Oxidative stress resistance gene

Details Oxidative stress resistance gene Oxidative stress resistance gene

1999-10-15

2003-05-13

Bui, Phuong T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C800S278000, C800S298000, C800S295000, C800S289000, C435S069100, C435S419000, C435S418000, C435S468000, C536S023100, C536S023600

Reexamination Certificate

active

06563019

ABSTRACT:

The present invention relates to plants, especially transgenic plants, plant parts and plant cells overproducing an iron binding protein (e.g. ferritin) and having an enhanced resistance against a wide range of abiotic and biotic oxidative stress conditions (e.g. against treatment with paraquat or fusaric acid and against viral, bacterial and fungal infections). The invention also comprises nucleic acid sequences encoding an alfalfa ferritin or functional variants thereof and the use of said sequences for rendering plants resistant against oxidative stress conditions.
The invention is useful for reducing environmental damages of crops caused by a wide variety of stress conditions.
With respect to the present specification and claims, we will use the following technical terms in accordance with the given definitions. With regard to the interpretation of the present invention, it shall be understood that the below defined terms are used in accordance with the given definitions even if said definitions might not be in perfect harmony with the usual interpretation of said technical term.
A “functional variant” of a protein is a polypeptide the amino acid sequence of which can be derived from the amino acid sequence of the original protein by the substitution, deletion and/or addition of one or more amino acid residue in a way that, in spite of the change in the amino acid sequence, the functional variant retains at least a part of at least one of the biological activities of the original protein that is detectable for a person skilled in the art. A functional variant is generally at least 50% homologous (i.e. the amino acid sequence of it is 50% identical), advantageously at least 70% homologous and even more advantageously at least 90% homologous to the protein from which it can be derived. Any functional part of a protein or a variant thereof is also termed functional variant.
The term “overproducing” is used herein in the most general sense possible. A special type of molecule (usually a polypeptide or an RNA) is said to be “overproduced” in a cell if it is produced at a level significantly and detectably higher (e.g. 20% higher) than natural level. Overproduction of a molecule in a cell can be achieved via both traditional mutation and selection techniques and genetic manipulation methods. The term “ectopic expression” is used herein to designate a special realisation of overproduction in the sense that, for example, an ectopically expressed protein is produced at a spatial point of a plant where it is naturally not at all (or not detectably) expressed, that is, said protein is overproduced at said point.
A plant, plant part, a plant tissue or a plant cell is said to have an “enhanced resistance” against a damaging effect, eg. damaging agent, if it can tolerate a significantly and detectably (e.g. at least 20%) stronger damaging effect, eg. dose or intensity of damaging agent, of the same type, without suffering any detectable damage, than its natural counterpart would do.
Within the framework of the present description a “ferritin protein” is defined, as it is usual in the art, as a protein capable binding iron ions (Theil E. C., 1987, Ann. Rev. Biochem. 56: 289-315). The members of the eucariotic ferritin family are highly conserved both in their amino acid sequence and three dimensional structure (Lobreaux, S. et al., 1992, Biochem. J. 288: 931-939).
The term “oxidative stress” is again used in very general sense comprising all kind of abiotic (e.g. treatment with different chemical agents or exposure to extreme weather conditions like high or low temperature or drought) and biotic (infection by different infectious agents) stress conditions in the manifestation of damaging effects of which oxidatively induced active radicals play a detectable role.
BACKGROUND OF THE INVENTION
During their different developmental stages plants are exposed to an extremely wide range of both biotic and abiotic stress conditions. It is, thus, a very important task of high economic significance to develop new breeding stocks of enhanced general stress resistance.
Under stress conditions such as high light intensity, UV-B irradiation, heavy metal contamination, high or low temperature, water deficiency, flooding, wounding, infection by viruses, bacteria, fungi, damage caused by insects and the like, oxygen toxicity can significantly contribute to the damage of crop plants. Reactive oxygen species as singlet oxygen, superoxide radical (O
2
), hydroxyl radical (OH
+
) and hydrogen peroxide (H
2
O
2
) play a key role in injury of stressed plants. There is good evidence that the biological damage attributed to superoxide and hydrogen peroxide is dependent on the presence of iron. The intracellular pool of free iron can react with H
2
O
2
or O
2
−
giving rise to the very reactive hydroxyl radical via Haber-Wiess or Fenton reaction (Halliwell and Gutteridge, 1984, Biochem. J. 219: 1-14). Intracellularly, most of the non-metabolised iron is sequestered in ferritin; therefore ferritin is able to restrict the availability of iron and so the generation of the very reactive hydroxyl radicals. cDNAs encoding ferritin have been isolated from variety of plant species. These proteins are highly conserved both in amino acids sequence and three dimension structure (Lobreoux S. et al. 1992, Biochem. J. 288: 931-939.). Ferritins are localised in chloroplasts and iron can activate their synthesis (Seckbach, J. 1982, J. Plant. Nutr. 5: 369-394; Lobreaux et al. 1992, Plant Mol. Biol. 19: 563-575). Under normal growth conditions ferritin is synthesised only in embryo and not in vegetative organs, like roots and leaves (Lobreaux and Briat: Biochem. J. 1991, 274: 601-606).
Significant antioxidant effect of ferritin molecules can be expected in systems where ferritin synthesis and degradation is released from the normal metabolic regulation. It has been demonstrated that during oxidative stress conditions, degradation of ferritin molecules occurs and the so released iron ions highly accelerate the production rate of the damaging radical species (Cairo et al. 1995, Journal of Biochemical Chemistry 270: 700-703). Numerous traditional plant breeding and genetic manipulation approaches are known in the art for improving the resistance of specific crops against preselected desired stress conditions (e.g. against cold, drought, UV light or pathogens). These known methods, however, will not be individually detailed herein as they are all highly different from the approach of the invention. The common feature of all these previously disclosed approaches is that they enhance the resistance of different plants against a single preselected stress condition (or against a limited groups of stress conditions of the same origin) e.g. by expressing a specific resistance gene. However, no specific approach providing plants with resistance against a wide range of both abiotic and biotic stress conditions is known in the art.
SUMMARY OF THE INVENTION
It is, thus, an object of the present invention to provide a novel and general method suitable to provide crops, especially transgenic crops, with enhanced resistance against a wide range of both abiotic and biotic stress conditions.
According to another aspect, it is also an object of the invention to provide crops and breeding material, advantageously transgenic, having increased resistance against a wide range of both abiotic and biotic stress conditions.
On the basis of the foregoing disclosure it has, thus, became clear to the inventors that a substantially new genetic manipulation approach is to be developed so as to achieve the above defined objects, possibly targeting a common step in the damaging mechanism of the different abiotic and biotic stress conditions.
The approach of the present invention is, thus, based on the novel theoretical concept that overproducing or ectopically expressing ferritin or other iron binding proteins, e.g. transferring, in different organs of plants will lower the intracellular iron concentration and, therefore, reduce the damaging effects of

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