Soluble sugar content of white spruce (Picea glauca) seeds during and after germination

Copyright © Physiologia Plantarum 2000PHYSIOLOGIA PLANTARUM 110: 1–12. 2000ISSN 0031-9317Printed in Ireland—all rights reser6ed

Soluble sugar content of white spruce (Picea glauca) seeds during andafter germination

Bruce Downiea,* and J. Derek Bewleyb

aDepartment of Horticulture, Uni6ersity of Kentucky, Lexington, KY, 40546-0091, USAbDepartment of Botany, Uni6ersity of Guelph, Guelph, ON, N1G 2W1, Canada*Corresponding author, e-mail: [email protected]

Received 26 April 1999; revised 25 November 1999; in final form 28 March 2000

In white spruce (Picea glauca [Moench.] Voss.) seeds, the then increasing. In seeds that did not complete germination,raffinose family oligosaccharides (RFOs) provide carbon re- the synthesis of RFOs at 4°C favored verbascose, so that at

the end of 14 (nondormant) or 35 (dormant) weeks, verbascoseserves for the early stages of germination prior to radicleprotrusion. Some seedlots contain seeds that are dormant, contents in megagametophytes exceeded the amount initially

present in the desiccated seed. This was also true in thefailing to complete germination under optimal conditions.Since dormancy may be imposed through a metabolic block in embryos of the dormant seedlot. In seed parts from bothreserve mobilization, the goal of this project was to identify seedlots after months of moist chilling, stachyose amountsany impediment to RFO mobilization in dormant relative to exceeded raffinose amounts. Upon radicle protrusion at 4°C,nondormant seeds. Desiccated seeds contain primarily, and in RFO contents decreased to amounts most similar to thoseorder of abundance on a molar basis, sucrose and the first 3 present in seeds that completed germination at 25°C. Hence,members of the RFOs, raffinose, stachyose and verbascose. the RFOs are utilized as a source of energy, regardless of theUpon radicle protrusion at 25°C, the contents of RFOs de- temperature at which white spruce seeds complete germina-

tion. Based on the similarity of sugar contents in seed partscreased to low amounts in all seed parts, regardless of priordormancy status and sucrose was metabolized to glucose and between dormant and nondormant seeds that did not complete

germination, differences in sugar metabolism are probably notfructose, which increased in seed parts. During moist chillingthe basis of dormancy in white spruce seeds.at 4°C, RFO content initially decreased before stabilizing and

stress tolerance in conifers (Mitcham-Butler et al. 1986,Fialho and Bucker 1996).

The role of RFOs, particularly regarding desiccation tol-erance, has been most intensively studied in seeds. RFOpresence has been correlated with the orthodox habit (seedsthat survive desiccation) (Lin and Huang 1994), the onset ofdesiccation tolerance during development (Koster andLeopold 1988, Leprince et al. 1993, Bewley and Black 1994,Horbowicz and Obendorf 1994, Black et al. 1996, Brenac etal. 1997a,b) and seed longevity in storage (Bernal-Lugo andLeopold 1992, Horbowicz and Obendorf 1994, Lin andHuang 1994, Bernal-Lugo and Leopold 1995). However,RFO concentration can be decreased to much below theconcentrations found in mature desiccated Arabidopsisseeds, and yet the mature seeds maintain desiccation toler-

Introduction

Likely ubiquitous in higher plants (Kerr et al. 1997), mem-bers of the raffinose family oligosaccharides (RFOs) aresecond only to sucrose among the nonstructural carbohy-drates with respect to abundance in the kingdom Planta(Lehle and Tanner 1973). Despite intensive investigation,the physiological significance of the RFOs remains con-tentious. In many plants they serve as storage (Keller andMatile 1985, Hendrix 1990) and transport (Hendrix 1968,Holthaus and Schmitz 1991, Madore 1992) metabolites.RFO accumulation has been correlated with enhanced cold,heat and/or desiccation tolerance from algae to angiosperms(Santarius 1973, Salerno and Pontis 1989, Hinesley et al.1992, Ashworth et al. 1993, Wiemken and Ineichen 1993,Bachmann et al. 1994). Additionally, RFOs correlate posi-tively with needle longevity as well as ozone- and sulfide-

Abbre6iations – DAI, days after imbibition; RFO, raffinose family oligosaccharides.

Physiol. Plant. 110, 2000 1

ance (Ooms et al. 1993). Still, in developing seeds, accumu-lated evidence supports a crucial role for the RFOs inproviding tolerance to water loss at stages before full matu-rity (Horbowicz and Obendorf 1994, Black et al. 1996,Brenac et al. 1997a,b). In mature orthodox seeds, the RFOsare probably vital at least for enhancement of longevity(Bernal-Lugo and Leopold 1992, 1995, Ooms et al. 1993,Lin and Huang 1994). These findings agree with the positivecorrelation between soybean longevity in accelerated agingstudies and the concentration of RFOs in the seeds (Main etal. 1983).

Decreasing to negligible amounts by the onset of radicleprotrusion, RFOs are thought to provide energy and carbonfor the initial stages of seed germination (Pazur et al. 1962,Abrahamsen and Sudia 1966, East et al. 1972, Hsu et al.1973, Amuti and Pollard 1977a,b, Maiti and Loewus 1978,Kuo et al. 1988, Nichols et al. 1993, Buckeridge and Di-etrich 1996, Dirk et al. 1999). This drastic decline in abun-dance during germination may provide a metabolic markerfor seed dormancy status if dormant seeds are incapable ofmobilizing these reserves. To ascertain whether differencesin sugar metabolism between seeds of a dormant and anondormant seedlot of white spruce were discernible, sugaridentities and contents were compared during germination,dormancy alleviation and seedling establishment at twodifferent temperatures.

Materials and methods

Plant material

Two white spruce (Picea glauca [Moench.] Voss.) seedlotswere obtained from open-pollinated stands (Slave Lake[nondormant]: 625 m above sea level [masl], 56°60%, 114°50%,collected in 1986; high level [dormant]: 341 masl, 58°28%,117°16%, collected in 1988). These seedlots were selected forthe study based on their germination response prior to andafter moist chilling, the standard dormancy alleviating treat-ment for this species (International Seed Testing Association1993). The germination of unchilled seeds and seeds thatwere moist chilled for 3 weeks, respectively, for the twoseedlots were 84 and 84% (nondormant seedlot) and 55 and88% (dormant seedlot). The enhancement of germinationpercentage for seeds of the dormant seedlot by moist chillingwas statistically significant (a=0.05). White spruce seedlotsfrom open-pollinated stands do not exhibit complete dor-mancy, and the latter was the most dormant available at thetime of the study.

Germination and moist chilling conditions

Throughout the present work, germination refers strictly tothe processes occurring in the seed from the time it takes upwater to the time the radicle protrudes (Bewley and Black1985). Germination (at 25°C) and moist chilling (at 4°C)took place in the dark on two layers of Whatman No. 1filter paper moistened with 6 ml distilled, deionized water(ddH2O) and 0.1% (w/v) Benlate fungicide (Lock et al. 1975)in 9×1-cm Petri dishes (International Seed Testing Associa-

tion 1993). A seed was considered to have completed germi-nation when its radicle was visibly protruding through thesurrounding structures (megagametophyte, nucellus andtesta).

For each seedlot, 3 replications of 20 seeds each weresampled for sugar content (Fig. 1). Seeds were sampled eachday for 8 days on water with or without a prior 3-weekmoist chilling treatment (Fig. 1; Expts 1 and 3). Seeds werealso sampled for the first 8 days moist chilling at 4°C andafter 21 days, 3 (nondormant) or 9 (dormant) months moistchilling (Fig. 1; Expts 2 and 4). Seeds of the nondormantseedlot were also sampled from days 0 to 4 on water at 25°Cand separated into the cotyledons, axis, micropylar andchalazal portions of the megagametophyte (Fig. 1; Expt 5).In all treatments permitting radicle protrusion, samples ofseeds that had and had not completed germination wereassessed (Fig. 1).

All embryos, megagametophytes or parts thereof wereplaced into preweighed, vented, 1.7-ml microfuge tubes onice until all 20 parts had been collected. Subsequently, thetubes and contents were weighed and plunged into liquidnitrogen, the contents lyophilized in a Savant SpeedVacattached to an FTS, Flexi-dry freeze dryer (Stone Ridge,NY, USA) for 24 h and the tubes and contents weighed andstored at −80°C until preparation and analysis.

Fig. 1. A flow chart depicting the treatments and sampling schemeused to examine sugar identity and content in white spruce seedsfrom a dormant and a nondormant seedlot. UG, live seeds failingto complete germination; G, live seeds that completed germination;M, megagametophyte; E, embryo.

Physiol. Plant. 110, 20002

Viability of ungerminated seeds

Since seed components from seeds that had not completedgermination were compared with seed components fromseeds that had completed germination, it was imperativeto ascertain that seeds not completing germination wereactually alive. The solid, white appearance of both theembryo and megagametophyte after imbibition weredeemed sufficient to classify a seed as alive, but this as-sumption was tested using tetrazolium dye (InternationalSeed Testing Association 1993). The results from the te-trazolium staining confirmed that all seeds from bothseedlots that had been classified as alive, ungerminated at7 days after imbibition (DAI), had embryos andmegagametophytes that stained an intense red color, signi-fying reducing capability, while those classified as dead ornecrotic usually did not stain, or were a lighter shade ofred.

Sample preparation

Sugar extraction followed the protocol of Foley et al.(1992), except that ice-cold 80% (v/v) ethanol and alterna-tive internal standards were used. At the time of assay,samples were retrieved from −80°C storage and pulver-ized in liquid nitrogen in a mortar and pestle. The powderwas ground in 5, 1-ml aliquots of 80% (v/v) cold ethanol.The first ml of ethanol (extractant A) contained 200 mMfucose and 200 mM lactose for recovery estimation and asinternal standards, while the 4 subsequent extractions usedunsupplemented 80% ethanol (extractant B). The aliquotswere pooled in 15 ml Falcon tubes (Fisher Scientific Co.,Springfield, NJ, USA), centrifuged at 10000 g in a Da-mon, IEC HN-S centrifuge (International Equipment Co.,Needham Heights, MA, USA) for 10 min and the super-natant collected, placed in 50 ml Falcon tubes andlyophilized to dryness. The resultant powder was subse-quently reconstituted in 1 ml ddH2O on ice with frequentagitation for 4 h just prior to analysis. Reconstituted sam-ples were transferred to 1.7 ml microfuge tubes and cen-trifuged at 16000 g for 10 min on a Brinkmann microfuge(Brinkmann Instruments Ltd, Rexdale, ON, Canada), thesupernatant collected and analyzed immediately or storedat −20°C (no more than 2 days) before analysis. Sampleswere centrifuged in lieu of filtration, since preliminarystudies indicated that up to 80% of the neutral sugarswere retained on pre-wetted, 0.45-mm filters.

