USE OF NON-PROLINE CYCLIC AMINO ACIDS TO INCREASE THE TOLERANCE OF PLANTS TO CONDITIONS OF OSMOTIC STRESS

Abstract

The present invention relates to the use of non-proline cyclic amino acids of general formula (I), wherein n, X, Y and Z have the meaning indicated in the description, to increase the tolerance of plants to conditions of osmotic stress, resulting from a lack of water in the environment. Non-proline amino acids used in the invention are of natural origin and are much more effective than other known amino acids used for the same purpose. This invention can therefore be considered very useful for preventing economic losses caused by a reduction in yield in agricultural crops.

##STR00001##

Claims

1.-4. (canceled)

5. A method to increase the tolerance of plants to conditions of osmotic stress, the method comprising administering to the plant an effective dose of at least one compound of formula (I): ##STR00005## wherein: n represents a whole number between 0 and 1, Y represents CO or CH.sub.2; X represents OH, OC.sub.1-4alkyl or NHC.sub.1-4alkyl; and Z represents H, OH, SH or SC.sub.1-4alkyl, with the condition that L-proline and D-proline are excluded from the definition of a compound of formula (I)

6. The method according to claim 5, wherein the compound of formula (I) is used in aqueous solution.

7. The method according to claim 5, wherein the compound of formula (I) uses an active ingredient which is selected from a nematicide, insecticide, acaricide, fungicide, bactericide and herbicide.

8. The method according to claim 5, wherein the osmotic stress is caused by a water deficit or by salinity.

9. The method according to claim 5, wherein the administration to the plant of the compound of formula (I) is carried out using a technique selected from spraying, injection, irrigation, immersion and application in substrate.

10. The method according to claim 5, wherein the administration of the compound of formula (I) is carried out with a dose between 0.1 M and 3 M.

11. The method according to claim 5, wherein the compound of formula (I) is selected from a compound of formula (II) and a compound of formula (III); and the administration is performed by immersion of the root system.

12. The method according to claim 5, wherein the compound of formula (I) is selected from a compound of formula (II) and a compound of formula (III); and and the administration is performed by seed immersion.

13. The method according to claim 5, wherein the compound of formula (I) is the compound of formula (II): ##STR00006##

14. The method according to claim 5, wherein the compound of formula (I) is the compound of formula (III). ##STR00007##

Description

BRIEF DESCRIPTION OF THE FIGURES

[0064] FIG. 1. Represents a diagram wherein it shows the growing conditions during the assay.

[0065] FIG. 2. Represents the weight of tomato plants after 7 days of growth in the different solutions. * Significant differences with respect to the reference group with the same treatment with a p value<0.05; ** Significant differences with respect to the reference group with the same treatment with a p value <0.01.

[0066] FIG. 3. Represents the relative water content of the different treatments after 10 days of assay. ** Significant differences with respect to its control with a p value <0.01.

[0067] FIG. 4. Represents the net photosynthesis measurements. ** Significant differences with respect to the drought reference with a p value <0.01

[0068] FIG. 5. Represents the efficiency in water use (Evapotranspiration/Net photosynthesis. Evapotranspiration=mol H.sub.2O s.sup.1; Net photosynthesis=mol CO.sup.2 m.sup.2 s.sup.1. ** Significant differences with respect to the drought reference with a p value <0.01

EXAMPLES

[0069] The conditions described below were used as the base for all examples included below. The growing conditions, such as the nutrient solution and the substrate used have been optimized to perform this type of experiments. Likewise, the dose of NaCl used has been optimized for this type of experiments since 50 mM makes it possible to evaluate if a treatment is capable of increasing tolerance to salinity (Jimnez-Arias et al., 2015. Environmental and Experimental Botany 120, 23-30).

[0070] A hydroponic culture system was used for the cultivation of the Arabidopsis thaliana plants necessary for the assays. This system was established in hydroponics trays with 1.9 L capacity (Araponics) wherein 18 plants were grown per container. A mixture of river sand with two different granulometries was used as physical substrate. The seeds were sowed in seed-holders, which were deposited during one week in a small greenhouse consisting of a high-density polyethylene tray with river sand (washed siliceous sand, with medium granulometry) with sterile distilled water covered with a transparent plastic sheet which was deposited in a growth chamber at 222 C., with a photoperiod of 16 hours of light (100-110 mol m-2 s-1 of PAR) and with 100% relative humidity. After one week, the seed-holders with the seedlings were transferred to the hydroponics trays in the same photoperiod and light intensity conditions but with 60-70% of relative humidity. The seedlings were maintained without aeration during the first week, after this the solution (Table 1) was generously aerated using aeration pumps and was renewed every 7 days.

TABLE-US-00001 TABLE 1 Hydroponic solution used in the experiments Macronutrients (mM) Micronutrients (M) KNO.sub.3 (1.25) H.sub.3BO.sub.3 (50) KH.sub.2PO.sub.4 (0.5) MnSO.sub.4 H.sub.2O (10) MgSO.sub.4 7H.sub.2O (0.75) ZnSO.sub.4 7H.sub.2O (2) Ca (NO.sub.3).sub.2 4H.sub.2O (0.75) CuSO.sub.4 5H.sub.2O (1.5) (NH.sub.4).sub.6Mo.sub.7O.sub.24 4H.sub.2O (0.075) Sequestrene (44.8)

Example 1. Effect of Alanine on the Response of A. thaliana to the Salt Stress Caused by the Addition of NaCl

[0071] To verify if the fundamental structure of the amino acids produces protective effects against salt stress, a simple amino acid, such as alanine, was used.

[0072] In this way, 21-day old plants were treated during 24 hours in nutrient solution enriched with a concentration of 2.5 mM of alanine. Subsequently, the plants were deposited in normal nutrient solution for 24 hours and they later grew for 7 days in nutrient solution with or without a supply of 50 mM of NaCl. This experiment was repeated twice, using 12 plants per experiment, with the value shown in table 2 being the average of 24 plants for each one of the conditions.

[0073] Subsequently, the wet weight of the aerial part of the plants was determined.

[0074] The following table (Table 2) shows the results of the amino acid alanine on the development of the plants.

TABLE-US-00002 TABLE 2 Effects of alanine on growth in optimal and saline conditions. Untreated Alanine Salt Alanine-salt Fresh weight (mg plant) 130 16 142 8 52 8** 36 2** RGR 0.53 0.58 0.21** 0.11** The data shown are the average of two independent experiments with 24 plants in total. The ** show significant differences with respect to the control group with a p < 0.01. RGR: Relative growth rate (R.G.R = (In Dw2 In Dw1)/T2 T1); In being the Napierian logarithm; Dw dry weight and T time).

[0075] As can be observed, the structure represented by the amino acid alanine was not capable of promoting growth, nor increasing tolerance to salinity, moreover, it seems that it harmed the plants with the same dose of salt.

Example 2. Effect of Pyroglutamic Acid or Pipecolinic Acid on the Response of A. thaliana to the Salt Stress Caused by the Addition of NaCl

[0076] 21-day old plants were treated during 24 hours in nutrient solution enriched with a concentration of 2.5 mM of pyroglutamic acid or pipecolinic acid. Subsequently, the plants were deposited in normal nutrient solution for 24 hours and they later grew for 7 days in nutrient solution with or without a supply of 50 mM of NaCl. This experiment was repeated twice, using 12 plants per experiment, with the value shown in table 2 being the average of 24 plants for each one of the conditions.

[0077] The fresh weight of the samples is shown in the following table (Table 3).

TABLE-US-00003 TABLE 3 Effects of pyroglutamic acid and pipecolinic acid on growth in optimal and saline conditions Pyroglutamic Pipecolinic Pyroglutamic Pipecolinic Untreated acid acid Salt acid acid-salt Fresh weight 158.3 20 183.9 15 145.9 22 90 18** 170.4 15 133.9 21 (mg plant) RGR 0.56 0.56 0.46 0.23 0.49 0.45 The data shown are the average of two independent experiments with 24 plants in total. The ** show significant differences with respect to the control group with a p < 0.01. RGR: Relative growth rate (R.G.R = (In Dw2 In Dw1)/(T2 T1); In being the Napierian logarithm; Dw dry weight and T time).

[0078] As can be observed, the salt again decreased growth considerably, as the fresh weight and relative growth rate show, after one week of being subjected to salt stress. This did not occur significantly in those plants that were previously treated with 2.5 mM of pyroglutamic acid or with 2.5 mM of pipecolinic acid, so that their effect on the increased tolerance to salt stress is proven.

