MXPA00004462A - Use of lysophosphatidylethanolamine (18:1) and lysophosphatidylinositol to retard senescene and to enhance fruit ripening - Google Patents

Abstract

The present invention relates to a method of enhancing fruit ripening and stability and of delaying senescence in fruit and other plant tissues. This method consists of applying an effective amount of a lysophospholipid, such aslysophosphatidylethanolamine (18:1) (hereinafter referred to as"LPE (18:1)") or lysophosphatidylinositol (hereinafter referred to as"LPI") to the fruit and other plant tissues. Lysophospholipids such as LPE (18:1) and LPI were found to be superior to other lysophospholipids in delaying senescence and in inhibiting phospholipase D, a key enzyme in mediating membrane deterioration during of plant senescence. LPE (18:1) and LPI are naturally occurring and environmentally safe. Their use could replace many environmentally toxic compounds that are currently being used to retard senescence of flowers, fruits and leaves and to enhance fruit ripening.

Description

USE OF LISOFOSFATIDILETANOLAMINA (18: 1) AND LISOFOSFATIDILINOSITOL TO DELAY AGING AND IMPROVE FRUIT MATURATION BACKGROUND OF THE INVENTION Currently, different chemical and biological compounds are used in the commercial cultivation of fruits to control the ripening time of the fruit. These compounds can be used for different purposes. One purpose is to synchronize the maturation of the fruit to help the efficient cultivation of the same. Another purpose is to avoid the fall of the fruit so that the fruit remains in the plant until the period of adequate maturation. 'Another purpose of the compounds for the ripening of fruits is to improve the development of color in the fruit so that it has a better color and more uniform as consumers expect it to retail fruit. In the United States it is a current practice that many types of fruit are treated with one or more of these compounds during the growing process. Some compounds previously used to control the ripening of fruits are purely synthetic compounds that were found with desired effects on the fruit in question. Unfortunately, due to the potential toxicity and oncogenicity aspects, some of these chemical, synthetic fruit ripening compounds have been banned and had to be quickly displaced due to commercial or consumer resistance to these products. The most common compounds currently used to improve fruit ripening is ethephon, a synthetic compound that is marketed under the name of Ethrel, a registered trademark of Rhone-Poulenc Ag. Co. (Research Triangle Park, NC). Although this compound simulates maturation, it also makes the fruit soften. Thus, the fruit treated with ethephon has a very poor shelf life. There is a very important need for a maturation compound that is environmentally safe and does not cause softening of the fruit. In addition, consumers agree to pay an optimal price for fruits ripened in the plant. However, the fruits ripened in the plant can not be transported over long distances because these fruits soften and have a poor storage life. Therefore, it would be beneficial to improve the storage life of the fruits ripened in the plant. There is also a great interest in the industry (especially in the fresh vegetable and cut flowers industries) to find a safe product for the environment that retards aging and favors life in storage or in the vase. At present, well-toxic compounds such as silver thiosulfate are being used to increase the life in the vase of cut flowers. However, the use of sodium thiosulfate is being withdrawn due to environmental aspects. Therefore, it is desired to develop alternatives for the silver thiosulfate that is more likely to be accepted by commercial interests and for the consuming public. Lysophosphatidylethanolamine (hereinafter referred to as "LPE") consists of a group of compounds that have shown promise in controlling fruit ripening, improving fruit stability during storage and increasing shelf life of fruits stored. Methods for using purified egg LPE (hereinafter referred to as "LPEegg" to improve fruit ripening and stability are described in U.S. Patent Nos. 5,126,155 and 5,100,341, which are incorporated herein by reference. It is obtained from phosphatidylethanolamine, a lipid normally found in cell membranes Phosphatidylethanolamine is a phospholipid with two fatty acid portions that is abundant in the yolk of the egg.The elimination of a fatty acid from phosphatidylethanolamine and phospholipase A2 produces LPE: LPE is also present naturally in plant and animal tissues, especially it is rich in egg yolk and brain tissue. It is available commercially from Avanti Polar Lipids, Inc. (Alabaster, Alabama). There are numerous different fatty acids that can be found in purified LPE from natural sources. Fatty acids can vary in length of a chain as well as the degree of establishment. However, the relative efficacy of the different species of LPE. and also of the different classes of Xisofosfolípidos besides LPE in the control of the maturation of the fruit and the improvement in the stability of the fruit have not been examined.

COMPENDIUM OF THE INVENTION The present invention refers to a method to retard aging in fruits or plant tissues. The method includes applying to the fruit and other plant tissues, before or after harvest, a composition containing a lysophospholipid and an activating agent. The composition contains an amount of a lysophospholipid that is effective in retarding aging in the fruit and other plant tissues. The preferred lysophospholipid contained in the composition is lysophosphatidylinositol and / or lysophosphatidylethanolamine (18: 1).

