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Extracellular amylase in myxomycete plasmodia and its fate during differentiation
Mycol. Res. 96 (11); 990-992 (1992)
990
Prinled in Greal Brilain
Extracellular amylase in myxomycete plasmodia and its fate during differentiation
N. MUBARAK ALI, JOSE MARY DAS AND INDIRA KAL YANASUNDARAM Centre for Advanced Study in Botany, University of Madras, Guindy Campus, Madras-600 025, India
When growing on starch-containing media in two-membered cultures with bacteria, vegetative plasmodia of myxomycetes secrete extracellular amylase. Plasmodia committed to sporulation, however, stop secreting amylase. The enzyme already synthesized is apparently held intracellularly during this period, because when actual sporulation occurs, the enzyme is again released into the medium. These observations are consistent with reported phenomena of cessation of assimilative metabolism during transition to reproductive phase, and the excretion of all unwanted material from the plasmodium at the time of sporulation.
In an earlier report, plasmodia of representative species of the orders Physarales and Stemonitales, namely Physarum f/avicomum and Stemonitis herbatica, were shown to produce extracellular amylase on starch agar plates, whereas their bacterial associates were not able to do so in pure culture (Mubarak Ali & Kalyanasundaram, 1991). In the present paper we present the results of our continuation of these studies.
MATERIALS AND METHODS The study was extended to include, besides the abovementioned cultures, seven additional cultures representing seven species, all belonging to the Physarales, in four genera (Table 1). All the cultures were from the culture collection being maintained by one of us (1. K.). These were mostly
Table 1. Details of cultures used and their starch-degrading abilities Starch degradation Bacterial associate
On
Plasmodium
starch agar
In fennentation medium
Culture no.
Species
Source
Bacterial associate
MUBL/IK/NMA/6
Physarum f/avicomum Berk. Physarum flavicomum Physarum melleum (Berk. & Br.) Massee Fuligo seplica (L.) Wiggers Physarella oblonga (Berk. & CurL) Morgan Didymium iridis (Ditmar) Fries Didymium ovoideum Nann.-Brem. Didymium squamulosum (Alb. & Schw.) Fries Slemonilis herbatica Peck
Cultured from spores
Bacillus sp"
Cultured from sclerotia Cultured from spores
Bacillus sp. Pseudomonas lemoignei Delafield eI al. Hafnia alvei Moller
+
Collected as plasmodia from nature Collected as plasmodia from nature Cultured from spores
Hafnia alvei
+
Bacillus polymyxa (Prazmowski) Mace Bacillus sp.
+
+
+
+
Cultured from spores
Pseudomonas lemoignei*
+
Cultured from spores
Pseudomonas lemoignei'
+
MUBL/IK/NMA/7 MUBL/IK/NMA/1O MUBL/IK/NMA/9 MUBL/IK/NMA/8 MUBL/IK/NMA/ 4 MUBL/IK/NMA/3 MUBL/IK/TVU/2 MUBL/IK/SH/5
, Species that replaced the original bacterial associate.
t Starch hydrolysis only during fructification.
Cultured from spores
+ ±t
+
+
N. Mubarak Ali,
J.
991
M. Das and Indira Kalyanasundaram
developed from spores by the method of I. K. (Indira, 1969) and occasionally from plasmodia or sclerotia collected in nature. Six of these plasmodia (MUBL/IK/NMA/7, 10, 9, 8, 4, 3) had retained their original bacterial associates. ln the remaining three (MUBL/IK/NMA/6, MUBL/IK/TVU/2, MUBL/IK/SH/S), contaminants have replaced the original bacterial associates. These are marked by an asterisk in Table 1. The methods were as described by Mubarak Ali & Kalyanasundaram (I991), wherein the plasmodia were grown on plates of Czapek-Dox agar substituting soluble starch for sucrose, and duplicate plates were tested daily with fresh iodine-potassium iodide solution, for starch degradation. Twenty plates of each culture were put up, and two plates were tested daily, from day 1 to day 10. The experiment was repeated, using the same nine cultures, after an interval of 2 yr.