Carbohydrate detection

Carbohydrates were identified and quantified by pulsedamperometric detection (PAD) (Townsend et al. 1988a,b)using a Dionex Series 4500 HPLC and a CarboPAC-PA1pellicular anion exchange column with guard column(Dionex Corp., Sunnyvale, CA, USA). The PAD electrodepotentials were set throughout the study to cycle at E1,0.05 V; E2, 0.6 V; and E3, −0.06 V with 500, 10 and 5ms durations. Column temperature, and the temperatureof the eluants, was maintained at approximately 23°C.

Elution protocols

Neutral sugars were eluted using a protocol modified fromClarke et al. (1991). This protocol resolved sugars isocrati-cally for 50 min following injection in 19.5 mM NaOHfollowed by a step gradient to 1 M sodium acetate con-taining 150 mM NaOH for 5 min to wash the column.The column was recharged with 150 mM NaOH for 10min following the wash prior to equilibrating with 19.5mM NaOH for 10 min before injecting the next sample.The flow rate throughout elution was 1.0 ml min−1.

Raffinose oligosaccharides were eluted isocratically in7.5 mM sodium acetate containing 147.5 mM NaOH for30 min (initial conditions) followed by a wash with 1 Msodium acetate containing 150 mM NaOH for 5 min. Thecolumn was then equilibrated to initial conditions for 10min prior to injection of the next sample. The CalibrateProgram of the Dionex Advanced Computer InterfaceChromatography Program (Dionex Corp.) was used to de-termine the range of linear response for each sugar. Re-sponses approaching the upper limit of linear detectabilitywere diluted and re-run.

Fucose and lactose were used to estimate recovery foreach sample and fucose was used with the Dionex Methodsoftware (Dionex Corp.) to indicate column performancefor neutral sugars and adjust expected retention times ac-cordingly. Lactose was used in a similar fashion with theprotocol for RFOs. Blank samples of extractant A (seeSample preparation) without tissue were placed in a coldmortar, ground as if they contained sample and re-ex-tracted with extractant B (see Sample preparation) 4 timesto estimate sample retention on the apparatus during thecentrifugations and during lyophilization.

As controls, embryos and megagametophytes that hador had not completed germination were first extractedwith cold 80% (v/v) ethanol. Immediately following extrac-tion, the samples were divided into two tubes and onesub-sample was boiled at 100°C in a water bath for 5 minthen plunged on ice. Once the boiled sub-sample was cool,both sub-samples were centrifuged and the ethanol super-natant transferred to a fresh tube and lyophilized. Nodifference in sugar identity or amount was evident be-tween embryos or between megagametophytes treated ineither cold or boiling ethanol. Since cold, 80% (v/v) etha-nol was subsequently used for extraction, we examined thepossibility that the extracted sugars could be metabolizedto different sugar species in vitro. Sugar extracts fromwhole, nondormant seeds at 4 DAI that had not com-pleted germination were digested with 0.4 U of either a-D-galactosidase (EC 3.2.1.22; Sigma, St Louis, MO, USA)or acid-invertase (EC 3.2.1.26; Sigma) in 10 ml added to100 ml of crude extract at 25°C for 24 h. A blank wasprepared by adding 10 ml ddH2O to a second 100-mlaliquot of the same samples. Extracts were then cen-trifuged at 16000 g for 10 min, the supernatant collectedand aliquots of unhydrolized and hydrolized sample di-luted for analysis by both neutral sugar and raffinose-se-ries protocols.


Verification of sugar identity

Monosaccharides, sucrose and the RFOs were identifiedby their co-elution with pure standards. Sucrose and theRFOs were further identified by their degradation prod-ucts after treatment with exogenous a-galactosidase oracid invertase (Dirk et al. 1999). The increase from unde-tectable quantities of the expected degradation products,galactose and sucrose in the first case, and of glucose(from the degradation of sucrose), fructose (from sucroseand RFOs), melibiose and other, larger oligosaccharides,in the second case, was taken as evidence that the sugarswere correctly identified and solely responsible for the de-tector response observed.

During the course of the experiment with white spruceseed parts, degradation products from endogenous inver-tase digestion of the RFOs were not detected in any ofthe samples. Hence, sucrose, glucose and fructose contentswere also unaffected by post-extraction degradation by in-vertase. Nor was there post-extraction degradation of theRFOs due to endogenous a-galactosidase activity sincegalactose was seldom detected and, when it was, it wasalways a trace constituent, unlike the large galactoseamounts detected when the samples were purposely sub-jected to enzymatic degradation.

Statistical analysis

The quantity of sucrose and RFOs (dependent variables)in parts from white spruce seeds were compared betweenthe dormant and nondormant seedlots (seedlot was theindependent variable) before and during incubation at25°C using analysis of variance (ANOVA) at a=0.05.One comparison was made using only seeds that failed tocomplete germination (0–8 DAI) and a second using onlyseeds from which the radicle had protruded (4–8 DAI).These comparisons between seedlots were performed bothbefore and after 3 weeks moist chilling. Once the seedshad commenced radicle protrusion at 25°C, the quantityof each sugar was compared between seeds that had andhad not completed germination within each seedlot. Thesugar contents among seed parts after no moist chilling,conventional moist chilling (3 weeks duration) andprolonged moist chilling (nondormant, 14 weeks; dormant,35 weeks), were tested for significantly deviating meansby analysis of variance (ANOVA) at a=0.05. Sugaramounts in seedparts were tested among days of in-cubation at 25°C (independent variable) prior to andafter 3 weeks moist chilling for each seedlot. This wascarried out for parts from seeds that had (3, 4–8 DAI)and had not (0–8 DAI) completed germination. Sugaramounts in seedparts were also tested among days ofmoist chilling at 4°C (0–8, 21). In all cases, if theANOVA proved to have a significant F-value (Fowler1990), significant differences among means were identifiedby Scheffe’s multiple pairwise comparison (a=0.05). Allanalyses were run using the Statistical Analysis Systemrelease 6.12 for Windows (Statistical Analysis System,Inc., Cary, NC, USA).

