A Survey of Bryophytes for Presence of Cholinesterase Activity

by Ashu Gupta, Rajendra Gupta, Prem L. Uniyal, Suman S. Thakur
A Survey of Bryophytes for Presence of Cholinesterase Activity
Ashu Gupta, Rajendra Gupta, Prem L. Uniyal, Suman S. Thakur
American Journal of Botany
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Department of Botany, University of Delhi, Delhi 110007, India

The neurotransmitter acetylcholine (ACh) is present in plants including bryophytes. The first biochemical evidence for ACh hydro- lysis by enzyme cholinesterase (ChE) in bryophytes is presented. Thirty-nine species belonging to 16 families of bryophytes were surveyed for ChE activity. Thirty species belonging to 13 families showed ChE activity. Of the bryophytes tested, Anoectangium bicolor showed the highest ChE activity. Widespread distribution of ChE in bryophytes indicates their suitability as a system to study the role of ACh in plants.

Key words: acetylcholine; Anoectangium bicolor; bryophytes; cholinesterase; plants; Pottiaceae.

Acetylcholine (Ach), the well-known neurotransmitter in higher animals, has been detected in all plants tested thus far (Fluck and Jaffe, 1974a; Hartmann and Gupta, 1989; Tretyn and Kendrick, 1991). The enzymes of ACh metabolism-ch~- line acetyltransferase and ChE (cholinesterase)-have als~ been reuorted from ulants (Fluck and Jaffe, 1974a, b: Miura et al., f982; ~artmkn and' Gupta, 1989; Roshchina and Se- menova, 1990; Tretyn and Kendrick, 199 1; Kashyap, 1996; Gupta and Gupta, 1997; Gupta, Vijayaraghavan, and Gupta, 1998). Acetylcholine has been reported only in one bryophyte, a hybrid of Funaria hygrometrica and Physcomitrium pirifor- me (Hartmann and Kilbinger, 1974), whereas its hydrolyzing enzyme ChE was not found either in Funaria hygrometrica or in Physcomitrium piriforme (see Fluck and Jaffe, 1974a). Until now ChE has been reported to be present in only one bryo- phyte, Marchantia polymorpha, where it was shown to be lo- calized in stomata by electron microscope studies (G6rska- Brylass and Smolinski, 1992). To discern the natural role of ACh system in plants, it is desirable to understand the distri- bution of ChE in the entire plant kingdom. Thus, ChE activity was tested in 219 plant species belonging to angiosperms, gymnosperms, pteridophytes, and algae in our laboratory (Kashyap, 1996; Gupta and Gupta, 1997; Gupta, Vijayarag- havan, and Gupta, 1998). In view of the lack of any substan- tive information on presence of ChE in bryophytes, the present work was undertaken to study the distribution of ChE in 6 liverworts and 33 mosses.


material-Gameto~h~tes of br~o~h~tes from

were "'lected places in India: Chandigarh (December, 1998), Delhi (January, 1999), Nainital (February, 1999) and Shimla (December, 1998) as indicated in the voucher specimen number-CB, DB, NB, and SB, respectively. Live bryophytes from outside Delhi were transported to the laboratory within 24 hr of collection and stored in a refrigerator at -10°C for a couple of days before experiments

' Manuscript received 20 October 2000; revision accepted 22 May 2001.

The authors thank Prof. S. C. Gupta, Department of Botany, University of Delhi, for use of the Shimadzu UV-VIS spectrophotometer; University Grants Commission, New Delhi for grant of a research project to RG; and Council of Scientific and Industrial Research, New Delhi for a senior research fellow- ship to AG. This paper is part three of the series "Cholinesterases in Plants"

(1: Gupta and Gupta, 1997; 2: Gupta, Vijayaraghavan, and Gupta, 1998). Author for reprint requests (e-mail: tree@vsnl.com).

were done. Bryophytes collected from Delhi and Nainital were used fresh, whereas those collected from Chandigarh and Shimla were used after rehy- dration. Identification was done by one of us (Prem La1 Uniyal), using stan- dard texts (Kashyap, 1929; Gangulee, 1974; Chopra, 1975). Voucher speci- mens were deposited in the Delhi University Herbarium and the voucher spec- imen numbers are indicated in

Ellman's test for ChE activity-Enzyme cholinesterase activity was mea- sured spectrophotometrically by employing a minor modification of the meth- od of Ellman et al. (1961) as described previously by Gupta and Gupta (1997). In the original method, the time course of the enzymatic activity was recorded, whereas in the present study the enzyme activity was measured after a fixed time of 180 min for each sample. The test is based on hydrolysis of acetyl- thiocholine (ATCh), a thiol analogue of ACh, to acetate and thiocholine, and the reaction of thiocholine with sulphydryl detection reagent 5 : 5'-dithiobis(2-nitrobenzoate) (DTNB) to yield a yellow-colored anion of 5-thio-2-ni- trobenzoate having a molar absorbance coefficient equal to 1.36 X lo4 (Ell- man et al., 1961). Besides thiocholine produced as a result of enzymatic hy- drolysis of acetylthiocholine iodide (ATChI), some other factors may also contribute to the yellow color produced in the test, e.g., autohydrolysis of ATChI to thiocholine, other thiol compounds, or some yellow pigments pres- ent in the samples. To check for the above factors, absorbance was recorded in controls containing a selective anti-ChE compound, neostigmine (Nst). Data of control sets were deducted from the corresponding test data. Experiments were canied out at 30°C. The reaction medium in a final volume of 5 mL contained 0.1 molL potassium phosphate buffer pH 8, 0.1 mmol/L DTNB, 1 mmol/L ATChI, and 100 mg plant material (gametophytes of bryophytes; 2 mm slices). Controls were preincubated with anti-ChE Nst 25 wmol/L for 60 min before addition of ATChI. The incubation medium was changed before adding ATChI. The fresh incubation medium also contained 25 pmol/L Nst. At 180 min after adding ATChI, the contents of tubes were filtered through a double layer of cheesecloth and absorbance was recorded at 412 nm (Model UV.260; Shimadzu, Kyoto, Japan), Each assay consisted of three replicates,


Of the 39 bryophytes representing 16 families we screened, 30 species belonging to 13 families showed detectable ChE activity (Table 1). Anoectangium bicolor, belonging to family Pottiaceae, showed highest ChE activity (240 pmol ATCh hy- dro1yzed.s-].g-' fresh mass). All the members of the Bryaceae, Entodontaceae, Funariaceae, Hypnaceae, Leskeaceae, Meteo- riaceae, Riccardiaceae, Ricciaceae, and Thuidiaceae tested positive. However, only one out of two members of Brachy- theciaceae, three out of four members of Marchantiaceae, three out of four members of Mniaceae, and seven out of nine mem-

fresh mass).   
 ATChI hydrolyzed (pmol ss'.g-' fresh mass)  Voucher specimen  
 Fam~lylSpecies  Gametophyte  no.  

Philonotis turneriana (Schwaegr.) Mitt.

Brachytheciaceae Brachythecium buchananii (Hook.) Jaeg. Brachythecium kamounense (Harv.) Jaeg. Homalothecium nilgheriense (Mont.) Robins.

Sample I Sample I1 Bryaceae Brachymenium ochianum Gangulee

Entodontaceae Entodon myurus (Hook.) Hamp. Entodon prorepens (Mitt.) Jaeg.

Fissidentaceae Fissidens viridulus (Sw.) Wahlenb.

Funariaceae Physcomitrium coorgense Broth. Physcomitrium cyathicarpum Mitt. Physcomitrium repandum (Griff.)

Hypnaceae Ectropothecium cyperoides (Hook.) Jaeg. Hypnum cupressiforme L. ex Hedw. Isopterygium distichaceum (Mitt.) Jaeg.

Sample I Sample I1 Leskeaceae Lindbergia koelzii Williams Meteoriaceae Papillaria sp. (C. Muell.) C. Muell.

Mniaceae Mnium lycopodioides Schwaeger. Mnium rhynchophorum Hook. Plagiomnium cuspidatum (Hedw.) T. Kop.

Sample I Sample I1 Plagiomnium integrum (Bosch and Lac.) T. Kop

Pottiaceae Anoectangium bicolor Ren. et Card. Anoectangium stracheyanum Mitt. Barbula constricta Mitt. Hyophila involuta (Hook.) Jaeg. Hymenostylium recurvirostre (Hedw.) Dix. Oxystegus cylindricus (Brid.) Hilp.

Sample I

Sample I1 Timmiella barbuloides (Brid.) Moenk. Tortella tortuosa (Hedw.) Limpr.

Sample I Sample I1 Weisia controversa Hedw. Thuidiaceae

Thuidium cymbifolium Doz. and Molk. Sample I Sample I1

Thuidium sparsifoliunz (Mitt.) Jaeg. Thuidium tamariscellum Bosch and Lac. Trachypodaceae Trachypodopsis auriculata (Mitt.) Fleisch. LIVERWORTS

Marchantiaceae Marchantia sp. L. Marchantia polymorpha L.

Sample I Sample I1


TABLE 1. Continued.


Conocephalum conicum (L.)Dumort. Sample I Sample I1

Plagiochasma appendiculatum L. et L. Riccardiaceae

Riccardia rnulti$da (L.) S.E Gray Ricciaceae

Riccia crystallina L.

ATChI hydrolyzed

(prno1.s-'.g ' fresh mass) Voucher specimen Gametophyte no.

22 NB-45 22 NB-48 63 NB-43

77 SB-6

26 DB-1

a Values of controls containing anti-ChE Nst were subtracted from the values in tests. Each value is a mean of three replicate determinations. (There is 10-33% variation within replicates in such tests performed with chopped tissues.)

bers of Pottiaceae showed ChE activity. Members of the fam- ilies Bartramiaceae, Fissidentaceae, and Trachypodaceae tested negative. The results indicate widespread distribution of ChE in bryophytes.


Inhibition of ATCh hydrolysis by 25 kmoVL Nst was used as a marker for presence of ChE activity. At this concentration, Nst is a potent anti-ChE agent in animals (Silver, 1974) and plants (Fluck and Jaffe, 1974b; Gupta and Maheshwari, 1980). In general, ChE activity in bryophytes is quite low compared to angiosperms, and it requires 5-6 times longer incubation period to get detectable activity. Enzyme cholinesterase activ- ity in Anoectangium bicolor, the bryophyte with highest ac- tivity, is quite comparable to ChE levels in angiosperms and -111 1 of that present in leaves of Physalis minima, the plant that has the highest ChE activity reported to date from any plant source (Gupta and Gupta, 1997). Of the 39 species screened, ChE activity was not detected in nine species. Lack of demonstrable ChE may not imply its absence for several reasons, including possible presence of decolorant or anti-ChE compounds (Fluck and Jaffe, 1974b; Gupta and Gupta, 1997). We have no evidence for presence of a decolorant in bryo- phytes as observed by Fluck and Jaffe (1974a) but we have observed that methanolic extracts of all "ChE-negative" brvo-


phytes inhibit electric eel acetylcholinesterase activity in vitro (Gupta et al., unpublished data). Because bryophytes are con- sidered to be an excellent system for experimental studies in plant physiology and biochemistry (Chopra and Kurnra, 1988), widespread occurrence of ChE in these plants suggests bryo- phytes be used to study the possible involvement of the ACh system in the signal transduction chain in plants.


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