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WO1996000789A1 - Plantes transgeniques productrices de trehalose - Google Patents

Plantes transgeniques productrices de trehalose Download PDF

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Publication number
WO1996000789A1
WO1996000789A1 PCT/FI1995/000377 FI9500377W WO9600789A1 WO 1996000789 A1 WO1996000789 A1 WO 1996000789A1 FI 9500377 W FI9500377 W FI 9500377W WO 9600789 A1 WO9600789 A1 WO 9600789A1
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WIPO (PCT)
Prior art keywords
trehalose
plant
gene
plants
promoter
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PCT/FI1995/000377
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English (en)
Inventor
John Londesborough
Outi Tunnela
Kjell-Ove Holmström
Einar Mäntylä
Björn Welin
Abul Mandal
E. Tapio Palva
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Alko Group Limited
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Application filed by Alko Group Limited filed Critical Alko Group Limited
Priority to NZ288635A priority Critical patent/NZ288635A/xx
Priority to HU9603608V priority patent/HU221613B/hu
Priority to US08/765,691 priority patent/US6130368A/en
Priority to GB9627137A priority patent/GB2303856B/en
Priority to AU27944/95A priority patent/AU699391B2/en
Priority to FI965132A priority patent/FI965132L/fi
Priority to JP8502849A priority patent/JPH10501978A/ja
Priority to EP95923355A priority patent/EP0763118A1/fr
Publication of WO1996000789A1 publication Critical patent/WO1996000789A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the present invention relates to the genetic engineering of plants to introduce a capacity to synthesise trehalose.
  • the invention concerns plants having an increased trehalose content and methods for producing them.
  • the present invention also relates to methods for increasing the tolerance of plants towards stresses such as cold and drought, as well as to methods for producing trehalose.
  • Trehalose ⁇ -glucopyranosyl- ⁇ -D-glucopyranose
  • Trehalose is a dimer of glucose molecules linked through their reducing groups. Because of its unusual combination of chemical properties compared to other sugars, including its lack of reducing groups, slow hydrolysis and ability to form a non- deliquescent glass, it is one of the most effective known preservatives of proteins, cellular membranes and other biological compounds in vitro. Also, living organisms that contain large amounts of trehalose are characteristically , those often exposed to osmotic, dehydration and heat stresses, such as insects, certain litoral animals and many microorganisms, including yeasts and bacteria.
  • trehalose is toxic to many plant tissues (Veluthambi et al. [1981] Plant Physiol. 68, 1369-1374) , especially those with little or no trehalase activity (trehalase is the enzyme that converts trehalose to glucose) .
  • At least one angiosperm Myrothamnus flabellifolia (another "resurrection” plant) , accumulates significant amounts of trehalose (Bianchi et al. [1993] Physiologia Plantarum 87, 223-226) , showing that there is not an absolute compatability barrier between trehalose and angiosperms.
  • trehalose makes its use in the dried food industry prohibitively expensive.
  • production of trehalose in the edible portion of certain plants could extend the shelf life of products such as tomatoes.
  • accumulation of trehalose in sensitive tissues could increase the tolerance of plants towards frost, drought, high salinity and similar stresses.
  • the present invention is based on the concept of trans ⁇ forming the plants of interest with structural genes for trehalose synthesis under the control of appropriate plant promoters in order to produce transgenic plants having increased trehalose contents.
  • the coding sequences of one or more genes encoding polypeptides of enzymes producing trehalose 6-phosphate or trehalose itself are, according to the invention, expressed in plants under the control of specific kinds of plant promoters.
  • the plant of interest is transformed at least with the coding sequence of a gene for trehalose-6-phosphate synthase (Tre6P synthase) fused appropriately to a plant promoter so that full expression of the gene is realised only as the plant matures or when it encounters specific environmental conditions.
  • the promoters are therefore preferably non- constitutive and chosen to allow temporal (e.g., diurnal), topological (e.g., tissue-specific) or stress-activated (or "stress-induced”) control over the expression of the genes.
  • the plant may be also transformed with one or more genes encoding a trehalose-6-phosphatase (Tre ⁇ Pase) or a regulatory polypeptide that interacts with the Tre6P synthase or the Tre6Pase or both.
  • Tre ⁇ Pase trehalose-6-phosphatase
  • a regulatory polypeptide that interacts with the Tre6P synthase or the Tre6Pase or both.
  • Transformation of the plant may be done by any of the methods available in the art, including infection with tranformed Acrrobacter tumefaciens and the direct introduction of foreign DNA by microinjection, electroporation, particle bombardment and direct DNA uptake.
  • the structural genes are preferably selected from the group comprising the yeast genes TPS1, TPS2 and TSL1 encoding respectively the 56 kDa Tre6P synthase, 102 kDa Tre6Pase and 123 kDa regulatory subunits of yeast trehalose synthase.
  • It is a third object of the invention to produce trehalose which comprises the steps of - transforming a plant with at least one structural gene for Tre6P synthase in order to produce a transgenic plant, - cultivating the transgenic plant under conditions which will induce the expression of trehalose synthesis in the plant, and - extracting the trehalose from the tissues of the plant.
  • a fourth object of the invention is to provide a method of protecting plants against adverse conditions such as drought, high salinity, temperature extremes and other stresses, which method comprises transforming the plant with at least the coding sequence of a gene for a trehalose-6- phosphate synthase fused appropriately to a plant promoter so that full expression of the gene is not realised until the plant encounters adverse conditions.
  • the plant may optionally be also cotransformed with one or more genes encoding trehalose-6-phosphatase and regulatory proteins that interact with the trehalose-6-phosphate synthase or trehalose-6-phosphatase or both.
