RESPIRATORY METABOLISM OF AMITROLE-INDUCED APLASTIDIC MESOPHYLL CELLS OF CANNA EDULIS KER. M.
University of Madras,
VIVIlKANANDAN
Department of Botany
Autonomous P.G. Centre, Tiruchiropalli, 620020. Tamil Nadu
Revised Nov. 30, 1980
SUMMARY
Sublethal concentration of amitrole (3-amino- 1,2, 4-triazole) spray induced
albinism in the young leaves of Canna edulis Ker. The bleached (aplastidic) mesophylJ
cells were devoid of well developed plastids. Amitrole treatment induced an enhanced
rate of O2 uptake of the treated leaves in the initial stages. The inhibition in the rate
of oxygen uptake of aplastidic cells was to the tune of 14% when compared to that
of green cells. The cellular mitochondrial function of these cells was intact and
more active than the green cells as measured by enhanced 02 uptake to the externally
added glucose and succinate. The aplastidic cells did not exhibt complete dependence
on external substrates, indicating probably that the levels of endogenous substrates
were only marginally limiting.
INTRODUCfION
Amitrole spray at sublethal concentrations induced the formation of apIa­
stidic leaves in Canna edulis (Vivekanandan, 1980). Amitrole induced aplastidic
mesophyll cells are quite similar to the green cells except for the absence of chloro­
plasts. It is therefore of interest to see how the absence of chloroplasts aft'ects the
other cellular metobolism and physiological functions such as mitochondrial respiration
and responses to uncouplers like DNP (dinitrophenol) and CCP (chlorocarbonyl .
cyanide phenyl hydrazone). Several antibiotics, herbicides. carcinogens and analogues,
used as inhibitors of chloroplast development seemed to lack the extent of specificity
to chloroplast and were shown to interfere also with more general metabolism asso­
ciated with cytoplasm and other organelles (Kirk and Juniper, 1963; Dobel, 1963;
Bonotto et al., 1969)~ But one of the herbicides, 3-amino-l,2,4-triazole produces
albinism-a state in which chloroplast development is completely inhibited-in many
higher plants without any marked eft'ect on the cellular metabolism other then
AMl11.toU! ON CANNA HDULIS
201
chloroplast associated ones or the morphology of the leaves (Hall et aT., 1953: Ashton
and Crafts, 1973). It appears to be very specific to chloroplasts in blocking their
development (Vivekanandan and Gnanam 1975 a, b). A more convenient and quick
responding system for study, such as isolated cells, a would be idea) for studies on
respiration. Canna edulis,a sturdy ornamental monocot plant offered a suitable
experimental material.
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The chloroplasts and mit~hondria are the major energy transducing orga­
nelles of the eucaryotic cells. These two organelles show a remarkable degree of
compartmentalization of the meatboIic pools, enzymes and other substances
treharne et aI., 1966). However, one can visualize a great deal of interaction
between the two organelles, since the starting products of photosynthesis and the
end products of respiration are similar and also because photosynthesis provides
substrates (or respiration. Respiratory metabolism of amitrole induced apiastidic
leaf cells was investigated to understand the possible interaction between
mitochondria and chloroplasts and also the cellular functioning of mitochondria when
one of the most important organelles namely, chloroplast, is totally blocked from
its development.
MATERIALS AND METHODS
PJant ntaterials : Canna (Canna edulis, Ker.) plants were raised from -rhizomes
in the university botanic garden. Fresh and wen developed leaves of canna were
conected and used for experiments. While choosing amitrole bleached leaves of
Canna, care was exercised to harvest leaves of the same age as that of the control
ones.
Amitrole treatment: Aqueous solution containing 5mM amitrole was applied
to canna plants either by foliar spray or by injection through the basal portion of the
older sheaths enclosing the shoot apex. Always IO-day old canna leaves, after
emergence, were chosen for AT spray.
Isolation of mesopyJl cells: The isolation of mesophyll cells from normal and
amitrole sprayed leaves was carried out following the method of Gnanam and KuJand­
aivelu (1969). The number of cells in suspension was determined by direct micros­
copic counting with Neubauer improved double Haemocytometer.
r
Respiratory measurements: Respiratory oxygen uptake was measured polar­
ographically at 30°C using Beckman oxygen analyser connected to a Heath Servo
Recorder Model-EU-20B in a final volume of 5.0 ml reaction mixture containing
,8f'mM phosphate buffer, pH 7.5, and 5mM MgC12'
RAroLTS
The effect of amitrole on respiratory . oxygen uptake of isolated green
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M. VIVBICANANDAH
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mesophyl1 cetIs is shown in figure 1. The rate of oxygen uptake was enhanced to an
extent of 20% at low concentrations and was inhibited beyond a critical level. This
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--'
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Fig. 1 Effect of amitrole on
oxygen uptake by isolated
mesophyll cells of amitrole
treated canna leaves.
Aqueous solution contain­
ing 5 mM amitrole was
sprayed on 10 day old
canna leaves, samples
were collected at specified
time intervals and respi­
ratory oxygen uptake was
measured polarographi­
cally.
•
40
20 -
TIM£:
IN "'OURS
kind of initial enhancement of oxygen uptake by isolated mesophyll cells from intact
plants on amitrole spray was also observed as shown in figure 2. At later stages of
development of the amitrole sprayed leaf the rate of oxygen uptake was drastically
inhibited.
Amitrole-induced aplastidic cells were morphologically similar to the normal
green cells except for the absence of any well developed chloroplasts. A compartive
respiratory study of the apJastidic and normal cells has been made and the results
are depicted in table I. The rate of oxygen uptake in amitroleinduced aplastidic
cells was 14% less when compared to the normal green cells. Of all the chemical
substances used in understanding the metabolism of respiration of -aplastidic cells,
quite interestingly the bleach",d cells utilize more efficiently the externally supplied
glucose and succinate as evidenced by the enhanced O2 uptake. Malic and citric acid
enhanced the respiratory O2 uptake in both green and aplastidic cells. The green
cells showed considerable increase in their respiratory rate when treated with 0.5 roM
..
