Effect of culture medium and illumination on the acid invertase activity in callus of Medicago...

ACTA

PHYSIOLOGIAE PLANTARUM

Vol. 21. No. 2. 1999:141-147

Effect of culture m e d i u m and i l lumination on the acid invertase activity

in callus of Medicago strasseri

Marta Medina *, Nieves Villalobos, Pedro J. de la Cruz, * * Ana Dorado ** *, Hilario Guerra*

Departamento de Fisiologia Vegetal, Facultad de Biologia, Universidad de Salamanca, Avda del Campo Charro s/n, E-37007. Salamanca, Spain

Key words: Medicago strasseri; Invertase (i3-D- fructofuranosidase); tissue culture; callus

Abstract

The different acid invertase activity (total, soluble, wall-bound and extracellular) in calli induced on explants (cotyledon, petiole, hypocotyl and leaf) originated from Medicago strasseri seedlings were evaluated. In cultures subjected to 16 h photoperiod, the highest total, soluble and extracellular activities were found in calli from leaves cultured in medium 12 (MS with 0.01 mg.dm -3 (0.045 ~tM) of TDZ), elevated amounts of total and wall-bound invertase being found in catli induced on

3 petioles in 12G medium (MS with 0.01 mg.dm- (0.045 ~M) TDZ and 3.104 mg.dm -3 glycerol). In cultures maintained in darkness, the activity detected was lower than that observed in cultures under light conditions. The highest amounts of enzyme was bound in calli cultured on medium 12 (total and extracellular invertase) -leaves- and medium 12D (MS with 0.001 rag.din -3 (0.0045 pM) TDZ) (soluble invertase) -using hypocotyls. In general, the different forms of invertase activity studied seem to appear in greatest amounts in calli induced under light conditions using leaves as explant and TDZ as growth regulator.

List of abbreviations: U, units; DW, dry weight; PH, photoperiod; D, darkness; TDZ, thydiazuron; 2,4-D,2,4-Dichlorophenoxyacet ic acid; KIN, kinetin.

Introduct ion

Medicago strasseri Greuter, Matth~is and Risse, was described in 1982 (Greuter et aL, 1982) as a new species by a German team directed by the Di- rector of the Botanical Gardens in Berlin, W. Greuter. In 1983, the Depar tment of Plant Biology of the Higher Technical School o f Agricultural En- gineering in Madrid received a small sample of seeds of this species f rom the Berlin Botanical Gar- dens. Medicago strasseri is a legume of great inter- est as a forage due to its resistance to cold and as an al temative for animal feed source in zones with continental climate. It has been proposed that large scale introduction of the species could serve to re- duce feed imports.

Invertases (g-D-fmctofuranosidases; E.C.3.2.1.26) play a crucial role in tissue culture since they hydro- lyse sucrose to hexose sugars and thus play a funda- mental role in the energy requirements for plant growth and maintenance (Klann et al., 1996).

Depend ing on the pH at which they act, invertases can be classified as acid and neutral (also called alkaline) with pH optima between pH 4.5-5.0 and 7.0-7.8, respect ively (Van den Ende and Van Laere, 1995). Both types of enzyme often occur in the same tissue, but acid invertase is generally found in

141

M. MEDINA, N. VILLALOBOS, P.J. ED LA CRUZ, A. DORADO & H. GUERRA

the cytoplasm and in the cell wall in the intact tissue and, depending on their location, as soluble and in- soluble, or wall-bound (Van den Ende and Van Laere, 1995). Enzymes bound to the cell wall are generally released by high concentrations of NaC1 but often considerable activity remains in the resid- ual cell wall after treatment with saline solutions (Masuda et al., 1988). In contrast to acid invertase activity, which is mainly found in immature tissues, neutral invertase activity seems to be confined to mature storage tissues (Van den Ende and Van Laere, 1995).

Masuda et al., 1988 (Masuda et al., 1988), suggest, in suspension cultures, that external sucrose is pref- erentially taken to the cell surface before it is hydro- lyzed by extracellular invertase. Thus, the sugar is probably degraded in the cell wall by the salit- released invertase and the products of degradation are finally transported to the cytoplasm as hexoses.