  • the invention provides a method of protecting plants bearing berries or other fruits against frost damage to blossom, which comprises transforming the plant with a gene for trehalose- 6-phosphatase fused appropriately to a plant promoter so that full expression of the gene is not realised until the plant encounters low temperatures.
  • a fifth object of the invention is to provide a method of producing transformed ornamental plants that do not require such intensive and expert care as the untransformed plant, which method comprises transforming the plant with a gene for trehalose-6-phosphate synthase appropriately fused to a plant promoter so that the plant contains trehalose in some of its tissues.
  • Figure 1 depicts the schematic structure of the chimeric gene construct containing the A. thaliana Rubisco small subunit promoter (patslA) fused to the TPS1 yeast gene, encoding the Tre6P synthase subunit, and the transcription stop signal from the nopaline synthase gene of Acrrobacterium tumefaciens (nos) . Only the part of the plasmid pKOH51 with the chimeric gene is shown. Unique restriction enzyme cleavage sites for the chimeric gene construction are shown.
  • Figure 2 gives the results of Western blot analysis of transgenic tobacco plants showing the 56 kDa TPS1 product indicated by an arrow.
  • Proteins were extracted from the transformed tobacco plants containing (lines 1, 3, 4, 5, 6, 8, 10, 12, 15, 16 and 19) the construct described in Fig 1, or (GUS) another chimeric gene with the Cauliflower mosaic virus 35S promoter fused to the -glucuronidase gene (UIDA) in the same vector or (SRI) from untransformed tobacco plants. Equal amounts of protein were loaded in each lane.
  • the antiserum used (anti-57) was raised against the 56 kDa Tre6P synthase subunit from yeast.
  • Figure 3 shows the chromatographic identification of trehalose.
  • Samples (20 ⁇ l ) of water extracts of tobacco leaves were analyzed by HPLC as described in General Materials and Methods. Extracts A and B contained 192 mg fresh weight of greenhouse-grown Transformant 19 ml "1 (A) before and (B) after treatment with trehalase. Extracts C and D contained 149 mg leaf ml "1 from (C) Transformant 4 or (D) a control plant transformed with plasmid pDElOOl lacking the TPS1 gene, both grown under sterile conditions.
  • Trehalose peaks are indicated with T.
  • the two large peaks at about 25 min are glucose and sucrose.
  • FIG. 4 Protein immunoblot showing tissue localization of TPSl-subunit analyzed in the atsl -TPSl transformed line 4.
  • Leaves of wild-type tobacco (SRI) were used as a negative control and 0.1 ⁇ g of Tre6P synthase subunit purified from yeast (TPS1) as a positive control.
  • the trehalose content of the same tissues is indicated in mg/g dry weight above the immunoblot. nt, not tested.
  • the following abbreviations are used: FB for flower buds, UL for upper leaves, ML for middle leaves, LL for lower leaves, US for upper stem, R for roots and EC for enzyme control.
  • Fig. 5. Enhanced drought tolerance in trehalose-producing plants .
  • A Six to eight weeks' old in vitro propagated whole plants were exposed to air-drying (D) for the times indicated. After 17 h of stress-treatment, the nontransformed control plants (SRI) and trehalose-producing transgenic plants of lines 4 and 8 were rehydrated (R) by placing them in water.
  • B Three week old seedlings of transgenic line 8 and both nontransformed (SRI) and vector- transformed (C) control plants were exposed to air-drying (D) for the times indicated. The seedlings were subsequently rehydrated (R) after 7 h of stress by placing them in water.
  • FIG. 7 SDS-PAGE analysis of fractions containing M. sme ⁇ matis Tre6P synthase eluted from the first Heparin- Sepharose column. Lanes 1 to 4 contain, respectively, samples of H33 (7.0 mU) , H35 (20 mU) , H36 (21 mU) and H37 (12 mU) (Table 3) . After SDS-PAGE on 8 % acrylamide, the gels were stained with Coomassie Brilliant Blue. The sizes of the molecular mass standards in lane 5 are shown in kDa. The position of the 55 kDa Tre6P synthase polypeptide is shown with an arrow.
  • FIG. 8 SDS-PAGE analysis of the peak fraction of M. sme ⁇ matis Tre6P synthase eluted from the second Heparin- Sepharose column. Fraction T21 (Table 3) was concentrated in a Centricon 10 tube and then mixed with half its volume of 3-fold concentrated SDS-PAGE sample buffer. The overall concentration was 6-fold. Samples containing (lane 1) 2.9 mU and (lane 3) 8.7 mU of Tre6P synthase were subjected to SDS- PAGE on 8 % acrylamide and stained with Coomassie Brilliant Blue. The molecular mass standards in lane 2 are, from top to bottom, 109, 84, 47, 33, 24 and 16 kDa.
  • FIG. 9 Western analysis of Tre6P synthase purified from M. smecrmatis.
  • An 8 % acrylamide gel was loaded with prestained molecular mass markers (lane 1: 200, 117, 80 and 47 kDa; lane 10: the only visible marker is 84 kDa) , pure Tre6P synthase from fraction T21 (Table 3) (lanes 7 and 8: 2.9 mU; lanes 3 ,4 and 9 : 8.7 mU) and the pooled active fractions from G100 Sephadex (Table 3) (lanes 6: 7.8 ⁇ g total protein; lanes 2 and 5 : 23 ⁇ g total protein) .
  • the nitrocellulose membrane was cut between lanes 3 and 4 and between lanes 7 and 8.
  • Lanes 1 to 3 were stained with Coomassie Brilliant Blue
  • lanes 4 to 7 were probed with anti-57 serum raised against the purified 56 kDa Tre6P synthase subunit from yeast and lanes 8 to 10 were probed with preimmune serum.
