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
2002-05-21
2004-09-28
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
C800S287000, C800S290000, C800S295000, C800S278000, C800S286000, C435S320100, C435S468000, C536S023600, C536S024500
Reexamination Certificate
active
06797861
ABSTRACT:
The present invention relates to DNA sequences, comprising nucleic acid fragments encoding dehiscence zone-selective proteins, particularly cell wall hydrolases such as polygalacturonases, the regulatory regions of the corresponding plant genes and their use for modifying dehiscence properties in plants, more particularly pod dehiscence properties in
Brassica napus.
BACKGROUND OF THE INVENTION
Loss of yield due to seed shedding by mature fruits or pods, also called pod dehiscence or pod shatter, as well as concomitant increase in volunteer growth in the subsequent crop year, are a universal problem with crops that develop dry dehiscent fruits. An economically important crop to which these adverse properties specifically apply is oilseed rape: up to 50% of the potential yield may be lost under adverse weather conditions.
Dry dehiscent fruits, also commonly called pods, may develop from a single carpel (such as the legume in many Fabaceae) or from more than one carpel (such as the silique in many Brassicaceae). In case of the silique, the pod consists of two carpels joined margin to margin. The suture between the margins forms a thick rib, called replum. As pod maturity approaches, the two valves separate progressively from the replum, eventually resulting in the shattering of the seeds that were attached to the replum.
Ultrastructural investigation have demonstrated that pod shatter is associated with the precise degradation of cell wall material at the site of pod valve separation (i.e., the suture). The degradation of the cell wall and loss of cellular cohesion prior to dehiscence is predominantly attributed to solubilization of the middle lamella of the cell wall. This middle lamella is found between primary cell walls and is the cement that holds the individual cells together to form a tissue. Cell separation is preceded by an ethylene climacteric, which temporally correlates with a tissue-specific increase in the activity of the hydrolytic enzyme cellulase (beta-1,4-glucanase) and this occurs specifically in a layer of cells along the suture, which is called the dehiscence zone. In contrast, the activity of the cell wall degrading enzyme polygalacturonase exhibits no correlation either temporally or spatially with pod dehiscence [Meakin and Roberts (1990),
J. Exp. Bot
. 41; 1003]. Pod dehiscence at an early stage of development is characteristic of infestation by the pod midge
Dasineura brassicae
. A localized enhancement of both polygalacturonase and cellulase activity has been observed. However, regulation of midge-induced and maturation-associated shatter was found to be different [Meakin and Roberts (1991),
Annals of Botany
67: 193].
At first sight, the process of pod dehiscence shares a number of features with abscission wherein plants shed organs, such as leaves, flowers and fruits. It has been observed that ethylene induces or accelerates abscission, whereas auxin inhibits or delays abscission. A decisive step in abscission is the highly coordinated expression, synthesis and secretion of cell wall hydrolytic enzymes in a discrete layer of cells, called the abscission zone. Cellulases (beta-1,4-glucanases) constitute one class of such cell wall hydrolases. Cellulase activity has been identified in various tissues including leaf abscission zones, fruit abscission zones, ripening fruit, senescent cotyledons and styles and anthers [Kemmerer and Tucker (1994),
Plant Physiol
. 104: 557 and references therein]. A second class of hydrolases involved in abscission of mainly fruits are polygalacturonases of which distinctive isoforms have been identified [Bonghi et al.(1992),
Plant Mol. Biol
. 20: 839; Taylor et al (1990)
Planta
183: 133].
Kadkol et al [(1986),
Aust. J. Biol
. 34: 79] reported increased resistance towards shattering in a single, Australian accession of rape. Variation in pod maturation has further been observed in mutants of rape stemming from irradiated seeds [Luczkiewicz (1987),
Proc
. 7
th Int. Rapeseed Congress
2: 463]. It can however be concluded that traditional methods for breeding have been unsuccessful in introducing shatter resistance into rape cultivars, without interference in other desirable traits such as early flowering, maturity and blackleg resistance [Prakash and Chopra (1990),
Genetical Research
56: 1].
Despite its economic impact very little is known concerning the molecular events and changes in gene expression that occur during oilseed pod dehiscence. At present, two pod-specific mRNAs whose expression is spatially and temporally correlated with pod development have been described. However, the function of the encoded proteins is unknown. [Coupe et al (1993),
Plant Mol. Biol
. 23: 1223; Coupe et al. (1994),
Plant Mol. Biol
. 24: 223]. PCT publication WO94/23043 in general terms describes an approach for regulating plant abscission and dehiscence.
Accordingly, it is an object of the present invention to provide dehiscence zone-selective genes in plants.
These and other objects are achieved by the present invention, as evidenced by the summary of the invention, description of the preferred embodiments and claims.
SUMMARY OF THE INVENTION
The present invention provides dehiscence zone(“DZ”)-selective genes of plants, cDNAs prepared from mRNAs encoded by such genes, and promoters of such genes. In particular this invention provides the cDNA of SEQ ID NO: 1 and the promoter of a gene encoding a mRNA wherein a cDNA of that mRNA has substantially the nucleotide sequence of SEQ ID NO: 1, particularly the promoter as contained within the 5′ regulatory region of SEQ ID NO: 14 starting at position 1 and ending at position 2,328.
In another aspect, the present invention also provides DZ-selective chimeric genes, that can be used for the transformation of a plant to obtain a transgenic plant that has modified dehiscence properties, particularly modified pod-dehiscence properties, when compared to plants that do not contain the DZ-selective chimeric gene, due to the expression of the DZ-selective chimeric gene in the transgenic plant.
In yet another aspect, the present invention thus provides a plant containing at least one DZ-selective chimeric gene incorporated in the nuclear genome of its cells, wherein said DZ-selective chimeric gene comprises the following operably linked DNA fragments:
a) a transcribed DNA region encoding:
1) a RNA which, when produced in the cells of a particular DZ of the plant, prevents, inhibits or reduces the expression in such cells of an endogenous gene of the plant, preferably an endogenous DZ-selective gene, encoding a cell wall hydrolase, particularly an endo-polygalacturonase (an “endo-PG”), or,
2) a protein or polypeptide, which when produced in cells of the DZ, kills or disables them or interferes with their normal metabolism, physiology or development,
b) a plant expressible promoter which directs expression of the transcribed DNA region at least in cells of the DZ, provided that if the transcribed DNA region encodes a protein or polypeptide, or encodes an antisense RNA or ribozyme directed to a sense RNA encoded by an endogenous plant gene that is expressed in the plant in cells other than those of the DZ, the plant expressible promoter is a DZ-selective promoter, i.e., a promoter which directs expression of the transcribed region selectively in cells of the DZ.
Preferably the transcribed DNA region encodes a protein or polypeptide which is toxic to the cells in which it is produced, such as a barnase; a protein or polypeptide that increases the level of auxins or auxin analogs in the cells in which it is produced, such as a tryptophan monooxygenase and/or a indole-3-acetamide hydrolase; a protein or polypeptide that increases the sensitivity to auxin in the cells in which it is produced, such as the roIB gene product; or a protein or polypeptide that decreases the sensitivity to ethylene in the cells in which it is produced, such as the mutant ETR1-1 protein or a another ethylene receptor protein.
In another pr
Borkhardt Bernhard
Botterman Johan
Child Robin
Petersen Morten
Poulsen Gert Bundgard
Bayer BioScience NV
Ibrahim Medina A.
Nelson Amy J.
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