RESUL TS AND DISCUSSION The results are summarized in Table 1. Plasmodia of 7 of the 9 isolates showed evidence of extracellular amylase, the zone of starch clearance increasing with time starting from 2-3 d, until the entire plate was cleared, usually between days 8 and 10. Didymium iridis was exceptional in that starch clearance did not start until day 8. However, in most cases, there was no close correlation between growth and starch clearance. As determined by measurement of area, plasmodial growth usually stopped after about 6 d, but starch clearance continued (Fig. 2). Only in the case of Slemonilis herbalica and Physarella
oblonga was there a definite correlation between growth and starch clearance. In Slemonitis herbatica, however, starch clearance was restricted to a narrow zone surrounding the larger veins. In the two remaining isolates, both of which were Physarum flavicomum, the inoculum for the test plates was taken from a
60 50
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30
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2 .:;. .c
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~
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'-
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10
2
4
6
8
10
Days of incubation Fig. 2. Relationship of starch hydrolysis to plasmodial growth; average values of seven isolates. 0--0, Starch clearance; 0---0, plasmodial growth. plasmodium already committed to sporulation, unlike the other seven isolates in which the inoculum came from vegetative plasmodia. In Physarum flavicomum, a plasmodium committed to sporulation is distinguishable from the vegetative plasmodium by its condensed and wavy veins, absence of a flat perforated protoplasmic sheet in the fans, greater intensity of the yellow pigment, restricted growth and restless migration over the agar surface. In plates of starch agar inoculated with these plasmodia, there was no starch clearance during plasmodial growth/migration. The cultures did not fruit even after 10 d and the starch plates remained intact, i.e. there was no secretion of amylase. Only in one experiment, one of the isolates (MUBL/IK/NMA/7) sporulated on day 9. Corresponding with the onset of
/
<.:: /'
8 day
9 day
10 day Bacterial isolate
Fig. 1. Starch hydrolysis by 'committed' plasmodium of Physarum flavicomum, delayed until onset of sporulation, marked by wide zone of starch clearance around the fructifications (arrow). The clearance zone at the edges of plates on days 5 and 6 is due to some contaminahng organisms.
992
Extracellular amylase in myxomycete plasmodia sporulation, starch hydrolysis started on day 7 and culminated at the end of sporulation on day 10, in a wide zone surrounding the sporangia (Fig. 1). As regards the bacterial associates, only the Bacillus spp. were found to degrade starch when tested in bacteriological fermentation broth; in starch agar plates, however, there was no evidence of starch degradation by any of the bacterial associates, which generally grew poorly on this medium (Table 1, Fig. 1). From the results of our study it is clear that the secretion of extracellular amylase must be widespread among the myxomycetes. However, it is not clear whether the products of this extracellular enzymatic degradation are used for plasmodial growth. Plasmodial growth in the present study was generally poor but amylase production continued even after cessation of growth (Fig. 2). It may be that the synthetic medium used by us lacked some essential nutrients and was not very conducive for plasmodial growth, or that the enzyme secreted during growth continued to diffuse through the agar. However. considering that plasmodia feeding on pulverized oats are usually found to contain abundant starch grains internally, the activity of intracellular amylase is implicit. When a plasmodium is committed to sporulation, however, it stops secreting amylase extracellularly. Since the production of extracellular amylase by the vegetative plasmodium has already been demonstrated in one of the two isolates of Physarum flavicomum used here (MUBL/IK/NMA/6: Mubarak Ali & Kalyanasundaram, 1991), it seems reasonable to infer that its cessation is associated with commitment to sporulation. This is understandable, since initiation of (Accepted 20 May 1992)
sporulation presupposes cessation of assimilative activities, representing the transformation from an assimilative to a reproductive phase. At the time of actual sporulation it appears that the endogenous reserves of the enzyme are excreted. A sporulating plasmodium is generally known to purify itself by excreting all unwanted materials, and this excretion is apparently related to the extrusion of water that occurs during sporulation (Gray & Alexopoulos, 1968). What is remarkable, however, is the permeability changes or other events that must occur in the plasmodial membrane - once when it enters the' committed state', and again at the onset of sporulation - first to hold back, and later to release those enzymes that are no longer required, while retaining the myriads of other enzymes that must be involved in the differentiation process. The authors thank the University Grants Commission, New Delhi, for the award of research project No. F3 / 44 / 86 to the third author and a Junior Research Fellowship to the first author.
REFERENCES Gray. W. D. & Alexopoulos, C. J. (1968). The Biology of Myxom!fcetes. New York: Ronald Press Co. Indira, P. U. (1969). In vitro cultivation of some myxomycetes. Nova Hedwigia 18. 627-636. Mubarak Ali. N. & Kalyanasundaram. 1. (1991). Amylase as an extracellular enzyme from plasmodia of myxomycetes. Mycological Research 95, 885-886.
