Bot. Bull. Acad. Sin. (2003) 44: 53-58

Barkosky and Einhellig Allelopathic interferenceof plant-water relationships

Allelopathic interference of plant-water relationships by para-hydroxybenzoic acid

Richard R. Barkosky1,* and Frank A. Einhellig2

1Minot State University, 500 University Avenue West, Minot, North Dakota 58707, USA

2Southwest Missouri State University, 901 South National Avenue, Springfield, Missouri 65804, USA

(Received April 9, 2002; Accepted August 15, 2002)

Abstract. Soybean seedlings were used as the test species to investigate the effect of phydroxybenzoic acid (pHBA) on growth and plant-water relationships. Plants were grown in nutrient solution under greenhouse conditions and were subjected to pHBA through amendments to the growth medium. Treatments were initiated 10 days after germination and continued for either 14 or 28 days with stomatal conductance, water potential, and water use monitored periodically. At harvest, effects on growth were determined and, in the 28-day study, the carbon isotope ratio (13C : 12C) of leaf tissue was analyzed as an indicator of integrated effects on plant-water status. Soybean growth was significantly reduced by 0.5 mM pHBA, or higher concentrations, with the degree of inhibition being concentration dependent. Plants treated with 0.75 mM pHBA had significantly lower stomatal conductance, lower water potential, and less discrimination against 13C. Similar trends were apparent in 0.5 mM pHBA-treated plants. Interference with plant-water balance appears to be one mechanism of action of pHBA causing a reduction in plant growth.

Keywords: Allelochemical; Allelopathy; Carbon isotopes; phydroxybenzoic acid; Soybean; Water status.

Introduction

The benzoic acid derivatives produced by higher plants have been frequently implicated in allelopathy (Rice, 1984). Para-hydroxybenzoic acid (pHBA; 4-hydroxybenzoic acid) has been isolated from a variety of crop residues and agricultural soils (Whitehead, 1964; Guenzi and McCalla, 1966; Chou and Patrick, 1976; Blum et al., 1991). It is one of several allelopathic Sorghum compounds found in aqueous extracts of plant material and associated with decomposing crop residues, germinating seeds, and root exudates (Abdul-Wahab and Rice, 1967; Hussain and Gadoon, 1981; Lehle and Putnam, 1983; Alsaadawi et al., 1986; Panasiuk et al., 1986; Einhellig and Rasmussen, 1989). Other plants implicated in allelopathic release of pHBA include Camelina alyssum and its influence on flax (Grummer and Beyer, 1960), several members of the genus Althaea (Gude and Bieganowski, 1990), and the grass Imperata cylindrica (Hussain and Abidi, 1991). Some of the reduction in root and coleoptile growth of wheat seedlings by wild oat (Avena fatua) root exudate is attributed to pHBA (Perez and Ormeno-Nunez, 1991).

Although several physiological effects of pHBA have been reported, its primary action affecting growth is obscure. Lee and Skoog (1965) found the hydroxybenzoic acids caused inactivation of indoleacetic acid. Effects on

respiratory metabolism have been suggested, as pHBA inhibited succinate hydrogenase and altered oxidation of NADH (Hulme and Jones, 1963; Lee, 1966). Glass (1973, 1974, 1975) demonstrated the inhibitory action of hydroxybenzoic acids on potassium and inorganic phosphate absorption of barley roots and related work established that membrane depolarization occurred (Glass and Dunlop, 1974). Stomatal effects from pHBA interaction with abscisic acid have been suggested (Purohit et al., 1991). Our previous work established that related phenolic compounds caused stomatal closure and other constraints on plant water relationships (Barkosky and Einhellig, 1993; Barkosky et al., 1999; Barkosky et al., 2000). Mechanism of action models proposed by Einhellig (1986) have suggested that membrane perturbations are probably a primary action of many phenolic allelochemicals.

This study was designed to determine the impact of pHBA on plant-water relationships and to evaluate the correspondence between these actions with effects on plant growth. As a part of the data collection, the relative concentration of stable carbon isotopes in leaf tissue was used as an indicator of water stress over time (Farquhar et al., 1982; Roeske and O'Leary, 1984; O'Leary, 1988; Berry, 1989; Tieszen, 1991). This analytical theory is based on the fact that several plant factors and environmental conditions, including water stress, contribute to the extent to which plants discriminate against carbon-13 (Farquhar et al., 1982, 1989; Ehleringer, 1989; Guy et al., 1989).

*Corresponding author. Telephone: 701-858-3116; Fax: 701-858-3163; E-mail: barkosky@minotstateu.edu


Botanical Bulletin of Academia Sinica, Vol. 44, 2003

Materials and Methods

Soybean [Glycine max (L.) Merr. var. Wells II] seedlings were used as the test species in these experiments. Soybeans have been shown to be sensitive to several phenolic allelochemicals and indices of water stress are conveniently measured in these seedlings (Einhellig and Rasmussen, 1979; Patterson, 1981; Colton and Einhellig, 1980; Einhellig and Schon, 1982; Einhellig and Eckrich, 1984; Barkosky and Einhellig, 1993). Soybeans were germinated and grown in a greenhouse which experienced the usual variations of summer greenhouse conditions.

