Planta (Berl.) 85, 299--302 (1969)
Short Communication
Sugar Transport: Occurrence of Trehalase Activity in Sugar Cane K . T. GLASZIOU ])avid North Plant Research Centre, Indooroopilly, Queensland K . R. GAYLnl~ Botany Department, University of Queensland, St. Lucia Received January 3, 1969
Summary. Trehalase activity was detected in extracts of roots, leaves, and stalk tissue from sugar cane. The enzyme was not bound to cell particulates, and had a pH optimum of 6.2 and a Michaelis constant for trehalose of 1 X 10-a M. The level of enzyme detected in mature stalk tissue was too low to account for glucose transport into tissue slices. The enzyme level was high in immature stalk tissue in which the vacuolar sugar pool turns over rapidly. Trehalose synthesis and breakdown may be part of a system for transport of hexose out from the vacuole. I t has been suggested t h a t glucose t r a n s p o r t from t h e g l o m e r u l a r f i l t r a t e in m a m m a l i a n k i d n e y tubules, a n d also across t h e i n t e s t i n a l mucosa is m e d i a t e d b y a n enzyme s y s t e m b y which trehalose p h o s p h a t e is f o r m e d a n d h y d r o l y s e d , firstly to trehalose ( l - ~ - D - g l u c o p y r a n o s y l ~-D-glucopyranoside), t h e n b y trehalose to glucose (SACKTO~, 1968; G~OSSMAN a n d SACKTOR, 1968). Trehalose was shown to be m e m b r a n e b o u n d , with a p i t o p t i m u m of 6.3, a n d a Michaelis c o n s t a n t for t r e h a lose of 3.5 • 10 -3 M. I n sugar-cane (Saccharum o//icinarum L.) stalks, sucrose is h y d r o lysed to glucose a n d fructose in t h e cell wall zone of storage p a r e n e h y m a cells, thence t r a n s p o r t e d into a m e t a b o h c c o m p a r t m e n t where sucrose p h o s p h a t e is formed, t r a n s p o r t e d across t h e v a c u o l a r m e m b r a n e , a n d a c c u m u l a t e d as sucrose in t h e vacuole (SAcI~I~ et al., 1963; IIAwK~g a n d HATCH, 1965). R e t u r n of sucrose from storage in t h e vacuoles of i m m a t u r e p a r e n c h y m a cells is p r e c e d e d b y h y d r o l y s i s b y v a c u o l a r invertase. Glucose a n d fructose m o v e r e a d i l y i n w a r d s t h r o u g h t h e cell m e m b r a n e a n d o u t w a r d s from t h e v a c u o l a r m e m b r a n e (SAckER et al., 1963), b u t t h e m e c h a n i s m of t r a n s p o r t is quite u n k n o w n . W e have investigated trehalase activity in sugar cane in an attempt to specify its possible role in sugar transport.
300
K. T. GLtszmv and K. R. GAYLER:
Materials and Methods Crude Enzyme Preparation. Tissue (10 g) was ground with pestle and mortar and the aid of a little sand in 25 ml 0.1 M sodium phosphate buffer, pH 8.0, including 0.02 M sodium dithionite, then squeezed through muslin and dialysed against distilled water for 24 hr. All operations were at 0 ~ The crude extract was stored at -- 20 ~ There was no significant loss of activity after 7 days storage. Enzyme Localisation. Media used for homogenizing tissue for subsequent fractionation by eentrifugation were either 0.5 M sorbitol, 2 X 10-3 M dithiothreitol, 1 X 10-8 M EDTA and 0.05 M potassium phosphate (pH 7.0), or 0.5 M sodium phosphate (pH 8.0) and 0.02 M sodium dithionite. Centrifugation, resuspension and dialysis of fractions were all carried out at 0 ~ Enzyme Assay. Trehalase was routinely assayed in a system containing 5 ~moles sodium 2-(N-morpholino)ethanesulfonate, pH 6.2, and 1.2 ~moles D-(A-)-trehalose per milliliter reaction mixture. Incubation was at 30 ~ Aliquots of the reaction mixture were added at suitable time intervals to 3 volumes ethanol to stop the reaction, and the glucose content of the ethanolie sample determined, after centrifugation, by the method of PA~K and JohNsoN (1949). Chemicals. All sugars used were obtained from Nutritional Biochemicals Corporation, Los Angeles, California. Th e p H o p t i m u m of 6.2 f o u n d for