# Patent

## Title
Methods for increasing the resistance of plants to hypoxic conditions

## Abstract

Methods are provided for increasing the resistance of plants to hypoxic or anoxic conditions. Such methods may be applied to increase the penetrance of plant roots in the growth medium or into soil. The methods according to the invention may include providing plants with a stress tolerance gene. Similar effects can be obtained by applying chemical compounds, including neonicotinoid compounds, to the plants.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage filing of International Application No. PCT/EP2006/005393, filed Jun. 6, 2006, which claims priority to EP 05076392.9, filed Jun. 15, 2005 and U.S. Provisional Patent Application No. 60/691,103, Jun. 16, 2005, the disclosures of each of which are hereby incorporated by reference.


Methods are provided for increasing the resistance of plants to hypoxic or anoxic conditions. Such methods may be applied to increase the penetrance of plant roots in the growth medium or into soil. The methods according to the invention may include modification of the genome of the plants by providing such plants with an exogenous stress tolerance gene or with a stress tolerant variant of an endogenous gene corresponding to such an exogenous gene. The methods according to the invention may also include applying neonicotinoid compounds, such as but not limited to imidacloprid, nitenpyram, acetamiprid, thiacloprid, thiamethoxam, clothianidin, and dinotefuran, to plants or their habitats, or to cells or seeds thereof. Particularly effective neonicotinoid compounds are neonicotinoid compounds which comprise a chloropyridine side chain, such as imidacloprid, nitenpyram, acetamiprid, and thiacloprid, particularly those during the degradation of which in plants 6-chloronicotininc acid (6-CNA) can be set free, like, e.g., imidacloprid and thiacloprid. The plants or their habitats can also be treated directly with 6-CNA.
BACKGROUND ART
Plants engineered to be stress tolerant are known in the art. Stress tolerance in plant cells and plants can, e.g., be achieved by reducing the activity or the level of the endogenous poly-ADP-ribose polymerases (PARP) or poly(ADP-ribose) glycohydrolases (PARG) as described in WO00/04173 and WO04/090140, respectively.
European patent application No. 04077624.7 describes that stress tolerance in plants and plant cells is achieved using nucleotide sequences encoding enzymes involved in the NAD salvage synthesis pathway and/or the NAD de novo synthesis pathway e.g. for overexpression in plants.
However, none of these documents disclose the possibility to use the stress tolerance genes mentioned therein for obtaining tolerance to hypoxic or anoxic conditions in plant cells and plants. Neither do they disclose the use of the stress tolerance genes described therein for the purpose of allowing the root system of the plant to penetrate deeper into the growth medium or the soil.
The application of compounds of the class of neonicotinoids on plants for purposes other than insect control is also known from the art (WO 01/26468, WO 03/096811).
WO 01/26468 discloses a method of improving the growth of plants comprising applying to the plants or the locus thereof at least one compound selected from the class of the neonicotinoids.
WO03/096811 describes that the yield and/or the vigor of an agronomic plant can be increased or improved in locations where the level of insect infestation below that indicating the need for the use of an insecticide for insect control purposes by treating a seed of the plant with a neonicotinoid compound. The method is deemed useful for non-transgenic plants and for plants having a foreign gene that encodes for the production of a modified Bacillus thuringiensis delta-endotoxin protein.
None of these documents however describe the use of compounds of the class of the neonicotinoids on plants for the purpose of increasing the tolerance of plant cells or plants to hypoxic or anoxic conditions or to allow the root system of the plant to penetrate deeper into the growth medium or the soil.
Thus, the art remains silent on methods to increase the depth of penetration of a root system or roots of a plant into the growth medium or soil, or to increase tolerance of plant cells or plants to hypoxic or anoxic stress conditions using stress tolerance genes or by application of a chemical compound of the neonicotinoid class to plants, or cells thereof as described hereinafter in the different embodiments and claims.
SUMMARY OF THE INVENTION
In one embodiment of the invention, a novel method of increasing the tolerance of plant cells or plants to hypoxic or anoxic conditions is provided comprising, providing the plant cells or plants with a stress tolerance enhancing transgene, wherein the stress tolerance enhancing transgene is selected from:

    
    
        a stress tolerance enhancing transgene capable of reducing the expression of plant endogenous PARP genes, particularly wherein the transgene codes for a PARP inhibitory RNA molecule
        a stress tolerance enhancing transgene capable of reducing the expression of plant endogenous PARG genes, particularly wherein the transgene codes for a PARG inhibitory RNA molecule; or
        a stress tolerance enhancing transgene coding for a plant-functional enzyme of the nicotinamide adenine dinucleotide salvage synthesis pathway selected from nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase or nicotinamide adenine dinucleotide synthetase.
    
    


In another embodiment, the invention relates to the use of such stress tolerance enhancing transgenes to increase the penetrance of plant roots in growth medium, including soil.
In yet another embodiment of the invention, a method for increasing the tolerance of plant cells or plants to hypoxic or anoxic conditions is provided comprising applying to the plant cell, plant or seed from which such plant is grown, or to the habitat thereof, an effective amount of a neonicotinoid compound.
In still another embodiment, the invention relates to the use of such compounds to increase the penetrance of plant roots in growth medium, including soil.
The invention further relates to a method for increasing the tolerance of plant cells or plants to hypoxic or anoxic conditions comprising the step of providing cells of said plant with an effective amount of 6-chloronicotinic acid.
The invention also relates to the use of 6-CNA for increasing the penetrance of plant roots into the growth medium.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Schematic representation of the assay to measure root depth of a plant growing in a agar solution. A container (1) with a seal (2) is filled with a transparent or translucent growth medium (3), such as 0.4% agar-water or 0.7% agar-water, to which additional test-compounds may be added. One pregerminated seed is added to the tube and allowed to grow for three weeks. After three weeks of growth in vertical position, the root depth (5) of the plant (4) is measured from the top of the medium to the lowest point of the roots.
FIG. 2: Boxplot representation of the root depth (mm) of Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule capable of reducing the expression of endogenous PARP2 genes compared to non-transgenic Arabidopsis thaliana plants (Col-0).
The following populations were analysed:

    
    
        Col-0: data points for wild-type Arabidopsis line
        427-16: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a weak tolerance to high light stress conditions
        427-20: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a weak tolerance to high light stress conditions
        427-20: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a moderate tolerance to high light stress conditions
    
    


The graph represents a typical boxplot (or box and whisker plot) on the left of each of the groups of values, summarizing the following statistical measures:

    
    
        median
        upper and lower quartiles
        minimum and maximum data values.
    
    


In addition, on the right hand for each of the groups of values, the mean and the standard error of the mean for those values is indicated.
The boxplot is interpreted as follows:

    
    
        the box itself contains the middle 50% of the data. The upper edge (hinge) of the box indicates the 75th percentile of the data set, and the lower hinge indicates the 25th percentile. The range of the middle two quartiles is known as the inter-quartile range.
        The line in the box indicates the median value of the data.
        The ends of the whiskers indicate the minimum and maximum data values, unless outliers are present in which case the whiskers extend to the nearest value points within a range of 1.5 times the interquartile range.
    
