1-Aminocyclopropane-1-Carboxylic Acid, its Malonyl Conjugate and 1-Aminocyclopropane-1-Carboxylate Synthase Activity in Needles of Damaged and Undamaged Norway Spruce Trees

J Plant Physiol.

Vol. 143. pp. 389-395 {1994)

1-Aminocyclopropane-1-Carboxylic Acid, its Malonyl Conjugate and 1-Aminocyclopropane-1-Carboxylate Synthase Activity in Needles of Damaged and Undamaged Norway Spruce Trees C.

YANG, W. WILKSCH,

and A.

WILD

Institute of General Botany, Johannes Gutenberg University, SaarstraEe 21, 55099 Mainz, Germany Received October 5, 1993 · Accepted November 16, 1993

Summary

Levels of free 1-aminocyclopropane-1-carboxylic acid (ACC), its malonyl conjugate (MACC) and ACC-synthase activity were significantly higher in the needles of damaged Norway spruce trees (Picea abies (L.) Karst) compared with those in undamaged trees during both the diurnal and seasonal courses. The concentrations of ACC and MACC, and ACC-synthase activity fluctuated much more intensively in the needles of damaged trees both in the diurnal and the seasonal courses than in those of undamaged ones, which implies that damaged trees respond more sensitively to the environmental conditions. ACC concentrations were the highest in summer and lowest in fall, while MACC concentrations seemed to increase during the year. The concentrations of ACC and MACC during the diurnal course present therelation of a power function. The results of the measurements on needles of different ages showed that the older the needles were, the more active the ACC-synthase was, and thereby more ACC and MACC were produced in the damaged trees.

Key words: Picea abies, 1-aminocyclopropane carboxylic acid (A CC), A CC-synthase, N-malonyl-1-aminocyclopropane carboxylic acid (MA CC}. Abbreviations: ACC = 1-aminocyclopropane carboxylic acid; CET = central European time; FW = fresh weight; HEPPS = N-[2-hydroxyethyl]piperazine-N' -[2-propanesulfonic acid]; MACC = N-malonyl-1-aminocyclopropane carboxylic acid. Introduction

Ethylene regulates many aspects of plant growth and development. It has been demonstrated that the direct precursor of ethylene biosynthesis is ACC (Yang and Hoffman, 1984), which is the key substance controlling ethylene synthesis. The malonyl conjugate of ACC (MACC) can also be produced from ACC together with ethylene (Amrhein et al., 1981). The synthesis .of ACC is regulated by two different types of factors. One type is promoted by the internal signals, as ethylene plays an important role in some developmental stages, e.g. seed germination (Ketring and Morgan, 1972; Logan et al., 1991), root and leaf growth (Burg and © 1994 by Gustav Fischer Verlag, Stuttgart

Burg, 1968; Chadwick and Burg, 1970), leaf abscission (Kao and Yang, 1983), and flower and fruit senescence (Burg, 1968). Ethylene enhanced in these developmental stages is recognized as «auxin induced» ethylene. Ethylene induced under stress conditions is defined as «stress ethylene» (Yang and Hoffman, 1984). Very pronounced stress ethylene is produced when the plants are under environmental stresses, e.g. water stress (Ben-Yehoshua and Aloni, 1974; Wright, 1977; McKeon et al., 1982; Hoffman et al., 1983), chilling stress (Elstner and Konze, 1976; Wang and Adams, 1982; Field, 1984), wounding (Yu and Yang, 1980; Hyodo and Nishino, 1981; Hoffman and Yang, 1982), the exposure to SOz (Peiser and Yang, 1979; Skorupka, 1985; Meyer et al.,