sugar-cane trehalase was similar to t h e m a m m a l i a n e n z y m e , b u t t h e Michaelis c o n s t a n t of 1.0 x 10 -4 M was an order of m a g n i t u d e lower. A f u r t h e r difference f r o m t h e m a m m a lian e n z y m e was f o u n d a f t e r f r a c t i o n a t i o n of tissue p a r t i c u l a t e s ; t h e sugar-cane e n z y m e was a l m o s t e n t i r e l y in t he s u p e r n a t a n t fraction after c e n t r i f u g a t i o n a t 120,000 X g. W h e n t e s t e d on g-methylglucoside, t h e cane e n z y m e h a d a b o u t 7 % of t h e a c t i v i t y o b s e r v e d on trehalose, a n d no a c t i v i t y was d e t e c t e d on lactose, melibiose, t u r a n o s e a n d cellobiose. Table. Distribution o] trehalase within the sugar-cane plant Tissue
Trehalase activity (~g trehalose hydrolysed hr -1 g fr. wt. -1)
Leaf Immature stalk Semi-mature stalk Mature stalk Root
70 195 4 7 43
As can be seen in t h e Table, trehalase was d e t e c t e d in e x t r a c t s of roots a n d leaves. I n stalk-tissue e x t r a c t s it was p r esen t in high levels in i m m a t u r e , e x p a n d i n g i n t e r n o d e tissue which contains high levels of v a c u o l a r i n v e r t a s e a n d a high r a t e of t u r n o v e r of t h e v a c u o l a r sugar c o n t e n t s ( S A c ~ et al., 1963), b u t was low in m a t u r e st al k tissue which does n o t c o n t a i n v a c u o l a r invertase.
Trehalase Activity in Sugar Cane
301
V a c u o l a r i n v e r t a s e is synthesised when tissue from i m m a t u r e internodes is placed in water, a n d synthesis m a y be repressed b y glucose or i n h i b i t o r s of p r o t e i n a n d I~NA synthesis (GLAszIou et al., 1966; GAYLnR a n d GLASZIOU, 1969). W h e n synthesis is blocked, v a c u o l a r i n v e r t a s e b r e a k s down with a half-life of 2 hr. D u r i n g a 16-hr i n c u b a t i o n of tissue slices in w a t e r w i t h 4-hr sampling i n t e r v a l s t h e v a c u o l a r i n v e r t a s e increased 3-fold, b u t t h e r e was no significant change in t h e level of t r e h a l a s e either in w a t e r or in the presence of glucose (0.11 M) or eycloh e x i m i d e (1 fzg/ml). B o t h these t r e a t m e n t s c o m p l e t e l y blocked the i n v e r t a s e increase. H e n c e t h e t u r n o v e r of trehalase a p p e a r s to be slow, synthesis is n o t s u b j e c t to e n d - p r o d u c t repression b y glucose, a n d t h u s this e n z y m e is d i s t i n c t l y different from the v a c u o l a r i n v e r t a s e a n d its control systems. The r a t e of glucose u p t a k e o b s e r v e d in tissue slices from t h e same sugar-cane v a r i e t y used in the c u r r e n t w o r k was more t h a n 600 ~zg glucose g-~ fresh weight hr -1 for tissue from b o t h i m m a t u r e a n d m a t u r e i n t e r n o d e s (SAcH~R et al., 1963; HAWKER a n d HATCH, 1965). These rates exceed b y factors of 3 to 85 t h e m a x i m u m rates of glucose formation from trehalose so far observed in e x t r a c t s from similar tissues. H e n c e trehalose synthesis a n d h y d r o l y s i s does n o t a p p e a r to h a v e a m a j o r role in glucose u p t a k e as o b s e r v e d in tissue slices. I n pulse-chase e x p e r i m e n t s w i t h tissue slices from i m m a t u r e internodes b a t h e d in glucose, i t was shown t h a t r a d i o a c t i v i t y d i s a p p e a r i n g from s t o r e d sucrose m o v e d t h r o u g h t h e hexoses, a n d thence o u t of t h e sugar pool, i n d i c a t i n g t h e occurrence of a t r a n s p o r t s y s t e m m o v i n g hexoses from t h e vacuole (GLASZIOV, 1961; SACHER et al., 1963). This s y s t e m m a y involve trehalose. If so, trehalase in sugar cane is p r o b a b l y a n u n b o u n d , c y t o p l a s m i c enzyme, a n d enzymes for synthesising trehalose should be f o u n d either in t h e vacuole or t h e v a c u o l a r m e m b r a n e .