Among the studies carried out by our team to obtain synthetic seeds we thought it important to gain insight into the variations occurring in invertase activity as the main factor responsible for the use of the carbon source. With the present study our aim was to elucidate the effects of certain growth regu- lating substances and carbon sources on the different invertase activities and their effects on later morphogenesis. Here we report a study on the activity of soluble acide wall-bound and extracellular invertases in calli derived from Medicago strasseri seedlings. Since we have found no other work of this kind in literature our aim was to determine whether differences exist in the type and activity of invertase as a function of the type of explant, culture medium and/or illumination conditions imple- mented during callus formation.

Materials and Methods

Plant material

Explants were obtained from cotyledons, petioles, hypocotyls and leaves of seedling obtained asepti- cally from seeds of Medicago strasseri (Greuter et al., 1982). These legumes are endemic to the prov- ince of Rethimnos (Crete, Greece). Plant material was provided by the Department of Plant Physiol-

ogy of the Higher Technical School of Agricultural Engineering in Madrid (Spain).

Seeds were germinated in Murashige and Skoog medium (Murashige and Skoog, 1962) without sucrose and with the mineral salts at one quarter strength. Seeds were kept in a dark chamber at 23_+2 °C. When hypocotyl reached a height of 10 cm, the plantlets were transferred to a chamber with a 16 h photoperiod (20 gE-m-2-s-1). Plantlets were kept under these conditions 20-30 days after which they were used to obtain explants.

The different explants ( lcm slices of cotyledons, petioles, hypocotyls and leaves) were incubated in chambers on a 16 h photoperiod (20 gE.m-Ls-1) or in complete darkness during four months at 23+2 °C in MS medium (Murashige and Skoog, 1962) with or without glycerol, with different cytokinins and/or 2,4-D, as specified in Table. After four months of subculture calli were obtained. These were sufficiently large to provide the necessary material for studies of acid invertases.

Preparation of invertases

The preparation of callus invertase was performed following the method reported by Burch et al. (1992). The whole process of invertase (g-D- fructofuranosidase E.C. 3.2.1.26) was carried out at -4 °C to avoid enzyme denaturation. Two g of calli (fresh weight) was ground in a mortar with 4 ml of extraction buffer (1M sodium acetate buffer, pH=5.0); 100 mM sodium sulphite). Extracts were further disrupted in a Polytron blender to break cell walls. Samples were then centrifuged for 15 min at 26,000 g in a Sorvall refrigerated centrifuge at 4 °C, after which the supernatant and pellet were subjected to different treatments:

Soluble acid invertase

This was collected with the supernatant. The supernatant was dialysed against water for 24 h at 4 °C using dialysis tubing (Medicell) (size 6, INF DIA 27/32", 21,5 mm or size 2 INF DIA 18/32", 14.3 mm).

Wall-bound acid invertase

Tubes containing the cell wall were washed with 5 mL of buffered extraction solution to which NaC1

142

EFFECT OF CULTURE MEDIUM ...

was added at a concentration of 1.3 M. The tubes were shaken for 12 h at 4 °C. Following this, they were centrifuged at 26,000 g for 20 min. The new supernatant was subjected to dialysis for 24 h as reported above.

Extracellular acid invertase

Precipitates from the previous centrifugation were resuspended in 2 cm 3 of extraction buffer with 1.3 M NaC1 evaluating invertase activity in this fraction.

Assay o f invertase activity

Invertase activity was determined using the modifi- cation of the method described by Miller and Ran- wala (Miller and Ranwala, 1994). In a reaction mix- ture that consisted of 40 mM sucrose (400 ~1), buffer (400 lal) (50 mM Na-acetate buffer, pH=5.0 for acid invertase, or 25 mM Tris-HC1 buffer, pH=8.0 and 1M Na-acetate buffer, pH=8.0, for alkaline invertase) and enzyme (200 ~1) in a total vol- ume of 1.0 mL. Buffer and enzyme were preincu- bated in a water bath at 30 °C for 5 rain prior to sub- strate addition. After sucrose was added, the mix- ture was incubated at 30 °C, after which the reaction was stopped in a 90 °C bath for 5 min. The amount of reducing sugars released was determined by the method of S omogyi ( 1952) and Nels on (1944). One unit of invertase activity was defined as the amount of enzyme that catalysed the production of 1 gmol of glucose per min at 37 °C. Owing to the oscilla- tions occurring in the fresh weight of the calli, due to variations in water content, enzymatic activities are expressed per dry weight this offering a more constant value.

