Short communication

Cellular evidence of allelopathic interference of benzoic acid to mustard(Brassica juncea L.) seedling growth

Harleen Kaur a, Inderjit b,*, Shalini Kaushik b

a Department of Botany, Panjab University, Chandigarh 160014, Indiab Department of Botany, University of Delhi, Delhi 110007, India

Received 27 October 2004; accepted 21 December 2004

Available online 18 January 2005

Abstract

Cellular changes in the roots of mustard (Brassica juncea L.) grown in soil treated with 1.09, 1.46 and 1.83 mg benzoic acid per g soil, aknown allelochemical, were analyzed after 7 days. The recoverable concentration of 1.09, 1.46 and 1.8 mg benzoic acid per g soil (measuredby high performance liquid chromatography) was 68, 150 and 250 µg benzoic acid per g soil, respectively. The benzoic acid treatmentssuppressed root growth by 30.5%, 58.8% and 81.1% with increasing concentrations. Transmission electron microscopy studies of rootsshowed irregular shaped cells arranged in a disorganized manner and disruption of cell organelles at cellular level. Root cells showed disso-lution of middle lamella (at 68 and 150 µg benzoic acid per g soil) but intact middle lamella with increased wall deposits was observed with250 µg benzoic acid per g soil. Damage to the mustard root at cellular level was evidenced by the changes in cell morphology and internalorganization.© 2005 Elsevier SAS. All rights reserved.

Keywords: Allelopathy; Benzoic acid; Mustard; Root growth inhibition; Transmission electron microscopy

1. Introduction

Benzoic acid, an allelochemical has been implicated inplant growth inhibition and also exploited after chlorinationas a commercial herbicide under the name dicamba[2,10,16,18]. Benzoic acid is reported to inhibit hydraulic con-ductivity and nutrient uptake by plant roots, thus resulting ingrowth inhibition [4]. Its mode of action might well be toinfluence the conformation of aquaporin water channels [4].To examine benzoic acid phytotoxicity in allelopathy research,data on root and shoot length and biomass are generally col-lected and compared with untreated controls [13]. Roots aregenerally more sensitive to phenolic acid than shoots [19].Recent studies showed that rye (Secale cereale L.) alle-lochemicals reduced the root growth of cucumber (Cucumissativus L.) by bringing changes in cellular ultrastructure [8].Since no data are available on the cellular changes inducedby the benzoic acid from allelopathy standpoint, moreresearch using ultrastructural tools is required. We hypoth-

esized that benzoic acid can cause changes at the cellular level.The objective of the present study was to examine cellularlevel changes in the root of mustard grown in soil treatedwith benzoic acid.

2. Material and methods

2.1. Growth experiments

Mustard (Brassica juncea L.) is a commonly grown cropand therefore selected as an assay species to assess the phy-totoxicity of benzoic acid.A 50 g soil (sandy-loam) was placedin 9-cm petri-dish, and irrigated with 30 ml of 15, 20 and25 mM benzoic acid mixed in double distilled water (hereaf-ter referred as water). The amount of benzoic acid thereforeadded in the soil was 1.09, 1.46 and 1.83 mg g–1 soil. Soilirrigated with water served as a control. Fifteen seedswere sown on the soil. Data on root length were collectedafter 7 days. Environmental conditions were: average tem-perature (day/night), 24/10 °C; 10-h light intensity was240 µmol m 2 s–1. Each experiment was replicated four times.Data were analyzed using one-way analysis of variance.

* Corresponding author.E-mail address: allelopathy@satyam.net.in (Inderjit).

Plant Physiology and Biochemistry 43 (2005) 77–81

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2.2. High performance liquid chromatography (HPLC)

Ten grams of soil treated with benzoic acid was extractedwith 10 ml of methanol. Since the objective of the presentstudy was to determine the concentration of benzoic acid (freeand reversibly-bound) that interacts with the plant, methanolwas selected as an extractant [16]. Both, water and methanol,extract similar amounts of phenolic acid [11], however, metha-nol is recommended as an extractant for HPLC quantifica-tion of phenolic acids [1,9,26]. The extracts were filteredthrough Whatman no. 44 and 30 µl was subjected to HPLC(Shimadzu LC-4A) using a wavelength UV detector set at275 nm. Reversed phase chromatography was carried out ina steel column (15 × 0.46 cm i.d.) with a flow rate of1 ml min–1. The solvent system used was methanol. To cal-culate the recovery of benzoic acid in treated soils, benzoicacid standard (1 and 5 mM) was subjected to HPLC. Benzoicacid was differentiated on the basis of its retention time andits concentration was calculated on the basis of total peakarea of standard and benzoic acid-treated soil.

