Comparison of sucrose metabolism during the rehydration of

desiccation-tolerant and desiccation-sensitve leaf material of

Sporobolus stapfianus

Anne Whittakera,b, Tommaso Martinellib, Adriana Bochicchiob, Concetta Vazzanab and Jill Farranta,*

aDepartment of Molecular and Cell Biology, University of Cape Town, Private Bag Rondebosch 7700, South AfricabDipartimento di Scienze Agronomiche e Gestione del Territorio Agroforestale; Universita di Firenze, Piazzale delle Cascine 18, I-50144Firenze, Italy*Corresponding author, e-mail: farrant@science.uct.ac.za

Received 17 December 2003; revised 9 March 2004

Sucrose accumulated during dehydration is a major potential

energy source for metabolic activity during rehydration. The

objective of the present study was to investigate aspectsof leaf sucrose metabolism during the rehydration of

desiccation-tolerant Sporobolus stapfianus Gandoger

(Poaceae) over a 10-day period. Comparison was then madeto sucrose metabolism during the rehydration of both

desiccation-tolerant excised leaf material (dehydrated

attached to the parent plant) and desiccation-sensitive leaf

material (dehydrated detached from the parent plant toprevent the induction of tolerance) over a 48-h period. The

pattern of sugar mobilization and glycolytic enzyme activity

during the rehydration of the desiccation-tolerant excised

leaves was similar to that in leaves attached to the parent

plants. Significant breakdown of sucrose was not apparent

in the initial phase of rehydration, suggesting the utilization

of alternate substrates for respiratory activity. Thedesiccation-tolerant excised tissues provided a suitable

control to compare the metabolism of rehydrating

desiccation-sensitive material. In contrast to the toleranttissues, sucrose breakdown in the sensitive leaves commenced

immediately after watering and the accumulation in

hexose sugars was inversely proportionate to the decrease in

sucrose content. Hexokinase (EC 2.7.1.1), PFK (ATPphosphofructokinase, EC 2.1.7.11), aldolase (EC 4.1.2.13),

enolase (EC 4.2.1.11), and PK (pyruvate kinase,

EC 2.7.1.40) activity levels were significantly lower in the

desiccation-sensitive material during rehydration.

Introduction

Resurrection plants have been defined as desiccation-tolerant angiosperms, which exhibit vegetative tissuetolerance to intensive dehydration, and subsequent rehy-dration from the air-dried state (Gaff 1971). During therehydration process, energy must be provided for therestoration of metabolic activity and the repair of anydesiccation-induced damage. From both chlorophyllfluorescence and gas exchange studies conducted on anumber of resurrection plants, it is evident that whereasmaximal photosynthetic activity is only restored close tofull rehydration (Schwab et al. 1989, Sherwin and

Farrant 1996, Di Blasi et al. 1998, Vander Willigen etal. 2001), respiration is rapidly reactivated followingwatering (Schwab et al. 1989, Tuba et al. 1998, VanderWilligen et al. 2001). The respiratory utilization of sub-strates stored during dehydration is a source of energy tometabolism during rehydration (Scott 2000).

Sucrose is the predominant sugar accumulated duringdehydration in resurrection plants (Kaiser et al. 1985,Ghasempour et al. 1998). Although sucrose is creditedas a major potential carbon source for the restoration ofmetabolic activity during rehydration (Scott 2000), todate there have been no studies detailing the metabolismof sucrose during rehydration. As a study of the

PHYSIOLOGIA PLANTARUM 122: 11–20. 2004 doi: 10.1111/j.1399-3054.2004.00346.x

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

Abbreviations – DM, dry mass; DS, desiccation-sensitive; GDH, glutamate dehydrogenase (EC 1.4.1.2); MDH, malate dehydrogenase (EC1.1.1.37); PFK, ATP-dependent phosphofructokinase (EC 2.1.7.11); PK, pyruvate kinase (EC 1.2.1.40); RH, relative humidity; RWC, relativewater content.

Physiol. Plant. 122, 2004 11

processes occurring during rehydration is important inunderstanding the phenomenon of desiccation tolerance,the first objective of the present study was to investigateaspects of leaf sucrose metabolism, with particular empha-sis on patterns of sugar mobilization and glycolytic enzymeactivity during the rehydration of the desiccation-tolerantspecies Sporobolus stapfianus Gandoger (Poaceae).