Fig. 2. Sucrose and raffinose content of embryos and megagameto-phytes (Meg.) from desiccated and imbibed seeds that had or hadnot completed germination from the nondormant seedlot prior to(A and D), during (B and E) or after (C and F) 3 weeks moistchilling at 4°C. A–C) Embryos; D–F) Megagametophytes. Seedcomponents from seeds during germination/moist chilling () andfrom seeds that completed (�) or failed to complete ( ) germina-tion at 25°C. DAI: days after imbibition. Each estimate is anaverage of 3 replicate samples and is accompanied by the standarderror of the mean.

Results

Sugars present in white spruce seeds

Glucose, galactose, fructose, mannose, ribose, sucrose,raffinose, stachyose and verbascose were detected in thechalazal and micropylar halves of white sprucemegagametophytes, as well as in the cotyledons and embry-onic axis. Mannose and galactose increased from unde-tectable to trace quantities in all seed parts as germinationprogressed.

Comparison of sugars in dormant versus nondormant seeds

Unimbibed seeds from the nondormant seedlot containedmegagametophytes that had approximately twice the su-crose, raffinose, stachyose and verbascose contents as thosefrom the dormant seedlot (a=0.05) (Figs. 2–5). Embryosfrom unimbibed seeds did not differ significantly in sugarcontent between the dormant and nondormant seedlots.When unchilled seeds imbibed at 25°C for 1 day or morethat had not completed germination were examined, the twoseedlots were statistically indistinguishable based on sugarcontent, regardless of the seed part examined (Figs. 2–5).When unchilled seeds that had completed germination werecompared between seedlots, megagametophytes from seeds


of the nondormant seedlot contained significantly greaterraffinose amounts up to 6 DAI, while embryos were statisti-cally indistinguishable (a=0.05) (Figs. 2–5).

Following moist chilling for 21 days but prior to incuba-tion at 25°C, megagametophytes from seeds of the nondor-mant seedlot had significantly greater amounts of RFOsthan megagametophytes from seeds of the previously dor-mant seedlot (a=0.05), while embryo sugar contents werestatistically identical. When seeds that had not completedgermination were compared between seedlots during thesubsequent 8 days of germination at 25°C, megagameto-phytes from moist chilled seeds of the nondormant seedlotmaintained greater amounts of sucrose until 6 DAI, whileembryos were statistically indistinguishable from 1 DAIonward (a=0.05) (Figs. 2–5). Sugar contents of seedpartsfrom moist chilled seeds that had completed germinationwere statistically identical between seedlots.

Effect of germination on seed sugar content

Differences between seeds that had or had not completedgerminationWhen sugar contents were compared between parts fromseeds that had or had not completed germination, the twoseedlots differed. While the unchilled, nondormant seedlothad embryos that had significantly less sucrose upon radicleprotrusion than embryos that did not protrude (a=0.05),the opposite was true of the dormant seedlot (a=0.05)(Figs. 2A and 3A). During incubation at 25°C, following 3

Fig. 4. Stachyose and verbascose content of seed components fromthe nondormant seedlot. Designations as in Fig. 2.

weeks moist chilling, sucrose contents from embryos of thenondormant seedlot that had completed germination andthose that had not were equal (a=0.05) (Fig. 2C). Embryosfrom the previously dormant seedlot that completed germi-nation at 25°C, following 3 weeks moist chilling, had signifi-cantly more sucrose than embryos from moist chilled seedsthat had not completed germination when incubated at 25°C(a=0.05) (Fig. 3C). For both seedlots, regardless of priormoist chilling treatment, megagametophytes from seeds thatfailed to complete germination had significantly greatersucrose amounts than those from seeds that had completedgermination (a=0.05) (Figs. 2D,F and 3D), except formegagametophytes from the previously dormant seedlot,moist chilled for 3 weeks, in which sucrose contents wereequal (Fig. 3F). Seedparts from both seedlots, regardless ofmoist chilling treatment, almost invariably had significantlygreater quantities of RFOs when they were from seeds thathad not completed germination (a=0.05) (Figs. 2–5). Theexceptions were stachyose contents, which were equal be-tween embryos from seeds of the nondormant seedlot thathad and had not completed germination after 3 weeks moistchilling (Fig. 4C). Verbascose contents in protruded andunprotruded embryos from unchilled seeds of the nondor-mant seedlot were also equal (Fig. 4A), and were signifi-cantly greater in embryos that had protruded than thosethat had not during incubation at 25°C, after 3 weeks moistchilling (a=0.05) (Fig. 4C). The single exception for thedormant seedlot was that verbascose contents were equalbetween protruded and unprotruded embryos after incuba-tion at 25°C following 3 weeks moist chilling (a=0.05) (Fig.5C).

Fig. 3. Sucrose and raffinose content of embryos and megagameto-phytes (Meg.) from desiccated and imbibed seeds that had or hadnot completed germination from the dormant seedlot prior to (Aand D), during (B and E) or after (C and F) 3 weeks moist chillingat 4°C. Designations as in Fig. 2.


