Plant Science, 72 (1990) 193--198 193 Elsevier Scientific Publishers Ireland Ltd.

Glycosidase activities and polysaccharide accumulation in sugar cane stalks during post-collection impairment

M a r i t z a M a r t i n e z a, M. Es t re l la Legaz b, M. P a n e q u e a, R. D o m e c h c, R. de A r m a s a,

Isabel M e d i n a a, C . W . R o d r i g u e z a a n d C. Vicente b

"Department of Plant Physiology, Faculty of Biology, La Havane University, La Havane (Cuba) bLaboratory of Plant Physiology, Faculty of Biology, Complutense University, 28040 Madrid (Spain) and cDepartment of Plant Physiology, Polytechnic Institute

Matanzas (Cuba)

(Received January 12th, 1990; revision received August 3rd, 1990; accepted August 3rd, 1990)

Sugar cane accumulates in its stalks sucrose and, later, polysaccharides including fructans. These polysaccharides are hydrolyzed by a fructanase which requires Mn 2÷ to act. Since these fructans are heteropolymers containing both fructose and galactitol, the enzyme must contain at least two different hydrolase activities. During the first 2 days of post-collection impairment, fructanase activity rapidly develops and, then, it decreases and stabilizes. The decrease in the amount of mid-molecular weight carbohydrates after the fourth day of post-collection impairment can be seen as a consequence of the stabilization of fructanase activity. This hydro- lase is inhibited by an excess of Mn 2° as well as by reducing sugars accumulated in the juices.

Key words: sugar cane; fructans; fructanase; glycosidase; manganese; post-collection impairment

Introduction

Sugar cane accumulates sucrose in its stalks, which reaches a maximum at a particular phase of the vegetative growth. During this phase, called 'industrial ripeness', the stalks contain a minimum of reducing sugars, mainly glucose and fructose, and they are cut to be industrially processed [1]. However, other carbohydrates different from suc- rose can also be accumulated, although their max- ima generally occur during post-ripening. This phase, when naturally occurs in the field, is called 'upright impairment'. Those carbohydrates are polysaccharides of variable chemical nature, including glucans [2], starch-like polymers [3,4], arabino-galactans [5], and fructans although these latter have also been found in many temperate C 3

Abbreviations: GLC, gas-liquid chromatography; HPLC, high performance liquid chromatography; MMWC, mid-molecular weight carbohydrates; SP, soluble polysaccharides.

grasses [6] and their accumulation occurs when the amount of sucrose exceeds that required to trans- port and metabolism [7,8].

In C 4 sugar cane, two fructans regarding their molecular mass have been described [9]. One of them, called mid-molecular weight carbohydrates, has a molecular mass varying from 0.7 to 10 kDa and its quantitative composition shows to be [Galactitols:Fructose4] n. The second class, called soluble polysaccharides, has a molecular mass higher than 10 kDa and a polymerization degree such as [Galactitol3:Fructose2] n. The linkage between galactitol and fructose can be defined as an ether bond, as derived from a strong peak at 1100 cm -I in the IR spectrum. Fructose and galac- titol have been chromatographically identified by GLC and HPLC [9]. Both SP and MMWC are hydrolyzed by a fructanase, the purification of which has been recently reported as well as some of its properties [9].

These fructans also occur as a consequence of

0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

194

the cut of stalks and a linear relationship between the number of cuts and the amounts of both MMWC and SP produced has been found [10]. However, production of some polysaccharides by sugar cane itself during post-collection impair- ment has been regarded as dubious. This is because some microorganisms, such as Leuconos- toc mesenteroides, which contaminate the stalks, can metabolize these polysaccharides [11].

Thus, we attempt in this work to probe that metabolism of MMWC during post-collection impairment is directed by a fructanase which is only produced by sugar cane cells.

Materials and Methods

Plant material Sugar cane (Saccharum officinarum L.), var.

Cuba 374-72, field grown, was used throughout this work.

Substrates preparation Stalks from 15.5-month-old plants were

mechanically crushed, immediately after cut, and the crude juices were brought to 5°70 (w/v) with trichloroacetic acid and centrifuged at 20 000 x g for 30 min at room temperature. The pellet was discarded and the supernatant was adjusted at pH 8.0 by adding a saturated solution of ammonium carbonate. The juice was centrifuged at 20 000 × g for 15 min at room temperature. The superna- tant was filtered through Whatman No 4 paper. Sodium ozide was added to the filtrate to obtain a final concentration of 0.02~70 (w/v).

