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Journal of Experimental Botany, Vol. 52, No. 358, pp. 961±969, May 2001

Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa Anne Whittaker1, Adriana Bochicchio2, Concetta Vazzana2, George Lindsey1 and Jill Farrant1,3 1

Department of Biochemistry, University of Cape Town, Private Bag Rondebosch 7700, South Africa Dipartimento di Scienze Agronomiche e Gestione del Territorio Agroforestale; UniversitaÁ di Firenze, Piazzale delle Cascine 18, I-50144 Firenze, Italia 2

Received 16 June 2000; Accepted 15 December 2000

Abstract

Introduction

The phosphorylation of glucose and fructose is an important step in regulating the supply of hexose sugars for biosynthesis and metabolism. Changes in leaf hexokinase (EC 2.7.1.1) activity and in vivo metabolite levels were examined during drying in desiccation-tolerant Sporobolus stapfianus and Xerophyta viscosa. Leaf hexokinase activity was significantly induced from 85% to 29% relative water content (RWC) in S. stapfianus and from 89% to 55% RWC in X. viscosa. The increase in hexokinase corresponded to the region of sucrose accumulation in both species, with the highest activity levels coinciding with region of net glucose and fructose removal. The decline of hexose sugars and accumulation of sucrose in both plant species was not associated with a decline in acid and neutral invertase. The increase in hexokinase activity may be important to ensure that the phosphorylation and incorporation of glucose and fructose into metabolism exceeded production from potential hydrolytic activity. Total cellular glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) levels were held constant throughout dehydration. In contrast to hexokinase, fructokinase activity was unchanged during dehydration. Hexokinase activity was not fully induced in leaves of S. stapfianus dried detached from the plant, suggesting that the increase in hexokinase may be associated with the acquisition of desiccation-tolerance.

Desiccation tolerance is the ability of cells to survive desiccation and revive from the air-dried state (Bewley, 1979). Resurrection plants are desiccation-tolerant angiosperms which exhibit vegetative tissue tolerance to intensive dehydration and subsequent rehydration from the air-dried state (Gaff, 1971). The synthesis and accumulation of soluble sugars during dehydration plays a major role in subcellular desiccation tolerance. Sugars are proposed to stabilize membranes, protect proteins, and contribute to cellular osmoregulation during stress (Hartung et al., 1998; Oliver et al., 1998). Sucrose is the predominant sugar accumulated in the dehydrated leaf tissue of all resurrection plants studied to date (Kaiser et al., 1985; Ghasempour et al., 1998; Scott, 2000). An understanding of the regulation of sucrose synthesis and accumulation is therefore an important requisite in understanding the physiology of desiccation-tolerance. Resurrection plants provide unique model systems to investigate sucrose metabolism in response to severe drought stress. Recently, it has been reported that both sucrose phosphate synthase (SPS, Ingram et al., 1997) and sucrose synthase (SuSy, Kleines et al., 1999) expression are induced during the period of sucrose accumulation in Craterostigma plantagineum. Since respiration is maintained during the early and intermediate phases of dehydration stress (Schwab et al., 1989; Hartung et al., 1998; Tuba et al., 1998; Farrant, 2001), the increase in SuSy was also proposed to be important in the supply of carbon from sucrose breakdown for glycolysis during dehydration anduor following rehydration (Kleines et al., 1999). The carbon requirement for sucrose accumulation (and metabolism) in dehydrating leaves is

Key words: Desiccation-tolerant, fructokinase, hexokinase, metabolites, resurrection plants. 3

To whom correspondence should be addressed. Fax: q27 21 65 04041. E-mail: [email protected]

