Pesticide Biochemistry and Physiology 97 (2010) 256–261

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Pesticide Biochemistry and Physiology

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Bentazone and bromoxynil induce H+ and H2O2 accumulation, and inhibitphotosynthetic O2 evolution in Synechococcous elongatus PCC7942 q

Palash Kumar Das, Suvendra Nath Bagchi *

Department of Biological Sciences, Rani Durgavati University, Jabalpur, MP 482001, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 October 2009Accepted 2 March 2010Available online 20 March 2010

Keywords:Antioxidant enzymesCyanobacteriaCytosolic acidificationHerbicideHydrogen peroxide productionPhotosynthesis

0048-3575/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.pestbp.2010.03.005

q Action of bentazone/bromoxynil on Synechococcou* Corresponding author. Fax: +91 761 4045389.

E-mail address: snbagchi_in@yahoo.com (S.N. Bag

Using a unicellular cyanobacterium, Synechococcous elongatus PCC7942, we have shown that cytosolicacidification, O2

��; H2O2 production and photosystem II-inactivation are the causes of cell death frombentazone/bromoxynil incubations. Butyric acid evoked solely pH lowering response and yet inhibitedPS II activity indicating that herbicide-caused acidification is sufficient to kill the cyanobacterial cells,but other factors like excess H2O2 production due to an imbalance in the peroxide sequestration machin-ery might be contributory. While the activities of superoxide dismutase and pyrogallol peroxidaseincreased consequent to herbicide incubations and displayed oligomeric states with mobility shift, cata-lase and glutathione peroxidase though present remained insensitive.

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1. Introduction

Post-emergence contact herbicides bentazone (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide) and bromoxynil(3,5-dibromo-4-hydroxybenzonitrile) have been recognized asphotosystem (PS) II inhibitors, though other mechanisms of plantkilling actions also exist [1,2]. As weak acids these lipophilic com-pounds enter plant cells in protonated forms facilitated by acidic toneutral external pH, and upon entry dissociate at cytosolic neutralpH releasing protons and bringing pH down [3]. Consequently, asshown in Chara corallina and higher plants, a number of pH-sensi-tive activities like chlorophyll fluorescence induction, CO2 fixation,oxidative phosphorylation and cytoplasmic streaming, have beenshown to be adversely affected by these herbicides owing to achange in cytosolic pH [3–5]. Most likely, acidification also leadsto the bentazone’s inhibitory action on RNA synthesis tested inPhaseolus vulgaris [6]. In rice field cyanobacterium Anabaena cylind-rica, bentazone’s action was shown to be targeted on photosyn-thetic and respiratory O2 exchanges and phycobiliproteinturnover [7]. Taking Synechococcous elongatus PCC7942 as a testmaterial we have shown that bentazone tolerance is accompaniedwith NaCl co-tolerance [1]. Generally, Synechococcous sp. is consid-ered to be low salt tolerant strain and external Na+ has been shownto cause light-dependent cytosolic acidification [8].

ll rights reserved.

s.

chi).

The phytotoxic action of the PS II herbicides bentazone and brom-oxynil is also manifest by reactive oxygen species (ROS) production,notably singlet oxygen (1O2) and superoxide anion (O2

��), producedfrom an inactivation of electron flow between primary and second-ary quinones, QA and QB, on D1 protein towards acceptor side and/orbetween H2O and reaction center chlorophyll, P680, at donor side [9].In cyanobacteria, in which several antioxidant enzymes are up-reg-ulated on account of diverse stress causing factors like UV, H2O2,heavy metals, etc. [10–13], whether any correlation exists betweenthe herbicide-caused ROS production and expression of the quench-ing enzymes, though expected, is not known. In S. elongatusPCC7942, besides Fe-superoxide dismutase (FeSOD), convertingO2�� to H2O2, the peroxide quenching enzymes – catalase–pyrogallol

peroxidase (CAT–POX), thioredoxin peroxidase [14–16], and gluta-thione peroxidase (GPX; Cyanobase, http://genome.kazusa.or.jp/cyanobase/SYNPCC7942) have been reported.

In this paper, we report that both bromoxynil and bentazonerapidly bring down the internal pH, produce peroxide and concom-itantly inactivate the PS II activity in S. elongatus PCC7942. Butyricacid was used as non-herbicide lipophile protonating compound toexamine whether solely acidification has any impact on PS II activ-ity, and on production and degradation of ROS.

