TREHALOSE TOXICITY IN CUSCUTA

a,a-Trehalose induced a rapid blackening of the terminal 2.5-centimeter region of excised Cuscuta relexa Roxb. vine. The incorporation of radioactivity from [I'C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confied to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A3, which promoted elongation growth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxygucose, which has been shown to be a potent inhibitor of ceD wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of ceDl wail polysaccharides, the chief component of which was presumed to be cellulose. The nonreducing diglucoside, a,a-trehalose, has not been found in the angiosperms, although constitutive trehalose-hydrolyzing activity (trehalase, EC 3.2.1.28) has been found in all plants examined (4-6, 14). The ability of plant tissues to utilize trehalose for growth in vitro has been demonstrated (6, 14). We observed a toxic effect of trehalose in an angiospermic plant, Cuscuta reflexa (dodder). This plant had an exceptionally low trehalase activity; as a result, trehalose accumulated in shoot tip cultured in vitro and induced its rapid blackening (14). A striking feature of trehalose toxicity in a cut vine was that blackening occurred only in the region of cell elongation although trehalose accumulated throughout the length of the vine (14). The toxicity symptom in shoot tips was delayed by the addition of a growth promotory sugar to the culture medium (15). These observations suggested that trehalose interferes in a distinctive metabolic process in the growing region of the vine which primarily involves the metabolic pathway of sugars. In this paper we present observations which suggest that trehalose may interfere with the synthesis of cell wall material. MATERIALS AND METHODS Culture Method. The shoot tips of C. reflexa Roxb. were cultured as described by Maheshwari et al. (10). The culture medium lacking sugar is designated as BM.3 Incorporation of ['4C]Glucose. Vines (8 cm) were marked with ink at 2 cm below the apex and placed with their cut ends dipped ' Supported in part by a fellowship from the Council of Scientific and Industrial Research to K. V. and a research grant from the University Grants Commission to S. M. and R. M. ' To whom reprint requests should be addressed. 'Abbreviation: BM, basal medium. in BM or BM + 2% trehalose. At the end of growth period, shoot tips from three identically treated vines were excised at the mark and placed upright in a test tube containing 0.1 ml BM + [U-14C] glucose at 30°C. After the incubation period, the shoot tips were used for analysis. The difference between the initial and the final radioactivity in the medium was taken as the uptake of [14C] glucose. Tissue Fractionation. Shoot tips were extracted three times with 10 ml portions of hot 80% ethanol (v/v). The residue was homogenized in 80%o ethanol, extracted at 80°C for 10 min, centrifuged and the extraction was repeated. All ethanol-extracts were combined. The alcohol-insoluble residue was fractionated by the method of Brett (2) to obtain alkali-soluble and alkali-insoluble fractions. The combined ethanol-extract was evaporated to dryness under reduced pressure between 40 and 45°C. The residue was dissolved in 0.4 ml water, mixed with I ml methanol + 0.5 ml chloroform and transferred to a centrifuge tube. To this, 0.5 ml chloroform and 0.5 ml water were added, vortexed and centrifuged. The lower chloroform phase was considered as the lipid fraction. The upper aqueous fraction was consecutively passed through small columns of Dowex 50 (H + form) and Dowex 1 (HCOOform). The columns were washed with 20 ml of deionized H20 and the eluate was designated as the neutral fraction. The basic fraction was eluted from the Dowex 50 column with 20 ml of 2 N NH40H, whereas the acidic fraction was eluted from the Dowex I column with 20 ml 6 N HCOOH. NH40H from the basic fraction and HCOOH from the acidic fraction were removed over H2SO4 and NaOH, respectively, in a vacuum desiccator. The neutral, acidic, and basic fractions were evaporated to dryness and dissolved in water. Solutions for radioactivity measurements were spotted on 1.8 x 1.8 cm Whatman No. 3 paper, dried, and counted in a toluenebased scintillation fluid containing methanol (13). Chemicals. All biochemicals used were bought from Sigma. [U-_4C]Glucose (114 mCi/mmol) was bought from Bhabha Atomic Research Centre, Trombay, India. [U4C]Trehalose (540 mCi/mmol) was bought from Radiochemical Centre, Amersham, England.