ß Society for Experimental Biology 2001

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proposed to originate from various potential sources. These include triose phosphates from photosynthesis, the complete utilization of carbohydrate storage reserves in source tissues, and reserve mobilization from sink tissues (Ghasempour et al., 1998; Hartung et al., 1998; Norwood et al., 1999; Scott, 2000). In the initial phase of drought stress in Sporobolus stap®anus, there is a concomitant increase in sucrose, fructose and glucose in leaf tissue (Ghasempour et al., 1998). Although the two hexose sugars are equimolar in fully hydrated tissue, glucose accumulation signi®cantly surpasses that of fructose as the plant is dried from 100% to 50% relative water content (RWC, Ghasempour et al., 1998). An increased glucose to fructose ratio is suggested to be indicative of increased amylolytic starch breakdown (HaÈusler et al., 1998; Veramendi et al., 1999). In the later stages of dehydration (below 50% RWC), both hexose sugars decline with a further accompanying increase in sucrose accumulation (Ghasempour et al., 1998). In addition to starch degradation, free hexose sugars in leaf material may also be generated from imported sucrose. Invertase as well as SuSy activity in leaf material may also produce glucose and fructose from the hydrolysisu cleavage of some storage sucrose. An increased expression of vacuolar acid invertase at the onset of drought stress has been reported in maize leaves (Pelleschi et al., 1997). The reaction catalysed by hexokinase is a key step regulating the entry of hexose sugars into primary carbon metabolism for both biosynthesis and sucrose storage. Thus, the reaction catalysed by hexokinase is likely to be important during dehydration. Hexokinases (EC 2.7.1.1) catalyse the conversion of glucose, and with a lower ef®ciency fructose, to hexose monophosphates using ATP as the preferred phosphoryl donor (Doehlert, 1989; Schnarrenberger, 1990; Renz and Stitt, 1993). Multiple forms of hexokinase occur in plant tissue, and include isozymes speci®c for either glucose (glucokinases) or fructose (fructokinases). Although the majority of information on hexose phosphorylating activities in plants is derived from studies on sink tissues, both hexokinases and fructokinases have been reported in leaf material (Baldus et al., 1981; Schnarrenberger, 1990; Renz et al., 1993). Studies suggest that in leaf tissue, hexokinase is predominantly located in the cytoplasm or bound to the outer envelope of chloroplasts (Baldus et al., 1981; Schnarrenberger, 1990; Wiese et al., 1999). The aim of the present study was to investigate hexokinase activity as well as the metabolite content during dehydration in the two monocotyledonous plant species Sporobolus stap®naus Gandoger (Poaceae) and Xerophyta viscosa Baker (Velloziaceae). Both plant species are considered fully desiccation-tolerant (Kuang et al., 1995; Sherwin and Farrant, 1998). In S. stap®anus, there is a partial loss of chlorophyll in leaf tissues during dehydration

(intermediate homiochlorophyllous, Quartacci et al., 1997), whilst in X. viscosa, thylakoid membranes are dismantled and chlorophyll is completely degraded within the intermediate phase of dehydration (poikilochlorophyllous, Sherwin and Farrant, 1998). In the present study, an increase in leaf hexokinase activity is reported during dehydration of both plant species.

Materials and methods Plant material and experimental conditions Plants of S. stap®anus were grown and maintained in a heated glasshouse in pots on sand containing 25% leaf mould. Plants were well watered prior to the dehydration experiments. X. viscosa was grown and maintained in a glasshouse as described earlier (Sherwin and Farrant, 1996, 1998). Dehydration stress was imposed on plants by withholding water. Non-senescent whole leaf samples from different plants were removed between 09.00 h and 10.00 h for sugar and enzyme analysis at different RWCs during dehydration. The leaf material was immediately frozen in liquid nitrogen and stored at 80 8C. At each sampling, duplicate leaf samples were removed for the determination of leaf RWC which was calculated according to the formula: RWC ˆ initial weight±dry weightufull turgor weight±dry weight (Ghasempour et al., 1998). Following dehydration plants were watered and allowed to rehydrate. All plants rehydrated within 72 h. Hexokinase extraction and measurement

Crude extracts were made by grinding leaf material (0.15±0.50 g) to a ®ne powder with liquid nitrogen in the presence of an equivalent mass of insoluble polyvinylpolypyrrolidone (PVP). The ground tissue was added to ice-cold extraction buffer containing 20 mM KH2PO4 (pH 7.5), 0.5 mM EDTA and 5 mM dithiothreitol (DTT) in a buffer volume to tissue mass ratio of 20 : 1. For tissue extracts sampled from plants in the late stages of drying, the buffer volume to tissue mass ratio was increased to 50 : 1. Extracts were homogenized for 1 min and then centrifuged for 20 min at 17 200 g. The protein extract was immediately desalted using 2.5 ml Sepahdex G-25 columns (particle size 50±150 mm) equilibrated with extraction buffer. Hexokinase activity was measured spectrophotometrically in a 1.0 ml volume. The standard reaction contained 100 mM KH2PO4 (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 0.4 mM NAD, 1 mM ATP, 1.0 IU glucose-6-phosphate dehydrogenase (G6PDH, from Leuconostoc mesenteriodes), 1.0 IU phosphoglucose isomerase (PGI), and 5 mM glucose or 5 mM fructose. Total protein was measured in crude leaf extracts according to the method of Bradford, using gamma globulin as a standard (Bradford, 1976). Invertase extraction and measurement