990
Prinled in Greal Brilain
Extracellular amylase in myxomycete plasmodia and its fate during differentiation
N. MUBARAK ALI, JOSE MARY DAS AND INDIRA KAL YANASUNDARAM Centre for Advanced Study in Botany, University of Madras, Guindy Campus, Madras-600 025, India
When growing on starch-containing media in two-membered cultures with bacteria, vegetative plasmodia of myxomycetes secrete extracellular amylase. Plasmodia committed to sporulation, however, stop secreting amylase. The enzyme already synthesized is apparently held intracellularly during this period, because when actual sporulation occurs, the enzyme is again released into the medium. These observations are consistent with reported phenomena of cessation of assimilative metabolism during transition to reproductive phase, and the excretion of all unwanted material from the plasmodium at the time of sporulation.
In an earlier report, plasmodia of representative species of the orders Physarales and Stemonitales, namely Physarum f/avicomum and Stemonitis herbatica, were shown to produce extracellular amylase on starch agar plates, whereas their bacterial associates were not able to do so in pure culture (Mubarak Ali & Kalyanasundaram, 1991). In the present paper we present the results of our continuation of these studies.
MATERIALS AND METHODS The study was extended to include, besides the abovementioned cultures, seven additional cultures representing seven species, all belonging to the Physarales, in four genera (Table 1). All the cultures were from the culture collection being maintained by one of us (1. K.). These were mostly
Table 1. Details of cultures used and their starch-degrading abilities Starch degradation Bacterial associate
On
Plasmodium
starch agar
In fennentation medium
Culture no.
Species
Source
Bacterial associate
MUBL/IK/NMA/6
Physarum f/avicomum Berk. Physarum flavicomum Physarum melleum (Berk. & Br.) Massee Fuligo seplica (L.) Wiggers Physarella oblonga (Berk. & CurL) Morgan Didymium iridis (Ditmar) Fries Didymium ovoideum Nann.-Brem. Didymium squamulosum (Alb. & Schw.) Fries Slemonilis herbatica Peck
Cultured from spores
Bacillus sp"
Cultured from sclerotia Cultured from spores
Bacillus sp. Pseudomonas lemoignei Delafield eI al. Hafnia alvei Moller
+
Collected as plasmodia from nature Collected as plasmodia from nature Cultured from spores
Hafnia alvei
+
Bacillus polymyxa (Prazmowski) Mace Bacillus sp.
+
+
+
+
Cultured from spores
Pseudomonas lemoignei*
+
Cultured from spores
Pseudomonas lemoignei'
+
MUBL/IK/NMA/7 MUBL/IK/NMA/1O MUBL/IK/NMA/9 MUBL/IK/NMA/8 MUBL/IK/NMA/ 4 MUBL/IK/NMA/3 MUBL/IK/TVU/2 MUBL/IK/SH/5
, Species that replaced the original bacterial associate.
t Starch hydrolysis only during fructification.
Cultured from spores
+ ±t
+
+
N. Mubarak Ali,
J.
991
M. Das and Indira Kalyanasundaram
developed from spores by the method of I. K. (Indira, 1969) and occasionally from plasmodia or sclerotia collected in nature. Six of these plasmodia (MUBL/IK/NMA/7, 10, 9, 8, 4, 3) had retained their original bacterial associates. ln the remaining three (MUBL/IK/NMA/6, MUBL/IK/TVU/2, MUBL/IK/SH/S), contaminants have replaced the original bacterial associates. These are marked by an asterisk in Table 1. The methods were as described by Mubarak Ali & Kalyanasundaram (I991), wherein the plasmodia were grown on plates of Czapek-Dox agar substituting soluble starch for sucrose, and duplicate plates were tested daily with fresh iodine-potassium iodide solution, for starch degradation. Twenty plates of each culture were put up, and two plates were tested daily, from day 1 to day 10. The experiment was repeated, using the same nine cultures, after an interval of 2 yr.
RESUL TS AND DISCUSSION The results are summarized in Table 1. Plasmodia of 7 of the 9 isolates showed evidence of extracellular amylase, the zone of starch clearance increasing with time starting from 2-3 d, until the entire plate was cleared, usually between days 8 and 10. Didymium iridis was exceptional in that starch clearance did not start until day 8. However, in most cases, there was no close correlation between growth and starch clearance. As determined by measurement of area, plasmodial growth usually stopped after about 6 d, but starch clearance continued (Fig. 2). Only in the case of Slemonilis herbalica and Physarella
oblonga was there a definite correlation between growth and starch clearance. In Slemonitis herbatica, however, starch clearance was restricted to a narrow zone surrounding the larger veins. In the two remaining isolates, both of which were Physarum flavicomum, the inoculum for the test plates was taken from a
60 50
'E
.:;.