The necessary protective mechanisms required forvegetative desiccation tolerance in angiosperm resurrec-tion species are suggested to be primarily laid downduring the course of dehydration (Kuang et al. 1995,Oliver et al. 1998, Farrant et al. 1999, Farrant 2000,Cooper and Farrant 2002). In most resurrection plantsstudied to date, the induction of desiccation toleranceproceeds only in leaves dried attached to intact plants(Gaff and Loveys 1992). However, metabolism duringthe rehydration of detached leaf material has not been aswell investigated. Hence the second objective of the studywas to examine aspects of sucrose metabolism in leafmaterial dehydrated attached to the parent plant, excisedand then rehydrated detached. The research was directedat determining whether leaf sucrose metabolism during amore rapid rehydration (48 h) in detached leaf materialwas comparable with that of intact material during aslower rehydration (10 days).

In S. stapfianus, desiccation tolerance in the leaf mater-ial is not induced during dehydration following excisionfrom the parent plant in the region between full hydra-tion or 100% relative water content (RWC) and 61%RWC (Gaff and Loveys 1992). The final objective of thestudy was aimed at investigating how sucrose metabo-lism differs in desiccation-sensitive tissue material, basedon the working premise that desiccation sensitivity isassociated with altered patterns of sugar mobilizationand glycolytic enzyme activity.

Materials and methods

Plant material and experimental conditions

Plants of Sporobolus stapfianus were grown in pots in soilcontaining 25% leaf mold and were maintained in agreenhouse. Dehydration stress was imposed by with-holding water for 18 days, after which the plants weremaintained in the dehydrated state for approximately 3weeks prior to watering. Rehydration of the desiccation-tolerant whole plant was achieved by spraying the plantswith water (to simulate rainfall) each evening, ensuringfull saturation of the soil. Over the experimental period,plants were exposed to full summer sunlight and theaverage daily temperature range, recorded within thegreenhouse, was 43.5–22.0�C. Non-senescent whole leafsamples were harvested from different plants at differentRWCs over a 10-day rehydration period. Only a portionof the leaf blade (15–20 cm) was used for the analyses.After discarding the leaf base (approximately 3 cm), andthe apical regions (beyond the 15–20 cm point), the leafsections were immediately frozen in liquid nitrogen andstored at �80�C. Well-irrigated plants of S. stapfianus

were maintained under the same environmental condi-tions, and the fully hydrated leaf material harvested atregular intervals for comparative purposes.

In a separate rehydration experiment, leaf materialwas dehydrated attached to the parent plant under green-house conditions, then detached from the parent plantand rehydrated over a 48-h period. Dehydrated, excisedleaf material (1.5–2.0 g, 15–20 cm leaf blade) was placedover a circular grid covered with moistened filter paper.Each grid was placed within a circular perspexcontainer (diameter�h¼ 14 cm� 6 cm) and the filterpaper surface kept moistened by wicks of filter papersuspended between the grid and water at the base ofthe container. The containers were maintainedunder controlled environmental conditions (27�C;100mmolm�1 s�1 photon flux density; 15 h light period).At various intervals over the rehydration period, theleaves were removed from the containers, frozen in liquidnitrogen and then stored at �80�C.

In a further experiment, aimed at preventing inductionof desiccation tolerance, leaf material at 95% RWCwas excised from the parent plant (Gaff and Loveys1992). The excised leaf material was placed upongrids (1.5–2.0 g per grid, 15–20 cm leaf blade) andsealed within circular perspex containers (diameter�h¼ 11 cm� 13 cm), and dehydrated suspended over asaturated K2SO4 solution for 48 h at a relativehumidity (RH) of 98% (Winston and Bates 1960). After48 h, the leaf material was dehydrated for a further 36 hover a solution of NH4NO3 at 68% RH (Winston andBates 1960). The dehydration was performed undercontrolled environmental conditions (27�C; 100mmolm�1 s�1 photon flux density; 15 h light period).Thereafter, the rehydration (48 h) of the detached leafmaterial was conducted as described for the leaves dehy-drated attached to the parent plant. The two excised leafrehydration experiments (leaves dried either attached ordetached from the parent) were performed simultan-eously.

At each sampling during the rehydration of all threetreatments, duplicate leaf samples were removed for thedetermination of leaf RWC. The RWC was calculatedaccording to the formula: RWC¼ (initial weight�dryweight)/(full turgor weight�dry weight). Full turgorweight was determined following a 24-h incubation ofthe tissues in sealed flasks containing water. Dry masswas determined after oven drying at 60�C for 2 days.

Viability test

At each sampling interval, duplicate leaf samples wereutilized for an assessment of relative viability, as deter-mined from the in vivo activity of the respiratory dehydro-genase complex (ISTA 1999). The principle is based oninteraction of the latter with 2,3,5-triphenyl tetra-zolium chloride, resulting in the development of a redcolour within the tissues (ISTA 1999). Leaf sections(0.5 cm) were excised from leaf blade and added to a1% (w/v) solution of 2,3,5-triphenyl tetrazolium

12 Physiol. Plant. 122, 2004

chloride. Each replicate contained a minimum of 150 leafsections representative of the entire leaf blade underinvestigation. Samples were incubated in the dark at37�C for up to 24 h (full colour development was notedwithin 6 h), after which leaf sections were scored for thepresence or absence of red staining.