2.3. Transmission electron microscope (TEM) studies

Roots of mustard seedlings were subjected to TEM stud-ies. Samples were fixed in modified Karnovsky’s fluid (2%paraformaldehyde + 2.5% glutaraldehyde HCl-sodium ca-codylate solution) buffered with 0.1 M sodium phosphatebuffer at pH 7.4 [20]. Fixation was for 10–18 h at 4 °C, afterwhich the tissues were washed in fresh buffer, and post-fixedfor 2 h in 1% osmium tetroxide in the same buffer at 4 °C.After several washings of 0.1 M sodium phosphate buffer,the specimens were dehydrated in graded acetone solutionsand embedded in CY 212 araldite. Ultrathin sections of60–80 nm thickness were cut using an ultracut E (ReichertJung) ultramicrotome and sections were stained in alcoholicuranyl acetate (10 min) and lead citrate (10 min) before exam-ining the grids in a TEM (Philips, CM-10) operated at60–80 kV.

3. Results and discussion

A significant (P < 0.05) reduction in root length wasobserved when mustard plants were grown in soil treated withbenzoic acid. The presence of 68, 150 and 250 µg benzoicacid per g soil caused 30.5%, 58.8% and 81.1% root growthinhibition of mustard, respectively. The recoverable concen-tration in soil treated with 1.09, 1.46 and 1.83 mg benzoicacid per g soil was 68, 150 and 250 µg benzoic acid per g soil,respectively (Table 1). This means that much of the benzoicacid is sequestered soon after its application to soil, and there-

fore not available to plant. There are several sink in the soiland lower recoverable concentration of benzoic acid in soilcould be due to its sorption on soil particles, and chemicaland/or microbial degradation [5,10,18,29]. As noted here, therecoverable concentration of benzoic acid in soil environ-ment is large enough to cause mustard root growth suppres-sion.

Ultrastructure studies revealed that the companion cells ofuntreated control mustard root phloem possess a distinctnucleus along with the nucleolus occupying a central posi-tion, several mitochondria, dense cytoplasm with smoothendoplasmic reticulum (SER), several small vacuoles, andwall protuberances at the sides connecting sieve elements(Fig. 1A). Root cells of mustard grown in soil treated withbenzoic acid were disorganized, distorted and deformed com-pared to those grown in control soil. This could be due toadverse effects of benzoic acid on cell division, mineraluptake, water balance, respiration or phytochrome activity[13]. Increase in lignin content has been associated withdecrease of soybean root growth [24], and this aspect needsfurther empirical evidence.

An increase in the SER profile, rough endoplasmic reticu-lum (RER), Golgi complex, elongated plastids with starchaccumulation and cell vacuolization was observed in com-panion cells of roots of mustard grown in soil with 68, 150 and250 µg benzoic acid per g soil (Fig. 1B, D, F). Lipid globuleprofile also increased in treated companion cells of roots ofmustard grown in soil treated with 150 and 250 µg benzoicacid per g soil (Fig. 1D, G). Burgos et al. [8] found highamounts of lipid globule in roots of 4-d old seedlings ofcucumber, which suggests that less energy is being utilizedby the roots of cucumber treated with rye allelochemicals.Root cells of Sinapis alba L. when treated with gramine andhordenine showed the lack of lipid degradation [22]. Sieveelements of root cell treated with benzoic acid showed accu-mulation of starch (Fig. 1F) when compared to control(Fig. 1A). This could be due to decreased translocation ofassimilate to growing points as benzoic acid inhibits auxinactivity (IAA) which is responsible for plant growth that gen-erates new cells for sustained growth [14]. Phloem paren-

Fig. 1. Transmission electron micrographs of root cells of mustard grown in untreated control soil (A, 1550X), and in soil with 68 (B, 6400X; C, 870X), 150 (D,1950X; E, 2650X) and 250 (F, 1550X; G, 6300X) µg benzoic acid per g soil. Abbreviations: GC, Golgi complex; l, lipid globule; m, mitochondrion; n, nucleus;nu, nucleolus; p, plastid; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; S, starch; v, vacuole; SE, sieve element; CC, companioncell; PP, phloem parenchyma; w, wall deposits, ML, dissolved middle lamella; sv, secretory vesicle.

