HHHHHHH|||||||IIIHIII USOO5268288A United States Patent (19) 11 Patent Number: 5,268,288 Pharr Et Al

HHHHHHH|||||||IIIHIII USOO5268288A United States Patent (19) 11 Patent Number: 5,268,288 Pharr et al. (45) Date of Patent: Dec. 7, 1993

54). MANNITOL OXIDOREDUCTASE SOLATED J. Everard and W. Loescher, Plant Physiol (Supple FROM WASCULAR PLANTS ment) 4, 2 (1991). R. Broglie et al., Plant Molecular Biology 3, 431-444 75 Inventors: David M. Pharr; Johan M. H. Stoop, (1984). both of Raleigh, N.C. P. Trip et al., Amer. Jour. Bot. 51, No. 8, 828-835 (1964). 73 Assignee: North Carolina State University, Grant & Hackh's chemical dictionary, p. 214, 1987, Raleigh, N.C. Fifth edition. (21) Appl. No.: 857,388 Primary Examiner-David M. Naff Assistant Examiner-Michael V. Meller (22 Filed: Mar. 25, 1992 Attorney, Agent, or Firm-Bell, Seltzer, Park & Gibson 51) Int. Cl...... C12N 9/04; C12N 5/00 57 ABSTRACT 52 U.S. C...... 435/190; 435/240.4 A mannitol oxidoreductase is isolated from vascular 58) Field of Search ...... 435/190, 240.4 plants which is NAD+ dependent and which converts 56) References Cited mannitol to mannose. A preferred source of the manni tol oxidoreductase is Apium graveolens, particularly U.S. PATENT DOCUMENTS Apium graveolens L. var. rapaceum (Mill.) Gaud. (cele 4,927,752 5/1990 Renacle ...... 435/8 riac) and Apium graveolens L. var. dulce (Mill.) Pers. (celery). DNA encoding of the mannitol oxidoreduc OTHER PUBLICATIONS tase may be used in the production of the enzyme. M. Tarczynski et al., Plant Physiol (Supplement) 889, 133 (1991). 7 Claims, 9 Drawing Sheets U.S. Patent Dec. 7, 1993 Sheet 1 of 9 5,268,288

5 s WEEK O 4 - E WEEK 53 O 2 E 1 O LEAF PETIOLE

5 4 3 2 3 S O FIG.1B i U.S. Patent Dec. 7, 1993 Sheet 2 of 9 5,268,288

i O YL YP ML MP SI TR OR RT PLANT PART FIG.2A

O YL YP ML MP SI TR OR RT PLANT PART FIG.2B

Yi YP ML MP SI TR OR RT PLANT PART FIG.2C U.S. Patent Dec. 7, 1993 Sheet 3 of 9 5,268,288

g ||T

U.S. Patent Dec. 7, 1993 Sheet 5 of 9 5,268,288

KINETIC PARAMETERS OF MDH

100 200 300 S (mM MANNITOL) FIG.5A

KINETIC PARAMETERS OF MOH

100 200 300 S (mM MANNITOL) FIG.5B U.S. Patent Dec. 7, 1993 Sheet 6 of 9 5,268,288

KINETIC PARAMETERS OF MDH 0.5

0.0 -0.04 0.00 0.04 0.08 0.12 1/S FIG.5C

KINETIC PARAMETERS OF MDH

0.5

0.0 -0.0 0.00 0.04 0.08 0.12 U.S. Patent Dec. 7, 1993 Sheet 7 of 9 5,268,288

KINETIC PARAMETERS OF MOH

S (mM) FIG.6A

KINETIC PARAMETERS OF MDH

U.S. Patent Dec. 7, 1993 Sheet 8 of 9 5,268,288

KINETIC PARAMETERS OF MDH

KINETIC PARAMETERS OF MOH

4. 12 1/S FIG.6D U.S. Patent Dec. 7, 1993 Sheet 9 of 9 5,268,288

100

2 O

O 0.1 0.2 0.5 NADH (mM) FIG.7 5,268,288 1. 2 MANNTOL OXDOREDUCTASE SOLATED SUMMARY OF THE INVENTION FROM VASCULAR PLANTS The present invention is based upon our discovery of a mannitol oxidoreductase in celeriac and celery. Inso FIELD OF THE INVENTION far as we are aware, this represents the first report of the The present invention relates to mannitol oxidore existence of a mannitol: mannose i-oxidoreductase in ductases in general, and particularly relates to the dis any living organism. covery of an NAD+-dependent mannitol: mannose A first aspect of the present invention is isolated l-oxidoreductase which can be isolated from vascular NAD+-dependent mannitol: mannose 1-oxidoreductase plants. O (mannitol oxidoreductase; mannitol dehydrogenase). The mannitol oxidoreductase may be obtained from BACKGROUND OF THE INVENTION plants, with a preferred source being Apium graveolens. Mannitol is by far the most abundant polyol in nature, A second aspect of the present invention is isolated occurring in bacteria, fungi, algae, lichens and vascular DNA encoding NAD+-dependent mannitol: mannose 15 1-oxidoreductase (mannitol oxidoreductase), as given plants. See R. Bieleski, in: Encyclopedia of Plant Physiol above. In one embodiment, the isolated DNA is se ogy, New Series Volume 13A. Plant Carbohydrates I. lected from the group consisting of: (a) isolated DNA p. 158-192 (1982); D. Lewis, in: Storage carbohydrates in which encodes Apium graveolens mannitol oxidoreduc vascular plants. Distribution, physiology and metabolism. tase; (b) isolated DNA which hybridizes to isolated p. 158-179 (1984). Although several physiological roles DNA of (a) above and which encodes a mannitol oxido have been proposed for mannitol, such as carbohydrate reductase as given above; and (c) isolated DNA differ storage, regulation of carbon partitioning, osmoregula ing from the isolated DNAs of (a) and (b) above in tion and cofactor regulation, little information exists on nucleotide sequence due to the degeneracy of the ge the enzymatic pathways of mannitol synthesis and utili netic code, and which encodes a mannitol oxidoreduc zation. See R. Bieleski, supra; W. Loescher, Physiol. 25 tase as given above. Plantarum 70:553-557 (1987); M. Rumpho et al., Plant Other aspects of the invention include vector DNA Physiol 73:869-873 (1983). Most information has come comprising a DNA sequence encoding a promoter op from studies of fungi and bacteria which contain a man erative in a plant cell operatively associated with nitol dehydrogenase that catalyzes the NAD-dependent NAD+-dependent mannitol: mannose 1-oxidoreductase or NADP dependent oxidation of D-mannitol to D (mannitol oxidoreductase), along with plants trans fructose. See, e.g., D. Quain et al., J. Gen. Microbiol. formed with such a vector. 133:1675-1684 (1987); W. Niehaus et al., Mycopathologia Mannitol oxidoreductase of the present invention is 107:131-137 (1989). useful for converting mannitol (not digestible by hu NAD-dependent mannitol-1-P dehydrogenase has mans) to mannose, which may then be further con been detected in bacteria, brown algae and fungi which 35 verted to other types of sugars by known enzymes. catalyzes the conversion of mannitol-1-P to fructose-6- DNA encoding mannitol oxidoreductase is useful for P. See, e.g., S. Horwitz et al., J. Biol. Chem. 239: genetically engineering plants which contain mannitol 830-838 (1964); Richter and Kirst, Planta 170:528-534 to convert that mannitol to mannose with the mannose (1987); R. Kiser et al., Arch. Biochem. Biophys. then optionally being converted to other sugars by 21 1:63-621 (1981). All three enzymes are 2-oxidore endogenous enzymes or enzymes introduced into that ductases and can catalyze the reaction in either direc plant by genetic engineering techniques. tion, toward synthesis or utilization of the polyol de The foregoing and other objects and aspects of the pending on the availability of oxidized or reduced co present invention are explained in detail in the drawings factor and the pH. herein and the specification below. Mannitol catabolism in higher plants is still poorly 45 understood. Several labeling studies using C-man BRIEF DESCRIPTION OF THE DRAWINGS nitol suggest that mannitol is utilized at a slow rate in FIG. 1 shows the hexose (A), sucrose (B) and manni vascular plants. When celery leaf discs were incubated tol (C) concentration of stressed celeriac cv. Monarch in C-mannitol, mannitol utilization was restricted to plants. Plants were transferred to 8' pots at week zero. young leaf tissue. See J. Fellmann et al., Physiol. Plan 50 Carbohydrate analysis was done at time of transfer tarum 69:337-341 (1987). Suspension cultures of Daucus (week zero) and one week after transfer (week one). carota L. and Pinus radiata D. or carrot root tissue had FIG. 2 shows the mannitol (A), sucrose (B) and hex a small uptake and metabolism of C-mannitol. See ose (C) concentration of different plant parts of celeriac M. Thompson et al., Physiol. Plant, 67:365-369 (1986); cv. Monarch. (YP, young petiole; YL, young leaf, MP, W. Cram, Physiol. Plant. 61:396-404 (1984). Mannitol 55 mature petiole; ML, mature leaf; SI, innermost part of respiration, as measured by 1 species and ranged RT, root tip). from very low rates (Avena sativa) to rates comparable FIG. 3, left (ethanol), shows ADH activity stain of to that of fructose or glucose (Fraxinus americana). BSA (lane B), knob (lane K), mature petiole (lane MP), White ash leaflets exposed to 14C-mannitol resulted in young petiole (lane YP) and root tip (lane RT). The the formation of a small amount of 14C-fructose after 2 right side (Mannitol) shows MDH activity stain of BSA days, perhaps suggesting that the first step of mannitol (lane B), knob (lane K), mature petiole (lane MP), utilization in this species may be oxidation to fructose. young petiole (lane YP) and root tip (lane RT). Five ul See P. Trip et al., Amer. Jour. Bot. 51:828-835 (1964). of extract was electrophoresed for each tissue, Although these labeling studies support the presence of 65 FIG. 4 shows a Fractogel DEAE ion-exchange col a mannitol utilizing enzyme in higher plants, insofar as umn profile. Alcohol dehydrogenase (ADH), Mannitol we are aware, no mannitol oxidizing enzyme has been dehydrogenase (MDH) and Phosphomanno isomerase isolated from a vascular plant. (PMI) activities were analyzed. Protein content was 5,268,288 3 4. monitored at A 280. Proteins were eluted with a linear vulgare L. (common privet); Olea europaea L. (olive); KCl gradient. Olea glandulifera, Olea lancea, Osmanthus aquifolium, FIG. 5 shows the kinetic parameters of mannitol Osmanthus armatus, Osmanthus jragrans, Syringa annu dehydrogenase of celeriac root tips (S= mannitol). Top: rensis, Syringa emodi. Syringa julianae Schr.; Syringa substrate saturation curves at pH 9.0 (A) and pH 7.5 (B). 