Example 3. Comparison of the Effect of Pyroglutamic Acid, Pipecolinic Acid or Hydroxyproline on the Response of A. thaliana to the Salt Stress Caused by the Addition of NaCl

[0079] With the aim of demonstrating that the use of these compounds is more effective than the use of other amino acids that have already been disclosed such as the case of hydroxyproline (US20090054241 A1), the effect of a treatment with a 2.5 mM dose of pyroglutamic acid or pipecolinic acid, or hydroxyproline was compared.

[0080] The plants were deposited in nutrient solution enriched with one of the compounds indicated above for 24 hours and they later grew for 7 days in nutrient solution with a supply of 50 mM of NaCl. As control, the plants grown on nutrient solution and NaCl were used. This experiment was repeated twice, using 12 plants per experiment, with the value shown in table 2 being the average of 24 plants for each one of the conditions.

[0081] Two measurements were used in order to illustrate the defence to salt stress. First, the values for the sensitivity index (S.I) described by Saadallah et al. (2001, Agronomie 21, 627-634) is shown.

[0082] The greater the negative value of the S.I., the greater the negative effect of salinity on the plant. The second measurement is a percentage of reduction in the relative a growth rate of the plant subjected to conditions of salt stress.

[0083] The results are shown in the following table (Table 4).

TABLE-US-00004 TABLE 4 Effects of pyroglutamic acid, pipecolinic acid or hydroxyproline on the sensitivity index Pyroglutamic Pipecolinic Control acid acid Hydroxyproline S.I 0.70 0.1** 0.07** 0.25** % reduction 62.00 10.22** 8.12** 24.34** RGR (S.I = DWs DWc/DWc; being Dry weight plants in saline conditions; Dry weight in control conditions) and the percentage of reduction in relative growth rate (% Red. R.G.R = (RGRs RGRc/RGRc) 100; with RGRs growth rate of plants grown in saline conditions; RGRc growth rate of plants grown in control conditions). The data shown are the average of two independent experiments with 24 plants in total. The ** show significant differences with respect to the control group with a p < 0.01.

[0084] As can be observed, the use of either of the two compounds considerably increased tolerance with respect to hydroxyproline, so that the use of these compounds at the same dose was more beneficial for the growth of the plant in salt stress conditions.

Example 4. Comparison of the Effect of Pyroglutamic Acid, Pipecolinic Acid or Hydroxyproline on the Response of Solanum lycopersicum (Tomato) to the Salt Stress Caused by the Addition of NaCl

[0085] This assay enables demonstrating that the use of the compounds of the invention is more effective than the use of other already disclosed amino acids, such as the case of the aforementioned hydroxyproline, for growing commercial plants. As an example, tomato (Solanum lycopersicum) of the variety Realeza pear was used. The experiment commenced with 3-week old tomatoes which were placed in hydroponic trays with 4 L of a mixture of nutrient solution (Table 1) and distilled water in a proportion of 1:1.

[0086] The trays were placed in a growth chamber at 222 C., with a relative humidity of 60-70%, using a photoperiod of 16 hours of light (100-110 mol m.sup.2 s.sup.1 of PAR). From the first day, the solution was generously aerated by air pumps (30 min per day). The plants were maintained during two days with this proportion of solution and distilled water, and then they were removed from the medium and placed, under the same conditions, in plastic containers during 24 hours with the different treatments in distilled water (table 5):

TABLE-US-00005 TABLE 5 Treatments used in the assay Treatment Reference Pyroglutamic (2.5 mM) Pipecolinic (2.5 mM) Hydroxyproline (OH-Pro) (2.5 mM) Proline (2.5 mM)

[0087] After 24 hours, the plants were again introduced in the trays with the same proportion of distilled water/nutrient solution stated above and they were maintained like this for 48 hours. Then, the nutrient solution was removed from all the trays and changed for 4 litres of a new solution with each one of the conditions under study (FIG. 1), keeping the plants in these conditions for 7 days (the quantity of water of each one of the trays was controlled every two days and, if necessary, it was corrected with a solution of NaCl in distilled water with the concentration established for the tray.

[0088] Then, the growth of treated and untreated plants was compared, in normal conditions and salinity conditions. For the statistical analysis, the data collected was subjected to the normality test of the Kolmogorov-Smirnov test with the Lilliefors correction. Levene's test was used to verify the homoscedasticity of the data. As the data behaved following a normal distribution, its measurements were compared by one-way ANOVA and the significant differences were calculated using the Bonferroni post hoc test. The statistical analyses were performed with the SSPS computer package, version 20 for Windows.