References G~u K. R., and K. T. GLASZIOV: Plant enzyme synthesis: Hormonal regulation of invertase and peroxidase synthesis in sugar cane. Planta (Berl.) 83, 185--194 (1969). GLASZIOU,K. T. : Accumulation and transformation of sugars in stalks of sugar cane. Origin of glucose and fructose in the inner space. Plant Physiol. 86, 175--179 (1961). - - J. C. W A L D R O N , and T. A. BULL: Control of invertase synthesis in sugar cane. Loci of auxin and glucose effects. Plant Physiol. 41, 282--288 (1966). GROSSMAI%I. W., and B. SACKTOR: Histochemieal localization of renal trehalase: Demonstration of a tubular site. Science 161, 5 7 1 ~ 7 2 (1968). H A W K E R , J. S., and M. D. HATC~: Mechanism of sugar storage by mature stem tissue of sugar cane. Physiol. Plantarum (Cph.) 18, 4 4 4 ~ 5 3 (1965).
302
K . T . GT,ASZ:[OVand K. R, G~YT,~zl~:Treh~lasc Activity ]n Sugar Cane
PARK, J . T . , and M. ,Y. Jo~,'so~: A sLJt~mi~odcLermhla.t.ion of glne~ose. J. biol. 6..ss.em. 181, 149--15][. (]949). SAc,.Fr,:~, d.A., M. D. HAr and K, T. GLAszIor: Sllgar a.ccumulat.~o~ cyole ia sugar cane. III. PhysieM and meta.bolic aspects of cyc.Ie in i m m a t , r e stor~gc i.igsues, f)lan~ Physiol. ,*I8, 348~-354 (1963). Sa~'• :B.: Trehalase and the t.ra.nsport of glucose in t.he ma.mmali~n lddney and int~tine. Proc. nat. Acad. Sei. (Wash.) 66, 1007--I014 (1968), Dr. :K. T. GLASZIOV Da.vid North Pla.nt. ~esea.rch Cvlltrc lndooroopilly, Qid,, hustr~fli~ 4068
Short Communication
Sugar Transport: Occurrence of Trehalase Activity in Sugar Cane K . T. GLASZIOU ])avid North Plant Research Centre, Indooroopilly, Queensland K . R. GAYLnl~ Botany Department, University of Queensland, St. Lucia Received January 3, 1969
Summary. Trehalase activity was detected in extracts of roots, leaves, and stalk tissue from sugar cane. The enzyme was not bound to cell particulates, and had a pH optimum of 6.2 and a Michaelis constant for trehalose of 1 X 10-a M. The level of enzyme detected in mature stalk tissue was too low to account for glucose transport into tissue slices. The enzyme level was high in immature stalk tissue in which the vacuolar sugar pool turns over rapidly. Trehalose synthesis and breakdown may be part of a system for transport of hexose out from the vacuole. I t has been suggested t h a t glucose t r a n s p o r t from t h e g l o m e r u l a r f i l t r a t e in m a m m a l i a n k i d n e y tubules, a n d also across t h e i n t e s t i n a l mucosa is m e d i a t e d b y a n enzyme s y s t e m b y which trehalose p h o s p h a t e is f o r m e d a n d h y d r o l y s e d , firstly to trehalose ( l - ~ - D - g l u c o p y r a n o s y l ~-D-glucopyranoside), t h e n b y trehalose to glucose (SACKTO~, 1968; G~OSSMAN a n d SACKTOR, 1968). Trehalose was shown to be m e m b r a n e b o u n d , with a p i t o p t i m u m of 6.3, a n d a Michaelis c o n s t a n t for t r e h a lose of 3.5 • 10 -3 M. I n sugar-cane (Saccharum o//icinarum L.) stalks, sucrose is h y d r o lysed to glucose a n d fructose in t h e cell wall zone of storage p a r e n e h y m a cells, thence t r a n s p o r t e d into a m e t a b o h c c o m p a r t m e n t where sucrose p h o s p h a t e is formed, t r a n s p o r t e d across t h e v a c u o l a r m e m b r a n e , a n d a c c u m u l a t e d as sucrose in t h e vacuole (SAcI~I~ et al., 1963; IIAwK~g a n d HATCH, 1965). R e t u r n of sucrose from storage in t h e vacuoles of i m m a t u r e p a r e n c h y m a cells is p r e c e d e d b y h y d r o l y s i s b y v a c u o l a r invertase. Glucose a n d fructose m o v e r e a d i l y i n w a r d s t h r o u g h t h e cell m e m b r a n e a n d o u t w a r d s from t h e v a c u o l a r m e m b r a n e (SAckER et al., 1963), b u t t h e m e c h a n i s m of t r a n s p o r t is quite u n k n o w n . W e have investigated trehalase activity in sugar cane in an attempt to specify its possible role in sugar transport.