In addition to containing the lysophospholipid, the composition may also contain an activating compound, such as ethanol, tergitol or sylgard 309. In addition, the present invention also relates to a method for improving ripening and fruit stability. The method includes applying to all plants before harvest a composition containing lysophospholipid and an activating compound. The composition contains an amount of lysophospholipid that is effective in improving the maturation and stability of the fruit. The preferred lysophospholipid contained in the composition is lysophosphatidylinositol and / or lysophosphatidylethanolamine (18: 1). In addition to containing the lysophospholipid, the composition may also contain an activating agent, such as ethanol, tergitol or sylgard 309.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the inhibition of partially purified pumpkin PLD activity by different concentrations of LPE with different acyl chains. Figure 2 is a graph showing the structural specificity of LPE (18: 1) for its inhibition of partially purified pumpkin PLD activity. Figure 3 is a graph showing the inhibition of partially purified pumpkin PLD activity as a function of the concentration of LPE.

Figure 4 is a graph showing the effect of substrate concentration on the inhibition of partially purified pumpkin PLD by LPE (18: 1). Figure 5 is a graph showing the effect of different lysophospholipids on partially purified pumpkin PLD activity. Figure 6 is a graph showing the relative chlorophyll content of the leaves treated with LPA, LPC, LPEegg, or LPI.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for improving ripening and fruit stability and retarding aging in fruit and other plant tissues using lysophospholipids, including, but not limited to, LPE (18: 1) and / or lysophosphatidylinositol (hereinafter referred to as "LPI"). As used herein, the term "lysophospholipids" refers to the derivatives of the. phospholipids having a single fatty acid removed by phospholipase A2. As used herein, the term "plant tissues" refers to any part or organ of a living plant. Examples include fruits, flowers, roots, stems, hypocotyls, leaves, petioles, petals, et cetera. The method of the present invention includes treating fruits and other plant tissues before or after harvesting with a composition containing a lysophospholipid having the formula: where Ri is selected from the group consisting of an acyloxy group of Cs-C22 and C5-C22 alkoxy; R 2 is selected from the group consisting of hydrogen, hydroxyl, a C 1 -C 5 acyloxy group and C 1 -C 5 alkoxy; and R3 is selected from the group consisting of hydrogen, choline, ethanolamine, glycerol, inositol and serine, wherein Ri and R2 are interchangeable with each other. Preferred compounds having the formula (I) above identified are LPE (18: 1) and LPI. Preferably, the composition contains an acceptable carrier for the lysophospholipid, such as water. However, it is also possible to use other carriers as organic solvents. The composition contains an amount of lysophospholipid which is effective in improving the ripening and stability of fruits and retarding the aging of the fruit and other plant tissues. More specifically, the amount of lysophospholipid in the composition can be from about 0.5 to about 1000 mg per liter of the composition, preferably from about 1 to about 500 mg per liter of the composition, most preferably from about 5 to about 250 mg per liter of the composition and even more preferably from about 5 to about 100 mg per liter of the composition. The composition can be applied to the fruit or plant tissues as a spray or simply in liquid form. In addition to containing the lysophospholipids, the composition may also contain one or more activating compounds. As used herein, the term "activating compounds" refers to compounds that improve the uptake of moisture and the efficacy of the active ingredient, which is lysophospholipid. Examples of the activating compounds that can be used in the method of the present invention include ethanol, tergitol and sylgard 309 '(available from Dow Corning Co., Midland, MI). The activating compounds are present in the amount from about 0.05 to about 2.0% v / v of the composition. The preferred lysophospholipid, LPE (18: 1), is a species of LPE having an 18-carbon fatty acid containing a single double bond. The LPE (18: 1) was found particularly superior to other species of LPE in the promotion of fruit ripening and retarding the aging of the fruit and plant tissues. The LPI has been found comparable with LPE (18: 1) and superior to the different LPE of LPE (18: 1) in the improvement of the fruit ripening and in the delay of the aging of the fruits and the vegetable tissues. As described in U.S. Patent Nos. 5,126,155 and 5,110,341, the LPE is effective in improving ripening and fruit stability. The exact mechanism by which these effects are achieved is only partially understood. It was described in U.S. Patent Nos. 5,126,155 and 5,110,341 that it was observed that the LPE stimulates the production of ethylene and suppresses respiration in the fruit. It was speculated that these effects could represent the improved maturation and stability of the fruit treated with LPE. The retardation of the aging of the fruit and vegetable tissues treated with LPE was correlated with less leakage of electrolytes through the membranes (5). Thus, the inventors suspect that LPE can regulate a key process of membrane deterioration in the aging and maturation of plants. The increased leakage of electrolytes during the aging of plants has been attributed to the breakdown of membrane phospholipids (1, 2). The reduced leakage of electrolytes in the leaves, flowers and fruits after harvest treated with LPE suggests that the LPE can protect the integrity of the membrane by inhibiting the degradation of the lipids of the membrane (3). Based on the release kinetics of the different lipolytic products in vivo and in vi tro, it has been proposed that phospholipase D (hereinafter referred to as "PLD") mediates the selective degradation of the membrane phospholipids, which is an event fast and early that occurs in senescent tissues (4-9). An increase in PLD expression was observed in tissues of senescent leaves and the expression of PLD was characterized by complex modes including an increase in PLD associated with the membrane, differential expression of PLD variants and changes in the amounts of the protein PLD and mRNA (10). As described herein, the inventors demonstrate that the lysophospholipid LPE can inhibit the activity of partially purified PLD in a highly specific form in plants. As the following examples demonstrate, the lysophospholipids LPE (18: 1) and LPI are particularly strong inhibitors of PLD. In addition, the treatment of plants with LPE (18: 1) or LPI is associated with reduced production of ethylene. LPI has been found particularly effective in delaying senescence in leaves, as evidenced by the high chlorophyll content of the senescent leaves treated with LPI, in relation to a control as well as compared to the leaves treated with LPE or LPC. Consequently, lysophospholipids, such as, but not limited to, LPE (18: 1) and LPI are particularly attive agents for retarding the aging of fruits and plant tissues. The inventors also demonstrate that LPE (18: 1) and LPI are particularly effective in improving ripening and fruit stability. By way of example and not as limitation, the examples of the present invention will now be provided.