Differences in sugar content in seed parts among days ofgerminationThe changes in sugar content were similar during germina-tion before or after moist chilling for the embryo andmegagametophyte. Generally, the amounts of monosaccha-rides present in mature, desiccated seeds were low and didnot increase appreciably prior to radicle protrusion. Uponradicle protrusion, monosaccharide quantities increasedabruptly. This increase was predominantly due to an in-crease in glucose and fructose contents. Embryo glucose andfructose contents in a seed from the nondormant seedlotwere: mature desiccated embryo, 090 and 0.690.3 nmol,respectively, increasing after radicle protrusion to a maxi-mum of 25.4916.2 and 29.8912.3 nmol, respectively. Inan embryo from a seed of the dormant seedlot, glucose andfructose contents were undetectable in mature desiccatedembryos but increased rapidly upon radicle protrusion to37.391.2 and 38.690.4 nmol per seed, respectively, by theend of the 8-day test. These values are the minimum andmaximum contents for glucose and fructose observed in allseed parts, regardless of moist chilling treatment. Regardlessof seedlot or prior moist chilling treatment, glucose andfructose amounts increased more in embryos and mega-gametophytes of seeds that had completed germination thanin those that had not, or those that were undergoing moistchilling (data not shown).

For unchilled seeds of the nondormant seedlot that didnot complete germination, only embryo RFO contents ofunimbibed seeds (0 DAI), when compared among daysduring incubation at 25°C (0–8 DAI), were significantlygreater than amounts in embryos from imbibed seeds (a=

0.05) (Figs. 2 and 4). No pattern was discernible in embryoor megagametophyte sucrose content changes during germi-nation after moist chilling (Fig. 2C,F). The content of RFOsin the embryo and megagametophyte from seeds of thenondormant seedlot, immediately after 21 days moist chill-ing, was significantly greater than the content of thesesugars after as little as 1 day of incubation at 25°C (a=0.05) (Figs. 2 and 4).

When the dormant seedlot was examined prior to moistchilling, sugar amounts in seedparts from seeds that had notcompleted germination for up to 8 days were statisticallyidentical (a=0.05) (Figs. 3 and 5). Sucrose amounts after 21days moist chilling also did not vary significantly during thecourse of germination, regardless of seed part (Fig. 3C,F).However, RFO amounts in embryos from seeds harvestedimmediately after 21 days moist chilling were significantlygreater (a=0.05) than RFO amounts in embryos frommoist chilled seeds that had been incubated at 25°C for aslittle as 1 day (Figs. 3 and 5C). In moist chilledmegagametophytes, the content of stachyose, immediatelyafter moist chilling and after 1 day incubation at 25°C, wassignificantly greater than the content of stachyose in seedsthat did not complete germination from 4 DAI onward(a=0.05) (Fig. 5F).

Effect of moist chilling on seed sugar content

Overall, regardless of dormancy status or seed part, sucrosecontents were statistically indistinguishable during 21 daysmoist chilling (a=0.05) (Figs. 2 and 3). On a molar basis,raffinose was the most prominent RFO present in whitespruce embryos and megagametophytes prior to moist chill-ing, regardless of dormancy status. There was no significantchange in any sugar quantity during 21 days moist chillingfor either megagametophytes or embryos, regardless of seed-lot (a=0.05) (Figs. 2–5).

Changes in sugar content in different embryo andmegagametophyte parts during germination

To ascertain whether there were quantitative differences inoverall sugar amounts among seed parts, sugars from thecotyledons, embryonic axis, chalazal and micropylar halvesof the megagametophyte were quantified. Quantities of eachsugar from the embryo or megagametophyte parts (Figs.6–9) were combined and compared with the quantitiesrecorded for the whole embryo or megagametophyte (Figs.2 and 4). Overall, the quantities of sugars detected fromwhole embryos or megagametophytes corresponded closelyto the sum of sugars from both halves of the embryo ormegagameotphyte. While there were discrepancies, theywere often associated with large standard errors and/or lowsugar contents. Three notable exceptions are embryo andmegagametophyte sucrose contents at 3 DAI and embryosucrose contents at 4 DAI. For these points, the whole,ungerminated embryos contained 40 and 30 nmol sucrose(Fig. 2A), while the sum of the sucrose content found in thecotyledons and axis was 60 and 70 nmol (combine amountsin Figs. 6 and 7), respectively. The discrepancy in the

Fig. 5. Stachyose and verbascose content of seed components fromthe dormant seedlot. Designations as in Fig. 2.


Fig. 6. Sugar content in the cotyledons of embryos from seeds ofthe nondormant seedlot during the first 4 days of imbibitionwithout prior moist chilling. Each estimate is an average of 3replicate samples and is accompanied by the standard error of themean. Gal, galactose; Glu, glucose; Frc, fructose; Man, mannose;Raf, raffinose; Sta, stachyose; Ver, verbascose; Suc, sucrose.

onic axis to 2 nmol axis−1 at 4 DAI, which was about 10times more abundant than in the cotyledons (Figs. 6 and 7).Galactose contents of the chalazal and micropylar halves ofwhite spruce megagametophytes were generally greater thanthose present in the embryo parts and tended to decreasefrom the amounts present at 0 or 1–4 DAI. Galactose wasalways at the lower limit of detection in both megagameto-phyte halves (Figs. 8 and 9). Mannose contents tended toincrease in both halves of the megagametophyte from 0 to 4DAI. Similar amounts of galactose were present in bothmegagametophyte parts and the same was true for mannose(Figs. 8 and 9). Both glucose and fructose quantities re-mained fairly stable throughout the time course in bothmegagametophyte halves. Both megagametophyte halveshad approximately equivalent amounts of glucose. The samewas true for fructose. Fructose amounts tended to trackglucose amounts in both megagametophyte componentswith glucose being the more abundant sugar (Figs. 8 and 9).