This clarified juice was then filtered through columns of Sephadex G-10 (15 cm × 2.5 cm) pre- equilibrated with 10 mM ammonium carbonate containing 0.02~70 sodium azide. Elution was car- ried out with the same solution. Fractions (1.0 ml) 1--20 were discarded. Fractions 20--32 were col- lected and filtered through Sephadex G-50 col- umns (30 cm x 2.5 cm) pre-equilibrated as above. Fractions 40--70 ml contained the soluble, high molecular weight polysaccharides (SP fraction) whereas mid-molecular weight carbohydrates (MMWC fraction) eluted from 70--120 ml [12]. HPLC analysis [13] showed the absence of sucrose and monosaccharides in fractions 40--120 ml.

Post-collection impairment Stalks cut in the field, in 40 cm length segments,

were maintained in air, at room temperature, for 12 days in the laboratory. As indicated, juices were obtained to analyze polysaccharides, Mn 2÷ content and fructanase activity.

Mn 2÷ measurement Mn 2÷ was measured in juices freshly obtained

by using a Pye Unicam SP9 computerized atomic absorption spectrophotometer. Measurement conditions were as follows: )~ = 279.5 min; slit, 0.2 nm; lamp current, 7.5 mA; oxidant, air, 34 mm/equiv.; fuel, acetylene, 24 mm/equiv.; burner height, 0.5 mm; burner angle, 0°; sensitiv- ity, 0.027 mg 1 -~.

Enzyme extraction and assay Crude juice from recently crushed stalks was

filtered through a column of Sephadex G-25 (30 cm x 3.0 cm) to remove low molecular weight metabolites. The filtrate in the void volume was used to fructanase assay. Hydrolase activity was measured in reaction mixtures containing 1.0 mg protein, 20/~mol sodium citrate (pH 6.0), 5.0/~mol Mn 2÷ (as manganese sulfate), and 2.0/~g MMWC or 8.0/~g SP in a final volume of 1.3 ml. Reactions were carried out for 30 min at 30°C and stopped by adding sufficient volume of 2 N NaOH to raise the pH to 8.0. Controls contained no substrate or protein. Fructose produced during reaction was determined with dinitrosalicylic reagent and the developed colour measured at 540 nm [14]. Absorbance was transformed by using a straight line calibration made with known concentrations of fructose. Protein was measured by the method of Lowry et al. [15] using bovine serum albumin as a standard. One unit of specific activity was defined as 1.0/zmol of fructose produced per mg protein per min.

When indicated, variable concentrations of fructose or galactitol were pre-incubated with enzyme and Mn 2÷ for 5 rain at 30°C before SP addition.

Sucrose and reducing sugars measurement Sucrose was estimated by polarimetric method

[16] used in sugar industry. Reducing sugars were

measured by reaction with dinitrosalicylic reagent [14] but they were twice quantified using the corre- sponding calibration curves made with either fruc- tose or glucose as standards.

Contamination test The presence of fructans-hydrolyzing microor-

ganisms was checked. Crude juice extracted from 6 days-deteriorated stalk segments was filtered through Whatman No. 4 paper and centrifuged at 9000 × g for 20 rain at room temperature. The supernatant was discarded and the pellet washed three times with sterile distilled water. The pellet was used to inoculate a medium composed by 1.16 g K2HPO 4 3 H20; 0.5 g KH2PO4; MgSO 4 7 H20; 4.0 mg NaCI; 0.4 mg nicotinic acid; 0.75 g (NH4)2SO 4 as the only nitrogen source and 10 mg SP or MMWC as sole carbon source per 100 ml distilled water [17]. Inoculated media were incu- bated at 30°C in a rotatory shaker. Microbial growth was estimated by measurement of dry weight of aliquots extracted at the indicated times.

Results

Figure 1 shows the time-course of sucrose and reducing sugars during post-collection impair- ment. Sucrose concentration slightly decreases

I

y : - 0 366x°225 ; r 2 : 0 9 4 £ i £

10 2 I 2 y = 0 . 2 7 8 x - 0 2 8 r =096 ~.o /

\ -...S. -/ , . -

. . . . o _ ~ _ . - ~ " ' ~ " y= 0132x-0.034~ r2= 0.97

02---.___-z~--- 0 0 2 4 6 e 10 12

days

Fig. 1. Time-course of sucrose ( I ) and reducing sugars, measured as equivalents of fructose (O) or glucose (A), accu- mulation in sugar cane stalk segments during post-collection impairment. Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols. Thick lines represent the values which are fitted by linear regression.

