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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.35 no.1 Santiago  2002

http://dx.doi.org/10.4067/S0716-97602002000100009 

Peroxidase and phenylalanine ammonia-lyase activities, phenolic acid contents, and allelochemicals-inhibited root growth of soybean

 

VANESSA HERRIG1, MARIA DE LOURDES L. FERRARESE1; LETÍCIA S. SUZUKI1; JOÃO DOMINGOS RODRIGUES2, AND OSVALDO FERRARESE-FILHO 1

1 Department of Biochemistry, University of Maringá, Av. Colombo, 5790, 87020-900, Maringá, PR, Brazil. 2 Department of Botany, IB, Unesp, P.O.B. 510, 18618-000, Botucatu, SP, Brazil

Corresponding Author: Osvaldo Ferrarese-Filho. Universidade Estadual de Maringá. Departamento de Bioquímica. Av. Colombo, 5790. 87020-900 Maringá _ PR. BRAZIL- Fax: (055) 44 2633655. e-mail: offilho@uem.br

Received: April 16, 2001. In revised form: December 11, 2001. Accepted: March 20, 2002

ABSTRACT

The influence of the allelochemicals ferulic (FA) and vanillic (VA) acids on peroxidase (POD, EC 1.11.1.7) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activities and their relationships with phenolic acid (PhAs) contents and root growth of soybean (Glycine max (L.) Merr.) were examined. Three-day-old seedlings were cultivated in nutrient solution containing FA or VA (0.1 to 1 mM) for 48 h. Both compounds (at 0.5 and 1 mM) decreased root length (RL), fresh weight (FW) and dry weight (DW) and increased PhAs contents. At 0.5 and 1 mM, FA increased soluble POD activity (18% and 47%, respectively) and cell wall (CW)-bound POD activity (61% and 34%), while VA increased soluble POD activity (33% and 17%) but did not affect CW-bound POD activity. At 1 mM, FA increased (82%) while VA reduced (32%) PAL activities. The results are discussed on the basis of the role of these compounds on phenylpropanoid metabolism and root growth and suggest that the effects caused on POD and PAL activities are some of the many mechanisms by which allelochemicals influence plant growth.


Key terms:
roots, phenolic acids, phenylalanine ammonia-lyase, peroxidase, soybean.

 

INTRODUCTION

Phenolic compounds constitute an important class of organic substances that are released into soil environment as root exudates, leaf leachates, and products of plant tissue decompose. These released chemicals are accumulated and persist for a period of time, imparting significant interfering effects on growth of neighboring plants, a phenomenon termed allelopathy (28). Deleterious or toxic effects of allelochemicals on germination and/or plant growth are well documented in different species, but the physiological or biochemical roles of many of them have not yet been examined rigorously. Furthermore, a characteristic of these effects is the variability among species, which does not have a defined rule that applies to all plants (10).

Among the types of chemical compounds identified as allelochemicals, cinnamic and benzoic acid derivatives, including FA and VA, are commonly found in soils at concentrations between 0.01 and 0.1 mM, and affect plant growth at concentrations of up to 10 mM (2, 18, 21). Previous investigations showed that plant exposure to cinnamic or benzoic acids inhibits foliar expansion and root elongation, reduces water and mineral uptake, photosynthesis, protein synthesis, enzyme activities and lipid mobilization (3, 4, 8, 20, 25, 26, 27).

Although a reasonable number of studies have been devoted to the effects of some allelochemicals on physiological and biochemical events in different species, less effort has been spent investigating these effects in soybean (4, 9, 22, 24). Surprisingly, no attention has been paid to the effects of benzoic or cinnamic derivatives on soybean POD, which is involved in various metabolic steps, as well as PAL, which is regarded as the primary enzyme that leads to phenylpropanoids, and consequently may protect plants against various biotic and abiotic stresses (14, 17). Due to this fact, the goal of the present work was to investigate the influence of FA, a cinnamic acid derivative, and VA, a benzoic acid derivative, on POD and PAL activities, PhAs contents and RL of soybean seedlings grown in nutrient solution.