Table. Composition of culture media used

Added compounds (mg/1) to MS medium 12

2,4-D

TDZ 0.01

KIN

Glycerol --

Culture medium

12D ~2G 11

2.00

0.001 0.01 1.00

30,000

Statistical analysis

Results were analysed statistically by analysis of variance using the SPSS programme. When analysis of variance showed treatment effects (p<0.01 and p<0.05), the least significant difference (Fisher LSD) test was applied to make comparisons between means at the 0.01 and 0.05 levels of signifi- cance.

2,4-D: 9.05, 13.57 pM; TDZ: 0.0045 to 4.54 gM; KIN: 4.65, 9.29 ~tM.

Results and Discuss ion

W e were unable to detect any neutral invertase activity even though the literature indicates that neutral invertases coexist with acid invertases in the same tissue (Kaur et al., 1992; Masuda et al., 1987; Ranwalaet al., 1991 ; Stommel and Simon, 1990). One reason for this could be that while our studies were carried out neutral invertase would still not have been synthesised since, as reported by Van den Ende and Van Laere (1995), acid invertase is mainly located in immature tissues while neutral invertase is present in mature storage tissues in Ci- chorium intybus. Thus, the results shown in Figures were obtained in studies performed on acid invertase.

To compare the results we used two-way analysis of variance (ANOYA). The factors involved in the experiment were: culture media (with six levels: 12, 12D, 12G, 1t, A and B)on 16 h photoperiod or darkness; explants (with four levels: cotyledons, petioles, hypocotyls and leaves), allowing between factors-interaction.

Culture under photoperiod conditions (16h light)

According to our results (Fig. 1), the total acid invertase activity was very high in calli formed in media with TDZ (media 12, 12D, 12G and 1t).

A B The utilization of hypocotyls 3.00 2.O0 and leaves as explants (cul-

tured in medium 12) and in those from petioles (cultured

1.00 2.00 in medium 12 G, which contained glycerol as well as TDZ) being outstanding. In general, the addition of TDZ

143


to the medium at a concentration of 0.01 mg-dm -3 (0.0045 pM) seemed to induce the highest acid invertase activity while media with kinetin (media A and B) had the lowest activity of this type (except for leaves in the case of the latter). On comparing the results obtained in the measurements of total callus invertase statistically for the different explants and culture media employed the interaction is highly significant (p<0.01).

Unlike what has been proposed by many authors (Masuda et al., 1988; Copping and Street, 1972) that the amounts of wall-bound invertase are higher than those of soluble and extracellular invertases, our results show that most of the acid enzyme activity evaluated was soluble invertase (Fig. 1). This form of enzyme is common in vacuoles of tissues that store sucrose (Boller and Wiemken, 1986) and may be responsible for the hydrolysis of sucrose stored in such tissues. In our work the highest amount of soluble acid invertase was found in calli from leaves cultured in medium 12 (which lacks 2,4-D and kinetin but contains a concentration of 0.01 mg.dm -3 TDZ) and the lowest amount in calli from cotyledons cultured in medium A (with 3.00 mg.dm -3 2, 4-D and 1.00 mg-dm -3 kinetin). Highly significant interaction was also found (p<0.01) be- cause we cannot interpret the principal effects.

The presence of invertase activity in plant cell walls is a widespread phenomenon (Masuda et al., 1988; Copping and Street, 1972). This enzyme plays a crucial role in callus formation and growth since sucrose in the medium is degraded extracellu- larly by this enzyme and the resulting hexoses are taken up by cells (B otha et al., 1992). In general, the wall-bound invertase detected in our calli showed high levels of enzyme activity when media with TDZ were used. Two media merit comment: medium 12 (only 0.0045 ~M TDZ) and medium 12G (medium 12+glycerol). This activity was very low (except in the case of calli obtained from leaves cultured in medium B) when kinetin, instead of TDZ was added to the culture medium (media A and B) (Fig. 1). calli from petioles cultured in medium B (0.021 U/g DW) and in medium A (0.017 U/g DW) showed the lowest levels of this form of the enzyme.