Gas exchange

Respiration was measured in the dark using a Ciras-Iinfrared gas analyser with a Parkinson’s Broad LeafCuvette (25�C, 100% RH, 0 p.p.m.CO2, 223 cm

3min�1

flow rate). Leaf samples, taken at different RWCs duringrehydration, were dark adapted for 15min and measure-ments of CO2 release made over a 20-min period.

Enzyme extraction and measurement

Enzymes were extracted as described previously(Whittaker et al. 2001), in an extraction buffer contain-ing 50mM KH2PO4 (pH7.5), 0.5mM EDTA, 2mMMgCl2, 10% (v/v) glycerol and 5mM dithiothreitol(DTT). Crude extracts were desalted using 2.5ml SephadexG-25 columns (particle size 50–150mm) equilibratedwith extraction buffer. Desalted extracts were main-tained at 4�C until assayed for enzyme activity. Activ-ity was measured spectrophotometrically (340 nm) in a1-ml volume. Hexokinase (EC 2.7.1.1) activity wasmeasured as previously reported (Whittaker et al.2001). ATP-dependent phosphofructokinase (PFK, EC2.1.7.11) was measured at pH7.1 according to Botha etal. (1988). Aldolase (EC 4.1.2.13) and pyruvate kinase(PK, 2.7.1.40) were assayed as described by Moorheadand Plaxton (1988), and enolase (EC 4.2.1.11) accord-ing to the protocol of Fox et al. (1995). The activity oftotal NAD-dependent malate dehydrogenase (MDH,EC 1.1.1.37), and glutamate dehydrogenase (GDH,EC 1.4.1.2) for the deamination reaction, was mea-sured according to Kumar et al. (2000).

Soluble protein was measured in the desalted extractsusing gamma globulin as a standard (Bradford 1976).

Soluble sugar and ATP analysis

Soluble sugars and ATP were extracted from leaves usinga modified alkaline extraction procedure based on themethod of Van Schaftigen 1985) as reported previously(Whittaker et al. 2001). The sugars and ATP were mea-sured enzymatically. Sucrose, glucose and fructose weredetermined using the Roche sugar food analysis kit(Bergmeyer and Bernt 1974) and ATP according to themethod of Trautschold et al. (1985).

Statistical analysis

Differences between two separate means were analysedby the Student’s-t-test. The least significance differencetest was performed to ascertain the difference among themeans (in excess of two) within a treatment.

Results

Assessment of rehydration and viability

The rehydration time course and viability of leaves fromthe three treatments were determined at the outset(Fig. 1). Leaves from whole plants were rehydrated over10 days, and excised leaves over a 48-h period. In theformer, the rehydration rate in the first 2 days was morerapid than in the remaining 8 days (Fig. 1A). Recoverywas complete. Excised leaf material (dehydrated intact ordetached) rehydrated rapidly over the first 6 h, with therate of rehydration slowing thereafter (Fig. 1B). How-ever, only leaves dried detached regained full turgorover the 48-h period, whereas the leaves dried attachedto the parent plant rehydrated to 75% RWC.

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Fig. 1. Relative water content (RWC) over time during therehydration of (A) leaf material rehydrated attached to the parentover 10 days. (B) RWC during the rehydration of excised materialdehydrated either intact (*) or excised (,) over 48 h. The initial10% RWC data point (intact leaves) and the 14.5% RWC datapoint (excised leaves) is representative of time zero immediatelyprior to watering. The control data point (&) is representative ofleaf material that had been maintained in the hydrated state forcomparative purposes. Values are the means of two replicates andeach replicate is representative of two to three separate plants. (C)Viability (%) over time during the rehydration of excised materialdehydrated intact (*) or excised (,) as assessed by tetrazoliumstaining. Values are the means� SD of three replicates and eachreplicate is representative of two to three separate plants.

Physiol. Plant. 122, 2004 13

The relative viability of the excised tissues was assessed(Fig. 1C) as based on the in vivo respiratory dehydrogen-ase complex activity (ISTA 1999). In the leaves driedattached, tissues were uniformly stained (100%) overthe entire 48 h rehydration period (Fig. 1C). Comparableresults were obtained from the pre-desiccated, fullyhydrated leaf material (henceforth referred to as ‘con-trol’) and the desiccation-tolerant leaf material rehy-drated attached to the parent plant (results not shown).In contrast, only 65% of the leaf sections were stained inthe material dehydrated detached, with a significantdecline in viability occurring with each consecutive sam-pling during rehydration (Fig. 1C).

