Relationship Between Invertase Enzyme Activities and Sucrose Accumulation in Sugarcane (Saccharum spp.)

Transcription

1 Ksetsrt J. (Nt. Sci.) 48 : (214) Reltionship Between Invertse Enzyme Activities nd Sucrose Accumultion in Sugrcne (Scchrum spp.) Bootsrin Tn 1,2, Sontichi Chnprme 1,2,3, Nongluck Tienseree 1,2,3 nd Sukuntros Tdkittisrn 4, * ABSTRACT The sucrose content, nd the ctivities of sugr metolism enzymes nmely, solule cid invertse (SAI), neutrl invertse (NI) nd cell wll invertse (CWI), of vrious developmentl stges of the stem nd the reltionships mong them were investigted in cultivted sugrcne (Scchrum officinrum L.), line nd wild sugrcne S. spontneum, line S Stems of oth sugrcne species 12 mth fter plnting (MAP) stems were collected nd exmined with smples consisting of three prts of the internode the top, middle nd ottom representing reltively immture, mturing nd mture internodes, respectively. The results showed tht sucrose ws the min sugr in the two species. Sugrcne stems of the two species reveled different ptterns of sucrose ccumultion nd ctivities of invertse (SAI, CWI nd NI). The sucrose content of line incresed from to 12 MAP nd ws highest in the ottom prt nd decresed in the middle nd top prts, respectively, wheres the sucrose content of line S decresed from to 12 MAP nd ws highest in the middle prt thn in the lower the ottom nd top prts. The ctivities of SAI, CWI nd NI in oth sugrcne species were higher in the top prt t ech growth stge, lower in middle nd lowest in the ottom prt of the stem. These ctivities decresed from to 12 MAP with line , wheres the ctivities incresed with incresing time fter plnting for line S There ws significntly negtive correltion etween the sucrose content nd the ctivity of SAI, CWI nd NI for line With line S98-244, the lowest concentrtion of sucrose ws significntly negtive only with SAI ctivity. The sucrose content correltion with CWI nd with NI ctivity showed negtive correltions ut they were not significnt. Keywords: solule cid invertse, neutrl invertse, cell wll invertse, sucrose, sugrcne INTRODUCTION Sugrcne (Scchrum officinrum L.) is the most importnt economic crop for the sugr industry, with pproximtely 5 7% of the sugr in the world derived from sugrcne (Crson nd Both, 22; Lkshmnn et l., 25). In Thilnd, ccording to Office of the Cne nd Sugr Bord (213), the plnted re of sugrcne in 213/214 ws 1.52 million h with n verge production of 1 million t of crushed stem with n verge commercil cne sugr (CCS) sugr 1 Center for Agriculturl Biotechnology, Ksetsrt University, Kmpheng Sen Cmpus, Nkhon Pthom 7314, Thilnd. 2 Center for Agriculturl Biotechnology (AG-BIO/ PERDO-CHE), Bngkok 19, Thilnd. 3 Deprtmnt. of Agronomy, Fculty. of Agriculture, Ksetsrt University, Kmpheng Sen Cmpus Nkhon Pthom 7314, Thilnd. 4 Ksetsrt Agriculturl nd Agro-Industril Product Improvement Institute (KAPI) Ksetsrt University, Chtuchk, Bngkok 19, Thilnd. * Corresponding uthor e-mil: psrt@ku.c.th Received dte : 17/7/14 Accepted dte : /11/14

2 87 Ksetsrt J. (Nt. Sci.) 48() percentge (sweetness) of 11.4%, which equtes to n verge sugr recovery of 1.25 kg.t -1 or n nnul mount of 1 million t of sugr. The CCS content is rther low when compred with its competitors such s Austrli nd Brzil where the CCS content is out 12 13% (Simturkit, 214). One of the importnt fctors cusing low sugr recovery is the limited numer of vrieties with the ility to ccumulte high sucrose levels nd this in turn is dependent on the rtio of sucrose synthesis to sucrose rek down (Office of the Cne nd Sugr Bord, 213). Sucrose is primry product which is synthesized y the photosynthesis in the photosynthetic tissue of the lef mesophyll nd is trnslocted to storge prenchym cells in the stem (Hlford et l., 21). Sucrose ccumultion in the sugrcne stem commences fter vegettive growth hs finished (Moore et l., 1997; Btt et l., 211). Sucrose