300
K. T. GLtszmv and K. R. GAYLER:
Materials and Methods Crude Enzyme Preparation. Tissue (10 g) was ground with pestle and mortar and the aid of a little sand in 25 ml 0.1 M sodium phosphate buffer, pH 8.0, including 0.02 M sodium dithionite, then squeezed through muslin and dialysed against distilled water for 24 hr. All operations were at 0 ~ The crude extract was stored at -- 20 ~ There was no significant loss of activity after 7 days storage. Enzyme Localisation. Media used for homogenizing tissue for subsequent fractionation by eentrifugation were either 0.5 M sorbitol, 2 X 10-3 M dithiothreitol, 1 X 10-8 M EDTA and 0.05 M potassium phosphate (pH 7.0), or 0.5 M sodium phosphate (pH 8.0) and 0.02 M sodium dithionite. Centrifugation, resuspension and dialysis of fractions were all carried out at 0 ~ Enzyme Assay. Trehalase was routinely assayed in a system containing 5 ~moles sodium 2-(N-morpholino)ethanesulfonate, pH 6.2, and 1.2 ~moles D-(A-)-trehalose per milliliter reaction mixture. Incubation was at 30 ~ Aliquots of the reaction mixture were added at suitable time intervals to 3 volumes ethanol to stop the reaction, and the glucose content of the ethanolie sample determined, after centrifugation, by the method of PA~K and JohNsoN (1949). Chemicals. All sugars used were obtained from Nutritional Biochemicals Corporation, Los Angeles, California. Th e p H o p t i m u m of 6.2 f o u n d for sugar-cane trehalase was similar to t h e m a m m a l i a n e n z y m e , b u t t h e Michaelis c o n s t a n t of 1.0 x 10 -4 M was an order of m a g n i t u d e lower. A f u r t h e r difference f r o m t h e m a m m a lian e n z y m e was f o u n d a f t e r f r a c t i o n a t i o n of tissue p a r t i c u l a t e s ; t h e sugar-cane e n z y m e was a l m o s t e n t i r e l y in t he s u p e r n a t a n t fraction after c e n t r i f u g a t i o n a t 120,000 X g. W h e n t e s t e d on g-methylglucoside, t h e cane e n z y m e h a d a b o u t 7 % of t h e a c t i v i t y o b s e r v e d on trehalose, a n d no a c t i v i t y was d e t e c t e d on lactose, melibiose, t u r a n o s e a n d cellobiose. Table. Distribution o] trehalase within the sugar-cane plant Tissue
Trehalase activity (~g trehalose hydrolysed hr -1 g fr. wt. -1)
Leaf Immature stalk Semi-mature stalk Mature stalk Root
70 195 4 7 43
As can be seen in t h e Table, trehalase was d e t e c t e d in e x t r a c t s of roots a n d leaves. I n stalk-tissue e x t r a c t s it was p r esen t in high levels in i m m a t u r e , e x p a n d i n g i n t e r n o d e tissue which contains high levels of v a c u o l a r i n v e r t a s e a n d a high r a t e of t u r n o v e r of t h e v a c u o l a r sugar c o n t e n t s ( S A c ~ et al., 1963), b u t was low in m a t u r e st al k tissue which does n o t c o n t a i n v a c u o l a r invertase.