Sucrose and RFO contents

The amount of sucrose in both the cotyledons and axistended to increase during the time course (Figs. 6 and 7),while it tended to remain unchanged (at least after 0 DAI)

Fig. 7. Sugar content in the embryonic axis (radicle and hypocotyl)from seeds of the nondormant seedlot during the first 4 days ofimbibition without prior moist chilling. Each estimate is an averageof 3 replicate samples and is accompanied by the standard error ofthe mean. Gal, galactose; Glu, glucose; Frc, fructose; Man, man-nose; Raf, raffinose; Sta, stachyose; Ver, verbascose; Suc, sucrose.

megagametophyte contents was 45 nmol, with the sum ofthe parts (Figs. 8 and 9) having a greater sucrose content.There were no qualitative differences in the identity of themonosaccharides, sucrose or RFOs in the different parts ofthe megagametophyte or embryo of white spruce. Sucrose,raffinose, stachyose and verbascose were the predominantsugars present in all tissues (Figs. 6–9).

Monosaccharide contents

There was no detectable mannose or galactose in either thecotyledons or the axis of white spruce embryos until 3 and4 DAI, respectively (Figs. 6 and 7). Similar quantities ofgalactose were present in both embryo parts. The same wastrue of mannose (Figs. 6–9). Galactose, when present, wasin trace amounts. Glucose amounts tended to remain thesame (cotyledons; Fig. 6) or increase after 3 DAI (axis; Fig.7) following an initial decrease from the amount present inthe mature, desiccated embryo. Glucose was 10 times moreabundant in the embryonic axis (4 nmol axis−1) than in thecotyledons (compare Fig. 6 with Fig. 7). Like glucose,fructose amounts were low in the cotyledons. Fructoseincreased after the second day of imbibition in the embry-


Fig. 8. Sugar content in the chalazal half of the megagametophytefrom seeds of the nondormant seedlot during the first 4 days ofimbibition without prior moist chilling. Each estimate is an averageof 3 replicate samples and is accompanied by the standard error ofthe mean. Gal, galactose; Glu, glucose; Frc, fructose; Man, man-nose; Raf, raffinose; Sta, stachyose; Ver, verbascose; Suc, sucrose.

were greatest in unimbibed seeds (a=0.05) and statisticallyindistinguishable in megagametophytes from seeds moistchilled for 3 or 14 weeks (a=0.05) (Fig. 10C). However,verbascose contents were significantly greater inmegagametophytes from seeds moist chilled for 14 weekswhen compared with megagametophytes from unchilled orseeds moist chilled for 3 weeks (a=0.05) (Fig. 10C).

Seeds from the dormant seedlot moist chilled for 0, 3 or35 weeks had embryos and megagametophytes in which thesucrose, raffinose and stachyose contents were statisticallyidentical (Fig. 11A,C). Verbascose contents in embryos andmegagametophytes, moist chilled for 35 weeks, were statisti-cally greater than in seedparts from unchilled or seeds moistchilled for 3 weeks (a=0.05) (Fig. 11A,C).

Figs. 10 and 11 compare sugar contents among embryosor among megagametophytes, regardless of whether theywere from seeds that had completed germination. Sucrosecontents were statistically identical or least in embryos fromseeds moist chilled for 3 weeks and incubated at 25°Cwithout radicle protrusion for 8 days (a=0.05) (Figs. 10and 11A,B). Sucrose contents were identical or least inmegagameophytes from seeds that had completed germina-tion during prolonged moist chilling (a=0.05) (Figs. 10 and

Fig. 9. Sugar content in the micropylar half of the megagameto-phyte from seeds of the nondormant seedlot during the first 4 daysof imbibition without prior moist chilling. Each estimate is anaverage of 3 replicate samples and is accompanied by the standarderror of the mean. Gal, galactose; Glu, glucose; Frc, fructose; Man,mannose; Raf, raffinose; Sta, stachyose; Ver, verbascose; Suc, su-crose.

in the megagametophyte until 4 DAI, when it decreased(Figs. 8 and 9). The increase in sucrose quantities in theembryo halves at 4 DAI coincided with the decrease insucrose pools in the megagametophyte parts. RFO contentswere comparable in both embryo halves and were rapidlymetabolized during germination so that, by 4 DAI, theywere at the lower limit of detectability (Figs. 6 and 7). RFOcontents were similar in both megagametophyte halves, atleast from 1 DAI onward, and rapidly declined duringgermination. By the fourth day of imbibition, with theexception of raffinose in the chalazal half of themegagametophyte, all RFOs were at the lower limit ofdetectability (Figs. 8 and 9). Both halves of themegagametophyte had greater quantities of RFOs than didthe embryo components (Figs. 6–9).

