Mitigation of Ethylene-promoted Leaf Senescence by a Natural Lipid, Lysophosphatidylethanolamine

Mitigation of ethylene promoted leaf senescence by lysophosphatidylethanolamine (LPE) was studied. Micropropagated ‘Russet Burbank’ potato (Solanum tuberosum L.,) plantlets were grown on MS media in sterile culture tubes. After 2 weeks of growth, tubes were sealed and ethylene gas was applied to obtain 5 nL·L fi nal concentration in the culture tubes. Observations and measurements were taken two weeks after ethylene injection. Potato plantlets treated with ethylene showed severe leaf senescence symptoms such as epinasty, lack of growth, yellowing and axillary shoot formation. These observations indicate that apical dominance has been lost with ethylene treatment. The same experiment was repeated with different concentrations of LPE in the MS medium. Inclusion of 50 or 100 mg·L of LPE in the medium mitigated the damage normally caused by applied ethylene. Leaves of plantlets exposed simultaneously to LPE and ethylene had signifi cantly higher chlorophyll content and more healthy leaves compared to plantlets grown on medium lacking LPE. Results of this study suggest that LPE may have the potential to retard ethylene-promoted leaf senescence and may mitigate ethylene induced loss in apical dominance of micropropagated potato plantlets. Lipids are known to be important in membrane structure and energy balance. It is now evident that lipids and lipid-derived metabolites also play a role in critical cellular processes. Studies have shown that membrane lipid-based signaling mediated by phospholipases, such as phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD) and their products constitutes a crucial step in plant growth and development (Exton, 1994; Liscovitch and Cantley, 1994; Nishizuka, 1992; Scherer, 1996; Ryu et al., 1997; Wang, 1999). Substances classifi ed among lipids (vitamins and their precursors, and a number of hormones) have intense biological activity. For example, PLA 2 -derived lysophospholipids were found to act as growth regulators that retard senescence of plant tissues (Farag and Palta, 1993; Ryu et al., 1997; Kaur and Palta, 1997; Ozgen et. al., 2005). Interestingly, PLA 2 products such as lysophosphatidylethanolamine (LPE) inhibited the activity of PLD (Ryu et al., 1997), a key enzyme thought to be responsible for membrane lipid breakdown leading to plant senescence (Ryu et al., 1997; Thompson, 1988). Ryu et al. (1997) concluded that lysophospholipids such as LPE serve as signaling molecule in retarding plant senescence. Foliar applications of LPE have also been found to retard senescence of in intact or detached leaves, fl owers, and fruits (Farag and Palta, 1993; Kaur and Palta, 1997; Ozgen et. al., 2005). Similarly, Ozgen and Palta (2002) found that LPE can protect cranberry fruit and foliage from injury by chlorothalonil-based fungicides. To date, no reports are available regarding the effects of LPE application through the root system, particularly in micropropagated plants. In a two-part study, we investigated the potential retardation of ethylene-promoted senescence by LPE using a model system of micropropagated potato plantlets. We aimed to stimulate leaf senescence through exposure to exogenous ethylene and to explore whether LPE in the rooting medium would affect plant response to ethylene. The role of ethylene in inducing leaf senescence has been well documented in many studies (Gepstein and Thimman, 1987; Lewington et al., 1967). Ethylene accelerates senescence in the leaves of most species and promotes processes characteristic of leaf senescence, such as decline in chlorophyll and photosynthesis, reduction in the levels of proteins and starch, and increase in the activities of many hydrolytic enzymes (Abeles et al., 1992; Ferguson and Simon, 1973; Mattoo and Aharoni, 1988). Obvious symptoms of ethylene induced leaf senescence include malformed leaves, thickened stems and leaves, lack of growth (stunting), and epinasty (curling and bending downward of leaves). This study marks the fi rst attempt to explore the inhibitory effects of LPE absorbed through the root system on ethylene action. Materials and Methods Plant material, medium, and growth conditions. MS media was prepared according to Murashige and Skoog (1962), with pH adjusted to 5.6 ± 0.02. Test tubes were then fi lled with 10 mL of media and capped before autoclaving at 132 °C for 15 min. LPE containing culture tubes were prepared by adding sterile LPE solutions with three different concentrations 0, 25, 50 and 100 mg·L when the medium had cooled to