 

METHODS

General procedures

Soybean (Glycine max (L.) Merr., cv BR-16) seeds were sterilized with 2% sodium hypochlorite for two minutes and washed extensively with deionized water. The seeds were grown between paper toweling placed in polystyrene containers (10 x 16 cm) with a small amount of deionized water in the bottom. The containers were incubated in a dark germination chamber at 25ºC (± 0.2) and 80% relative humidity. Three-day-old seedlings of uniform size were transferred to containers (10 x 16 cm) filled with 200 ml of full-strength Hoagland's solution (11) with or without FA or VA (0.1; 0.5 or 1 mM). Nutrient solution was buffered with 17 mM potassium phosphate buffer and adjusted to pH 6.0. Each container held 25 uniform seedlings suspended in the solution by floating styrofoam boats. The containers were kept in a growth chamber at 25ºC under fluorescent light (280 µmol m-2 s-1) on a 12-h dark/12-h light cycle. The nutrient solution was aerated continuously by air bubbling. The roots were exposed to allelochemicals for 48 h with the nutrient solution completely renewed after the first 24 h. Roots were measured before and after experiments. Roots FW were determined gravimetrically immediately after 48 h and DW was determined after oven drying at 80°C for 24 h. Phenolic compounds used in this investigation were purchased from Sigma Chemical Co (USA). All other reagents used were of the purest grade available or chromatographic grade.


Enzymatic activities and determination of the PhAs contents

After 48 h, all the roots of treated or untreated seedlings were detached and used for enzyme extraction and PhAs quantitation. For POD, fresh roots (0.25 g) were extracted with 2.5 ml of 67 mM phosphate buffer (pH 7.0) as described by Shann and Blum (32). The extract was centrifuged at 10,000 x g for 15 min at 4ºC and the supernatant was used to determine the activity of soluble POD. To isolate CW-bound POD, the pellet was washed with deionized water (at 4ºC) until no activity of soluble POD was detected in the supernatant. The pellet was washed twice with 1 ml of 1 M NaCl (less than 7% of POD activity remained in the pellet). At each step of extraction, the homogenate was centrifuged at 10,000 x g for 15 min. The supernatants of each fraction were pooled and considered to be the CW-(ionically) bound POD. Guaiacol-dependent activities of the soluble and CW-bound POD were determined according to Cakmak and Horst (7) with some modifications. The reaction mixture (3 ml) contained 25 mM sodium phosphate buffer (pH 6.8), 2.58 mM guaiacol and 10 mM H2O2. The reaction was initiated by the addition of diluted enzyme extract in phosphate buffer. Guaiacol oxidation was followed for 5 min at 470 nm, and the enzyme activity was calculated using the extinction coefficient (25.5 mM-1 cm-1) for tetraguaiacol. The reaction mixture without enzyme extract was used as a blank, and this value was subtracted from those with enzyme extract. POD activities were expressed as µmol min-1 g-1 FW.

PAL was extracted following the Havir method (15) with some modifications. Fresh roots (2 g) were ground at 4ºC in 0.2 M sodium borate buffer (pH 8.8). The homogenates were centrifuged at 12,000 x g for 15 min, and the supernatant was used as enzyme preparation. For the PAL activity assay, the reaction mixture (100 µmoles sodium borate buffer (pH 8.7) and a suitable amount of enzyme extract in a final volume of 1.55 ml) was incubated at 40ºC for 5 min, started by the addition of 15 µmoles of L-phenylalanine and stopped after one hour of incubation by the addition of 50 µl of 5 N HCl (12). Samples were filtered through a 0.45 µm disposable syringe filter and analyzed (20 µl) using a Shimadzu® Liquid Chromatograph equipped with a LC-10AD pump, a Rheodine® injector, a SPD-10A UV detector, a CBM-101 Communications Bus Module, and a Class-CR10 workstation system. A reversed-phase Shimpack® GLC-ODS (M) column (150 x 4.6 mm, 5 µm) was used at room temperature in conjunction with the same type of pre-column (10 x 4.6 mm). The mobile phase was methanol:water (70:30) with a flow rate of 0.5 ml min-1 for an isocratic run of 10 min. Absorbance of the samples as well as of the standard were detected at 275 nm, and data collection and integration were performed with the Class-CR10 software (Shimadzu®). t-Cinnamate, the product of the PAL reaction, was identified by comparison of its retention time with that of the standard. Simultaneous controls without L-phenylalanine or with t-cinnamate (added as internal standard in the reaction mixture) were made as described elsewhere (12). PAL activity was expressed as µmol t-cinnamate min-1 g-1 FW.

To quantify PhAs, roots treated with working solutions were thoroughly washed with deionized water. Fresh roots were boiled for 30 min in 5 ml of 2 N HCl as described by Heimler and Pieroni (16). After cooling the homogenate was filtered through a Whatman filter, and the filtrate was used to determine the PhAs contents. Samples (5 ml) were mixed with 0.75 ml of 1.9 M Na2CO3 and 0.25 ml of Folin-Ciocalteau phenol reagent. The mixture was allowed to stand in darkness at room temperature (23°C - 25°C) for one hour and the absorbance was measured at 750 nm against deionized water as blank (11). PhAs contents were expressed as µmol g-1 FW.

Statistical Analysis

Statistical tests were performed using InStat package, version 1.12 (GraphPAD Software, USA). The statistical significance of the difference between parameters was evaluated by means of Student's t-test. The results are given in the text as p, the probability values, and p£0.05 was adopted as criterion of significance. Data in the text and figures are expressed as means of separate experiments ± SEM.

 

RESULTS

The effects of FA and VA (0.1 to 1 mM) on RL, FW and DW were examined 48 h after the root treatment (Table I). It is clear that FA or VA, at the highest concentration, prompted a significant decrease in the RL compared with the control roots (61% and 54%, respectively). At 0.5 mM, FA and VA reduced RL by about 23% and 46%, respectively. On the other hand, at the lowest concentration, no significant (p>0.05) alteration on RL occurred after FA or VA treatments. The two compounds also affected FW and DW. At the highest phenolic acid concentration, root FW diminished 34% and 32% in the presence of FA and VA, respectively. Under this same condition, both compounds reduced the root DW by 33% and 18%. At 0.5 mM, FA and VA reduced FW (17% and 27%, respectively) and DW (15% and 26%). Unlike the results above, no appreciable changes in FW or DW were recorded when roots were cultivated in the lowest concentration (0.1 mM). The same Table reveals significant (p­0.05) differences between the results obtained with each compound: the effect of 0.5 mM FA on RL was less pronounced than VA and more effective at 1 mM. In turn, differences with respect to the effects on FW and DW were not significant (p>0.05) at any concentration.

Figure 1: Effects of ferulic (FA) and vanillic (VA) acids on soluble POD activity (A), CW-bound POD activity (B), PAL activity (C) and PhAs contents (D). Three-day-old seedlings (25) were cultivated in 200 ml of full-strength Hoagland's solution (pH 6.0, 25ºC, 80% relative humidity, 12-h photoperiod, 280 µmol m-2 s-1), with or without FA or VA (0.1; 0.5 or 1 mM). After 48 h, all roots were detached and used for enzyme extraction and PhAs quantitation. POD activities and PhAs contents were carried out by colorimetric assays, and PAL activity was determined by HPLC.

* Significantly different from control. ` Significantly different between the two compounds at the same concentration. Student's t-test (p£0.05).


In order to scrutinize the effects of FA or VA (0.1 to 1 mM) on POD activity, seedlings were cultivated with exogenous treatment of each allelochemical for 48 h (Fig. 1). It was found that the soluble POD activity gradually increased against FA concentration (Fig. 1A) since significant results over the control roots occurred at 0.5 mM (18%) and 1 mM (47%). On the other hand, there is no relationship between POD activity and VA concentration since the effects were more pronounced at 0.5 mM (33% of control) than at 1 mM (17%). Furthermore, it is important to note a significant (p£0.05) difference between the two compounds at 1 mM. The same Figure 1B shows that FA was able to stimulate the CW-bound POD activity by approximately 61% at 0.5 mM and by 34% at 1 mM. However, in contrast with the results obtained with FA, VA did not alter CW-bound POD activity. Consequently, the significant (p£0.05) difference between FA and VA at 0.5 mM or 1 mM is evident.

Effects of the phenolic compounds on PAL activity are summarized in Figure 1C.

Both FA and VA affected PAL activity significantly (p£0.05) but only in the highest concentration. Under this condition, a great increase (82%) in activity was found after FA treatment, in comparison with the control roots. On the contrary, a significant decrease (32%) in PAL activity was detected in roots exposed to 1 mM VA. The difference between the compounds is quite clear.

Another fact revealed in this work is that RL, FW and DW decreased in the presence of both phenolic compounds (Table I), while values of the PhAs content increased (Fig. 1D). With the exception of 0.1 mM, the PhAs content increased in other concentrations in comparison with the control roots. For example, FA and VA increased PhAs by 13% and 40%, at 0.5 mM, and by 57% and 59%, at 1 mM, respectively. Treatment of data did not show any significant (p>0.05) differences between the two compounds.

