Active substances for increasing the stress defense in plants to abiotic stress, and methods of finding them

Abstract

The invention relates to a method of finding compounds which increase the tolerance of plants to abiotic stress factors acting on this plant, such as, for example, temperature (such as chill, frost or heat), water (such as dryness, drought or anoxia), or the chemical load (such as lack of or excess of mineral salts, heavy metals, gaseous noxious substances) by increasing the expression of plant-endogenous proteins, and to the use of these compounds for increasing the tolerance in plants to abiotic stress factors.

Claims

1. A method of increasing the yield of crop plants that are exposed to abiotic stress, which comprises treating the seeds to be grown to said crop plants that will be exposed to abiotic stress with a composition comprising 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide, thereby increasing the yield of said crop plants as compared to crop plants grown from the same seeds untreated with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide and grown under the same environmental conditions, wherein (i) the abiotic stress is selected from the group consisting of chill, frost, heat stress, and drought, and (ii) said seeds or crop plants are treated with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide as the sole active substance.

2. The method of claim 1, where the crop plants are maize, wheat, barley, rye, oats, rice, soya, sunflower, oilseed rape, or sugar beet.

3. A method of increasing the yield of crop plants that are exposed to abiotic stress, which comprises treating the crop plants by foliar spray or by soil application with a composition comprising 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide, thereby increasing the yield of said crop plants, wherein (i) the abiotic stress is selected from the group consisting of chill, frost, heat stress, and drought, and (ii) said crop plants are treated with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide as the sole active substance.

4. The method of claim 3, where the crop plants are maize, wheat, barley, rye, oats, rice, soya, sunflower, oilseed rape, or sugar beet.

5. The method of claim 3, where the crop plants are maize.

6. The method of claim 3, wherein the plants are treated pre-emergence with the composition.

7. A method of increasing tolerance of crop plants to abiotic stress factors acting on the plants, comprising treating the plants by seed dressing, foliar spray or soil application with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide, wherein (i) the abiotic stress factors are chill stress conditions, frost stress conditions, heat stress conditions, and/or drought stress conditions, and (ii) seeds of crop plants or crop plants are treated with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide as the sole active substance.

8. The method of claim 7, where the crop plants are maize.

9. The method of claim 7, where the abiotic stress factors acting on the plants are drought stress conditions.

10. The method of claim 7, where the abiotic stress factors acting on the plants are heat stress conditions.

11. The method of claim 7, where the abiotic stress factors acting on the plants are chill stress conditions.

Description

EXAMPLE 1

(1) Proof of the activity of safeners on plants which had been exposed to specific drought-stress conditions, by means of gene expression profiling (GFP):

(2) Abiotic Wtress Factor=Drought Stress

(3) Maize seeds cv. Lorenzo were dressed with the compound 4-cyclopropylamino-carbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (=VIII-3). To this end, 10 g of seeds were incubated with 20 mg of active substance dissolved in 2 ml of methylene chloride, with gentle shaking, until the solvent had evaporated (approx. 30 minutes). The seeds of the control group were only dressed with solvent. Thereafter, the treated seeds were placed into pots with compost (diameter 10 cm, in each case 10 seeds per pot), and the maize seedlings were raised for 10 days in a controlled-environment chamber under defined light, moisture and temperature conditions [white light, long day (16 hours light, 8 hours dark), 70% atmospheric humidity, 24 C.]. In each case 210 pots were used for the control groups and for the drought stress experiment. While the plants were raised, they were watered from below for 20 minutes every 2 days by raising the water level in a tray. 10 days after the seeds had germinated, the maize plants were exposed to the drought stress. To this end, the plants of control group 1 (without dressing with active substance) and of the test group (with dressing with active substance) were only irrigated every 7 days as described above. In the case of the plants of control group 2 (without dressing with active substance) and of the test group 2 (with dressing with active substance), the normal irrigation regime was reclaimed. After 3 weeks of drought stress conditions, the experiment was evaluated as follows. The aerial plant parts were cut off and dried overnight at 50 C. On the next day, the foliar biomass was determined in [g] (dry matter) per pot.