    


FIG. 3: Boxplot representation and standard error of the mean for the measured values for root depth (mm) of Arabidopsis thaliana cv. Col-plants comprising a transgene encoding a dsRNA molecule capable of reducing the expression of endogenous PARP genes.
The following populations were analyzed:

    
    
        Col-0: data points for wild-type Arabidopsis line
        427-22: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a high tolerance to high light stress conditions
        427-24: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a low tolerance to high light stress conditions
    
    


FIG. 4: Boxplot representation and standard error of the mean for the measured values for root depth (mm) of Arabidopsis thaliana cv. C24-plants comprising a transgene encoding a dsRNA molecule capable of reducing the expression of endogenous PARP genes.
The following populations were analyzed:

    
    
        C24: data points for wild-type Arabidopsis line
        1599: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a high tolerance to high light stress conditions
        1463: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a moderate tolerance to high light stress conditions
        1681: data points for A. thaliana transgenic line comprising anti-PARP1 gene with a moderate tolerance to high light stress conditions
        1690: data points for A. thaliana transgenic line comprising anti-PARP1 gene with a moderate tolerance to high light stress conditions
    
    


FIG. 5: Boxplot representation and standard error of the mean for the measured values for root depth (mm) measured on an A. thaliana Col-0 population segregating for the anti-PARP2 transgene.
The following populations were analyzed:

    
    
        Azygous: data points for A. thaliana plants from the population which do not contain an anti-PARP2 gene
        Transgenic: data points for A. thaliana plants from the population which contain an anti-PARP2 gene
    
    


FIG. 6: Boxplot representation and standard error of the mean for the measured values for root depth (mm) of Arabidopsis thaliana cv. C24 plants treated with various concentrations of imidacloprid as compared to untreated Arabidopsis thaliana cv. C24 plants.
The following populations were analyzed:

    
    
        0: untreated A. thaliana C24 plants
        50: A. thaliana C24 plants treated with 50 mg/L imidacloprid
        100: A. thaliana C24 plants treated with 100 mg/L imidacloprid
    
    


FIG. 7: Boxplot representation and standard error of the mean for the measured values for root depth (mm) of Arabidopsis thaliana cv. C24 plants treated with various concentrations of 6-chloronicotinic acid as compared to untreated Arabidopsis thaliana cv. C24 plants.
The following populations were analyzed:

    
    
        0: untreated A. thaliana C24 plants
        1: A. thaliana C24 plants treated with 1 mg/L 6-chloronicotinic acid
        5: A. thaliana C24 plants treated with 5 mg/L 6-chloronicotinic acid
    
    





DETAILED DESCRIPTION
The current invention is based on the realization that plants comprising stress tolerance genes, such as the chimeric genes encoding dsRNA targeted for silencing the expression of parp1 or parp2 genes of plants, developed a root system with roots that protruded deeper into the growth medium than the roots of control plants. The deeper penetrance of the roots has remained unnoticed with the plants comprising stress tolerance genes as described in the prior art and required the development of a particular assay, as described herein, to analyze statistically the root penetrance into the medium.
Although not intending to limit the invention to a particular mode of action, it is thought that the stress tolerance genes increase the tolerance of plant cells, including the plant cells of roots to hypoxic or anoxic conditions, thereby allowing the roots comprising such a stress tolerance gene to grow in less favourable oxygen conditions, as can be found in the deeper areas of a growth medium or the deeper soil layers, where the oxygen tension is lower. The increased penetrance of the root system of plants into the deeper layers of the soil, provides a partial explanation for the increased drought resistance under field conditions observed for plants comprising the stress tolerance genes as described herein, such as the dsRNA encoding genes silencing the expression of the endogeneous parp1 or parp2 genes.
A similar effect on the root protrusion could be observed in described assay after addition of compound of the neonicotinoid class or 6-chloronicotinic acid. The effect of the application of neonicotinoids on root growth depth is independent of the presence of insects which are the targets of the above-mentioned neonicotinoids. Accordingly, the effect is also connected with the biochemical improvement of stress tolerance, particularly hypoxia- or anoxia-related stress tolerance, of a plant or plant cell or the seed from which it is grown.
Accordingly, in a first embodiment, the invention is directed towards the use of a stress tolerance enhancing transgene to increase the tolerance of a plant cell, plant or seed to hypoxic or anoxic conditions.
As used herein, “hypoxic or anoxic conditions” refer to conditions to which plant cells, plants or parts of such plants are exposed wherein the availability of oxygen is low to very low. Anoxic conditions refer to conditions where there is almost no oxygen available. Typically, conditions wherein the dissolved oxygen concentration is below about 2 mg/L, are indicated as hypoxic (0.1 mg/L to 2 mg/L), conditions wherein the dissolved oxygen is below 0.1 mg/L, particularly below 0.05 mg/L are indicated as anoxic. Normal dissolved oxygen concentration in water is about 8 mg/L. Hypoxic conditions in soil refer to those conditions where the oxygen tension is low, particularly where oxygen drops below 5% in the soil atmosphere.
Hypoxic conditions may occur e.g. upon flooding of the plants or parts of the plants. Hypoxic conditions may also occur where oxygen consumption is high, such as in soil layers comprising a lot of organic debris in the process of metabolization by microorganisms. Furthermore, hypoxic conditions occur in the deeper layers of a growth medium where diffusion of oxygen occurs from the surface. Hypoxic conditions also occur in the deeper layers of the soil as oxygen diffusion and consequently oxygen tension decreases from the surfaces. The rate of decrease in oxygen depends on the compactness of the soil (whereby the more compact the soil, the less soil atmosphere is present), the presence of decomposing organic material, water content etc.
As used herein, “a stress tolerance enhancing transgene” refers to a transgene which when introduced or expressed in a plant cell or plant, provides the cell or the plant with a better tolerance to stress which is brought on a plant, e.g., by the application of chemical compounds (e.g., herbicides, fungicides, insecticides, plant growth regulators, adjuvants, fertilizers), exposure to abiotic stress (e.g., drought, waterlogging, submergence, high light conditions, high UV radiation, increased hydrogen peroxide levels, extreme (high or low) temperatures, ozone and other atmospheric pollutants, soil salinity or heavy metals, hypoxia, anoxia, etc.) or biotic stress (e.g., pathogen or pest infection including infection by fungi, viruses, bacteria, insects, nematodes, mycoplasmas and mycoplasma like organisms, etc.).
Such a stress tolerance enhancing transgene may be a transgene capable of reducing the expression and/or the activity of poly(ADP-ribose)polymerase (PARP) gene in the plant cells or plants as described in WO 00/04173 or EP 04077984.5 (herein incorporated by reference).
Poly(ADP-ribose) polymerase (PARP), also known as poly(ADP-ribose) transferase (ADPRT) (EC 2.4.2.30), is a nuclear enzyme found in most eukaryotes, including vertebrates, arthropods, mollusks, slime moulds, dinoflagellates, fungi and other low eukaryotes with the exception of yeast. The enzymatic activity has also been demonstrated in a number of plants (Payne et al., 1976; Willmitzer and Wagner, 1982; Chen et al., 1994; O'Farrell, 1995).
PARP catalyzes the transfer of an ADP-ribose moiety derived from NAD+, mainly to the carboxyl group of a glutamic acid residue in the target protein, and subsequent ADP-ribose polymerization. The major target protein is PARP itself, but also histones, high mobility group chromosomal proteins, topoisomerase, endonucleases and DNA polymerases have been shown to be subject to this modification.
As a particular embodiment, the stress tolerance enhancing transgene may comprise the following operably linked DNA fragments:

    a) a plant-expressible promoter;
    b) a DNA region which when transcribed results in an RNA molecule capable of reducing the expression of the endogenous PARP encoding genes of a plant (a PARP inhibitory RNA molecule);
    c) a DNA region involved in transcription termination and polyadenylation.