It is possible that after four months under culture conditions wall-bound invertase activity could be- come depleted since the enzyme already played its role in the degradation of sucrose in the medium (Masuda et al., 1988) and the hexoses released may have been utilized as substrates for respiration, for the synthesis of structural polysaccharides and, possibly, for starch formation (Sung et al., 1989: Venkataramana et al., 1991; Xu et al., 1986). In this

~̂ 0,5 ISOLUBLE 0,4

0,0 IZ I2D I~G 11 A B

Culture medium

,., 0,6

~ 0,4

~o.2}

0,0 '

WALL-BOUND

12 12D 12G I I A B Culture medium

~ o'°/EXTRACELLULAR ]~ ° 'T ITOTAL

!0, 12 12D 12G 11 A B 12 12D 12G II

Culture medium A B

Culture medium

[ ] Cotyledon

[ ] Petiole

[ ] Hypoeotyl

[ ] Leaf

Fig. 1. Acid invertase activity of cultures grown under 16 h light conditions. Each point shows the average of three replicates: experiments were repeated three times. Vertical bars indicate SE.

144


sense, Goldner e t al. (1991 ) reported that high levels of acid wall-bound invertase activity are only found at the beginning of the culture cycle.

The analysis of variance for the whole set of data corresponding to the amount of wall-bound acid in- vertas revealed that the differences regarding the type of medium and type of explant shows again the presence of a signiicant interaction (p<0.01).

A s reported in the literature (Masuda e t al . , 1988; Masuda and Sugawara, 1980), whatever the extraction method employed after salt extraction of wail- bound enzyme, a slight amount of invertase activity remains retained in the wall. Currently, it is not known whether wall-bound invertase released by salt treatment is the same or different from the enzyme remaining in the wall after treatment. It is not known whether the physiological functions of both differ (Van den Ende and A. Van Laere,1995; Masuda et a l . , 1988). Despite this, the invertase remaining in the cell wall seems to be able to participate in the degradation of extracellular sucrose (Masuda e t al . , 1988). We used the term extracellular activity to refer to the enzyme remaining in the cell wall after saline treatment and refer to the enzyme obtained prior to performing fractionation into the different activities as total activity.

Extracellular invertase activity (Fig. 1) was low in comparison to that obtained for the other invertase forms studied. The highest activities were found in calli from cotyledons (0.0057 U/g DW), hypocotyls (0.051 U/g DW), and leaves (0.059 U/g DW) cultured in the Medium 12. It is possible that such wall-retained activity might also participate in the hydrolysis of sucrose in the medium in support of wall-bound invertases (Masuda e t al. 1988). The analysis of variance revealed again the presence of interaction (p<0.01)

Taken together, our results show that media with TDZ induce greater invertase activity in calli than media with kinetin, 0.0045 plVI being the TDZ concentration with the greatest inducing power.

C u l t u r e in d a r k n e s s

A s reported in the literature (Raj Bhansali e t al . ,

1990; Zheng e t al . , 1996), micropropagation or somatic embryogenesis require a first step in callus induction in the darkness before the calli are subjected to light conditions. In view of the data reported in the above references, we carried out culture in the darkness and then compared the results obtained on invertase activity with those obtained under photoperiod conditions. It should be stressed that under conditions of darkness it is only possible to achieve callus formation using media 12, 12D

.-~ 0.6 ̧

v 0.4

m ~ 0 ,2 :

g

,-- 0,6

v 0.4 .=

0,2

m

COTYLEDON~ ~ 0,4

0,3

Pll PH PIT D -- 12 12D 12G

Conditions-Culture medium

HYPOCOTYL ~ 0,6 O

~ 0,4

0,2

Ptl ~ 0.0 12 12D 12G

Condi t ions-Cul ture medium

'PETIOLE

PH D PH D PH D 12 12D 12G


LEAF"

PH D 1311 D PIt D 12 121) 12G


• Soluble Inver tase

[ ] Vai l -Bound Inver tase

ml Ext raoo l lu la r Inver tase

[ ] Tota l Inver ta~e

Fig. 2. Invertase acid activity of cultures grown under 16 h light (PH) or under continuous darkness (D). Each point shows the average of three replicates: experiments were repeated three times. Vertical bars indicate SE.

