Effect of GA3 and Light on Polysaccharide Levels and Metabolism in Germinating Coffee Seeds

Abstract

The free sugars and polysaccharides extractable from coffee seeds during germination in light or darkness increase greatly when these seeds are treated with gibberellic acid (GA3) which inhibits germination. The major changes are observed in the content of free mannose and mannose containing polysaccharides. The levels of polysaccharidases are also enhanced by GA3 treatment. The possible role of these events in the inhibition of coffee seed germination by GA3 is discussed. INTRODUCTION The germination of coffee seeds is influenced by several factors as storage conditions (Bacchi, 1958), light (Valio, 1976), and endogenous and exogenous levels of hormones (Saenz, 1959; Velasco and Gutierrez, 1974; Valio, 1976). Exogenous gibberellic acid (GA3) inhibits coffee seed germination, the inhibition being greater in seeds placed in continuous light than in darkness (Valio, 1976). With GA3 treatment there is an increase in peroxidase activity, which is associated with the appearance of a peroxidase isoenzyme band that is also detectable in untreated seeds at a later stage of germination (Takaki and Dietrich, 1979a). Recently, Takaki and Dietrich (19796) observed that GA3-treated coffee seeds produced several droplets of exudate on their surface. Analysis of this exudate revealed the presence of large quantities of carbohydrates and amino acids, the former consisting largely of mannose, with traces of glucose, fructose, and galactose. The effects of GA3 and light have been related to the activity of certain hydrolytic enzymes (Sacher, Hatch, and Glasziou, 1963; Brian, 1966; Taiz and Jones, 1970; Carter, Garrard, and West, 1973). Thus the activity of these enzymes and levels of carbohydrates have been examined in coffee seeds given GA3 and/or light treatments. MATERIALS AND METHODS Seed supply and germination Fresh seeds of Coffea arabica L. cv. Mundo Novo were supplied by the Department of Genetics of the Instituto Agronomico in Campinas. Fresh decoated seeds were allowed to imbibe in distilled This content downloaded from 207.46.13.149 on Wed, 28 Sep 2016 04:17:13 UTC All use subject to http://about.jstor.org/terms 1644 Takaki and Dietrich—Polysaccharide Metabolism in Coffee Seeds water or GA3 (0-5 or 1-0 mM) for 48 h and placed to germinate at 30 °C under light (5400 lx) or darkness in Petri dishes lined with filter paper moistened with water (Valio, 1976). Extraction and analysis of carbohydrates Batches of 30 seeds at different periods of germination were homogenized and extracted with 70% (v/v) ethanol under reflux for 60 min in a proportion of 1:7 (by vol.) (fraction 1). The residue after centrifugation was extracted according to the method of Matheson and Saini (1977), yielding the following polysaccharide fractions: soluble in cold 10 mM HgCl2 (fraction 2); soluble in hot 0-5% (w/v) NH4-oxalate (fraction 3); soluble in 5% (w/v) NaOH (fraction 4). Total carbohydrates were estimated in each fraction by the anthrone method (Umbreit, Burris, and Stauffer, 1964). Reducing sugars were determined directly in fraction 1 and after hydrolysis in the other three fractions according to Nelson (1944). Fructose was analysed in fraction 1 according to Roe (1934). The polysaccharides were hydrolysed with 2 N HC1 for 5 h at 100 °C in sealed vials. The ethanolic extracts (fractions 1) as well as the polysaccharide hydrolysates were chromatographed on paper using butan-l-olipyridine:H20 (6:4:3, by vol.) as solvent system A (Chargafif, Levine, and Green, 1948) and ethyl acetate : pyridine : acetic acid :H20 (60:15 :15:10, by vol.) as system B (Jarvis and Duncan, 1974). Aliquots of equivalent amounts of seed extracts for each period and each treatment were used for chromatography. The reducing sugars were stained with alkaline silver nitrate reagent (Trevelyan, Procter, and Harrison, 1950) and the intensity of the spots was measured with a EEL scanning densitometer. Free fructose and combined fructose (sucrose and raffinose) were detected with the p-anisidine reagent (Lewis, Chen, Woods, and Culpin, 1972). Extraction and assay of enzymes Random samples of 30 seeds were taken at different periods of germination for each treatment. Acetone powders of seeds were prepared according to Oelshlegel and Stahmann (1973). The enzyme extractions were based in the method described by McCleary and Matheson (1974), 1 of powder being used for each extraction. The enzymic extracts were incubated with the following substrates: laminaran (Koch Light), coffee seed galactomannan (obtained according to McLeary and Matheson, 1974), carboxymethyl cellulose (Koch Light). The incubation conditions were those described as optimal for these enzymes (Taiz and Jones, 1970; Beaugiraud and Percheron, 1968; Maxemiuc and Dietrich, 1973, respectively). Each incubation mixture contained 5 mg of the substrate, sodium acetate buffer of the desired ionin strength and molarity, and enzyme solution equivatent to 1 mg protein (determined according to Lowry, Rosebrough, Farr, and Randall, 1951). After the appropriate period of incubation the products were measured by reducing sugar determination (Nelson, 1944) and the products characterized by paper chromatography as descri bed above.