D epartment of Bot an y, Sri Venkateswa ra Uni ver sity, T irupat i - 517 502, Andhra Pradesh,In dia

Herbricide-inhibited Phosphoenolpyruvate Carboxylasein Leaves of six Nonsucculent Scrub Species')

1. M ADHUSUDANA R Ao , P. M . S WAMY and V. S. R. D AS

Wit h 1 figure

Received D ecember 14, 1979 . Accepte d February 11, 1980

Summary

The influence of two herbic ides, par aquat or 2,4,5- T on th e in vitro as well as in vivoactivi ty of phosphoenolpyruvat e car boxylase from leaves of six nonsucculent scrub specieswas investigated. The plants, all nat ive and important constitu ents of the scrublandvegetation, included Carissa splnarum L., Maba buxifolia CL., Flacourt ia sepiaria ROXB. ,Cbomelia asiatica O. K ZE. , Gymnosporla emarginata LAWS., and Dodonaea v iscosa L. Th e inv itro activ ity of th e enzy me from unt reat ed leaves of all species was inh ibi ted when thepur if ied leaf extr act s were incuba ted in herb icide solut ions. Foliar ap plica tion of 100 mg ofpar aqu at or 2000 mg of 2,4,5-T per liter also inhibited th e enzy me activity of purif ied leafextracts at 72 h after herbicide tr eatment in all the species tested. T he inhibition of th e inv itro as well as in v iv o enzyme act ivi ty wa s more prom inent with par aquat than with 2,4,5-Ttreatm ent . It is suggested th at th e inh ibit ion of thi s ma jor carboxylat ing enzyme activ ity maybe involved in the herbicide inhibit ion of carbon dioxide fixa tion in leaves.

K ey w ords : H erbicides, foliar application, PEP-c arboxyl ase, scrub species.

Introduction

We have demonstrated the occurrence of some charac teristics of crassulacean acidmetabo lism (CAM) in five nonsucculent scrub species, as an adaptation for survivalunder semi-ar id environment ( RAo et al., 1979 a). The occurrence of CAM in thesespecies was characterized by the scotoactive stomatal behav iour, nocturnal carbonassimilation and high activity of phosphoenolpyruvate carboxylase (PEP-case)resulting in high dark acidification. PEP-case is one of the two key enzymes in thecarbon assimilation pathways of higher plants. Th ere is strong evidence thatfi-carboxylation of phosph oenolpyruvate catalysed by PEP-case is the initiating

1) Th e result s fo rm a part of the Ph. D. thesis of 1. M. R. approve d by Sri VenkateswaraUniversity , Tirupat i. Th is investigat ion was suppo rted by a grant of the United Sta tesD epartment of Agriculture Un der USPL- 480 Project FG -IN-544 (IN -ARS-27) to ProfessorV. S. R. D AS.

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70 1. MADHUSUDANA RAo, P. M. SWAMY and V. S. R. DAS

reaction of the CAM pathway. Feed-back inhibition of PEP-case by malateaccumulated during the night was assumed to be one of the key factors controllingthe PEP-case activity during the day/night cycle (QUEIROZ, 1968; TING, 1968;KLUGE and OSMOND, 1972; QUEIROZ and MOREL, 1974). The inhibition of PEP-caseby malate was demonstrated in leaf extracts of some succulents exhibiting CAM(TING, 1968; KLUGE and OSMOND, 1972).

Though the inhibition of CO2 fixation in leaves by foliar applied herbicides wasreported earlier (ASHTON and CRAFTS, 1973; VAN OORSCHOT, 1976), the inhibition ofone of the major carboxylases, PEP-case by herbicides is hitherto unnoticed. Thepresent study provides evidence on herbicide inhibition of PEP-case from a numberof some nonsucculent scrub species exhibiting some CAM characteristics.

Materials and Methods

Plant materials

From the scrub formation located at the foot of Tirumala hills, near Tirupati sixnonsucculent scrub species were selected for the present study. They were in the followingdecreasing order of dominance: Carissa spinarum L., Maba buxifolia CL., Flacourtia sepiariaRoxa., Chomelia asiatica O. KZE., Gymnosporia emarginata Lxws., and Dodonaea viscosa L.Fully mature leaves were used for all the experiments.