'E
'" u
40
''""
30
::: ~
OJ
.c
2 .:;. .c
~
8eo
~
~
:;
'-
s:'"
0
~
~
o 20 'oc"::
N
10
2
4
6
8
10
Days of incubation Fig. 2. Relationship of starch hydrolysis to plasmodial growth; average values of seven isolates. 0--0, Starch clearance; 0---0, plasmodial growth. plasmodium already committed to sporulation, unlike the other seven isolates in which the inoculum came from vegetative plasmodia. In Physarum flavicomum, a plasmodium committed to sporulation is distinguishable from the vegetative plasmodium by its condensed and wavy veins, absence of a flat perforated protoplasmic sheet in the fans, greater intensity of the yellow pigment, restricted growth and restless migration over the agar surface. In plates of starch agar inoculated with these plasmodia, there was no starch clearance during plasmodial growth/migration. The cultures did not fruit even after 10 d and the starch plates remained intact, i.e. there was no secretion of amylase. Only in one experiment, one of the isolates (MUBL/IK/NMA/7) sporulated on day 9. Corresponding with the onset of
/
<.:: /'
8 day
9 day
10 day Bacterial isolate
Fig. 1. Starch hydrolysis by 'committed' plasmodium of Physarum flavicomum, delayed until onset of sporulation, marked by wide zone of starch clearance around the fructifications (arrow). The clearance zone at the edges of plates on days 5 and 6 is due to some contaminahng organisms.
992
Extracellular amylase in myxomycete plasmodia sporulation, starch hydrolysis started on day 7 and culminated at the end of sporulation on day 10, in a wide zone surrounding the sporangia (Fig. 1). As regards the bacterial associates, only the Bacillus spp. were found to degrade starch when tested in bacteriological fermentation broth; in starch agar plates, however, there was no evidence of starch degradation by any of the bacterial associates, which generally grew poorly on this medium (Table 1, Fig. 1). From the results of our study it is clear that the secretion of extracellular amylase must be widespread among the myxomycetes. However, it is not clear whether the products of this extracellular enzymatic degradation are used for plasmodial growth. Plasmodial growth in the present study was generally poor but amylase production continued even after cessation of growth (Fig. 2). It may be that the synthetic medium used by us lacked some essential nutrients and was not very conducive for plasmodial growth, or that the enzyme secreted during growth continued to diffuse through the agar. However. considering that plasmodia feeding on pulverized oats are usually found to contain abundant starch grains internally, the activity of intracellular amylase is implicit. When a plasmodium is committed to sporulation, however, it stops secreting amylase extracellularly. Since the production of extracellular amylase by the vegetative plasmodium has already been demonstrated in one of the two isolates of Physarum flavicomum used here (MUBL/IK/NMA/6: Mubarak Ali & Kalyanasundaram, 1991), it seems reasonable to infer that its cessation is associated with commitment to sporulation. This is understandable, since initiation of (Accepted 20 May 1992)
sporulation presupposes cessation of assimilative activities, representing the transformation from an assimilative to a reproductive phase. At the time of actual sporulation it appears that the endogenous reserves of the enzyme are excreted. A sporulating plasmodium is generally known to purify itself by excreting all unwanted materials, and this excretion is apparently related to the extrusion of water that occurs during sporulation (Gray & Alexopoulos, 1968). What is remarkable, however, is the permeability changes or other events that must occur in the plasmodial membrane - once when it enters the' committed state', and again at the onset of sporulation - first to hold back, and later to release those enzymes that are no longer required, while retaining the myriads of other enzymes that must be involved in the differentiation process. The authors thank the University Grants Commission, New Delhi, for the award of research project No. F3 / 44 / 86 to the third author and a Junior Research Fellowship to the first author.
REFERENCES Gray. W. D. & Alexopoulos, C. J. (1968). The Biology of Myxom!fcetes. New York: Ronald Press Co. Indira, P. U. (1969). In vitro cultivation of some myxomycetes. Nova Hedwigia 18. 627-636. Mubarak Ali. N. & Kalyanasundaram. 1. (1991). Amylase as an extracellular enzyme from plasmodia of myxomycetes. Mycological Research 95, 885-886.