Oligosaccharide contents during germination at 4°C

When white spruce seeds were subjected to prolonged moistchilling, some completed germination (14 weeks, nondor-mant; 35 weeks, dormant). The sugar content of embryosfrom seeds of the nondormant seedlot, moist chilled for 0, 3or 14 weeks, did not vary significantly (Fig. 10A).Megagametophyte sucrose, raffinose and stachyose contents


Fig. 10. A: Sucrose and RFO contents of whole embryos from thenondormant seedlot that have not completed germination before,during or after moist chilling at 4°C. B: Same as A but for seedsthat have completed germination. C: Same as A but for themegagametophytes. D: Same as B but for the megagametophytes.W, weeks of moist chilling at 4°C followed by . . . D, days ofincubation at 25°C and then separated into either. . . UG, seeds thathad not completed germination or G, seeds that had completedgermination.

et al. 1999). Murphy et al. (1992) found that glucose andfructose contents in pinyon pine embryos were positivelycorrelated with acid invertase activity, and high rates ofexpansive growth have been positively correlated with highglucose amounts in a variety of plant tissues (Morris andArthur 1984). Although the sucrose content in the embryoalso increased during the initial increase in glucose andfructose (5–8 DAI), the contribution to the pool of sucrosein the embryo by its transport from the megagametophytewould help replenish that lost due to invertase activity. Thereason that embryos from the two seedlots varied by anorder of magnitude in glucose and fructose amounts afterradicle protrusion is not apparent, but this discrepancy waslargely relieved after moist chilling (data not shown).

Sucrose and RFO contentThe fact that raffinose was the predominant RFO present inwhite spruce seeds makes them more similar to sugar pine(Pinus lambertina) and castor bean (Ricinus communis) thanother gymno- or angiosperms for which RFO contents havebeen reported (Hattori and Shiroya 1951, Durzan andChalupa 1968, Nyman 1969, Kuo et al. 1988, Murphy andHammer 1988). The predominant RFO in dry seeds doesnot appear to be correlated with the type of major carbonstorage reserve (lipid or starch) utilized by the seed. Noreports of RFO contents in spruce seeds were found in the

Fig. 11. A: Sucrose and RFO contents of whole embryos from thedormant seedlot that have not completed germination before, dur-ing or after moist chilling at 4°C. B: Same as A but for seeds thathave completed germination. C, Same as A but for themegagametophytes. D, Same as B but for the megagametophytes.Designations as in Fig. 10.

11C,D). When seeds had completed germination duringprolonged moist chilling or during incubation at 25°C priorto or after 3 weeks moist chilling, embryo sucrose contentswere equal statistically (Figs. 10 and 11A,B). Among seedsthat had completed germination, raffinose contents weregreatest in embryos that completed germination during pro-longed moist chilling, regardless of seedlot (a=0.05) (Figs.10 and 11B). Frequently, stachyose contents in both em-bryos and megagametophytes, among seeds that had com-pleted germination, were significantly greater from seedsthat had completed germination during prolonged moistchilling (a=0.05) (Figs. 10 and 11B,D).

Discussion

Sugar content of dormant and nondormant seed componentsduring germination, early establishment and moist chilling

Monosaccharide contentThe low to undetectable amounts of glucose and fructose inmature, desiccated seeds prior to the completion of germina-tion, and their increase afterward, agrees well with reportsfor other species (Hattori and Shiroya 1951, Durzan andChalupa 1968, Nyman 1969, Kao and Rowan 1970, Kao1973, Murphy and Hammer 1988, Murphy et al. 1992, Dirk


literature. All RFOs decreased during the first 24 h ofimbibition, regardless of seedlot or treatment. This patternhas been found in all seeds in which these oligosaccharidesare present (see surveys by Amuti and Pollard 1977a, Kuoet al. 1988). The current theory regarding the role of theRFOs in seeds and roots is that they are stable storageforms of sucrose, positively correlated with longevity (Mainet al. 1983, Bernal-Lugo and Leopold 1992, Lin and Huang1994), but probably not essential for desiccation toleranceper se (Ooms et al. 1993, Bochicchio et al. 1994). The factthat sucrose contents increase during the initial decline inRFOs invites the speculation that degradation of RFOs wasby a-galactosidase and not invertase. Cleavage of galactosefrom RFOs to free sucrose for transport and furthermetabolism is a common metabolic scenario in the seeds ofmany angiosperms (Kuo et al. 1988).

It is possible to explain the decrease in RFO amounts bypresuming that sucrose was metabolized extensively by theembryo and either metabolized or transported directly tothe embryo from the megagametophyte in both seedlots.This would account for the observed declines in RFOs tosupplement the pool of sucrose and result in very lowamounts of RFOs after the radicle had protruded. A rolefor the RFOs as an energy source for events early in seedgermination has been disputed in the past for dormant wildoat caryopses artificially induced to complete germination(Foley et al. 1992, 1993), but it remains a well-substantiatedelement of normal seed germination (Pazur et al. 1962,Abrahamsen and Sudia 1966, East et al. 1972, Hsu et al.1973, Amuti and Pollard 1977a,b, Maiti and Loewus 1978,Kuo et al. 1988, Nichols et al. 1993, Buckeridge and Di-etrich 1996, Dirk et al. 1999).

After an initial decline, seed components during moistchilling had a net accumulation of sucrose and RFOs.Although not statistically significant, the apparent increasein sucrose content during moist chilling is consistent withthe findings of Kao and Rowan (1970) studying seedmetabolism during moist chilling of radiata pine (Pinusradiata). Moist chilling occurs in nature every winter at thesubnivian layer, and the seeds may or may not be subse-quently dehydrated in spring. One signal necessary for ger-mination is water; another is accumulated degree days. Incold conditions, both seed components began to synthesizeRFO reserves after about 6 DAI and synthesis continueduntil the seeds completed germination at 4°C (14 weeksnondormant; 35 weeks dormant). The enzymes responsiblefor RFO synthesis are activated by low temperature inconiferous roots (Wiemken and Ineichen 1993) and needles(Kandler et al. 1979). Transcription and/or mRNA stabilityof galactinol synthase, the rate-limiting enzyme in the syn-thesis of RFOs, as well as galactinol synthase enzyme activ-ity itself are enhanced by low temperature (Liu et al. 1998).

