Copyright © Physiologia Plantarum 2000PHYSIOLOGIA PLANTARUM 108: 361–369. 2000Printed in Ireland—all rights reser6ed ISSN 0031-9317

Carbohydrate metabolism in Nerine sarniensis bulbs developing in liquidculture

Jane Vishnevetskya, Eli Zamskia and Meira Ziva,b,*

aDepartment of Agricultural Botany, The Hebrew Uni6ersity of Jerusalem, P.O. Box 12, Reho6ot 76100, IsraelbThe Warburg Center for Biotechnology in Agriculture, The Hebrew Uni6ersity of Jerusalem, P.O. Box 12, Reho6ot 76100, Israel*Corresponding author, e-mail: meira@agri.huji.ac.il

Received 1 November 1999

stages of development. The activity of ADP-glucose pyrophos-Anatomical and physiological changes accompanying en-hanced Nerine sarniensis cv. Salmon Supreme bulb growth in phorylase (EC 2.7.7.27), a key enzyme of starch synthesis,vitro were examined. Small bulbs, 2–3 mm in diameter, grown increased just before the increase in starch accumulation.in vitro on a semi-solid medium were subcultured to liquid Sucrose was the dominant soluble sugar in the bulbs, onlymedium with elevated levels of sucrose (Suc) and inorganic traces of glucose and fructose were detected. The activity of

alkaline invertase (INV, EC 3.2.1.26) was higher than that ofphosphate. Bulbs’ fresh and dry weights, carbohydrate con-acid INV during the growth period. Sucrose synthase (ECtents and the activities of enzymes related to carbohydrate2.4.1.13) exhibited the highest Suc-degrading activity duringmetabolism were determined at different stages of bulb devel-

opment. Starch was the dominant storage carbohydrate in bulb growth. Suc was hydrolyzed in the medium by the cellwall bound acid INV during the growing period. The resultsthese bulbs, and the leaf bases parenchyma cells were the

principal storage tissue. During the first month of bulb are discussed in relation to enhanced nerine bulb growth anddevelopment in vitro.growth, only small changes in starch content were detected.

However, an increase in starch level was observed at later

1997b). However, despite the fact that this enhanced devel-opment is well documented in the literature (Santos et al.1998), carbohydrate metabolism during different stages of invitro bulb development has never been reported.

Flower bulbs contain a variety of reserve carbohydrates,including soluble sugars (sucrose [Suc], glucose [Glc], fruc-tose [Fru]), starch, glucomannans and fructans, which allcontribute to bulb development (Miller 1992). Limited infor-mation is available on the biochemistry of carbohydratemetabolism in ornamental geophytes, or on the regulationof carbohydrate partitioning. Several reports discusschanges in carbohydrate levels in geophytic organs in vitro(Davies and Kempton 1975, Taeb and Alderson 1990).Theron and Jacobs (1996) studied the carbohydrate compo-sition of different bulb components after the development offlowering-size Nerine bowdenii bulbs. They found that the

Introduction

Nerines are autumn flowering bulbous plants belonging tothe Amaryllidaceae in which bulb morphology and develop-ment have been well documented (Rees 1985, Van Brenkand Benshop 1993, Vishnevetsky et al. 1997a). Their storageorgan is a perennial tunicate bulb (Bryan 1989). On theoutside of the bulb are a few papery scales followed by anumber of fleshy scales which are the bases of either dry orgreen leaves. Carbohydrate reserves stored in fleshy bulbleaf bases, fleshy scales or corms are important for the initialgrowth of these and other geophytic plants (Miller 1992).

Nerine bulbs micropropagated by conventional methodsrequire several periods of growth in vitro to reach theappropriate size before transferring to soil and a few yearsbefore they can flower. Enhanced storage-organ develop-ment in liquid culture significantly shortens this period andcan contribute to early flowering (Vishnevetsky et al.