2. Materials and methods

2.1. Chemicals

Fine chemicals at their purest grade including bentazone (CASregistry number 25057-89-0, purity 99.3%) and bromoxynil (CAS

P.K. Das, S.N. Bagchi / Pesticide Biochemistry and Physiology 97 (2010) 256–261 257

registry number 1689-84-5, purity 98.0%) were purchased fromAccustandard Inc. (USA). All other general purpose chemicals includ-ing DCMU, butyric acid and catalase were from Hi-Media, India.

2.2. Strain, growth conditions, preparation of cell suspensions/spheroplasts and photosynthesis measurements

Axenic culture of S. elongatus PCC7942 was grown in BG-11medium at 30 �C under constant illumination of 60 lE photons/m2s using cool fluorescent lamps (Philips, India) as previously de-scribed [17]. Periodic examination of bacterial contamination, har-vesting of cells by centrifugation from 500 mL exponential culture(OD750nm = 0.7–0.75), corresponding to 0.1 lL packed cells/mL,chlorophyll (Chl) and protein estimations, preparation of cell sus-pension and measurement of overall photosynthetic O2 evolutionin presence of 15 mM NaHCO3 and cells equivalent of 10–20 lg/mL Chl at pH 5.0 and 6.0 (54 mM 2-(N-morpholino)ethanesulfonicacid–NaOH), 7.0 and 8.0 (54 mM N-(2-hydroxyethyl)piperazine-N0-(2-ethanesulfonic acid–NaOH)), and 9.0 (54 mM 2-(N-cyclohexyl-amino)ethanesulfonic acid–NaOH) and at incident illumination ofphotosynthetically active radiation of 650 lE photons/m2s, werecarried out as shown in Ref. [18]. For spheroplast preparation, cellsfrom 1 L cultures were harvested (5000g) and cells equivalent to80 lL packed volume or 225 lg Chl were suspend in 10 mL of10 mM Hepes–NaOH buffer pH 7.5, containing 0.5 M sorbitol,5 mM Na2HPO4, 5 mM NaH2PO4, 12.5 mM Na2–EDTA and 10 mMMgCl2. Lysozyme (from chicken egg white; Sigma, L2879) was addedto a final concentration of 1 mg/mg Chl. The mixture was incubatedat 37 �C for 1 h. Spheroplasts were spun at 2000g, washed in abovebuffer and the Chl adjusted to 0.8–1 mg/mL. For measurement oflight dependent-2,6-dichlorophenolindophenol (DCPIP) reductionin a reaction mixture consisting of 50 mM Hepes–NaOH buffer pH7.0, 50 mM CaCl2, 0.4 mM sucrose, 60 lM DCPIP and 15 lg Chl, theprocedure of Ray et al. [19] was adopted. Different concentrations

Fig. 1. Overall photosynthetic O2 evolution rate in S. elongatus PCC7942 as a function of (6.0 (N), 7.0 (�), 8.0 (s) and 9.0 (d).

of bentazone and bromoxynil (range 20–150 lM) from 0.1 M freshlyprepared ethanolic stocks or ethanol solely (<1% v/v final concentra-tion), and aqueous butyric acid (0.5 M) were added to the reactionmixture 3–4 min prior to start of the assays.

2.3. Measurement of cytosolic pH changes

A dual-wavelength absorbance technique of Thomas et al. [20]was modified as follows: the spheroplasts harvested by centrifuga-tion were re-suspended in 26 mM Hepes–NaOH buffer pH 7.3 con-taining 0.166 M mannitol, 0.133 M CaCl2 and 10% (v/v) percoll, andChl was adjusted to 60–80 lg/mL. About 3 mL of spheroplasts sus-pension was transferred in four glass cuvettes containing each:spheroplast alone, spheroplasts + 20 lM 6-carboxyfluoresceindiacetate (CFA2), spheroplasts + CFA2 + 4 mM butyric acid, andspheroplasts + CFA2 + 4 mM butyric acid + 20 lM 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO). The absorbance difference(A490–465) was monitored at every 1 min interval for 30 min usinga Beckman DU spectrophotometer. Assays in which butyric acidand dye were added to spheroplasts, it took 20 min to achievethe maximum absorbance difference, and DA490–465 (0–20 min)was recorded as being the maximum pH change in the given assay.Butyric acid, DCMU, bromoxynil and bentazone concentration-dependent DA490–465 was recorded as above by adding the desig-nated concentrations from respective stock solutions (in case ofDCMU, 0.1 M in ethanol). Addition of ethanol alone (<2% v/v) didnot significantly affect the DA490–465 values.