Soluble invertase was extracted using the same protocol described for hexokinase extraction. Crude extracts were desalted and soluble invertase activity measured according to Albertson et al. (Albertson et al., 2001). Soluble invertase activity was initially measured at 0.5 pH increments from pH 3.0±8.0. Peak activities were recorded at pH 5.0 (acid invertase) and pH 7.0±7.5 (neutral invertase). Thereafter, invertase activity

Carbohydrate metabolism in resurrection plants measurements on the dehydrating leaf material of both plant species were recorded at pH 5.0 and pH 7.0. Soluble sugar and metabolite analysis

Soluble sugars and metabolites were extracted using a modi®ed alkaline extraction procedure, based on the method of van Schaftigen (van Schaftigen, 1985). Ground tissue (0.15±0.35 g) was extracted with 100 mM NaOH in 50% (vuv) ethanol to denature proteins. Chloroform was then added to 15% (vuv) of the total extraction volume. After incubation on ice for 10 min, tissue extracts were adjusted to pH 7.5 with 100 mM (vuv) glacial acetic acid in 100 mM HEPES. The total buffer volume to tissue mass ratio ranged from 8 : 1 (hydrated tissue) to 20 : 1 (dehydrated tissue). Samples were centrifuged for 20 min at 28 000 g, the supernatants removed and the pellet fractions extracted for a second time. The two supernatants from each sample were combined. Chloroform was added to 15% of the volume and the extracts centrifuged. Metabolite measurements were made immediately after extraction. The soluble sugars and metabolites were measured enzymatically. Sucrose, glucose and fructose were determined using the Boehringer Mannheim sugar food analysis kit (Bergmeyer and Bernt, 1974). Sequential measurements of glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) were made in a 1.0 ml reaction volume, according to a modi®cation of the method by Lang and Michal (Lang and Michal, 1974). The standard reaction contained 100 mM KH2PO4 (pH 7.5), 5 mM MgCl2, 0.33 mM NAD, 1% (wuv) soluble PVP, 0.5 IU G6PDH (from L. mesenteriodes), and 0.7 IU PGI. ATP determinations were made using hexokinase, based on the method described previously (Trautschold et al., 1985). The standard 1.0 ml reaction contained 100 mM KH2PO4 (pH 7.5), 6.6 mM MgCl2, 0.33 mM NAD, 1% (wuv) soluble PVP, 0.5 IU G6PDH (from L. mesenteriodes), 50 mM glucose, and 1.8 IU hexokinase. ADP was determined in a 1.0 ml reaction using pyruvate kinase (PK), according to the modi®ed method of Jaworek and Welsch (Jaworek and Welsch, 1985). The assay mixture contained 100 mM KH2PO4 (pH 7.0), 100 mM KCl, 33 mM MgSO4, 0.18 mM NADH, 1% (wuv) soluble PVP, 2.5 IU lactate dehydrogenase (LDH), and 2.0 IU PK. The extraction ef®ciency for each metabolite was determined from the recovery of exogenous metabolite added to the both hydrated and dehydrated leaf samples of S. stap®anus and X. viscosa. The percentage recovery of added metabolites in both plant species was: sucrose, 106.7"11.3; glucose, 104.4"10.1; fructose, 101.8"10.0; Glc-6-P, 108.5"17.2; Fru-6-P, 94.6"9.1; ATP, 93.5"11.1; ADP, 83.3"10.1.