  • Immunoreactive bands were visualised using goat anti-rabbit IgG-alkaline phosphatase conjugate from Promega according to the manufacturer's instructions, with colour development times of 2.8 minutes in both cases.
  • the diagonal lines across lanes 4 to 7 are due to accidental creasing of the membrane during transfer to nitrocellulose.
  • constitutive plant promoter refers to plant promoters that cause the continuous and general expression of their associated coding sequences, so that the products of these sequences are found in all cells of the plant and at all phases of growth.
  • Non- constitutive promoters are activated by specific internal or external events, such as the differentiation of cells to form distinct tissues as a plant developes and matures or changes in the plant's environment.
  • Many kinds of environmental change are known to activate particular promoters . Examples include the light induced activation of promoters for the small subunit of ribulose- 1, 5-bisphosphate carboxylase (Krebbers et al . [1988] Plant. Mol. Biol .
  • the biosynthesis of trehalose in transgenic plants can be subjected to temporal control (i.e., it only occurs at certain times) , topological control (i.e., it is limited to certain parts of the plant) or both.
  • the present invention is concerned with the genetic engineering of plants to introduce a heterologous capacity for trehalose synthesis (in the present context also called a "novel" capacity for trehalose synthesis) .
  • a heterologous capacity for trehalose synthesis in the present context also called a "novel" capacity for trehalose synthesis
  • the increase in trehalose required, compared to that in the untransformed plant grown under identical conditions, is that which either causes a useful improvement in stress-tolerance or can be profitably extracted from the plant for commercial use.
  • Tre6P trehalose-6-phosphate
  • Saccharomvces cerevisiae contains a trehalose synthase complex comprising 56, 102 and 123 kDa subunits (Londesborough & Vuorio [1993] Eur. J. Biochem. 216, 841- 848) which condenses uridinediphosphoglucose (UDPG) and glucose-6-phosphate (Glc6P) first to Tre6P (the Tre6P synthase reaction) and then to free trehalose (the Tre6Pase reaction) .
  • UDPG uridinediphosphoglucose
  • Glc6P glucose-6-phosphate
  • Tre6P synthase and Tre6Pase activities reside in the 56 and 102 kDa subunits, respectively, and the 123 kDa subunit confers regulatory properties on the complex and appears to stabilise it.
  • Other microbial systems include those of Candida utilis (Soler et al [1989] FEMS Microbiol Letters 61, 273-278) , E. coli (Glaever et al [1988] J. Bacteriol . 170, 2841-2849), Dictvostelium discoideum (Killick [1979] Arch. Biochem. Biophys. 196, 121-133) and Mvcobacterium smegmatis (Lapp et al [1971] J. Biol. Chem. 246, 4567-4579), the latter two systems being able to use adeninediphosphoglucose (ADPG) as an alternative to UDPG.
  • ADPG adeninediphosphoglucose
  • Multicellular organisms that make trehalose including nematodes, insects and resurrection plants, may also be assumed to contain enzymes with Tre6P synthase and Tre6Pase activity.
  • Tre6P synthases transfer of any of these Tre6P synthases to plants would give the plants the novel capacity to synthesise Tre6P. It is disclosed that some plants, such as tobacco, have an inherent capacity to convert Tre6P to trehalose, so that, surprisingly, transformation of, e.g., tobacco with a gene for Tre6P synthase alone leads to efficient production of trehalose itself. However, if the endogenous conversion of Tre6P to trehalose is slow or absent, a Tre6Pase from any suitable source may be also transferred.
  • Tre6P synthase and Tre6Pase are subunits of a trehalose synthase
  • the trehalose formed in the transgenic plants can then be extracted or could confer increased tolerance to certain stresses.
  • the production of trehalose in plants transformed in this way with certain yeast genes has already been described (PCT/FI93/00049) .
  • a plant promoter (a promoter is a part of a gene that promotes transcription of the coding sequence) that does not permit full expression of the gene(s) causing trehalose synthesis until the plant is mature or encounters environmental conditions, including drought and low temperature, in which the benefits of trehalose outweigh its possible disadvantages to the plant.
  • Several examples of such non-constitutive plant promoters are known to those familiar with the art, including the small subunit ribulose-1,5-bisphosphate carboxylase (Rubisco) promoter, which drives the light-induced expression of the small subunit of RUBISCO (Krebbers et al. [1988] Plant Mol. Biol. 11, 745-759).
  • Tobacco and Arabidopsis plants transformed with the coding sequence (open reading frame, ORF) of the yeast TPS1 gene correctly fused to the atslA promoter of a Rubisco small subunit gene are disclosed.
  • TPS1 encodes the 56 kDa subunit of yeast trehalose synthase.
  • the transformed plants are healthy and fertile and contain trehalose in their leaves. Untransformed tobacco or tobacco transformed with a similar vector lacking the TPS1 gene do not contain trehalose.
  • the disclosed transgenic plants were obtained using A.
  • tunefaciens to mediate the transformation, but any method available in the art may be exploited, including the direct introduction of DNA by microinjection, electroporation or particle bombardment (Gasser & Fraley [1989] Science 244, 1293) .
  • Transformant 4 One of these transformed tobacco plants (Transformant 4) is shown to contain Tre6P synthase activity.
  • the free 56 kDa subunit is known to be unstable when isolated from the intact trehalose synthase complex of yeast (Londesborough & Vuorio [1993] loc cit) .
  • Methods are described by which a person skilled in the art can co-transform plants with the TPS1 gene and one or both of the other yeast trehalose synthase genes (TPS2 and TSL1) under the control of plant promoters, e.g. the atslA promoter or some convenient promoter that may be constitutive.
  • Such co-transformation may increase the trehalose content of the plants compared to that of plants containing only TPS1, because the subunits encoded by TPS2 and TSL1 will stabilise the 56 kDa subunit.
