121-147. The proline biosynthesis route in plants is not as well known but is thought to follow the biosynthesis route in bacteria. In Escherchia coli, the synthesis starts with the phosphorylation of glutamate by gamma-glutamyl kinase, hereinafter gamma-GK, which is encoded by the proB gene. The gamma-glutamyl phosphate thus formed is reduced to glutamic-gamma-semialdehyde, hereinafter GSA, by GSA dehydrogenase (GSD) which is encoded by the proA gene. The GSA thus produced spontaneously cyclizes to delta.sup.1 -pyrroline-5-carboxylate (P5C) which is delta.sup.1 -pyrroline-5-carboxylate reductase, hereinafter P5CR, which is encoded by the proC gene, to thereby form proline.
With the exception of P5CR which has been recently characterized (Krueger et al., Plant Physiol., 82, 1986, pp. 890-903; Rayapati et al., Plant Physiol., 91, 1989, pp. 581-586; Delauney and Verma, Mol. Gen. Genet., 221, 1990, pp. 299-305; Verbruggen et al., Abstract, Third International Congress, International Society for Plant Molecular Biology, Tucson, Ariz., Oct. 6-11, 1991), however, little is known about the other enzymes in this pathway in plants.
Therefore, it would be desirable to use genetic engineering of the proline production pathway in plants to counter osmotic stress to alter the level of a known osmoprotectant to thereby lead to a significant enhancement of crop performance under conditions of salt and drought stress.
BRIEF DISCLOSURE OF INVENTION
The invention is directed to the isolation and identification of a mothbean (Vigna aconitifolia) cDNA clone encoding a bifunctional enzyme, delta.sup.1 -pyrroline-5-carboxylate synthetase, hereinafter P5CS, with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in plant proline production, also called biosynthesis. By "bifunctional" herein is meant the ability of a single enzyme to perform two different catalyses and encode for gamma-GK and GSD.
The present invention was achieved by direct complementation of specific E. coli mutants with a Vigna nodule cDNA expression library. The isolated cDNAs have been fused to a strong plant promoter, and transferred to plants by Agrobacterium-mediated transformation. Transgenic Vigna roots have demonstrated overproduction of proline.
It is one object of the present invention to provide the complete nucleotide sequence of a P5CS cDNA clone, pVAB2.
Another object of the present invention is to provide a cDNA clone encoding a bifunctional enzyme, i.e., an enzyme that catalyzes both gamma-GK and GSD.
Yet another object of the present invention is to provide a method to overproduce proline and thus increase sodium chloride tolerance and drought resistance in a plant by the introduction into the plant of the P5CS cDNA clone encoding a bifunctional enzyme that catalyzes both gamma-GK and GSA production in said plant.
Yet another object of the present invention is to provide a method to increase glutamine synthetase (GS) activity in plants, thereby increasing the amount of substrate available for P5CS (glutamate) in the production of proline.
BRIEF DESCRIPTION OF THE FIGURES
In describing the preferred embodiment of the invention, which is illustrated in the FIGURES, specific terminology will be resorted to for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose. The words used to describe the invention are not literal limitations but include elements recognized as being equivalent by those skilled in the art.
FIG. 1 is the complete nucleotide sequence of a P5CS cDNA clone, pVAB2, and primary sequence of the encoded V. aconitifolia delta.sup.1 -pyrroline-5-carboxylate synthetase. The polypeptide region homologous to the E. coli gamma-GK (ProB) domain is underlined. The carboxy half of P5CS corresponds to GSA dehydrogenase (ProA).
FIG. 2 is a comparison of the V. aconitifolia P5CS amino acid sequence with A) the E. coli gamma-GK and B) GSA dehydrogenase sequences. Identical amino acids are indicated by a vertical line and similar amino acids are indicated by colons and periods. The conserved aspartic acid residues implicated in proline feedback inhibition are boxed, as are the leucine zipper sequences and the potential phosphorylation sites.
FIG. 3 is a graph illustrating feedback inhibition of delta.sup.1 -pyrroline-5-carboxylate synthetase by proline. Ammonium sulfate-fractionated extracts from E. coli CSH26 ( ), E. coli CSH26 harboring pVAB2 (.omicron.), the proB74 allele ( ), and the wild-type proB allele (.quadrature.) were assayed for gamma-GK activity by the method of Hayzer and Leisinger, J. Gen. Microbiol., 118, 1980, pp. 287-293.
FIG. 4 illustrates the proline synthetic pathways in bacteria and plants, starting from glutamate. In bacteria, separate proB and proA steps are found for encoding gamma-GK and GSD, respectively, to produce GSA, whereas in plants, the glutamate is converted directly to GSA by the bifunctional enzyme, P5CS.
DETAILED DESCRIPTION
By the present invention, E. coli proB, proA, and proBA auxotrophic mutants were transformed by electroporation with a Vigna aconitifolia nodule cDNA expression library. A transformation efficiency of 10.sup.9 -10.sup.10 transformants/ug of DNA was routinely obtained by the present invention, which is about two orders of magnitude higher than that obtained by classical transformation methods. Thus electroporation significantly enhances the recovery of complemented mutants. One group of clones of the present invention was isolated by selection for growth of transformants on minimal media without proline. It has been determined by the present invention that these clones encode a novel bifunctional enzyme, P5CS, which has both gamma-GK and GSD activities. The P5CS clones produced according to the present invention efficiently complemented E. coli proB, proA, and proBA, but not proC mutants. The predicted amino acid sequence of the enzyme produced by the present invention revealed the presence of two domains with significant homology to the E. coli proA and proB proteins. The recombinant P5CS protein of the present invention demonstrated GSD-dependent gamma-GK enzyme activity.
The complete nucleotide sequence of a P5CS cDNA clone, pVAB2, is shown in FIG. 1. The 2417 bp sequence contains a single major open reading frame encoding a polypeptide of 73.2kD, assuming that translation is initiated at the first ATG codon from the 5' end of the coding strand. Sequence comparison with the E. coli ProB and ProA proteins (FIG. 2) indicate that V. aconitifolia P5CS polypeptide has two distinct domains. The amino terminal domain of the P5CS protein showed 33.3% identity and 55.5% overall similarity to the E. coli ProB protein. A domain with 35.7% identity and 57.9% similarity to the ProA protein is located at the carboxyl end of the nucleotide sequence. An unexpectedly high level (42.4%) of the sequence similarity was found between the E. coli ProB and ProA proteins leading the inventor to the conclusion that the proB and proA of an ancestral gene; the encoded proteins may have later acquired domains conferring the respective kinase and reductase activities of the present-day enzymes. It has been surprisingly discovered in the present invention that the corresponding plant genes have evidently fused to create the bifunctional enzyme identified herein from V. aconitifolia.
FIG. 4 illustrates the comparison between the proline production pathways in bacteria and plants and the genes involved. The key difference is the fusion or merging of the proB and proA catalytic steps of the bacteria into the single gene in the plant pathway to form GSA. Both the bacteria and the plant proline production pathways start with glutamate, then proceed through GSA to P5C which, in the presence of P5CR, is converted or reduced to proline. However, in bacteria, the glutamate is phosphorylated in the presence of gamma-GK encoded by proB to produce glutamyl phosphate (GP). The GP is then converted to GSA by GSD encoded by proA. In the plant, however, it has been discovered herein that the glutamate is converted directly to GSA by the bifunctional enzyme P5CS, shown in FIG. 1. The P5CS therefore has the combined properties of proB and proA in the proline synthetic pathway.
Interestingly, it has also been discovered herein that a "leucine zipper" sequence (see FIG. 2) is present in each of the enzymatic domains of Vigna P5CS. The leucine zippers mediate protein:protein dimerization in structural proteins of both eucaryotes and procaryotes as well as in transcriptional regulatory proteins. It is believed that the leucine zippers in P5CS function intramolecularly to maintain the proper tertiary structure of the two domains of this enzyme, and homodimer or heterodimer formation may occur through the leucine zippers to allow close association between the originally separate enzymes (proB and proA) fused in P5CS. It is also possible that these zippers are a relic of the time when the gamma-GK and GSA-dehydrogenase enzymes were separate and had to be brought together to form a functional complex, as in E. coli. Thus these leucine zippers may facilitate inter- intra-molecular interactions. In addition, P5CS contains a potential phosphorylation site (FIG. 2). P5CS has GSA dehydrogenase and gamma-GK activities, and the gamma-GK activity is feedback inhibited by proline. To confirm that the presently claimed sequence, pVAB2 of FIG. 1, encoded P5CS activity, ammonium sulfate-fractionated extracts of E. coli CSH26 and CSH26 harboring pVAB2 were assayed for GSA dehydrogenase-dependent gamma-GK enzyme activity. As shown by the essentially horizontal line () in FIG. 3, no gamma-GK activity was detected in E. coli strain CSH26, whereas CSH26 containing pVAB2 exhibited a high level of gamma-GK activity (see curve (.omicron.)). Because proline production is regulated by end-product inhibition of gamma-GK in bacteria, the plant enzyme P5CS was similarly subjected to feed-back control testing. FIG. 3 shows that the Vigna enzyme is 50% inhibited by 6 mM proline. The wild-type E. coli gamma-GK (see curve (.quadrature.) of FIG. 3) was 30 times more sensitive than the Vigna enzyme, i.e., it shows 50% inhibition at 0.2 mM proline, while a mutant form of the bacterial enzyme (encoded by the proB74 allele, curve ()) is approximately 200-fold less sensitive to end product inhibition. Thus, the sensitivity of the recombinant Vigna enzyme (curve (.omicron.)) to feedback inhibition is much less than that of the wild-type E. coli enzyme (curve (.quadrature.)) but more than that of the mutated gamma-GK enzyme. The mutation responsible for the resistance to end-product inhibition of the gamma-GK in the proB 74 allele involves a substitution of an A for a G nucleotide, resulting in a change from aspartic acid to asparagine residue at position 107. It is to be noted, however, that this aspartate residue is conserved in the Vigna P5CS enzyme of the present invention, raising the possibility that changing this residue to asparagine by site-directed mutagenesis may result in further diminution of feedback inhibition of the plant enzyme. In spite of the conserved aspartic acid residue implicated in feedback control, the Vigna enzyme is less sensitive to inhibition by proline than is the E. coli enzyme suggesting that other regions of the P5CS enzyme may also be important for feedback regulation.