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no further change in sucrose content below 29% RWC. The increase in glucose and fructose was equimolar between 100% and 85% RWC (Fig. 1). Over the 3 d period following the decline in water content from 85% to 64% RWC, glucose accumulation (29.9 nmol min 1 g 1 DW) exceeded that of fructose (6.39 nmol min 1 g 1 DW). From 64% to 29% RWC both hexose sugars declined (Fig. 1), with a net glucose removal rate of 20.9 nmol min 1g 1 DW and a net fructose removal rate of 6.95 nmol min 1 g 1 DW. The sum total of the rate of net glucose and fructose removal was equivalent to the rate of sucrose accumulation. In X. viscosa, sucrose content was consistent over the ®rst 5 d of dehydration as water content declined from 100% to 89% RWC (Fig. 2). Sucrose content then accumulated 2-fold from 89% to 55% RWC. The rate of accumulation over the 4 d period was 16.3 nmol min 1 g 1 DW. There was no further signi®cant increase in the sucrose levels below 55% RWC. In contrast with S. stap®anus, glucose and fructose levels were low. Glucose peaked at 78% RWC and then declined (Fig. 2) at a rate of 6.5 nmol min 1 g 1 DW. Fructose content was unchanged between 100% and 78% RWC, before declining to the same level as glucose at 55% RWC (Fig. 2).

Results To determine the relationship between sugar content and invertase and hexokinase activity, plants of S. stap®anus and X. viscosa were dried over an 18 d and 21 d period, respectively. Measurements were then made on the leaf material at various RWCs during dehydration. The sucrose content in S. stap®anus was unchanged in the ®rst ®ve days of dehydration as water content declined from 100% to 85% RWC (Fig. 1). Thereafter, there was a 5-fold increase in the sucrose content between 85% and 29% RWC. The rate of sucrose accumulation over the 8 d period was 27.0 nmol min 1 g 1 DW. There was

Fig. 1. Sucrose ($), glucose (j) and fructose (m) in leaf material of Sporobolus stap®anus at different RWCs over an 18 d dehydration period. Values are the means"SD of 4±6 replicates. Each replicate is representative of 2±3 separate plants.

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Fig. 2. Sucrose ($), glucose (j) and fructose (m) in leaf material of Xerophyta viscosa at different RWCs over a 21 d dehydration period. Values are the means"SD of 4±6 replicates. Each replicate is representative of 2±3 separate plants.

The change in total hexokinase and fructokinase activity was examined in relation to sugar content during dehydration. To ensure accurate comparisons between enzyme activity and sugar content, hexokinase and fructokinase were measured in the same source of leaf material used to determine the sugar content. Comparisons were made on dry mass basis. Hexokinase activity in S. stap®anus was shown to increase signi®cantly between 85% and 29% RWC on a dry mass basis (Fig. 3A). The level of activity increased 2.4-fold from 85% to 64% RWC (during the transient increase in hexose sugars) and a further 4.5-fold from 64% to 29% RWC (coinciding with the net removal of the hexose sugars, compare Fig. 1 and Fig. 3A). Within the region of hexose sugar removal, there was a rapid 3-fold induction in hexokinase activity from 37% to 29% RWC. The trend in hexokinase activity expressed on a soluble protein basis (speci®c activity) was similar to the trend on a dry mass basis shown in Fig. 3A. However, the increase in speci®c hexokinase activity between 37% and 29% RWC was lower due to a 1.5-fold higher protein content in the leaf material between 29% and 8% RWC (results not shown). To con®rm the signi®cant increase in hexokinase activity during the late stages of dehydration, a separate drying experiment was performed. Plants were dried from the hydrated state to 18% RWC over

Fig. 3. Hexokinase activity (glucose substrate) in (A) Sporobolus stap®anus and (B) Xerophyta viscosa leaf tissue at different RWCs over an 18 d and a 21 d dehydration period, respectively. Enzyme activity is expressed as a function of dry mass. Values are the means"SD of 4 replicates, each from 2±3 plants.

a 27 d period. Leaf hexokinase activity was shown to increase approximately 3-fold from 72% to 43% RWC and a further 2-fold to 28% RWC. Consistent with the result in Fig. 3A, activity then declined 1.5-fold to 18% RWC (results not shown). In S. stap®anus, the rate of hexokinase activity on a dry mass basis was signi®cantly higher than the rate of sucrose accumulation and the rate of net hexose sugar removal (compare Fig. 1 and Fig. 3A). In X. viscosa, hexokinase activity increased 2-fold between 89% and 55% RWC (Fig. 3B). There was no signi®cant increase in activity below 55% RWC. The same 2-fold increase was evident when hexokinase activity was expressed on a soluble protein basis. The rate of hexokinase activity in X. viscosa exceeded both the rate of sucrose accumulation between 89% and 55% RWC, and the rate of net hexose sugar removal in the