ccumultion in the stem cn rech up to 2% of the dry weight or 27% of the fresh weight (Bull nd Glsziou, 193; Schdev et l., 211). The sugr recovery percentge is minly determined y the ccumultion of sucrose in the stem (Btt et l., 211). The ility of sucrose ccumultion in the sugrcne stem depends on the ility of sucrose synthesis nd sucrose rek down y the plnt itself nd the sucrose clevge in sugrcne is ctlyzed y two enzymes (sucrose synthse nd invertse) tht cleve the sucrose to hexose (Strum nd Tng, 1999). Invertse (et-fructofurnosidse, E.C ) is the key enzyme involved in sucrose metolism, y hydrolyzing sucrose to glucose nd fructose s n energy source for cell growth nd elongtion nd other metolic processes (Strum, 1999; Roitsch nd Gonzlez, 24). Invertse is clssified, y sucellulr loction, optimum ph, soluility nd the isoelectric point, into three types of invertse nmely, vcuolr invertse or solule cid invertse (SAI), cell wll invertse (CWI) nd cytoplsmic invertse or neutrl invertse (NI) (Quick nd Schffer, 199; Tymowsk nd Kreis, 1998; Link et l., 24; Chndr et l., 212). In sugrcne, sugr in the form of sucrose is trnslocted through the phloem to the sinks, where it is used for cell growth, metolism, respirtion or storge (Re et l., 25; Ayre, 211; Wng et l., 213). Severl different physiologicl functions hve een proposed for invertses, tht is, to provide growing tissue with hexoses s source of energy, to generte sucrose concentrtion grdient nd to prtition sucrose etween source nd sink tissues, s well s to id sucrose trnsport (Rees, 1974; Eschrich, 198; Chndr et l., 212.). The im of this reserch ws to investigte the sucrose ccumultion pttern, the ctivities of different invertses nd the reltionship of the sucrose ccumultion nd invertse ctivities t different stges of development nd in different prts of the stem of cultivted sugrcne line nd the wild species. MATERIALS AND METHODS Plnt mterils Sugrcne (Scchrum officinrum L.) line (high sucrose ccumultion) nd wild sugrcne (S. spontneum) line S (low sucrose ccumultion) were plnted. All culturl prctices undertken followed the recommendtions of Deprtment of Agriculture (29). Sugrcne stems were collected t, 8, 1 nd 12 mth fter plnting (MAP) corresponding to the internodl lte elongtion, the ripening nd the mturity stges of crop development. The stem smples were lwys collected erly in the morning t 9 hours to minimize the diurnl vritions in the enzyme ctivity nd sugr level. The stems were cut into three equl prts nmely, top, middle nd ottom representing the reltively immture, mturing nd mture storge tissues, respectively (Btt et l., 22; Joshi et l., 213). The smples were peeled nd cut into smll pieces nd frozen in liquid nitrogen for lter use. The first experiment involved sucrose content nlysis using high performnce liquid chromtogrphy

3 Ksetsrt J. (Nt. Sci.) 48() 871 (HPLC) nd the second experiment consisted of enzyme ctivity ssy for SAI, NI nd CWI. Sugr extrction nd determintion Sugr ws extrcted using the protocol of Lu et l. (211). The frozen tissue smples (1 2 g) were ground to powder in liquid nitrogen. Then, 1 ml of 8% ethnol ws dded nd incuted t 8 C in wter th for 3 min. The smples were centrifuged t 12, revolutions per minute (rpm) t 4 C for 2 min. The superntnt ws removed nd the volume ws recorded; the residues were extrcted with 1 ml of 8% ethnol y incuting t 8 C in wter th for 3 min nd centrifuging t 12, rpm for 2 min. The superntnt ws removed nd comined with the first superntnt nd recorded s the finl volume. All the ethnol ws removed from the superntnt y incuting t 95 C in wter th for min. The sucrose content ws determined using HPLC with reflective index detector set t 8 C nd n Aminex HPX-87P column (3 7.8 mm), the moile phse ws deionized wter t flow rte of. ml.min -1. Enzyme extrction nd ssy Enzymes were extrcted using the protocol of Lontom et l. (28). Frozen