(4) The data were averaged over the in each case 10 pots of the plant group. The numerical values shown in table 1 are relative values in [%] relative to the data obtained with the control group 2 (without dressing with active substance, normal irrigation regime).

(5) TABLE-US-00001 TABLE 1 Drought stress experiment with maize plants without and with dressing with active substance Relative dry Plant group: Treatment: matter [%]: Control group 1 S/+D 50 Control group 2 S/D 100 Test group 1 +S/+D 80 Test group 2 +S/D 100 S = compound VIII-3 (=4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide). D = drought stress

(6) Without stress conditions, the average dry matter is the same plants from undressed seeds and from dressed seeds (control group 2, test group 2).

(7) On average, the plants from the group which had been dressed with the compound VIII-3 (=4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide) showed a more compact habit than the plants from the control group which, however, had no effect on the dry matter. Under drought stress, however, the average foliar biomass (dry matter) of the active-substance-dressed plants was significantly increased over the undressed control plant (control group 1, test group 1).

EXAMPLE 2

(8) Abiotic Stress Factor=Heat Stress

(9) Maize seeds cv. Lorenzo were dressed as in example 1 with the compound 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (=VIII-3) or treated only with solvent without active substance. The seedlings were raised for 10 days in the controlled-environment chamber under defined conditions, likewise as described in example 1.210 pots with maize plants were used for the heat stress experiment. The control group consisted of undressed plants (solvent), the test group of plants which had been dressed with active substance. To apply the heat stress conditions, both plant groups were placed for 2 days into a controlled-environment cabinet at 45C, white light, long day (16 hours fight, 8 hours dark) and 70% atmospheric humidity. To avoid desiccation as the result of the high temperature, the plants were irrigated once per day from below by raising the water level in a tray. After the heat stress, it was observed thatespecially in the control groupthe shoots of many plants had collapsed and that the leaves were lying flat on the ground.

(10) The experiment was valued quantitatively, taking into consideration the following criteria.

(11) After the heat treatment, the plants which had collapsed were counted and the result per pot was assessed:

(12) TABLE-US-00002 <20% of the emerged plants collapsed: some damage 20-50% of the emerged plants collapsed: medium damage >50% of the emerged plants collapsed: severe damage .circle-solid.

(13) Thereafter, all plants were grown on for 2 weeks under standard conditions. Then, the length increment of the individual plants was measured, and the survival rate of the plants per pot was determined:

(14) TABLE-US-00003 >50% survival rate: some damage 20-50% survival rate: medium damage <20% survival rate: severe damage .circle-solid.

(15) The results of the evaluation of the experiment are compiled in table 2.

(16) The undressed control plants were severely damaged by the heat stress. What was noticeable was in particular the collapse of the shoots in the case of most plants and the poor survival rate. The test plants which had been dressed with active substance were distinguished in particular by considerably better standing. While the final score highlights the damage caused by the severe heat stress even in those plants, their survival rate was significantly higher than in the control group.

(17) TABLE-US-00004 TABLE 2 Dried-stress experiments with maize plants with or without dressing with active substance Plant group: Treaetment: Control group S/+H Interim score (collapsed plants): .circle-solid..circle-solid..circle-solid..circle-solid..circle-solid..circle-solid..circle-solid. Final score (survival rate): .circle-solid..circle-solid..circle-solid..circle-solid..circle-solid..circle-solid..circle-solid..circle-solid..circle-solid. Test group +S/+H Interim score (collapsed plants): Final score (survival rate): .circle-solid..circle-solid..circle-solid..circle-solid..circle-solid. S = compound VIII-3 (=4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide). H = heat stress

EXAMPLE 3

(18) Abiotic Stress Factor=Chill Stress (Greenhouse)

(19) Maize seeds cv. Lorenzo were sown into 10-cm-pots into compost at a rate of 10 seeds per pot. All experimental groups consisted of in each case 4 pots. The sown seeds of test groups 1 and 2 were sprayed pre-emergence with 50 and 100 [g a.i./ha], respectively, of the compound 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (=VIII-3). The seeds of the control group remained untreated. The plants were raised under controlled conditions in a controlled-environment chamber [white light (long day: 16 h light, 8 h dark), 22 C. day-time temperature, 14 C. night-time temperature, 60% atmospheric humidity].

