J. PlantPhysiol. Vol. 134. pp. 232-236{1989}

Fructan Metabolism: Reversal of Cold Acclimation

J. A. TOGNETTI, P. L. CALDER6N, and H. G. PONTIS

Centro de Investigaciones Biol6gicas, Fundaci6n para Investigaciones Biol6gicas Aplicadas (F.I.B.A.) and Instituto de Investigaciones Biol6gicas, Universidad Nacional de Mar del Plata, Casilla de Correo 1348,7600 Mar del Plata, Argentina

Received June 10, 1988 . Accepted October 30,1988

Summary

Wheat seedlings grown at 23°C were acclimated for 15 days at 4°C and then transferred back to 23°C. The activtiy of sucrose synthase (SS), sucrose phosphate synthase (SPS), invertase, sucrose sucrose fruc­tosyl transferase (SST), fructan hydrolase (FH) and UDPase were measured at different intervals after sub­jecting the plants to the higher temperature. The activities of SS, SPS and SST, which had increased dur­ing the cold period, steadily decreased to reach the levels before the cold acclimation, while the invertase and FH activities significantly increased at 23°C. SS and SPS activities also fluctuate diurnally by having two peaks of activity during the light period.

Arrhenius plots of SS and SPS showed no discontinuity between 4 and 30°C, suggesting no changes in protein conformation as a result of modifications in temperature.

Key words: Triticum aestivum, cold acciimation,jructan, sucrose metabolizing enzymes.

Abbreviations: DP, degree of polymerization; FS, fructosyl sucrose; FW, fresh weight; FH, fructan hy­drolase; SPS, sucrose phosphate synthase; SS, sucrose synthase; SST, sucrose sucrose fructosyl transferase; TBA, thiobarbituric acid.

Introduction

Plants respond to temperature stresses in a variety of ways (Levitt, 1980). Exposure of grasses from temperate and cool climatic zones to chilling temperatures leads to an accumula­tion of fructan (Eagles, 1967; Pont is and del Campillo, 1985), which is preceded by an increase in the level of sucrose in the plant cell (Pontis, 1970; Chandorkar and Collins, 1974). This increment may be caused by the low demand for photo­synthates at low temperatures (Pollock et a1., 1983). During growth under these conditions, the metabolism undergoes an adaptation process, and the plants acquire «hardiness» (Le­vitt, 1980). When wheat seedlings were exposed to chilling temperatures, sucrose synthase (SS), one of the sucrose meta­bolizing enzymes, started to increase its activity within one hour after the beginning of the stress (Calderon and Pontis, 1985), while the activities of all other enzymes related to sucrose biosynthesis did not change. Similarly the enzymes of fructan metabolism were affected by a cold shock (Wag­ner et a1., 1983; Pollock, 1984).

© 1989 by Gustav Fischer Verlag, Stuttgart

We wondered what would happen to plants adapted to these hardy conditions when the cold stress disappeared, and if there could be a temperature effect on the enzymes acti­vities due to altered protein conformation. This paper re­ports studies carried out in order to answer these queries.

Materials and Methods

Wheat seeds (Triticum aestivum L. cv. San Agustin INTA) were kindly supplied by the Instituto Nacional de Tecnologia Agrope­cuaria, EERA Balcarce, Argentina. Wheat plants were grown in a chamber at 23°C with a day/night regime of 14/10 h. Light inten­sity at the plant canopy was 100Wm- 2

• Seven days following emergence seedlings were transferred to 4°C with the same day/ night regime and light intensity. Chilling treatments were always started at the beginning of day time (07.00 h). Similarly plants were always returned to 23°C at the same time of the day to minimize the effect of diurnal fluctuations in enzyme activity.

Procedures for leaf sampling, preparation of homogenates, and determination of SS, SPS, invertase, UDPase, sucrose and fructans

were as previously reported (Calder6n and Pont is, 1985). SST was prepared in a similar way. Conditions for preparation of ho­mogenates were selected in all cases to give maximum enzyme activ­ity. The only alteration in the previous procedure was the use of li­quid nitrogen to prepare leaf powders for the SS and SPS determinations instead of acetone. Fructan hydrolase was assayed by incubating in a total volume of 0.05 ml, 10/tmol (as fructose) of fructans (DP> 5), 5/tmol Tris acetate buffer pH 5.5, and enzyme at 30°C for 60 min. The fructose formed was determined by the So­mogyi-Nelson method (Spiro, 1966). Values obtained were cor­rected for possible invertase action on the terminal sucrose by de­termining the glucose formed using the glucose oxidase method (Spiro, 1966). SST was assayed by incubating in 0.05 ml total volume, 10/tmol 14C-sucrose (6,000 bq/ /tmol specific activity), 5/tmol acetate buffer pH 5.5 and an aliquot of enzyme extract at 30°C for 3 hs. Reaction was stopped by depositing aliquots of reac­tion mixture on Whatman N 4 paper. FS formed was separated from sucrose by paper chromatography on ethyl acetate-pyrydi­ne-water (8: 2: 1). The area corresponding to FS was cut and counted by liquid scintillation in a Beckman spectrometer. Blanks without sucrose or enzyme were also incubated. All incubations performed were linear with time and amount of enzyme. Fructans used as substrates for FH were extracted from wheat plants which had been kept at 4 °C for at least 15 days. Leaves were cut, extracted at 100°C with 10 mM Tris buffer pH 8.0. The extracts were freeze dried, the powders redissolved in distilled water and the fructans were fractionated in Biogel P-I0 columns. Fractions of the gel filtra­tion chromatography were analyzed by paper chromatography. All fructans of DP > 5 were pooled and the solutions were freeze dried.