145


and 12G. With media 11, A and B, in the darkness, calli do begin to form but then degraded rapidly.

With respect to total invertase activity (Fig. 2), very high levels of activity were not observed. When petioles were used as the explants, maximum levels of activity were observed in the calli from the three culture media used.

In the determination of soluble acid invertase (Fig. 2), the results obtained when petioles were used as explants are also interesting: in calli obtained in the three culture media soluble activities greater taller than in the rest of explant can be detected. The leaf is the explant to induce the shortest enzymatic activities in the three culture media.

According to what was suggested by Masuda et al (1988) in some calli obtained in the darkness, wall- bound invertase activity showed higher levels than those obtained for the soluble fraction (Fig. 2). The media with glycerol (media 12G) induced calli on any of the explants used, with very low wall-bound enzyme activity. Cotyledons afforded the explants that induced calli with the lowest degree of enzyme activity in all three culture media. On the other explants, the addition of 0.0045 laM TDZ to the culture medium (medium 12) produced the highest activities of this form of the enzyme.

Regarding extracellular invertase activity (Fig. 2), calli obtained from petioles in the three culture media showed the highest activity of this form of the enzyme.

The ANOVA in each case (total invertase activity, soluble acid invertase, wall-bound invertase and extracellular invertase activity), reveals highly significant interaction (p<0.01) for the four variables in the study.

Comparison of cultures carried under photoperiod conditions and in the darkness

W e used three-way analysis of variance (ANOVA) to compare the factors involved in the experiment which were: culture media (with three levels: 12, 12D and 12G and photoperiod/darkness (with two levels); explants (with four levels: cotyledons, petioles, hypocotyls and leaves), allowing between factors-interaction (double and triple).

For all the enzymatic activities evaluated (total and extracellular invertases, soluble acid invertase, wall-bound invertase) statistical analysis revealed the presence of triple interaction in the three cases. We studied it and summarized the results as follows: The results obtained for calli cultured in the darkness show that under these conditions invertase activity was decreased with respect to that observed under photoperiod conditions (Fig. 2). In ~this case, the results obtained when petioles were cultured in medium 12G were outstanding in the sense that they were higher than those obtained for calli cultured under photoperiod conditions. Only for leaves as explant on medium 12D no differences were found as regards treatment with or without light (p<0.05).

The activity of soluble invertase (Fig. 2) was lower in calli formed in the darkness, although there were no differences between light and darkness for petiol derived callus in medium 12D (p<0.05) or those obtained from cotyledons and hypocotyls in medium 12G (p<0.05).

Wall-bound invertase activity was also lower in calli induced in the darkness (Fig. 2). Only three cases were outstanding in this sense: leaves cultured in media with TDZ but without glycerol, and petioles cultured in a medium such as 12D, which contained a 0.045 pM TDZ and whose invertase activity was higher than that obtained from calli cultured under photoperiod conditions.

Extracellular invertase activity was generally lower in the darkness than under photoperiod conditions (Fig. 2).

Overall, from the foregoing it may be inferred that with a few exceptions culturing in the darkness in- duces low invertase activity in calli. In cases depart- ing from this trend, it is not possible to draw firm conclusions since none of the explants or culture media was found to induce higher activity.

Although there are no references in the literature concerning differences in invertase activity in calli obtained in vitro as a function of the explant, of the culture medium and of the illumination conditions employed, we hope that the present results will help in the search for further insight into callus metabolism and, in due course, that they will shed light on the processes possibly involved in cytodifferentia- tion.

146


Acknowledgements

This work was supported by a grant f rom the Direc- ci6n General de Investigaci6n Cientifica y T6cnica, Spain, N ° PB94-1403.