  • Tre6P synthase In plants cotransformed with a Tre6P synthase gene controlled by an inducible promoter together with one or more genes for Tre6Pase or regulatory proteins (such as the TSL1 product) controlled by constitutive promoters, the production of trehalose will be regulated by the inducible promoter, since Tre6P synthase catalyses the first unique step of trehalose biosynthesis.
  • the water-stress tolerance of the disclosed transgenic plants is surprising, because the amounts of trehalose found in their tissues seem too small to provide osmotic buffering of the intracellular contents. Possibly the trehalose acts by protecting specific sites, such as membranes, or possibly trehalose or its precursor, Tre6P, perturb the plant's metabolism to cause secondary changes that protect the cells against stress.
  • transgenic plants synthesised trehalose although they were transformed with only the yeast TPS1 gene encoding Tre6P synthase, and not with the TPS2 gene, encoding Tre6Pase.
  • some plants have an endogenous capacity to convert Tre6P into trehalose. It may be advantageous in some cases to cotransform the plant also with a gene, such as TPS2, encoding a Tre6Pase, to increase the rate of conversion of Tre6P to trehalose.
  • TPS2 encoding a Tre6Pase
  • Tre6P synthase from Mycobacterium smecrmatis is disclosed to be an about 55 kDa polypeptide that cross-reacts with anti-serum raised against the purified 56 kDa Tre6P synthase subunit of yeast trehalose synthase.
  • Amino acid sequences of tryptic peptides from the M. smecrmatis enzyme are disclosed, revealing close sequence homology to the yeast enzyme.
  • Plants containing trehalose as a result of transformation with one or more genes for trehalose synthase can be used in several ways.
  • trehalose can be extracted from the plants on a commercial scale.
  • Such trehalose would be cheap enough to be used in bulk applications, including the preservation of the flavour and structure of food stuffs during drying.
  • the trehalose would preferably be accumulated in a storage organ, such as the tuber of a potato, the root of a sugar beet or turnip or the bulb of an onion.
  • the present application could also be realised for example, in the stem and leaves of a transformed sugar cane or the fruit of a banana.
  • Plant promoters are known in the art (e.g., the patatin promoter) that cause expression specifically in a storage organ.
  • the coding sequence of a gene for Tre6P synthase, such as TPS1 is fused to such a promoter in the same way as the TPS1 coding sequence was fused to the atslA promoter, and suitable plants are transformed with these DNA constructions.
  • the trehalose accumulated in the storage organs may then be extracted.
  • the trehalose accumulated in the tissues of a plant may improve the storage properties after harvesting.
  • the detached leaves of transformed tobacco lost water more slowly than those of untransformed tobacco, and even after loosing water did not become discoloured (Fig. 5) .
  • This aspect is applicable both to edible plants and to ornamentals.
  • the shrivelling and discolouration of the various kinds of commercial lettuces that occur after harvesting is a serious economic burden especially to retail outlets. The ultimate cost is passed on to the consumer.
  • Trehalose-producing lettuces will provide the consumer with both cheaper and more attractive salads. Similar considerations apply to other food plants, especially leafy products such as cabbages, broccoli, spinach, dill or parsley and other green vegetables such as peas and runner beans.
  • the Tre6P synthase gene is fused to a plant promoter chosen so that trehalose accumulates in the plant parts to be harvested.
  • the atslA promoter causes expression of Tre6P synthase in the leaves, upper stem and flower buds of tobacco, and trehalose accumulates in these parts.
  • the inventors disclose that plants such as tobacco transport trehalose from its site of synthesis to tissues that do not synthesise trehalose. Thus, smaller amounts of trehalose were also found in the roots of these transgenic tobacco plants, although the non-constitutive atslA promoter did not cause expression of Tre6P synthase in the roots.
  • edible parts of transformed plants containing trehalose such as tomatoes, berries and other fruits, are processed to make purees, pastes, jellies and jams that have a fresher and richer flavour because of their trehalose content. It has been shown (WO 89/00012) that adding trehalose to such food stuffs improves the preservation of their flavour, particularly when the processing involves a drying step.
  • the present invention circumvents the need to add trehalose, by providing a plant that already contains trehalose. It may be an advantage in this aspect of our invention to use promoters, such as the patatin promoter, that direct the synthesis of trehalose primarily to the storage organ of the target plant.
  • transformed tobacco that produces trehalose has increased drought resistance.
  • transformed plants containing trehalose may be more resistant to drought, frost, high salinity and other stresses than the untransformed plants.
  • drought, frost and osmotic stress all distress plants primarily by withdrawing water from within cells, so causing damage to membranes and proteins that trehalose is known to alleviate in vitro (Crowe et al [1992] Annu. Rev. Physiol. 54, 579) .
  • the plant promoter used may be one that is induced by stress.
  • Such promoters are known in the art, e.g. LTI78 (Nordin et al . [1993] Plant Mol. Biol.
  • This aspect has applications not only for food plants, but also for ornamental plants intended for gardens or indoor display.
  • the transformed stress-tolerant ornamental plants would require less intensive and less expert care than the corresponding untransformed plants.
  • the slower growth of some trehalose-producing plants and minor morphological changes observed at least in the primary transformants may not be a disadvantage with ornamental plants: slower growth and novel appearance can be attractive characteristics especially for indoor ornamentals.