390

C. YANG, W. WILKSCH, and A. WILD

1987), ozone (Elstner et al., 1985; Rodecap and Tingey, 1986; Mehlhorn and Wellburn, 1987; Meyer et al., 1987; Langebartels et al., 1991) or other pollutants (Hogsett et al., 1981; Fuhrer, 1982). The enhancement of ethylene production, together with the changes in concentrations of other kinds of plant hormones, provides the plants with the mechanisms in adapting or avoiding the environmental stress. Quite a few papers have been published about the changes in the concentrations of ACC and MACC in stressed plant tissues. Many observations demonstrate that the level of ACC in stressed plants increases many times more than that under normal conditions (Boller and Kende, 1980; Yu and Yang, 1980; Kende and Boller, 1981; Rodecap et al., 1981; Field, 1984; Elstner et al., 1985; Langebartels et al., 1991). Yet, quite different results about the changes in the concentration of MACC were published. Fuhrer {1985) found elevated levels of MACC and ACC in the needles of injured fir trees. Elstner et al. {1985) observed that levels of MACC were five times higher in yellow green needles of spruce trees injured by photooxidants compared with dark green needles. However, Skorupka (1985) measured no accumulation in the concentrations of MACC in the needles of both the spruce and fir trees exposed to ozone for 5 weeks, as well as the damaged ones exposed to ozone for a longer time. Chen and Wellburn (1989) did not find any increase in MACC concentrations in damaged spruce trees compared with that in undamaged trees. The changes in the concentrations of ACC and MACC, and ACCsynthase activity in the needles of damaged and undamaged spruce trees both in short-term and long-term observations at a natural site are presented in this paper. The comparison of the relations of ACCMACC concentrations during diurnal and seasonal courses and the possible factors regulating the diurnal course are also discussed.

Materials and Methods Description ofthe site The observations were performed in the northern Black Forest, on the Schollkopf mountain, on a plateau of sandstone 820- 830 m above sea level. It is part of the Forest District Vordersteinwald (Division IITI12a4), Forest Office Freudenstadt. This area is characterized by a high ozone concentration, which reaches 125 11g m -l (monthly mean) during summer. The emission and the climatic data were observed by the IVD (Institut fiir Verfahrenstechnik and Dampfkesselwesen), Department of Air Pollution Prevention, University of Stuttgart, at a measuring station situated at a distance of 500 m from the stand. A detailed description of the sites has been presented by Wessler and Wild (1993) and Wild et al. (1993).

Plant materials and chemicals The spruce trees studied were about 45 years old. The needles of the second generation (grown in the previous year) from the sixth to the eight whorl were sampled for the investigation on variations in the concentrations of ACC and MACC, and ACCsynthase activity during both diurnal and seasonal courses in damaged and undamaged trees. The needles of the current and third generations (grown in the present year and the year before the previous year, respectively) were also sampled for the investigations on concentra-

tions of ACC and MACC, and on ACCsynthase activity in needles of the different ages. The samples were taken from two groups of trees, six damaged (damage class 2-3) (Bundesminister fiir Ernahrung, Landwirttschaft und Forsten, 1992) and six undamaged (damage class 0) spruce trees, respectively. After sampling, the twig pieces were immediately put into liquid nitrogen and needles were removed from the twig by stirring theiil. The needles of damaged and undamaged trees were mixed separately, except for those that were used for the measurements of the data for each single tree to evaluate the validity of the measurement of the mixed samples. The deepfrozen needles were finally stored at - 80 °C. The concentrations of ACC and MACC measured from the needles of the six single trees separately and the results measured from the mixed samples of the six trees were compared. They coincide with each other very well (data omitted). The sampling for investigation of seasonal changes was performed four times from April to October in 1991, and three times from August to October in 1992. Attention was paid to the uniformity of the harvest time (14:00) during the day in order to avoid the disturbance of strong diurnal variations in the analysis of seasonal changes. On October 9, 1992, the sampling for the measurement of the diurnal course was performed from 8 to 17 o'clock (CET). The standard MACC was a gift from Dr. Amrhein, ETH Ziirich. All other chemicals (analytical grade) were obtained commercially.

Measurements ofthe concentrations ofA CC and AM CC One g of deep-frozen needles was ground in a microdismembrator (B. Braun, Melsungen, Germany) at a speed of 1,800 RPM for 2 min. Eighty percent (v/v) methanol was used to extract ACC and MACC at 4 °C for 2 h; 4% (w/v) insoluble PVP was added to the homogenate to remove phenolic compounds. The homogenate was centrifuged (1000 xg, 5min, Heraeus-Christ, Minifuge GL, Hanau, Germany). The pellet was extracted using the same method once more. The supernatants were collected and evaporated to dryness in a vacuum at 38 °C, then resolved and stirred in 1 mL chloroform and 3 mL water, and centrifuged again (15,000 x g, Beckman J2-21, Miinchen, Germany). The water phase was used for the assay of ACC and MACC concentration. An aliquot (200 11L) of the extract was employed for the ACC assay according to Lizada and Yang (1979). Another aliquot (200 11L) was hydrolysed in 2 N HCl at 100 °C for 3 h to liberate ACC (Hoffman et al., 1983) and neutralized with NaOH. The resulting hydrolysate was assayed for ACC as described above. The content of MACC was calculated by subtracting the content of ACC before hydrolysis from the content of ACC after hydrolysis. The measurement was repeated three times on each extract. Ethylene was measured on a gas chromatograph (Schimadzu, GC9A, Kyoto, Japan) equipped with an alumina column (temperature 80 °C} and a flame ionization detector. The whole procedure was repeated three to four times.