about 70 °C. Stem cuttings of potato plantlets were then, micropropagated in these culture tubes. The 2nd and 3rd nodes of one month old micropropagated ‘Russet Burbank’ potato (Solanum tuberosum L.) plantlets grown on MS medium were excised as single node cuttings and returned to sterile culture tubes (20 × 150 mm, Pyrex no. 9820) containing MS medium with or without LPE. LPE derived from egg lecithin was used in our experiments and was obtained from Doosan Serdary Research Lab (Englewood, N.J.). LPE was suspended in water by using a sonicator (Sonic Dismembrator model 550; Fisher Scientifi c, Pittsburgh, Pa.). Leaf senescence was stimulated by the treatment with exogenous ethylene after 2 weeks of growth. Tubes were sealed and ethylene gas was applied to obtain 5 nL·L fi nal concentration in the culture tubes. The study was conducted in two steps: In step 1, the damaging effects of ethylene on the plantlets were observed to confi rm that the expected action of ethylene was evident in our system. In step 2, the potential retardation of leaf senescence by different concentrations of LPE was assessed. Observations were taken on axillary bud growth, leaf chlorophyll content and number of healthy leaves 2 weeks after ethylene injection. Cultures were arranged in a randomized complete block design under continuous light with about 60 μmol·m·s photosynthetic photon fl ux from cool white fl uorescent lamps. Each treatment consisted of 15 plantlets. Experiments were repeated three times and similar results were obtained. Results from one experiment are summarized here. Chlorophyll determination. Leaf disk samples from fi ve plantlets in each of three replications were collected for chlorophyll extraction. Samples were incubated in vials containing 10 mL of absolute methanol. Vials were continuously shaken in a covered water bath at ambient temperature to facilitate chlorophyll extraction. Absorbance was read at 650 and 665 nm and chlorophyll concentration was calculated (25.5 [A650] + 4.0 [A665]) as mg chlorophyll/g fresh weight (Crafts-Brandner et al., 1984; Holden, 1976). Data collection. In step 1 of the experiment, micropropagated potato plantlets grown on regular MS media were incubated in 0 and 5 nL·L ethylene concentrations. The number of axillary buds and healthy (no epinastic appearance) leaves were counted after two weeks of incubation. In stage 2 of the experiment, single node cuttings of potato plantlets were grown on different concentrations of LPE containing media. After two weeks of growth, these plantlets were incubated in 5 nL·L ethylene concentrations. Two weeks later, the number of axillary shoots and healthy leaves were counted and chlorophyll content of leaves was determined. HORTSCIENCE 40(5):1166–1167. 2005. Received for publication 29 Nov. 2004. Accepted for publication 8 Mar. 2005. Former graduate research assistant. Current address: Department of Horticulture, University of Gaziosmanpasa, Tasliciftlik Tokat, 60240, Turkey. Professor. To whom reprint request should be addressed; e-mail [email protected]. AugustBook.indb 1166 6/14/05 12:07:23 PM 1167 HORTSCIENCE VOL. 40(5) AUGUST 2005 Results Effect of ethylene on leaf senescence. Exposure to ethylene in step 1 of the experiment signifi cantly affected potato plantlets in the culture tubes (Table 1). Potato plantlets treated with 5 nL·L ethylene showed severe leaf senescence symptoms including thickened stems and leaves, axillary shoot growth, lack of growth, yellowing and epinasty (curling and bending downward of leaves) after two weeks. About 60% of the leaves of each plantlet were damaged by exposure to ethylene. Ethylene application also resulted in formation of an average of 3.7 axillary shoots per plantlet while untreated control plants had only 0.9 axillary shoots per plantlet. Effect of LPE on ethylene-promoted leaf senescence. Incorporation of LPE into the rooting medium mitigated ethylene-promoted leaf senescence in micropropagated potato plantlets (Table 2). Plantlets grown in medium containing 50 or 100 mg·L LPE had showed signifi cantly higher chlorophyll content compared to control and plant grown on medium containing 25 mg·L LPE, after 2 weeks of ethylene exposure. Also, these plantlets had signifi cantly more healthy leaves and less axillary shoot formation compared to plantlets grown in medium lacking LPE. Plantlets grown on medium containing 50 mg·L LPE had three times less axillary shoot formation, more than twice the number of healthy leaves and 98% higher chlorophyll content in their leaves than plantlets grown on medium lacking LPE but exposed to ethylene.