  • a gene for Tre6P synthase is appropriately fused to a plant promoter (e.g., LTI78 or RAB18) that is activated by a specific event or set of conditions (e.g. cold or drought stress) so that accumulation of commercially extractable amounts of trehalose in the plant can be triggered to occur in the mature plant shortly before harvesting, avoiding any deleterious effects of trehalose on the early development of certain plants
  • a plant promoter e.g., LTI78 or RAB18
  • a specific event or set of conditions e.g. cold or drought stress
  • the transgenic plants according to the invention can be onocotyledonous plants, such as corn, oats, millet, wheat, rice, barley, sorghum, amaranth, onion, asparagus or sugar cane, or dicotyledonous plants such as alfalfa, soybean, petunia, cotton, sugarbeet, sunflower, carrot, celery, cabbage, cucumber, pepper, tomato, potato, lentil, flax, broccoli, tobacco, bean, lettuce, oilseed rape, cauliflower, spinach, brussel sprout, artichoke, pea, okra, squash, kale, collard greens, tea or coffee.
  • onocotyledonous plants such as corn, oats, millet, wheat, rice, barley, sorghum, amaranth, onion, asparagus or sugar cane
  • dicotyledonous plants such as alfalfa, soybean, petunia, cotton, sugarbeet, sunflower, carrot, celery, cabbage, cucumber, pepper, tomato, potato,
  • yeast gene TPS1 and its product are compatible with the biochemical machinery of tobacco: the gene was highly expressed and the 56 kDa subunit caused the appearance of trehalose.
  • plant genes often have lower A+T ratios than, e.g., microbial genes, and that the expression level of heterologous genes in plants can be increased by altering the codon usage, particularly near the start of the coding sequence, towards that found in plants (Perlak et al . [1991] 88, 3324-3328) .
  • these and similar modifications of genes may be useful in the present invention.
  • TPS1 includes all DNA sequences homologous with TPS1 (or TPS2 or TSL1) that encode polypeptides with the desired functional or structural properties of the 56 kDa (or 102 kDa or 123 kDa) subunit of yeast trehalose synthase.
  • the plants used were Nicotiana tabacum cv. SRI and Arabidopsis thaliana L. Heynh. ecotype C-24.
  • the yeast genes TPSl (formerly called TSSl) , encoding the 56 kDa Tre6P synthase subunit of trehalose synthase, and TSL1, encoding the 123 kDa regulatory subunit of trehalose synthase, were obtained from the plasmids pALK752 and pALK754 described by Vuorio et al . ([1993] Eur. J. Biochem. 216, 849-861) .
  • the yeast gene TPS2 was obtained from a plasmid supplied by Drs Claudio De Virgilio and Andres Wiemken, Botanisches Institut der Universitat Basel, Switzerland and containing the TPS2 gene cloned into the Sad site of plasmid YCplaclll.
  • This gene (described in De Virgilio et al . [1993] Eur. J. Biochem. 212, 315-323) encodes amino acid sequences derived from the 102 kDa subunit as shown in Table 1 of Vuorio et al . (1993, loc cit) .
  • Antisera prepared against yeast trehalose synthase (anti-TPS/P) and the 56 kDa subunit (anti-57K) and general biochemicals were from the sources cited in Vuorio et al . (1993, loc cit) .
  • Vacuolar trehalase was partially purified as described by Londesborough & Varimo ( [1984] Biochem. J. 219, 511-518) from a suc " gal " mel " mal " yeast strain (ALK02967) and did not hydrolyse sucrose, maltose or melibiose.
  • the atslA promoter fragment lacking the sequence for the transit peptide, was amplified by PCR from the plasmid pGSFR401.
  • Synthetic oligonucleotide primers were used to create an EcoRI site at the 5'end and an Xbal site at the 3'end of the amplified fragment.
  • the PCR amplification product was digested with the appropriate restriction enzymes and, following purification on an agarose gel, ligated into an EcoRI and Mlul digested pUC19 plasmid.
  • the yeast TPSl gene was amplified from the plasmid pALK752 described above. The resulting fragment contained 5' Mlul and 3' Xbal sites.
  • axenic growth sterilised explants from Nicotiana tabacum (SRI) were planted in glass jars containing solidified MS (Murashige & Skoog [1962] Physiol. Plant 15, 473-497) medium supplemented with 2 % sucrose (MS-2) . These jars were then placed in a controlled growth environment in a culture chamber where plants were allowed to grow at 22 °C with a 16 h photoperiod. Explants were regularly transferred to new jars and MS-2 medium for a continuous growth of axenic material. Greenhouse plants were grown in soil in pots and watered daily.
  • Transformed A_j_ thaliana plants were first grown axenically in baby-food jars in a controlled environment as described for tobacco above, but were later transferred to soil in pots in the greenhouse for seed production. Seeds from the primary transformants were either directly planted in soil for new seed production or surface sterilised and grown axenically in 24-well tissue culture plates for molecular analysis.
  • Tobacco and A. thaliana were both transformed according to the following protocol, with starting material being excised leaves of tobacco and roots of A. thaliana.
  • the transformation and tissue culture were essentially as described by Valvekens et al ([1988] Proc. Natl. Acad. Sci. U.S.A. 85, 5536-5540) with the following modifications. Isolated roots or leaves were preincubated on solidified callus-inducing medium (CIM) for 4 days, roots were cut into small segments (1 - 2 mm) and leaves were cut into larger pieces (10 - 20 mm) and transferred into 20 ml liquid CIM.
  • CIM callus-inducing medium
  • 3' , 5' -Dimethoxy-4'hydroxyacetophenone was added (0.2 mg/1) prior to Agrobacterium (C58C1 rif R ) infection.
  • the bacteria used for infection were propagated overnight in YEB medium (Vervliet et al . [1975] J. Gen. Virol. 26, 33-48) containing appropriate antibiotics at 28 °C, and collected by centrifugation. The bacterial pellet was then resuspended in 10 mM MgS04, added to the plant tissue and mixed gently for about 15 min. Excess liquid was poured off and the roots blotted on sterile filter paper.
  • the plant tissue was rinsed 3 - 4 times with liquid CIM to wash off bacteria, and transferred to 22 selective shoot induction medium (SIM) . After 7 days of growth, explants with differentiated morphogenic sectors were transferred to fresh SIM.
  • SIM selective shoot induction medium
  • Tre6P synthase Assays About 500 mg of frozen plant material was weighed and then ground to a fine powder with a mortar and pestel on solid C0 2 . The powder was transferred to 0.7 ml of 50 mM HEPES/KOH pH 7.0 containing 1 mM benzamidine, 2 mM MgCl 2 , 1 mM EDTA and 1 mM dithiothreitol (HBMED) containing 1 mM PMSF, and 10 ⁇ g/ml each of pepstatin A and leupeptin and allowed to melt.
  • HEPES/KOH pH 7.0 50 mM HEPES/KOH pH 7.0 containing 1 mM benzamidine, 2 mM MgCl 2 , 1 mM EDTA and 1 mM dithiothreitol (HBMED) containing 1 mM PMSF, and 10 ⁇ g/ml each of pepstatin A and leupeptin and allowed to melt.
  • Trehalose Assays About 500 mg of frozen plant material was quickly weighed into a glass tube. Hot, distilled water (1 ml) was added and the mixture was boiled for 20 min, the leaf material being broken up at intervals with a blunt glass rod. The liquid phase was collected with a pasteur pipette and the solid re-extracted with 0.5 ml of water. The combined liquid phases were clarified by centrifugation. Combined solid residues were dried to constant weight at 107 °C (the dry weights of leaves averaging 5.1 % of the fresh weights) . The supernatant was analysed using a Dionex DX-300 liquid chromatograph equipped with a Dionex pulsed electrochemical detector (PED-2) .
  • PED-2 Dionex pulsed electrochemical detector
  • Tobacco transformants and control plants were grown up both in sterile, "in vitro" , conditions and in a greenhouse. Mature transformants had no obvious phenotype compared to the untransformed controls or controls transformed with the vector pDElOOl (lacking TPSl) . Leaves were collected at 0900 h, frozen and stored at or below -70 °C prior to analysis.
  • Fig 4 shows that tissues in which Tre6P synthase was expressed also contained trehalose. In addition, smaller amounts of trehalose were found in the roots, indicating that transgenic tobacco transports trehalose from its site of synthesis to other tissues.
  • results also disclose that tobacco plants expressing TPSl under the atslA promoter and accumulating trehalose in their green tissues during daylight are healthy and normal in appearance.
  • primary transformants containing the chimeric TPSl gene had some minor morphological alterations, such as lancet-shaped leaves, reduced apical dominance and reduced height (see Figs 5 & 6) , most of the changes were not exhibited in self-pollinated TPSl-positive progeny that still produced trehalose (see Fig 6) .
  • the morphological changes in the primary transformants appear to be artefacts of the tissue culture rather than results of trehalose production.
  • the trehalose contents of the best transformants in Table 1 are already at least 20 % of the level at which a clear improvement in thermotolerance is observed in yeast (De Virgilio et al [1990] FEBS Letters 273, 107-110) .
  • TPSl-transformants and controls were assayed for Tre6P synthase activity.
  • the results shown in Table 2 are means + the extreme ranges from duplicate zero and 15 or 30 min assays.
  • Tre6P synthase activity did not differ from zero.
  • Transformant 4 an acid- and alkali- stable carbohydrate accumulated in the presence of UDPG and Glc6P. This accumulation required Glc6P, but not fructose 6- phosphate (Fru6P; this hexose phosphate activates the native trehalose synthase complex of yeast) and was prevented when UDPG was replaced by ADPG (the enzyme purified from yeast also cannot use ADPG) .
  • Tre6P is synthesised by extracts of Transformant 4 faster than it is converted to trehalose.
  • TPS2 which encodes the Tre6Pase subunit.
  • the Tre6P synthase activity of yeast extracts found by the method used in Table 2 agreed with that found by measuring the appearance of UDP as described by Londesborough & Vuorio ([1991] J. Gen. Microbiol. 137, 323-330) .
  • yeast extracts measured in the presence of extracts of tobacco plants were not inhibited.
  • the absence of activity in the control plants in Table 2 is not due to interference by some factor present in tobacco extracts.
  • Tre6P synthase activity found in Transformant 4 was labile. With some extracts, the activity disappeared during a few hours storage on ice. However, the specific band seen in Western analyses was still present at nearly its original strength in extracts stored for 24 h at room temperature. Thus, it is probable that the conformation of the Tre6P synthase subunit changes during storage of tobacco extracts. These results indicate that increased Tre6P synthase activity will be achieved by transforming the tobacco simultaneously with TPSl and one or more of the other subunits of yeast trehalose synthase, thereby increasing the conformational stability of the Tre ⁇ P synthase subunit.
  • Example 2. Transgenic Arabidopsis thaliana.
  • A. thaliana plants containing TPSl under the atslA promoter were constructed in the same way as the tobacco transformants described above. These transformed Arabidopsis plants are also healthy and normal in appearance and produced fertile seed. Western analysis of plants grown up from seeds of the primary transformants showed that they contained the 56 kDa subunit of yeast trehalose synthase (Fig 10) . It is an obvious expectation that these plants accumulate trehalose in their green tissues.
  • Example 3 Dry resistance of trehalose-producing tobacco plants.
  • Enhanced drought survival was also manifested in young seedlings (Fig. 6B) .
  • Exposure of 3-week old seedlings from transgenic line 8 together with nontransformed and vector transformed control seedlings to air drying (50 % RH) demonstrated clear differences in drought tolerance between the trehalose-producing and control plants.
  • the transgenic trehalose-positive line 8 showed both delayed loss of turgor and enhanced survival of dehydration stress as compared with the control plants (Fig. 6B) .
  • TPS2 and TSL1 under the control of the atslA promoter and use them to transform tobacco, Arabidopsis and other plants by the methods described in General Materials and Methods and Example 1.
  • Plants simultaneously transformed with TPSl and one or both of the other genes, TPS2 and TSL1 can be obtained by cross ⁇ breeding of individual transformants, by further transformation of one transformed plant with a second gene, or by transformation with vectors containing two or three of the genes linked to appropriate promoters: for example, TPSl can be linked to the non-constitutive atslA promoter, to provide control over trehalose synthesis, and the other gene(s) driven by constitutive promoters.
  • the TPSl coding sequence can be replaced by the coding sequence of some other Tre6P synthase structural gene, the TPS2 sequence by that of some other Tre6Pase gene and the TSL1 sequence by that of some other gene encoding a polypeptide that confers regulatory properties or stability upon the Tre6P synthase and Tre6Pase in the same way as the TSL1 product confers such properties upon the other subunits of native yeast trehalose synthase.
  • Plant promoters such as LTI78 (Nordin et al [1993] Plant Mol. Biol. 21, 641-653) and RAB18 (Lang & Palva [1992] Plant Mol. Biol. 20, 951-962) , are known that are induced in response to drought and cold stress.
  • the advantage is that levels of trehalose that might be deleterious to certain tissues of certain plants and which can also represent a yield-decreasing diversion of photo-synthetic capacity and possibly retard the growth of the plant, would accumulate only (1) when the plant is exposed fortuitously to stress (the benefits of the protection afforded by the trehalose then overcoming any deleterious effects) or (2) when the plant is deliberately exposed to stress in order to cause the accumulation of trehalose which will then be extracted from the harvested plant.
  • Tre6P the main synthetic pathway
  • the key enzyme in this pathway is Tre6P synthase, since, as disclosed above, once Tre6P has been made, many cells will be capable of dephosphorylating it to free trehalose.
  • the key concept in the present invention is to introduce Tre6P synthase activity into the target plant. It is not of primary importance where this activity comes from.
  • yeast enzyme which happens to be a subunit of the yeast trehalose synthase complex, which contains at least two other subunits.
  • Tre6P synthase subunit may require the presence of one or more of the other subunits, though evidently the 56 kDa Tre6P synthase subunit from yeast functions effectively in tobacco, without the 102 and 123 kDa subunits.
  • Mvcobacterium smecrmatis contains a heparin-activated Tre6P synthase which has been partially purified and studied by Elbein's group (Liu et al, [1969] J. Biol. Chem. 244, 3728- 3791; Lapp et al [1971] J. Biol. Chem. 246, 4567-4579; Elbein & Mitchell [1975] Arch. Biochem. Biophys. 168, 369- 377; Pan et al, [1978] Arch. Biochem. Biophys. 186, 392- 400) .
  • the enzyme purified by these workers had a specific activity of 0.8 U/mg protein at 37 °C, with Glc6P and UDPG as substrates (the enzyme can use a spectrum of nucleoside- diphospho-glucose derivatives) and in the presence of optimal heparin.
  • the preparation contained two polypeptides with SDS-PAGE molecular weights of about 45 and 90 kDa.
  • We modified the authors' purification procedure by including protease inhibitors and other protein protecting agents in the buffers used, and adding a final chromatographic step in the presence of the non-ionic detergent, Triton X-100. Our final procedure was as follows:
  • M. smecrmatis cells (28 g fresh weight grown for 3 days in Luria broth and stored frozen) were allowed to melt in 40 ml of 50 mM HEPES/KOH pH 7.5 containing 1 mM benzamidine, 2 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride (PMSF) and 10 ⁇ g pepstatin
  • Tre6P synthase was assayed as described by Londesborough and Vuorio (1993, loc.cit.) but at 35 °C and in the presence of 0.25 ⁇ g heparin/ml.
  • Fig 9 shows that this polypeptide is recognised by antiserum raised against the 56 kDa Tre6P synthase subunit of yeast trehalose synthase but not by pre-immune serum, showing that the Tre6P synthase of M. smegmatis shares antigenic determinants with the yeast enzyme.
  • the nature of the other immunoreactive bands present in relatively crude preparations of the enzyme has not been investigated: not all of them are artefacts due to the large protein load, but represent related proteins.
  • the antiserum raised against the yeast enzyme can be used to detect and isolate positive clones from host cells transformed with a M. smegmatis gene bank.
  • the amino acid sequence data in Table 4 can be used to check the sequence of the isolated gene.
  • the immunological and amino acid sequence similarities between the enzymes from yeast and M. smegmatis indicate that nucleotide probes designed from the TPSl gene may also be successfully used to screen for the M. smegmatis gene.
  • MOLECULAR TYPE Peptide
  • HYPOTHETICAL no
  • MOLECULAR TYPE Peptide
  • HYPOTHETICAL no

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Abstract

L'invention porte sur des plantes transgéniques productrices de tréhalose et sur un procédé pour accroître la teneur de plantes en tréhalose. Selon cette invention, les plantes concernées sont transformées à l'aide d'une séquence d'un gène codant pour la tréhalose-6-phosphate synthase fondue en un promoteur non constitutif de la plante qui assure un contrôle temporel, topologique et fonction des agressions de l'expression du gène. L'invention peut servir à protéger les récoltes de plantes de première nécessité contre la sécheresse, la forte salinité, les températures extrêmes, et pour améliorer les propriétés de stockage des récoltes, y compris des légumes verts, des fruits cueillis et des plantes ornementales.