The mentioned DNA region may result upon transcription in a so-called antisense RNA molecule reducing in a transcriptional or post-transcriptional manner the expression of a PARP encoding gene in the target plant or plant cell, comprising at least 20 or 21 consecutive nucleotides having at least 95% to 100% sequence identity to the complement of the nucleotide sequence of a PARP encoding gene present in the plant cell or plant.
The mentioned DNA region may also result in a so-called sense RNA molecule comprising reducing in a transcriptional or post-transcriptional manner the expression of a PARP encoding gene in the target plant or plant cell, comprising at least 20 or 21 consecutive nucleotides having at least 95% to 100% sequence identity to the nucleotide sequence of a PARP encoding gene present in the plant cell or plant.
However, the minimum nucleotide sequence of the antisense or sense RNA region of about 20 nt of the PARP coding region may be comprised within a larger RNA molecule, varying in size from 20 nt to a length equal to the size of the target gene. The mentioned antisense or sense nucleotide regions may thus be about from about 21 nt to about 5000 nt long, such as 21 nt, 40 nt, 50 nt, 100 nt, 200 nt, 300 nt, 500 nt, 1000 nt, 2000 nt or even about 5000 nt or larger in length. Moreover, it is not required for the purpose of the invention that the nucleotide sequence of the used inhibitory PARP RNA molecule or the encoding region of the transgene, is completely identical or complementary to the endogenous PARP gene the expression of which is targeted to be reduced in the plant cell. The longer the sequence, the less stringent the requirement for the overall sequence identity is. Thus, the sense or antisense regions may have an overall sequence identity of about 40% or 50% or 60% or 70% or 80% or 90% or 100% to the nucleotide sequence of the endogenous PARP gene or the complement thereof. However, as mentioned antisense or sense regions should comprise a nucleotide sequence of 20 consecutive nucleotides having about 100% sequence identity to the nucleotide sequence of the endogenous PARP gene. Preferably the stretch of about 100% sequence identity should be about 50, 75 or 100 nt.
For the purpose of this invention, the “sequence identity” of two related nucleotide sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (×100) divided by the number of positions compared. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970) Computer-assisted sequence alignment, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wis., USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3.
It will be clear that whenever nucleotide sequences of RNA molecules are defined by reference to nucleotide sequence of corresponding DNA molecules, the thymine (T) in the nucleotide sequence should be replaced by uracil (U). Whether reference is made to RNA or DNA molecules will be clear from the context of the application.
The efficiency of the above mentioned transgenes in reducing the expression of the endogenous PARP gene may be further enhanced by inclusion of DNA elements which result in the expression of aberrant, unpolyadenylated PARP inhibitory RNA molecules. One such DNA element suitable for that purpose is a DNA region encoding a self-splicing ribozyme, as described in WO 00/01133 A1.
The efficiency of the above mentioned transgenes in reducing the expression of the endogenous PARP gene of a plant cell may also be further enhanced by including into one plant cell simultaneously a transgene as herein described encoding a antisense PARP inhibitory RNA molecule and a transgene as herein described encoding a sense PARP inhibitory RNA molecule, wherein said antisense and sense PARP inhibitory RNA molecules are capable of forming a double stranded RNA region by base pairing between the mentioned at least 20 consecutive nucleotides, as described in WO 99/53050 A1.
As further described in WO 99/53050 A1, the sense and antisense PARP inhibitory RNA regions, capable of forming a double stranded RNA region may be present in one RNA molecule, preferably separated by a spacer region. The spacer region may comprise an intron sequence. Such a transgene may be conveniently constructed by operably linking a DNA fragment comprising at least 20 nucleotides from the isolated or identified endogenous PARP gene, the expression of which is targeted to be reduced, in an inverted repeat, to a plant expressible promoter and 3′ end formation region involved in transcription termination and polyadenylation. To achieve the construction of such a transgene, use can be made of the vectors described in WO 02/059294 A1.
Current nomenclature refers to the classical Zn-finger-containing polymerases as PARP1 proteins (and corresponding parp1 genes) whereas the structurally non-classical PARP proteins are currently referred to as PARP2 (and corresponding parp2 genes) and “PARP encoding genes” as used herein, may refer to either type.
The following database entries (herein incorporated by reference) identifying experimentally demonstrated and putative poly ADP-ribose polymerase protein sequences, parts thereof or homologous sequences, could be used according to the current invention: BAD53855 (Oryza sativa); BAD52929 (Oryza sativa); XP—477671 (Oryza sativa); BAC84104 (Oryza sativa); AAT25850 (Zea mays); AAT25849 (Zea mays); NP—197639 (Arabidopsis thaliana); NP—850165 (Arabidopsis thaliana); NP—188107 (Arabidopsis thaliana); NP—850586 (Arabidopsis thaliana); BAB09119 (Arabidopsis thaliana); AAD20677 (Arabidopsis thaliana); Q11207 (Arabidopsis thaliana); C84719 (Arabidopsis thaliana); T51353 (Arabidopsis thaliana); T01311 (Arabidopsis thaliana); AAN12901 (Arabidopsis thaliana); AAM13882 (Arabidopsis thaliana); CAB80732 (Arabidopsis thaliana); CAA10482 (Arabidopsis thaliana); AAC79704 (Zea mays): AAC19283 (Arabidopsis thaliana); CAA10888 (Zea mays); CAA10889 (Zea mays); CAA88288 (Arabidopsis thaliana).
As a particular embodiment of the invention, the PARP gene expression reducing gene may comprise the following operably linked DNA fragments:

    a) a plant expressible promoter
    b) a DNA region which when transcribed yields an RNA molecule, the RNA molecule comprising:
    
        a. An antisense nucleotide sequence comprising at least about 20 consecutive nucleotides having about 96% sequence identity to a nucleotide sequence of about 20 consecutive nucleotides selected from the nucleotide sequences of SEQ ID 1 (Arabidopsis parp1 coding region) SEQ ID 2 (Arabidopsis parp2 coding region) SEQ ID 3 (Zea mays parp1 coding region), SEQ ID 4 (another Zea mays parp1 coding region), SEQ ID 5 (Zea mays parp2 coding region) or SEQ ID 6 (cotton parp2 partial cDNA) or from nucleotide sequences encoding proteins with similar or identical amino acid sequences as encoded by the mentioned nucleotide sequences.
        b. A sense nucleotide sequence comprising at least about 20 nucleotides which are complementary to the antisense nucleotide sequence. The sense nucleotide sequence may thus comprise a sequence of at least about 20 consecutive nucleotides having about 96% sequence identity to a nucleotide sequence of about 20 consecutive nucleotides selected from the nucleotide sequences of SEQ ID 1 (Arabidopsis parp1 coding region) SEQ ID 2 (Arabidopsis parp2 coding region) SEQ ID 3 (Zea mays parp1 coding region), SEQ ID 4 (another Zea mays parp1 coding region), SEQ ID 5 (Zea mays parp2 coding region) or SEQ ID 6 (cotton parp2 partial cDNA) of from nucleotide sequences encoding proteins with similar or identical amino acid sequences as encoded by the mentioned nucleotide sequences;
        
            whereby the sense and antisense nucleotide sequence are capable of forming a double stranded RNA molecule (dsRNA);
        
        
    
    
    c) A DNA region for transcription termination and polyadenylation.