195

5'.

.I

3 0,12 o °

3.10

, ° ° "

I / • D,04 i"

/ o" 0.02

I I I I I 0 2 z. 6 8 110 12

days

Fig. 2. Time-course of SP (O) and MMWC ( • ) accumula- tion in sugar cane stalk segmetns during post-collection impair- ment. Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols.

from the second day impairment whereas reducing sugars increase from the beginning of stalks stor- age. However, the rate of increase of these latter is lower than that of sucrose disappearance (0.278 g • day -] and 0.366 g" day -~ per 100 ml, respectively) as derived from the slopes of the respective straight lines.

The amount of SP linearly increases for the complete time of storage but that of MMWC decreases rapidly for the first 2 days and slowly from this time to increase for the last 2 days (Fig. 2). On the other hand, Mn 2÷ content shows to be low and slightly increases for the fourth day. From this time the amount of Mn 2÷ rapidly increases to reach a maximum at the 8th day and, then, it stabilizes (Fig. 3).

Table I. Fructanase activity in juice from 2 days deteriorated sugar cane stalks as depending upon Mn z° concentration in reaction mixtures.

Filtration Mn 2÷ concentration Spec. act." though in reaction (munits) G-25 mixtures

Yes 0 0 No 2.5 mM 0 Yes 2.5 mM 0.29 + 0.03 Yes 3.gmM 0.16 + 0.02 Yes 5.0 mM 0

• Values are the mean of three replicates.

196

I

OJ I I I I I i 0 2 4 6 8 10 12

days

Fig. 3. Time-course of M n 2÷ recovered in juices from sugar cane stalk segments during post-collection impalEment. Values are the mean of there replicates. Vertical bars give standard e r r o r w h e r e l a r g e r than the symbols.

0 . 4 0

• \

~ 0.3 oo

~ ou\' ,~X_~...~e y---O.O18x+0.275; r2=0.84 ® 0.2 \

o = , \ , " 0.1

\ y = - 0 . 1 2 x * 0 . 3 8 ; r 2 : 0 . 9 7 \\

0 o 2 4 6 8 10

[fructose] or [gatactitot] mM

Fig. 5. In vitro inhibition of fructanase activity by fructose ( • ) and galacfitol (O) when the enzyme uses SP as substrates.

Fructanase activity is not detectable in crude juices but it is easily measurable after juice filtra- tion through Sephadex G-25 (Table I). In addi- tion, enzyme obligatorily requires Mn 2. but cation concentration higher than 5.0 mM completely inhibits fructanase activity (Table I). Using the optimal Mn 2÷ concentration, the time-course of total fructanase activity using both SP and MMWC as substrate shows a maximum at the

I • 31.6

.R 8

E ~I '9 3~.2 18.4 29.3

-> .... \ / ' ' ' . ;

'~ 2 ' , . . . . . . . . b . . . . . . . o . . . LS0

-~ / " ' ' 6

OO I I I I I "'6 0 2 4 6 8 10 12

days Fig. 4. Time-course of fructanase activity, measured using SP (O) or M M W C ( • ) as substrates, during post-collection impa i rmen t of sugar cane stalks. Values are the mean of three replicates. Vertical bars give standard error where larger than the symbols.

second day of post-collection impairment to decrease later. From the fourth day of stalk stor- age, fructanase activity against MMWC stabilizes to the end of treatment. However, it is levelled off at 12 days when SP is used as substrate (Fig. 4).

Both fructose and galactitol behave as fructan- ase inhibitors in in vitro conditions, although about 15 times more fructose than polyol is required to the complete inhibition of the enzyme (Fig. 5).

Microorganisms obtained from sugar cane stalks after 6 days of post-collection impairment show to be unable to grow on both SP and MMWC as sole carbon sources, as it is shown in Table II.

Table 11. Growth of microorganisms, isolated f r o m 6 days deteriorated sugar cane stalks, on SP and MMWC.

