Lipids of Senescent Leaf Tissue Induced by Inhibition of Synthesis and Acceleration of Breakdown

Tissues were made senescent by inhibiting synthetic reactions with abscisic acid and by accelerating protein catabolism with L-serine in combination with kinetin. Squash (Cucurbita maxima) leaf discs were floated on the above-mentioned solutions, harvested, and the lipids extracted therefrom. Fatty acids of the acyl lipids were determined by gas chromatography, galactolipid concentrations were determined by sugar analyses, and phospholipid concentrations were determined by phosphorus analyses. All of the lipids were separated by thin-layer chromatography prior to analysis. Those tissues floated on abscisic acid or L-serine with kinetin contained less chlorophyll, less total fatty acids — especially linolenic acid, less glycolipid material, and less phospholipid material. Abscisic acid caused a decline in all of the cellular lipids as indicated by acyl group analysis and by analysis of the parental lipids. Possible effects of these compounds on chloroplast thylakoid stacking are discussed. OHIOJ . SCI. 83 (1): 50-54, 1983 INTRODUCTION molecules and other molecules decrease The relative amounts of many biological during t h i s P h a s e o f development. The compounds change in tissues that are unh P l d P r o f l l e o f photosynthetic tissue is dergoing senescence. Some of the macrodependent upon the developmental state of the tissue (Newman et al. 1973). In this study we investigated the lipids of photo'Manuscript received 8 December 1981 and in synthetic tissues that had been treated to revised form 25 March 1982 (#81-49). either enhance protein breakdown or to inJ. r l ti t f i l i l i ti t t r i . f t r 1 anuscri t recei e r 81 revised form 25 arch 1982 (#81-49). during this phase of develop ent. he lipid profile of photosynthetic tissue is dependent upon the develop ental state of the tissue ( e an et al. 1973). In this study e investigated the lipids of photosynthetic tissues that had been treated to either enhance protein breakdown or to inOhioJ. Sci. LIPIDS OF SENESCENT LEAF TISSUE 51 hibit both DNA and RNA synthesis and consequently protein synthesis. It is assumed that ABA causes an inhibition of protein synthetic reactions as well as other effects (Thimann 1980) and that the application of L-serine with kinetin causes increased protein breakdown (Martin and Thimann 1972). The lipid profiles of the treated and nontreated tissues were then compared. Martin and Thimann (1972) suggested that L-serine is incorporated into the active center of a protease which in turn results in a vigorous promotion of senescence. METHODS AND MATERIALS Blue Hubbard Squash (Cucurbita maxima Duchesne) was grown 5 wk under continuous light (13,000 lux; using Sylvania Cool White bulbs) at room temperature (ca 25 C). The first and second nodal leaves were surface sterilized with 10% Clorox; 1 cm leaf discs were removed. Forty leaf discs were placed on 25 ml of either water or on aqueous solutions of 3.78 X 10" M ABA, 50 X 10~ M L-serine, or L-serine plus 2.32 X 10" M kinetin. The discs were incubated 6 days in continuous light. The discs then were boiled 3 min in chloroform-methanol (2:1, v/v) and homogenized in a ground-glass homogenizer. The sample was filtered, and a portion of the filtrate was removed for chlorophyll analysis. Three replications of each treatment were analyzed for the various components. The chlorophyll analysis was done according to Arnon (1949). The chloroform-methanol was evaporated, and the sample was redissolved in 80% acetone. Absorbancies were read at 645 and 663 nm. The remaining portion of each sample was washed 3 times with acidified water (2 drops 5 N H2SO4 in 70 ml water). The chloroform phase was then dried with anhydrous Na2SC>4 and stored under N2 at 0 C. A portion of the sample was used for twodimensional thin-layer chromatography (Gurr and James 1971). The first dimension was developed in chloroform-methanol-7 N ammonia (65:25:4, v/v), and the second dimension was developed in chloroform-methanol-acetic acid-water (170:15: 15:2, v/v). Lipophilic spots on the thin-layer plates were visualized with I2 vapors and identified by standards and by comparison with results found in the literature (Gurr and James 1971). The spots were eluted from the plates and analyzed for phosphorus and galactose content. The phosphorus analysis was done according to Marinetti (1962). The galactose analysis consisted of heating the sample at 100 C in 2 ml of 1 N H2SO4 for 1 hr, then adding 12 ml of cold 98% H2SO4, and heating for an additional 5 min. The absorbance was measured at 322 nm. A third portion of the sample was used for fatty acid analysis. The samples were transesterified with methanolic-HCl (2.5% HC1, w/w) under reflux for 1.5 hr in an atmosphere of N2 (Kates 1964). The methyl esters were purified by passing the sample through silicic acid columns. The fatty acid methyl esters were resolved by gas chromatography on a 2.5-m column of 10% DEGS (diethylene glycol succinate) on acid-washed Chromosorb W. Separations were made isothermally at 185 C; the chromatograph contained a thermal conducitivity detector. RESULTS AND DISCUSSION From the time the leaf discs were removed to the time of harvest, the chlorophyll concentration in the leaf discs of those tissues floated on water alone decreased 47%. The changes in the chlorophyll content after floating on the various solutions for 6 days are given in table 1. Leaf tissue exposed to 2.32 X 10~M kinetin contained about the same amount of chlorophyll as that exposed to water alone (controls). In contrast, those tissues exposed to either ABA or to L-serine with kinetin contained much less chlorophyll. Serine alone (without added kinetin) does not cause the marked reduction in leaf chlorophylls (Martin and Thimann 1972). Using the chlorophyll content as an indication of the degree of senescence, both ABA and L-serine with kinetin accelerated the senescence process. When the tissue was harvested after a 6-day exposure period, the ABA-treated tissue was quite yellow (visually) but turgid; whereas the