Measurements ofA CC.synthase activity One and a half g of deep-frozen needles was ground as described above, and then extracted at 4 °C for 1 h with 100 mM HEPPSKOH buffer (pH 8.5), containing 4 mM dithioerythritol (DTE) and 4J.LM pyridoxal phosphate. The homogenate was centrifuged at 36,000 g for 25 min (Beckman J2-21, Miinchen, Germany). The pellet was extracted and centrifuged once again. The supernatant was collected and dialyzed at 4 °C over night against dialysis buffer (2mM HEPPS, 0.1mM DTE, 0.2J.LM PAL pH 8.5). An aliquot (400 J.LL) of the dialysed extract was used for the enzyme activity assay. The measurement of each extract was repeated three times. The reaction was started by the addition of 0.055 mM SAM. After an incubation period of 30 min at 30 °C, the reaction was stopped with

ACC, MACC and ACC-synthase in spruce 111M HgCb solution and then the mixture was assayed for the concentration of ACC with the method described above. The measurement was repeated twice.

391

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Statistical analysis The arithmetic mean of two to four repeated measurements was used as the value of every determination. The standard deviations, which are indicated as the bars in the figures, reflect the dispersion of the mean. A two-tailed independent Student's t-test was employed to analyse the difference between the diurnal courses of damaged and undamaged trees. The arithmetic means of determinations of each hour present the average levels of parameters, and the standard deviations of them express the fluctuation of the parameters during the day.

Results

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Fig.2A,B,C: Concentrations of ACC (A) and MACC (B) and the ACC-synthase activity (C) in needles of the second generation in undamaged (D) and damaged (•) spruce trees at the Freudenstadt site in 1992. The bars indicate the standard deviation of three to four replicates (ACC and MACC) or two replicates (ACC-synthase activity).

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The results of four measurements of the content of ACC and MACC in 1991 are presented in Fig. 1 A, B, respectively. During the summer, the concentration of ACC reached its maximal level and then decreased in the fall (Fig. 1 A). The concentrations of MACC increased all the time during the year in both damaged and undamaged trees (Fig. 1 B). It can also be seen clearly that both levels of ACC and MACC in the needles of damaged trees underwent great fluctuations in the course of one year; there were no such great changes in the needles of undamaged trees. Figure 2 A, B, C shows the levels of ACC and MACC, and ACC-synthase activity, respectively, from August to Octo-

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ber in 1992. The determination of ACC showed the same decrease in fall observed in 1991 in the needles of damaged trees (Fig. 2 A). The changes of MACC concentrations did not show the exact same tendency as in 1991, as the determination of the last harvest in the needles of damaged trees was somewhat lower. It can be seen that both the concentrations of ACC and MACC in damaged trees are significantly higher than those in undamaged ones. ACCsynthase activity in the needles of both damaged and undamaged trees increased along with the time. ACC-synthase activity is much higher in needles of damaged trees. The diurnal courses of ACC, MACC and ACC-synthase activity in the needles of damaged and undamaged trees are shown in Fig. 3 A, B, C, respectively. In damaged trees, the concentration of ACC showed great fluctuation in the first 3 h; it kept decreasing until afternoon and then increased towards evening. There was no such great variation in the concentrations of ACC in undamaged trees. This is also the case in two other diurnal courses that we measured at another natural site (Yang et al., 1993).

392

C. YANG, W. WILKSCH, and A. Wn.o

Table 1: The average of the data and the standard deviation (X±Sd), the maximal (max.) and the minimal (min.) values of the concentrations of ACC and MACC {nmol/g FW), and the ACC-synthase activity [nmollh g FW] of the diurnal courses at the Freudenstadt site measured on October 9, 1992 in the needles of undamaged and damaged spruce trees.

ACC

MACC

ACC -syn

undamaged

damaged

t-test

X±Sd max. mm.

0.24±0.07 0.35 0.13

7.33±4.68 15.73 2.00

P<0.001

X±Sd max.

23.21±7.72 40.08 15.15

P<0.001

mm.

7.48±0.96 8.62 6.04

X±Sd max. mm.