Prolonged moist chilled seeds from both seedlots hadaltered amounts of RFOs relative to unimbibed seeds. Fol-lowing prolonged moist chilling, stachyose was the predom-inant RFO in both seedlots in all seed parts, followed byraffinose (embryo) or verbascose (megagametophyte), andthen verbascose (embryo) or raffinose (megagametophyte)(Figs. 10 and 11). It is possible that other enzymes involvedin the biosynthesis of the RFOs (raffinose, stachyose and

verbascose synthase) are up-regulated by cold temperaturesand resulted in the accumulation of higher order RFOsfrom raffinose precursors, thereby altering the relative mo-lar ratios of these RFOs from that present in the mature,desiccated seed. The physiological significance of accumu-lating stachyose rather than raffinose, which was the pre-dominant RFO in mature desiccated white spruce seeds, isunknown.

Sugar content during germination of different embryo andmegagametophyte parts of a nondormant seedlot

Monosaccharide contentThe increase in mannose in white spruce seed parts prior toradicle protrusion may be attributed to the partial break-down of mannan-containing hemicelluloses in the cell wallsof white spruce (Downie et al. 1997). There was very littlegalactose build-up in the seeds although RFOs were hy-drolized extensively. This is not surprising since galactose isa metabolic poison (Yamamoto et al. 1988), necessitatingdetoxification by rapid phosphorylation and isomerizationin plant cells (Main et al. 1983). Galactose was not detectedin mature, desiccated or germinating seeds of jack pine(Durzan and Chalupa 1968) or Scots pine (Nyman 1969),both of which mobilized large reserves of galactosyl-su-croses. It may be significant that high amounts of osmoti-cally active substances (sugars) were found in theembryonic axis at approximately the commencement ofrapid elongation and associated water uptake.

There was no less than 10 times the amount of glucoseand fructose in the embryonic axis than in the cotyledonsduring and after germination of seeds from the nondormantseedlot. Such a disparity of glucose and fructose amountsbetween the axis and the cotyledons during germinationand early establishment has been reported previously andattributed to higher catabolism of sucrose in the axis (Kuoet al. 1988), more specifically, the hypocotyl in coniferembryos/seedlings (Murphy et al. 1992). In fenugreek, thisdisparity arises due to the conversion of transported sugarsfrom the endosperm into a transient starch deposit. Thisoccurs predominantly in the cotyledons depleting glucoseand fructose amounts far below that present in the axis(Dirk et al. 1999). Not surprisingly, starch transiently in-creases in white spruce embryos after radicle protrusion (B.Downie and J.D. Bewley, unpublished data). In addition,sucrose contents were lower in the embryonic axis than inthe cotyledons.

Sucrose and RFO contentThe decrease in sucrose amounts in the chalazal and mi-cropylar parts of the megagmetophyte, and subsequent in-crease in both the cotyledons and axis, is typical of thetransfer of sucrose from storage organs into the embryo ofangiosperm seeds, most studied in the leguminosae, prior toand after the completion of germination (Buckeridge andDietrich 1996). The initial source of sucrose are the RFOs,which decline to very low amounts in all seed parts prior toradicle protrusion providing sucrose to the embryo (Reid1971, Main et al. 1983, Buckeridge and Dietrich 1996).


Oligosaccharide metabolism during germination at 4°CThe ability of white spruce seeds to complete germination atlow temperature allowed us to ascertain whether the RFOsaccumulated during cold stress (Kandler et al. 1979, Wiemkenand Ineichen 1993, Takahashi et al. 1994, Liu et al. 1998)would be metabolized as an energy source at 4°C as they areduring germination at 25°C. Completing germination at 4°C,white spruce embryos and megagametophytes of both seed-lots metabolized the RFOs to amounts more similar to thosepresent in seeds that had completed germination at 25°C thanthose that had not completed germination at either 4 or 25°C.Hence, the RFOs are utilized as an energy source, regardlessof the temperature at which they complete germination.

Conclusions

There does not appear to be any obvious deficiency in solublesugar metabolism responsible for seed dormancy in whitespruce. White spruce seeds exhaustively metabolize RFOsprior to radicle protrusion. Amounts of RFOs and sucrose incomponents of unimbibed seeds from a nondormant seedlotexceed those in components of unimbibed seeds from adormant seedlot. During moist chilling, RFO amounts declineinitially but then accumulate to amounts intermediate be-tween those in the mature, desiccated seed and their lowestpoint during moist chilling. The accumulation of RFOsduring moist chilling is such that stachyose becomes the mostpredominant RFO on a molar basis, supplanting raffinose themost predominant RFO prior to moist chilling. However,upon radicle protrusion at 4°C, the RFO content in seedcomponents declines to amounts most similar to those presentin components from seeds that complete germination at 25°C.We therefore conclude that the RFOs are utilized as an energysource, regardless of the temperature at which they completegermination.

Acknowledgements – Dr Donald J. Nevins, Department of Veg-etable Crops, UC Davis, allowed the use of his Dionex HPLC fora part of this work. Dr Sunitha Gurusinghe, Department of Veg-etable Crops, UC Davis, extracted and analyzed some of the sugarsamples for prolonged moist chilled seeds, for which we are grate-ful. A previous version of this manuscript was much improved byeditorial suggestion from Dr Robert Geneve, Department of Horti-culture, Drs. Dennis Egli and Dennis TeKrony, Department ofAgonomy, University of Kentucky, Lexington.

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Soluble sugar content of white spruce (Picea glauca) seeds during and after germination

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