tissue smples, ech 1 2 g, were ground to powder in liquid nitrogen. Solule enzymes were extrcted in 8 ml of extrction uffer I ( mm (4-(2- Hydroxyethyl)piperzine-1-ethnesulfonic cid sodium slt, [HEPES-NOH] t ph 7.5,.5% (weight per volume, w/v) Triton-X, 5 mm Nethylenediminetetrcetic cid (EDTA), 5 mm dithiothreitol (DTT) nd 1 mm MgCl 2 ). The cell wll residue ws removed y centrifugtion t 12, rpm t 4 C for 2 min. The superntnt volume ws recorded. The solule enzymes were concentrted using cetone precipittion. The pellet ws re-soluilized in 3 µl of extrction uffer II ( mm HEPES-NOH t ph 7.5,.5 mm, N-EDTA, 5 mm MgCl 2 nd 2.5 mm DTT); these smples were prepred for SAI nd NI ssy, respectively. Insolule enzyme ws soluilized y incuting the cell wll residue in 2 ml of extrction uffer III (1 mm HEPES-NOH t ph 7.5,.5% (w/v) Triton-X, 5 mm N-EDTA, 1 mm MgCl 2, 5 mm DTT nd 1 M NCl) overnight t 4 C; this smple ws prepred for CWI ssy. SAI, NI nd CWI were ech ssyed in finl volume of 1 µl of rection mixture tht consisted of 25 µl of mm N-citrte uffer t ph 3.5 for CWI ctivity ssy, or 25 µl of mm N-citrte t ph 5. for SAI ctivity ssy or 25 µl of mm potssium phosphte uffer t ph 7.5 for NI ctivity ssy, nd lso µl of 125 mm sucrose nd 25 µl of smple, with n incution temperture of 37 o C. The rections were stopped t nd 2 min y 1 µl of lkline regent followed y oiling for 15 min nd then keeping on ice for 3 min. The reducing sugr products were determined using Nelson s rsenomolydte regent method (Nelson, 1944). The totl solule protein ws determined using Lowry s method (Lowry et l., 1951). One unit of invertse enzyme ws defined s the mount of enzyme used to hydrolyse sucrose to reducing sugr t 1 nmol. min -1. Sttisticl nlysis A fctoril completely rndomized design ws used for ech experiment. Correltions etween the sucrose content nd enzyme ctivity were determined using Person s correltion using the R-Progrm for Windows, version (R Core Tem, 214). RESULTS AND DISCUSSION Sucrose content The sucrose contents in different prts of the internode tht is, the top (immture) reltive to the middle (mturing) nd ottom (mture) t different ges fter plnting in oth species were different (Figure 1). In cultivted sugrcne line , the verge sucrose

4 872 Ksetsrt J. (Nt. Sci.) 48() content ( mg g -1 fresh weight, fr.wt.) ws 2-fold higher thn tht of the wild sugrcne, line S98-244, (2.1 mg.g -1 fr.wt.). For line , the sucrose content incresed with incresing months fter plnting (MAP) from MAP (7.81 mg.g -1 fr.wt.) to 12 MAP (153.1 mg.g -1 fr.wt). However, the sucrose content of every prt did not significntly increse fter 8 MAP. In ech prt of the internode, the sucrose content in the ottom prt ( mg g -1 fr.wt.) ws not significntly different from the middle prt (14.42 mg g -1 fr.wt.) ut ws significntly greter thn tht of the top prt (88. mg g -1 fr.wt.). In line S98-244, the sucrose content incresed grdully from MAP (74.33 mg g -1 fr.wt.) to 8 MAP nd fter this period, it significntly decresed until 12 MAP (44.99 mg g -1 fr.wt.). The sucrose content ws highest in the middle internode prt (74.75 mg g -1 fr.wt.) nd lowest in the top prt (51.8 mg g -1 fr.wt.), prticulrly t 1 nd 12 MAP. The sucrose content ws not significntly different etween the middle nd ottom prts (Figure 1). As expected, the sucrose content in line (high-sucrose ccumultion) ws higher thn in line S (low-sucrose ccumultion). The increse in the sucrose content with ge in line nd the decrese in S oserved in the current experiment were similr to those reported y Verm et l. (211) where the sucrose content in high sucrose-ccumulting cultivr incresed during the period 24 to 39 DAP ut decresed in low sucrose-ccumulting cultivrs. Furthermore, Btt et l. (211) found tht the sucrose content in the stem incresed grdully with ge from 44. to 1.5 to mg g -1 fr.wt t stem elongtion, stem ripening nd mturity, respectively. Gutierrez-Miceli et l. (22) lso found tht the sucrose content in the stem chnged over time depending on the vriety. Invertse ctivity The ctivities of SAI in different prts of the internode nd t different ges fter plnting in oth species were different (Figure 2). In S98-244, the verge enzyme ctivity ws 2. fold (4.48 nmol min -1 mg -1 protein) higher thn tht of line (18.12 nmol min -1 mg -1 protein). With line , the SAI ctivity grdully decresed with incresing time fter plnting from MAP to 12 MAP. In ech prt of the internode, the men SAI ctivity of the middle prt (22.5 nmol min -1 mg -1 protein) ws not significntly different from 2 2 Top Middle Bottom Top Middle Bottom 2 2 c Months fter plnting Months fter plnting Figure 1 Sucrose content mesured on fresh weight (fr.wt.) sis in top, middle nd ottom prts of sugrcne stems t different months fter plnting of: () Cultivted sugrcne line ; nd () Wild sugrcne line S Error rs represent men ± SE of three replictes; mong sets of dt t ech time fter plnting, different lowercse letters indicte significnt difference t P <.5.

5 Ksetsrt J. (Nt. Sci.) 48() 873 the top prt (21.12 nmol min -1 mg-1 protein) ut ws significntly greter thn the ottom prt (1.8 nmol min -1 mg-1 protein) except t 1 MAP when the middle prt hd the lowest SAI ctivity. With line S98-244, the men SAI ctivity incresed with incresing months fter plnting from MAP (8.39 nmol min -1 mg-1 protein) to 12 MAP (75.88 nmol min -1 mg-1 protein) nd ws gretest in the top prt (88.51 nmol min - 1 mg -1 protein) nd lower in the middle nd the ottom prts (42.21 nd 8.72 nmol min-1 mg-1 protein, respectively). The greter SAI ctivity in the immture internode (top) compred with the mture internode (ottom) in oth cultivted lines ws in greement with Verm et l. (211) who reported tht the SAI ctivity ws higher in the immture internode compred to the mture internode. The SAI ctivity decresed with incresed DAP indicting tht decresed SAI ctivity ws correlted with mturing sink tissue (Kuo et l., 21; Verm et l., 211). The SAI my ply role in the remoiliztion of stored sucrose from the vcuole (Scher et l., 193) nd its ctivity ws high in the elongtion phse. Venktrmn et l. (1991) found tht the SAI ctivity ws high when growth of sugrcne ws rpid nd SAI ctivity ws high, usully in tissue tht ws rpidly growing during cne growth nd development, such s the immture internode (Htch nd Glsziou, 193). The ctivities of cell wll invertse (CWI) in different prts of the internode nd t different ges fter plnting in oth sugrcne lines were different (Figure 3). The verge CWI ctivity of line (48.47 nmol min-1 mg-1 protein) ws out 2-fold higher thn tht of line S (24.33 nmol min -1 mg-1 protein). With line , the men CWI ctivity decresed with incresed period fter plnting from MAP (131.9 nmol min -1 mg-1 protein) to 12 MAP (8.21 nmol min -1 mg-1 protein). The CWI ctivity of the top internode prt (.1 nmol min -1 mg -1 protein) ws not significntly different from the middle prt (57.94 nmol min -1 mg-1 protein) ut it ws generlly greter in the ottom prt (33.9 nmol min -1 mg-1 protein). With line S98-244, the men CWI ctivity incresed from MAP (12.53 nmol min -1 mg-1 protein) to 12 MAP (4.43 nmol min -1 mg-1 protein) nd ws gretest in the top prt (39.95 nmol min -1 mg-1 protein) nd decresed in the middle nd ottom prts (27.4 nd 5.4 nmol min -1 mg-1 protein, respectively), SAI ctivity (nmol. min -1. mg -1 protein) 2 2 Top Middle Bottom Top Middle Bottom SAI ctivity (nmol. min -1. mg -1 protein) Months fter plnting Months fter plnting Figure 2 Solule cid invertse (SAI) ctivity in top, middle nd ottom prts of sugrcne stems t different months fter plnting of: () Cultivted sugrcne line ; nd () Wild sugrcne line S Error rs represent men ± SE of three replictes; mong sets of dt t ech time fter plnting, different lowercse letters indicte significnt difference t P <.5.

6 874 Ksetsrt J. (Nt. Sci.) 48() prticulrly t 1 nd 12 MAP (Figure 3). The results for line were in ccordnt with Btt et l.(28) in tht the mximum CWI occurred t the stem elongtion stge (175 DAP) nd declined therefter in ech prt of stem. CWI is loclized in the poplst, is ioniclly linked to the cell wll nd the ctivity remins mesurle during different stges of plnt development (Xu et l., 199). Vorster nd Both (1998) lso referred to n poplstic solule form of cid invertse tht ws elieved to e present in sugrcne. The cell-wll-ssocited isoform function is the hydrolysis of sucrose to glucose nd fructose in the poplst, yielding monoscchrides tht re ctively trnsported cross the prenchym plsmlemm (Roitsch nd Gonzlez, 24). The neutrl invertse (NI) ctivity of different prts of the internode t different months fter plnting etween line nd line S were not significntly different (Figure 4). With line , the NI ctivity decresed from MAP (74.1 nmol min -1 mg-1 protein) to 12 MAP (2.95 nmol min -1 mg-1 protein). In ech prt of the internode, the verge NI ctivity ws greter in the top prt (59.31nmol min-1 mg-1 protein) nd lower in the middle (34.47 nmol min - 1 mg -1 protein) nd lowest in the ottom prt (18.55 nmol min -1 mg-1 protein). Similr results were reported y Dendsy et l. (1995) nd Rossouw et l. (21) nmely, tht NI ctivity ws low in the mture storge tissue of mny high-sugr storing, erly-mturing vrieties of sugrcne. With line S98-244, the men NI ctivity t different cne growth stges ws reltively stle t MAP (3.8 nmol min -1 mg-1 protein) to 8 MAP (33.77 nmol min -1 mg -1 protein) nd ws highest t 1 MAP (47.35 nmol min-1 mg-1 protein) nd less t 12 MAP (42.34 nmol min -1 mg -1 protein). In ech prt of the internode, the men NI ctivity ws greter in the top prt (88.81 nmol min -1 mg-1 protein), lower in the middle prt (27.17 nmol min -1 mg -1 protein) nd lowest in the ottom prt (5.4 nmol min -1 mg-1 protein) s shown in Figure 4. Zhu et l. (1997) suggested tht the highest SAI ctivity ws ssocited with low levels of sucrose in immture stem internodes nd, conversely, the lowest levels of SAI ctivity were ssocited with high levels of sucrose in mture internodes. Siswoyo et l. (27) found negtive nd significnt correltion etween NI nd SAI with sucrose ccumultion. CWI ctivity (nmol. min -1. mg -1 protein) 2 2 Top Middle Bottom Top Middle Bottom c c CWI ctivity (nmol. min -1. mg -1 protein) c c Months fter plnting Months fter plnting Figure 3 Activities of cell wll invertse (CWI) in top, middle nd ottom prts of sugrcne stems t different months fter plnting of: () Cultivted sugrcne line ; nd () Wild sugrcne line S Error rs represent men ± SE of three replictes; mong sets of dt t ech time fter plnting, the sme lowercse letter indicte no significnt difference t P <.5.