Seeds were germinated in vermiculite flats and after 7 days the seedlings were individually transplanted to opaque plastic vials containing 120 mL of nutrient medium. The seedlings were supported upright by a hole in the lid of the vial. The nutrient medium was a modified Hoagland's solution (Hoagland and Arnon, 1950) containing 5 mM Ca(NO3)2, 5 mM KNO3, 2 mM MgSO4, 0.9 mM NH4H2PO4, 0.1 mM (NH4)2HPO4, standard Hoagland's micronutrients, and 72 M iron supplied as sodium ferric diethylene-triamine pentaacetate (Sequestrene 330). Transplanted seedlings were allowed to acclimate three days before treatments with pHBA were initiated (day 10).

Treatments were initiated by replacing the nutrient medium with nutrient solution amended with pHBA. Subgroups of plants in the first experiment were treated over a 14-day period with zero, 0.25 mM, 0.5 mM, and 1.0 mM pHBA. These results provided data demonstrating the threshold concentration at which pHBA inhibited soybean seedling growth. The intent of the second experiment (28-days treatment) was to subject seedlings to longer-term exposure to pHBA levels near the inhibition threshold. The treatments in this 28-day experiment were 0.50 mM pHBA, 0.75 mM pHBA, and a control.

On the day of treatment, a subset of six seedlings was harvested to establish leaf area and dry weight baseline data. Adequate numbers of seedlings were used in each treatment group to provide a minimum of six plants per group at the final harvest after the sacrifice of some seedlings for water potential determinations during the treatment period. On day 10 following treatment, plants were transferred into 350 mL containers, and in the 28-day ex

periment soybeans were subsequently transferred to 750 mL containers on day 15. The treatment solution was replaced every three days during the experiments to ensure the plants had a constant nutrient supply and allelochemical exposure. In the 28-day experiment, the amount of solution used was recorded.

In both experiments, stomatal conductance was measured every second day on six randomly chosen plants per treatment group. Measurements were obtained from the abaxial surface of the unifoliate leaves using a Li-Cor LI-1600 steady-state porometer. In the 28-day experiment, leaf water potential was obtained once a week from four plants in each pHBA level. A 7-mm diameter disk was punched from the center of a leaflet in the most fully developed trifoliate, leaf samples were equilibrated for 2 h in Wescor C-52 sample chambers, and water potential was determined using a Wescor HR 33 dewpoint microvoltmeter. Plants from which a leaf disk was cut were discarded. Stomatal conductance and water potential were obtained between 1300 and 1500 h.

At the termination of an experiment, plants were harvested by separating the leaves from the plant, obtaining the leaf area, and oven drying all tissue at 104C for 48 h. These data were used to compute the leaf to plant weight ratio (LWR; mg mg-1), specific leaf weight (SLW; mg cm-2), and relative growth rate (RGR; mg mg-1 day-1) (Evans, 1972; Bhowmik and Doll, 1983). Calculation of RGR over the treatment period utilized the initial plant weights from the subsample harvested at the time of treatment (T1;W1), compared to weights at harvest (T2;W2), using equation 1.

In the 28-day experiment, leaf tissue of the second and third trifoliate leaves was analyzed to determine the carbon isotope ratio, 13C:12C. Oven-dried tissue was ground in a Cyclone sample mill fitted with a 0.4 mm screen and the carbon isotope ratio was determined by mass spec


Barkosky and Einhellig Allelopathic interferenceof plant-water relationships

trometry (Augustana College, Sioux Falls, SD). These results are expressed as the delta (d) 13C value in per mill () units (Equation 2).

All the data were analyzed using one-way analysis of variance (ANOVA) with means separated by Duncan's Multiple Range Test using the Statistical Analysis System (SAS).

Results

Soybeans were slightly stimulated by 0.25 mM pHBA in the growth medium as evidenced by the leaf area and dry weight of plants after 14 days treatment (Table 1). Stunting of plants was noted after several days of treatment with 0.5 mM and 1.0 mM pHBA, and the latter were more severely affected. When harvested, these plants had significantly lower plant dry weight, leaf weight, leaf area, and RGR than controls (Table 1). Leaf growth was more markedly impacted than overall plant growth as evidenced by the smaller LWR. Soybeans exposed to 0.5 and 1.0 mM pHBA regularly had lower stomatal conductances (Figure 1).

Since the threshold for growth inhibition in the first trial was approximately 0.5 mM pHBA, the second experiment focused on effects of chronic treatments of pHBA at and slightly above this level. The data demonstrate that 0.5 mM pHBA caused even more deleterious effects over 28 days treatment, as compared to 14 days, with soybeans achieving only about half the leaf area, leaf weight, and plant weight of controls (Table 2). Other than their depressed growth, these plant did not show overt signs of damage from the pHBA treatment. Plants grown with 0.75 mM pHBA for 28 days achieved less than 20% of the leaf area and biomass of untreated seedlings, indicating the importance of the concentration response.

Figure 1. Effects of pHydroxybenzoic acid (pHBA) on leaf conductance in soybean over 14 days of treatment. Each data point is the mean of measurements taken on six plants; *: indicates significance from controls (P<0.05, with Duncan's Multiple Range Test). Control (~); 0.25 mM pHBA (); 0.5 mM pHBA (p); 1.0 mM pHBA (q).

Figure 2. Effects of pHydroxybenzoic acid (pHBA) on leaf conductance in soybean over 28 days of treatment. Each data point is the mean of measurements taken on six plants; *: indicates significance from controls (P<0.05, with Duncan's Multiple Range Test). Control (~); 0.5 mM pHBA (); 0.75 mM pHBA (p).


Botanical Bulletin of Academia Sinica, Vol. 44, 2003

Throughout the 28-day treatment, rates of stomatal conductance were significantly depressed in soybeans treated with 0.75 mM pHBA (Figure 2). A significant reduction in stomatal conductance was also noted in 0.5 mM pHBA-treated plants on most measurements after the first week of treatment. The week by week water potentials in both pHBA treatments showed a trend toward lower values (Table 3). The water potentials in leaf samples from 0.5 and 0.75 mM pHBA-treated plants at the second week (14-day interval) and fourth week (28-day interval) were significantly below those of untreated plants. The fourth week water potentials were -0.27 and -0.37 MPa lower than controls for the 0.5 and 0.75 pHBA-treated plants, respectively.

Analysis of the carbon isotope ratio in leaf tissue at the termination of the 28-day experiment showed significantly less discrimination (less negative d13C) had occurred against 13C in plants grown with 0.75 mM pHBA and some indication of this effect was shown at the reduced pHBA treatment (Table 4). Conversely, the transpiration ratio computed over the duration of the treatments was significantly higher in the presence of 0.75 mM pHBA.

Discussion

The effects of pHBA on soybean growth fit the pattern of an allelochemical effect in that inhibition was concentration dependent and there was a slight stimulatory effect at a sub-toxic level. At the seedling growth-inhibition threshold of approximately 0.5 mM pHBA, plants treated for 28 days were more inhibited than those treated 14 day, achieving 86% and 53%, respectively, of the control biomass. Undoubtedly, both the duration of exposure to pHBA and variations in environmental conditions between the two experiments contributed to this difference. Due

to periodic replacement of the growth medium, the longer treatment gave a higher total amount of pHBA exposure, a situation that influences the results in bioassays (Weidenhammer et al., 1987). Higher greenhouse temperatures and solar irradiance occurred during the 28-day experiment. These conditions resulted in more rapid growth in control plants and greater inhibition by pHBA. Einhellig and Eckrich (1984) reported that in combination with high temperature stress ferulic acid was more inhibitory to soybean growth.

Interestingly, even the highest concentrations of pHBA were not lethal. This may be due to the glucosylation of pHBA by enzyme systems within the plant. Scholten et al. (1991) found that cell cultures of Datura innoxia and Scopolia carniolica are able to glucosylate hydroquinone, vanillin, and pHBA acid by an enzyme-mediated, concentration dependent, bioconversion. Tabata et al. (1988) revealed that certain plant cell cultures can glucosylate a wide variety of phenolics including pHBA. Reduction of toxicity by glycosylation could allow soybean survival even through their growth is suppressed.

In all pHBA treatments which inhibited growth, lower stomatal conductances indicate the plants had some stomatal closure. Further evidence of water stress is shown by the reduced water potential values found in pHBA-treated plants in the 28-day experiment. Tissue from these plants also had relatively more 13C than untreated seedlings, which supports an integrated view of pHBA resulting in higher stomatal resistances over time. Other phenolic acids allelochemicals, including p-coumaric, caffeic, ferulic, and salicylic acid, are known to cause water stress in plants (Einhellig et al., 1985; Booker et al., 1992; Barkosky and Einhellig, 1993).


Barkosky and Einhellig Allelopathic interferenceof plant-water relationships

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The fact that higher transpiration ratios and thus lower water use efficiency was recorded under pHBA treatment is not readily explained. This apparent conflict with the data showing decreased stomatal conductance and enrichment of 13C, typically associated with higher water use efficiencies, may be due to other actions of pHBA. Glass and Dunlop (1974) reported pHBA depolarizes membranes, and it likely that membrane perturbations will have multiple effects on metabolism and the efficiency of plant processes.

The data from this study shows good correspondence between the changes in plant-water parameters and inhibition of plant growth, and we conclude that the impact of pHBA on water relationships is an important mechanism of action of this allelochemical.

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