Since leaf material dried attached to the parent plantremained viable regardless of the manner of rehydration,these treatments are henceforth referred to as DT(desiccation-tolerant). The DT-intact leaves are represen-tative of material rehydrated attached to the parentplant, whereas the DT-excised are those leaves rehy-drated detached from the parent plant. Desiccation-sensitive excised leaves are referred to as DS-excised.To ensure accurate comparisons between sugar contentand enzyme activity, measurements were made on thesame source of leaf material. Comparisons were madeon a dry mass basis.

Sugar content

Sucrose content of DT-intact, DT-excised and DS-excised leaf material during the course of rehydration isprovided in Fig. 2A and B. In the DT-intact leaf tissue, atapproximately 50% RWC, sucrose breakdown com-menced, with no further significant breakdown evidentafter 74% RWC (Fig. 2A).

In the DT-excised leaf material (Fig. 2B), sucrose con-tent was not significantly different between 14.5 and47% RWC (time 0–12 h of rehydration, t¼ 2.62,d.f.¼ 4), despite the initiation of breakdown after 30%RWC. Significant breakdown occurred between 47 and70% RWC (Fig. 2B). In the DS-excised leaf material,sucrose content was two-fold lower than in DT-excisedmaterial (Fig. 2B). Sucrose breakdown in the DS-excisedmaterial was immediate following watering and sucrosecontent declined with increasing leaf RWC (14.5–83%,Fig. 2B).

Since the hexose sugars are the products of bothsucrose hydrolysis activity by invertase (glucose andfructose) and cleavage activity by sucrose synthase (fruc-tose), hexose sugar content was examined in relation tosucrose content during rehydration. In the DT-intactmaterial (Fig. 2C), glucose and fructose were equivalentand comparable with that of fully hydrated material.Coinciding with the period of sucrose breakdown(Fig. 2A), hexose sugar levels remained constant(Fig. 2C).

During the more rapid rehydration of DT-excised leafmaterial, hexose sugar content increased between 14.5and 48% RWC (Fig. 2D). Between 47 and 70% RWC,the hexose sugar levels remained constant (Fig. 2D). In

the DS-excised material, the increase in glucose andfructose content (Fig. 2D) was inversely proportionateto the decrease in sucrose content (Fig. 2B). Thesucrose content at time 0 (14.5% RWC) was179� 8.2 mmol g�1DM. After 30 h of rehydration (81%RWC), the sum total of sucrose and fructose (one fruc-tose molecule originating from the breakdown of eachsucrose molecule) was 168� 25 mmol g�1DM. This sug-gests no net incorporation of the hexose sugars (levels ofglucose were comparable to that of fructose) fromsucrose breakdown into metabolism. The decrease inhexose sugars between 81 and 100% RWC (between 30and 48 h after watering) cannot be attributed to incor-poration into metabolism since leaching of cellular com-ponents was noted. To a lesser extent, the decline inhexose sugars after 70% RWC in the DT-excised tissuesmay similarly reflect the same phenomenon.

Glycolytic enzyme activity

HexokinaseTo ascertain whether the trends in hexose sugars duringrehydration were correlated to hexose phosphorylating

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Fig. 2. Sucrose content in (A) desiccation tolerant intact (DT-intact) leaf material of Sporobolus stapfianus at different RWCs overa 10-day rehydration period, and (B) desiccation-tolerant excised(DT-excised; *) and desiccation-sensitive excised (DS-excised; ,)leaf material over a 48-h rehydration period. (C) Glucose (&) andfructose (&) content in DT-intact leaf material at different RWCsover a 10-day rehydration period, and (D) glucose and fructose inDT-excised (* and *) and DS-excised (. and ,) leaf materialover a 48-h rehydration period. The initial data points arerepresentative of time zero immediately prior to watering. Thecontrol data point (&) is representative of hydrated leaf material.Values are the means � SD of three to five replicates. Each replicateis representative of two to three separate plants.

14 Physiol. Plant. 122, 2004

activity, hexokinase (glucose substrate) and fructokinase(fructose substrate) were measured.

In the DT-intact leaves, hexokinase activity at theinitiation of the experiment was 4.5-fold higher thanthat of the control (Fig. 3A) indicating that an up-regulation in activity levels had occurred during dehydra-tion. During the 10-day rehydration period, hexokinaseactivity levels remained high (Fig. 3A). Similarly, inDT-excised leaves, hexokinase activity levels wereequivalent to that of the intact leaves (compareFig. 3A and B) and were constant over the 48 h rehy-dration period (Fig. 3B). Incomplete up-regulation ofhexokinase activity was evident in the DS-excised leafmaterial (compared to the control in Fig. 3A). Levels ofhexokinase activity were constant over the first 30 h ofrehydration (Fig. 3B) and significantly lower than thatreported for the two desiccation-tolerant experimentaltreatments (Fig. 3A and B).