Herbicide treatment

The herbicides used in the present study were paraquat (1,1'-dimethyl-4,4-'bipyridiniumion) and 2,4,5-T [(2,4,5-trichlorophenoxy) acetic acid].

Based on the preliminary morphological observations, they were applied as a foliar sprayto the drip point using aqueous emulsions containing 100 mg of paraquat and 2000 mg of2,4,5-T per liter, and prepared from the commercial formulations. Ten plants of each specieswere treated. The concentrations selected for studies on the in vitro activity of the enzymefrom untreated leaves were 10 mg of paraquat and 100 mg of 2,4,5-T per liter. They werealso prepared from commercial formulations. The untreated plants were simultaneouslysprayed with deionised water.

Sampling

Mature leaves of similar symptoms were excised at 24, 48 and 72 h after treatment. Theywere thoroughly washed with tap water followed by deionized water and then blotted dry.The injury after paraquat treatment consisted of progressive leaf drying and desiccation. Thesymptoms after 2,4,5-T treatment were chlorosis, leaf tip necrosis and finally also desiccation.The leaves sampled during three days after herbicide treatment received and absorbedapproximately the same amount of herbicide per unit leaf area.

Extraction of PEP-case (EC 4.1.1.31)

Leaf material (1-2 g of fresh weight) was cut into about 1 ern" pieces and groundvigorously in a prechilled mortar for 90 sec with 4 vols. of extraction medium along with apinch of sand. The extraction medium was 50 mM rris-HCl buffer, pH 7.8, containing 5 mMdirhiorhreitol (DTT), 1 mM EDTA, 2 mM MgCI2, and 10 mM 2-mercaptoethanol. Theextract was filtered through four layers of muslin cloth and an aliquot was set aside forchlorophyll estimation according to the method of ARNON (1949). During extraction allmaterials were kept in the cold (0 + 2 Qq. The extracts were partially purified by

Z. Pflanzenphysiol. Ed. 99. S. 69-74. 1980.

Herbicide-inhibited PEP carboxylase 71

ammonium sulphate fractionation and by passing through a Sephadex G-25 column to removemalic acid and other enzyme activators and inhibitors before assay (RAO et al., 1979 a). Thein vitro as well as in vivo activity of the enzyme was assayed as given below.

Enzyme assay

The enzyme was assayed by estimating 14C incorporation into acid stable products(RAGHAVENDRA and DAS, 1975). Since oxaloacetate, the reaction product, was unstable underordinary conditions, the reaction was stopped with 2,4-dinitrophenylhydrazine in HC\' Thereaction mixture (2 ml) contained 50 mM tris-H'Cl buffer, pH 8.0; 2 mM MgCI2 ; 2 mMNaH14C03 (1.6 mCi/mmole); 1 mM PEP and the enzyme. The reaction was stopped after 3min by the addition of an equal volume of 1 N HCI saturated with 2,4-dinitrophenyl­hydrazine. An aliquot was examined for incorporated radioactivity, using a continuous gasflow proportional counter (RAO, 1978).

Besides purified leaf extracts from herbicide-sprayed plants, also extracts from untreatedleaves were incubated for 5 min in herbicide solution. The enzyme activities are expressed permg of chlorophyll but given as percentages of untreated in the figure. The enzyme values ofuntreated leaves are given in Table 1.

Table 1: PEP-case activity of the untreated leaf extracts of 6 scrub species (100 % values,averaged from those given in the figure, here expressed in mmoles per mg chlorophyll per h).Values are means of five observations ± S.E.

Plant species

Carissa spina rumMaba buxifoliaGymnosporia emarginataFlacourtia sepia riaDodonaea viscosaChomelia asiatica

mmoles(mg chlorophyllt1 hr

1.68±0.063.64±0.192.22±0.125.11 ±0.321.87±0.080.11 ± 0.01

Results

The in vitro acuvity of the PEP-case was inhibited when purified leaf extractsfrom untreated leaves of all species were incubated in herbicide solutions. Theinhibition was more prominent with paraquat than with 2,4,5-T in all the six speciestested (Fig. 1).