0.12±0.02 0.17 0.09

0.36±0.19 0.78 0.16

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Table 1 presents the averages and standard deviations, maximal and minimal values and the results of the t-test for the diurnal courses. It can obviously be seen that in damaged trees the concentrations of ACC and MACC, and the ACC synthase activity are significantly higher throughout the whole day compared with those in undamaged ones. The standard deviations of the means of the concentrations of ACC and MACC, and ACCsynthase activity in the diurnal course are much higher in damaged spruce trees compared with undamaged ones, which means that the concentrations of ACC and MACC, and ACCsynthase activity fluctuate to a greater extent in the needles of damaged trees during the day. The variation in the concentration of MACC in shortterm observations does not show that MACC accumulates continuously, although it does in the seasonal changes, especially in damaged trees. The concentrations of MACC fluctuate along with the changes of the concentrations of ACC both in damaged and undamaged trees throughout the day. The relation of ACC and MACC measured at the Freudenstadt site can be fitted into a power function (formula 1), which indicates that MACC increases along with the level of ACC.

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When analysing the differential equation of formula 1, we can see that the increment of MACC slows down when the level of ACC increases, which clearly means that levels of MACC change more sensitively along with variations in the levels of ACC in undamaged trees. One possible reason could be that damaged trees produced more ethylene and less MACC from ACC. The concentrations of ACC and MACC, and ACCsynthase activities in the needles of the current to third generations sampled at three different times in 1992 were also measured in order to investigate the difference among them due to the needle age. Figure 4 A, B, C shows the concentrations of ACC and MACC, and ACC-synthase activity in the needles of the current to third generations. It can be seen that older needles have higher levels of ACC and MACC, and ACC-synthase activity, which is particularly distinct for the damaged trees. In addition, Fig. 4 demonstrates that the concentrations of ACC and MACC, and the ACCsynthase activity are significantly higher in the needles of all different generations of damaged trees than those of undamaged ones.

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Fig.3A,B,C: The diurnal courses of concentrations of ACC {A), MACC (B) and ACC-synthase activity (C) in needles of the second generation in undamaged ( 0- - - - -0) and damaged · {• - - • ) spruce trees at the Freudenstadt site measured on October 9, 1992. The bars indicate the standard deviation of three to four replicates (ACC and MACC) or two replicates {ACC-synthase activity).

Discussion Our results clearly demonstrate that significantly higher values appear not only in levels of ACC and ACCsynthase activity, but also in levels of MACC in the needles of damaged trees compared with those of undamaged ones, both in short-term and long-term observations, which confirm the observations in other research with different plant materials (Elstner and Konze, 1976; Kende and Boller, 1981; McKeon et al., 1982; Hoffman et al., 1983; Elstner et al., 1985; Kacperska and Kubacka-Zebalska, 1989; Langebartels et al.,

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Fig.4A,B,C: Concentrations of ACC (A), MACC (B) and the ACC-synthase activity (C) in the needles of different ages, {needles of the current generation {0), needles of the second generation {lilil), needles of the third generation (•)), in undamaged and damaged spruce trees at the Freudenstadt site in 1992. The bars indicate the standard deviation of three to four replicates {ACC and MACC) or two replicates {ACC-synthase activity). nd = not detected.

1991). Kende and Boller (1981) proposed from the experiment with wounded tomato fruits that the rate of ethylene production rose parallel to the levels of ACC, which is controlled by ACC-synthase activity. The high levels of ACC and ACCsynthase activity in needles of the damaged trees imply a high rate of ethylene production in the plant tissue. The concentrations of ACC in the needles of damaged trees showed clear changes, while in undamaged trees there was no such great change in the course of the year. In view of the fact that ozone is the main air pollutant in this area, the highest concentrations of ACC in the needles of damaged spruce trees could be the result of the high 03 pollutant stress, which reaches its highest level during summer in combination with drought stress and light stress. Our result also shows that damaged trees respond to environmental condi-