PCT/FI1995/000377 1992-02-14 1995-06-29 Plantes transgeniques productrices de trehalose WO1996000789A1 (fr)

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NZ288635A NZ288635A (en) 1994-06-29 1995-06-29 Transgenic plants producing trehalose
HU9603608V HU221613B (hu) 1994-06-29 1995-06-29 Trehalózt termelő transzgenikus növények
US08/765,691 US6130368A (en) 1992-02-14 1995-06-29 Transgenic plants producing trehalose
GB9627137A GB2303856B (en) 1994-06-29 1995-06-29 Transgenic plants producing trehalose
AU27944/95A AU699391B2 (en) 1994-06-29 1995-06-29 Transgenic plants producing trehalose
FI965132A FI965132L (fi) 1994-06-29 1995-06-29 Trehaloosia tuottavat transgeeniset kasvit
JP8502849A JPH10501978A (ja) 1994-06-29 1995-06-29 トレハロース産生トランスジェニック植物
EP95923355A EP0763118A1 (fr) 1994-06-29 1995-06-29 Plantes transgeniques productrices de trehalose

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FI943133A FI943133A0 (fi) 1994-06-29 1994-06-29 Transgena vaexter
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WO1997026365A3 (fr) * 1996-01-19 1997-09-12 Dekalb Genetic Corp Mais transgenique a teneur accrue en mannitol
EP0784095A3 (fr) * 1996-01-12 1997-12-29 Mogen International N.V. L'accumulation de tréhalose améliorée dans des plantes
WO1997042327A3 (fr) * 1996-05-08 1998-01-08 Univ Mexico METHODE PERMETTANT D'ACCROITRE LA TENEUR EN TREHALOSE DES ORGANISMES PAR TRANSFORMATION AVEC L'ADNc DE LA TREHALOSE-6-PHOSPHATO SYNTHETASE/PHOSPHATASE DE SELAGINELLA LEPIDOPHYLLA
ES2110918A1 (es) * 1996-07-04 1998-02-16 Univ Poltecnica De Valencia Obtencion de plantas tolerantes al estres osmotico mediante manipulacion genetica del metabolismo de carbohidratos.
WO1997042326A3 (fr) * 1996-05-03 1998-03-12 Mogen Int Regulation du metabolisme par modification du taux de trehalose-6-phosphate
WO1998050561A1 (fr) * 1997-05-02 1998-11-12 Mogen International N.V. Metabolisme regulateur modifiant le niveau de trehalose-6-phosphate par inhibition des niveaux de trehalase endogene
WO1999023225A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Nouveaux micro-organismes a fermentation elevee
WO1999023233A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Plantes presentant une sterilite male nucleaire, production de ces plantes et restauration de leur fertilite
WO1999023234A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Inhibition de la remobilisation des composes stockes avant et apres recolte
WO1999024558A3 (fr) * 1997-10-30 1999-07-15 Mogen Int Micro-organismes pour fermentation haute
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EP1002867A1 (fr) * 1998-10-15 2000-05-24 K.U. Leuven Research & Development Modification génétique spécifique de l'activité de tréhalose-6-phosphate synthase et son expression dans un environnement homologue et hétérologue
WO2000004164A3 (fr) * 1998-07-17 2000-06-08 Norika Nordring Kartoffelzucht Gene de glucosylglycerolphosphate synthase pour la production de glucosylglycerine faiblement moleculaire comme substance de protection et son utilisation
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EP1563060A4 (fr) * 2002-11-06 2006-03-22 Cornell Res Foundation Inc Genes chimeres et transformants vegetaux de la tps
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US7288403B2 (en) 1993-08-25 2007-10-30 Anderson Paul C Anthranilate synthase gene and method for increasing tryptophan production
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US7064248B2 (en) 1990-01-22 2006-06-20 Dekalb Genetics Corp. Method of preparing fertile transgenic corn plants by microprojectile bombardment
US7288403B2 (en) 1993-08-25 2007-10-30 Anderson Paul C Anthranilate synthase gene and method for increasing tryptophan production
US6960709B1 (en) 1993-08-25 2005-11-01 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
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US6833490B1 (en) 1996-05-03 2004-12-21 Mogen International N.V. Regulating metabolism by modifying the level of trehalose-6-phosphate
WO1997042327A3 (fr) * 1996-05-08 1998-01-08 Univ Mexico METHODE PERMETTANT D'ACCROITRE LA TENEUR EN TREHALOSE DES ORGANISMES PAR TRANSFORMATION AVEC L'ADNc DE LA TREHALOSE-6-PHOSPHATO SYNTHETASE/PHOSPHATASE DE SELAGINELLA LEPIDOPHYLLA
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US6229069B1 (en) * 1996-10-24 2001-05-08 Japan Tobacco Inc. Method for controlling water content of plant
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US7186824B2 (en) 1997-03-04 2007-03-06 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Reduction inhibitory agent for active-oxygen eliminating activity
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AU739946B2 (en) * 1997-10-30 2001-10-25 Syngenta Mogen Bv Nuclear male sterile plants, method of producing same and methods to restore fertility
WO1999023234A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Inhibition de la remobilisation des composes stockes avant et apres recolte
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HUP9603608D0 (en) 1997-02-28
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JPH10501978A (ja) 1998-02-24
AU699391B2 (en) 1998-12-03
GB2303856B (en) 1998-12-30
CA2193861A1 (fr) 1996-01-11
AU2794495A (en) 1996-01-25
HUT75659A (en) 1997-05-28
NZ288635A (en) 1998-12-23
GB2303856A (en) 1997-03-05
FI943133A0 (fi) 1994-06-29
CN1159833A (zh) 1997-09-17
EP0763118A1 (fr) 1997-03-19
HU221613B (hu) 2002-11-28
CZ294329B6 (cs) 2004-12-15

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