Abbre6iations – ADP-GlcPPase, adenosine diphosphate glucose pyrophosphorylase; AP, alkaline phosphatase; BA, benzyladenine; BFJ,baby-food jar; DIECA, diethyldithiocarbamic acid, sodium salt; Glc-1,6-bisP, glucose-1,6-bisphosphate; Glc-6-P DH, glucose-6-phosphatedehydrogenase; INV, invertase; IBA, indole-3-butyric acid; MS, Murashige and Skoog minerals; NAA, a-naphthaleneacetic acid; 3-PGA,3-phosphoglycerate; PGM, phosphoglucomutase; PPi, pyrophosphate; SuSy, sucrose synthase; TCA, trichloroacetic acid.

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developing inflorescence had a noticeable influence onsource-sink relationships.

Given that Suc is the main translocated carbohydrate ingeophytes, its breakdown and metabolism are critical forgrowth of the storage organs, the inflorescence, and othersinks (Miller 1992). It is generally accepted that the break-down of Suc in sink tissues occurs through the action ofinvertase (INV, EC 3.2.1.26) or sucrose synthase (SuSy, EC2.4.1.13) (Avigad 1982), but this has never been verified inbulbous plants.

INV activity in vascular plants is appreciable and wide-spread. The activities of acid and alkaline INVs vary inde-pendently during development (ap Rees 1984).Neutral/alkaline INV, with a pH optimum of 7.5, is usuallyconfined to the cytosol, whereas acid INV, with a pHoptimum of 3.5–5.0, is soluble in the vacuole and bound tothe cell wall (Avigad 1982, Krishnan et al. 1985).

SuSy is also involved in Suc breakdown, but has neverbeen studied in geophytes (Miller 1992). Geigenberger andStitt (1993) have shown that SuSy can carry out bothcleavage and synthesis of Suc in vivo. However, the direc-tion in which SuSy acts (Suc synthesis or breakdown) iscontrolled by the subsequent metabolism of the reactionproducts, rather than by the enzyme itself (Quick andSchaffer 1996).

A number of biochemical and genetic studies have shownthat ADP glucose serves as the main precursor for starchsynthesis. Evidence for the important role of ADP glucosepyrophosphorylase (ADP-GlcPPase, EC 2.7.7.27) in starchsynthesis was first provided by mutants in which a starchdeficiency, in maize grains (Tsai and Nelson 1966), wasassociated with a loss of enzyme activity. Different data,demonstrating a direct relationship between the activity ofthe ADP-GlcPPase and starch accumulation in several plantspecies, support the hypothesis that the ADP-GlcPPase isthe major regulatory step in starch synthesis (Prioul et al.1994, Nakata and Okita 1995, Brangeon et al. 1997, Geigen-berger et al. 1998).

The aim of the present work was to examine the morpho-logical, anatomical and physiological changes that accom-pany enhanced bulb growth under in vitro conditions.Specifically, changes in endogenous carbohydrate contentwere monitored, and INV, SuSy and ADP-GlcPPase activi-ties were characterized along with changes in the weight anddiameter of bulbs grown under optimal conditions in vitro.

Materials and methods

Plant material

Microbulbs of Nerine sarniensis cv. Salmon Supreme wereoriginated from twin-scaled, in vivo-grown bulbs importedfrom New Zealand and subcultured on the semi-solid Mu-rashige and Skoog minerals (MS) medium (Murashige andSkoog 1962) supplemented with 10 mM indole-3-butyric acid(IBA), 6% (w/v) Suc and 0.25% (w/v) activated charcoal(basic medium). Fast propagation from in vitro-grown bulbswas carried out by dissecting the 7–9-mm diameter bulbsinto two halves and growing them on a semi-solid MS

medium supplemented with 1 mM a-naphthaleneacetic acid(NAA), 1 mM benzyladenine (BA) and 3% (w/v) Suc. Ad-ventitious bulbs were originated after 30–50 days of cultiva-tion in the dark, with the subsequent transition to the basicmedium. For the experiment discussed in the present paper,small bulbs, 2–3 mm in diameter, from the basic mediumwere subcultured in 125 ml volume baby-food jars (BFJ,V8630, Sigma, St Louis, MO), 5 bulbs per vessel. Each jarcontained 12 ml liquid medium supplemented with 0.1 mMNAA, 6% (w/v) Suc and twice the level of phosphate(relative to the MS medium). Raising the level of phosphatein the medium up to 340 mg l−1 KH2PO4 enables moreefficient utilization of Suc from the medium (Vishnevetsky etal. 1997b). Plantlets were cultured under controlled condi-tions of 25/22°C day/night, 16/8 h light/dark on a gyratoryshaker at 80–100 rpm. Plantlets were harvested from 4 BFJand fresh and dry weights, bulb diameter, carbohydratecontent and enzyme activity were measured every 2 weeksfor 8 weeks. The experiments were carried out twice.

Carbohydrate determination

Samples of whole bulbs were dried in the oven at 70°C andthen ground with a mortar and pestle. Dried tissues, rangingfrom 20 to 80 mg, were extracted 3 times in 80% ethanol at70°C for a total of 2.5 h. Combined supernatants from eachsample were evaporated to dryness and the precipitate wasdissolved in 3 ml water, centrifuged at 10000 g for 10 min,filtered through a 0.45-mm filter and assayed for solublecarbohydrates (Suc, Glc and Fru) by HPLC, as describedbelow. The insoluble material was assayed for starch asfollows: starch was gelatinized in an autoclave at 121°C for1.5 h and determined after amyloglucosidase (Sigma) treat-ment (Thivend et al. 1972), followed by analysis of releasedGlc by HPLC. The amyloglucosidase reaction mixture wasas follows: 8.5 ml of a water-bulb extract plus 0.5 ml acetatebuffer at pH 4.8 plus 1 mg of enzyme (20.8 units) dissolvedin 1 ml of double-distilled water. The mixture was incubatedat 55°C for 18 h.

Sugar analysis was carried out in a HPLC system (WatersCorporation, Milford, MA, USA) consisting of the follow-ing: a 501 pump, a Sugar Pak I column placed in a 90°Coven, a 410 differential refractometer and a 740 data moduleintegrator. The mobile phase consisted of double-distilledwater at a flow rate of 0.5 ml min−1. Suc, Glc and Fru wereidentified by their retention times and quantified accordingto standards.

Level of exogenous sugars in the medium

Aliquots of the incubation medium were collected duringthe growth period at 2-week intervals. Cells were pelleted bycentrifugation at 10000 g for 10 min. The supernatant wasfrozen and kept at −20°C. Suc, Glc and Fru in the mediumwere analyzed by HPLC as described. The Glc, Fru and Succoncentrations in the medium were calculated and summa-rized as total Suc concentration. The amount of Suc dissipa-tion from the medium was expressed as a percentage of theinitial Suc concentration (60 g l−1). We tried to evaluate the

Physiol. Plant. 108, 2000362

relationships between sugar uptake by the tissue and theincrease in tissue fresh weight throughout the investigation.Total Suc dissipation from the medium was calculated asthe difference between the initial Suc in the medium and thetotal sugar every 2 weeks in a single BFJ, and this value wasthen divided by the total fresh weight (expressed in g) of the5 bulbs grown in each BFJ.

Enzyme extraction and assays

Soluble acid and alkaline INV and SuSy activities (in theSuc cleavage direction) were measured. Extraction proce-dures were modified from Schaffer et al. (1987) and Mironand Schaffer (1991). Briefly, approximately 250–500 mgfresh weight frozen tissue (kept at −70°C until the proce-dure commenced) was pulverized in liquid nitrogen with amortar and pestle. All subsequent procedures were carriedout on ice. Powdered tissues were homogenized in extractionbuffer (2.5–5 ml) containing 25 mM HEPES, 7.4 mMMgCl2, 0.5 mM Na2EDTA, 3.2 mM DTT and 2 mMDIECA (pH 7.5). Insoluble material was removed by cen-trifugation at 12000 g for 40 min. Supernatants were dia-lyzed overnight against the buffer (12.5 mM HEPES, 3.7mM MgCl2, 0.25 mM Na2EDTA, pH 7.5), with one bufferchange, and used as the crude soluble enzyme extract. Theinsoluble pellet was washed up with extraction buffer and,after centrifugation, homogenized in 3 ml of extractionbuffer containing 25 mM HEPES, 7.4 mM MgCl2, 0.5 mMNa2EDTA, 3.2 mM DTT and 2 mM DIECA (pH 7.5). Anattempt to solubilize the ‘insoluble’ INV activity was carriedout with NaCl (1 M final concentration) added to the initialextraction buffer.

For the determination of INV activity, 200 ml of crudeenzyme extract plus 200 ml of 1 M Suc and 600 ml of 100mM citrate phosphate buffer at a pH of 5 or 7 werecombined to give a final volume of 1 ml reaction mixture.The mixtures were incubated on a shaker at 37°C for 60min. The reaction was terminated by adding 1 ml dinitrosal-icylic acid reagent (Miller 1959) and boiling for 5 min.Enzyme extract was also added after a 60-min incubationand used as a blank control. The amount of producedreducing sugars (Glc and Fru) was determined spectropho-tometrically at 550 nm. Reaction mixtures with insolubleenzyme suspensions were centrifuged before spectrophoto-metric reading.

To determine SuSy activity, 200 ml of crude enzymeextract plus 200 ml of 1 M Suc, 600 ml of 100 mM citratephosphate buffer at pH 7 and 50 ml of 100 mM UDP werecombined to give a final volume of 1.05 ml reaction mixture.The mixtures were incubated on a shaker at 37°C for 60min. The addition of 1 ml of dinitrosalicylic acid reagent(Miller 1959) with subsequent boiling for 5 min terminatedthe reaction. The amount of Fru was determined spec-trophotometrically at 550 nm.

To assay ADP-GlcPPase, frozen plant tissue was ex-tracted in a chilled extraction buffer containing 50 mMHEPES, 1 mM MgCl2, 5 mM Na2EDTA, 1 mM DTT and3 mM DIECA (pH 7.7). Homogenates were centrifuged at12000 g for 40 min at 4°C and the protein concentration

was determined. The crude enzyme extract was heated for 4min at 60°C, in order to increase enzyme activity (Schafferand Petreikov 1997), centrifuged (12000 g, 5 min) and thesupernatant used for the assay. ADP-GlcPPase was assayedspectrophotometrically by monitoring the pyrophosphate(Ppi)-dependent production of Glc-1-P via a continuousenzyme-linked assay coupling Glc-1-P to Glc-6-P (withphosphoglucomutase [PGM]) and monitoring the glucose-6-phosphate dehydrogenase (Glc-6-P DH)-catalyzed produc-tion of NADH at 340 nm (Schaffer and Petreikov 1997).The assay buffer contained 50 mM HEPES-NaOH (pH 7.8),5 mM MgCl2, 1 mM ADP-Glc, 1 mM NAD+, 1 unit ofGlc-6-P DH, 1 unit of PGM (Sigma), 10 mM of glucose-1,6-bisphosphate (Glc-1,6-bisP), 10 mM 3-phophoglycerate (3-PGA), 10 mM NaF and 200 ml of the enzyme extract.Following a 6-min incubation at 37°C, the reaction wasinitiated with the addition of 1 mM PPi to a total volume of1 ml.

Protein determination

Protein for each of the enzyme extracts was determinedcolorimetrically at 595 nm as described by Bradford (1976)using a Sigma Protein Assay Kit with bovine serum albumin(BSA) as the standard (0–15 mg).

Electrophoresis and immunoblotting

Protein was extracted in 20 mM MOPS (pH 7.5) andprecipitated with trichloroacetic acid (TCA) as described byBathgate et al. (1985). The precipitated protein redissolvedin protein gel loading buffer: 62.5 mM Tris, 2% SDS, 5%b-mercaptoethanol, 25 mM EDTA, 15% Suc and 5%bromphenol blue. Extracts were maintained at 4°C and allprocedures were performed as rapidly as possible to preventproteolysis.

Proteins were separated by denaturing SDS-PAGE(Laemmli 1970), with a final acrylamide concentration of12% (w/v) separating gel and 4.5% (w/v) stacking gel (20 mgprotein per lane). Proteins were blotted onto nitrocelluloseusing a Bio-Rad transfer apparatus (Hercules, CA, USA).Western blots were performed with antibodies (diluted1:3000) raised against ADP-GlcPPase from tomato fruit(kindly supplied by B.Y. Chen, Rutgers University, NewBrunswick, NJ, USA) and the central part of vacuolar acidINV (diluted 1:400) from tomato fruit (kindly supplied byA.B. Bennett, University of California, Davies, CA, USA).Cross-reacting bands were identified using anti-rabbit IgGalkaline phosphatase (AP)-conjugated (Sigma) secondaryantibodies (1:2000) and the reaction was visualized using5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetra-zolium (Sigma) according to the manufacturer’sinstructions.

Microscopy

Bulb scales were fixed in 2.5% (v/v) glutaraldehyde in 100mM phosphate buffer, pH 7.2, for 18 h and dehydrated in agraded ethanol series. Fixed material was embedded in

Physiol. Plant. 108, 2000 363

‘hard-grade’ L-R (London resin) White by heat-curing at60°C for 24 h. Sections, 1 mm thick, prepared in an ultrami-crotome (LKB produkter, Stockholm, Sweden), weremounted on ProbeOn microscope slides (Fisher Scientific,Pittsburg, PA, USA) for immunolocalization and detectionunder the light microscope (Olympus BH-2, Tokyo, Japan).

Immunodetection of ADP-GlcPPase

Immunolocalization of ADP-GlcPPase was performed onslide-mounted sections. Immunodetection was carried outaccording to standard protocols using primary anti-ADP-GlcPPase or no first antibodies, followed by secondaryantibodies conjugated to AP.

Microscope slides were processed in large Petri dishes, andeach slide was covered with 150-ml solution volumes. Sectionson slides were blocked in 3% (w/v) low-fat milk for 30 minand challenged for 1 h with anti-ADP-GlcPPase serum(diluted 1:100). Secondary antibodies, anti-rabbit IgG conju-gated to AP (Sigma), were applied to slides (1:500) and thereaction was visualized according to the manufacturer’sinstructions. At the end of the procedure, slides were washedwith distilled water, dried and sealed with Permount (FisherScientific).

Results

Bulb growth in liquid culture and starch accumulation

In general, the anatomical structure of the bulb scales isidentical to that of the photosynthetic parts of the leaves. Aninner and outer epidermal layer, several parenchymal layersand the vascular bundles embedded among them were de-tected in all examined scales. The number of parenchymallayers varied. Small bulbs, taken from semi-solid solidmedium, consisted of 3 scales with their upper green portionsand one or two inward leaves. The outer scale was thinnerwith bigger cells than the two inner scales with smaller cells.Starch accumulated within amyloplasts in the parenchymacells of the scales. Amyloplasts (seen as dark granules in Fig.1A) were larger and more abundant in the outer cell layersof the external scales. The volume of the scale parenchymacells increased 16.6 times (from 0.055×10−3 mm3 to 0.913×10−3 mm3) within 4 weeks in the liquid medium and increasedup to 42 times (to 2.325×10−3 mm3) during a 6-week period.The volume of the amyloplasts of these cells increasedapproximately 3.4 times during 4 weeks (compare Fig. 1A andB) and up to eight times after 6 weeks in culture (Fig. 1C).

After a slight increase during the first 4 weeks, the plant’s(whole and bulb) fresh (Fig. 2A) and dry (Fig. 2B) weightsincreased substantially. The largest increment in bulb andplantlet weight was between weeks 4 and 6 of culture. Thiswas correlated with a concomitant increase in starch concen-tration. At 8 weeks, the plants developed 3–4 green leaves andthe bulb scales increased in size.

Carbohydrate content during bulb growth

Sucrose hydrolysis was observed after autoclaving themedium, decreasing Suc level by 5%. After plantlets inocula-

tion, Glc and Fru concentrations increased gradually until theend of the experiment concomitantly with a decrease in Succoncentration (Fig. 3). The most noticeable Suc dissipationfrom the medium occurred during the first 2 weeks ofincubation and represented approximately 22% of the exoge-nous Suc. At the same time, only negligible increases in thebulb and leaf dry weights were observed (Fig. 4). Sucrose inthe medium was hydrolyzed to Glc and Fru and after 8 weeks,the total concentration of Suc, Glc and Fru (total solublesugars [TSS]) decreased by approximately 35% from 60 g l−1

to approximately 39 g l−1 (calculated from the data shownin Fig. 3).

Changes in endogenous Suc and starch content, measuredover 8 weeks of bulb growth, are shown in Fig. 5. Only tracesof Glc and Fru were detected in the bulbs throughout theexperiments. Sucrose content in the bulbs, expressed on afresh weight basis, showed no changes during the first 2 weeks;however, on a dry weight basis, there was a slight increase inSuc content. Starch content, expressed on a fresh weight basis,decreased during the first 2 weeks; however, there was nochange on a dry weight basis. This discrepancy in the results,when expressed on a fresh and a dry weight basis can be dueto H2O uptake by the plantlets when subcultured from thesemi-solid to the liquid medium. Sucrose content graduallydecreased from week 4 to 8. Starch accumulation increasedbetween weeks 4 and 6 and remained constant until the endof the experiment (Fig. 5).

Enzyme activity

The cytosolic enzyme, SuSy, exhibited the highest Suc-degrad-ing activity in the bulbs. Its activity increased approximately42% during the first 2 weeks, after transferring the bulbs tothe liquid medium (Fig. 6A). Alkaline INV activity was higherthan that of acid INV. Acid INV activity, detected in thesoluble fraction (probably vacuolar), was lower than alkalineINV activity throughout the incubation period (Fig. 6A).Alkaline INV activity, detected in the soluble fraction (prob-ably cytosolic), decreased slightly during 6 weeks of culture,and increased to the original level after week 8. Cell wallbound INV, detected in the insoluble fraction activity (notshown), was approximately 96918.8 nmol RS g−1 freshweight min−1 during the whole growing period. No INVactivity was found in the medium.

The activity of ADP-GlcPPase decreased during the first 2weeks of incubation in the liquid medium, increased approx-imately 7-fold to a maximum after a month and then droppeddown to the lower value after week 8 (Fig. 6B). The increasein the enzyme activity occurred prior to the increase in starchcontent in the bulbs. Amyloplasts reached their maximum sizeafter 6 weeks (approximately 20 mm in diameter) and starchaccumulation ceased with the decrease in ADP-GlcPPaseactivity (Figs. 5 and 6B).

ADP-GlcPPase localization in parenchyma cells of the bulbscale

Bulbs grown in liquid culture were harvested, fixed, embed-ded, sectioned and challenged with either anti-ADP-GlcPPase

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serum or controls with no primary antibodies. Fig. 7A showsmany precipitates on the starch grains surface, representingspecific binding of antibodies with ADP-GlcPPase and somenon-specific binding in the cytoplasm. No cross-reactionappeared in the vacuole and in the control sections (Fig. 7B).

ImmunoblottingProteins extracted from the bulbs were separated bySDS-PAGE. Immunoblots of protein from bulbs grown for8 weeks in liquid culture, using antibodies against the centralpart of the INV molecule, INV G2 and against ADP-Glc-

Fig. 1. Cross-sections of the bulb at different stages of growth. (A) Two scales from the bulb grown on the solid MS medium (at the startof the incubation period); (B) outer scale of the bulb grown for 4 weeks in the liquid medium; (C) outer scale of the bulb grown for 6 weeksin the liquid medium. Bar=100 mm.

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Fig. 2. Changes in fresh (A) and dry (B) weights of plantlets andbulbs during 8 weeks of growth in the liquid medium.

Fig. 4. Total soluble sugars (TSS) in the medium and changes indry weight of bulbs and leaves of 5 plantlets from one BFJ during8 weeks of growth in the liquid medium. BDW, bulb dry weight;LDW, leaf dry weight.

slightly after transfer of the bulbs to liquid medium andthen remained relatively constant during the following 8weeks (Fig. 8).

Discussion

Nerine bulbs were examined and characterized throughout 8weeks of growth in liquid medium. Starch was the dominantstorage carbohydrate in N. sarniensis, and the leaf baseswere the principal bulb structures in which it was stored.Similar results were also found in N. bowdenii grown in vivo(Theron and Jacobs 1996). The nerine bulb represents astorage organ with a high sink strength. The volume ofstarch grains increased 8-fold during 6 weeks of growth inliquid culture. The differences in amyloplast numbers in theparenchyma cells, seen in Fig. 1A–C, probably stemmedfrom changes in the cell volume. The bigger the cells, thesmaller the chance of cutting through the amyloplasts. Thecell volume increased exponentially throughout the growthperiod and paralleled the increase in dry weight.

PPase, are presented in Fig. 8. Acid INV antibodies showedspecific immunogenic responses with crude extracts. Theantibodies recognized two polypeptides of approximately 63and 75 kDa. No visual changes were detected in the enzymequantity of bulbs from different growing periods when equalquantities of proteins were loaded; however, the totalprotein content in the tissue decreased after week 4 until theend of week 8 (Fig. 9).

The 53-kDa band of ADP-GlcPPase was detected in thebulbs at different stages of development. Since equalamounts of protein were loaded per lane, the amount of thisenzyme, as part of a total protein quantity, decreased

Fig. 5. Starch and sucrose (Suc) content in the bulbs during 8weeks of growth.

Fig. 3. Different sugar concentrations in the liquid medium during8 weeks of bulb growth. Suc, sucrose; Glc, glucose; Fru, fructose.

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Fig. 6. Enzyme activity in the bulbs during 8 weeks of growth inthe liquid medium. (A) Sucrose synthase (SuSy) and soluble inver-tases (INV); (B) ADP-GlcPPase. RS, reducing sugars; alk., alkaline.

week 2 could be mainly attributed to photosynthesis ratherthan to sugars taken from the medium. This was previouslyreported in tobacco plants grown in vitro under photo-mixotrophic conditions (Ticha et al. 1998). The increase indry weight between weeks 6 and 8 can be explained by celldivision activity and the initiation of new leaves inside thebulb, since no additional starch content was found.

Suc degradation by SuSy was much higher than that ofthe INVs. This situation is characteristic of non-Suc-accu-mulating tissues. As shown in potato tubers, more than halfof the degraded Suc is catalyzed by SuSy while INVs playonly a minor, if any, role (ap Rees and Morrell 1990).

Our results show that alkaline INV activity was higherthan that of the acid (vacuolar) INV, the latter being verylow. A similar relationship between different INVs has beenobserved in developing potato tubers (Merlo et al. 1993).The increase in alkaline INV activity towards the end of thegrowing period was concomitant with the decrease in Succontent.

The antibodies raised against the central part of the INVmolecule recognized two polypeptides of approximately 63and 75 kDa in nerine bulb tissue. In tomato fruit tissue,these antibodies recognize proteins at 52- and 48-kDa bands(Schaffer and Petreikov 1997). The significance of thesedifferences was not investigated. However, the results sug-gest the existence of different INV isoforms in tomato fruitsand nerine bulbs. Various INV isoforms exist in differentplant tissues (Leigh et al. 1979, and references cited therein).Most INVs that have been analyzed for molecular mass arein the range of 40–70 kDa (denatured protein), and theenzyme has been reported to function as a monomer in thenative state (Burch et al. 1992, Michaud et al. 1993, Walkerand Pollock 1993). To the best of our knowledge, there hasbeen no characterization of INVs from bulb tissues.

Enhanced leaf and bulb growth was observed betweenweeks 4 and 6 of culture. Concomitantly, starch accumula-tion in the bulbs reached its maximum. Subsequently, nofurther starch accumulation was detected. Sucrose contentin the bulbs decreased from the beginning of the experimentto the end of week 8.

The results show that ADP-GlcPPase activity (Fig. 6B)decreased during the first 2 weeks (probably due to adapta-tion to the new culture conditions), increased in the follow-ing 2 weeks and dropped significantly at the end of week 8.The amount of total protein in the bulbs (Fig. 9) somewhatdecreased during the adaptation stage, increased during thefollowing 2 weeks and significantly decreased at weeks 6 and8. Western blots, however, show (Fig. 8) that when equalamounts of protein (20 mg), obtained from 2–8-week-oldbulbs, were loaded on each lane, no significant change in theamount of ADP-GlcPPase, or INV, was noticed. This sug-gests that if the amount of the enzymes remained constantin total protein content, then when the total protein dropsduring weeks 6 and 8 (Fig. 9), the amount of the enzymesdecreased as well. ADP-GlcPPase exhibited increased activ-ity 2 weeks before starch accumulation had begun anddecreased after the starch content reached its peak. Thisenzyme became highly active prior to starch synthesis. We-ber et al. (1995) noted a similar increase of ADP-GlcPPase

When plantlets were transferred from the semi-solid to theliquid medium, they were exposed to a temporary stress,such as the direct contact with the medium or low oxygensupply. As the plants were transferred from an aeratedenvironment to a submerged one, they went through anadaptation stage. The immediate contact with the liquidmedium enabled the cells to take up nutrients and H2O.There was an approximately 46% increase in net Suc contentin the bulbs during the first 2 weeks (48–70 mg g−1 dryweight). However, on a fresh weight basis, as can be seen inFig. 5, there was no significant change in Suc content duringthis period. This is probably due to a noticeable wateruptake by the cells.

Sucrose hydrolysis was detected in the medium at thebeginning of the experiment (Fig. 3) and the concentrationsof Glc and Fru were similar throughout the experiment. NoINV activity was found in the medium, but Suc degradationin the medium occurred by the insoluble cell wall boundINV. The disappearance of the TSS from the mediumduring the first 2 weeks of culture was not totally compen-sated by an increase in the plantlet’s dry weight, suggestingthat there was a loss in net carbon due to respiration.Conversely, the increase in the plantlet’s dry weight after

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Fig. 7. ADP-GlcPPase localization in parenchyma cells of the bulb scale grown in vitro. Specific (seen as dark spots on the starch grain [sg]surface) and non-specific (in the cytoplasm) binding of ADP-GlcPPase antigens were visualized using AP-conjugated secondary antibodies(A). No cross-reaction was observed when cells were not treated with primary antibodies (B). Bar=10 mm.

Fig. 8. Immunoblots of proteinsextracted from bulbs grown in liquidculture for 8 weeks challenged withtomato antibodies to acid INV andto ADP-GlcPPase followed byvisualization with AP. 0, 2, 4, 6 and8 weeks of incubation in liquidmedium; Ab, antibody.

activity in broad bean (Vicia faba L.) cotyledons duringstarch accumulation. However, Jenner and Hawker (1993)did not find any correlation between ADP-GlcPPase activityand starch synthesis in wheat endosperm. We detectedADP-GlcPPase protein on the surface of starch grains in theparenchyma cells (Fig. 7). Identical localization of ADP-GlcPPase has been demonstrated by Kim et al. (1989) inpotato tuber amyloplasts.

The nerine bulb ADP-GlcPPase (53 kDa) cross-reactedwith antibodies raised against the tomato protein. However,the purified enzyme from tomato fruit showed two bandswith molecular masses of 50 and 51 kDa (Chen and Janes1997). There are two reasons that could account for thedifferences: (1) tomato antibodies recognized only one ADP-GlcPPase subunit; or (2) the ADP-GlcPPase enzyme innerine bulbs is composed of two similar molecular-weightsubunits. The plant ADP-GlcPPase is a heterotetramericprotein, assumed to be composed of two different, butrelated subunits (Morell et al. 1987, Okita et al. 1990).Depending on the plant species, the size of the two smallsubunits ranges from 50 to 56 kDa, whereas the size of thetwo large subunits lies between 51 and 60 kDa (Copelandand Preiss 1981, Sowokinos and Preiss 1982, Morell et al.1987, Lin et al. 1988).

Plantlets taken from the semi-solid medium and used forthe experiments consisted of 3 scales and one to two inner

leaves. During 8 weeks in liquid culture the upper greenportions of the scales extended nearly 4 times (9.4–34.4mm). The two inward leaves emerged from the bulbs, turnedgreen and extended to approximately 20 mm during 8 weeksin liquid culture. When transferred to the semi-solidmedium, the green portions of the outer scales degeneratedand the new leaves and roots developed after 2–3 weeks.

Fig. 9. Changes in protein content in the soluble fraction of thebulbs grown in liquid culture for 8 weeks.

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The plants were ready for planting into the soil. To the bestof our knowledge, this is the first report on carbohydratemetabolism in bulbs grown in liquid culture. The resultspresented show that there was a strong correlation betweenthe levels of different carbohydrates and the related enzymeactivities at the different stages of bulb development.

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Edited by R. Scheibe

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