5 pekinensis, Syringa persica, Syringa pubescens Turcz.; Bottom: Lineweaver-Burk plots at pH 9.0 (C) and pH Syringa vulgaris L. (common lilac); Orobanche cumana, 7.5 (D). Orobanche ramosa L. (hemp broomrape); Pinus radiata FIG. 6 shows the kinetic parameters of mannitol D. Don (Monterey pine); Platanus orientalism L. (orien dehydrogenase of celeriac root tips (S=NAD). Top: tal planetree); Cyclamen europaeum, Punica granutum substrate saturation curves at pH 9.0 (A) and pH 7.5 (B); 10 L. (pome granate); Aconitum napellus L. (aconite, bottom: Lineweaver-Burk plots at pH 9.0 (C) and pH monkshood); Delphinium consolida Prunus lauracerasus 7.5 (D). L. (cherry, laurel); Coffea arabica L. (coffee); Gardenia FIG. 7 shows NADH inhibition as affected by NAD brasiliensis, Gardenia florida L.; Gardenia jasminoides concentration. Ellis (Cape, jasmine); Gardenia turgida, Linaria vulgaris 15 Mill. (yellow toadflax); and Veronica (speedwell). DETALED DESCRIPTION OF THE These species are representative of the following plant INVENTION families: Apiaceae, Arecaceae, Asteraceae, Brass The term "plant' herein refers to higher plants (i.e., icaceae, Bromeliaceae, Buxaceae, Cactaceae, Canel vascular plants). Mannitol oxidoreductase of the present laceae, Combretaceae, Compositae, Convolvulaceae, invention may be that of any plant, including both gym 20 Cucurbitaceae, Cucurbita, Euphorbiaceae, Fabaceae, nosperms and angiosperms (including both monocots Gnetaceae, Gramineae, Liliaceae, Oleaceae, Oroban and dicots). Preferred sources are plants which have chaceae, Pinaceae, Platanaceae, Primulaceae, Punica been reported to contain mannitol, and particularly ceae, Ranunculaceae, Rosaceae, Rubiacea, and Scro preferred sources are plants which have been reported phulariaceae. to both contain and utilize mannitol. Numerous such 25 Plants reported to both contain and metabolize Man plants are known. See, e.g., S. Barker, in Modern Meth nitol include the following: Apium graveolens L. var. ods of Plant Analysis, Vol. 2, p. 62-67 (K. Paech and M. rapaceum (Mill.) Gaudich (celeriac); Apium graveolens Tracev eds. 1955); E. Bourne, in Encyclopedia of Plant L. var dulce (Mill.) Pers. (celery); Brassica oleracea L. Physiology 6:345-372 (1958); A. Cranswick and J. Zab Botrytis group (cauliflower); Ananas comosa (L.) Merr kiewicz, J. Chromatography (1979); M. Thompson et al., 30 (pineapple); Lactuca sativa L. (lettuce); Asparagus offic Physiol Plant, 67:365-369 (1986); P. Trip et al., Amer. inalis L. (asparagus); Fraxinus americana L. (white ash); Jour. Bot. 51: 828-835 (1964); M. Zimmermann and H. Fraxinus ornus L. (flowering ash, manna); Ligustrum Ziegler, in Encyclopedia of Plant Physiology, Vol. 1, vulgare L. (common privet): Syringa vulgaris L. (com New-Series, pp. 408-503 (1975). mon lilac): Coffea arabica L. (coffee); and Gardenia Plants reported to contain mannitol include the fol 35 florida L. These species are representative of the follow lowing: Apium graveolens L. var. rapaceum (Mill.) ing families: Apiaceae, Brassicaceae, Bromeliaceae, Gaud. (celeriac); Apium graveolens L. var dulce (Mill.) Compositae, Liliaceae, Oleaceae, and Rubiacea. Pers. (celery); Daucus carota L. (carrot); Oenanthe Isolated mannitol oxidoreductase of the present in crocata, Oenanthe crocata, Pastinaca satiya L. (parsnip, vention may be produced in the manner described be wild p.); Cocus nucifera L. (coconut); Scorzonera hispan low, or variations thereof which will be apparent to ica L. (black Salsify); Brassica oleracea L. Botrytis group those skilled in the art. In one embodiment, the isolated (cauliflower); Brassica oleracea L. Capitata group (cab mannitol oxidoreductase is essentially free from alcohol bage); Ananas comosa (L.) Merr (pineapple); Buxus dehydrogenase and phosphonannose isomerase. Iso Sempervitens L. common boxwood); Opuntia vulgaris lated mannitol oxidoreductase of the invention (e.g., Mill; Canella alba Murr.; Laguncularia racemosa (L.) 45 isolated Apium graveolens L. var. rapaceum (Mill.) Gaertn. f. (white mangrove); Terminalia arjuna Bedd Gaud. NAD+ dependent mannitol: mannose 1-oxidore Terminalia chebula (Gaertn.) Retz.; Terminalia myri ductase (celeriac mannitol oxidoreductase), as de ocarpa Heurck et Muell.-Arg; Terminalia oliveri Bran scribed below) is further purified, and/or purified suffi dis; Lactuca satiya L. (lettuce); Ipomaea purga (Wender.) ciently for amino acid sequencing, in accordance with Hayne. Gjalap); Citrillus vulgaris (watermelon); Cucur 50 known techniques, such as with high performance liq bita pepo L. (pumpkin, squashes); Manihot utilissima uid chromatography (HPLC), affinity chromatography Pohl (cassava, manioc); Cercissiliquastrum L.; Glycyrh (e.g., with antibodies to the enzyme or with an NAD iza glabra L. (common licorice); Gymnocladus canaden affinity column), comprehensive two-dimensional high sis L.; Phaseolus vulgaris L. (green bean, French bean); performance liquid chromatography coupling ion ex Pisum (pea); Spartium junceum L. (Spanish broom); 55 change chromatography and size exclusion chromatog Ephedra distachya, Agropyron repens, Andropogon annu raphy (see M. Bushey and J. Jorgenson, Analytical latus, Asparagus offinalis L. (asparagus); Forestiera acu Chemistry 62: 161-167 (1990)), two-dimensional minata (Michx.) Poir. (swamp privet); Forestiera neo HPLC/Capillary zone electrophoresis (see, e.g., M. mexicana, Fraxinus americana L. (white ash); Fraxinus Bushey and J. Jorgenson, Analytical Chemistry 62: exelsior L. (European ash); Fraxinus julandifolia, Frax 60 978-984 (1990) and variations thereof (i.e., where other inus oregona Nutt. var. glabra; Fraxinus ornus L. (flow liquid chromatography columns such as reverse phase ering ash, manna); Fraxinus oxycarpa Willd.; Fraxinus chromatography columns, ion exchange chromatogra pistaciaefolia, Fraxinus rotundifolia, Jasminum abys phy columns, adsorption chromatography columns, and sinicum, Jasminum beesianum, Jasminum fruticans, Jas affinity chromatography columns are used; or where minum heterophyllum, Jasminum humile, Jasminum 65 other capillary electrophoresis apparatus such as capil nudiflorum Lindl. (winter jasmine); Jasminum odoratis lary gel electrophoresis, capillary isotachophoresis, simum, Jasminum officinale L. (poet's jessamine); Ligus micellar electrokinetic capillary chromatography, and trum ibota, Ligustrum japonicum Thunb.; Ligustrum capillary isoelectric focusing are used), other multidi 5,268,288 5 6 mensional separation systems (see J. Giddings, J. High homologous, 85% homologous, or even 90% homolo Resolut. Chromatogr. Chromatogr. Commun. 10:319-323 gous or more with the sequence of Apium graveolens (1987)), SDS-polyacrylamide gel electrophoresis, two mannitol oxidoreductase. Further, DNA sequences dimensional polyacrylamide gel electrophoresis such as which code for Apium graveolens mannitol oxidoreduc disclosed in R. Broglie et al., Plant Mol. Biol. 3:431-444 tase, or sequences which code for a mannitol oxidore (1984), etc. Thus, the present invention provides iso ductase coded for by a sequence which hybridizes to lated mannitol oxidoreductase having specific activities the DNA sequence which Apium graveolens mannitol of 1, 10, 50, 100, or even 200 or more, in Units per oxidoreductase, but which differ in codon sequence Milligram of Protein, as described in greater detail be from these due to the degeneracy of the genetic code, low. 10 are also an aspect of this invention. The degeneracy of Isolated DNA encoding Apium graveolens mannitol the genetic code, which allows different nucleic acid oxidoreductase is produced from isolated Apium gra sequences to code for the same protein or peptide, is veolens mannitol oxidoreductase in accordance with well known in the literature. See e.g., U.S. Pat. No. known techniques. Amino acid sequences or partial 4,757,006 to Toole et al. at Col. 2, Table 1. amino acid sequences are generated from isolated man 5 Hybridization procedures are available which allow nitol oxidoreductase in accordance with known tech for the isolation of cDNA clones whose mRNA levels niques, such as the automated sequencing of tryptic are as low as about 0.05% of poly(A)RNA. See M. digests of isolated mannitol oxidoreductase as described Conkling et al., Plant Physiol 93, 1203-1211 (1990). In above. From the amino acid sequence or sequences, one brief, cDNA libraries are screened using single-stranded or more degenerate oligonucleotide probes are gener 20 cDNA probes of reverse transcribed mRNA from plant ated and labelled and used to screen an appropriate tissue (i.e., roots and leaves). For differential screening, DNA library (e.g., a genomic DNA library produced a nitrocellulose or nylon membrane is soaked in from a source which contains DNA encoding a manni 5X SSC, placed in a 96 well suction manifold, 150 L of tol oxidoreductase or a cDNA library produced from a stationary overnight culture transferred from a master suitable source enriched in mRNA encoding the manni 25 plate to each well, and vacuum applied until all liquid tol oxidoreductase) to isolate a DNA sequence encod has passed through the filter. 