(20) After germination, when the plants had attained a length of approx. 1 cm, 2 pots from each group were incubated for 6 h in a different controlled-environment chamber under chill-stress conditions at 2 C. Thereafter, these plants were returned to the others in the first controlled-environment chamber.

(21) After a further 24 hours under standard conditions, the experiment was evaluated. It was observed that the chill stress caused chloroses at the leaf tips of the seedlings of the untreated control group. These symptoms were either absent or only very weakly pronounced on the plants which had been treated with the active substance.

(22) None of the plants from the test groups of the control group which had been kept exclusively under standard conditions without chill stress showed any damage symptoms whatsoever.

(23) To evaluate the experiment quantitatively, the plants with chloroses of the leaf tips were counted. The total number of the plants per test group and cold stress treatment was 20, provided over 2 pots in each case.

(24) The results of the evaluation of the experiment are compiled in table 3.

(25) TABLE-US-00005 TABLE 3 Treatment with active substance (pre- Number of damaged Plant group: emergence) [g a.i./ha]: plants (chloroses): Control group 0 9 Test group 1 50 1 Test group 2 100 0 Chill-stress experiment (greenhouse) with maize plants without and with treatment with the active substance, compound VIII-3 (= 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide), pre-emergence. All plants were exposed to chill-stress treatment. The total number of plants per group was 20.

(26) The results demonstrate that the treatment with the active substance VIII-3 (=4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide) can markedly reduce the damage symptoms which are the result of chill stress, or, at the higher dosage rate, completely prevents the occurrence of these symptoms.

EXAMPLE 4

(27) Abiotic Stress Factor=Chill Stress (Field)

(28) Maize seeds (dent com) were dressed with 0.003 mg and 0.03 mg of the compound 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (=VIII-3) per g of seeds and sown into test plots, each of which measure 34 m.sup.2 in size. One control plant contains untreated seed. Approximately 8 days after the seed emerged, the seedlings were in the one-leaf stage and were exposed for 5 days to the following temperature conditions:

(29) TABLE-US-00006 Maximum: Minimum: Day 1: 16.1 C. 7.2 C. Day 2: 17.8 C. 2.7 C. Day 3: 16.7 C. 0.6 C. Day 4: 16.7 C. 1.1 C. Day 5: 22.8 C. 12.2 C.

(30) After this chill period, the test plots were scored. For this purpose, all plants were assessed individually, and plants with at least 20% chill symptoms based on the total leaf area (burns and/or chloroses) were considered to be damaged.

(31) The results are compiled in table 4. In the control plot without dressing with active substance, all plants (100%) showed the above-described chill symptoms. In the test plots with dressing with active substance, the chill damage was significantly reduced. Here, only approximately 12% of the plants showed damage symptoms. The maximum frost-protection effect was attained in the range of the active substance quantities which had been used for the dressing, as shown in the table.