However, it will be clear that other PARP gene expression reducing genes as described in WO00/04173 or EP 04077984.5 may be used.
In another embodiment of the invention, the stress tolerance enhancing transgene may be a transgene capable of reducing the expression and/or the activity of the PARG encoding genes of the plants or plants cells, as described e.g. in WO 2004/090140 (herein incorporated by reference).
PARG (poly(ADP-ribose) glycohydrolase; E.C.3.2.1.143) converts poly(ADP-ribose) polymers to free ADP-ribose by its exoglycosidase and endoglycosidase activity (PARG).
In plants, a poly(ADP-ribose) glycohydrolase has been identified by map-based cloning of the wild-type gene inactivated in a mutant affected in clock-controlled transcription of genes in Arabidopsis and in photoperiod dependent transition from vegetative growth to flowering (tej). The nucleotide sequence of the gene can be obtained from nucleotide databases under the accession number AF394690 (Panda et al., 2002 Dev. Cell. 3, 51-61; SEQ ID No 7)
Nucleotide sequences of other plant PARG encoding genes from plants can be found in WO 2004/090140 A2, such as the PARG gene from Solanum tuberosum (SEQ ID No 8); Oryza sativa (SEQ ID No 9) or Zea mays (SEQ ID No 10) as well as methods to isolate additional PARG encoding genes and variants thereof from other plants.
Thus, in one embodiment, the plants or plant cells engineered to be stress resistant may comprise the following operably linked DNA fragments:

    a) a plant expressible promoter
    b) a DNA region, which when transcribed yields an inhibitory RNA molecule, the RNA molecule comprising
    
        i. a antisense nucleotide region comprising at least 20 consecutive nucleotides having at least 96% sequence identity to a nucleotide sequence of about 20 nucleotides selected from the complement of a nucleotide sequence encoding a plant PARG protein, such as the nucleotide sequences of SEQ ID 7, SEQ ID 8, SEQ ID 9 or SEQ ID 10 or nucleotide sequences encoding proteins with similar or identical amino acid sequences as the nucleotide sequences mentioned; or
        ii. a sense nucleotide region comprising at least 20 consecutive nucleotides selected from a nucleotide sequence encoding a plant PARG protein, such as the nucleotide sequences of SEQ ID 7, SEQ ID 8, SEQ ID 9 or SEQ ID 10 or nucleotide sequences encoding proteins with similar or identical amino acid sequences as the nucleotide sequences mentioned; or
        iii. a antisense and sense nucleotide sequences as mentioned sub i) or ii) whereby said antisense and sense nucleotide sequence are capable of forming a double stranded RNA molecule;
    
    
    c) A DNA region involved in transcription termination and polyadenylation.


It will be immediately clear to the skilled artisan that additional parameters of length of sense and antisense nucleotide sequences or dsRNA molecules, and sequence identity for the ParG inhibitory RNA molecules can be used as mentioned above for the PARP inhibitory RNA molecules.
In yet another embodiment of the invention, the stress tolerance enhancing transgene may a transgene coding for a plant-function enzyme of the nicotineamide adenine dinucleotide salvage synthesis pathway. Accordingly, the stress tolerance enhancing gene may comprise the following operably linked DNA molecules:

    a) a plant-expressible promoter;
    b) a DNA region coding for a plant-functional enzyme of the nicotinamide adenine dinucleotide salvage synthesis pathway selected from nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase or nicotinamide adenine dinucleotide synthetase; and
    c) a 3′ end region involved in transcription termination and polyadenylation,

as described in EP 04077624.7 (herein incorporated by reference).



As used herein, “a plant-functional enzyme of the nicotinamide adenine dinucleotide salvage synthesis pathway” is an enzyme which when introduced into plants, linked to appropriate control elements such as plant expressible promoter and terminator region, can be transcribed and translated to yield a enzyme of the NAD salvage synthesis pathway functional in plant cells. Included are the enzymes (and encoding genes) from the NAD salvage synthesis, which are obtained from a plant source, but also the enzymes obtained from yeast (Saccharomyces cereviseae) or from other yeasts or fungi. It is thought that the latter proteins may be even more suitable for the methods according to the invention, since these are less likely to be subject to the enzymatic feedback regulation etc. to which similar plant-derived enzymes may be subject.
Enzymes involved in the NAD salvage synthesis pathway comprise the following

    
    
        Nicotinamidase (EC 3.5.1.19) catalyzing the hydrolysis of the amide group of nicotinamide, thereby releasing nicotinate and NH3. The enzyme is also known as nicotinamide deaminase, nicotinamide amidase, YNDase or nicotinamide amidohydrolase
        Nicotinate phosphoribosyltransferase (EC 2.4.2.11) also known as niacin ribonucleotidase, nicotinic acid mononucleotide glycohydrolase; nicotinic acid mononucleotide pyrophosphorylase; nicotinic acid phosphoribosyltransferase catalyzing the following reaction

Nicotinate-D-ribonucleotide+diphosphate=nicotinate+5-phospho-α-D ribose 1-diphosphate

        Nicotinate-nucleotide adenylyltransferase, (EC 2.7.7.18) also known as deamido-NAD+ pyrophosphorylase; nicotinate mononucleotide adenylyltransferase; deamindonicotinamide adenine dinucleotide pyrophsophorylase; NaMT-ATase; nicotinic acid mononucleotide adenylyltransferase catalyzing the following reaction

ATP+nicotinate ribonucleotide=diphosphate+deamido-NAD+

        NAD-synthase (EC 6.3.1.5) also known as NAD synthetase; NAD+ synthase; nicotinamide adenine dinucleotide synthetase; diphosphopyridine nucleotide synthetase, catalyzing the following reaction

Deamido-NAD++ATP+NH3=AMP+diphosphate+NAD+

    
    