150 lull of denaturing ing a mannitol oxidoreductase of the invention. The solution (0.5M NaOH, 1.5M NaCl) is placed in each construction of oligonucleotide probes, DNA libraries, well using a multiple pipetter and allowed to sit about 3 and the hybridization screening of such DNA libraries minutes. Suction is applied as above and the filter re with oligonucleotide probes is carried out in accor 30 moved and neutralized in 0.5M Tris-HCl (pH 8.0), 1.5M dance with known techniques. See generally J. Sam NaCl. It is then baked 2 hours in vacuo and incubated brook et al., Molecular Cloning. A Laboratory Manual with the relevant probes. By using nylon membrane (2d ed. 1989)(Cold Spring Harbor Laboratory Press). In filters and keeping master plates stored at -70° C. in the alternative, PCR primers are generated from the 7% DMSO, filters may be screened multiple times with amino acid sequences and used to either screen a DNA 35 multiple probes and appropriate clones recovered after library directly or produce a larger oligonucleotide several years of storage. probe for use in screening a DNA library, also in accor The term "operatively associated,' as used herein, dance with known techniques, to isolate a DNA se refers to DNA sequences on a single DNA molecule quence encoding a mannitol oxidoreductase of the in which are associated so that the function of one is af. vention. See, e.g., U.S. Pat. No. 4,683,202 to Mullis and fected by the other. Thus, a promoter is operatively U.S. Pat. No. 4,683, 195 to Mullis et al. associated with a structural gene (i.e., a gene encoding Isolated DNA encoding mannitol oxidoreductase mannitol oxidoreductase as given above) when it is may be obtained from isolated DNA encoding Apium capable of affecting the expression of that structural graveolens mannitol oxidoreductase in accordance with gene (i.e., the structural gene is under the transcrip known hybridization techniques. Thus, DNA sequences 45 tional control of the promoter). The promoter is said to which hybridize to DNA which encodes mannitol oxi be "upstream' from the structural gene, which is in turn doreductase of the invention and which code on expres said to be "downstream' from the promoter. sion for a mannitol oxidoreductase of the invention are Numerous promoters operable in plant cells are also an aspect of this invention. Conditions which will known which may be employed in practicing the pres permit other DNA sequences which code on expression 50 ent invention, including a broad variety of plant viral for a mannitol oxidoreductase of the invention to hy promoters and promoters associated with cloned plant bridize to the DNA sequence of Apium graveolens man genes, such the Cauliflower mosaic virus 35S promoter, nitol oxidoreductase (e.g., the DNA sequence encoding inducible heat shock protein promoters such as the HSP Apium graveolens L, var. rapaceum (Mill.) Gaud. (cele 70 promoter, light inducible promoters such as the ribu riac) or Apium graveolens L. var. dulce (Mill.) Pers. 55 lose bisphosphate carboxylase small subunit promoter (celery) mannitol oxidoreductase) can be determined in and the chlorophyll AB binding protein promoter, a routine manner. For example, hybridization of such stress response proteins such as the PR protein pro sequences may be carried out under conditions of re moter, the Agrobacterium tumefaciens nos promoter, and duced stringency or even stringent conditions (e.g., root-specific promoters such as those disclosed in R. conditions represented by a wash stringency of 0.3M Croy et al., PCT Application Publication Number NaCl, 0.03M sodium citrate, 0.1% SDS at 60° C. or WO91/13992, S. Gurr et al., Mol. Gen. Genet. 226: even 70° C. to DNA encoding Apium graveolens manni 361-366 (1991), and M. Conkling et al., Plant Physiol. 93: tol oxidoreductase in a standard in situ hybridization 1203-1211 (1990). assay. See J. Sanbrook et al., Molecular Cloning, A DNA constructs, or "expression cassettes,' of the Laboratory Manual (2d Ed. 1989)). In general, sequen 65 present invention include, 5'-3' in the direction of tran ces which code for a mannitol oxidoreductase of the scription, a promoter as discussed above, a structural invention and hybridize to the DNA encoding Apium gene operatively associated with the promoter, and, graveolens mannitol oxidoreductase will be at least 75% optionally, a termination sequence including a stop 5,268,288 7 8 signal for RNA polymerase and a polyadenylation sig ing hygromycin resistance, and the mutated aroA gene, nal for polyadenylase (e.g., the nos terminator). All of providing glyphosate resistance, these regulatory regions should be capable of operating The various fragments comprising the various con in the cells of the tissue to be transformed. The 3' termi structs, expression cassettes, markers, and the like may nation region may be derived from the same gene as the be introduced consecutively by restriction enzyme transcriptional initiation region or may be derived from cleavage of an appropriate replication system, and in a different gene. sertion of the particular construct or fragment into the Structural genes are those portions of genes which available site. After ligation and cloning the DNA con comprise a DNA segment coding for a mannitol oxido struct may be isolated for further manipulation. All of reductase as given above, optionally including a ribo 10 these techniques are amply exemplified in the literature Somal binding site and/or a translational start codon, and find particular exemplification in Maniatis et al., but lacking a promoter. This term can also refer to Molecular Cloning: A Laboratory Manual, Cold Spring copies of a structural gene naturally found within a cell Harbor Laboratory, Cold Spring Harbor, N.Y., 1982. but artificially introduced. The structural gene may Vectors which may be used to transform plant tissue encode a protein not normally found in the plant cell in 15 with DNA constructs of the present invention include which the gene is introduced or in combination with the both Agrobacterium vectors and ballistic vectors, as promoter to which it is operationally associated, in well as vectors suitable for DNA-mediated transforma which case it is termed a heterologous structural gene. tion. Structural genes which may be operationally associated Agrobacterium tumefaciens cells containing a DNA construct of the present invention, wherein the DNA with a promoter of the present invention for expression construct comprises a Tiplasmid, are useful in methods in a plant species may be derived from a chromosomal of making transformed plants. Plant cells are infected gene, cDNA, a synthetic gene, or combinations thereof. with an Agrobacterium tunefaciens as described above Where the expression product of the structural gene to produce a transformed plant cell, and then a plant is is to be located in a cellular compartment other than the 25 regenerated from the transformed plant cell. Numerous cytoplasm, the structural gene may be constructed to Agrobacterium vector systems useful in carrying out include regions which code for particular amino acid the present invention are known. For example, U.S. Pat. sequences which result in translocation of the product No. 4,459,355 discloses a method for transforming sus to a particular site, such as the cell plasma membrane, or ceptible plants with an Agrobacterium strain containing may be secreted into the periplasmic space or into the 30 the Ti plasmid. The transformation of woody plants external environment of the cell. Various secretory with an Agrobacterium vector is disclosed in U.S. Pat. leaders, membrane integration sequences, and translo No. 4,795,855. Further, U.S. Pat. No. 4,940,838 to Schil cation sequences for directing the peptide expression peroort et al. discloses a binary Agrobacterium vector product to a particular site are described in the litera (i.e., one in which the Agrobacterium contains one ture. See, for example, Cashmore et al., Bio/Technology 35 plasmid having the vir region of a Tiplasmid but no T 3, 803-808 (1985), Wickner and Lodish, Science 230, region, and a second plasmid having a T region but no 400-407 (1985). vir region) useful in carrying out the present invention. The expression cassette may be provided in a DNA Microparticles carrying a DNA construct of the pres construct which also has at least one replication system. ent invention, which microparticles are suitable for the For convenience, it is common to have a replication ballistic transformation of a plant cell, are also useful for system functional in Escherichia coli, such as Col. 1, making transformed plants of the present invention. The pSC101, pACYC184, or the like. In this manner, at each microparticle is propelled into a plant cell to produce a stage after each manipulation, the resulting construct transformed plant cell, and a plant is regenerated from may be cloned, sequenced, and the correctness of the the transformed plant cell. Any suitable ballistic cell manipulation determined. In addition, or in place of the 45 transformation methodology and apparatus can be used E. coli replication system, a broad host range replication in practicing the present invention. Exemplary appara system may be employed, such as the replication sys tus and procedures are disclosed in Sanford and Wolf, tems of the P-1 incompatibility plasmids, e.g., pRK290. U.S. Pat. No. 4,945,050 (applicants intend that the dis In addition to the replication system, there will fre closure of all U.S. Patent references cited herein be quently be at least one marker present, which may be 50 incorporated herein by reference), and in Agracetus useful in one or more hosts, or different markers for European Patent Application Publication No. 0 270 individual hosts. That is, one marker may be employed 356, titled Pollen-mediated Plant Transformation. When for selection in a prokaryotic host, while another using ballistic transformation procedures, the expres marker may be employed for selection in a eukaryotic sion cassette may be incorporated into a plasmid capa host, particularly the plant host. The markers may be 55 ble of replicating in the cell to be transformed. Exam protection against a biocide, such as antibiotics, toxins, ples of microparticles suitable for use in such systems heavy metals, or the like; provide complementation, by include 1 to 5 um gold spheres. The DNA construct imparting prototrophy to an auxotrophic host: or pro may be deposited on the microparticle by any suitable vide a visible phenotype through the production of a technique, such as by precipitation. novel compound in the plant. Exemplary genes which Plant species may be transformed with the DNA may be employed include neomycin phosphotransferase construct of the present invention by the DNA (NPTII), hygromycin phosphotransferase (HPT), chlo mediated transformation of plant cell protoplasts and ramphenicol acetyltransferase (CAT), nitrilase, and the subsequent regeneration of the plant from the trans gentamicin resistance gene. For plant host selection, formed protoplasts in accordance with procedures well non-limiting examples of suitable markers are beta 65 known in the art. glucuronidase, providing indigo production, luciferase, Any plant tissue capable of subsequent clonal propa providing visible light production, NPTII, providing gation, whether by organogenesis or embryogenesis, kanamycin resistance or G418 resistance, HPT, provid may be transformed with a vector of the present inven 5,268,288 10 tion. The term "organogenesis,' as used herein, means a thereby eliminating sorbitol contamination in the man process by which shoots and roots are developed se nito substrate. quentially from meristematic centers; the term "embry ogenesis,' as used herein, means a process by which B. Purification of mannitol:mannose 1-oxidoreductase shoots and roots develop together in a concerted fash Enzyme extraction. Celeriac root tips were har ion (not sequentially), whether from somatic cells or vested, pooled and washed in distilled water. Fresh gametes. The particular tissue chosen will vary depend roots were ground in a chilled mortar using a 1:4 tissue ing on the clonal propagation systems available for, and to-buffer ratio. Theextraction buffer contained 50 mM best suited to, the particular species being transformed. Mops (pH 7.5, 5 mM MgCl2, 1 mM EDTA, 5 mMDTT Exemplary tissue targets include leaf disks, pollen, em 10 and 1% Triton X-100, Homogenates were centrifuged bryos, cotyledons, hypocotyls, megagametophytes, at 20,000 g for 15 min at 4 C. The crude extract con callus tissue, existing meristematic tissue (e.g., apical sisted of supernatant that was desalted by centrifugal neristems, axillary buds, and root meristems), and in filtration on a Sephadex G-25-50 column equilibrated duced meristem tissue (e.g., cotyledon meristem and with 50 mM Mops-NaOH (pH 7.5), 5 mM MgCl2 and 5 hypocotyl meristem). 5 Plants of the present invention may take a variety of mM DTT prior to assay for MDH activity. forms. The plants may be chimeras of transformed cells Purification. Supernatant from the root extraction and non-transformed cells; the plants may be clonal was brought to 45% saturation with ammonium sulfate transformants (e.g., all cells transformed to contain the at 4 C. and centrifuged at 20,000 g for 15 min. The expression cassette); the plants may comprise grafts of 20 supernatant was retained and brought to 65% saturation transformed and untransformed tissues (e.g., a trans by further addition of (NH4)2SO4. After centrifugation formed root stock grafted to an untransformed scion in the supernatant was discarded and the pellet was sus citrus species). The transformed plants may be propa pended in a minimum volume of 20 mM Mops (pH 7.5), gated by a variety of means, such as by clonal propaga 2 mM MgCl2 and 1 mM DTT. The dissolved pellet was tion or classical breeding techniques. For example, first 25 filtered through a 0.4 um filter and applied to a Frac generation (or T1) transformed plants may be selfed to togel (R) DEAE ion-exchange column (10 cm x 1.5 cm) give homozygous second generation (or T2) trans equilibrium with 20 mM Mops (pH 7.5), 2 mM MgCl2 formed plants, and the T2 plants further propagated and 1 mM DTT. Protein eluting from the DEAE col through classical breeding techniques. A dominant se umn was monitored by absorbance at 280 nm and pro lectable marker (such as npt II) can be associated with 30 tein in pooled fractions with Bio-Rad reagent. Proteins the expression cassette to assist in breeding. were eluted with a linear KCl salt gradient, OM to 0.4M The present invention is explained in greater detail in KCl, in running buffer. Flow rate equaled 1.25 ml/min. the following non-limiting examples. In these examples, and two ml fractions were collected. Fractions contain 'ppm' means parts per million, "nM' means millino ing MDH which was free from alcohol dehydrogenase lar, "M" means Molar, "ma" means milliamps, "mil" 35 (ADH) or phosphomannose isomerase (PMI) were means milliliters, "umol” means micromoles, “nn' pooled and further concentrated by ammonium sulfate means nanometers, "U" means units, "Mops' means precipitation (90% saturation), and the pellet was resus 3-(N-Morpholino) propanesulfonic acid, “EDTA' pended in a small volume of 20 mM Mops (pH 7.5), 2 means ethylenedianinetetraacetic acid, “DTT' means mM MgCl2 and 1 mM DTT. The concentrated enzyme dithiothreitol, 'h' means hours, "min.' means minutes, 40 was desalted and stored at -80 C. "g' means gravity, "rpm' means revolutions per min ute, "MDH' means mannitol dehydrogenase, and tem C. Enzyme Assays peratures are given in degrees centigrade unless other Spectrophotometric assay. Mannitol dehydrogenase wise indicated. (MDH) activity was assayed by monitoring the 45 oxidation-reduction of NADH/NAD+ spectrophoto EXAMPLE 1. metrically at 340 nm. Assays were done at 25 C. in a Isolation of NAD+-Dependent Mannito: Mannose total volume of 1 ml. Optimum conditions (buffer, pH Oxidoreductase and substrate concs) were determined. The reaction I. Materials and Method mixture contained 100 mM Bis-Tris (pH 9.0), 2 mM 50 NAD, enzyme extract and 50 mM D-mannitol. The A. Materials reactions were initiated with mannitol. One unit of ac Celeriac (Apium graveolens var. rapaceum) cultivar tivity was defined as the amount of enzyme which cata "Monarch' was seeded and reached transplant stage lyzes the reduction of 1 mol NAD+ per hour approximately three months later. Some seedlings were (unol/h). For the reverse reaction (mannose reduction) kept in seeding flats (1" by 1' containers) for a period of 55 the enzyme extract was incubated in a mixture 0.1 mM 4 weeks, in order to intentionally stress the young plants NADH, enzyme extract, 20 mM substrate (D-mannose, before being transplanted to 8' pots. Celeriac cv. 'Mar D-fructose or D-glucose) and 100 mM MOPS at ble Ball' transplants, acquired from DNA-Plant Tech pH = 7.5 in a total volume of 1 ml. nology Inc., were transplanted in 10" pots containing The alcohol dehydrogenase (ADH) assay was similar soilless media (Promix) and grown in a greenhouse for 60 to the MDH assay using 5 mM ethanol as the substrate. about five months. Plants were watered daily and fertil The phosphomannose isomerase (PMI) assay contained ized twice a week with a soluble fertilizer solution (200 the following reaction mixture: 100 mM Hepes (pH 7.5), ppm N, 100 ppm P2O5, 200 ppm K2O). Substrates and 2 mM NAD+, enzyme extract and 150 mM D-man nucleotides were obtained from Sigma Chemical Co. or nitol. from Pharmacia Fine Chemical, Inc. Because the high 65 Detection of enzyme activity by staining. Enzyme est purity of mannitol obtained from Sigma Chemical extract was loaded on a 7.5% T, 2.6% C polyacryl Co. still contained ca. 1% sorbitol, mannitol was puri amide gel, electrophoresed at constant voltage (200 V, fied by two successive recrystilizations from H2O 150 mA) for 40 to 45 min. on a Mini-Protean II gel 5,268,288 11 12 apparatus and stained for MDH activity. MDH activity were done at 25 C. in a total volume of 1 ml. The stain contained 50 ml of 0.2M Bis-Tris pH 9.0, 150 mM product determination is based ultimately on the Glu mannitol, 2 mM NAD+, 1 ml of MTT (3,04,5-demethy cose-6-P dehydrogenase reaction which reduces thiazolyl-2)-2,5-diphenyltetrazolium bromide Sigma) NAD+ to NADH and can be monitored spectrophoto (10 mg/ml) and 0.5 ml of PMS (2 mg/ml) phenazine 5 metrically at 340 nm. The key enzyme for mannose methosulphate (Sigma). detection is phosphomannose isomerase (PMI). By comparing the amount of NAD+ reduction in the pres D. Product Purification and Identification ence (+PMI) and absence (-PMI), it is possible to Four different MDH reactions either containing the determine if fructose or mannose is present in a particu complete reaction mixture (100 mM Bis-Tris (pH =9.0), O lar sample. If mannose is the sample, the (-PMI) reac 2 mM NAD+, 10 ul partially purified enzyme and 150 tion will exhibit no NAD+ reduction while the (+PMI) mM mannitol); the complete reaction (-mannitol); the will. If both (--PMI) and (-PMI) result in equal NAD complete reaction (-NAD+) or the complete reaction reduction fructose and or glucose is in the sample. (-enzyme) extract were run for 2 hours, heat killed in boiling water for 60 seconds and centrifuged at 10,000 g 15 E. Carbohydrate Analysis for 1 min. Because the buffer in the reaction mixture Ethanolic extractions from each of the samples were co-chromatographed with and masked several sugars used for measurements of sucrose, hexose sugars and including mannose and fructose it was necessary to polyols. The procedures for ethanolic extraction were purify the reaction product away from its reaction followed as described by N. Hubbard et al., Plants buffer prior to analysis of the product on a HPLC sys 20 Physiol 91:1527-15 (1989). Carbohydrate content was tem. An ion-exchange column was developed which determined on a HPLC system using a Waters Sugar was composed of an anion-exchanger (Bio-Rad AG Pak I (Ca column). The column was preceded by a 1-X8, Hit-form) separated from a cation-exchanger Waters Bondapak C18/Corasil guard and a set of anion (Dowex-50W, formate form) by Watman #1 paper. and cation cartridges (Bio-rad Laboratories). Total column volume equaled 2 ml. Columns were spun 25 to dryness for 4 min. at 1,000 rpm in a table top centri II, Results fuge. When this column was loaded with 1 ml of MDH Discovery of mannitol dehydrogenase. When cele reaction mixture and centrifugally passed through, the riac cv. 'Monarch' transplants were intentionally column effectively exchanged the highly concentrated stressed for a period of four weeks by growth in seeding buffer, Bis-Tris, with a lower concentration of formic 30 flats and then transplanted to 8 inch pots, both leaf and acid. The latter had the great advantage, that on the petiole hexose, sucrose and especially mannitol concen chromatogram, formic acid had a different retention trations decreased drastically by one week after trans time (RT = 4.26 min.) than mannose (RT = 11.20 min.) planting (FIG. 1). Transplants were stressed and or fructose (RT = 12.00 min.). When mannose was stunted as compared to the control plants grown in 15 added to the MDH reaction mixture, loaded on the 35 gallon pots in the same greenhouse environment. Total ion-exchange column and analyzed by HPLC on a Bio shoot weight increased only slightly over the one week Rad Fast Carbohydrate column (Pb column) a distinct period after transplanting, while root volume increased mannose peak (RT =3.64 min.) could be observed. Be drastically (data not shown), suggesting the probability cause the Waters Sugar-Pak I column (Ca column) had that mannitol was translocated to the root where it much longer retention times (RT) for most of the sug could be metabolized. When root tips of these stressed ars, the Fast Carbohydrate column with shorter RT plants were analyzed for mannitol dehydrogenase values was used to hasten the process of product purifi (MDH), an activity of 7.22 rumol/h/g fresh weight cation. The MDH product peak was collected from (fr.wt.) was observed indicating that celeriac root tips numerous injections, freeze dried and suspended in 1 ml contained a MDH which catalyzed the NAD-depend of distilled H2O. 45 ent oxidation of mannitol. No activity was observed if The purified MDH product resulting from the proto NADP was used as the substrate. In order to determine col above was then analyzed on three different HPLC the tissue specificity of MDH, a crude extract of the systems: 1) An HPLC system containing a Bio-Rad Fast root, tip, older more fibrous root, young petiole and Carbohydrate column (Pb column) 2) A HPLC system mature petiole of stressed celeriac transplants was ana with high resolution conditions consisting of a Waters 50 lyzed for MDH activity. The highest MDH activity Sugar-Pak I calcium column which was not preceded was observed in root tips (7.22 umol/h/g fr.wt.). Fi by a Waters Bondapak C18/Corasil guard or anion and brous roots, young petioles and mature petioles had a cation cartridges (Bio-Rad Laboratories). Elimination much lower activity of 1.15, 0.82 and 0.33 umol/h/g of the guards resulted in very high resolution chroma fr.wt. respectively. When celeriac cv. 'Monarch' was tography with particular improvement in the resolution 55 grown under normal, non-stressed conditions mannitol between mannose and fructose. 3) A Dionex High Per concentration was high in the foliage, knob and older formance Anion Exchange Chromatography system root but mannitol concentration was very low in the using pulsed amperometric detection. The purified root tip. Foliar sucrose concentration was almost 10 product was also identified with an enzymatic link sys fold lower as compared to mannitol concentrations and tem using commercial reagent enzymes so that the pres about 50% lower than the foliar hexose concentration ence of mannose could be differentiated from fructose. (FIG. 2). This enzymatically linked spectrophotometric assay FIG. 3 is a gel showing the MDH and ADH activity consisted of 5 U/ml hexokinase, 0.047 U/ml phos stain of different plant parts. Five ul of extract for each phomannose isomerase, 1 U/ml phosphoglucoisome tissue was electrophoresed on a PAGE gel. FIG. 3 rase and 1 U/ml glucose-6-P dehydrogenase, 1 mM 65 supports the previous finding that the MDH is most NAD+, 1 mM ATP, 1 mM MgCl, 100 mM MOPS active in root tissue. A low activity was observed in buffer at pH = 7.5 and was initiated with 20 ug/ml sub young petioles and the knob. Alcohol dehydrogenase strate (mannose, fructose or purified product). Assays (ADH) activity was observed when the gel was incu 5,268,288 13 14 bated with 25 mM ethanol. The ADH activity in this nose and not fructose or glucose. Furthermore, only 45-65% ammonium sulfate cut on the gel was lower mannose and not fructose or glucose was reduced by than the MDH activity but was present in all parts of MDH when assayed in the reverse direction (Table 3). the celeriac plant. The MDH and ADH bands seemed to have a similar migration pattern, particularly in root 5 tissue, indicating that these bands might represent a TABLE 2 single enzyme, such as an ADH which recognizes man Enzymatic determination of MDH product nitol as a substrate. Alternatively, the enzyme prepara - A340 tion might contain both MDH and ADH. In order to Substrate -PMI -PM support or contradict one of these hypotheses, the 1O Product 0.007 0.624 MDH extract was further purified. (20 g/ml) Purification of MDH. Ammonium sulfate fraction Mannose std. 0.003 0.702 (20 g/ml) ations were carried out at 45-65% saturation resulting Fructose std. 0.708 0.722 in over 100% recovery of the MDH activity and an (20 g/ml) elimination of 88% of the proteins (Table 1). It became 5 apparent that MDH was inhibited by some substance(s) in the crude extract as indicated by the greater than TABLE 3 100% recovery of MDH activity. Reverse reaction of mannitol TABLE 1 dehydrogenase:mannose reduction Purification table of NAD 20 - p - 6.0 7.5 9.0 dependent mannitol dehydrogenase - mol/h/ng protein SA % fold Units/ (mg/ (U/mg recov. purifi Substrate (mM) protein Step ins Iml ml) protein) ery cation Mannose 20 0.497 0.810 O.124 Fructose 20 0,000 al Crude 88.0 0.82 .585 0.517 100 o 25 Extract Glucose 20 2 0.000 a (NH4)2SO4 8.0 9.08 2,073 9.204 2 17.8 na: not analyzed, 45-65% saturation Fractogel 10.2 10.68 0.393 27.76 5 52.6 Enzyme stability. The 45-65% (NH4)2SO4 fraction DEAE 30 could be stored overnight at 4 C. or at -80 C. for ion-exchange several weeks without loss of activity. The partially (NH4)2SO4 2.2 35.92 .80 30.44 110 58.9 purified enzyme was concentrated with 90% 90% (NH4)2SO4 and remained stable at -80 C. for several saturation weeks. The enzyme was always stored in the presence of 1 to 2 nM DTT. When the Fractogel (R) DEAE fractions were assayed 35 Effect of pH on enzyme activity and kinetic con for MDH, alcohol dehydrogenase (ADH) and phos stants. Initially, assays of MDH at 150 nM mannitol and phomannose isomerase (PMI) activity three distinct peaks could be observed (FIG. 4). At high protein load 1 mM NAD+ revealed that the enzyme activity was not ing, ADH was not retained on the column and eluted at greatly affected by variations in pH between 7.5 and 9.0. the start of the salt gradient. The MDH peak eluted at Subsequently, the effect of pH on kinetic constants, Km 0.16 mM KCL and PMI eluted at 0.26 mM KCl. It was and Vmax, was investigated. Substrate saturation clear from this profile that a MDH pool could be ob curves and Lineweaver-Burk plots with mannitol as the tained free from ADH and PMI. Purification through substrate are shown in FIG. 5. Mannitol concentrations the DEAE step resulted in a 52-fold purification of the ranged from 0 to 250 mM. Substrate saturation curves enzyme from the crude extract. This is, however, an 45 and Lineweaver-Burk plots with NAD+ as the sub apparent value, since the crude extract had inhibitory strate are shown in FIG. 6. Kinetic parameters were activity. The MDH pool was then concentrated with a based on Lineweaver-Burk plots (highest R2 compared 90% (NH4)2SO4 saturation step resulting in a final 58 to other classic kinetic plots) and are summarized in fold purification of the enzyme from the crude extract. Table 4. Normal Michaelis-Menten kinetics are exhib Product Purification and Identification. The purified 50 ited for both mannitol and NAD+ giving Kim values of MDH product resulting from the protocol described in 72 mM and 0.26 mM respectively at pH =9.0. The materials and methods was analyzed on three different Vmax is 40.14 umol/h/mg protein for mannitol synthe HPLC systems: 1) A HPLC system containing a Bio sis at pH =9.0. At pH = 7.5 the substrate saturation Rad Fast Carbohydrate column (Pb column), 2) A curve for mannitol does not reach a saturating level HPLC system with high resolution conditions consist 55 resulting in a Km value of 228.69 mM and Vmax of ing of a Waters Sugar-Pak I calcium column and 3) A 85.64 umol/h/mg. These values are much higher than Dionex High Performance Anion Exchange Chroma at pH =9.0 indicating that the saturating mannitol con tography system using pulsed amperometric detection. centration at pH =9.0 is different and lower than at In all three chromatographic systems the product had a pH = 7.5. This is also reflected in the kinetic data of retention time indistinguishable from that of reagent NAD at pH 7.5 where a higher Kim value was observed D-mannose and different from that of D-fructose and as compared to pH =9.0. D-glucose (data not shown). The purified product was TABLE 4. also identified with an enzymatic link system using com Kinetic parameters of mannitol dehydrogenase mercial reagent enzymes so that the presence of man Vmax nose could be differentiated from fructose (see materials 65 (umol/h/mg and methods). Results of the enzymatic analysis are Substrate pH KM (mM) protein) shown in Table 2. All four independent analyses indi Mannitol 9.0 2.26 40.14 cated that the MDH product corresponded with man NAD- 9.0 0.27 3455 5,268,288 15 16 TABLE 4-continued TABLE 7-continued Kinetic parameters of mannitol dehydrogenase Mannitol dehydrogenase (MDH) regulation Vmax MDH (umol/h/mg activity Substrate pH KM (mM) protein) Reaction (unol/h/mg Mannitol 7.5 228.69 85.64 conditions Effector (mM) protein) NAD- 7.5 0.46 35.59 Limiting --- O 17.39 conditions Pi 1 18.43 (200 mM PPi 1 20.52 Substrate specificity. MDH was very specific for O mannitol + ADP l 12.87 mannitol but showed low activity with sorbitol and 0.2 mM NAD) ATP l 18.79 galactitol (Table 5). The apparent substrate specificity Limiting - O 24.00 conditions Pi 1 25.74 was higher at pH 7.5 as compared to pH 9.0. However, (75 mM PP 1 25.04 when a 99% pure sorbitol preparation was used at high mannitol + ADP 1 25.04 concentration, a significant amount of contaminants 15 2 mM NAD) ATP 24.35 could be detected by HPLC chromatography (data not shown). When sorbitol was used at 150 mM, the manni tol contamination wa high enough to contribute to the III. Summary and Conclusions apparent oxidation of sorbitol. Therefore the actual The data presented herein is the first evidence of the specificity of the enzyme for mannitol might be greater 20 pressure of a mannitol oxidizing enzyme in a higher than apparent from the data. NADP+ was not a sub vascular plant. Based upon purification away from strate for activity in either the crude extract nor in the ADH and PMI, product identification and reverse reac partially purified enzyme. tion data, it is concluded that this enzyme is a NAD TABLE 5 dependent mannitol:mannose 1-oxidoreductase. The Substrate specificity of mannitol dehydrogenase 25 enzyme is not unique to celeriac but has also been found MDH (uno/h/mg protein) to be present in celery, Apium graveolens var. dulce (mM) pH = 7.5 pH = 9.0 (data not shown). Mannitol 150 1.30 (100%) 30.96 The conversion of mannitol to mannose by this plant Sorbitol 50 3.13 (10%) 6.77 (21%) enzyme is uniquely different from the mannitol oxida Galactitol 75 1.74 (5%) 2.20 (7%) 30 tion occurring in lower organisms. Fungi, bacteria, Myo- 150 0.00 (0%) 0.00 (0%) lichens and algae oxidize mannitol to fructose either inositol directly (N. Morton et al., J. Gen. Microbiol. 131:2885–2890 (1985)) or through a phosphorylated MDH effectors. When the partially purified mannitol intermediate (R. Kiser et al., Arch. Biochem. Biophys. dehydrogenase was assayed in the presence of some 35 21 1:613-621 (1981)) by a NAD+ or NADP+ dependent potential effectors such as hexose or hexose-P, no inhi 2-oxidoreductase. Mannitol synthesis is also different bition of the enzyme was observed (Table 6). The en between lower organisms and vascular plants. Celery zyme was strongly inhibited by NADH and was sensi synthesizes mannitol by a NADPH-dependent man tive to alterations of NAD/NADH ratio (FIG. 7). Inor nose-6-P reductase which reduces mannose-6-P to man ganic phosphate, PPi, ADP and ATP did not substan 40 nitol-1-P whereas mannitol in lower organisms is syn tially affect ADH activity (Table 7). ADP may be par thesized from fructose or fructose-6-P. It remains to be tially inhibitory at low NAD+ concentrations. determined if mannitol to mannose interconversions, TABLE 6 may occur, but have escaped detection in lower organ isms. Potential Effectors of Mannitol 45 Dehydrogenase (MDH) Activity Mannitol metabolizing plants may possess a unique MDH advantage over non-polyol metabolizing plants in that activity both mannitol synthesis and mannitol utilization result Reaction (unol/h/mg % in a more efficient overall metabolism. Thus, genetic Conditions Effector (mM) prot.) inhibition 50 transformation of plants for expression of the mannose Saturating - O 32.69 - 6-P reductase in photosynthetic tissues and the manni conditions Mannose 5 31.65 0 (15 mM Sucrose 20 32.68 0 tol: mannose 1-oxidoreductase in sink tissues may pro mannitol -- 2 Mannose-6-P 5 36.18 O vide a novel way to increase photosynthetic rates and mM NAD) Fructose-6-P 5 35.83 O biomass production in agricultural crops. Glucose-6-P 5 32.68 O Glucose-1-P 5 35.83 0 5 5 EXAMPLE 2 NADH 0.3 4.87 85 Affinity Purification of Mannitol Oxidoreductase The partially purified desalted (NH4)2SO4.90% satu TABLE 7 ration fraction of mannitol oxidoreductase prepared as Mannitol dehydrogenase (MDH) regulation described in Example 1 above was further purified by MDH affinity chromatography as described below. activity The mannitol oxidoreductase, with a specific activity Reaction (umol/h/mg of 64.19 umol/h/mg protein, was loaded on a f3 conditions Effector (mM) protein) Nicotinamide Adenine Dinucleotide agarose column Saturating m O 32.69 65 conditions Pi 1 31.65 (Column characteristics: Sigma N 1008, 1 ml volume, (200 mM PP 1 36.18 spacer 9, attachment C8). The column was washed with mannitol -- ADP 1. 35.83 10 bed volumes of distilled water followed by 10 bed 2 mM NAD) ATP 32.68 volumes of running buffer (RB) prior to addition of 5,268,288 17 18 protein. (Running buffer (RB) contained: 20 mM Bis The foregoing examples are illustrative of the present Tris pH =9.0 and 1 mM DTT, Eluting buffer (EB) invention, and are not to be construed as limiting contained: 20 mM Bis-Tris pH =9.0, 2 mM NAD and 1 thereof. The invention is defined by the following mM DTT). At the time of protein (partially purified claims, with equivalents of the claims to be included mannitol:mannose 1-oxidoreductase) loading, elution 5 therein. solvent was collected in 1 ml fractions and the column That which is claimed is: was washed with running buffer until protein concen 1. Isolated mannitol oxidoreductase which: tration in fractions (measured by A280) returned to (a) is NAD+ dependent; baseline levels (approximately after 5 bed volumes). (b) converts mannitol to mannose; and The column was then washed with eluting buffer which 10 (c) is isolated from a vascular plant. 2. The mannitol oxidoreductase according to claim 1 contained 2 mM NAD and which eluted the oxidore isolated from a plant selected from the group consisting ductase. One ml fractions were collected and assayed of Apiaceae, Arecaceae, Asteraceae, Brassicaceae, for mannitol dehydrogenase (MDH) activity and pro Bromeliaceae, Buxaceae, Cactaceae, Canellaceae, Com tein content (Biorad method). The mannitol:Inannose 5 bretaceae, Compositae, Convolvulaceae, Cucur i-oxidoreductase eluted in 2 ml and had a specific activ bitaceae, Cucurbita, Euphorbiaceae, Fabaceae, Gneta ity of 201 umol/h/mg protein and a 73.79% recovery of ceae, Gramineae, Liliaceae, Oleaceae, Orobanchaceae, the amount of the enzyme activity which was loaded on Pinaceae, Plantanaceae, Primulaceae, Punicaceae, the column. This "NAD column" step therefore repre Ranunculaceae, Rosaceae, Rubiaceae, and Scro sents an additional 3.13 fold purification as compared to 20 phulariaceae. the "(NH4)2SO4.90% saturation' step and a 186 fold 3. The mannitol oxidoreductase according to claim 1 purification as compared to the crude extract (see Puri isolated from a plant selected from the group consisting fication Table 7). of Apiaceae, Brassicaceae, Bromeliaceae, Compositae, TABLE 7 Liliaceae, Oleaceae, and Rubiaceae. 25 4. The mannitol oxidoreductase according to claim 1 Purification Table isolated from an Apiaceae. DESALTED FOLD 5. The mannitol oxidoreductase according to claim 1 FRACTIONS SA(U/MG PROT) PURIFICATION isolated from Apium graveolens. Crude Extract 1079 45-65% (NH4)2SO4 9.705 8.99 6. The mannitol oxidoreductase according to claim 1 saturation 9 30 isolated from a plant selected from the group consisting Fractogel DEAE - of Apium graycolons L. var. rapaceum (Mill.) Gaud. 90% (NH4)2SO4 64.19 59.49 (celeriac) and Apium graveolens L. var. dulce (Mill.) NAD column 201.00 186.28 Pers. (celery). one unit (U) equals one unol product/h. 7. Isolated mannitol oxidoreductase which: 35 (a) is NAD+ dependent; This highly purified fraction was stored and stable at (b) converts mannitol to mannose; and -80 C. with only a small loss (< 12%) occurring dur (c) is isolated from coloriac. ing storage at -80 C. sk sk k z

65 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 5,268,288 DATED O7 December 1993 INVENTOR(S) : David M. Pharr and Johan M. H. Stoop It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

column 1, Line 56, correct "(12CO)" to read --(14CO2)--. Column 3, Line 57, correct "offinalis" to read --officinalis--. Column 10, Line 8, correct "Theextraction" to read --The extraction--. column 12, Line 12, correct "NAD" to read --NAD'--. Column 15, Line 41, correct "ADH" to read --MDH--. Column 16, Line 20, correct "pressure" to read --presence--. Column 18, Line 31, correct "gravcolons" to read --graveolens--.

Signed and Sealed this Fifth Day of July, 1994 (a teen BRUCELEMAN Attesting Officer Commissioner af Patents and Trademarks