Protein-level expression and localization of sucrose synthase in the sugarcane culm

Protein-level expression and localization of sucrose synthase in the

sugarcane culm

Wolfgang E. Schafera,*, Johann M. Rohwerb and Frederik C. Bothac

aInstitute for Plant Biotechnology, University of Stellenbosch, Private Bag X1, 7602 Matieland, South AfricabDepartment of Biochemistry, University of Stellenbosch, Private Bag X1, 7602 Matieland, South AfricacSouth African Sugar Experiment Station, 170 Flanders Drive, Private Bag X02, Mount Edgecombe, 4300, South Africa*Corresponding author, e-mail: [email protected]

Received 22 September 2003; revised 11 December 2003

No comprehensive studies on the localization of sucrose

synthase (SuSy, EC 2.4.1.13) in sugarcane internodes havebeen reported. The expression and localization of SuSy in

young (internode 3) to mature (internode 9) internodes of

sugarcane (Saccharum spp. hybrids) var. N19 was investi-gated. Enzyme activity in the top and bottom, as well as the

peripheral and core parts of the internodes suggested that

SuSy is present ubiquitously but that levels can differ signifi-

cantly in different parts of the internodes and with maturity.This was also confirmed by immunohistochemistry, which

showed that both vascular and storage parenchyma tissues

contain SuSy in young and mature internodes. The ratio ofsucrose breakdown to synthesis activity increased approxi-

mately 12-fold from an average of 0.12 in internode three to

1.4 in internode nine. This indicates that different forms ofSuSy are present in young and mature internodes, or that the

ratios of different isoforms differ between young and mature

internodes. Immunoblotting showed that at least one form of

SuSy present in young tissue was absent, or present belowdetection limits, in mature culm tissue.

Introduction

Sucrose synthase (SuSy, UDP-glucose: D-fructose 2 a-D-glucosyltransferase, EC 2.4.1.13), catalysing the reversi-ble conversion of sucrose and UDP to UDP-glucose andfructose, is a central enzyme of carbohydrate metabolismin all plant species. SuSy is implicated in a wide variety ofprocesses, which include nitrogen fixation (Gordon et al.1999), starch synthesis (Chourey et al. 1998, Ricard et al.1998), cellulose biosynthesis (Amor et al. 1995), phloemtransport (Geigenberger et al. 1993, Martin et al. 1993,Nolte and Koch 1993) and the ability of storage organsto act as carbon sinks (Huber and Akazawa 1986, Nolteand Koch 1993, Zrenner et al. 1995). Of particular inter-est is the fact that in almost all plants sucrose is the mainform of translocated carbon, and in addition it is also themain storage carbohydrate in some plants, for examplethe tap root of sugar beet and the mature internodes ofsugarcane (Saccharum species hybrids).

Enzymes of sucrose metabolism are of particular interestin sugarcane as sucrose is the main storage carbohydrateand sugarcane accounts for about 60% of the world’ssucrose production (Grivet and Arruda 2001). In contrastto tomato fruit, where SuSy activity is drastically reducedin mature fruit, mature sugarcane internodes still containappreciable amounts of activity (Botha and Black 2000).SuSy is associated with vascular bundles (Yang andRussel 1990, Tomlinson et al. 1991, Nolte and Koch 1993),and there has been some speculation in the literature onwhether the SuSy activity in mature sugarcane tissues isassociated exclusively with vascular bundles (Buczynskiet al. 1993). It was noted that without tissue printing orstaining data one cannot assume that SuSy is only presentin vascular bundles in mature tissue. Information on thelocalization of SuSy is important for the study of sucroseaccumulation, and for programmes for its improvement.

PHYSIOLOGIA PLANTARUM 121: 187–195. 2004 DOI: 10.1111/j.1399-3054.2004.00316.x

Printed in Denmark – all rights reserved Copyright# Physiologia Plantarum 2004

Abbreviations – SuSy, sucrose synthase.

Physiol. Plant. 121, 2004 187

Metabolic models of sucrose accumulation (Rohwer andBotha 2001) need to take into account enzyme localizationin order to have additional utility. It also needs to bepointed out that localization and expression of enzymesoften depend on the developmental stage of plants andtheir organs. For example, SuSy is phloem-associatedin mature maize leaves (Nolte and Koch 1993) but inyoung leaves this is not the case (Hanggi and Fleming2001). Hence, both structural and kinetic data need tobe incorporated into models, which need to be carefullydefined in order to approximate the system to be modelledas accurately as possible.

Evidence exists that a membrane-associated form ofSuSy could be involved in the biosynthesis of celluloseand callose (Amor et al. 1995). In sugarcane this has notyet been investigated. This aspect is important, because ifthe purpose of plasma membrane associated SuSy is toprovide the UDP-glucose precursor for the synthesis ofglucans (Amor et al. 1995), then only part (maybe verylittle or nothing) of this UDP-glucose would be releasedinto the cytosolic compartment, as the glucan synthasesare membrane associated enzymes (Delmer 1999). Infact, it is possible that SuSy and cellulose synthase mayinteract, resulting in metabolite channelling (Ovadi1991). This hypothesis is supported by the fact thatcellulose synthase contains cytosolic domains that havehigh homology to domains in animal proteins that areknown to be involved in protein–protein interaction(Delmer 1999). The implications are clear: assumingthat all measured SuSy activity is cytosolic potentiallyoverestimates the activity of SuSy in this compartment.Therefore, knowing the partitioning of SuSy betweenmembrane fractions and the cytosol would be useful formetabolic modelling, as well as for providing clues aboutits physiological roles in specific tissues.

The tissue localization of SuSy protein has only beenstudied in a limited number of species. Isoforms of SuSywere preferentially expressed in specific tissues, e.g. rice(Oryza sativa) RSus3 was immunolocalized predominantlyin endosperm cells and therefore is thought to provideprecursors for starch synthesis (Wang et al. 1999). The RSus1and RSus2 isoforms were more widely expressed andwere found in both leaves and roots. The RSus1 isoformwas localized in mesophyll cells in leaves, but in roots itoccurred in the phloem, indicating differences in SuSygene regulation between these organs (Wang et al. 1999).

As SuSy occurs in a variety of organs, tissues andsubcellular locations, clarifying functional relationshipsis difficult. This is especially true where there is a lack oftissue or organ specificity between isoforms, as in thecase of RSus1 and RSus2 in rice. Nevertheless, localiza-tion data and experiments on specific plant organs andSuSy isoforms have provided insight into SuSy func-tions, such as the above-mentioned membrane associa-tion study, while antisense inhibition of a tuber-specificSuSy showed that SuSy activity is highly correlated withthe sink strength of potato (Solanum tuberosum) tubers,with reduced tuber size and starch content in the anti-sense plants (Zrenner et al. 1995). In tomato (Lycopersicon

esculentum) fruit, SuSy activity was found to be highduring the phase of rapid fruit growth, but during thesugar accumulation and ripening stage SuSy activity wasmuch lower (N’tchobo et al. 1999). Significantly, intomato fruit the mechanism of phloem unloadingswitches from symplastic in young, fast-growing fruit toapoplastic in maturing, sugar-accumulating fruit (Ruanand Patrick 1995, N’tchobo et al. 1999). Therefore, theSuSy present in cells surrounding vascular bundles inyoung tomato fruit (Wang et al. 1994) may be involvedin symplastic phloem unloading. The value of bothenzyme activity and localization data to elucidateenzyme function is clear. There are currently no immu-nolocalization data in the literature for sugarcane SuSy.

The aim of this study was to investigate the localiza-tion and expression of SuSy enzyme in young to maturesugarcane internodal tissue. The results show that SuSyis present in both vascular and storage parenchyma tis-sue in young and mature internodes. An increase insucrose breakdown/synthesis ratio occurs with increasinginternode maturity. No membrane association in matureinternodal tissue was evident.

Materials and methods

Materials

Sugarcane (Saccharum spp. hybrids) var. N19 plants thathad been field-grown at the University of Stellenboschexperimental farm were used. Internode 1 was taken asthe internode attached to the leaf with the first exposeddewlap (Van Dillewijn 1952).

Tris buffer, DTT, coupling enzymes and alkalinephosphatase-conjugated goat antirabbit antibody wereobtained from Roche (Basel, Switzerland). UDP-glucosepyrophosphorylase was obtained from Sigma (St. Louis,MO). Merck (Darmstadt, Germany) provided the otherchemicals.