209
AMITROLE ON CANNA EDULJS
1
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Pig. 2 Effect of various concenUations of amitrole on
respiratory oxygen uptake of normal canna leaf cells.
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AMITR'OL
Table I. Comparative respiratory studies on normal and amitro/e- induced
ap/astidic leaf cells (The figures given are the average of three different experiments)
Substrate
None
Glucose
Succinate
Malic acid
Citric acid
lAA
GA
2,4-D
DNP
CCCP
(lOmM)
(2mM)
(2 mM)
(2mM)
(0.5 mM)
(0.5mM)
(0.5 mM)
(1 mM)
(0.1 mM)
lAA - Indole acetic acid
GA - Gibb~ellic acid
;Z, 4-Dichlorophenox{' acetic aci4
Green cells
(control)
67
67
87
82
10
89
97
73
51
48
Aplastidic
cells
moles 0./107 cells/min
%control
100.00
100.00
129.85
122.38
104.48
132.83
144.71
108.95
76.11
71.64
86.56
132.75
153.54
124.11
%control
58
71
89
12
71
43
52
48
49
29
122.13
74.14
89.65
82.15
84.48
50.00
DNP • Dinitrophenol
CCCP - ChIorocarbonyl cyanide phenyl
hydrazon~
210
M. VIVEItANANDAN
concentration of IAA, GA and 2,4-D. No such stimulation was observed in the
aplastidic cells. In fact they were found to inhibit the O2 uptake marginally.
The response of these cells to uncoupleJ.!s like DNP(dinitrophenol) and CCCP
(chlorocarbonyl cyanide phenyl hydrazone) was very much similar to green cells.
DrscosslON
The rate of oxygen uptake of amitrole.sprayed young leaves was enhanced
i.InQlediately after spray. This might be due to enhancement of oxidation of subst­
rates. as evidenced by low level of total soluble sugars in amitrole-induced aplastidic
leaf cells (Vivekanandan and Gnanam, 1975) and increased oxygen uptake to the
externally added glucose, when compared to the green cells. This is further suppor­
ted by the finding of Vivekauandan and Gnanaru (1975) that aplastidic cel1s are
capable of utilizing laC-glucose more efficiently than the green cells. From the present
study on the physiological properties of aplastidic cells with those of green cells, it
appears that the cellular mitochondrial function of aplastidic cells was more active as
measured by O2 uptake. They were able to utilize the externally added substrates
like glucose and succinate more efficiently than the green cells for oxidation as
evidenced by an increased 03 uptake. albeit they did not show complete dependence
on external substrate indicating that the endogenous levels of substrates were only
marginally limiting. The complete inhibition of chloroplast development by amitrole.
in any way did not aff'ect the cellular functioning of mitochondria. It is very
difficult to conclude from the preliminary investigation about the mechanism of action
of amitrole on mitochondria. although it is opined that herbicides in general suppress
glycolytic and mitochondrial energy production which might modulate transport of
materials across the membranes and atrect integrity of membranes resulting in
altered biosynthetic and regulatory activities (Moreland, 1980).
ACKNOWLBDGEMBNT
The author expresses his deep sense of gratitude to prof. A. Gnanam for
help and su~estions during the investigation of this work and to Prof. K Periasamy
for having gone through the manuscript.
Ashton, F. M. and Crafts, A.S. (1973). Mode of Action of Herbicides.
Sons. New York. John Willey and Bonotto, S. Gotfeau, A., Janowski, M., Van Den Driessche, T. and Brachet, J. (1969). Effects of various inhibitors of protein synthesis on Acetobularla medllerranea Biochem. Biophys. Aeta (Amst.) 174 : 704-1~. "
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AMITIlOLH ON CANNA mULlS
211
Dobel, P. (1963). Untersuchung der Wirkung von Streptomycinchloramphenicol-und 2-ThioUl'8CiI
Behandluns auf die plastidenetwicklung von Lycoper.icon esculerrtum Miller.
BioI. Zbl. 82: 275-25.
Ganam, A. and G. Kulandaivelu. (1969). Photosynthetic studies with leaf cell suspensions from
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Hall, W. C., Johanson, S. P. and Leinweber, C.L. (1953). Chemical defoliation and growth
inhibition in cottoD. Texas Agri. Exp. Sta. Bull. 759.
Kirk, J. T. O. and Juniper, B.B. (1963). The effecl of streptomycin on the mitochondria and
plastids of barley. Exp. Cell. Res 30 : 621-23.
Ann., Rev. Plant Physiol 31:
MorelaRd, D.E. (1980), Mechanism of action of herbicides.
591-638.
Treharne, K.J., Mercer, E.I· Goodwin, T.W. (1966). Incorporation of (laC) carbon dioxide and
(2_1IC) mevalonic acid into terpenoids of blgher plants during chloroplast develop­
ment. Biochem. J. 99 : 239-45.
Vivekanandan, M. and Gnanam, A. (1915). Studies of the mode of action of aminotriazole in the
induction of chlorosis., Plant PhysioJ., 55: 526-31.
Vivek:mandan, M. and Gnanam, A. (1915). Studies on the mechanism of action of amitrole on c'lloroplastdevelopment. Ind. J.Biochem. Biophys•• 12: 314-78. Vivekanandan. M. (1980). Selective induction of chlorosis in young leaves of Canna edulis Ker. Proc. Indian Acad Sei.,89 : 395-400.
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