2.4. Estimation of H2O2 production, lipid peroxidation and ROSaccumulation

S. elongatus PCC7942 cells from 1 L cultures were harvested bycentrifugation (5000g, 15 min) and suspended in 10 mL ofHepes–NaOH buffer pH 7.0 to get a final cell density of �7.5 lL

A) bentazone, (B) bromoxynil and (C) butyric acid concentrations and at pH 5.0 (j),

258 P.K. Das, S.N. Bagchi / Pesticide Biochemistry and Physiology 97 (2010) 256–261

packed cells/mL. To 3 mL cell suspension different concentrationsof herbicides/butyric acid with/without catalase (7 U, from bovineliver) were added, while parallel controls were run in which <1%(v/v) ethanol was added. The cells were illuminated at 650 lE pho-tons/m2s, and at given time intervals cells were removed at 5000gfor 1 min in a microfuge. The extracellular H2O2 production wasmeasured in the supernatant using the H2O2-dependent decay ofscopoletin fluorescence catalysed by horseradish peroxidase (LifeTechnologies India Pvt., Ltd.) [21]. Lipid peroxidation as malonyldialdehyde (MDA) formation and cellular H2O2 content were deter-mined by adapting the method given in Ref. [22], and for estimat-ing superoxide content that converted hydroxylamine to NO2

� inunit time, the procedure shown in Ref. [23] was applied. The cellsfrom 500 mL cultures, previously incubated with 150 lM each ofbentazone or bromoxynil, 5 lM DCMU or 4 mM butyric acid, wereharvested at the indicated time by centrifugation (5000g, 10 min)and cells corresponding to �15 lL packed cell/mL were suspendedin 3 mL of 50 mM K-PO4 buffer pH 7.8. For O2

�� determination, thecells were broken using a MSE, Soniprep 150 sonicator (3 � 2 min,5 min interval at 4 �C) and then supernatant of 10,000g was takenfor the estimations.

2.5. Determination of antioxidant enzymes’ activity and in-gel assays

CAT (EC 1.11.1.6), SOD (EC 1.15.1.1), POX (EC 1.11.1.7) andGPX (EC 1.11.1.9) activities in 50 mM Na-PO4 buffer pH 7.0 wereestimated, respectively, according to the methods given in Refs.

Fig. 2. (A) Change in CFA2 A490–465 representing DpH of spheroplasts without (s) or withHQNO. (B) DA490–465 (20–0th min) of CFA2 in spheroplasts with DCMU (d), butyric acid

[22,24–26]. One unit of SOD inhibited 50% of the photochemicalreduction of nitrobluetetrazolium. For this, harvested cellsas above were adjusted to 10 lL packed cells/mL in 50 mMNa-PO4 buffer pH 7.0, and sonicated as above to get crude en-zyme extracts. From this, extract equivalent to 75 lg proteinwas supplemented to the reaction mixtures of the respectiveenzymes.

The enzyme activities were also determined by in-gel assays.Briefly, 75 lg protein equivalent extracts were applied to nativePAGE using 7.5% running gel according to Ref. [27]. Electrophoresiswas carried out for 6 h at 4 �C and the gels were stained as follows:CAT-gel was incubated in 3.27 mM H2O2 for 25 min and after rins-ing with water it was soaked in freshly prepared solutions of 1%each FeCl3 and K3Fe(CN)6 until the achromatic bands on a prussianblue background appeared [22], SOD-gel was immersed in 50 mMTris–HCl buffer pH 8.5, containing 10 mg methylthiazolyl tetrazo-lium, 6 mg phenazine methosulphate and 15 mg MgCl2. The en-zyme bands were located as colourless zones on a dark bluebackground [22], POX – the gel was pre-incubated with 50 mMNa-PO4 buffer pH 7.0 for 15 min followed by additional 20 minincubation in 4 mM H2O2 and 20 mM pyrogallol until dark brownbands appeared [28]; and GPX-gel was washed in sequence with2.5% Triton X-100 for 15 min and distilled water for 15 min. Thegel was immersed in 10 mM K-PO4 buffer pH 7.2 containing2 mM o-dianisidine dihydrochloride for 1 h and shifted for15 min to another solution containing buffer and 0.1 mM H2O2.Brown bands against pale yellow background appeared [29].