 

DISCUSSION

In agreement with the results of the present investigation (Table I), several researchers have reported alterations in roots treated with phenolic compounds in different plant species. Vaughan and Ord (35) verified that root growth and FW of pea (Pisum sativum L.) were inhibited by 1 mM FA (> 70%) or 1 mM VA (> 60%). Application of 1 mM FA caused a considerable decrease in the growth of maize (Zea mays L.) seedlings, affecting both shoots (> 60%) and roots (> 40%), as reported by Devi and Prasad (8). FA (at 0.4 mM) reduced RL (21%) and root DW (30%) in sorghum (Sorghum bicolor L.) seedling (9). At 1 mM FA, primary RL and root FW of canola (Brassica napus L.) were drastically reduced by 55% and 37% respectively (2). In soybean, Patterson (24) has demonstrated that FA and VA, at 1 mM, significantly reduced total DW (FA, 45% and VA, 49%). As can be seen, the results reported here (Table I) agree with those obtained by the authors cited above.

TABLE I

Changes in the root length (RL), root fresh weight (FW) and root dry weight (DW) of soybean
seedlings treated with ferulic (FA) or vanillic (VA) acids for 48 h. The data represent mean
values of three to ten independent experiments ± SEM.


  RL
(cm)
  FW
(g)
  DW
(g)
 
 
mM FA VA FA VA FA VA

0 7.0 ± 0.14
(n = 9)
7.0 ± 0.14
(n = 9)
4.1 ± 0.17
(n = 10)
4.1 ± 0.17
(n = 10)
0.27 ± 0.01
(n = 9)
0.27 ± 0.01
(n = 9)
0.1 7.3 ± 0.60
(n = 8) ns
6.9 ± 0.42
(n = 10) ns
4.4 ± 0.07
(n = 4) ns
4.1 ± 0.16
(n = 10) ns
0.28 ± 0.01
(n = 4) ns
0.25 ± 0.01
(n = 8) ns
0.5 5.4 ± 0.24
(n = 3) *'
3.8 ± 0.15
(n = 10) *'
3.4 ± 0.16
(n = 4) *
3.0 ± 0.08
(n = 4) *
0.23 ± 0.01
(n = 4) *
0.20 ± 0.01
(n = 6) *
1.0 2.7 ± 0.16
(n = 4) *'
3.2 ± 0.12
(n = 10) *'
2.7 ± 0.06
(n = 9) *
2.8 ± 0.07
(n = 10) *
0.18 ± 0.06
(n = 10) *
0.22 ± 0.01
(n = 4) *

Means followed by (*) are significantly different from control. Means followed by (`) are significantly different between the two compounds at the same concentration. ns (not significant). Student's t-test (p£0.05).

 

With respect to POD (Fig. 1), various lines of evidence indicate that the soluble form, localized in the apoplastic space and cytosol, has been involved in the catalysis of most of the peroxidative reactions (1), while the bound form is responsible for the oxidative polymerization of monolignols to produce lignin (29). In these aspects, some researchers reported alterations in POD activity under action of both allelochemicals. For example, in cucumber roots treated with FA (0.5 or 1 mM), the soluble and bound forms of POD activities increased significantly, while VA did not affect these same forms (25, 32). Application of 1 mM FA caused a significant increase in both soluble and bound POD activities in maize roots and correlated with a pronounced decrease in root length (8). Baziramakenga et al. (4) exploited the effects of benzoic and cinnamic acids (but not their derivatives) on soluble POD activity of hydroponically-grown soybean roots. The results showed dual behavior: an increase in activity with 0.05 mM cinnamic acid (no alteration with benzoic acid) and a similar decrease under the action of the compounds at 0.2 mM. Based on these results, the researchers concluded that the effects of phenolic compounds were due to the production of free radicals. In fact, phenolic compound oxidation generally leads to the production of quinones, which are toxic compounds responsible for the generation of reactive oxygen species (1). These radicals are extremely dangerous to cells because they can cause enzyme inactivation, membrane lipid peroxidation, and strand breaks in DNA (1, 4). Although soybean cells possess other antioxidant enzyme systems (4, 7) to scavenge oxygen radicals, the possibility that the capacity of these systems has been exceeded cannot be discarded (4). At the same time, an essential role of CW-bound POD in the stiffening of the cell wall has been postulated (29). Indeed, the dimerization of ferulic acid in pine (Pinus pinaster Aiton) hypocotyl, due to the oxidative capacity of CW-bound POD, was inversely related to the growth capacity (29). It therefore seems feasible that these facts could explain the increases of soluble and CW-bound POD activities observed here (Fig. 1), at least with respect to FA in relation to decreased root growth capacity (Table I).