Fructokinase (fructose substrate) activity levels in theDT-intact leaves at time 0 (10% RWC) are not differentfrom the fully hydrated control (open symbol, Fig. 3C;t¼ 2.15, d.f.¼ 4). Activity levels were variable in the DT-intact tissues. Fructokinase activity in the control (Fig. 3C)was also not significantly different from that at 14.5%RWC for the DT-excised (t¼ 0.6, d.f.¼ 6) and DS-sensitive

(t¼ 0.7, d.f.¼ 5) leaf material (Fig. 3D). Activity levelsin the DT-excised and DS-excised tissues (Fig. 3D) werecomparable, and were unchanged over rehydration.

ATP contentGiven that total cellular fructose and glucose were notpotentially limiting for hexose phosphorylating activityin the DS-excised material over the first 24 h (14.5–80.5% RWC), levels of the second substrate of hexosephosphorylating activity, namely ATP, were next exam-ined. The levels were then compared with those of theDT-excised material. Table 1 shows that despite a reduc-tion in the in vitro activity level of hexokinase in thesensitive material, the total ATP content was not signifi-cantly different from the DT-excised material prior towatering (t¼ 1.07, d.f.¼ 4) and after 24 h rehydration(t¼ 2.7, d.f.¼ 4).

PFK, aldolase, enolase and PKTo ascertain whether there was a correlation betweenglycolytic enzyme activity and the trends in sugars, themaximum catalytic activities of various glycolyticenzymes were measured. The enzymes selected includedPFK and PK, shown to be key regulatory enzymes fromclassical studies (Plaxton 1996), and those catalysingnear equilibrium reactions such as aldolase and enolase.

In the DT-intact leaf material (Fig. 4A), PFK activityprior to watering was higher than that of the control(Fig. 4A) indicating that an up-regulation in activitylevels had occurred during dehydration. During the 10-day rehydration period, PFK activity levels declined withthe increase in RWC from 14.5 to 45%, after whichlevels were constant (Fig. 4A). In the DT-excised tissues,PFK activity prior to watering was similarly higher thanthe control (Fig. 4B) and after the initial decline in activ-ity (31% RWC), PFK activity levels were variable butnot different (LSD¼ 0.32; P� 0.05). In the DS-excisedleaf material there was no apparent up-regulation ofPFK activity levels during dehydration since the activityprior to watering was comparable to the control (t¼ 2.2,d.f.¼ 6). PFK activity declined two-fold as water contentincreased to 36% RWC, after which levels remained lowover the duration of the rehydration period (Fig. 4B).Aldolase activity levels were variable and there was nodifference in the levels between the DT-intact leaves overthe 10-day period and the control (LSD¼ 0.63; P� 0.05)(Fig. 4C). Additionally, there was no significant differ-ence in aldolase activity in the DT-excised leaves over the

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Fig. 3. (A) Hexokinase activity (glucose substrate) and (C)fructokinase activity (fructose substrate) in DT-intact leaf materialat different RWCs over a 10-day rehydration period. (B)Hexokinase activity and (D) fructokinase activity in DT-Excised(*) and DS-excised (,) leaf material at different RWCs over a 48-h rehydration period. The initial RWC data point is representativeof time zero immediately prior to watering. The control data point(&) is representative of hydrated leaf material, which had beenmaintained in the hydrated state. Values are the means � SD of threeto five replicates. Each replicate is representative of two to threeseparate plants.

Table 1. Leaf ATP content at time 0 and after 24-h rehydration indesiccation-tolerant and -sensitive excised leaf material. Values arethe means� SD of three replicate extractions, each from three tofour separate plants.

DT-excised leaves DS-excised leaves

RWC ATP RWC ATP(%) (mmol g�1DM) (%) (mmol g�1DM)

14.5 3.55� 0.93 14.5 4.03� 0.7764 2.73� 0.33 80.5 4.65� 1.70

Physiol. Plant. 122, 2004 15

48-h rehydration period (LSD¼ 0.64; P� 0.05) (Fig. 4D).A decrease in the maximal catalytic rate of aldolase dur-ing dehydration occurred during drying of DS-excisedleaves (compare control in Fig. 4A and time 0 inFig. 4D). Levels in the DS-excised leaf material declineda further six-fold over the course of rehydration (Fig. 4D).Enolase activity in the DT-intact leaf material (Fig. 4E)was comparable with that in the DT-excised material(Fig. 4F) during rehydration. In the dehydrated DT-intact (Fig. 4E) and DT-excised (Fig. 4F) material priorto watering, enolase activity was higher than the control(Fig. 4E), indicating up-regulation of maximal catalyticlevels during dehydration. However, in the sensitivematerial, activity levels declined 2.5-fold over the courseof rehydration and were lower than that of the DT-excised leaves (Fig. 4F).