EXAMPLE 1: Specific inhibition of PLD and LPE (18: 1) and LPI Example: Chemical materials and plants _ The purified natural lysophospholipids of egg yolk, bovine liver, and soybeans, and the synthetic LPE with different acyl chains (14: 0, 16: 0, 18: 0, 18: 1 ) were obtained from Avanti Polar Lipids (Alabaster, Alaba a). The other chemical compounds phospholipids and materials used were obtained from Sigma (Saint Louis, MO). The phospholipids and fatty acids were dissolved in chloroform: methanol: KOH (1M) (95: 5: 1, v / v). After water was added, the organic solvents were expelled by flowing nitrogen gas. The concentrations of the standard solution were adjusted to ImM with water before being added to the PLD reaction mixture. The LPE solution to treat the fruit and vegetable tissues was prepared in volume by sonification of the LPE powder suspended in water without the use of organic solvents. The partially purified pumpkin PLD, which has commonly been used to investigate the biochemical and physiological aspects of PLD (11, 12), was dissolved in 50 mM Tris (pH 8.0) and added to a reaction mixture with a final concentration of 0.6 μg / ml to examine the effect of LPE on the activity of PLD. In addition to partially purified pumpkin PLD, the researchers also investigated the effect of LPE on the activities of membrane-associated PLD and soluble PLD that were obtained from two plant sources, namely pumpkin (Brassica olerácea L. Blue Vintage) and Castor bean (Ricinus communis L. cv Hale). The castor seed plants were grown in plastic pots containing a mixture of vermiculite and parlayed (1: 1, v / v), which were sub-irrigated at 22 ° C with Hoagland nutrient solution under cold white fluorescent lights ( 150. μmol min -1 m-2) with a light period of 14 hours (10). The pumpkin was obtained fresh from the market.

Example lb: Fractionation of the tissue Fully extended leaves of two-month castor bean and squash plants were harvested, rapidly frozen in liquid nitrogen and homogenized with ice-cooled mortar and pestle (13). A buffer solution for extraction containing 50 mM Tris-HCl (pH 8.0) 10 mM KCl, 1 mM EDTA, 0.5 mM PMSF and 2 mM DTT was added to the powder samples. After triturating for an additional 5 minutes, the homogenate was centrifuged at 6000 g for 10 minutes to remove the remains. The supernatant was centrifuged at 100,000 g for 30 minutes to fractionate the extract into soluble, membrane-associated PLD. The resulting supernatant was collected as the soluble fraction and the package as the membrane fraction. The membrane fraction was washed once with buffer solution for the extract in order to remove the soluble contaminants. The samples of soluble PLD and membrane-associated PLD were added to the reaction mixtures in final concentrations of 100 μg / ml and 10 μg / ml, respectively.

Example: Study of PLD activity Pumpkin PLD activity, partially purified, was assessed by measuring the content of phosphorus contained in phosphatidylethanol (hereinafter referred to as "PEOH") and phosphatidic acid (hereinafter referred to as "PA") released from the substrate phosphatidylcholine (hereinafter referred to as "PC") (13). For this test, 200 μmol of egg PC in chloroform was dried under nitrogen gas stream. The lipid was emulsified in 1 ml of H20 by sonification at room temperature. A standard enzyme assay mixture contained 100 mM Mes / NaOH (pH 6.5), 50 mM CaCl2, 0.5 mM SDS, 20 μl substrate (0.4 μmol PC), 1% ethanol and 20 μl PLD in a total volume of 200 μl (14). The assay mixture was then incubated at 30 ° C for 30 minutes in a water bath. The reaction was interrupted by adding 750 μl of chloroform: methanol (1: 2). Chloroform (200 μl) was added to the mixture followed by 200 μl of KCl (2 M). After shaking with vortex, the chloroform and the aqueous phases were separated by centrifugation at 12,000 g for 5 minutes.The chloroform phase was collected and dried.The dried samples were dissolved in 50 μl of chloroform before being placed in spots on TLC plate (gel of silica G) The plate was developed with chloroform solvent: methanol: NHOH (65: 35: 5) The lipids on the plates were visualized by exposure to iodine vapor, the spots corresponding to the lipid PE standards. QH, PA and PC were scraped into vials and the quantities were quantified by measuring the phosphorus content as described in Rouser et al. (15) .The PEOH, the product of the transphosphatidylation reaction was used as the indicator of the PLD activity instead of PA, the product of the hydrolytic reaction, since the former is not easily metabolized PLD activity associated with the membrane and the soluble fractions obtained from zucchini tissues Cassava seeds and castor were measured by quantifying the release of radiolabelled PEOH and PA from the PC substrate (10). For this purpose, 0.4 μCi of L-3-phosphatidylcholine, 1,2-dα [14 C] palmitoyl (Amersham (Arlington Heights, IL)) was mixed with 20 μmol of egg PC in chloroform. The conditions of the assay and the separation of the reaction product were the same as already described. The radioactivity in PEOH, PA and PC scraped from the TLC was quantified by scintillation spectroscopy.