By observing the sensitivity of the mothbean enzyme to end-product inhibition in proline synthesis, it has been discovered in the present invention that regulation of the pathway in plants is exerted primarily at the P5CS step.
To demonstrate the expression levels of the Vigna P5CS gene in different tissues and under conditions of osmotic stress, RNA from nodules, leaves, and roots, as well as roots from plants watered with 200 mM sodium chloride, was probed on a northern blot with the pVAB2 cDNA. The results showed that the P5CS transcript in Vigna leaves was more abundant than in the roots and nodules. The level of P5CS transcripts in roots was enhanced by treatment of the plant with 200 mM sodium chloride. An increase in the level of transcript of P5CS and P5CR during osmotic stress will therefore facilitate increased proline production from glutamate.
To demonstrate that P5CR activity is not a rate limiting step in proline production in plants, the soybean P5CR cDNA was placed under the control of cauliflower mosaic virus (CaMV-35S) promoter and introduced into Vigna via T-DNA mediated transformation. Regenerated plants showed 10 to 100 fold increase in the amount of P5CR activity in the leaves. Measurement of proline and P5C levels in the transgenic plants indicated that proline synthesis was limited by P5C production rather than the activity of P5CR. All P5C in control and transgenic plants was converted to proline. No significant accumulation of proline occurred in transgenic plants expressing high levels of P5CR because the specific activity of the P5CR of both stressed and unstressed Vigna cell cultures is hundreds of folds greater than the proline synthesis rate, and the enzyme of the present invention functions at only a small fraction of its V.sub.max. Therefore, P5CR is not rate limiting in the proline production pathway in plants.
Using the procedures described in Miao et al., The Plant Cell, Vol. 3, January 1991, pp. 11-12, the isolated cDNA of the present invention was transferred into transgenic Vigna plants to demonstrate the effect therein on proline production. Table I illustrates a comparison of control versus transgenic Vigna plants. The results illustrate that the proline content of the control plants (samples 1, 2 and 3) was low (0.76 to 2.42 micromoles of proline per gram of root tissue) relative to the proline content of the transgenic Vigna plants of the present invention (samples 4-8), i.e. 3.9 to 13.2 micromoles of proline per gram of root tissue. This represents a 10 to 100 fold increase in proline production in the transgenic plants as a result of the transference of the nucleotide sequence of the present invention.
Thus, the invention provides a method of inducing both P5CS and P5CR genes by salt stress. The cloned plant gene of the present invention encodes the two enzymes in the plant which catalyze the first two reaction steps in the production of proline, long believed to provide a crucial role in osmoregulation. As demonstrated by the present invention, it is now possible to remove feedback control on proline production in plants and to produce the overexpression of proline from P5CS to confer salt and drought tolerance on a crop plant.
The invention further relates to increasing GS activity in plants thereby increasing the amount of substrate available for P5CS (glutamate) in the production of proline. This increase is achieved by introduction to the plant of the GS gene obtained by the procedures described in Miao et al., The Plant Cell, Vol. 3, January 1991, pp. 11-12. By this invention, the GS activity in the plant is increased relative to the level before introduction of the GS gene, whereby proline production is increased.
EXPERIMENTAL
Bacterial Growth Conditions
E. coli mutant cells were grown at 37 degrees Centigrade in LB medium or in Minimal A medium (see Delauney and Verma, Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht, 1990, pp. A14: 1-22) supplemented with 0.2 mM of required amino acids and 0.05 mM of thiamine. Ampicillin was added at 100 mg/l. The osmotic medium was increased by the addition of NaCl as indicated.
Plasmids and Bacterial Strains
Plasmids carrying wild-type proB (pDU1) and mutant proB74 (pDU101) allele were obtained from A. Dandekar (University of California, Davis). E. coli strains #X340, #W4032, #CSH26and #X342 were obtained from B. Bachman, E. coli Genetics Stock Center, Yale University.