PK activity levels over the course of rehydration werenot significantly different within the DT-intact(LSD¼ 0.71; P� 0.05, apart from the 50% RWC datapoint) and DT-excised (LSD¼ 0.70; P� 0.05) tissues.There was no significant difference in PK activitybetween the control and DT-excised leaves prior towatering (t¼ 1.08, d.f.¼ 5). Activity levels between theDT-intact and DT-excised material were also compar-able (compare Fig. 5A and B). PK activity in the DS-excised material declined five-fold over the rehydrationperiod (Fig. 5B).

MDH and GDH activityNAD-dependent MDH activity, responsible for catalys-ing the interconversion of OAA and malate in the cytosoland mitochondria, as well as mitochondrial GDH, sug-gested to regulate the entry of amino acids into the TCA

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Fig. 4. Activity of (A) PFK, (C) aldolase and (E) enolase in DT-intact leaf material of S. stapfianus at different RWCs over a 10-dayrehydration period. Activity of (B) PFK, (D) aldolase and (F)enolase in DT-excised (*) and DS-excised (,) leaf material atdifferent RWCs over a 48-h rehydration period. The initial RWCdata point is representative of time zero immediately prior towatering. The control data point (&) is representative of hydratedleaf material. Values are the means � SD of three to five replicates.Each replicate is representative of two to three separate plants

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Fig. 5. Activity of (A) PK, (C) MDH and (E) GDH in DT-intactleaf material of S. stapfianus at different RWCs over a 10-dayrehydration period. Activity of (B) PK, (D) MDH and (F) GDH inDT-excised (*) and DS-excised (,) leaf material at differentRWCs over a 48-h rehydration period. The initial RWC data pointis representative of time zero immediately prior to watering and thecontrol data point (&) is representative of fully hydrated tissues.Values are the means � SD of three to five replicates. Each replicateis representative of two to three separate plants.

16 Physiol. Plant. 122, 2004

cycle under conditions of carbon limitation from glyco-lysis (Robinson et al. 1991) were measured.

NAD-dependent MDH activity in the DT-intact mater-ial was variable over the period of rehydration (Fig. 5C).Activity in the DT-excised and DS-excised material wascomparable over the first 24 h after watering, after whichlevels declined two-fold near full hydration in the DS-excised leaves (Fig. 5D).

After an initial decline in activity after 18% RWC(1.7-fold), GDH levels were constant throughout therehydration (Fig. 5E). Levels of GDH activity were com-parable between the DT-excised and DS-excised leafmaterial (Fig. 5F).

Protein contentSoluble protein contents were included to demonstratethat the lower glycolytic enzyme activities in the DStissues over the rehydration period, in comparison tothe DT tissues, were not attributable to a correspondinglower level of protein (Table 2). There is an increase inprotein content in DT dry material in comparison withthat of fully hydrated control tissues. After watering,there is an overall decline in protein content in all threeexperimental treatments (leaching was evident after81.2% in DS tissues).

Respiration

Dark respiration was measured only in the DT- and DS-excised leaves (Fig. 6). In the DT-excised leaf material,the rate of dark respiration increased immediately afterwatering (Fig. 6). Increasing rates of respiratory activitywere also evident during the first 24 h of watering in theDS-excised material (Fig. 6). Thereafter, negligible levelsof respiration were recorded.

Discussion

The pattern of sucrose breakdown and associated glyco-lytic enzyme activity levels during the rapid rehydration(48 h) of DT-excised leaves is similar to that in intactleaves from whole plants rehydrated over 10 days.Furthermore, the rehydration of DT-excised tissue was

shown to provide a suitable control with which to com-pare sucrose metabolism in DS-excised material (rehy-drated under the same conditions), thereby providingadditional information into the processes associatedwith the phenomenon of desiccation tolerance.

In DT material, whether rehydrated intact or excised,significant breakdown of sucrose is not initiated immedi-ately after rewatering. A rapid metabolism of sucrose dur-ing rehydration, as reported by Scott (2000) for otherresurrection plants, is not evident for S. stapfianus, suggest-ing that sucrose is not an immediate energy source duringthe initial phase of rehydration. The possibility that inS. stapfianus, a significant delay in the breakdown ofsucrose may serve as an effective protective mechanism toensure that sucrose is only metabolized at a point wherewater availability for full rehydration has been guaranteedwill require investigation. Previous investigations haveindicated that the role of sucrose in this species may pri-marily reside in the protection of proteins and the func-tional integrity of enzymes, in stabilizing membranes andcontributing to cellular osmoregulation during dehydra-tion (Schwab and Gaff 1990, Ghasempour et al. 1998,Hartung et al. 1998, Oliver et al. 1998).