In one embodiment of the invention, the DNA regions coding for a plant functional enzyme of the NAD salvage pathway may comprise a nucleotide sequence from SEQ ID Nos 11, 12, 13, 14 or 15 or a nucleotide sequence encoding a protein with similar or identical amino acid sequences as the proteins encoded by the above mentioned nucleotide sequences.
As described by Hunt et al., 2004, plant homologues of these enzymes have been identified and these DNA sequences may be used to similar effect (Hunt et al., 2004, New Phytologist 163(1): 31-44). The identified DNA sequences have the following Accession numbers: for nicotinamidase: At5g23220 (SEQ ID No 16); At5g23230 (SEQ ID No 17) and At3 g16190 (SEQ ID No 18); for nicotinate phosphoribosyltransferase: At4g36940 (SEQ ID No 19), At2g23420 (SEQ ID No 20), for nicotinic acid mononucleotide adenyltransferase: At5g55810 (SEQ ID No 21) and for NAD synthetase: At1g55090 (SEQ ID No 22).
However, it will be clear that the plants engineered to be stress resistant may also comprise variants of these nucleotide sequences, including insertions, deletions and substitutions thereof. Equally, homologues to the mentioned nucleotide sequences from species different from Saccharomyces cerevisea can be used. These include but are not limited to nucleotide sequences from plants, and nucleotide sequences encoding proteins with the same amino acid sequences, as well as variants of such nucleotide sequences.
Variants of the described nucleotide sequence will have a sequence identity which is preferably at least about 80%, or 85 or 90% or 95% with identified nucleotide sequences encoding enzymes from the NAD salvage pathway, such as the ones identified in the sequence listing. Preferably, these variants will encode functional proteins with the same enzymatic activity as the enzymes from the NAD salvage pathway.
Having read the above description of the use according to the invention of stress tolerance enhancing transgenes to increase tolerance of plant cells, plants or seeds to hypoxic or anoxic conditions, the skilled person will immediately realize that similar effects can be obtained using variants of an endogenous gene corresponding to such a stress tolerance enhancing transgene, which variant results in higher stress tolerance of the plant cells or plants harbouring such a variant. By way of example, variants of an endogenous parp2 gene of a plant, having a low expression level and providing the harbouring plant with increased stress tolerance could be used in a similar way as a transgene reducing the expression of the endogenous parp2 gene. Such variants gene can be introduced into plant cells or plants by breeding techniques.
A person skilled in the art will also be aware that expression of the different stress tolerance enhancing genes or transgenes may lead to a population of different events, which exhibit a distribution of effects ranging from almost no effect to a very pronounced effect. However, a person skilled in the art will clearly be able to distinguish, identify or isolate those representatives of a population that best suit the needs.
In another embodiment, the invention provides a method for increasing the penetrance of the roots of a plant into growth medium or soil comprising the step of providing the plant with a stress tolerance enhancing transgene, or with a endogenous variant of such stress tolerance enhancing transgene, as herein described in its different embodiments.
As used herein, “protrusion of plant roots” or “the penetrance of the roots of a plant into growth medium or soil” refers to the depth of the growth of roots in solid growth medium, including soil, as measured from the surface of the growth medium to the lowest point of the roots (see also FIG. 1).
As a rule, an “increase in the protrusion or penetrance of plant roots” means at least a statistically significant increase in the depth of the growth of roots in growth medium as measured from the surface of the medium to the lowest point of root growth, which can be measured either as a difference in a comparison of the root depth of wild-type reference plants versus the root depth of plants engineered to be stress tolerant, or as a difference in a comparison of the root depth of plants treated with particular chemical compounds versus the root depth of untreated plants.
For a correct understanding of the invention, it is important to realize that deeper penetration of a root system of a plant into the growth medium or soil, achieved by the methods according to the invention, is not to be equalled with an increase of the root system in volume or dry or fresh weight. Indeed, the volume of a root system may be increased significantly, while the roots all remain quite superficial below the surface of the growth medium or soil. By contrast, roots of plants treated according to the invention may be equal in size, volume, weight or even length, yet protrude much deeper below the surface of the growth medium or the soil.
As used herein, “growth medium” is intended to refer to any medium suitable for plant growth including soil. Such media may include solidified or gellified liquids, such as water-agar, peat, turf, different types of soil etc.
In another embodiment, the invention is directed towards the use of a compound of the neonicotinoid class to increase the tolerance of a plant cell, plant or seed to hypoxic or anoxic conditions. Thus, a method is provided to increase the tolerance of a plant cell, plant or seed to hypoxic or anoxic conditions comprising the step of applying an effective amount of a neonicotinoid compound of the formula (I) to the plant cells, plants or seeds or to the habitat of the plants, or to the growth medium.




wherein

    Het represents a heterocycle which is in each case optionally mono- or polysubstituted by fluorine, chlorine, methyl or ethyl, which heterocycle is selected from the following group of heterocycles:
    
        pyrid-3-yl, pyrid-5-yl, 3-pyridinio, 1-oxido-5-pyridinio, 1-oxido-5-pyridinio, tetrahydrofuran-3-yl, thiazol-5-yl,
    
    
    A represents C1-C6-alkyl, —N(R1)(R2) or S(R2),
    
        in which
        R1 represents hydrogen, C1-C6-alkyl, phenyl-C1-C4-alkyl, C3-C6-cycloalkyl, C2-C6-alkenyl or C2-C6-alkynyl, and
        R2 represents C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, —C(═O)—CH3 or benzyl,
    
    
    R represents hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, —C(═O)—CH3 or benzyl or together with R2 represents the groups below:
    
        —CH2—CH2—, —CH2—CH2—CH2—, —CH2—O—CH2—, —CH2—S—CH2—, —CH2—NH—CH2—, —CH2—N(CH3)—CH2—, and
    
    
    X represents N—NO2, N—CN or CH—NO2.


Saturated or unsaturated hydrocarbon radicals, such as alkyl or alkenyl, can in each case be straight-chain or branched as far as this is possible, including in combination with heteroatoms, such as, for example, in alkoxy.
These compounds are known to have insecticidal activity (see, for example, EP-A1-192 606, EP-A2-580 533, EP-A2-376 279, EP-A2-235 725).
Compounds of the formula (I) which may be mentioned are the neonicotinoids listed in “The Pesticide Manual”, 13th Edition, 2003 (British Crop Protection Council).
One compound is imidacloprid of the formula




known, for example, from EP A10 192 060.

Another compound is nitenpyram of the formula




known, for example, from EP A2 0 302 389.

Another compound is acetamiprid of the formula




known, for example, from WO A1 91/04965.

Another compound is thiacloprid of the formula




known, for example, from EP A2 0 235 725.

Another compound is thiamethoxam of the formula




known, for example, from EP A2 0 580 553.

Another compound is clothianidin of the formula




know, for example, from EP A2 0 376 279.

Another compound is dinotefuran of the formula




known, for example, from EP A1 0 649 845.

Particularly suited for the current inventions are compounds of the formula (I) wherein the substituent “Het” represents chloropyridyl




such as imidacloprid, nitenpyram, acetamiprid, and thiacloprid.

Particularly preferred compounds are imidacloprid and thiacloprid.
6-Chloronicotinic acid can be set free during the degradation of the above mentioned neonicotinoids which carry this group, such as imidacloprid, nitenpyram, and thiacloprid. For example, imidacloprid is degraded stepwise to the primary metabolite 6-chloronicotinic acid, which eventually breaks down into carbon dioxide. It was found that this metabolite also increases the stress tolerance and health of a plant or plant cell or seed from which such plant is grown and which is engineered to be stress tolerant and can be also be used according to the methods of the current invention.
One way of determining whether 6-CNA is set free during the degradation of the above mentioned neonicotinoids in plants or in particular plants is described by Placke and Weber (Pflanzenschutz-Nachrichten Bayer 46/1993, 2 109-182).
Thus, in another embodiment of the invention, a method is described which is useful to increase the tolerance of plants cells or plants or parts thereof to hypoxic or anoxic conditions comprising the step of providing to said plant, to a plant cell or to seed from which said plants are grown an effective amount of 6-chloronicotinic acid (niacin, CAS NO: 5326-23-8) of the formula (3)