Example Id: Treatment of LPE and production of ethylene in fruit The post-harvest treatment of fruit tissues with LPEegg (which is purified from egg and consists mainly of 16: 0 LPE and 18: 0 LPE) was found previously, it delays aging and improves the shelf life of fruits (3, 16). However, the impact of the different acyl chains of the LPE on the aging of the fruit has not been investigated. In the current study, complementary to the effect of the acyl chains other than the LPE on the activity of the PLD, the inventors investigated the effect of the different acyl chains of the LPE on the ethylene production of the blueberry fruits. The fully ripened blueberry fruits (Vaccinium macrocarpon Ait.? Stevens') were harvested during the autumn season and kept in a cold room. Cranberry fruits after harvest, randomly selected (5 fruits per sample) were immersed in LPE solutions with different acyl chains (100 μM) for 30 minutes, then air-dried and left at room temperature (26 ± 2 ° C) C). After two days, the fruits were incubated in a covered glass jar for 24 hours to measure the ethylene production. The ethylene was quantified with a gas chromatograph equipped with a flame ionization detector (Shimadzu 9AM, Shimadzu Corporation, Kyoto, Japan) (3).

Example: Effect of lysophospholipids on the chlorophyll content of the leaves To evaluate the relative efficacy of the different species of lysophospholipids in the retardation of aging in the leaves, treatment solutions containing lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), LPE , LPI or water were applied to the leaves. The chlorophyll content of each sample was measured by normal methods (10) after an aging of 8 days. The relative chlorophyll contents were expressed as the percentage of the control. 4 Example lf: Results and discussion _ _ _ _ __ Inhibition of PLD activity by LPE with 5 different acyl chains The inventors studied whether the LPE, a phospholipid found in nature, acts as a biologically mediated lipid active inhibiting the activity of the PLD in vitro in a specific form. The inhibitory effects of LPE inhibitors on partially purified pumpkin PLD were tested using PC as a substrate. The activity of PLD was inhibited by LPE with different acyl chains at concentrations of 40 and 200 μM (Figure 1) . The degree of inhibition increased with length and establishment of the acyl chains. The LPE with an acyl chain of 18: 1 was the most effective inhibitor among the species tested and the activity of the resulting PLD was 16% and 11% gt of the control at the LPE concentrations of 40 and 200 μM, respectively. On the other hand, the LPE 14: 0, which is rare Once present in plant tissues, it had very little effect. The effects of LPE with other acyl chains including 18: 2 and 18: 3 would be interesting to try but these forms of LPE are not available in commerce today. A drastic inhibition of PLD by LPE (18: 1) compared With other LPE molecules tested, it suggests that a specific configuration of LPE is necessary for this inhibitory effect.

Structural specificity of LPE (18: 1) for its inhibition of PLD The effect of different components of the LPE molecules on PLD activity was tested to determine if any structural specificity for the inhibition of LPE was necessary. The main group (ethanolamine) and the acyl chain (fatty acid 18: 1) by themselves had no inhibitory effect on the activity of PLD (Figure 2). These results indicate that only the intact LPE molecule is capable of inhibiting PLD, and a loss of any structural component gives rise to complete inefficiency, thus indicating its structural specificity. In fact, phosphatidylethanolamine (PE) had some simulating effect on the activity of PLD. In the presence of 2 μM of PE, the activity of the PLD was 126% of the control (Figure 2). Given that the PE itself is a preferential substrate of the PLD (17), the increase in the activity of the PLD could be explained by its direct stimulating effect on PLD and / or a preferential hydrolysis of PE by PLD.

Dependence on the dose and inhibition kinetics of PLD by LPE (18: 1) The inhibition of PLD by LPE was dose dependent (Figure 3). The LPE (18: 1) showed a drastic inhibitory effect at the concentration of 10 μM, giving rise to 50% of the control activity and a gradual increase of the inhibition with increasing concentrations up to 200 μM. The LPE concentrations of 10 and 200 μM reflect 0.5 and 10 mol% of total phospholipid in the reaction mixtures, respectively. To characterize the inhibition of PLD, the effects of substrate concentration on the inhibition of PLD were analyzed in the presence and absence of LPE (Figure 4). The normal conditions of the assay use the saturation concentration of the substrate (2 mM PC). The inhibitory effect of LPE (18: 1) is still maintained at the substrate concentration of 4 mM (Figure 4). The apparent Km for PLD was 1.7 mM and did not change in the presence of LPE However, the presence of LPE (18: 1) gave rise to a drastic decrease in Vmax (2.9 μmol min -i mg-i protein), compared to the control (Vmax of 20.0 μmol min -1 mg-1). of protein). These results suggest non-competitive inhibition of PLD by LPE.