7 Ksetsrt J. (Nt. Sci.) 48() 875 NI ctivity (nmol. min -1. mg -1 protein) 2 2 Top Middle Bottom Top Middle Bottom 1 1 NI ctivity (nmol. min -1. mg -1 protein) Months fter plnting Months fter plnting Figure 4 Activities of neutrl invertse (NI) in top, middle nd ottom stem prts of sugrcne stems t different months fter plnting of: () Cultivted sugrcne line ; nd () Wild sugrcne line S Error rs represent men ± SE of three replictes; mong sets of dt t ech time fter plnting, different lowercse letters indicte significnt difference t P <.5. Reltionship of invertse ctivity to sucrose content There ws significntly negtive correltion in oth sugrcne lines etween the sucrose content nd invertse enzyme ctivity (Figure 5). As the plnt mtured, the invertse enzyme ctivity incresed ut the sucrose content decresed. Plotting the ctivity of the different enzymes versus the concentrtion of sucrose gve significntly negtive correltion for SAI (correltion coefficient, r = , P <.5), CWI (r = -.952, P <.1) nd NI (r = , P <.1) with line Similrly, with line S98-244, the concentrtion of sucrose gve significntly negtive correltion for SAI (r = , P <.5); however, the concentrtion of sucrose ws not significntly different compred to CWI (r = , P >.5) nd NI (r = , P >.5). The results of the experiments showed tht the ccumultion of sucrose in the stem ws low, where the ctivity of SAI ws high. SAI ctivity is usully high in tissues tht re rpidly growing in the immture stem internode (Zhu et l., 1997; Gutierrez-Miceli et l., 22). Lontom et l. (28) showed tht the sucrose content of sugrcne during ripening ws negtively correlted with the SAI ctivity in two studied cultivrs (r = nd -.3, respectively), nd Pn et l. (29) found negtive correltion for the sucrose content with oth the SAI nd NI ctivity. The results of the present study indicted tht the SAI ctivity ws lowest in the sugrcne cultivr line with high sugr ccumultion nd ws highest in the sugrcne line with low sugr ccumultion. This my indicte the importnt role of SAI with regrd to the totl sugr content of the sugrcne cultivr s M et l. (2) stted tht SAI hd negtive correltion with nd ws importnt in the ccumultion of sucrose. CONCLUSION In cultivted sugrcne line , the verge sucrose content ws higher thn tht of wild sugrcne in ll stem segment types regrdless of the month fter plnting. There ws negtive nd generlly significnt correltion etween the sucrose content nd enzyme ctivity (SAI, CWI nd NI) in oth sugrcne species. As the plnt ws mturing, the invertse enzyme ctivity incresed

8 87 Ksetsrt J. (Nt. Sci.) 48() ut the sucrose content decresed. In the cultivted line , the ctivities of SAI, CWI nd NI decresed with incresing of months fter plnting nd decresed with incresing mturtion of the internode. With line S98-244, the enzyme ctivity incresed with incresing time fter plnting nd the ctivity levels of SAI, CWI nd NI were significntly nd negtively correlted with sucrose ccumultion. Although, the ctivities of SAI, CWI nd NI for line S were negtively correlted with sucrose ccumultion, only SAI ws significnt. The dt from these studies supported the hypothesis tht invertse enzymes, especilly SAI, ply key role in determining the sucrose concentrtion during ripening. The results lso provide references for further reserch on regulting the sucrose ccumultion in the sugrcne stlk t the moleculr level y = x+15. r = P <.5 * y = -.27x+71.3 r = P <.5 * 1 d 2 SAI ctivity (nmol. min -1. mg -1 protein) SAI ctivity (nmol. min -1. mg -1 protein) y = -.551x+. r = P <.1 *** y = -.34x+7.88 r = P >.5 ns 2 4 e 8 1 CWI ctivity (nmol. min -1. mg -1 protein) CWI ctivity (nmol. min -1. mg -1 protein) y = -.94x r = P <.1 *** 1 NI ctivity (nmol. min -1. mg -1 protein) c 2 y = -.228x+71.1 r = P >.5 ns Figure 5 Reltionship etween sucrose content nd specific enzyme ctivity of solule cid invertse (SAI), cell wll invertse (CWI) nd neutrl invertse (NI) in sugrcne line (, nd c, respectively) nd line S (d, e nd f, respectively). (r = Correltion coefficient; * nd *** = Significnt t the P <.5 nd P <.1 levels of proility, respectively; ns = Not significnt) NI ctivity (nmol. min -1. mg -1 protein) f

9 Ksetsrt J. (Nt. Sci.) 48() 877 ACKNOWLEDGEMENTS This reserch ws prtilly supported y the Center for Agriculturl Biotechnology, Postgrdute Eduction nd Reserch Development Office, Commission on Higher Eduction, Ministry of Eduction nd the Ksetsrt University Reserch nd Development Institute (KURDI), Ksetsrt University, Bngkok, Thilnd. LITERATURE CITED Ayre, B.G Memrne-trnsport systems for sucrose in reltion to whole plnt cron prtitioning. Mol. Plnt. 4: Btt, S.K., S. Kur nd A.P.S. Mnn. 22. Sucrose ccumultion nd mturity ehvior in sugrcne is relted to invertse ctivities under sutropicl conditions. Sugrcne Int Btt, S.K., N.C. Pnt, K.S. Thind nd S.K. Uppl. 28. Sucrose ccumultion nd expression of enzyme ctivities in erly nd mid-lte mturing sugrcne genotypes. Sugr Tech. 1(4): Btt, S.K., B. Kur, J.S. Sitl, S.K. Sndhu nd S.K. Uppl.211. Sucrose ccumultion nd internodl solule invertse isoenzymes in plnt nd rtoon crops of sugrcne. Sugr Tech. 13(1): Bull, T.A. nd K.T. Glsziou The evolutionry significnce of sugr ccumultion in Scchrum. Aust J. Biol. Sci. 1: Crson, D.L. nd F.C. Both. 22. Genes expressed in sugrcne mturing internodl tissue. Plnt Cell Rep. 2: Chndr, A., R. Jin nd S. Solomon Complexities of invertse controlling sucrose ccumultion nd retention in sugrcne. Curr. Sci. 12(): Dendsy, J.P.S., P. Singh, A.K. Dhwn nd H.L. Sehtiy Activities of internodl invertses during mturtion of sugrcne stlks. Sugrcne. : Deprtment of Agriculture. 29. Sugrcne Plnting. Deprtment of Agriculture, Bngkok. Thilnd. 12 pp. Eschrich, W Free spce invertse, its possile role in phloem unloding. Ber. Dtsch. Bot. Ges. 93: Gutierrez-Miceli, F.A., M.A. Rodriguez- Mendiol, N.Ocho-Alejo, R. Mendez-Sls, L. Dendooven nd C. Aris- Cstro. 22. Reltionship etween sucrose ccumultion nd ctivities sucrose-phosphthse, sucrose synthse, neutrl invertse nd solule cid invertse in micropropgted sugrcne plnts. Act Physiol. Plnt. 24 (4): Hlford, N.G., T.Y. Curtis, N. Muttucumru, J. Postles nd D.S. Mottrm. 21. Sugr in crop plnts. Ann. Appl. Biol. 158: Htch, M.D. nd K.T. Glsziou Sugr ccumultion cycle in sugrcne. II. Reltionship of invertse ctivity to sugr content nd growth rte in storge tissue of plnts grown in controlled environments. Plnt Physiol. 38: Joshi, J.B., S. Krishnveni, D. Vijylkshmi, R. Sudhgr nd M. Rveedrn Activities of enzyme involved in synthesis nd degrdtion of sucrose in populr sugrcne vrieties. Asin J. Exp. Biol. Sci. 4(2): Kuo, T., I. Hohjo nd S. Hirtsuk. 21. Sucrose ccumultion nd its relted enzyme ctivities in the juice scs of Stsum mndrin fruit from trees of different crop lods. Sci. Hortic. 91: Lkshmnn, P., R.J. Geijskes, K.S. Aitken, C.P.L. Grof, G.D. Bonnett nd G.R. Smith. 25. Sugrcne iotechnology: the chllenges nd opportunities. In Vitro Cell. Dev. Biol. Plnt. 41: Link, M., T. Rusch nd S. Greiner. 24. In Aridopsis thlin, the invertse inhiitors AtC/VIF1 nd 2 exhiit distinct trget enzyme specificities nd expression profiles. FEBS Lett. 573(34):

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