The effective amount of 6-chloronicotinic acid may be provided to the plant cell, plant or seed by applying directly the compound of the formula (3) to the plant cell, plant, seed and/or the habitat thereof. However, the 6-CNA may also be provided to the plant by providing a compound which can be metabolized by the plant to yield 6-CNA as a metabolite, such as the compounds mentioned above.
It will be immediately clear that the above described compounds can also be used to increase the penetrance of the roots of a plant into growth medium or soil comprising the step of providing an effective amount of a neonicotinoid compound of the formula (I), such as a neonicotinoid compound of the formula (I) comprising a chloropyridine side chain, particularly those neonicotinoids of the formula I comprising a chloropyridine side chain which can be metabolized in plants to yield 6-CNA, including imidacloprid or thiacloprid or providing 6-CNA to the plant cells, plants or seeds or to the habitat of the plants or to the growth medium.
One of the advantages of the present invention is that the systemic properties of the compounds according to the invention and compositions comprising said compounds mean that treatment of the seed of plants with these compositions is sufficient to increase the protrusion of the roots of the germinating plant and the resulting plant after emergence.
In another embodiment of the invention, a method is described which is useful to increase the protrusion of the roots of a plant, comprising applying to said plant and/or its habitat, to a plant cell or to seed from which said plants are grown an effective amount of a composition comprising the compounds of the formula (I).
Accordingly, the invention also relates to compositions comprising the compounds of the formula (I) for the use of such compositions according to the invention.
The compounds of formula (I) can be used also in a mixture with other active compounds, for example, insecticides, bactericides, miticides, fungicides, etc. in the form of their commercially useful formulations or in the application forms prepared from such formulations. This can be done to obtain compositions which in addition to increasing the protrusion of plant roots according to the invention also to combat pests which may be present. Insecticides which can be used are, for example, organophosphorous agents, carbamate agents, carboxylate type chemicals, chlorinated hydrocarbon type chemicals, insecticidal substances produced by microbes, etc.
In many cases, this results in synergistic effects, i.e. the activity of the mixture exceeds the activity of the individual components. Such formulations and application forms are commercially and ecologically especially useful as generally lower amounts of active ingredients can be used. A synergist, however, must not necessarily be active itself, as long as it enhances the action of the active compound.
A mixture with other known active compounds, such as herbicides, or with safeners, fertilizers and growth regulators is also possible.
Treatment according to the invention of the plants and plant parts with the active compounds is carried out directly or by allowing the compounds to act on their surroundings, environment or storage space by the customary treatment methods, for example by immersion, spraying, evaporation, fogging, scattering, painting on and, in the case of propagation material, in particular in the case of seed, also by applying one or more coats.
The active compounds can be converted into the customary formulations, such as solutions, emulsions, wettable powders, suspensions, powders, dusts, pastes, soluble powders, granules, suspension-emulsion concentrates, natural and synthetic materials impregnated with active compound, and microencapsulations in polymeric substances.
The content of the active compounds of the present invention in a commercially useful formulation or application form can be varied in a wide range. The active-compound content of the use forms prepared from the commercial formulations can vary within wide limits.
These formulations are produced in a known manner, for example by mixing the active compounds with extenders, that is liquid solvents and/or solid carriers, optionally with the use of surfactants, that is emulsifiers and/or dispersants, and/or foam-formers.
If the extender used is water, it is also possible to employ for example organic solvents as auxiliary solvents. Essentially, suitable liquid solvents are: aromatics such as xylene, toluene or alkylnaphthalenes, chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethylenes or methylene chloride, aliphatic hydrocarbons such as cyclohexane or paraffins, for example petroleum fractions, mineral and vegetable oils, alcohols such as butanol or glycol and also their ethers and esters, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar solvents such as dimethylformamide and dimethyl sulphoxide, and also water.
As solid carriers there are suitable: for example ammonium salts and ground natural minerals such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth, and ground synthetic minerals, such as highly disperse silica, alumina and silicates; as solid carriers for granules there are suitable: for example crushed and fractionated natural rocks such as calcite, marble, pumice, sepiolite and dolomite, and also synthetic granules of inorganic and organic meals, and granules of organic material such as sawdust, coconut shells, maize cobs and tobacco stalks; as emulsifiers and/or foam-formers there are suitable: for example nonionic and anionic emulsifiers, such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, for example alkylaryl polyglycol ethers, alkylsulphonates, alkyl sulphates, arylsulphonates and also protein hydrolysates; as dispersants there are suitable: for example lignin-sulphite waste liquors and methylcellulose.
Tackifiers such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or lattices, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids such as cephalins and lecithins, and synthetic phospholipids, can be used in the formulations. Other additives can be mineral and vegetable oils.
It is possible to use colorants such as inorganic pigments, for example iron oxide, titanium oxide and Prussian Blue, and organic dyes, such as alizarin dyes, azo dyes and metal phthalocyanine dyes, and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.
The formulations generally comprise between 0.1 and 98% by weight of active compound, preferably between 0.1 and 90% and particularly preferably between 0.5 and 70% by weight of active compound.
The effect of the neonicotinoid compounds and 6-CNA on root growth depth is particularly strongly pronounced at certain application rates. However, the application rates of the active compounds can be varied within relatively wide ranges. In general, the rates of applications are from 1 g to 1600 g of the active compound per hectare, preferably from 10 g to 800 g of the active compound per hectare, and particularly preferably from 10 g to 600 g of the active compound per hectare
As mentioned before, the invention relates to methods which are useful to increase the protrusion of the roots of a plant into the growth medium or to increase the tolerance to hypoxia conditions, comprising applying to the plant propagation material including seed from which the plant is grown an effective amount of a composition comprising the compounds of the formula (I). The plant propagation material may be treated before planting, for example seed may be dressed before sowing. The compounds according to the invention may also be applied to seed grains either by impregnating the grains with a liquid formulation or by coating them with a solid formulation. The composition may also be applied to the planting site when the propagation material is being planted, e.g. during sowing.
In connection with the treatment of plant propagation material such as seeds, favourable rates of application are in general 0.1 to 1000 g, in particular 1 to 800 g, preferably 10 to 500 g of one of the neonicotinoid compounds or 6-CNA per 100 kg of material to be treated.
All plants and plant parts can be treated in accordance with the invention. Plant parts are to be understood to mean all above-ground and underground parts and organs of plants, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers and rhizomes. The plant parts also include harvested material, and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, offsets and seeds. They also include plant cells, such as may be used or result from the transformation of a plant cell in accordance with the invention. It is also possible to apply the aforementioned compounds onto or into the soil, e.g. before planting or sowing to achieve the effect described, e.g. to enhance the stress tolerance of the plants after planting and the emerging plant which grows from a seed which has been sown into treated soil.
It will also be immediately clear that the methods of the invention comprising the use of stress tolerance enhancing transgenes or stress tolerance enhancing endogenous variants can be combined with the methods of the invention comprising the use of a neonicotinoid compound or 6-CNA, to yield additive and synergistic effects in increasing the tolerance to hypoxic or anoxic conditions or in increasing the root depth of a plant in a growth medium or soil.
The method of the current invention may be suitable for any plant, both dicotyledonous and monocotyledonous plants including but not limited to cotton, Brassica vegetables, oilseed rape, wheat, corn or maize, barley, sunflowers, rice, oats, sugarcane, soybean, vegetables (including chicory, lettuce, tomato), tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, but also plants used in horticulture, floriculture or forestry, cereal plants including wheat, oat, barley, rye, rice, turfgrass, sorghum, millet or sugarcane plants. The methods of the invention can also be applied to any plant including but not limited to cotton, tobacco, canola, oilseed rape, soybean, vegetables, potatoes, Lemna spp., Nicotiana spp., sweet potatoes, Arabidopsis, alfalfa, barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat, asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash, tomato, zucchini, almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut and watermelon.
As used herein “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A transgene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.
Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR—Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
Throughout the description and Examples, reference is made to the following sequences:

    SEQ ID No. 1: parp1 coding region from Arabidopsis thaliana. 
    SEQ ID No. 2: parp2 coding region from Arabidopsis thaliana. 
    SEQ ID No. 3: parp1 coding region 1 from Zea mays. 
    SEQ ID No. 4: parp1 coding region 2 from Zea mays. 
    SEQ ID No. 5: parp2 coding region from Zea mays. 
    SEQ ID No. 6: parp2 partial coding region from cotton.
    SEQ ID No. 7: parG coding region from Arabidopsis thaliana. 
    SEQ ID No. 8: parG coding region from Solanum tuberosum. 
    SEQ ID No. 9: parG coding region from Oryza sativa. 
    SEQ ID No. 10: parG coding region from Zea mays. 
    SEQ ID No. 11: nucleotide sequence of the nicotinamidase from Saccharomyces cereviseae (PNC1).
    SEQ ID No. 12: nucleotide sequence of the nicotinate phosphoribosyltransferase from Saccharomyces cereviseae (NPT1) (complement)
    SEQ ID No. 13: nucleotide sequence of the nicotinic acid mononucleotide adenyl transferase 1 (NMA1) from Saccharomyces cereviseae. 
    SEQ ID No. 14: nucleotide sequence of the nicotinic acid mononucleotide adenyl transferase 2 (NMA2) from Saccharomyces cereviseae. 
    SEQ ID No. 15: nucleotide sequence of the NAD synthetase (QNS1) from Saccharomyces cereviseae. 
    SEQ ID No. 16: nucleotide sequence of the nicotinamidase from Arabidopsis thaliana (isoform 1).
    SEQ ID No. 17: nucleotide sequence of the nicotinamidase from Arabidopsis thaliana (isoform 2)
    SEQ ID No. 18: nucleotide sequence of the nicotinamidase from Arabidopsis thaliana (isoform 3)
    SEQ ID No. 19: nucleotide sequence of the nicotinate phosphoribosyltransferase from Arabidopsis thaliana (isoform 1).
    SEQ ID No. 20: nucleotide sequence of the nicotinate phosphoribosyltransferase from Arabidopsis thaliana (isoform 2).
    SEQ ID No. 21: nucleotide sequence of the nicotinic acid mononucleotide adenyl transferase from Arabidopsis thaliana. 
    SEQ ID No. 22: nucleotide sequence of the NAD synthetase from Arabidopsis thaliana. 


EXAMPLES
Example 1
Protocol for Measurement of Depth of Arabidopsis Root Growth in Growth Medium
Media
Germination medium: Half concentrated Murashige and Skoog salts; B5 vitamins; 1.5% sucrose; pH 5.8; 0.4% Difco agar.
Arabidopsis Plants
Sterilization of Arabidopsis seeds: 2 min. 70% ethanol; 10 min. bleach (6% active chlorine)+1 drop Tween 20 for 20 ml solution; wash 5 times with sterile tap water; sterilization is done in 2 ml eppendorf tubes. Arabidopsis seeds sink to the bottom of the tube, allowing removal of the liquids by means of a 1 ml pipetman.
Pregermination of seeds: In 9 cm Optilux Petridishes (Falcon) containing 10 ml sterile tap water. Low light overnight to 24 hours.
Growing of Arabidopsis plants: Seeds are sown in 25×150 mm glass tubes (Sigma C5916) with natural (transparent) colored closure (Sigma C5791) containing ±34 ml germination medium: 1 seed/tube. The tubes are put in the two outer rows of tube holders for 40 tubes (VWR nalg5970-0025) wrapped in aluminium foil so that the roots can grow in the dark. Plants are grown at 23° C. 30-50 μEinstein s−1m−2. 12 hours light-12 hours dark. (See FIG. 1)
Measuring Root Depth
After three weeks, the root depth is measured from the surface of the medium to the lowest point of root growth. (See FIG. 1).
Example 2
Analysis of Depth of Root Growth of Arabidopsis Plants Comprising a Transgene which Enhances Stress Tolerance
Arabidopsis thaliana plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP1 or PARP2 genes, as described in WO 00/04173 A1, (e.g. in Example 8 thereof) were grown as described in Example 1.
After three weeks, the depth of the roots of the transgenic plants was measured and compared to the depth of the roots of non-transgenic control plants or to non-transgenic isogenic plants grown in a similar manner.
In a first experiment, various populations of Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes (with lines 427-16 and 427-20 showing weak tolerance to high light stress and line 427-19 showing a moderate tolerance to high light stress) were compared with a population of non-transgenic Arabidopsis thaliana cv. Col-0 plants.
The results of the measurements were subjected to statistical analysis, summarized in Table 1, which also represents the mean, standard deviation and confidence intervals.
The roots of transgenic Arabidopsis thaliana plants with the highest tolerance to high light stress conditions (line 427-19) protruded statistically significant deeper (at 99% confidence level) into the growth medium than non-transgenic Arabidopsis thaliana cv. Col-0 control plants (see FIG. 2 and Table 1)







TABLE 1







Root depth (mm) of Arabidopsis thaliana cv. Col-0 plants comprising a


transgene encoding a dsRNA molecule which is capable of reducing the


expression of endogenous PARP2 genes as compared to non-transgenic


Arabidopsis thaliana cv. Col-0 plants












Col-0
427-16
427-19
427-20















mean
25.173611
26.388889
27.42
24.942029


Standard dev.
3.64166
3.422548
3.361185
3.69174


Standard error
0.429174
0.46575
0.388116
0.444433


95% Confidence
0.858348
0.94128
0.776233
0.888866


99% Confidence
1.141602
1.259388
1.032389*
1.182192





*p < 0.01






In a further experiment, a population of transgenic Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes and which are tolerant to high light stress (line 427-22) were compared to a transgenic line containing a similar transgene but which was sensitive to high light stress (line 427-24), as well as to non-transgenic Arabidopsis thaliana cv. Col-0 control plants.
The results of the measurements were subjected to statistical analysis, summarized in Table 2, which also represents the mean, standard deviation and confidence intervals.
The roots of transgenic Arabidopsis thaliana cv. Col-0 plants of the stress tolerant transgenic line (line 427-22) protruded deeper into the growth medium (comprising 0.7% Difco agar instead of 0.4%) than the roots Arabidopsis thaliana cv. Col-0 control plants and than the roots of stress-sensitive transgenic Arabidopsis thaliana cv. Col-0 plant line (line 427-24) (see FIG. 3 and Table 2).







TABLE 2







Root depth (mm) of Arabidopsis thaliana cv. Col-0 plants comprising a


transgene encoding a dsRNA molecule which is capable of reducing the


expression of endogenous PARP2 genes as compared to non-transgenic


Arabidopsis thaliana cv. Col-0 plants











Col-0
427-22
427-24
















Mean
18.291339
21.146154
17.384058



Standard dev.
2.928677
5.368981
2.740149



Standard error
0.259878
0.66594
0.329875



95% Confidence
0.514558
1.33188
0.65975



99% Confidence
0.680101
1.771401*
0.877468







*p < 0.01






In another experiment, the following populations were analyzed:
C24: wild-type Arabidopsis line; line 1599: A. thaliana transgenic line comprising anti-PARP2 transgene with a high tolerance to high light stress conditions; line 1463: A. thaliana transgenic line comprising anti-PARP2 transgene with a moderate tolerance to high light stress conditions; line 1681: A. thaliana transgenic line comprising anti-PARP 1 gene with a moderate tolerance to high light stress conditions; and line 1690: A. thaliana transgenic line comprising anti-PARP1 gene with a moderate tolerance to high light stress conditions. The stress tolerance of line 1599 is very high, the stress tolerance of lines 1463, 1681 and 1690 varies from moderate to high.
The results of the measurements were subjected to statistical analysis, summarized in Table 3, which represents the mean, standard deviation and confidence intervals.
The roots of transgenic Arabidopsis thaliana cv. C24 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP1 genes (lines 1681 and 1690) or PARP2 genes (lines 1599 and 1463) protruded deeper in the growth medium than the non-transgenic Arabidopsis thaliana cv. C24 control plants (see FIG. 4 and Table 3)







TABLE 3







Root depth (mm) of Arabidopsis thaliana cv. C24 plants comprising a


transgene encoding a dsRNA molecule which is capable of reducing the


expression of endogenous PARP1 or PARP2 genes as compared to non-


transgenic Arabidopsis thaliana cv. C24 plants













C24
1599
1463
1681
1690















mean
16.18
18.125
17.405063
17.433333
17.897727


Standard
2.192181
2.992481
2.16036
2.112246
1.885007


dev.