(�) dye, and with dye + 4 mM butyric acid in absence (h) or presence (D) of 20 lM(�), bentazone (N) and bromoxynil (j). Bars represent means ± SD (n = 3).

P.K. Das, S.N. Bagchi / Pesticide Biochemistry and Physiology 97 (2010) 256–261 259

Statistical analysis was carried out by two way-ANOVA withherbicides/butyric acid and time as two fixed factors. Mean valueswere compared by using Tukey’s HSD Post hoc test at threshold pvalue of 0.05.

Table 1Effect of bentazone, bromoxynil, DCMU and butyric acid incubations for 6–24 h onlipid peroxidation as MDA produced and ROS content in 100 lL packed cells of S.elongatus PCC7942.

0 h 6 h 12 h 24 h

BentazoneLipid peroxidation (lmol) 9 ± 0.6 48 ± 2.4* 75 ± 3.9* 84 ± 12.6*

H2O2 (lmol) 5 ± 0.7 16 ± 0.8* 20 ± 2* 26 ± 2.6*

O2�� (lmol h�1) 4 ± 0.4 8 ± 0.4* 14 ± 1.4* 20 ± 2*

BromoxynilLipid peroxidation 15 ± 1.5 45 ± 2.1* 72 ± 3.6* 66 ± 3.3*

H2O2 4 ± 0.2 15 ± 0.7* 23 ± 1.1* 28 ± 1.4*

O2�� 3 ± 0.4 6 ± 0.3 12 ± 0.6* 18 ± 0.8*

DCMULipid peroxidation 6 ± 0.3 24 ± 1.2* 36 ± 1.8* 48 ± 2.4*

H2O2 4 ± 0.2 5 ± 0.3 4 ± 0.2 4 ± 0.2O2�� 5 ± 0.7 10 ± 0.5* 16 ± 0.8* 18 ± 0.9*

Butyric acidLipid peroxidation 12 ± 1.2 15 ± 0.6 12 ± 0.6 12 ± 1.2H2O2 3 ± 0.2 4 ± 0.2 3 ± 0.2 4 ± 0.2O2�� 4 ± 0.2 5 ± 0.3 6 ± 0.3 5 ± 0.7

Values are means ± SD (n = 3).* Significantly different from 0 h values at p < 0.05.

3. Results and discussion

S. elongatus PCC7942 was grown in BG-11 medium and wholecell photosynthetic O2 evolution was measured at varied pH withthe addition of increasing concentrations of bentazone and brom-oxynil. Results show that O2 evolving activity was completely lostat 80 lM of both the herbicides at pH 8.0 (Fig. 1A and B). Decreas-ing the pH to 5.0 during the assays without the herbicides had nodetectable effect on O2 evolution, though it rendered the cells moresensitive to the herbicides, and lesser concentrations were found tobe inhibitory at acidic pH (100% inhibition at 20 lM). On the otherhand, there was no apparent effect of the herbicides at pH 9.0. In anearlier finding the pH-dependent action of bromoxynil in C. coral-lina and Lilium longiflorum pollen tubes was attributed to the cyto-solic acidification [5] and in S. elongatus PCC7942 too, thisexplanation seems to hold true. Moreover, it was shown that cyto-solic acidification can be achieved in Zea mays plasmodesmata byadding butyric acid [30]. It was a matter of interest to examinewhether a non-herbicide butyric acid would lower the intracellularpH in S. elongatus PCC7942, and thus influence the O2 evolution.The data suggests that in acidic to neutral pH, butyric acid alonewas sufficient to inhibit the O2 evolution activity in a concentra-tion-dependent and pH-dependent manner just like the herbicides(Fig. 1C). Our next attempt was to ascertain whether butyric acid orthe herbicides had any effect on intracellular pH. As shown inFig. 2A neither the spheroplasts alone nor the spheroplasts mixedwith dye affected time-course of A490–465. When spheroplasts anddye were incubated with 4 mM butyric acid the A490–465 within20 min decreased (0.078 U). The addition of an uncoupler, HQNO,completely reversed this trend, suggesting that A490–465 on accountof addition of butyric acid was due to intracellular H+ accumulationas was interpreted for higher plants [31]. In the next course ofinvestigation DA490–465 was recorded at increasing concentrationsof butyric acid, bentazone and bromoxynil. As shown in Fig. 2Bconcentration dependence of DA490–465 was clearly discernablefor butyric acid and the herbicides, the later being more effective.To make an assessment whether the effect of butyric acid and