Tissue preparation

Core and peripheral parts of the internodes wereobtained by punching out progressively larger diametercylinders with a cork borer in three steps, starting fromthe centre. The tissue from step two was discarded. Corkborer sizes were chosen so that almost the whole inter-node was used, up to about 3 mm from the edges.

Protein extraction

Tissue was ground to powder in liquid nitrogen andextracted in a 1 : 2 (w/v) ratio of 300 mM Tris-HCl(pH 7.5) buffer containing 10% (v/v) glycerol, 1% (v/v)a-mercapto-ethanol, 2 mM MgCl2, 2 mM EDTA andRoche Complete1 protease inhibitor at the recom-mended concentration. The homogenate was filteredthrough a double-layered nylon cloth, centrifuged at10 000 g for 10 min, and the pellets discarded. The pro-teins in the supernatant were precipitated by adding 25%

188 Physiol. Plant. 121, 2004

(w/v) PEG 6000 and recovered by centrifugation at10 000 g for 10 min. The pellets were re-suspended in asmall volume of 100 mM Tris-HCl (pH 7.5) buffer con-taining 2 mM MgCl2, 2 mM DTT and 2 mM EDTA(buffer A). Glycerol was added to 20% (v/v) and thesamples were then rapidly frozen by submersion in liquidnitrogen, followed by storage at �80�C. Enzyme assaysusing these samples were conducted within 1 month fromthe protein extraction. Enzyme samples treated in thisway lost about 2.5% enzyme activity over a 1-monthperiod under these storage conditions.

For isolation of membrane-bound SuSy the supernatantfrom the first centrifugation step was centrifuged at 10 000 gfor 10 min, the supernatant transferred to clean tubes, andthe centrifugation repeated. This supernatant was ultra-centrifuged for 60 min at 100 000g in order to obtainthe microsomal fraction. The pellet was re-suspendedin a small volume of buffer A containing 5% (v/v)Triton X1001. Samples were frozen in liquid nitrogen andstored at �80�C. Contamination from cytosolic proteinsin the microsomal fraction was assessed by assayingfor pyrophosphate-dependent phosphofructokinase (PFP),which only occurs in the cytosol. The real cytosolic andmicrosomal SuSy activities were calculated as follows:SuSycytosolic (real)¼ SuSycytosolic (measured)� (x1 y)/x andSuSymembrane (real)¼ SuSymembrane (measured) � SuSycytosolic

(measured)� (y/x), where x¼PFP activity in cytosolicfraction and y¼PFP activity in microsomal fraction.The average contamination of microsomal fractionswith cytosolic PFP was about 1.5%.

Enzyme assays

Activity in the sucrose synthesis direction was measured in100 mM Tris-HCl (pH 7.0) buffer (Zeng et al. 1998). Thesucrose formed was measured by the anthrone bindingmethod (Van Handel 1968). Activity in the sucrose break-down direction was measured in an assay containing100 mM Tris-HCl (pH 7.0), 2 mM MgCl2, 2 mM NAD1,1 mM pyrophosphate and appropriate concentrations ofsucrose and UDP. UDP-glucose pyrophosphorylase,phosphoglucomutase and Leuconostoc glucose-6-phos-phate dehydrogenase were added to a final activity of4 U ml�1. NADH production was monitored at 340 nm.

Sucrose breakdown/synthesis ratios were determinedfrom reactions with 320 mM sucrose and 1.5 mM UDPfor the sucrose breakdown reaction, and 10 mM UDP-glucose and fructose in the sucrose synthesis reaction, atzero initial product concentrations in each case.

PFP activity in the direction of fructose-1,6-bisphosphatesynthesis was assayed in buffer containing 50 mM Tris-HCl(pH 7.2), 2 mM MgCl2, 0.1 mM NADH, 5 mM fructose-6-phosphate, 10mM fructose-2,6-bisphosphate, 1 U aldolase,1 U glycerol-3-phosphate dehydrogenase and 10 U triose-phosphate isomerase per reaction. Pyrophosphate wasused to initiate the reaction. Reactions were carried out ina 96-well microtitre plate and NAD1 formation wasmonitored at 340 nm in a Bio-Tek Instruments PowerWaveX spectrophotometer (Bio-Tek, Winooski, VT).

Electrophoresis

SDS-PAGE was performed at room temperature in aBio-Rad Mini-PROTEAN II1 electrophoresis cell(Bio-Rad, Hercules, CA). The separating gel contained7.5% polyacrylamide; the stack gel 4%, with a 37.5 : 1acrylamide/bisacrylamide ratio (Laemmli 1970).

Native PAGE was performed similarly at 4�C, but thegel and buffers did not contain SDS.

Preparation of antigen, immunoblotting and immuno-

inactivation

Leaf roll tissue was ground to powder in liquid nitrogenand extracted, filtered and centrifuged as for the proteinextraction. The proteins in the supernatant were precipi-tated by 80% saturation with ammonium sulphateand recovered by centrifugation at 10 000 g for 10 min.The pellets were re-suspended in 100 mM Tris-HCl(pH 7.5) buffer containing 2 mM MgCl2, 2 mM DTT and2 mM EDTA (Buffer A). The protein extract was thendesalted by passage through an Amersham (AmershamBiosciences, Little Chalfont, Buckinghamshire) PD-10(Sephadex G25) column and the eluant was diluted twicewith buffer A. The desalted extract was applied to a 5-mlAmersham Hi-trap Q anion exchange column that hadpreviously been equilibrated with buffer A. The columnwas eluted at 4�C with a linear KCl gradient at a flowspeed of 1 ml min�1 and fractions containing 20% ormore of maximum activity were pooled. Active fractionsfrom the column were dialysed against buffer A.

Affinity chromatography was also performed at 4�Cusing a 2-ml bed volume of UDP-glucuronic acid agar-ose (Sigma). The sample was circulated through the col-umn for at least five column volumes at 0.5 ml min�1,followed by washing with five column volumes buffer Aand elution with buffer A plus 100 mM UDP-glucose.Active fractions from the column were dialysed againstbuffer A.

The dialysed active fractions from the affinity columnwere used for native gel electrophoresis. The part con-taining SuSy activity (of the two major bands containingSuSy activity, the one with the higher electrophoreticmobility was used) was excised, crushed in liquid nitro-gen and the resulting powder extracted with water. Aftercentrifugation the supernatant was used to immunize arabbit.

Immunoblotting was performed after SDS gel electro-phoresis and transfer to a nitrocellulose membrane(Hybond1-C Extra, Amersham Biosciences) using aBio-Rad Transblot1 SD semi-dry transfer cell and trans-fer buffer [48 mM Tris-HCl (pH 8.6), 39 mM glycine,20% (v/v) methanol, 0.0375% (w/v) SDS]. The mem-brane was blocked for 2 h at room temperature withgentle agitation with a TBST buffer [20 mM Tris-HCl(pH 7.6), 137 mM NaCl, 0.1% (v/v) Tween 20, 3%(w/v) BSA]. Anti-SuSy antiserum was diluted 1 : 2000 inTBST buffer with 3% (w/v) BSA and used to probe themembrane for 1 h. After rinsing and washing three times


for 15 min with TBST buffer, a 2000 times diluted alka-line-phosphatase conjugated goat antirabbit IGG anti-body was added and the membrane incubated for 1 h.The membrane was washed as before and developed witha solution consisting of a Roche NBT/BCIP tablet dis-solved in deionized water. Development was stoppedwith running tap water.

Immuno-inactivation incubation mixtures contained0.1% (w/v) BSA, in 20 mM Tris-HCl (pH 7.5) bufferedsaline (TBS), with day 0 or day 39 serum in a totalvolume of 50 ml. Different serum volumes were compen-sated for with TBS. After addition of crude extract, thecontents were mixed and the tubes incubated at 4�C for45 min. After centrifugation at 13 000 g for 5 min thesupernatants were assayed for SuSy in the sucrose break-down direction with the UDP-glucose pyrophosphory-lase, phosphoglucomutase and Leuconostoc glucose-6-phosphate dehydrogenase coupled assay.

Immunohistochemistry

Cylindrical pieces of tissue were bored out of internodeswith a cork borer. The cylinders were bisected length-ways and left overnight at 4�C in fixing solution, con-sisting of PBS buffer with 2% (w/v) paraformaldehydeand 2 mM DTT. The next day sections about 1 mm thickwere cut by hand from the tissue with a blade. Thesections were rinsed in PBS buffer and then washed for15 min at room temperature with gentle agitation. Sec-tions were then blocked in 100 mM Tris-HCl (pH 7.5)buffer containing 150 mM NaCl, 15 mg ml�1 gelatineand 10 mg ml�1 BSA for 2 h at 37�C with gentle agita-tion. The block buffer was replaced with block buffercontaining anti-SuSy antiserum and pre-immune serumat 1000 times dilution, and sections incubated for 1 h asbefore. Sections were rinsed and then washed three timesfor 15 min in PBS buffer containing 0.5 ml Tween.20 ml�1. Block buffer with 2000 times diluted alkaline-

phosphatase conjugated goat antirabbit IGG antibodywas added and the sections incubated for 1 h as before.Sections were washed as before. For detection, an NBT/BCIP tablet (Roche) dissolved in deionized water contain-ing 10% (w/v) polyvinylalcohol was used. Colour devel-opment was monitored at 5 min intervals until satisfactory(about 1 h). The sections were washed carefully underrunning tap water until all visible traces of detectionsolution was gone. The sections were then stored in tapwater containing 0.1M EDTA to prevent further staining.

The sections were studied with a Nikon Eclipse E400microscope and photographed with a Nikon Coolpix 990digital camera (Nikon, Kawasaki, Kanagawa, Japan).

Protein determinations

Protein concentrations were determined using mouseimmunoglobulin G as a protein standard (Bradford 1976).

Results

SuSy activity in different regions of internodes

The level of SuSy activity in the different parts withineach internode was similar for internodes 5–9. Internode5 had the highest overall activity at an average of40 nmol min�1, whereas the average activity in internodes7 and 9 was 22 and 26 nmol min�1, respectively. Inter-node 3 exhibited the highest differences between differentregions, with the lowest activity in the top core region at6.5 nmol min�1, and the highest activity in the bottomcore region, at 37 nmol min�1 (Fig. 1).

Breakdown/synthesis ratios and SuSy isoforms

The ratio of maximum sucrose breakdown to synthesisactivity increased with internode maturity, from inter-node 3 to internode 9 (Fig. 2). There was a positive

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3TC 3BC 3TP 3BP 5TC 5BC 5TP 5BP 7TC 7BC 7TP 7BP 9TC 9BC 9TP 9BP

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Fig. 1. SuSy activity � SE (n¼ 3) in the sucrose breakdown direction in different parts of young to mature internodes. TC, top core; BC,bottom core; TP, top periphery; BP, bottom periphery. Higher numbers indicate more mature internodes; for clarity, bars are differentlyshaded for different internodes.


correlation between sucrose breakdown/synthesis ratioand sucrose content, with previous experiments, whichhave shown sucrose levels in internode 9, at about 50%of dry mass, to be roughly five times higher than ininternode 3 (Botha and Black 2000). Anion exchangechromatography indicated the presence of at least twoSuSy isoforms in internode 9 (Fig. 3).

Immunological analyses

An immunoblot with crude extracts from internodes 3–9(Fig. 4) using the polyclonal antiserum detected a poly-peptide of about 90 kDa, which is in agreement with thecalculated molecular mass of monomeric sugarcane SuSy(Lingle and Dyer 2001). From the immunoblot it isevident that the antiserum discriminates between thedenatured SuSy polypeptides from the different inter-

nodes. This is shown by the lack of signal for internode9, even though enzyme activity was similar to internode 7(Fig. 1). The SuSy from internode 3 was best detected,whereas internodes 5 and 7 gave similar signals.

All SuSy activity in crude extracts from both theyoung internode 3, and the mature internode 9, wasimmuno-inactivated by a polyclonal antiserum, whereaspre-immune serum had no effect (Fig. 5).

SuSy localization

SuSy protein was present in vascular and storage par-enchyma tissue in young, intermediate and mature inter-nodes (Fig. 6). There was a very low (3% of total)

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3TC 3BC 3TP 3BP 5TC 5BC 5TP 5BP 7TC 7BC 7TP 7BP 9TC 9BC 9TP 9BP

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Fig. 2. Breakdown/synthesis ratios � SE (n¼ 3) in different parts of young to mature internodes. TC, top core; BC, bottom core; TP, top periphery;BP, bottom periphery. Higher numbers indicate more mature internodes; for clarity, bars are differently shaded for different internodes.

Fig. 3. Separation of two SuSy isoforms during chromatography onan anion exchange column using internode 9 tissue. Dashed lineindicates KCl gradient.

Fig. 4. Immunoblot of crude protein extracts from internodes threeto nine. Ten micrograms of protein per lane. Lane 1, int. 3; lane 2,int. 5; lane 3, int. 7; lane 4, int. 9.


amount of SuSy in the microsomal fraction in internode2, but none in internode 9 (Fig. 7).

Discussion

The aim of this study was to compare differences andestablish trends in SuSy enzyme activity, properties and

tissue localization between internodes differing in matur-ity. Internode 3 was chosen as the youngest internodeand internode 9 as the most mature for the activitydeterminations in different parts of the internodes andimmunohistochemistry. It is known that both sucrosecontent and sucrose accumulation rate are very close tomaximum levels by internode 9 of the N19 sugarcanevariety (Botha and Black 2000).

Data indicate that SuSy is present in all regions of youngand mature sugarcane internodes. Sucrose breakdownactivity determinations on core and peripheral regions ofinternodes (Fig. 1) clearly indicated that the enzyme activ-ity is not only present in vascular or storage parenchymatissue. If there had been preferential or exclusive vascularor storage parenchyma localization of SuSy, one wouldhave expected significant differences in activity betweenperipheral and core regions of internodes because of thedistribution of vascular bundles, the occurrence of whichdeclines towards the centre of internodes. It has to bementioned that the vascular bundles on the extreme per-iphery (outer 3 mm) of sugarcane internodes sometimes donot contain phloem (Jacobsen et al. 1992), but this part ofthe internodes was discarded in this study. All vascularbundles in tissue sections stained positive for SuSy. Thebiggest difference in activity was found between the topand bottom core parts of internode 3, in which activity inthe bottom was about six times higher than in the top part.It is proposed that the lower sucrose content in the bottomparts of sugarcane internodes (most evident in very young

Fig. 5. Immuno-inactivation of SuSy in crude extract frominternode 3 (filled circles) and internode 9 (open circles). The twoseparate points to the right indicate activity with 15 ml pre-immuneserum. Activities were measured in the sucrose breakdown directionas described in ‘Materials and methods’.

Fig. 6. Internode immunohistochemistry sections incubated with 1000 times diluted pre-immune serum (columns 1 and 3) and anti-SuSy serum(columns 2 and 4) at 400� magnification. Row 1, internode 3; row 2, internode 5; row 3, internode 9. Columns 1 and 2 show storageparenchyma cells, and columns 3 and 4 show storage parenchyma cells with adjacent vascular bundles. Cell types: PV, pitted vessel; AV,annular vessel; S, sclerenchyma; P, storage parenchyma; Pm, phloem (note large sieve elements and much smaller companion cells).


internodes) indicates a metabolically more active environ-ment in terms of respiratory and growth processes (Roseand Botha 2000). Growth and elongation is shown tooccur mostly in the bottom parts of internodes (Jacobsenet al. 1992). This is consistent with a higher demand forhexoses and precursors for cellulose synthesis in the bot-tom part of the internode, which could both be providedby SuSy. Sucrose content in the bottom of internode 3 isabout half that in the top section, with this difference insucrose content between different parts of the same inter-node the highest among the young, maturing and matureinternodes tested (Rose and Botha 2000).

The SuSy sucrose breakdown/synthesis ratios differedbetween internodes varying in maturity, showing anincreasing trend as internodes mature (Fig. 2). This pointsto the presence of different SuSy isoforms in differentinternodes. Consistent with this is that a polyclonal anti-serum against a purified leaf roll SuSy isoform discrimi-nated between the SuSy polypeptides from internodesdiffering in maturity (Fig. 4), to the point that there wasno signal for internode 9 on the immunoblot (even thoughSuSy sucrose breakdown activity in internode 9 was similarto that in internode 7). However, the antiserum efficientlyimmuno-inactivated all SuSy activity in crude extractsfrom both young and mature internodes, with pre-immuneserum having no effect (Fig. 5). The discrepancy betweenthe fact that the antiserum recognizes native SuSy frominternode 9, but does not detect it when it is bound to anitrocellulose membrane, may be attributable to the tetra-meric quaternary structure of SuSy in vivo [all SuSys whichhave been analysed by gel filtration were tetramers, includ-ing those from maize, rice and sugarcane (this author, datanot shown)]. The antiserum used in this investigation wasraised against a native, tetrameric enzyme from leaf roll.Possibly, the antiserum recognizes an epitope(s) on theinternode 9 SuSys which is made up of polypeptide sidechains from more than one subunit. On the nitrocellulose

membrane, only approximately 90 kDa monomers are pre-sent, so these epitopes would not be present here. In addi-tion, epitopes on SuSy polypeptides from youngerinternodes recognized on the immunoblot must also beabsent on the monomers from internode 9 SuSys boundto the nitrocellulose membrane. An explanation could bethat the SuSy isoforms in younger internodes may be moreresistant to denaturation than those present in older inter-nodes and hence still possess at least some of the epitopesof the native, tetrameric enzyme. Some plant enzymes,such as peroxidases, are well-known to resist denaturationby SDS and can even exhibit enzyme activity when boundto a nitrocellulose membrane following ‘denaturing’ elec-trophoresis (W.E. Schafer, own observation). The fact thatthe antiserum produces a signal in the immunohistochem-istry sections is consistent with the immuno-inactivation ofnative SuSys.

The immunohistochemistry data (Fig. 6) indicates thepresence of SuSy throughout young and mature inter-nodes. It is not possible to judge localization on thesubcellular level with the tissue fixation and sectioningmethods used, because of physical disturbance of thecellular contents during sectioning, but it is clear thatSuSy is present in vascular, as well as storage parenchymatissue in internodes 3–9.

A preliminary investigation showed that SuSy activityin the microsomal fraction of crude extracts from culmtissue was below detection limits for internode 9, whereasin internode 2, only 3% of the total SuSy sucrose break-down activity was present (Fig. 7). Some membrane-associated SuSy may be expected in younger, activelygrowing tissue, where there is a greater demand forUDP-glucose for cellulose synthesis (Amor et al. 1995).We concluded that membrane-associated SuSy does notconstitute a significant portion of overall SuSy activity inthe sugarcane culm and can therefore be disregarded asfar as this tissue is concerned.

Fig. 7. Distribution of SuSy between cytosolic and microsomal fractions in a very young internode (2) and mature internode (9).


The sucrose concentration in sugarcane storage par-enchyma is slightly lower than in the phloem (Komor2000), which may be as a result of SuSy functioning hereto lower sucrose concentration to assist phloem unload-ing. The promotion of phloem unloading in this way islikely to function in addition to other mechanisms, suchas bulk flow. The current model for sucrose uptake intothe vacuoles of sugarcane storage parenchyma cells is afacilitated diffusion one (Preisser and Komor 1991), so afunction for SuSy similar to that in phloem, namelyindirectly driving active transport processes, looks unli-kely. Sucrose breakdown by SuSy in storage parenchymatissue of sugarcane culm may be favoured over neutralinvertase because of the likely low oxygen content of thistissue; hence, this ATP-conserving route for sucrosebreakdown may be preferred. SuSy is known to functionin an ‘alternative’ route for sucrose breakdown, utilizingPPi, instead of ATP, for feeding into glycolysis (Huberand Akazawa 1986). In sugarcane it is known that SuSydoes not participate in sucrose synthesis in mature inter-nodes, as a study using [U-14C]-glucose showed thatlabelling in the glucose and fructose moieties was equal,pointing to sucrose synthesis exclusively throughsucrose-phosphate synthase (SPS). In younger internodesboth SuSy and SPS were implicated in sucrose synthesis,because of higher labelling in glucose (Botha and Black2000). This is consistent with the lower sucrose break-down/synthesis ratio of SuSy in younger internodes, asfound in this study.

An exciting possibility, which has not yet beenaddressed in sugarcane, is whether different SuSy isoformsfunction in vascular and storage tissue. In potato, theSus3 isoform was expressed mainly in stems and rootsand so appears to provide a vascular function, while theSus4 isoform was expressed chiefly in the storage andvascular tissue of tubers (Fu and Park 1995). In otherwords, there seems to be a distinction between sink andvascular function in potato rather than vascular and sto-rage. No conclusions about whether different isoforms ofSuSy function in sugarcane vascular and storage parench-yma tissue can be drawn from this study. However, bothnative gel electrophoresis and anion exchange chromato-graphy (Fig. 3) show that at least two forms occur inmature culm tissue. Therefore the theoretical possibilityfor isoform-specific sink and vascular functions exists andshould be further investigated, particularly with possibleimprovements to sucrose accumulation in commercialsugarcane varieties in mind. N-terminal sequencing ofsugarcane SuSy isoforms proved infeasible, because theproteins were blocked at the N-terminal. Up to now it hasbeen assumed that only the SS1 isoform occurs in matureculm tissue (Buczynski et al. 1993), but this assumptionneeds revision in the light of the findings in this study andalso data on other crops that show expression of morethan two SuSy genes (Wang et al. 1992, Carlson et al.2002, Komatsu et al. 2002).

Investigating the expression and localization of SuSyin sugarcane internodes differing in maturity has parti-cular relevance for efforts to quantify the contribution of

specific enzymes to sucrose accumulation. A kineticmodel of sucrose accumulation has been published(Rohwer and Botha 2001). For obvious reasons, actuallocalization of enzymes considered in such a modelshould be confirmed for the particular tissue modelled.Furthermore, the differential expression of kineticallydifferent isoforms between tissues, such as that observedin sugarcane internodes of differing maturity, needs to beintegrated with the localization of these isoforms. Thisstudy is a first step in that regard, and shows: (a) that theassumption made in the above-mentioned model thatSuSy is present in storage parenchyma tissue of inter-node 5 is correct, and (b) that a model describing sucroseaccumulation in the more mature internode 9, whereboth sucrose content and sucrose accumulation rate areat near maximum levels, must also take into account thepresence of SuSy in storage parenchyma cells. Apartfrom their localization, the distinct kinetic parametersof different SuSy isoforms present in internodes of differ-ing maturity will also impact differently on factorsimportant for sucrose accumulation, such as the degreeof sucrose breakdown and resynthesis (futile cycling) andthe net sucrose accumulation rate. In this regard, kineticmodels that can calculate the coefficients of metaboliccontrol analysis (MCA) (Kacser and Burns 1973, Hein-rich and Rapoport 1974) and also the direction of rever-sible enzyme reactions, such as that catalysed by SuSy,are very useful. MCA can be used to quantify the con-tribution of individual reaction steps to the pathway fluxor steady-state metabolite concentrations, as in pioneer-ing experiments to determine the contribution of indivi-dual enzymes to mitochondrial respiration (Groen et al.1982), and has been reviewed from a plant metabolismperspective (Ap Rees and Hill 1994). The enzymes (orenzyme isoforms) that have, for example, the highestcontrol coefficients for futile cycling would therefore begood candidates for manipulation in order to increasesucrose content. Hence, it can be seen that localization,identification and characterization of all SuSy isoformsin sucrose accumulating tissue would enhance the accu-racy of metabolic models and therefore contribute tostrategies for increasing sucrose content.

In conclusion, we have shown that SuSy is present inboth vascular bundles and storage parenchyma of youngand mature internodal tissue. Although localization wassimilar between these tissues, the increase in the sucrosebreakdown/synthesis ratio from young to mature tissueindicates a change in the expression of SuSy isoformsbetween young and mature tissues. With the exception ofinternode 3, SuSy activity was similar in different partsof internodes. At least one isoform present in youngtissue is absent in mature tissue, but more than oneisoform is present in mature tissue. No significantmembrane association was evident in internodal tissue.The question whether different isoforms are presentin vascular and storage tissue could potentially beaddressed using monoclonal antibodies for immuno-histochemistry, or with in situ hybridization with veryspecific probes.


Acknowledgements – Support from the South African Sugar Asso-ciation and the South African National Research Foundation isgratefully acknowledged.

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Protein-level expression and localization of sucrose synthase in the sugarcane culm

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