Time (h) D r y wt . (mg) a

S P MMWC

0 11.1 + 1.3 9 .4 + 0 .83

24 8 .2 + 0 .74 9.1 + 0 .79

42 7 .4 + 0 .66 6 .8 + 0.61

• Values are the mean of three replicates.

Discussion

Amounts of sucrose obtained from sugar cane stalks are in agreement with those reported for the end of the industrial ripeness, as well as the low content of reducing sugars [1], and support the hypothesis that fructans accumulate after produc- tion of an excess of sucrose [7,8]. However, instead of the increase of reducing sugars with the time, their concentrations are lower than those expected from both sucrose and MMWC hydro- lysis (Figs. 1 and 2). This can be due to the respira- tory activity of sugar cane cells during impairment or, even, to that of contaminating bacteria.

The initial amount of MMWC is very high (Fig. 2), as corresponding to sugar cane plants which start the upright impairment [12]. However, the rapid decrease in the content of these polysacchar- ides is in agreement with the apppearance of fruc- tanase activity (Fig. 4) in a way that the minimal amount of MMWC is coincident with the maximal enzyme activity for the second day of impairment. But a maximum of MMWC accumulation is found for the fourth day of impairment, just when fructanase activity is decreasing. This fact is not easily explained. MMWC could be produced by partial hydrolysis of SP achieved by fructanase [18] or by degradation of healing polymers accu- mulated on the cell walls as a response to the cuts [10,19]. In this last process, fructanase activity could not be involved. This could explain even the increase in both SP and MMWC for the latter days of impairment (Fig. 2). On the contrary, the slow decrease in MMWC concentration after the fourth day could be due to the low, stabilized fruc- tanase activity. Quantitative analysis of both SP and MMWC hydrolysis indicates that 6 molecules of fructose per each one of galactitol are enzymat- ically removed from MMWC whereas two mole- cules of galactitol per each one of fructose are removed from SP [18]. This could indicate that fructanase preferentially hydrolyzes fructose-fruc- tose bonds, relatively more abundant in MMWC (88070) than in SP (66070). Hydrolysis of ether bond is, as yet, an unknown mechanism, although this implies that the highly purified protein contains at least two different enzyme activities, one of them hydrolyzing the fructosyl linkages and the second

197

one the linkages of galactitol. However, sponta- neous breakdown of SP catalyzed by free Mn 2÷ cannot be rejected.

The decrease of fructanase activity to stabilize later can be explained in two different ways. Firstly, soluble Mn 2+ content in juice strongly increases from the fourth day of stalks storage. Impairment obviously mobilized particulate Mn 2÷ and high concentrations of free cation are recovered in juices. However, the maximal con- centration of Mn 2÷ in juice (Fig. 3 and Table I) is at most (2 ppm) far from that required to inhibit fructanase (over 2.5 mM). Alternatively, it is pos- sible that fructanase could be regulated through a feed-back inhibition. This is suggested by the fact that it is necessary to remove low molecular weight metabolites, including reducing sugars, to detect fructanase activity in crude juices, freshly pre- pared (Table I). As indicated in Fig. 1, the increase in the concentration of reducing sugars with the time of post-collection impairment could be responsible of this effect. Both fructose and galac- titol, the hydrolysis products of MMWC, really inhibit fructanase activity (Fig. 5), although 15 times more concentration of fructose than that of hexitol is required for the complete inhibition of the enzymes.

An interesting point about this regulation process concerns the complete loss of hydrolase activity against SP at the end of the storage period of stalks (Fig. 4). Purification of hydrolase [9] reveals that the fraction with major activity against both SP and MMWC is desorbed from calcium phosphate gel with 15 mM citrate. How- ever, little amount of protein able to hydrolyze only MMWC is desorbed with 5 mM citrate [20]. This implies that several isoforms of fructanase can be involved in polysaccharide metabolism and explains that two activities separate at 12 days of post-collection impairment.

On the other hand, bacterial populations con- taminating detached sugar cane stalks are not responsible of the enzymatic disappearance of MMWC, since they are unable to use these poly- saccharides as growth substances (Table II). This does not necessarily imply that contaminating bac- teria do not use sucrose, reducing sugar, or other polysaccharides produced after ripening [11].

198

Acknowledgements

This work was supported by a grant from the Direccion General de Cooperacion con Iberoam- erica, Ministerio de Educacion y Ciencia (Spain) to carry out a Joint Action between Complutense and La Havane Universities.

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