In the DT-intact and -excised leaf tissues, the hexosesugars do not accumulate following sucrose breakdown,suggesting incorporation into metabolism. Additionally,the dehydrogenase complex activity, influenced by the invivo physiological conditions of the tissue, is shown to befunctional during the course of dehydration in both DT-intact and -excised leaf tissues. Although detached leavescannot sustain metabolism, the DT-excised leaves(within the 48-h period) follow a similar trend in sugarpatterns and enzyme activity to that of the intact tissues,in which in vitro activity levels of hexokinase, aldolase,enolase PK, MDH and GDH activity are comparableand sustained over the period of rehydration. Therefore,metabolism during rehydration in DT-excised leaves, asinvestigated by the present study, appears independent ofproposed hormonal control originating from the roots,which contrasts with the situation during dehydration(Gaff and Loveys 1992).

The sucrose content in the dry DT-excised tissues ismore comparable with that reported for dry S. stapfianusleaf tissue in a previous study (Whittaker et al. 2001) than

Table 2. Soluble protein content at different RWCs during the rehydration of DT-intact leaf material over 10 days, and DT-excised and DS-excised leaf material over 48 h. The initial RWC data point (intact) is representative of time zero immediately prior to watering. The controldata point is representative of leaf material, which had been maintained in the hydrated state for comparative purposes. Values are the means� SD of three to four replicates. Each replicate is representative of two to three separate plants.

DT-intact leaves DT-excised leaves DS-excised leaves

RWC Protein content RWC Protein content RWC Protein content(%) (mg g�1DM) (%) (mg g�1DM) (%) (mgg�1DM)

10 47.4� 4.9 14.5 54.3� 5.26 14.5 34.0� 9.1020 39.4� 10.3 31.1 55.9� 19.5 36.3 37.4� 5.2745 34.9� 2.06 48.4 54.5� 13.0 51.7 36.8� 3.2950 35.8� 1.82 47.0 48.1� 4.69 69.7 31.1� 1.2274 32.0� 9.14 63.6 54.5� 6.67 80.5 30.8� 2.4383 32.5� 8.85 70.1 40.0� 8.15 81.2 23.6� 3.07Control 24.8� 8.0 75.2 41.9� 6.71 100 ND

Physiol. Plant. 122, 2004 17

that of the DT-intact tissues. Levels in sucrose contentaccumulated during dehydration have been shown tovary both within and among different desiccation-tolerantspecies, and this difference is not shown to influence theability of the respective species to revive following rehydra-tion (Ghasempour et al. 1998,Whittaker et al. 2001). In theDT-excised leaf tissues, sucrose content was not brokendown to the basal levels reported for the DT-intact leaves.It is probable that within a more rapid time period, theextent of sucrose catabolism may have been restricted bythe catalytic activity rates of the enzymes required to meta-bolize the products. In the dehydration process, thechanges implemented to induce tolerance are dependenton the rate of dehydration (Farrant et al. 1999, Farrant2000, Cooper and Farrant 2002). Though the more rapidrate of rehydration imposed on the DT-excised tissues wasnot shown to affect viability, enzyme activity or the processof sucrose catabolism (at least within the initial 48 h), therate of the latter would be subject to the activities of therespective enzymes. Fructokinase and PFK activitiesbetween the DT-intact and excised tissue are not consistentand the physiological significance of this is not known.

Respiratory activity is initiated and proceeds rapidlyfollowing watering in desiccation-tolerant leaf materialof S. stapfianus. Similar results have been obtained forother resurrection species (Schwab et al. 1989, Hartunget al. 1998, vander Willigen et al. 2001). Interestingly,since sucrose is not significantly broken down immedi-ately, it is evident that respiratory mitochondrial respir-ation must also be reliant on the utilization of alternatesubstrates during the initial phase of rehydration as neg-ligible levels of hexose sugars are present in the dry DTtissues. Amino acids, which are shown to accumulateduring drought stress (Lawlor and Cornic 2002) andalso during the dehydration of resurrection plants(Tymms and Gaff 1978, Gaff and McGregor 1979,Hartung et al. 1998) may serve as important substratesfor the initial induction of respiratory activity in DT leafmaterial. Metabolism surrounding the accumulationand subsequent utilization of amino acids during dehy-

dration and rehydration, respectively, is currently underinvestigation.