Carbohydrate metabolism in resurrection plants

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78% to 55% RWC period (compare Fig. 2 and Fig. 3B). The 2-fold increase in hexokinase activity was con®rmed in a separate drying experiment. Fructokinase activity in S. stap®anus was not signi®cantly different, with the exception of the 29% RWC interval (Table 1). However, speci®c fructokinase activity at 29% RWC was not signi®cantly different from the hydrated control (results not shown). There was no signi®cant change in fructokinase activity during dehydration in X. viscosa. In the initial stages of dehydration, the fructokinase : hexokinase ratio averaged 5 : 1 (100± 85% RWC) in S. stap®anus and 2 : 1 (100±89% RWC) in X. viscosa. Coinciding with the peak in hexokinase activity levels during dehydration, the fructokinase : hexokinase ratio decreased to 0.8 : 1 in S. stap®anus and to 1 : 1 in X. viscosa. To verify the involvement of increased hexokinase activity in the observed trend of sugar content (Figs 1, 2), it was necessary to measure soluble acid and neutral invertase activity during dehydration. Invertase activity produces equimolar quantities of glucose and fructose from sucrose in both the vacuole (acid invertase) and cytoplasm (neutral invertase). Acid and neutral invertase activities were measured in the same source of leaf material used to determine the sugar content and hexokinase activity. In S. stap®anus, from 100% to 85% RWC, there was a signi®cant increase in both soluble acid and neutral invertase activity concomitant with the increase in hexose sugars (compare Fig. 1 and Table 2). Acid invertase activity was consistently higher than neutral invertase activity in both the hydrated and in dehydrating tissues. During the period of net sucrose accumulation in S. stap®anus (85% to 29% RWC), levels of both acid and neutral invertase activities were maintained and were not signi®cantly different, respectively (compare Fig. 1 and Table 2). In X. viscosa, both acid and neutral invertase activities were not signi®cantly different and

invertase remained unaltered during the period of sucrose accumulation (compare Fig. 2 and Table 2). Leaves of S. stap®anus dried detached from the parent plant are desiccation-sensitive (Kuang et al., 1995). In order to determine whether increased hexokinase activity is associated with the acquisition of drought tolerance, hexokinase activity was measured in leaves dried detached from the parent plant (Fig. 4). The detached leaves were dried to a RWC below 40% since the highest levels of hexokinase activity were recorded in this stage of dehydration in plants dried intact. Hexokinase activity in detached leaves dried over a 6 h period to 25% RWC was not signi®cantly different from the hydrated control (Fig. 4). Hexokinase activity was 2-fold higher in leaves dried to 32% RWC over a slower drying period of 55 h. The substrate, product and cofactor contents of the hexokinase reaction were determined to investigate changes in the in vivo reaction components. A separate drying experiment was conducted and the metabolite contents determined in the leaf material at various RWC intervals during dehydration. In S. stap®anus, glucose and fructose content were shown to increase, peak at 50% RWC and then decline (Fig. 5A, B). Glc-6-P and Fru-6-P contents were not signi®cantly different during dehydration (Fig. 5C, D). The mean cellular Glc-6-PuFru-6-P quotient was 2.0. ATP content increased and was signi®cantly higher at 50% RWC than in the control (Fig. 5E). Thereafter, the ATP and ADP content declined (Fig. 5E, F). The total cellular ATPuADP quotient averaged 2.6 between 100% and 26% RWC, and declined to 2.2 at 8% RWC. In X. viscosa, glucose and fructose levels declined from 100% to 8% RWC (Fig. 6A, B). Glc-6-P and Fru-6-P content was unchanged during dehydration (Fig. 6C, D) and the total cellular Glc-6-PuFru-6-P quotient was 2.1. Similar to S. stap®anus, ATP and

Table 1. Leaf fructokinase activity on a dry mass basis at various leaf RWC intervals in Sporobolus stap®anus and Xerophyta viscosa

Soluble invertase activity was measured in the same leaf extracts as hexokinase activity. Values are the means"SD of 4 replicate extractions, each from 2±3 separate plants.

Fructokinase activity was measured in the same leaf extracts as hexokinase activity. Values are the means"SD of 4 replicate extractions, each from 2±3 separate plants.