Fig. 3. Spheroplastic photosynthetic DCPIP reduction in presence of butyric acid(s), bentazone (�), bromoxynil (N) and (d) DCMU. Bars represent means ± SD(n = 3).

the herbicides on photosynthetic activity is related to pH reduc-tion, the concentration range of the herbicides and butyric acidused for cytosolic pH estimations was applied to PS II assaymixture and spheroplastic DCPIP reduction was monitored. Ascan be seen in Fig. 3, at the given concentrations of bentazone,bromoxynil and butyric acid, the DCPIP reduction rates were sig-nificantly lowered alongside the decrease in pH. We wanted toknow whether herbicide DCMU would also cause the same effecton spheroplast pH and PS II activity as the other herbicides did.The PS II activity was instantaneously and completely inhibitedat 5 lM DCMU, while DA490–465 was rendered unaffected at givenconcentration (cf. Fig. 2B). This suggests that the action of bentaz-one/bromoxynil (range 5–100 lM) on S. elongatus PCC7942 is dueto cytosolic acidification that corresponded to a similar effect ofbutyric acid, albeit at higher concentrations (1–6 mM). The actionis therefore not similar to that of DCMU.

Fig. 4. Effects of (A) bromoxynil and (B) bentazone with/without catalase, (C)DCMU and (D) butyric acid, and no additions (controls) on extracellular H2O2

production.

260 P.K. Das, S.N. Bagchi / Pesticide Biochemistry and Physiology 97 (2010) 256–261

The modus operandi of bentazone and bromoxynil is believed tobe similar to that of ‘‘classical” herbicide DCMU, operating at the QB

pocket binding site at D1 protein. We wanted to know whetherboth class of herbicides evoke responses that induce ROS produc-tion in S. elongatus PCC7942, and whether acidification itself canalso do the same. To ascertain this, we compared the oxidative ef-fect of the three herbicides in terms of lipid peroxidation, H2O2 andO2�� production. In the long-term incubation experiments whose

results are presented in Table 1, only the herbicides and not butyricacid significantly enhanced the lipid peroxidation activity over un-treated controls. Further, the cellular level of O2

�� also exhibited asimilar increasing trend. However, the cellular H2O2 content wentup only when cells were incubated with bentazone/bromoxyniland not DCMU. In order to determine whether H2O2 productionstarts from the beginning of the incubation, S. elongatus PCC7942cells were incubated with bentazone/bromoxynil/DCMU/butyricacid, and extracellular H2O2 release was monitored (Fig. 4). Therewas a concentration-dependent quenching of scopoletin onlywhen bromoxynil or bentazone was supplemented, and this attri-bute completely reverted upon addition of catalase. This data sug-gests that in addition to acidification, bentazone and bromoxynil

Table 2Effect of bentazone, bromoxynil, DCMU and butyric acid incubations for 6–24 h onactivities of SOD (U/min �mg protein) and other enzymes (lmol product/min �mgprotein) in S. elongatus PCC7942.