Table 1Recoverable concentrations of soil treated with benzoic acid and their effecton root growth of mustard seedlings

Soil treated with benzoic acid(mg g–1)

Recovery(µg g–1)

Relative root growthinhibition (%)a

1.09 68 30.5*1.46 150 58.8*1.83 250 81.1*

Asterisk indicates that values are significantly different from control at thelevel of P < 0.05.

a Root growth inhibition compared to control. Soil treated with water ser-ved as control.

79H. Kaur et al. / Plant Physiology and Biochemistry 43 (2005) 77–81

chyma of treated root cells showed an increase in RER, SER,Golgi bodies, and lipid globules profiles and elongated plas-tids with starch accumulation (Fig. 1C, E). The observedchanges in the shapes of plastids in treated root cells could bedue to their higher activity [28]. Ferulic acid is reported toincrease the contents of saturated and unsaturated fatty acidsof the polar and non-polar lipid fractions and xylose, fructoseand sucrose in soybean root [15]. Irregular shaped cellsarranged in a disorganized manner has been observed in ben-zoic acid treatment (Fig. 1C–E). Root cells of mustard grownin soil with benzoic acid (68 and 150 µg benzoic acid per gsoil; Fig. 1C–E) showed dissolution of middle lamella, butintact middle lamella with increased wall deposits wasobserved in root cells of mustard grown in soil with 250 µgbenzoic acid per g soil (Fig. 1F, G). Benzoic acid promotesIAA decarboxylation, which results in reduced availabilityof IAA [14]. The lower concentration of IAA promotes cellwall loosening, which might have attributed to uneven thick-ening and dissolution of middle lamella in benzoic acid-treated roots [7,27].

An increase in the profile of endoplasmic reticulum wasobserved in root cells of mustard treated with benzoic acid.Allelochemicals applied to plants results in hypertrophy ofSER ultimately detoxified by increased activity of monooxy-genase enzyme [12]. The increase in the profiles of ER andGolgi complex contributes to detoxification releasing secre-tory vesicles, and lipid globules synthesis [12]. Detoxifiedbyproducts are either compartmentalized in vacuoles or depos-ited to the cell wall [23,25]. Such compartmentalization wasobserved in root cells of mustard grown in 25 mM benzoicacid (Fig. 1F, G). Mitochondria of treated companion cellsswelled, showed decrease in the profile of lipid globules andeven lost their stratiform structure. Hence, benzoic acid-treated cells showed ultrastructural changes in mitochondriathat could contribute to enhanced respiration. Secalonic acidF causes ultrastructural damage to mitochondria responsiblefor enhanced respiration in corn (Zea mays L.) [30].

Amount of benzoic acid added to the soil (1.09, 1.46 and1.83 mg g–1 soil) may be considered high from natural eco-system standpoint. From allelopathy standpoint, the concen-tration of an allelochemical detected in the soil is consideredecologically important compared to what is added to the soil[17]. As showed in this work, the concentration of benzoicacid in soil treated with 1.09, 1.46 and 1.83 mg benzoic acidper g soil was 68, 150 and 250 µg benzoic acid per g soil,respectively (Table 1). Therefore any growth response or cel-lular changes observed in mustard seedlings grown in soiltreated with 1.09, 1.46 and 1.83 mg benzoic acid per g soilare actually due to 68, 150 and 250 µg benzoic acid recover-able from per g soil. Blum [3] studied the recovery of ferulicacid in soil ranging from 75 to 200 µg g–1 soil. Although theamount of phenolic acid incorporated in soil may be high,the amount of phenolic acid available in soil to interact withplant seedlings is considered ecologically relevant. The con-centration of benzoic acid recovered from soil is well withinrange often tested in bioassays for allelopathy [3,6]. More-

over, in natural situations, high amount of phytotoxic debrismay be incorporated at one place, and little may be incorpo-rated at another place [21]. This is particularly observed whenfarmers incorporate wheat (Triticum aestivum L.)/rice (Oryzasativa L.) straw into soil. Therefore different concentrationsof benzoic acids were taken.

Differences in soil pH after the addition of benzoic acidcould be another reason of differences in observed phytotox-icity. The pH of untreated control (7.34 ± 0.14) was not dif-ferent from soil treated with 1.09 (7.27 ± 0.09), 1.46 (7.27 ±0.09) and 1.83 (7.16 ± 0.04) mg benzoic acid per g soil. Blum[4] reported that some phenolic acids (p-hydroxybenzoic,p-coumaric and ferulic acids) inhibit hydraulic conductivityand nutrient uptake by roots of plants, result in growth inhi-bition. Benzoic acid inhibits root growth of mustard seed-lings and caused damage at cellular level. The present studyreports about the relationship between inhibition of rootgrowth by benzoic acid in mustard and cellular changes.

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