RWC (%)

Sporobolus stap®anus

Xerophyta viscosa

RWC (%) Fructokinase activity RWC (%) Fructokinase activity (mmol min 1 g 1 DW) (nmol min 1 g 1 DW) 100 85 64 37 29 8

440"134 342"63.0 295"75.4 343"15.7 808"6.64 338"24.6

100 89 78 55 33 7

0.83"0.26 1.34"0.27 1.13"0.31 1.56"0.25 1.37"0.19 1.68"0.12

Table 2. Leaf acid invertase (pH 5.0) and neutral invertase (pH 7.0) on a dry mass basis at various leaf RWC intervals in Sporobolus stap®anus and Xerophyta viscosa

Acid invertase activity (nmol min 1 g 1 DW)

Sporobolus stap®anus 100 46.3"4.9 85 147"32.3 64 138"35.5 37 119"32.7 29 195"49.8 Xerophyta viscosa 100 27.5"3.7 89 39.0"6.4 78 36.2"10.5 55 32.7"4.1 33 27.1"5.1

RWC (%)

Neutral invertase activity (nmol min 1 g 1 DW)

100 85 64 37 29

19.7"2.6 51.7"5.5 62.9"15.5 39.2"7.5 62.7"35.0

100 89 78 55 33

25.8"4.3 32.9"7.7 28.2"4.4 23.7"4.5 20.0"3.0

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Fig. 4. Comparison of hexokinase (glucose substrate) activity in detached hydrated leaves (control) and leaves dried detached from Sporobolus stap®anus. Leaves were dried over silica gel in a desiccator (100 mmol m 2 s 1 photon ¯ux density and 27 8C) to 45% RWC and 25% RWC for 3 h and 6 h. Leaf material dried for 55 h was incubated (100 mmol m 2 s 1 photon ¯ux density and 27 8C) over saturated K2SO4 for 48 h and then NH4NO3 for 7.5 h. Values are the means"SD of 3 replicates from 3 plants.

ADP content increased signi®cantly from 100% to 59% RWC, and then remained constant (Fig. 6E, F). The total cellular ATPuADP quotient was 2.8 (between 100% and 59% RWC) before declining to 2.0 (between 43% and 13% RWC).

Discussion

Fig. 5. Levels of metabolites in dehydrating leaf tissue from Sporobolus stap®anus at 100%, 64%, 50%, 26%, and 8% RWC. (A) Glucose, (B) fructose, (C) Glc-6-P, (D) Fru-6-P, (E) ATP, and (F) ADP. Results are the means"SD of 4 replicates, each comprising 2±3 plants.

From the present investigation, hexokinase activity is shown to increase signi®cantly during the period of sucrose accumulation in both S. stap®anus and X. viscosa. Since neither sucrose nor hexose sugars accumulate extensively in source tissues, hexokinases were traditionally considered of minor importance in leaf material (Schnarrenberger, 1990) and were therefore not as well investigated as in sink tissues (Schnarrenberger, 1990; Veramendi et al., 1999). However, the accumulation of sucrose does occur in resurrection plants and is important in the acquisition of desiccation tolerance (Ghasempour et al., 1998; Hartung et al., 1998; Scott, 2000). Additionally, the transient accumulation of glucose and fructose evident in S. stap®anus is suggested to contribute to osmoregulation during the intermediate stages of dehydration (Ghasempour et al., 1998). This contrasts with the situation in X. viscosa, where sucrose accumulation is shown to be complete by the intermediate stage of dehydration. The lower level of glucose and fructose accumulation in X. viscosa may re¯ect differences in the rates of starch degradation and extent of sucrose cycling anduor differences in the rate at which the hexose sugars are

phosphorylated for incorporation into metabolism. The amount of sucrose accumulated in the leaf material of S. stap®anus from this study is three times higher than that reported previously (Ghasempour et al., 1998). This discrepancy may be explained by comparing differences in environmental growth conditions. In the present investigation, both plant species were maintained prior to and during dehydration at near full sunlight in midsummer. The photon ¯ux density recorded previously (Ghasempour et al., 1998) was low (96 mE m 2 s 1) by comparison. Hence, an increased level of photosynthetic products and an increased amount of reserve material may have been available for the production of leaf sucrose both prior to and during the early stages of dehydration in plants from the present investigation. Although the relative contribution of triose phosphates and hexose sugars to hexose monophosphate (and subsequent sucrose) production was not determined during dehydration, the importance of hexose phosphorylation during dehydration is re¯ected by an increase in hexokinase activity which is con®ned to the period of sucrose accumulation. The highest levels of hexokinase