0 h 6 h 12 h 24 h

BentazoneCAT 32 ± 4.9 30 ± 4.5 34 ± 5.1 28 ± 4.2POX 90 ± 13.5 92 ± 13.8 182 ± 27.3* 100 ± 15*

SOD 55 ± 8.2 99 ± 14.8* 180 ± 27* 120 ± 18*

GPX 524 ± 78.6 485 ± 72.8 495 ± 74.3 501 ± 75.2

BromoxynilCAT 35 ± 5.2 34 ± 5.1 30 ± 4.5 36 ± 5.4POX 88 ± 13.2 92 ± 13.8 212 ± 31.8* 99 ± 14.8*

SOD 65 ± 9.7 120 ± 18* 210 ± 31.5* 145 ± 21.7*

GPX 598 ± 89.7 550 ± 82 587 ± 88 620 ± 93*

DCMUCAT 28 ± 4.2 34 ± 5.1 30 ± 4.5 33 ± 4.9POX 82 ± 12.3 68 ± 10.9 61 ± 9.5 55 ± 8.2SOD 61 ± 9.13 74 ± 11.1* 119 ± 17.8* 133 ± 19.9*

GPX 543 ± 81.5 425 ± 63 450 ± 67.5 350 ± 52.5

Butyric acidCAT 30 ± 4.5 31 ± 4.6 29 ± 4.4 32 ± 4.8POX 80 ± 12 82 ± 12.3 80 ± 12 83 ± 12.5SOD 55 ± 8.3 52 ± 7.8 51 ± 7.7 56 ± 8.4GPX 514 ± 77.1 501 ± 75 523 ± 78.5* 498 ± 74.7

Values are means ± SD (n = 3).* Significantly different from 0 h values at p < 0.05.

Fig. 5. Activity staining of CAT, POX, SOD and GPX of cyanobacterial cells tr

also enhanced concomitant H2O2 production and sustained highcellular level at later growth period. Further, the data also suggeststhat butyric acid only acidified the cytoplasm and did not triggerany oxidative response, yet it inhibited the PS II activity. DCMUwithout causing any pH change evoked ROS production and inhib-ited the PS II activity (cf. Figs. 2B and 3).

As a consequence of ROS production, the level of antioxidantenzymes generally increases [11]. It was a matter of interest toexamine the fate of O2

�� and H2O2 degrading enzymes already re-ported in S. elongatus PCC7942, once the cells were incubated withthe above chemicals. It can be seen that butyric acid had practicallyno effect on the antioxidant enzyme activities tested spectrophoto-metrically (Table 2) and in-gel (Fig. 5). SOD in all herbicide incuba-tions and POX in only bentazone and bromoxynil incubationsexhibited enhancement of activities up to 12 h. In the gel assays,as revealed from the new bands, only bentazone and bromoxynilinduced oligomeric states of CAT, POX and SOD, exhibiting mobilityshifts. Notwithstanding, CAT activity did not significantly changeunder similar incubations (Table 2). Paradoxically, SOD activitywhich increased upon DCMU incubation, displayed no oligomericstates. Apparently, mobility shifts do not necessarily account forenhanced activities.

4. Conclusion

Here, we show that two structurally distinct herbicides, bentaz-one and bromoxynil, exhibited analogous mode of action on S.elongatus PCC7942. Both brought down the internal pH, producedROS mainly H2O2, and inhibited the PS II activity. Whether all thethree events are interrelated or are independent processes occur-ring simultaneously and whether there are other physiological tar-gets affected, is presently not clear. From butyric acid treatmentresults it appears that acidification alone is sufficient to antagonizePS II, and this seems to be the primary action also for bentazoneand bromoxynil. Nonetheless, the herbicides perhaps also evokeoxidative stress, particularly due to instant and sustained peroxideproduction which is not being able to be sequestered by alreadyup-regulated POX and the other H2O2 quenching enzymes. Thesource of H2O2 in herbicide treated cells is not known but can beassumed to be from an inactivation of PS II particularly at the watersplitting complex [32], which we have earlier shown to be a plau-sible site of bentazone’s action in S. elongatus PCC7942 [18]. Iflight-induced ROS production from triplet state of P680 is responsi-ble for herbicide phytotoxicity independent of cytoplasmic acidifi-cation, then it would be worthwhile to include a component ofvarying light intensity in such analysis. Moreover, this study canbe extrapolated in heterocystous filamentous cyanobacteria. Given

eated with bentazone, bromoxynil, DCMU and butyric acid for 6–24 h.

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the continuity of periplasm [33], uniform distribution of CFA2 andherbicides along the length of filaments can be expected.

Acknowledgments

The authors thank the Head, Dept. Biological Sciences, R.D. Uni-versity, Jabalpur (India) for lab facilities and the Department of Sci-ence & Technology (Govt. of India), New Delhi for financialassistance vide Project No. SR/SO/PS-28/06.

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