In situ inhibition of PLD by LPE (18: 1) _ _ _ Since PLD is present not only in a soluble form in the cytosol but also in a form associated with the membrane, the inventors determined the in situ inhibition of LPE on the membrane-associated PLD extracted from pumpkin and seed leaves. castor The specific activities of membrane - associated PLD and soluble pumpkin were decreased to 59% and 51% of control in. presence of LPE (18: 1), respectively (Table 1). The activities of the PLD of castor bean-associated and soluble also decreased to 31% and 30% of the control, respectively, these results indicate "that the activities of PLD associated with membrane and soluble are inhibited by LPE. The PLD associated with the membrane and the soluble fractions was, however, less pronounced than the inhibition of partially purified squash PLD by LPE (Figure 1 and Table 1.) This may be due to the presence of some factors " interferers or the presence of other forms of PLD that are less sensitive to LPE. The partial purification of PLD, therefore, has been suggested as crucial in the characterization of the PLD regulatory mechanism (18). For this reason, in the current study the inventors have used partially purified pumpkin PLD which is commercially available. However, the observed inhibitory effect of LPE on the membrane-associated and soluble PLD extracted from the leaf tissues supports the results obtained with the partially purified PLD.

Table 1. Inhibition of soluble and membrane-associated PLD activities (nmol min -1 mg-1 protein) per LPE (18: 1). The data are average ± SD of the two separate extractions (duplicate experiments for each extraction) prepared from pumpkin and castor leaves.

PLD soluble PLD associated with the membrane Castor Pumpkin Castor Ricino Control 15.2 ± 3.5 10.2 ± 0.1 368.8 ± 6.5 153.8 ± 8.5 LPE 18: 1 23.1 ± 1.6 3.1- + 0.1 217.0 ± 13.0 47.0 + 3.6 (200 μM) Proportion 0.51 0.30 0.59 0.31 (LPE / control) Inhibition of fruit ethylene production by LPE with different acyl chains Previously it was found that LPEegg (egg yolk extract) retards fruit aging as indicated by the reduced rates of ethylene production when compare with the control (3). Since the inventors found that the inhibitory efficacy of LPE on PLD was dependent on the length and unsaturation of the acyl chain of LPE (Figure 1), the effects of LPE with different acyl chains on fruit aging were reviewed. Cranberry fruits were tested with LPE with chain lengths of 14: 0, 16: 0, 18: 0 and 18: 1, and 'the ethylene production of these fruits. The inhibition of ethylene production increased with the length of the acyl chains and the establishment of LPE (Table 2). The LPE (18: 1) gave rise to a greater drastic decrease (40%) in the production of ethylene two days after the treatment. It is interesting to note that this pattern of inhibition of ethylene production by different types of LPE was similar to the pattern of PLD inhibition by different types of LPE (Figure 1). These results indicate that the inhibition of PLD activity and ethylene production depends in a manner consistent with the. length of the acyl chains and the installation of LPE. These results suggest that LPE (18: 1) is superior to the other LPET species reviewed to inhibit PLD and retard fruit aging.

Table 2. Inhibition of ethylene production in blueberry fruits by LPE (100 μM) with different acyl chains. The values are average + SD of three replicas.

Control LPE14: 0 LPE16: 0 LPE18: 0 LPE18: 1 Ethylene 1.78 ± 0.38 1.78 ± 0.11 1.65 ± 0.05 1.27 ± 0.05 1.06 ± 0.13% relative 100 100.0 92.7 71.3 59.6 Structurally selective regulation of PLD P ° r lysophospholipids To check whether PLD inhibition can occur across a broad range of lysophospholipids, the inhibitory effect of LPE on PLD was compared for inhibition by related lysophospholipids present in plant cells (Figure 5). Lysophosphatidylcholine (hereinafter referred to as "LPC"), lysophosphatidylglycerol (hereinafter referred to as "LPG") and lysophosphatidylserine (hereinafter referred to as "LPS") did not significantly affect the activity of PLD. However, the LPI showed inhibitory effects somewhat similar to those of LPE. Whereas, lysophosphatidic acid (hereinafter referred to as "LPA") significantly increased the activity of PLD (Figure 5). For example, at a concentration of 200 μM of LPI and LPA, the activity of the PLD was 31% and 169% of the control, respectively. The only synthetic lysophospholipid tested in Figure 5 was LPA. The other lysophospholipids were from natural sources containing mainly 16: 0 or 18: 0 fatty acids. In addition to LPA (16: 0) (Figure 5). The inventors also reviewed LPA (18: 1) and found similar results of the two types of APL. At present, LPE but not LPC had a strong inhibitory effect on PLD (Figure 5). These results indicate that the regulatory effect of the individual lysophospholipids on the PLD enzyme is very specific and structurally selective. In addition to LPE, the results suggest that LPI can also be a lipid mediator to slow aging in plants.