Carbohydrate metabolism in resurrection plants

Fig. 6. Levels of metabolites in dehydrating leaf tissue from Xerophyta viscosa at 100%, 59%, 43%, and 13% RWC. (A) Glucose, (B) fructose, (C) Glc-6-P, (D) Fru-6-P, (E) ATP, and (F) ADP. Results are the means"SD of 4 replicate extractions, each comprising 4 separate plants.

activity coincide with the decline of glucose and fructose. However, it was also observed that sucrose accumulation and the concomitant removal of the hexose sugars during dehydration is not associated with a decline in either vacuolar acid or cytosolic neutral invertase in both plant species. Similar to SuSy in C. plantagineum (Kleines et al., 1999), the maintenance of acid and neutral invertase during dehydration is likely to be important when sucrose is hydrolyseducleaved to support metabolism during rehydration. This then raises the interesting question of how sucrose accumulation (and the decline of hexose sugars) is accomplished in the presence of hydrolytic activity. Hence, the importance of a continual increase in hexokinase activity during this period may be necessary to ensure that incorporation of both hexose sugars into metabolism (towards sucrose biosynthesis) exceeds potential sucrose hydrolytic activity in the respective tissues. Possibly the larger increase in hexokinase activity in S. stap®anus is necessary since both acid and neutral invertase activities are induced early in dehydration and held constant over the entire period of sucrose

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accumulation in this species. In S. stap®anus, the increased invertase activity may also have contributed to the higher level of both hexose sugars in the earlier stages of dehydration compared with that of X. viscosa. The rates of hexokinase activity in the hydrated tissues of both resurrection grasses are comparable to that reported for leaf material in other plant species (Schnarrenberger, 1990; Renz et al., 1993; Veramendi et al., 1999). Although the fructokinase : hexokinase ratio is higher in S. stap®anus than in X. viscosa, the ratios are within the range (1 : 1.5±1 : 5.3) of other plant species reported (Schnarrenberger, 1990; Renz et al., 1993). Within the period of sucrose accumulation, the initial increase in hexokinase activity coincides with the transient increase in glucose in both plant species. The further signi®cant increase in hexokinase activity (more speci®cally in S. stap®anus) then spans the region of net glucose (and fructose) removal. Additionally, the fructokinase : hexokinase ratio is shown to equalize. During potato tuber development, hexokinase activity was reported to increase and exceed fructokinase activity during sprouting, a condition where starch degradation is the major source of carbohydrate to metabolic activity (Renz et al., 1993). Increased hexokinase activity has also been documented under conditions of increased hydrolytic starch degradation in tobacco leaf material (HaÈusler et al., 1998). The higher rate of glucose production in S. stap®anus and X. viscosa is potentially indicative of hydrolytic starch degradation (HaÈusler et al., 1998; Veramendi et al., 1999). The only source of fructose would have been as a consequence of sucrose import anduor sucrose cycling, which would also have contributed to some of glucose measured. It is of interest that when S. stap®anus leaf tissue is dried detached from the parent plant, starch degradation is not complete (Quartacci et al., 1997). Whether this phenomenon is related to the incomplete induction of hexokinase activity, as seen from the present study will require further investigation. The present investigation shows that the maximal catalytic rate of both hexokinase and fructokinase is very far in excess of the rate of net sucrose accumulation and net hexose sugar removed. Despite existing high levels of activity in the hydrated tissues, the large induction and maintenance of increased hexokinase activity during sucrose accumulation is likely to be related to the balance between sucrose synthetic versus hydrolytic activity. However, additional factors are likely accountable for the increase in hexokinase activity. In S. stap®anus, desiccation tolerance is only acquired in leaf material if dried on the plant (Kuang et al., 1995). The hormone abscisic acid (ABA) accumulates to a greater extent in leaf material of S. stap®anus when dried attached to the parent plant (Gaff and Loveys, 1992). Endogenous leaf ABA has been reported to peak between 40% and 15%