Protein was estimated according to Lowry et al. (1951). The re­sults presented are means of at least three different experiments.

Results

SS and SPS activities, temperature effect and diurnal fluctuations

Previous reports have shown that when plants are sub­mitted to a chilling shock there is an increase in the level of activity of some enzymes (Calderon and Pont is, 1985). Be­cause the measurement of these enzyme activities was carried out at 30°C, the question arises whether the differences found in enzyme activity could be ascribed to a temperature­induced modification of the enzyme conformation. In an at­tempt to clarify this point, we selected SS and SPS, two key enzymes of sucrose metabolism. The enzymes were ex­tracted from plants grown at 4 and 23°C. Enzyme activities were determined at various temperatures between 4 and 23 °C, and Arrhenius plots were drawn. Straight and parallel lines with no discontinuity resulted, indicating that there was no change in the activation energy of either enzyme with temperature modifications. Slopes, intercepts and cor­relation coefficients are presented in Table 1.

Table 1: Slopes, intercepts and correlation coefficients of the Ar­rhenius plots of SS and SPS (y = a + b x).

a b r

SS (Plants grown at 4°C) 22.5 -6.30' 103 0.999 SS (Plants grown at 23°C) 22.2 -6.96' 103 0.989 SPS (Plants grown at 4 0C) 25.6 -7.51' 103 0.997 SPS (Plants grown at 23°C) 24.3 -7.74.103 0.988

0.5

- Q25 1:

....

Reversal of cold acclimation 233

-~ 0 i--+-----l---t----l--+-----+--I c -1.0

0.5

-Time (hours)

Fig. 1: Diurnal fluctuation of SS and SPS. Enzymes extracted from plants grown at 4°C. Similar curves were obtained from plants at 23°C.

Diurnal variation of sucrose metabolizing enzymes was in­vestigated to determine if the time of the day that plants are transferred has any influence on enzyme activities. This variation, if any, depends on the species under study (Huber and Israel, 1982; Huber et aI., 1985). In wheat, SS and SPS fluctuate diurnally at 4 °C as well as at 23°C, showing two peaks of activity per 24 hours period, at the beginning and end of the light period (Fig. 1). Fluctuations in SS activity have been previously observed only in cotton leaves; how­ever, one peak was detected (Hendrix and Huber, 1986). On the other hand, diurnal variations in SPS activity were re­ported in cotton, soybean, corn and peas (Rufty et aI., 1983).

Reversal of cold adaptation

Wheat plants grown at 23°C were divided into three groups seven days following emergence. The first group was maintained at 23 °C throughout the experiment; the second was transferred to 4°C and kept at this temperature during the remaining period of the experiment. The third group was transferred to 4 °C for 15 days and then moved back to 23°C.

The low temperature treatment resulted in an increase in the level of sucrose, FS and fructans (DP>3) (Table 2). These results were in agreement with previous reports (Pollock and Ruggles, 1976; Wagner et aI., 1983). When plants were moved back to 23°C following 15 days at 4°C a dramatic drop in the level of fructans (DP> 3) occurred. A decrease in the level of sucrose during the first 8 hours at 23°C was also

234 J. A. TOGNETTI, P. L. CALDER6N, and H. G. PONTIS

Table 2: Carbohydrate levels in wheat plants grown under different temperature conditions.

Growing conditions

10 days 23°C 10 days 23 °C, 15 days 4 °C 10 days 23°C, 15 days 4°C + 2 hs 23°C 10 days 23 °C, 15 days 4°C, 8 hs 23°C 10 days 23 °C, 15 days 4°C, 32 hs 23°C 10 days 23 °C, 15 days 4°C, 7 days 23°C 32 days 23 °C 10 days 23°C, 22 days 4°C

~0.3 a

Carbohydrates /,moles of fructose g - 1 FW

Sucrose FS F ructans (DP>3)

4.15 10.48 6.92 8.07 4.23 3.82 4.62

11.95

0.14 0.93 0.90 0.78 0.25 0.19 0.11 1.00

1.25 10.63 4.50 3.90 1.59 0.58 0.87

15.84

observed. Initially, the level of FS remained quite high. However, after one day at 23°C levels of sucrose and FS ap­proached those of plants that had not been exposed to 4°C.

