Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2001-06-28
2004-02-03
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S284000, C800S288000, C800S287000, C800S317000, C435S320100
Reexamination Certificate
active
06686516
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to expression of trehalose biosynthetic genes and drought resistance in plants.
BACKGROUND OF THE INVENTION
Trehalose (a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside) is a disaccharide commonly found in lower organisms such as bacteria, fungi and insects where it often accumulates in resting or stationary phase cells and organs. Two enzymatic activities are required for trehalose biosynthesis: a trehalose-6-phosphate synthase catalyses the condensation of UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate and a trehalose-6-phosphate phosphatase phosphorylates trehalose-6-phosphate to trehalose.
Although trehalose can serve as a storage form for reduced carbon, it may play a more significant role as a protectant against the deleterious effects of various abiotic stresses, notably heat and desiccation. Both in vivo and in vitro, trehalose accumulation is correlated with protection of biological macromolecules (particularly membranes and proteins) from dessication, temperature extremes, and osmotic shock. Trehalose produced by fermentation is used commercially in the preservation of enzymes and is registered as a food additive for the stabilization of dehydrated and processed foods.
While it has long been recognized that trehalose may occur in plants as a product of symbiotic microorganisms, as a rule vertebrates and higher plants were thought not to be capable of synthesizing trehalose. The near-ubiquitous occurrence of specific trehalose-catabolizing enzymes (trehalases) in higher plant families was a biological curiousity ascribed mainly to the presence of exogenous trehalose entering plant cells from symbiotic or epiphytic microbial and fungal sources. Notable exceptions are the lower plants and angiosperms grouped loosely into the category of “resurrection plants” which are capable of surviving extraordinarily prolonged periods of dessication. These plants, including species of Selaginella and Myrothamnus, can accumulate as much as 10% trehalose by dry weight following the onset of droughting.
In view of trehalose's association with drought resistance and the historically poor economics of microbial trehalose fermentation, attempts have also been made to engineer transgenic plants to accumulate this disaccharide. Although such plants have been obtained, it has become apparent that constitutive trehalose production in the plant cytosol is accompanied by significant deleterious effects. These phenotypes (stunted growth, abnormal leaves, undeveloped roots) are particulary severe when trehalose expression occurs in root tissue or during early development, as the use of green-tissue specific plant promoters to drive trehalose producing genes ameliorates some, but not all, of these effects.
Given these facts, an inducible expression system for the trehalose biosynthetic genes, which allows for trehalose accumulation and results in drought resistance but without deleterious effects to the plant is of great practical use and economic interest.
SUMMARY OF THE INVENTION
The present invention thus relates to expression of trehalose biosynthetic genes and drought resistance in plants. In particular, this invention addresses the issue of trehalose accumulation and drought resistance in higher plants and novel ways to engineer such trait. It also addresses the need for improved storage properties of harvested plants, improved shelf-life of fruits and flowers, as well as stabilization of foreign proteins expressed in transgenic plants. In a prefered embodiment, the invention describes the expression of the trehalose biosynthetic genes in plants, preferably under the control of an inducible promoter, which allow for drought resistance without the deleterious effect associated with uncontrolled accumulation of trehalose. A prefered promoter is a chemically inducible promoter, such as the tobacco PR-1a promoter, which can be activated by foliar application of a chemical inducer.
Additionally, the invention describes expression of the trehalose biosynthetic genes expressed in different cellular compartments. In a first embodiment the trehalose biosynthetic genes are expressed in the plant cytoplasm. In a further embodiment, the trehalose biosynthetic genes are expressed from the plant nuclear genome and the trehalose biosynthetic enzymes encoded therefrom are targeted to the plastids, e.g. by using a plastid transit peptide. In a further embodiment, the trehalose biosynthetic genes are expressed from the plant plastid genome. In a preferred embodiment, vectors containing the trehalose biosynthetic genes fused to a promoter capable of directing the expression of the trehalose biosynthetic genes in plant plastids are transformed into the plastid genome. In a preferred embodiment, vectors containing a phage promoter fused to the trehalose biosynthetic genes are transformed into the plastid genome. The resulting line is crossed to a transgenic line containing a nuclear coding region for a phage RNA polymerase supplemented with a plastid-targeting sequence and operably linked to a plant promoter, such as an inducible promoter, a tissue-specific promoter or a constitutive promoter. In another preferred embodiment, a promoter capable of directing the expression of the trehalose biosynthetic genes in plant plastids is a promoter transcribed by a RNA polymerase normally present in plastids, such as a nuclear-encoded polymerase or a plastid-encoded polymerase. Such promoters are e.g. but not limited to a clpP promoter, a 16S r-RNA gene promoter, a psbA promoter or a rbcL promoter.
In the present invention, trehalose biosynthetic genes from
E. coli
are preferably used, but trehalose biosynthetic genes from other organisms including but not limited to yeast, other lower organisms or higher organisms, such as plants, are suitable. In a preferred embodiment, a nucleotide sequence encoding a trehalose phosphate synthase and a nucleotide sequence encoding a trehalose phosphate phosphatase are both expressed in the plant. In another preferred embodiment, a nucleotide sequence encoding a trehalose phosphate synthase is expressed in the plant, or a nucleotide sequence encoding a trehalose phosphate phosphatase is expressed in the plant. The present invention also relates to the expression from the plastid genome of two trehalose biosynthetic genes transcribed from a single promoter in an operon-like polycistronic gene.
The invention thus provides:
A plant expressing a nucleotide sequence encoding a trehalose biosynthetic enzyme, for example a plant comprising nucleotide sequence coding for the trehalose 6-phosphate synthase and/or trehalose 6-phosphate phosphatase, for example the
E. coli
OtsA and/or
E. coli
OtsB genes. Such nucleotide sequences are for example stably integrated in its nuclear or plastid genome, under the control of a promoter capable of directing the expression of the trehalose biosynthetic genes in said plant, e.g. under the control of an inducible promoter, e.g., a wound-inducible or chemically inducible promoter, such as the tobacco PR-1a promoter or Arabidopsis PR-1 promoter, or, a transactivator-regulated promoter wherein the corresponding transactivator is under the control of a promoter capable of directing the expression of the transactivator in said plant, e.g. an inducible promoter, a tissue-specific promoter or a constitutive promoter, e.g., a wound-inducible or chemically inducible promoter, such as the tobacco PR-1a promoter or Arabidopsis PR-1 promoter;
also including the seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag with instructions for use. In particular, the invention provides:
A plant comprising in its nuclear genome a first heterologous expression cassette or parts thereof comprising a nucleotide sequence encoding a trehalose 6-phosphate synthase under control of an inducible promoter and a second heterologous expression cassette or parts thereof comprising a nucleotide sequence encoding a trehalose-6-phosphate phosphatase under
Goff Stephen Arthur
Heifetz Peter Bernard
Lebel Edouard Guillaume
Kakefuda Mary
Kubelik Anne
Nelson Amy J.
Syngenta Participations (AG)
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