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RWC (Gaff and Loveys, 1992), which coincides with the highest levels of hexokinase in this species. The role of ABA as a hormonal signal in the induction of hexokinase activity is currently being investigated. In detached leaves of S. stap®anus dried over a 6 h period, hexokinase activity is unaltered. While the dehydration rate was rapid, signi®cant anaerobic stimulation of hexokinase activity has been reported to occur within only 3 h in leaf material (coleoptiles) of Echinochloa phyllopogon (Fox et al., 1998). Although not considered a general stress protein, the increase in hexokinase activity was suggested to support respiratory activity during anaerobiosis (Fox et al., 1998). It is of interest that the increase in hexokinase activity was evident in the ¯oodresistant E. phyllopogon but not in the ¯ood-sensitive E. cruspavonis (Fox et al., 1998). In the present study, the extension of the dehydration time to 55 h stimulated only a 2-fold induction in activity, which is a signi®cantly lower level of response to dehydration when compared to leaves dried intact. Since leaf material dried detached from the parent plant is desiccation-sensitive, it is possible that the substantial induction of hexokinase activity is associated with the acquisition of desiccation tolerance. Examining the metabolite content, it is evident that removal of hexose sugars during dehydration had no effect on the total cellular Glc-6-P and Fru-6-P content. Since net glucose removal during drying is signi®cantly higher than that of fructose in both plant species, a higher incorporation of carbon into the hexose monophosphate pool in the form of Glc-6-P may likely occur. Equilibrium of the Glc-6-P with Fru-6-P (the substrate for SPS activity) would be important for sucrose synthesis (and metabolism). The consistent Glc-6-PuFru-6-P quotient of 2.0 suggests that overall equilibrium of Glc-6-P with Fru6-P is maintained during dehydration. A total Glc-6-Pu Fru-6-P quotient of 2.0 for leaf material has been reported previously (Gerhardt et al., 1987). From the present study, there is no build-up of the two hexose monophosphates, suggesting that overall incorporation of phosphorylated hexoses into metabolism was not perturbed. This may also suggest that no major product inhibition by hexose monophosphates on in vivo hexokinase activity is likely to occur as a consequence of dehydration. Since the af®nity of both fructokinases and hexoinases for fructose and glucose is in the micromolar concentration range (Schnarrenberger, 1990; Renz and Stitt, 1993; Martinez-Barajas and Randall, 1998) there was not likely to be substrate limitation during dehydration. It is, however, possible that hexokinase enzymes were substrate limited at the completion of sucrose accumulation. Determination of the precise subcellular sugar and metabolite concentrations in vivo is not possible due to a lack of suf®cient reliable data on the volumes occupied by the cytosol, mitochondria and chloroplasts. How these subcellular volumes might change as a result

of dehydration stress in the two resurrection grasses is also not known. Total cellular ATP content doubles during the intermediate stages of dehydration in both plant species. Maintenance of respiratory activity down to 20% RWC has been reported for several desiccation-tolerant plant species (Schwab et al., 1989; Hartung et al., 1998; Tuba et al., 1998; Farrant, 2001) and may explain the increased ATP content. The cellular ATPuADP quotient of 2.8 is similar to that reported for leaf material (Stitt et al., 1982). The decline in the ATPuADP quotient (2.0±2.2) in the ®nal stages of dehydration implicates a perturbation in metabolism. The precise effect of changing metabolite levels on enzyme activity is dif®cult to assess since multiple forms of both hexokinase and fructokinase, each with differing kinetic properties, exist in plant tissue (Baldus et al., 1981; Doehlert, 1989; Schnarrenberger, 1990; Renz and Stitt, 1993; Renz et al., 1993; MartinezBarajas and Randall, 1998). However, common to all hexokinases and fructokinases is an inhibitory effect by a decline in the ATPuADP ratio. At an ATPuADP quotient of 3.0, both enzymes are reported to be inhibited by 50% (Renz and Stitt, 1993; Martinez-Barajas and Randall 1998). It is feasible that changing adenylate content may modulate in vivo regulation of catalytic activity in the two resurrection grasses. Acknowledgements The project was funded by a grant from the National Research Foundation (NRF) to Jill Farrant and George Lindsey, and a bursary to Anne Whittaker. The project was also funded by the Italian Ministry of University and Scienti®c Technological Research. Work collaboration with the University of Florence (Adriana Bochicchio and Concetta Vazzana) is gratefully acknowledged. Thanks are extended to John and Sandie Burrows for the collection of X. viscosa from the Buffelskloof Private Nature Reserve in South Africa. The authors also wish to thank Frikkie Botha at the Institute of Plant Biotechnology in Stellenbosch for informative discussions and constructive criticism.

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