Construction of cDNA Expression Library
RNA was prepared from 21 day old mothbean (Vigna aconitifolia, L.) nodules by the method of Verwoerd et al., Nucl. Acid Res., 17, 1989, p. 2362, and poly(A).sup.+ RNA was isolated by chromatography on an oligo(dT)-cellulose column. Double stranded cDNA was synthesized, ligated to BstXI linkers, and size-fractionated on an agarose gel. Molecules greater than 500 bp in size were electroeluted from the gel, cloned into the BstXI-linearized pcDNAII plasmid vector (Invitrogen Corp., San Diego, Calif., and E. coli DHI.alpha. cells were transformed. cDNA molecules inserted within the polylinkers of pcDNAII in the correct orientation and reading frame can be expressed under the control of the lacZ promoter.
Functional Complementation of E. coli Auxotrophs
E. coli proline auxotrophs were transformed by electroporation using Electro Cell Manipulator 600 apparatus (BTX, San Diego, Calif.) according to the manufacturers instructions. Briefly, cells were grown to late log phase in LB broth, pelleted by centrifugation, washed twice in ice-cold 10% glycerol in water solution, resuspended in cold 10% glycerol, and stored in 200 ul aliquots at -80.degree. C. For electroporation, 20 ng of the pooled library DNA purified by double cesium chloride centrifugation was added to 200 ul of competent cells in a 2 mm cuvette and subjected to a 5 msec pulse at 2.5 kV/cm. The cells were immediately added to 10 ml of LB broth and incubated at 37.degree. C. with shaking for 1 hour. The cells were then pelleted by centrifugation, washed with Minimal A medium, and plated into Minimal A plates containing ampicillin (100 ug/ml), IPTG (1 mM) and the amino acid supplements other than proline required by the respective mutants. To determine the transformation efficiencies, aliquots of electroporated cells were plated on the same media supplemented with proline (1 mM). Plates were incubated at 37.degree. C. for two days. Plasmid DNA, prepared from single colonies grown in minimal media containing ampicillin, was used to retransform the mutants to confirm the complementation.
DNA Sequencing and Analysis
DNA sequencing was performed on CsCl-purified, double-stranded plasmid DNA by the dideoxynucleotide chain termination method using Sequenase II (U.S. Biochemicals, Cleveland, Ohio) according to the manufacturer's instructions. Deletions of the plasmid template were generated using Exonuclease III and mung-bean nuclease.
P5CS Enzyme Assay and Determination of End-product Feedback Inhibition
E. coli CSH26 cells and CSH26 harboring different plasmids were grown aerobically at 37.degree. C. for about 16 hours. The cells were harvested by centrifugation at 4,000.times.g for 10 minutes, and the cell paste resulting was suspended in 4 ml of buffer A (50 mM Tris-HCl 1 mM dithiothreitol) at 40.degree. C. Following sonication, (Benson Sonifier, Type 450) at 50 watts, the slurry was centrifuged at 4,000.times.g for 10 minutes and the supernatant fractionated by ammonium sulfate precipitation. For E. coli CSH26 containing pVAB2, the fraction precipitating between 0 and 30% saturation of ammonium sulfate was used for enzyme assay. The E. coli CSH26 and CSH26 harboring the proB74 or wild-type proB alleles, a fraction of 40 to 60% saturation was used. Ammonium sulfate precipitated fraction was dissolved in buffer A and dialyzed against the same buffer. Gamma-GK activity was assayed as a function of proline concentration (0.01 to 100 mM) to determine if proline synthesis is subject to end-product inhibition in plants.
Northern Blot Analysis
Poly(A).sup.+ RNA from mothbean nodules, leaves and roots, as well as roots from plants watered with 200 mM NaCl , were electrophoresed in a 1.2% agarose-2.2M formaldehyde gel and transferred by vacuum blotting to a Gene Screen membrane (DuPont NEN, Boston, Mass.). The pVAB2 cDNA insert was labeled with .sup.32 P-dCTP to a specific activity of 8.times.10.sup.8 dpm/ug using a random primer labeling system (Amersham, Arlington Heights, Ill). Hybridization was performed by the method of Delauney and Verma, Mol. Gen. Genet., 221, 1990, pp. 299-305, and filters were exposed to x-ray film at -70.degree. C.
While certain preferred embodiments of the present invention have been disclosed in detail, it is to be understood that various modifications may be adopted without departing from the spirit of the invention or scope of the following claims.
Claims
We claim:
1. An isolated DNA molecule having the sequence of SEQ ID NO.: 1.
2. A cDNA clone encoding the enzyme of SEQ ID No. 1, a bifunctional enzyme that has both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities.
Patent Citations (1)
| Patent | Date | Inventor | Cited By |
|---|---|---|---|
| US4594323 | 1986-06-01 | Csonica et al. |
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