Delayed leaf aging by LPI The chlorophyll content of the senescent leaves treated with LPI was found much higher than in leaves treated with LPA, LPC or LPEegg (Figure 6). This result indicates that LPI is particularly effective in delaying the senescence of the leaves. In the summary of Example 1, according to the knowledge of the inventors, this is the first study showing a specific inhibitory regulation of PLD by LPE (18: 1) and LPL, which directly targets the activity of the PLD enzyme. This is an important finding since there are no known specific inhibitors of PLD in plants and animals (19). It has also been shown that the treatment of fruit plants with LPE (18: 1) reduces the production of ethylene more than the LPE species with shorter lengths of acyl chains or a higher degree of saturation. Because ethylene and PLD are associated with plant aging, it is reasonably expected that LPE (18: 1) and LPI will be particularly effective in retarding the aging of fruits and plant tissues compared to other species of plants. LPE EXAMPLE 2: Delay of aging and improvement of storage life of the flowers Spikes in dragonfly bloom (Antirrhinum majus L. cv. Potomac White) were harvested and obtained from a commercial grower overnight (20). Upon receipt, the ends of the stems of the ears were cut out under distilled water and allowed to rehydrate for two hours. After rehydration, the ears were cut to a length of 40 cm and the leaves were separated from the lower 18 cm of the spike. This prevented the leaves from becoming a source of bacteria and fungal contamination in the vase. All spikes were then combined and randomly selected for treatment. LPE (18: 1), LPI and LPEegg were prepared in distilled water. Sonification was used to facilitate the dissolution of LPE and other lysophospholipids in the water. For the treatment with LPE, the cut ends of the spikes were kept for 24 hours in a solution of LPE at different concentrations. After, these were transferred to distilled water and kept in this water for three weeks. The spikes were observed for the opening of the flower buds and also for the symptoms of aging (wilting and brown color). If the "neck" of the flower withered, the flower was considered non-commercial. It was considered a commercial spike as long as it remained turgid (not withered) and when more than 50% of the flowers remained healthy. At the end of the study, the water content of the leaves of the spike was determined as an indicator of the pomposity and health of the leaves by measuring the ratio of fresh weight to dry weight. As seen later in Table 3, treatment with LPEegg (purified egg LPE) could retard aging compared to the control; in the first 37-52% of the spikes showed wilting while in the control 76% of them withered after 7 days of treatment. The treatment with LPE (18: 1) and LPI was particularly effective in increasing the life_ in the vase of dragonberry flowers; only 30-39% and 15-22% of the ears withered, respectively, in these two treatments. The LPE (18: 1) and LPI not only increased the life of the flower in the vase but also improved the sensitivity of the flowers for these lipids. For example, 5 mg / l of LPI and LPE (18: 1) produced more prolongation of the flower's life in the vase than the 25 mg / l of LPEegg, indicating that LPI and LPE (18: 1) are more active among the lysophospholipids to retard aging. Flowers treated with LPI and LPE (18: 1) remain commercial until 13 days while flowers treated with water and flowers treated with LPEegg remain commercial for 4 days and 7 days, respectively. The water content of the LPE leaves (18: 1) and the LPI treated spikes was higher than in the spikes treated with LPEegg and with water 18 days after the treatment. These data are consistent with the improvement in the life of flowers during storage treated with LPE (18: 1) and LPI. These data support the theory that LPE (18: 1) and LPI are superior to LPEegg.

Table 3 Treatment Spikes with Life water content of flowers withered leaves after spikes after 7 days 18 days (vase weight (% of total) fresh / dry weight) (days) ** Control Prom. ± DE * Prom. ± DE * (water) 75.5 ± 10.3 5.79 ± 0.18 4 LPE 5 mg / l 52.3 + 9.5 6.77 ± 0.44 10 50.0 ± 15.5 25 36.7 ± 5.55 LPE18: 1 5 mg / l 34.5 ± 5.5 7.81 + 0.22 10 30.0 ± 5.5 12 25 38.7 ± 8.5 LPI 5 mg / l 21.6 ± 7.0 7.57 + 0.52 10 15.0 ± 11.5 13 25 17.8 ± 7.5 The data are mean ± SD of two independent experiments. Each experiment was performed with 12-spikes for the treatment. ** Life in the vase: days when > 50% of the spikes remained commercial.

Carnation spikes in flowering (Dianthus caryophyllus L. cv. White Sim) obtained from a commercial grower were treated with different lipids as described above for dragoncillo. As with the dragonlets, the LPI and LPE (18: 1) at 25 mg / l were superior to LPEegg and the control in prolonging the life of the carnations in the vase (Table 4). LPEsoy (purified LPE from soybeans) also provided better shelf life compared to-LPEegg (purified egg LPE). The LPEsoy consists of 64% unsaturated LPE, such as LPE (18: 1), LPE (18: 2) and LPE (18: 3) and 30% saturated LPE such as LPE (16: 0) and LPE. (18: 0), and 2% LPI (available from Avanti Polar Lipids Inc., Alabaster, Alabama), while LPEegg contains mainly (> 94%) saturated LPE such as LPE (16: 0) and LPE (18: 0) This result supports the conclusion of the inventors that the LPE (18: 1) and LPI are superior to the other species of LPE in prolonging the life of the flowers in the vase.

Table 4 Treatment of commercial flowers after 6 days of treatment (% of total) * average ± SD Control (water) 27.5 ± 3.3 LPE 30.8 + 3.3 LPE18: 1 41.7 ± 8.3 LPI 44.2 ± 8.3 LPE 41.7 ± 8.3 * data are average ± DE of 36 flowers for treatment. (9 flowers / duplicates).

Example 3: Retardation of fruit aging Green, ripe tomato fruits (Lycopersicon esculentum cv H9144) were harvested from three-month-old plants. The harvested fruits were immersed in the lysophospholipid solutions indicated in Table 5 below, at the concentration of 100 mg / l in 1% ethafor 20 minutes. The control tomatoes were immersed in distilled water containing 1% etha After immersing them, the fruits were stored at room temperature for three weeks. The production of ethylene gas was measured 7 days after the treatment. The speed of ethylene production by the fruits increased gradually as the fruits began to mature. Although the green green at 0 days had no ethylene production, untreated control fruits produced ethylene at a rate of 1.26 nl / g.h after 7 days of treatment (see Table 5 below). The fruits treated with LPEegg showed ethylene production similar to the control. Meanwhile, the fruits treated with LPE (18: 1) and LPI showed suppression of the ethylene production, and this rate was only- approximately half of the control, in fruits treated with LPEegg, the suppression of the ethylene production was correlated with the prolongation of the life of the fruits in storage. Consistent with this explanation, the percentages of rotten fruits after three weeks of incubation also indicate that LPE (18: 1) and LPI are particularly more effective than LPEegg and "control in_ prolonging the life of tomatoes in storage. that LPJSsoy is better than LPEegg in terms of prolonging the shelf life of fruits (rotten fruits 24% in LPEegg and 15% in LPEsoy).

Table 5 Treatment * Production of rotten fruits ethylene after 3 weeks 7 days (nl / g. H "-1) (% of total) Control 37.2 1.26 ± 0.21 LPE 1.22 ± 0.22 24.4 LPE18: 1 0.70 ± 0.20 7.7 LPI 0.71 ± 0.40 17.5 LPE 0.74 ± 0.10 15.0 * all solutions were prepared at 1% in ethanol (v / v) ** the data are average ± SD of two independent experiments.Each experiment had 9 fruits per treatment.

Example 4: Retardation of leaf aging induced by ethephon Ethephon, also known as Ethrel, (Ethrel is a trademark of Rhone-Poulenc Ag. Co. (Research Triangle Park, NC)) is an aqueous formulation that decomposes to ethylene and is widely used to maximize the yield of ripe tomato fruits in harvesting operations with shallow revision. The present invention demonstrates that LPE (18: 1) is superior to LPEegg and other lysophospholipids in the protection of leaves from leaf aging induced by ethephon. Tomato plants cv. H9144 were grown in a greenhouse for two and a half months to serve as a source of leaf samples. The plants were sprayed to runoff with ethephon at 1000 mg / L with ethephon plus mixtures of lysophospholipids as shown below in Table 6. Lysophospholipid solutions at 50 mg / L were prepared in 1% (v / v) ethanol and mixed with ethephon (1000 mg / l). The control plants were sprayed with ethephon alone in 1% (v / v) ethanol. The aging of the treated leaves was quantified 10 to 14 days after the treatment by measuring the content of chlorophyll and protein. The tissue of the leaf sprayed with ethephon showed drastic loss in the content of chlorophyll and protein as shown in Table 6 below. LPEegg significantly retarded ethephon induced aging. The LPE (18: 1) showed much better retardation of leaf aging caused by ethephon. LP1 had a little retarding effect on leaf aging induced by ethephon. These results demonstrate that LPE (18: 1) works even better than other forms of LPE for this purpose.

Table 6 Treatment * Content of chlorophyll content (mg / g protein (mg / g dry weight) dry weight) average + SD ** average ± SD ** + 3.65 ± 0.25 58.8 ± 14.7 E + LPE 5.88 ± 2.04 81.9 ± 17.6 E + LPE18: 1 8.40 ± 2.70 100.0 + 20.0 E + LPI 4.12 ± 0.55 60.0 ± 14.7 * all solutions were prepared in 1% ethanol (v / v) ** the data are average ± SD of three independent experiments. Data were collected 10 to 14 days after treatment.

Example 5: Improvement of fruit ripening This experiment was carried out to compare the effects of LPEegg, LPE (18: 1) and LPI on the ripening of the fruits. Tomato plants cv. H9478 were grown in pots for two and a half months under fluorescent lights. Whole plants having about 10% of their fruits in the maturation stage were sprayed with a solution containing 100 mg / l of different lysophospholipids such as LPEegg, LPE (18: 1) or LPI. All solutions contained 1% ethanol and 0.05% sylgard 309 (Dow Corning Co., Midland, MI) as activating agents. The control plants received distilled water containing 1% ethanol and 0.05% sylgard 309. The fruits were harvested 10 days after treatment and graduated in green, red and red (indicating complete maturation). The LPEegg significantly improved the ripening of the fruits compared to the control as described above in US Patents Nos. 5,126,155 and 5,110,341 (see Table 7). However, LPI and LPE (18: 1) were more effective than LPEegg. LPI and LPE (18: 1) also improved fruit stability by prolonging storage life of post-harvested fruits compared to control and LPEegg (see Table 7).

Table 7 Treatment * In the collection Three weeks Red green partial red after harvest% in weight of total Soft fruits (non-commercial) Control% in 33.2 13.2 53.6 47.8 weight of total LPE 22.8 15.9 61.3 36.6 LPE18: 1 22.9 11.7 65.4 33.0 LPI 18.3 11.7 70.1 25.6 * all solutions were prepared in ethanol at 1% (v / v) and 0.05 and [sic] sylgard 309. Spray applications were made 10 days before collection. ** the data are average representing three independent experiments.

REFERENCES Borochov, A., Halevy, A.H. & Shinitzky, M. (1982) Plant Physiol. 69, 296-299. Fobel, M "Lynch, D.V. & Thompson, J.E. (1987) Plant Physiol. 85, 204-211. 3. Farag, K.M. & Palta, J.P. (1993) Physiol. Plant. 87, 515-524. 4. Paliyath, G., Lynch, D.V. & Thompson, J.E. (1987) Physiol. Plant. 71, 503-511. Thompson, J.E., Paliyath, G., Brown, J.H. & Duxbury, C.L. (1987) in Plant Senescence: Its Biochemistry and Physiology, eds. • 10 Thompson, W.W. & Northnagel, E.A., Huffaker, R.C. (The American Society of Plant Physiologists, Rockville, MD), pp. 146-155. Cheour, F., Arul, J., Makhlouf, J. & Willemot, C. (1992) Plant Physiol. 100, 1656-1660. 7. McCormac, D.J., Todd, J.F., Paliyath, G. & Thompson, J.E. 15 (1993) Plant Physiol. Biochem. 31, 1-8. 8. Samama, A.M. & Pearce, R.S. (1993) /. Exp. Bot. 44, 1253-1265. Voisine, R., Vezine, L.-P. & Willemot, C. (1993) Plant Physiol. 102, 213-218. 10. Ryu, S.B. & Wang, X. (1995) Plant Physiol. 108, 713-719. 20 11. Abousaiham, A., Riviere, M., Teissere, M. & Verger, R. (1993) Biochim. Biophys. Acta 1158, 1-7. 12. Lee, J.E. & Choi, M.U. (1996) Bull. Korean Chem. §oc. 17, 905-908. 13. Ryu, S.B. & Wang, X. (1996) Biochim. Biophys. Minutes 1303, 25 243-250. 14. Ryu, S.B. Zheng, L. & Wang, X. (1996) J. Am. Oil Chem. Soc. 73, 1171-1176. 15. Rouser, G., Fleisher, S. & Yamamoto, A. (1970) Lipids 5, 494-496. 16. Farag, K.M. & Palta, J.P. (1993) HortTechnology 3, 62-65. 17. * Dyer, J.H., Ryu, S.B. & Wang, X. (1994) Plant Physiol. 105, 715-724. 18. Kim, J.H., Suh, Y.J., Lee, t.G., Kim, Y., Bae, S.S., Kim, M.J., Lambeth, J.D., Suh, P.-G & Ryu, S.H. (1996) J. Biol. Chem. 271, 25213-25219. 19. Ryu, S.B., Karlsson, B.H. , Ozgen, M.J. Palta J.P. (1997) Proc. Natt. Acad. SCT. USA 94, 12717-12721. 20. Kaur, M.N. & Palta, J.P. (1997) HortScience. 32, 888-890. fifteen

Claims (13)

1. A method for delaying aging in fruits and other plant tissues, the method comprises the step of applying to the fruit and other plant tissues a composition containing an activating compound and a lysophospholipid having the formula: where Ri is selected from the group consisting of acryloxy group of Cs-C22 and C5-C22 alkoxy; R2 is selected from the group consisting of hydrogen, hydroxyl, C1-C5 acyloxy group and C1-C5 alkoxy; and R3 is selected from the group consisting of hydrogen, choline, ethanolamine, glycerol, inositol and serine, wherein Ri and R are interchangeable.

2. The method of claim 1, wherein the lysophospholipid is lysophosphatidylinositol and / or lysophosphatidylethanolamine (18: 1).

3. The method of claim 1, wherein the composition is an aqueous solution.

4. The method of claim 1, wherein the composition is applied before or after harvesting.

5. The method of claim 1, wherein the composition contains an effective amount of lysophospholipid to retard aging in fruits and other plant tissues.

6. The method of claim 5, wherein the composition contains from about 0.5 to about 1000 mg per liter of lysophospholipid.

The method of claim 1, wherein the activating agent is ethanol, tergitol or sylgard 309.

8. A method for improving ripening and fruit stability, the method comprises the step of applying before harvest to all plant tissues a composition containing an activating compound and a lysophospholipid having the formula: where Ri is selected from the group consisting of acyloxy group of Cs ~ C2 and C5-C22 alkoxy; R2 is selected from the group of hydrogen, hydroxyl, C1-C5 acyloxy group and C1-C5 alkoxy; and R3 is selected from the group consisting of hydrogen, choline, ethanolamine, glycerol, inositol and serine, wherein Ri and R2 are interchangeable.

9. The method of claim 8, wherein the lysophospholipid is lysophosphatidylinositol and / or lysophosphatidylethanolamine (18: 1). The method of claim 8, wherein the composition is an aqueous solution. The method of claim 8, wherein the composition contains an effective amount of lysophospholipid to improve the maturation and stability of the fruits. The method of claim 11, wherein the composition contains from about 0.5 to about 1000 mg per liter of lysophospholipid. The method of claim 8, wherein the activating agent is ethanol, tergitol or sylgard 309.