Trehalose Toxicity in Cuscuta reflexa1 SUCROSE CONTENT DECREASES IN SHOOT TIPS UPON TREHALOSE FEEDING

Trehalose, an a,a-diglucoside, induced a rapid blackening and death of shoot tips of Cuseuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fail in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from j'4Clglucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as weOl as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding. In a previous paper (13) we reported that the nonreducing disaccharide, a,a-trehalose, induced a rapid blackening of excised shoot tips of Cuscuta reflexa (dodder) cultured in vitro. This effect of trehalose was specific insofar as several mono-, di-, and trisaccharides tested were not toxic and these generally supported good growth of shoot tips. The addition of a utilizable sugar delayed the blackening of the shoot tips. This observation suggested that trehalose may interfere in some metabolic pathway involving sugars. With a view to understanding the mechanism of trehalose toxicity in Cuscuta, we have investigated some aspects of carbohydrate metabolism in shoot tips following trehalose feeding. We have found a decrease in the contents of carbohydrates and, in particular, of sucrose upon trehalose feeding. MATERIALS AND METHODS In Vitro Culture of Cuscuta. The culture medium (6) was the same as that used earlier except that medium lacking sugar is designated as BM.3 Shoot tips of Cuscuta were cultured by two methods. Method I, unless mentioned otherwise, is the same as described earlier (6); 2.5-cm long shoot tips were cut from primed and surface-sterilized tissue and cultured aseptically. In method II, 3.5-cm long shoot tips were marked with indelible ink 2.5 cm below the apex and used without priming and surface-sterilization. ' Supported in part by a research grant from the Department of Science and Technology, Government of India. 2To whom corespondence should be addressed. 3 Abbreviation: BM, basal medium. The tips were placed singly in tubes containing 0.3 ml medium which was changed daily to minimize microbial growth. At the end of the growth period the shoot tips were cut at the mark and the terminal portion assayed for carbohydrates. Extraction and Estimation of Starch and Sugars. Starch was extracted from the tissue by the method of ap Rees et al. (1). An aliquot of the extract was incubated for 3 h at 50°C with amyloglucosidase (0.75 unit) and a-amylase (65 units) in 50 mm sodium acetate buffer (pH 5.6) in a total volume of 1 ml. Glucose formed was estimated by glucose oxidase-peroxidase method (2). Starch content was determined from a standard curve of glucose obtained by the hydrolysis of known amounts of starch by the same procedure. Ethanolic extracts rendered free of lipids, were estimated for total sugars by the method of Yemm and Willis (14) and for reducing sugars by the method of Somogyi (11). Trehalose was estimated using trehalase as described earlier (13). Sucrose in the neutral fraction (13) was estimated using invertase from Candida utilis. For this, an aliquot of the neutral fraction was incubated for 2 h at 40°C with invertase (28 units) in a total volume of 1 ml made up with 50 mm sodium acetate buffer (pH 5.5). The reaction was stopped by immersing the tubes in a boiling water bath and glucose formed was estimated by glucose oxidase-peroxidase method. Sucrose content was calculated by multiplying the glucose value by 1.9. Measurement of Invertase Activity. Cell-free homogenate of shoot tips was prepared as described previously (13). Invertase was assayed by glucose oxidase-peroxidase method after hydrolysis of sucrose. The reaction mixture contained 0.2 ml 200 mm sucrose and a suitable volume ofthe homogenate in a total volume of 1 ml made up with 50 mm sodium acetate buffer (pH 5.6). The mixture was incubated for 20 min at 30°C and the reaction was stopped by placing the tubes in a boiling water bath for 2 min. Protein was estimated by the method of Potty (9). Respiration Measurements. 02 uptake was measured with an Aminco Warburg apparatus by standard procedures. Respiration was also studied by measuring the "CO2 released from [U-'4C1 glucose. Five-mm segments from 5 shoot tips were placed in the main compartment of the Warburg flask containing 2 ml BM. The center well contained 0.3 ml 10% KOH and 0.5 ml 5% TCA was placed in the side-arm. After the addition of 100 ,ul [U-"4C]glucose solution (about 650,000 cpm) the flasks were incubated for 2 h at 30°C on a metabolic shaker. Respiration was terminated by tipping TCA from the side-arm into the main compartment. After 20 min, radioactivity in KOH solution was measured by the method of Madsen (5). Isotope Experiments. For study of trehalose uptake, shoot tips were cultured in tapered tubes containing 0.1 ml medium. [U-'4C] trehalose was added to culture media. At 12-h intervals, radioactivity remaining in the culture medium was determined and the difference between the initial and final radioactivity was taken as a measure of [14C]trehalose taken up by the tissue. 1247 www.plantphysiol.org on July 21, 2017 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 69, 1982