393

tions more sensitively and undergo more intensive reactions. Langebartels et al. {1991) reported that treatment of 03 to the ozone-sensitive tobacco cultivate enhances the ACC production five times. Mehlhorn and W ellburn {1987) proposed that formation of stress ethylene determines the sensitivity of plants to atmospheric levels of ozone. They have observed that plants that produce a high level of ethylene are more sensitive to atmospheric levels of ozone. When hardened with 0 3 fumigation, the pea seedlings reduce the production of stress ethylene and thereby avoid the visible injury. The high concentrations of ACC measured in damaged trees at the Freudenstadt site during the summer could be one of the phenomena that indicate that plants could be injured from environmental stress. This result also agrees with the long-term observations of other parameters made on the same trees. Richter and Wild (1992) reported that phenolic compounds, a group of substances that increases as a consequence of stress, which causes membrane damage, reached their highest concentrations in the needles of the same trees during summer. Tenter and Wild (1991) observed the variations in the concentrations of polyamines during the seasonal changes at the Freudenstadt site. They demonstrated that the concentrations of putrescine increased in the needles of damaged trees but there was a minimal level of putrescine during summer. Polyamines and ethylene are two types of phytohormones that exert opposite functions. Polyamines delay senescence, while ethylene promotes it. They are both synthesized from the same precursor and mutually inhibit each other's synthesis (Apelbaum et al., 1981; Evans and Malmberg, 1989; Langebartels et al., 1991). The protecting function of polyamines under stress conditions is to stabilize membranes (Altman, 1982; Naik and Srivastava, 1986), while the rate of ethylene emission is often connected with the visible injury of plant tissue (Mehlhorn and Wellburn, 1987). In 1991, the concentrations of MACC in both damaged and undamaged trees showed a similar tendency in the course of the year. MACC concentration reached its maximal value at the last harvest. In 1992, MACC measured in damaged trees did not completely repeat the tendency of MACC variation in 1991. The value for the last harvest date was somewhat lower, perhaps because it was snowing during the harvest. It remains obscure whether the higher value of MACC concentration in winter reflects the accumulation in concentration of MACC, or a higher activity in ACC N-malonyltransferase. Research on the diurnal courses of the concentrations of ACC and MACC, and ACC-synthase activity demonstrates that the variations in the concentrations of ACC and MACC are connected closely to the activity of ACC-synthase. When comparing the sum of the levels of ACC and MACC in the diurnal courses in the needles of undamaged and damaged trees, it is like-wise clear that they have similar tendencies that also coincide with the changes in ACC-synthase activity. Because other diurnal courses of ACC and MACC concentrations that we measured in both damaged and undamaged spruce trees at another natural site presented almost the same pattern as shown in Fig. 3 {Yang et al., 1993), it is reasonable to say that some pattern of diurnal variance does exist in the needles of the spruce trees.

394

C. YANG, W. WILKScH, and A. Wn.o

Rikin et al. (1984) and Ievinsh and Kreicbergs (1992) demonstrated that ethylene emission is an endogenous rhythmical process in higher plants. Rikin et al. (1984) proposed that the conversion of ACC to ethylene is not affected by the rhythm, but directly by light, while an earlier step in the conversion of methionine to ethylene is controlled by an endogenous rhythm. According to his prediction and the result of our measurement, it is quite possible that ACCsynthase is not only the key enzyme in stress ethylene production but also in the rhythmical changes of ethylene emission. It is interesting to see that the relationships between the concentrations of MACC and ACC are different in the diurnal course and in the seasonal changes. In the course of the year, there is no correlation between the concentrations of ACC and MACC. MACC increased during the year while ACC increased until summer and then decreased in fall. In the diurnal course, MACC did not accumulate continuously but fluctuated along with the levels of ACC. This phenomenon does not confirm the common idea that MACC is an inactive end product, and that it accumulates continuously in the plant tissue. It seems that the concentrations of ACC and MACC and perhaps the emission of ethylene keep a dynamic balance, but the state of this dynamic balance is determined by the condition of the plant, whose variations can normally be seen in long-term observation. As to the reason for the fluctuation in the concentration of MACC in the diurnal course, it is not yet clear whether it is hydrolized to ACC, or translated to other plant tissues. Jiao et al. (1986) suggested that MACC can be hydrolysed to ACC when its concentration in plant tissue is very high. Further research on the mechanism of fluctuation of MACC concentration during the day is still needed to clarify whether MACC is really an inactive end product or whether it also serves as a storage substance. Levels of both ACC and MACC are significantly higher in damaged trees than in undamaged trees at the Freudenstadt site both in diurnal course and seasonal changes. The diurnal course of the concentrations of ACC and MACC could be regulated by the physiological endogenous rhythm. The relation of concentrations of ACC and MACC and the emission of ethylene are perhaps determined by the conditions of the plants. More work is necessary to elucidate the mechanisms of the interaction of polyamines and ethylene, and the physiological functions of MACC. Acknowledgements

This study was supported by the KfK.-PEF (K.ernforschungszentrum Karlsruhe - Projekt Europliisches Forschungszentrum fiir Maf!nahmen zur Luftreinhaltung), grant no.: 88/007/1A, Thanks are also due to Prof. Dr. Amrhein for providing standard MACC; and Dr. W. Ruehle for the suggestion in the statistical analysis.

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