Changes in the activities of SS, SPS, invertase, SST, FH and UDPase in response to temperature modifications are pre­sented in Figs. 2 and 3. While SS and SPS levels increased dur­ing the cold period, invertase went down. When activity is expressed on a per gram FW, invertase levels remained con­stant. This agrees with our previous work (Calderon and Pontis, 1985). However, SPS activities were different from those previously we reported, when there was no increase in activity after 7 days at 4 dc. We have now found that SPS, when extracted from acetone powders, is very unstable. It

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Fig. 2: Changes in the' enzyme actiVIties after cold adaptation: SS (--0--), SPS (--e--) and invertase (--\1--). Vertical bars: one standard error. Control of plants kept all the time at 23°C or 4°C are indicated by the same symbol followed by the respective temperature. Standard er­rors of the controls were always below 15 % of the values (not shown). Inset: protein levels during the deacclimation (-6-).

Fig. 3: Changes in enzyme activIties after cold adaptation: SST (--\1--), FH (-0-) and UDPase (-e-). Vertical bars: one standard error. Control of plants kept all the time at 23°C or 4°C are indicated by the same symbol followed by the respective temperature. Standard er­rors of the controls were always below 15 % of the values (not shown).

appeared that our previous results could be due to this fact. When plants were moved back to 23°C, SPS and SS acti­vities went down steadily while invertase activity began to rise after 24 hours. In all cases enzyme activities returned to those of controls kept at 23°C.

The enzymes closely associated with fructan metabolism, SST and FH showed a different pattern. SST activity rose sig­nificantly during the 4 °C period and then decreased at a warmer temperature. FH activity decreased sharply during the cold treatment and then rose steadily during the first day at 23 °C reaching a plateau corresponding to the enzyme level of plants kept at 23°C. On the other hand, UDPase, an enzyme not related directly with fructan metabolism, was not affected by the changes in temperature. Enzyme acti­vities were expressed on a protein basis in order to eliminate the influence of the change in amount of protein (Fig. 2, inset} on the patterns of enzyme activities.

Discussion

The influence of temperature on enzyme actlvltles in wheat shows close relationship between sucrose and fructan metabolisms in agreement with results reported in Compo­sitae (Pontis, 1966; Edelman and Jefford, 1968; Frehner et aI., 1984) and in other Gramineae (Pollock, 1979, 1984; Wagner et aI., 1983; Volenec and Nelson, 1984). Particularly the two sucrose metabolizing enzymes, SS and SPS, appear to have a key role during the cold acclimation and its reversal. The in­crease in SS activity after a chilling shock has led Calder6n and Pontis (1985) to suggest that this enzyme may be asso­ciated with the sucrose transport into the vacuole. In con­trast with such a hypothesis, Thorn and Maretzki (1985) as­signed this role to SPS. Further work is necessary to ascertain the roles of each synthase regarding sucrose trans­port into the vacuole, specially in light of the experiments of Salerno et ai. (1989), which showed that in wheat leaf sec­tions supplied with exogenous sugars, SS activity levels are not affected while SPS activity as well as sucrose and fructan levels increase several fold.

It appears that SPS and SS behave similarly to temperature changes, reaching both of them, at the end of the deacclima­tion period, activity levels that are very similar to those in plants maintained at 23°C. SST and FH activities also show a response to temperature changes. During cold acclimation SST increases while FH decreases. Reversal of cold adapta­tion was associated with a reversal in the enzyme activities. Concomitantly, fructan accumulated during cold acclima­tion are hydrolized when the plants are returned to warm temperatures.

In conclusion, young wheat plants seem to be a good system in which to study the factors that govern plant ad­aptation to environmental stresses.

Acknowledgements

The authors are indebted to their colleagues in the Centro de In­vestigaciones Biolagicas for helpful discussions and criticism, and to Mrs. Clara Fernandez and Miss Carmen Rodriguez for their able technical assistance.

Reversal of cold acclimation 235

H. G. P. is Careerlnvestigator andJ. A. T. is a fellow of the Con­sejo Nacional de Investigaciones Cientificas y Tecnicas (CON­ICET), Argentina. This paper is part of a dissertation of J. A. T. at the Universidad Nacional de Mar del Plata. This work was sup­ported by grants of the CONICET, by SECyT and by the Comi­sian de Investigaciones Cientificas de la Provincia de Buenos Aires.

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