The in vivo activity of the PEP-case of purified leaf extracts fromherbicide-sprayed leaves was also markedly inhibited when followed for 72 h afterfoliar application of paraquat or 2,4,5-T. This is true for all species and treatmentsexcept Chomelia asiatica where 2,4,5-T treatment caused a slight enhancement in theenzyme activity at 24 and 48 h. However, at 72 h after treatment the enzyme activitywas inhibited in all the six species tested. The inhibition of the enzyme activity wasmore prominent with paraquat than with 2,4,5-T treatment (Fig. 1).

Z. Pilanzenpbysiol. Bd. 99. S. 69-74. 1980.

72 I. MADHUSUDANA RAO, P. M. SWAMY and V. S. R. DAS

150 GYMNOSPORIA

EMARGINATA150 FLACOURTIA

SEPIARIA

100 100

50 50

--J0150c;f-zoU 100

LLof- 50zwU0:::W0...

150

MABAEilJXIF0 L IA

CARISSASPINARUM

150

100

50

150

DODONAEA

~

CHOMELIAASIATICA

100 2 100

7248

50

o

50

24 48 72 0 24

HOURS AFTER TREATMENT

Fig. 1: Effects of paraquat (Li, .) and 2,4,5-T (0, .) on the PEP-case activity of leaves(control: 0). Open symbols represent the enzyme activity of leaf extracts incubated in her­bicide solutions and closed symbols those from treated plants after 24, 48 and 72 h (stan­dard errors are given by the vertical bars).

Discussion

The present investigation provides evidence that paraquat and 2,4,5-T are effectiveinhibitors of PEP-case isolated from six nonsucculent scrub species. Paraquat or2,4,5-T treatment inhibited the in vitro activity of PEP-case from untreated leavesand also the in vivo activity of the enzyme from herbicide-sprayed leaves.

BOLHAR-NoRDENKAMPF (1970) in his experiments on the effects of atrazine on gasexchange of plants stated that atrazine seems to inhibit one of the two enzymes,carbonic anhydrase or ribulose-1-5-diphosphate carboxylase, thereby stopping CO2

uptake. Inhibition of CO 2 fixation by foliar application of paraquat or 2,4,5-Twasreported in the six plants tested here (RAO et aI., 1978). The inhibition of CO2

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Herbicide-inhibited PEP carboxylase 73

fixation by paraquat or 2,4,5- T observed in the test plants confirms the observationsof earlier workers (WEDDING et a!', 1954; WORT, 1964; VAN OORSCHOT, 1970, 1976;HARRIS and DODGE, 1972).

The herbicide-inhibited CO2 fixation in leaves has been attributed to the consistentinhibition of NADP reduction of isolated chloroplasts (ASHTON and CRAFTS, 1973;MORELAND and HILTON, 1976). However, chloroplasts isolated from herbicidesprayed leaves did not show consistent inhibition of NADP reduction by eitherparaquat or 2,4,5- T (RAO et a!', 1979 b). In spite of the operation of normal NADPreduction activity in the chloroplasts of herbicide sprayed leaves, the photosyntheticcarbon fixation was inhibited in all the test plants (RAO et a!', 1978). A few earlierworkers (ALLAWAY and MANSFIELD, 1967; MEIDNER and MANSFIELD, 1968; ZELITCH,1969) were of the opinion that the inhibition of CO2 fixation by herbicides was dueto stomatal closure. They attributed the stomatal closure to the accumulation of CO2

in intercellular spaces as a result of inhibition of photosynthesis.In contrast to the assumption of the above workers, considerable stomatal opening

was observed in the present test plants treated with paraquat or 2,4,5- T (RAO et a!',1977), although the CO2 fixation was completely inhibited (RAO et a!', 1978).Therefore, it is believed that fully open stomata and sufficient availability ofNADPH need not allow normal CO2 fixation. The present investigation suggeststhat the inhibition of the major carboxylating enzyme, PEP-case may be involved inthe herbicide-inhibition of CO2 fixation in leaves.

Acknowledgements

Paraquat (Gramoxones) was supplied by the Alkali and Chemical Corporation of IndiaLimited, and 2,4,5- T (Weedone®) was from the Agromore Limired, India. We express oursincere appreciation to Dr. HERBERT M. HULL, USDA-ARS Western Region, Tucson,Arizona, USA and Professor Dr. M. KLUGE of Botanisches Institut, 61 Darmstadt, WestGermany for their suggestions and criticism during this investigation.

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Z. Pflanzenphysiol. Bd. 99. S. 69-74. 1980.