Another situation is that the accumulation of phenolic compounds and increase of the lignification of the cell walls accompany the increase of the POD activity, as the plant's response to stress (33, 34). In maize roots treated with FA, Devi and Prasad (8) observed that the increased activity of the soluble or bound-POD correlated with pronounced increase in contents of total PhAs and lignin. Politycka (26, 27) verified that treatment of cucumber roots with FA and VA stimulated the increase of free and glucosylated phenols. Similar behavior can be seen here; both FA and VA provoked an increase of PhAs contents (Fig. 1D). However, it is important to emphasize that this work likewise reports changes in total PhAs, and consequently, the mechanism by which FA or VA produces PhAs in the roots cannot be inferred from the available results. Since changes in soluble or wall-bound phenolics may occur in short time periods as a result of plant growth or stress condition, the precise identification of these PhAs will be necessary. At present, there are no reports of the effects of exogenous FA or VA on production of specific PhAs.

As mentioned earlier, PAL is the entry enzyme into the phenylpropanoid pathway that is responsible for the synthesis of a diverse array of phenolic metabolites such as ferulic, caffeic, p-coumaric, sinapic acids, flavonoids, tannins and the structural polymer lignin. These compounds are often induced and play specific roles in plant protection against biotic and abiotic stresses (14, 17). In addition to POD, PAL activity increases in different circumstances, but the few studies reported on the effects of exogenous PhAs have been controversial. For example, the inhibition of the PAL activity by cinnamic acid and derivatives was well documented in different sources of pure enzyme with the exception of FA (VA was not tested), which was ineffective in sweet potato (Ipomea batatas L.) and pea (30). Similarly, Shann and Blum (32) revealed that the PAL activity was unaffected by FA in cucumber roots. In contrast, Politycka (27) demonstrated that FA (but not VA) increased PAL activity and PhAs contents, while decreasing cucumber root growth.

In the light of these observations, the results presented here indicate a differentiated behavior of the soybean roots, as FA increased and VA decreased PAL activities. For FA, the high PAL activity reported (Fig. 1C) strengthens the hypothesis of wall-stiffening by lignin synthesis (8, 27, 29, 32) and subsequently reduction in the root growth (Table I). Moreover, FA or PhAs (Fig. 1D) may be esterified with CW polysaccharides, incorporated into lignin structures or form bridges that connect lignin with polysaccharides, thus leading to rigidifying the CW and restricting cell growth.

On the other hand, a final question that emerges is concerned with the mechanism that underlies the effect of VA. Although it can be difficult to discuss the inhibitory effect of 1 mM VA on PAL activity, a possible explanation might be the fact that this inhibition was not complete, allowing lignin synthesis and growth reduction. Another interpretation is that PhAs produced in the later stages of the phenylpropanoid pathway (5, 6, 14) may provoke this inhibition, or the PhAs biosynthesis from L-phenylalanine may occur by more than one route, even at the stages of that pathway. There is evidence to support these suggestions, as the production of phenolic compounds is often observed when levels of phenylpropanoid pathway intermediates or end products are increased. For example, the addition of exogenous cinnamic acid to alfalfa (Medicago sativa L.) cell cultures leads to the accumulation of cinnamate conjugates (23). In cucumber roots, Politycka (26, 27) has pointed out that exogenous VA affected other enzymes involved in phenolic metabolism, such as phenol-ß-glucosyltransferase and ß-glucosidase (6). Finally, the possibility of those direct or indirect effects of VA on the early or late stages of the phenylpropanoid pathway cannot be ruled out. In this aspect, recent reports have shown that down-regulation of PAL, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase resulted in reduced levels of lignin accompanied by an altered syringyl/guaiacyl monomer ratio (13, 19, 23, 31). Because it is assumed that different pathways may lead to the activation of plant defense reaction (14), the results reported here may be only part of an even more complex regulatory network, and the PAL reaction is one of these pathways. Based on these ideas, and to gain insight into the possible mechanism of VA, the determination of the PAL activity under action of inhibitors (36), and of the cinnamyl alcohol dehydrogenase, a highly specific marker for lignification (5, 6) as well as the lignin production are necessary to give a clear-cut answer to this question. This is the challenge of a new study in progress.

 

ACKNOWLEDGEMENTS

This work was made possible by grants from the CNPq and from the Fundação Araucária (PR), Brazil. Vanessa Herrig and Maria de Lourdes L. Ferrarese thank Capes for their scholarships. We thank Dr. S. R. Böing for correcting the English version. We are especially grateful to the anonymous reviewer of Biological Research for his critical and helpful comments.


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