References

Boiler T., Wiemken A. 1986. Dynamics of vacuolar compartmentation. Annu. Rev. Plant Physiol. 37, 137- 164.

Botha F.C., M.M. O'Kennedy M.M., du Plessis S. 1992. Activity of key enzymes involved in carbohydrate metabolism in Phaseolus vulgaris cell suspension cultures. Plant Cell Physiol 33,477-483.

Butch L.R., Davies H.V., Cuthbert E.M., Machray G,C., Hedley P., Waugh R. 1992. Purification of soluble invertase from potato. Phytochem. 31, 1901-1904.

Copping L.G., Street H.E, 1972. Properties of the invertases of cultured sycamore cells and changes in their activity during culture growth. Physiol. Plant. 26, 346- 354.

Goldner W., ThomM., Maretzki A. 1991. Sucrose metabolism in sugarcane cell suspension cultures. Plant Sci. 73, 143-147.

Greuter W., Matth~s U., Risse H, 1982. Notes on Car- daegean plants. 3. Medicago strasseri, a new legumi- nous shrub from Kriti. Willdenowia 12, 201-206.

Kaur N., Jain H., Mann P., Gupta A.K., Singh R. 1992. A comparison of properties of invertases and inuli- nase from chicory. Plant Physiol. Biochem. 304, 445- 450.

Klann E.M., Hall B., Bennett A.B. 1996. Antisense acid invertase (TIV 1) gene alters soluble sugar composition and size in transgenic tomato fruit. Plant Physiol. 112, 1321-1330.

Masuda H., Sugawara S. 1980. Purification and some properties of cell wall-bound invertases from sugar beet seedlings of mature roots. Plant Physiol. 66, 93-96.

Masuda H., Takahashi T., Sugawara S. 1987. The oc- curence and properties of alkaline invertase in mature roots of sugar beets. Agric. Biol. Chem. 51,2309-2314.

Masuda H., Takahashi T., Sugawara S. 1988. Acid and alkaline in suspension cultures of sugar beet cells. Plant Physiol. 86, 312-317.

Miller W.B., Ranwala A.P. 1994. Characterization and localization of three soluble forms from Lilium longiflo- rum flower buds. Physiol. Plant. 92, 247-253.

Murashige T., Skoog F. 1962. A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant. 15, 473-497.

Nelson N. 1944. A photometric adaptation of the So- mogyi method for the determination of glucose. J. Biol. Chem. 153,375-380.

Raj Bhansali R., Driver J.A., Durzan D.J. 1990. Rapid multiplication of adventitious somatic embryos in peach and nectarine by secondary embryogenesis. Plant Cell Rep. 9, 280-284.

Ranwala A.P., Iwanami S.S., Masuda H. 1991. Acid and neutral invertases in the mesocarp of developing muskmelon Cucumis melo L. cv. Prince fruit. Plant Physiol. 96, 881-886.

Somogyi M. 1952. Notes on sugar determination. J. Biol. Chem. 195, 19-23.

Stommel J.R., Simon P. 1990. Multiple forms of invertase from Daucus carota cell cultures. Phytochem. 29, 2087-2089.

Sung S.S., Xu D.-P., Black C.C. 1989. Identification of actively filling sucrose sinks. Plant Physiol. 89, 1117- 1121.

Van den Ende W., Van Laere A. 1995. Purification and properties of a neutral invertase from the roots of Cicho- rium intybus. Physiol. Plant. 93,241-248.

Venkataramana S., Naidu K.M., Singh S. 1991. Inver- tases and growth factors dependent sucrose accumula- tion in sugarcane. Plant Sci. 74, 65-72.

Xu D.-P., Sung S.S., Alvarez C.A., Black C.C. 1986. Pyrophosphate-dependent sucrose metabolism and its activation by fructose 2,6-bisphosphate in sucrose im- porting plant tissues. Biochem. Biophys. Res. Commun. 141,440-445.

Zheng, Q., Dessai, A.P., Prakash, C.S. 1996. Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis. Plant Cell. Rep. 15, 381-385.

Received September 18, 1998; accepted December 21,1998

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