Additionally, since hexose sugars originating fromsucrose breakdown are not incorporated into meta-bolism in DS leaf material, it is also feasible that aminoacids may have served as respiratory substrates duringthe initial phase of rehydration in the latter. The max-imum catalytic levels of the enzymes associated with themitochondria (MDH and GDH) were comparablebetween the DT and DS tissues over the first 24 h ofrehydration for MDH, and over the entire period forGDH. The maintenance of MDH and GDH activitymay be associated with the presence of respiratory activ-ity, evident within the first 24 h of rehydration in thesensitive tissues. In particular, the deamination of gluta-mate by GDH is shown to support aminotransferaseactivity in funnelling carbon from glutamate into theTCA cycle (Aubert et al. 2001). A role for GDH insustaining TCA cycle activity under stress has been pro-posed (Robinson et al. 1991, Kumar et al. 2000).

In corroboration with earlier reports, leaf material ofS. stapfianus dried excised from the parent plant wasshown not to be viable (Gaff and Loveys 1992, Quartacciet al. 1997). Desiccation sensitivity, became increasinglymore evident as rehydration proceeded in DS leaves. Thepresent study specifically outlines differences in sucrosemetabolism in sensitive tissues and clearly demonstratesa reduction in glycolytic enzyme activity followingdehydration (and subsequent rehydration), perhapsdue to various factors which may have included thecessation of probable hormonal signalling between theroot and shoot system responsible for the induction ofdesiccation tolerance (Gaff and Loveys 1992) and areduction in the pattern of total in vivo protein changesduring dehydration (Kuang et al. 1995). The possibilityof this altered metabolism also being a consequence ofwounding prior to dehydration of these tissues cannot benegated.

The present study indicates that despite an immediatebreakdown of the sucrose in the DT-excised tissues,glucose and fructose are not incorporated into the hexosemonophosphate pool. Although the in vitro fructokinaseactivity levels among the DT and DS tissues duringrehydration are comparable, hexokinase activity is sig-nificantly lower in the sensitive tissues. The presentresults confirm earlier findings showing an incompleteinduction of hexokinase activity levels in leaf materialdried detached from the plant (Whittaker et al. 2001).The reduced level of hexokinase activity in conjunctionwith the associated lack of hexose sugar phosphorylationin the DS-excised tissues suggests an important role ofhexokinase (being the entry point of hexose sugars intoglycolysis) during rehydration. Inhibition of hexokinaseactivity has previously been shown to be associated withlower cellular levels of ATP (Bouny and Saglio 1996). Incontrast, in the present study it was shown that totalATP content over the period of hexose sugar accumula-tion in the DS leaf material was equivalent to that of thetolerant material. The maintenance of constant ATP

00 20 40 60

Relative water content (%)

80 100

10

20

CO

2 re

leas

e (n

mol

s–1

g–1

DM

)

Fig. 6. Dark respiration in DT-excised (*) and DS-excised (,) leafmaterial at different RWCs over a 48-h rehydration period. Valuesare the means � SD of three replicates. Each replicate isrepresentative of two to three separate plants.

18 Physiol. Plant. 122, 2004

levels during rehydration despite the increase in respir-ation may reflect an ATP-increased utilization.

There is a positive correlation between reduced max-imal catalytic levels of hexokinase and glycolytic enzymeactivity in the DS leaf tissues. A significant level ofproposed control over glycolytic activity has beenascribed to hexokinase (Tretheway et al. 1998), however,without specifically down-regulating hexokinase activity,it is not possible to predict a cause and effect relationshipbetween reduced hexokinase and glycolytic activity in theDS tissues. In the DS tissues, the level of activity (PFK,aldolase and enolase) in the dehydrated state prior torehydration was already lower than in the DT tissue,with rehydration simply exacerbating the further declinein activity levels. The lower rates of glycolytic enzymeactivity over the course of rehydration in the DS tissuesis not attributable to a corresponding lower level ofprotein since protein contents in the intact and DStissues are comparable.

In conclusion, the present study shows that leafsucrose metabolism was similar between DT materialrehydrated either excised or attached to the parentplant, suggesting that mechanisms controlling sucroseutilization and metabolism are laid down during dehy-dration. In contrast, desiccation-sensitivity is correlatedwith an inability to metabolize hexose sugars fromsucrose breakdown and a significant decline in glycolyticenzyme activity.

Acknowledgements – The project was funded by a grant from theNational Research Foundation (NRF) to J.f. in South Africa, andby funding to A.B. and C.V. from the Italian Ministry of Educa-tion, University and Research. Thanks are extended to the ClaudeLeon Harris Foundation for a postdoctoral bursary to A.W. inSouth Africa, and to the University of Florence for a contractworking position in Italy.

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Edited by J. J. Schjørring

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