Standard
0.219218
0.451133
0.243059
0.27269
0.284175


error


95%
0.438436
0.911741
0.486119
0.551106
0.574319


Confidence


99%
0.58312
1.219865*
0.646538*
0.737353*
0.76841*


Confidence





*p < 0.01






In a further experiment, a 1:1 segregating population of transgenic Arabidopsis thaliana cv. Col-0 line comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes and with high tolerance to high light stress, was analyzed. The presence of the transgene was verified by PCR analysis.
Plants comprising the transgene had roots which protruded deeper into the growth medium than the azygous Arabidopsis thaliana cv. Col-0 plants derived from line 427-19 (see FIG. 5 and Table 4)







TABLE 4







Root depth (mm) of Arabidopsis thaliana cv. Col-0 plants comprising a


transgene encoding a dsRNA molecule which is capable of reducing the


expression of endogenous PARP2 genes as compared to the azygous


Arabidopsis thaliana cv. Col-0 plants










Azygous
Transgenic



427-19
427-19















Mean
25.296512
27.38172



Standard dev.
3.573907
3.244894



Standard error
0.385384
0.33648



95% Confidence
0.770769
0.67296



99% Confidence
1.025122
0.895036*







*p < 0.01






Example 3
Analysis of Depth of Root Growth of Arabidopsis Plants after Application of Imidacloprid
Arabidopsis thaliana cv. C24 plants were grown as described in Example 1 on germination medium (with 0.7% Difco agar in stead of 0.4%) comprising various concentrations of imidacloprid (0, 50, and 100 mg/l). After three weeks, the depth of the roots of the plants treated with 50 and 100 mg/l imidacloprid was measured and compared to the depth of the roots of untreated plants grown in a similar manner.
The roots of the treated Arabidopsis plants protruded deeper in the growth medium than the roots of the non-treated Arabidopsis plants (see FIG. 6 and Table 5)







TABLE 5







Root depth (mm) of Arabidopsis thaliana cv. C24 plants treated with 50


and 100 mg/l imidacloprid as compared to Arabidopsis thaliana cv. C24


plants not treated with imidacloprid











0 mg/L
50 mg/L
100 mg/L
















Mean
1.9475
2.1875
2.2725



Standard dev.
0.289714
0.353508
0.456072



Standard error
0.037402
0.045638
0.058879



95% Confidence
0.075589
0.092234
0.118994



99% Confidence
0.101135
0.123404*
0.159208*







*p < 0.01






Example 4
Analysis of Depth of Root Growth of Arabidopsis Plants after Application of 6-Chloronicotinic Acid (6-CNA)
Arabidopsis thaliana cv. C24 plants were grown as described in Example 1 on germination medium comprising various concentrations of 6-CNA (0, 1, and 5 mg/l). After three weeks, the depth of the roots of the plants treated with 1 and 5 mg/l 6-CNA was measured and compared to the depth of the roots of the plants not treated with 6-CNA grown in a similar manner.
The roots of the treated Arabidopsis plants protruded deeper in the growth medium than the roots of the non-treated Arabidopsis plants (see FIG. 7 and Table 6)







TABLE 6







Root depth (mm) of Arabidopsis thaliana cv. C24 plants treated with 1


and 5 mg/l 6-CNA as compared to Arabidopsis thaliana cv. Col-0


plants not treated with 6-CNA











0 mg/L
1 mg/L
5 mg/L
















Mean
2.031034
2.179661
2.118367



Standard dev.
0.189373
0.186418
0.237762



Standard error
0.024866
0.02427
0.033966



95% Confidence
0.050254
0.049049
0.068645**



99% Confidence
0.067237
0.065625*
0.091844







*p < 0.01



**p < 0.05

## Claims

1. A method for increasing the tolerance of a plant cell or plant to hypoxic or anoxic conditions, comprising:
a) applying an effective amount of a compound of formula (I)




wherein
Het represents a heterocycle which is either mono- or polysubstituted by fluorine, chlorine, methyl or ethyl, wherein said heterocycle is:
pyrid-3-yl, pyrid-5-yl, 3-pyridinio, 1-oxido-5-pyridinio, 1-oxido-5-pyridinio, tetra-hydrofuran-3-yl, or thiazol-5-yl,

A represents C1-C6-alkyl, —N(R1)(R2) or S(R2),
in which
R1 represents hydrogen, C1-C6-alkyl, phenyl-C1-C4-alkyl, C3-C6-cycloalkyl, C2-C6-alkenyl or C2-C6-alkynyl, and
R2 represents C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, —C(═O)—CH3 or benzyl,

R represents hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, —C(═O)—CH3 or benzyl or together with R2 represents the groups below:
—CH2—CH2—, CH2—CH2—CH2—, —CH2—O—CH2—, —CH2—S—CH2—, —CH2—NH—CH2—, —CH2—N(CH3)—CH2—, and

X represents N—NO2, N—CN or CH—NO2 
on said plant cell or plant or on its locus, or on seeds of said plant; and
b) identifying a plant or plant cell which has an increased tolerance to hypoxic or anoxic conditions compared to a plant cell or plant where said compound was not applied to the plant, its locus or its seeds.

2. A method for increasing the penetrance of roots of a plant into a growth medium, comprising:
a) applying an effective amount of a compound of formula (I)




wherein
Het represents a heterocycle which is either mono- or polysubstituted by fluorine, chlorine, methyl or ethyl, wherein said heterocycle is:
pyrid-3-yl, pyrid-5-yl, 3-pyridinio, 1-oxido-5-pyridinio, 1-oxido-5-pyridinio, tetra-hydrofuran-3-yl, or thiazol-5-yl,

A represents C1-C6-alkyl, —N(R1)(R2) or S(R2),
in which
R1 represents hydrogen, C1-C6-alkyl, phenyl-C1-C4-alkyl, C3-C6-cycloalkyl, C2-C6-alkenyl or C2-C6-alkynyl, and
R2 represents C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, —C(═O)—CH3 or benzyl,

R represents hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, —C(═O)—CH3 or benzyl or together with R2 represents the groups below:
—CH2—CH2—, —CH2—CH2—CH2—, —CH2—O—CH2—, —CH2—S—CH2—, —CH2—NH—CH2—, —CH2—N(CH3)—CH2—, and

X represents N—NO2, N—CN or CH—NO2 
on said plant or on its locus, or on seeds of said plant; and
b) identifying a plant which has an increased penetrance of roots into a growth medium compared to a plant where said compound was not applied to the plant, its locus or its seeds.

3. The method of claim 1 or 2, wherein said compound of formula (I) is a neonicotinoid compound.

4. The method of claim 1 or 2, wherein said heterocycle represented by Het of said compound of formula (I) is a pyrid-3-yl heterocycle substituted by chlorine.

5. The method of claim 4, wherein said compound of formula (I) is imidacloprid, or thiacloprid.

6. A method for increasing the tolerance of a plant to hypoxic or anoxic conditions, comprising the step of: providing said plant with an effective amount of 6-chloronicotinic acid, whereby said 6-chloronicotinic acid is applied directly to said plant or the habitat thereof, or said plant's seed, and the tolerance of said plant to hypoxic or anoxic conditions is increased.

7. A method for increasing the penetrance of roots of a plant into a growth medium comprising the step of: providing said plant with an effective amount of 6-chloronicotinic acid, whereby said 6-chloronicotinic acid is applied directly to said plant or the habitat thereof, or said plant's seed, and the penetrance of roots of said plant into a growth medium is increased.

8. The method of claim 1 or 2, wherein said compound is applied to said plant, said locus, or said seeds when planting said plant or seed.