(32) TABLE-US-00007 TABLE 4 Chill Plant group: damage [%]*: Untreated 100 Dressed with 0.003 mg of (VIII-3 = 12 4-cyclopropylaminocarbonyl-N- (2-methoxybenzoyl)benzenesulfonamide) per seed Dressed with 0.03 mg VIII-3 (= 4- 12 cyclopropylaminocarbonyl-N- (2-methoxybenzoyl)benzenesulfonamide) per seed Chill stress experiment (field) with maize plants without and with dressing with the active substance VIII-3 (=4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide) *Number of plants with chill damage > 20% best of the total number of plants in the test plot

EXAMPLE 5

(33) Characterization of Genes which are Induced by Test Substances Under Abiotic Stress Conditions, Measured by Gene Expression Profiling (GEP):

(34) Maize seeds cv. Lorenzo were dressed as described in example 1 with the compound VIII-3 (=4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide) or with solvent. The plants were raised for 10 days in a controlled-environment chamber (conditions: see example 1).

(35) Thereafter, the plants were exposed to the following stress conditions: (1) Heat stress: 6 h at 45 C. (2) Drought stress: 7 days without irrigation, 24 C.

(36) The control plants of the specific experimental group were kept under the standard conditions described in example 1 (temperature, irrigation).

(37) After the stress treatment, the leaves of the stressed plants and of the unstressed control plants were harvested, shock-frozen in liquid nitrogen and stored at 80 C. until processed. All experiments were carried out in replications of in each case 2 pots.

(38) The labeled RNA probes for the DNA chip hybridization were prepared as described in the protocols (Expression Analysis. Technical Manual) from Affymetrix (Affymetrix Inc., 3380 Central Expressway, Santa Clara, Calif., USA). First, total RNA was isolated from in each case 500 mg of the harvested leaves. In each case 10 g of total RNA were used for the cDNA first- and second-strand synthesis. The cDNA was amplified with T7 polymerase and simultaneously labeled with biotin-UTP. In each case 20 g of this biotinylated cDNA were employed for the hybridization of the maize genome array from Affymetrix. This DNA microarray contains DNA sequences whose totality represents 13339 genes. Thereafter, the DNA microarrays were washed in the Affymetrix Fluidics Station, stained with streptavidin/phycoerythrin (Molecular Probes, P/N S-866) and scanned with the appropriate Agilent Laser Scanner (Agilent Gene Array Scanner). The fluorescence data obtained were analyzed using Affymetrix's Microarray Suite 5 software. After the quality assurance had been performed, all DNA chip analyses were stored in a database. To determine relevant expression values (induction factors, repression factors), the absolute expression values of the genes from the respective stress experiments were compared with the values of the respective control experiments (i.e. without abiotic stress and solvent-dressing only), based on the scoring function predetermined by the Affymetrix software. The resulting 4 expression values per gene were averaged by calculating the median value. These median values are shown in the results tables as induction factors. Similarity comparisons of expression profiles of various experiments and cluster analyses were carried out using the genedata expressionist software from Genedata (Genedata, Maulbeerstr. 46, CH-4016 Basel, Switzerland).

(39) The analysis of the expression profiles specifically searched for genes which are induced by the test substances only in conjunction with abiotic stress, but not by the substances or by stress alone. Such genes can be considered as indicators for additional anti-stress effects of the substances which exceed the known safener effect. The results from the analyses are shown in the tables which follow. The induction patterns of the indicator genes described permit the targeted finding of active substances for increasing the abiotic stress tolerance in crop plants. a) Under heat stress conditions, i.e. the tested maize plants (dressed with 2 mg a.i. 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide/g seeds) were exposed to a temperature of 45 C. for 6 hours 7 days after germination.

(40) An overview over the induced gene groups revealed the following pattern, which is shown in table 5:

(41) TABLE-US-00008 TABLE 5 Sample Set No. Condition A Condition B Condition C Zm.11840.1.A1_at 1.74 1.75 4.10 Zm.4274.1.S1_at 1.32 1.22 1.93 Zm.3040.1.S1_at 1.52 1.33 2.48 Zm12587.1.S1.s_at 1.30 1.45 2.33 Zm18994.2.A1_at 1.16 1.46 2.66 Zm.13498.1.S1_at 2.56 1.73 4.45

(42) The respective sample set no. corresponds to:

(43) TABLE-US-00009 Zm.11840.1.A1_at: putative N-carbamyl-L-amino acid amidohydrolase Zm.4274.1.S1_at: cytochrome P450 Zm.3040.1.S1_at uricase II (E.C.1.7.3.3); nodule specific uricase Zm12587.1.S1.s_at: glycosyltransferase Zm18994.2.A1_a_at: putative serine transferase Zm.13498.1.S1_at: membrane protein Condition A: heat stress (6 hours, 45 C.) Condition B: Seeds dressed with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (VIII-3)/NO heat stress Condition C: Seeds dressed with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (VIII-3)+heat stress (6 hours, 45 C.).

(44) Ignoring a slight basal induction of the analyzed gene activities, a pronounced increase in the gene expression was observed in all cases and is, in the case of the genes mentioned here, in the range of from 1.5 to 2.35 (expression under condition C/expression under condition A). If the test compound VIII-3 was tested on its own, i.e. without heat stress, the measured expression levels were in the range of the range induced by heat stress, or below or slightly above the range induced by heat stress.

(45) The induction patterns derived from table 5 and which are shown directly by the resulting expression values show characteristic inductions by the action of the compound 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (=VIII-3), the effect on the putative N-carbamyl-L-amino acid amidohydrolase [Zm.11840.1.A1_at] and on the putative serine carboxypeptidase [Zm18994.2.A1-at] being most pronounced. b) Under dried stress conditions, i.e. the tested maize plants (dressed with 2 mg a.i. 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide/g seeds) were exposed to a temperature of 24 C. for 7 hours 7 days after germination.

(46) An overview over the induced gene groups revealed the following pattern, which is shown in table 6:

(47) TABLE-US-00010 TABLE 6 Sample Set No. Condition A Condition B Condition C Zm.818.1.A1_at 1.06 1.12 8.47 Zm.3633.4.A1_at 1.12 0.74 3.03 Zm.18273.1.S1_at 1.66 0.95 2.91 Zm.13229.1.S1_at 1.55 1.02 3.39 Zm.12035.1.A1_at 1.86 0.90 3.66 Zm.485.1.A1_at 0.89 1.00 5.49 Zm.818.2.A1_at 0.93 1.10 5.40 Zm.10097.1.A1_at 1.23 1.27 3.29 Zm.18682.1.A1_at 1.25 1.12 4.19

(48) The respective sample set no. corresponds to:

(49) TABLE-US-00011 Zm.818.1.A1_at universal stress protein Zm.3633.4.A1_at wound induced protein (fragment) Zm.18273.1.S1_at regulatory protein-like Zm.13229.1.S1_at NBS-LRR type disease resistance protein O2 (fragment) Zm.12035.1.A1_at similar to AT3G10120 Zm.485.1.A1_at non-symbiotic hemoglobin (HBT) (ZEAMP GLB1) Zm.818.2.A1_at expressed protein Zm.10097.1.A1_at expressed protein Zm.18682.1.A1_at unknown protein Condition A: drought stress (7 days, 24 C.) Condition B: Seeds dressed with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (VIII-3)/NO drought stress Condition C: Seeds dressed with 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide (VIII-3)+drought stress (7 days, 24 C.).

(50) Ignoring a slight basal induction of the analyzed gene activities, a pronounced increase in the gene expression was observed in all cases and is, in the case of the genes mentioned here, in the range of from 1.75 to 8.0 (expression under condition C/expression under condition A). If the test compound VIII-3 was tested on its own. i.e. without heat stress, the measured expression levels were in the range of the range induced by dried stress, or in some cases even below the expression of unstressed plants (at values<1.0).

(51) The induction patterns derived from table 6 and which are shown directly by the resulting expression values show characteristic inductions in the presence of the compound 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide, the effect on the universal stress protein [Zm.818.1A1_at] and non-symbiotic hemoglobin (HBT) (ZEAMP GLB1) [Zm.485.1A1_at] being most pronounced.