Am. J. Bot. Cross-Journal Searching
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gupta, A.
Right arrow Articles by Gupta, R.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Gupta, A.
Right arrow Articles by Gupta, R.
Agricola
Right arrow Articles by Gupta, A.
Right arrow Articles by Gupta, R.
(American Journal of Botany. 2001;88:2133-2135.)
© 2001 Botanical Society of America, Inc.

Physiology and Biochemistry

A survey of bryophytes for presence of cholinesterase activity1

Ashu Gupta, Suman S. Thakur, Prem L. Uniyal and Rajendra Gupta2

Department of Botany, University of Delhi, Delhi 110007, India

Received for publication October 20, 2000. Accepted for publication May 22, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The neurotransmitter acetylcholine (ACh) is present in plants including bryophytes. The first biochemical evidence for ACh hydrolysis 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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—choline acetyltransferase and ChE (cholinesterase)—have also been reported from plants (Fluck and Jaffe, 1974a, b ; Miura et al., 1982 ; Hartmann and Gupta, 1989 ; Roshchina and Semenova, 1990 ; Tretyn and Kendrick, 1991 ; 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 piriforme (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 bryophyte, Marchantia polymorpha, where it was shown to be localized in stomata by electron microscope studies (Górska-Brylass and Smolinski, 1992 ). To discern the natural role of ACh system in plants, it is desirable to understand the distribution 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, Vijayaraghavan, and Gupta, 1998 ). In view of the lack of any substantive information on presence of ChE in bryophytes, the present work was undertaken to study the distribution of ChE in 6 liverworts and 33 mosses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material
Gametophytes of bryophytes were collected from several 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 were done. Bryophytes collected from Delhi and Nainital were used fresh, whereas those collected from Chandigarh and Shimla were used after rehydration. Identification was done by one of us (Prem Lal Uniyal), using standard texts (Kashyap, 1929 ; Gangulee, 1974 ; Chopra, 1975 ). Voucher specimens were deposited in the Delhi University Herbarium and the voucher specimen numbers are indicated in Table 1.


View this table:
[in this window]
[in a new window]
  		Table 1. Neostigmine inhibited hydrolysis of acetylthiocholine (ATChI) in bryophytes. ND = not detected (<10 pmol ATChI hydrolyzed·s–1·g–1 fresh mass)

 
Ellman's test for ChE activity
Enzyme cholinesterase activity was measured spectrophotometrically by employing a minor modification of the method 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 acetylthiocholine (ATCh), a thiol analogue of ACh, to acetate and thiocholine, and the reaction of thiocholine with sulphydryl detection reagent 5 : 5'-dithio-bis(2-nitrobenzoate) (DTNB) to yield a yellow-colored anion of 5-thio-2-nitrobenzoate having a molar absorbance coefficient equal to 1.36 x 104 (Ellman et al., 1961 ). Besides thiocholine produced as a result of enzymatic hydrolysis 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 present 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 carried out at 30°C. The reaction medium in a final volume of 5 mL contained 0.1 mol/L 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 µmol/L for 60 min before addition of ATChI. The incubation medium was changed before adding ATChI. The fresh incubation medium also contained 25 µmol/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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
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 hydrolyzed·s–1·g–1 fresh mass). All the members of the Bryaceae, Entodontaceae, Funariaceae, Hypnaceae, Leskeaceae, Meteoriaceae, Riccardiaceae, Ricciaceae, and Thuidiaceae tested positive. However, only one out of two members of Brachytheciaceae, three out of four members of Marchantiaceae, three out of four members of Mniaceae, and seven out of nine members of Pottiaceae showed ChE activity. Members of the families Bartramiaceae, Fissidentaceae, and Trachypodaceae tested negative. The results indicate widespread distribution of ChE in bryophytes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Inhibition of ATCh hydrolysis by 25 µmol/L 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 activity in Anoectangium bicolor, the bryophyte with highest activity, is quite comparable to ChE levels in angiosperms and ~1/11 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 bryophytes as observed by Fluck and Jaffe (1974a) but we have observed that methanolic extracts of all "ChE-negative" bryophytes inhibit electric eel acetylcholinesterase activity in vitro (Gupta et al., unpublished data). Because bryophytes are considered to be an excellent system for experimental studies in plant physiology and biochemistry (Chopra and Kumra, 1988 ), widespread occurrence of ChE in these plants suggests bryophytes be used to study the possible involvement of the ACh system in the signal transduction chain in plants.