Trehalase Activity in Sugar Cane
301
V a c u o l a r i n v e r t a s e is synthesised when tissue from i m m a t u r e internodes is placed in water, a n d synthesis m a y be repressed b y glucose or i n h i b i t o r s of p r o t e i n a n d I~NA synthesis (GLAszIou et al., 1966; GAYLnR a n d GLASZIOU, 1969). W h e n synthesis is blocked, v a c u o l a r i n v e r t a s e b r e a k s down with a half-life of 2 hr. D u r i n g a 16-hr i n c u b a t i o n of tissue slices in w a t e r w i t h 4-hr sampling i n t e r v a l s t h e v a c u o l a r i n v e r t a s e increased 3-fold, b u t t h e r e was no significant change in t h e level of t r e h a l a s e either in w a t e r or in the presence of glucose (0.11 M) or eycloh e x i m i d e (1 fzg/ml). B o t h these t r e a t m e n t s c o m p l e t e l y blocked the i n v e r t a s e increase. H e n c e t h e t u r n o v e r of trehalase a p p e a r s to be slow, synthesis is n o t s u b j e c t to e n d - p r o d u c t repression b y glucose, a n d t h u s this e n z y m e is d i s t i n c t l y different from the v a c u o l a r i n v e r t a s e a n d its control systems. The r a t e of glucose u p t a k e o b s e r v e d in tissue slices from t h e same sugar-cane v a r i e t y used in the c u r r e n t w o r k was more t h a n 600 ~zg glucose g-~ fresh weight hr -1 for tissue from b o t h i m m a t u r e a n d m a t u r e i n t e r n o d e s (SAcH~R et al., 1963; HAWKER a n d HATCH, 1965). These rates exceed b y factors of 3 to 85 t h e m a x i m u m rates of glucose formation from trehalose so far observed in e x t r a c t s from similar tissues. H e n c e trehalose synthesis a n d h y d r o l y s i s does n o t a p p e a r to h a v e a m a j o r role in glucose u p t a k e as o b s e r v e d in tissue slices. I n pulse-chase e x p e r i m e n t s w i t h tissue slices from i m m a t u r e internodes b a t h e d in glucose, i t was shown t h a t r a d i o a c t i v i t y d i s a p p e a r i n g from s t o r e d sucrose m o v e d t h r o u g h t h e hexoses, a n d thence o u t of t h e sugar pool, i n d i c a t i n g t h e occurrence of a t r a n s p o r t s y s t e m m o v i n g hexoses from t h e vacuole (GLASZIOV, 1961; SACHER et al., 1963). This s y s t e m m a y involve trehalose. If so, trehalase in sugar cane is p r o b a b l y a n u n b o u n d , c y t o p l a s m i c enzyme, a n d enzymes for synthesising trehalose should be f o u n d either in t h e vacuole or t h e v a c u o l a r m e m b r a n e .
References G~u K. R., and K. T. GLASZIOV: Plant enzyme synthesis: Hormonal regulation of invertase and peroxidase synthesis in sugar cane. Planta (Berl.) 83, 185--194 (1969). GLASZIOU,K. T. : Accumulation and transformation of sugars in stalks of sugar cane. Origin of glucose and fructose in the inner space. Plant Physiol. 86, 175--179 (1961). - - J. C. W A L D R O N , and T. A. BULL: Control of invertase synthesis in sugar cane. Loci of auxin and glucose effects. Plant Physiol. 41, 282--288 (1966). GROSSMAI%I. W., and B. SACKTOR: Histochemieal localization of renal trehalase: Demonstration of a tubular site. Science 161, 5 7 1 ~ 7 2 (1968). H A W K E R , J. S., and M. D. HATC~: Mechanism of sugar storage by mature stem tissue of sugar cane. Physiol. Plantarum (Cph.) 18, 4 4 4 ~ 5 3 (1965).
302
K . T . GT,ASZ:[OVand K. R, G~YT,~zl~:Treh~lasc Activity ]n Sugar Cane
PARK, J . T . , and M. ,Y. Jo~,'so~: A sLJt~mi~odcLermhla.t.ion of glne~ose. J. biol. 6..ss.em. 181, 149--15][. (]949). SAc,.Fr,:~, d.A., M. D. HAr and K, T. GLAszIor: Sllgar a.ccumulat.~o~ cyole ia sugar cane. III. PhysieM and meta.bolic aspects of cyc.Ie in i m m a t , r e stor~gc i.igsues, f)lan~ Physiol. ,*I8, 348~-354 (1963). Sa~'• :B.: Trehalase and the t.ra.nsport of glucose in t.he ma.mmali~n lddney and int~tine. Proc. nat. Acad. Sei. (Wash.) 66, 1007--I014 (1968), Dr. :K. T. GLASZIOV Da.vid North Pla.nt. ~esea.rch Cvlltrc lndooroopilly, Qid,, hustr~fli~ 4068