[0089] As FIG. 2 indicates, under control conditions only those plants treated with hydroxyproline show a growth significantly different to that of the reference. They show a decrease in their growth (of 35%), for which reason we can establish that root treatment with hydroxyproline in our conditions has been harmful for the plant.

[0090] As regards growth in saline conditions, it can be observed how the plants of the reference group decreased their growth by 32%. This reduction was increased in the plants treated with proline via 24-hour root treatment (42% with respect to the growth in the control solution) although there are no significant differences when compared with the reference in saline conditions, for its part hydroxyproline only decreased its growth 17%. As regards the plants treated with pipecolinic and pyroglutamic acid, the growth decreased only by 7 and 9% respectively, with this fresh weight value being very significant with respect to its counterpart in the control group. This involves an increase in tolerance of 72% (pyroglutamic) and 78% (pipecolinic) if we compare with the control, and if we compare with the proline treatment it is 78% (pyroglutamic) and 83% (pipecolinic).

Example 5. Comparison of the Effect of Pyroglutamic Acid, Pipecolinic Acid or Hydroxyproline on the Response of Solarium lycopersicum (Tomato) to Drought in Greenhouse Conditions (Water Deficit)

[0091] The crop used was tomato (Solanum lycopersicum) of the Gransol capa negra variety. The experiment was performed in a glass greenhouse with four tables measuring 10 metres in length by 2 metres in width. Drip irrigation was used, supplied by self-compensated drippers to control the flow of water at all times.

[0092] 5-week old seedings were transplanted to 2-litre capacity pots using a mixture of peat and river sand to facilitate drainage. In total, 120 tomato plants were used. Four treatments were performed on 40 plants after the transplant (with 15 days between each treatment), adding 50 ml of a 2.5 mM concentration of Pyroglutamic to each pot.

[0093] Another 40 plants were treated via root applications using 2.5 mM of pipecolinic acid. The remaining plants randomly distributed throughout the greenhouse were treated with distilled water by way of control. The drought assay commenced on completing the four treatments, which was performed by removing the dripper from the 20 plants randomly chosen for each one of the treatments. The plants were deprived of water for 10 days.

[0094] The relative water content of the leaf was estimated after 10 days of drought for each one of the treatments, in control conditions and drought conditions, using 15 leaves. The following formula was used for this:

R.W.C(%)/=[(FwDw)/(TwDw)]100

[0095] With Fw: fresh weight; Dw: dry weight; Tw: turgor weight. RWC is the measurement most commonly used to estimate the possible water deficit a plant leaf has.

[0096] Furthermore, photosynthesis was estimated with the Lc-pro sd system (ADC Bioscientific). To do this, the tomato leaves were stimulated with a light source at 1000 mol m-2 s-1, with this light intensity being previously estimated to achieve the maximum photosynthesis in our crop and conditions. To do this, the parameters of net photosynthesis (A) and evapotranspiration (E) were determined, calculating with them the efficiency in water use (A/E) as described by Lambers et al. (2008. Plant Physiological Ecology. 2nd edition. Springer, New York). The measurements were taken after 10 days of assay from six plants for each one of the treatments.

[0097] The data of the different variables were subjected to the normality test using the Kolmogorov-Smirnov test with the Lilliefors correction. Levene's test was used to verify the homoscedasticity of the data. As the data behaved following a normal distribution, its measurements were compared by one-way ANOVA and the significant differences were calculated using the Bonferroni post hoc test. The statistical analyses were performed with the SSPS computer package, version 20 for Windows.

[0098] The results of the statistical analysis are shown in FIG. 3, wherein it can be observed how after ten days of drought the reference plants significantly decrease their relative water content (10.2%), whilst the plants treated with four treatments at 2.5 mM of pyroglutamic did not show this decrease. The plants treated with pipecolinic acid did show a decrease with respect to their control, but this was less than that in the reference plants (5.1%); however, these differences were not significant with respect to their control.

[0099] These data were corroborated by the gas exchange measurements. As shown in FIG. 4, the plants treated with pyroglutamic or pipecolinic had a net photosynthesis greater than the drought reference plants.

[0100] The analysis of the net photosynthesis and evapotranspiration measurements revealed the efficiency in the water use was significantly greater for the treated plants (FIG. 5). This efficiency was almost double in the treated plants than in the drought reference.