View this table:
[in this window]
[in a new window]
  		Table 1. Continued

 

   FOOTNOTES
 
1 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 fellowship to AG. This paper is part three of the series "Cholinesterases in Plants" (1: Gupta and Gupta, 1997 ; 2: Gupta, Vijayaraghavan, and Gupta, 1998 ). Back

2 Author for reprint requests (tree{at}vsnl.com ). Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Chopra R. N. P. K. Kumra 1988 Biology of bryophytes. Wiley Eastern Limited, New Delhi, India

Chopra R. S. 1975 Taxonomy of Indian mosses. Council of Scientific and Industrial Research, New Delhi, India

Ellman G. L. K. D. Courtney V. Andres Jr. R. M. Featherstone 1961 A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7: 88-95[CrossRef][ISI][Medline]

Fluck R. A. M. J. Jaffe 1974a The acetylcholine system in plants. Current Advances in Plant Science 5(11): 1-22

Fluck R. A. M. J. Jaffe 1974b The distribution of cholinesterase in plant species. Phytochemistry 13: 2475-2480[CrossRef][ISI]

Gangulee H. C. 1974 Mosses of eastern India and adjacent regions. Council of Scientific and Industrial Research, New Delhi, India

Górska-Brylass A. D. J. Smolinski 1992 Ultrastructural localization of acetylcholinesterase activity in stomata of Marchantia polymorpha L. Electron Microscopy 3: 439-440

Gupta A. R. Gupta 1997 A survey of plants for presence of cholinesterase activity. Phytochemistry 46: 827-831[CrossRef][ISI]

Gupta A. M. R. Vijayaraghavan R. Gupta 1998 The presence of cholinesterase activity in marine algae. Phytochemistry 49: 1875-1877[CrossRef][ISI]

Gupta R. S. C. Maheshwari 1980 Preliminary characterization of a cholinesterase in Bengal gram—Cicer arietinum L. Plant and Cell Physiology 21: 1675-1679[ISI]

Hartmann E. R. Gupta 1989 Acetylcholine as a signaling system in plants. In W. F. Boss and D. J. Morre [eds.]. Plant biology: second messengers in plant growth and development, vol. 6, 257–288. Alan R. Liss, New York, New York, USA

Hartmann E. H. Kilbinger 1974 Gas-liquid-chromatographic determination of light dependent acetylcholine concentrations in moss callus. Biochemical Journal 137: 249-252[ISI][Medline]

Kashyap S. R. 1929 Liverworts of the western Himalayas and the Punjab plain. University of Punjab, Lahore, India

Kashyap V. 1996 Distribution of cholinesterases in plants. M.Phil. thesis, University of Delhi, Delhi, India

Miura G. A. C. A. Broomfield M. A. Lawson E. G. Worthley 1982 Widespread occurrence of cholinesterase activity in plant leaves. Physiologia Plantarum 56: 28-32[CrossRef]

Roshchina V. V. M. N. Semenova 1990 Plant cholinesterases: activity and substrate-inhibitor specificity. Journal of Evolutionary Biochemistry and Physiology 26: 487-493[ISI]

Silver A. 1974 The biology of cholinesterases. North-Holland Research Monographs, Frontiers of biology, vol. 36. North-Holland Publishing, Amsterdam, The Netherlands

Tretyn A. R. E. Kendrick 1991 Acetylcholine in plants: presence, metabolism and mechanism of action. Botanical Review 57: 33-73





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gupta, A.
Right arrow Articles by Gupta, R.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Gupta, A.
Right arrow Articles by Gupta, R.
Agricola
Right arrow Articles by Gupta, A.
Right arrow Articles by Gupta, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS