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J. Appl. Genet. 43(4), 2002, pp. 451-462
Biochemical differences in
Cannabis sativa L.
depending on sexual phenotype
depending on sexual phenotype
Elena T
RU1, Elvira GILLE2, Ecaterina TÓTH2, Marilena MANIU3
1
Department of Genetics, Institute of Biological Research, Iai, Romania
2
Department of Genetics, Centre of Research “Stejarul”, Piatra Neamþ, Romania
3
Department of Genetics, Faculty of Biology, University “Al. I. Cuza”, Iai, Romania
Abstract
. Hemp (Cannabis sativa L.) is a species considered as having one of the most
complicated mechanisms of sex determination. Peroxidase and esterase isoenzymes in
leaves of the two sexual phenotypes of hemp were studied. Significant differences
in isoperoxidase and isoesterase patterns were found between male and female plants,
both in the number and stain intensity of bands. For both esterase and peroxidase,
the isoenzymatic spectrum is richer for staminate plants. Also, some differences are obvious
between the two sexes concerning catalase and peroxidase activities, as well as
the level of soluble protein. The quantitative analysis of flavones, polyholozides
and polyphenols emphasized differences depending not only on sex, but also on tested
organ.
complicated mechanisms of sex determination. Peroxidase and esterase isoenzymes in
leaves of the two sexual phenotypes of hemp were studied. Significant differences
in isoperoxidase and isoesterase patterns were found between male and female plants,
both in the number and stain intensity of bands. For both esterase and peroxidase,
the isoenzymatic spectrum is richer for staminate plants. Also, some differences are obvious
between the two sexes concerning catalase and peroxidase activities, as well as
the level of soluble protein. The quantitative analysis of flavones, polyholozides
and polyphenols emphasized differences depending not only on sex, but also on tested
organ.
Key words
: electrofocusing, hemp, isoenzymatic pattern, secondary metabolites, sexual
phenotype.
phenotype.
Introduction
The chemical composition of hemp (
Cannabis sativa L.) is very complex, including
about a hundred of compounds isolated from hemp organs: flavonoids, fatty
acids, phenolic spiroindans, dihydrostilbenes, nitrate substances (amines, ammonium
salts, spermidine-derived alkaloids), etc. The hemp flavour is due to volatile
terpenic compounds of essential oils, monoterpenes representing 47.9-92.1%
and sesquiterpenes 52-48.6% of total terpenes. Compounds like friedeline,
epifriedelinol, -sitosterol, carvone and dihydrocarvone were isolated from roots
(SETHI et al. 1978). Seeds contain oils (PETRI 1988), while among plant organs
about a hundred of compounds isolated from hemp organs: flavonoids, fatty
acids, phenolic spiroindans, dihydrostilbenes, nitrate substances (amines, ammonium
salts, spermidine-derived alkaloids), etc. The hemp flavour is due to volatile
terpenic compounds of essential oils, monoterpenes representing 47.9-92.1%
and sesquiterpenes 52-48.6% of total terpenes. Compounds like friedeline,
epifriedelinol, -sitosterol, carvone and dihydrocarvone were isolated from roots
(SETHI et al. 1978). Seeds contain oils (PETRI 1988), while among plant organs
Received: February 12, 2002. Accepted: July 2, 2002.
Correspondence: E. T
Correspondence: E. T
RU, Department of Genetics, Biological Research Institute Bd. Carol I 20A,
6600 Iai, Romania, e-mail: elentru@email.ro
6600 Iai, Romania, e-mail: elentru@email.ro
flowers are richer in oils than leaves (L
EMBERKOVICS et al. 1979). The fatty acid
composition of fruits is of great interest, because of their use for nutritive
and pharmaceutical purposes. If the complete fruit and seed are similar in this aspect,
some differences are in the outer layer (MÖLLEKEN, THEIMER 1997). Although
we have not found any systematic study on flavonoid synthesis in
composition of fruits is of great interest, because of their use for nutritive
and pharmaceutical purposes. If the complete fruit and seed are similar in this aspect,
some differences are in the outer layer (MÖLLEKEN, THEIMER 1997). Although
we have not found any systematic study on flavonoid synthesis in
the genus
Cannabis there are a few papers regarding these compounds in hemp
(BATE-SMITH 1962, PARIS et al. 1975, 1976, PARIS, PARIS 1976, SEGELMAN
(BATE-SMITH 1962, PARIS et al. 1975, 1976, PARIS, PARIS 1976, SEGELMAN
et al. 1978), but the findings are contradictory, having a limited systematic value,
because of the use of different analytic methods or of different plant organs or
of various provenances.
The hemp-specific substances, cannabinoids, include more than 70 substances,
such as
because of the use of different analytic methods or of different plant organs or
of various provenances.
The hemp-specific substances, cannabinoids, include more than 70 substances,
such as
9-THC (tetrahydrocannabinol), CBD (cannabidiol) and CBN
(cannabinol), which are the criteria distinguishing between the hemp chemotypes
(especially 9-THC and CBD and THC/CBD ratio).
Although the hemp is a dioecious species, as a consequence of intensive
improvement, a lot of sexual phenotypes are cultivated, the most frequent being
the monoecious forms, classified in more categories, on a five-point scale, depending
on female flowers/male flowers ratio. Cannabis sativa L. has a very complex
genetic constitution and heredity, which explains the dioicism, amplitude
of phenotypical variability, polymorphism and the great biological plasticity
of this species.WESTERGAARD (1958) considered the sex inheritance in hemp as
being one of the most complicated mechanisms among all dioecious plants.
For hemp we could not find any consistent study on differentiation between sexual
phenotypes, regarding morphological, physiological or biochemical traits, in spite
of some disparate data. It is known that, by specific reactions, it becomes possible
to make the distinction between male and female individuals of Populus, as well
as between the – and + Mucor hyphae (SINNOTT 1960). Certain differences also
exist between staminate and pistillate plants of Lychnis dioica (STANFIELD 1944,
cited in SINNOTT 1960). For genera Cannabis and Spinacia, variable levels of cellular
extract pH are cited, depending on sex (CHEUVART 1954). In other plant species,
the analyses evidenced different values of oxidase activities in female and
male individuals (AITCHINSON 1953, cited in SINNOTT 1960).
(cannabinol), which are the criteria distinguishing between the hemp chemotypes
(especially 9-THC and CBD and THC/CBD ratio).
Although the hemp is a dioecious species, as a consequence of intensive
improvement, a lot of sexual phenotypes are cultivated, the most frequent being
the monoecious forms, classified in more categories, on a five-point scale, depending
on female flowers/male flowers ratio. Cannabis sativa L. has a very complex
genetic constitution and heredity, which explains the dioicism, amplitude
of phenotypical variability, polymorphism and the great biological plasticity
of this species.WESTERGAARD (1958) considered the sex inheritance in hemp as
being one of the most complicated mechanisms among all dioecious plants.
For hemp we could not find any consistent study on differentiation between sexual
phenotypes, regarding morphological, physiological or biochemical traits, in spite
of some disparate data. It is known that, by specific reactions, it becomes possible
to make the distinction between male and female individuals of Populus, as well
as between the – and + Mucor hyphae (SINNOTT 1960). Certain differences also
exist between staminate and pistillate plants of Lychnis dioica (STANFIELD 1944,
cited in SINNOTT 1960). For genera Cannabis and Spinacia, variable levels of cellular
extract pH are cited, depending on sex (CHEUVART 1954). In other plant species,
the analyses evidenced different values of oxidase activities in female and
male individuals (AITCHINSON 1953, cited in SINNOTT 1960).
For these reasons, the principal objective of this study was to identify the existence
of some biochemical differences (enzymes, secondary metabolites)
in hemp, depending on sexual phenotype.
of some biochemical differences (enzymes, secondary metabolites)
in hemp, depending on sexual phenotype.
Material and methods
The studied material was collected from female and male plants of hemp (
Cannabis
sativa L. subsp. sativa var. sativa), randomly chosen from a population grown
in the experimental field of the Botanical Garden of University “Al. I. Cuza” Iai.
The seeds belonging to a fiber hemp cultivar were provided by the Agricultural
sativa L. subsp. sativa var. sativa), randomly chosen from a population grown
in the experimental field of the Botanical Garden of University “Al. I. Cuza” Iai.
The seeds belonging to a fiber hemp cultivar were provided by the Agricultural
452 E. Tru
et al.
Research Centre of Secuieni at Neam
. To estimate in vivo catalase and peroxidase
activities, as well as soluble proteins, leaves of female and male hemp plants were
used. These determinations were carried out individually, in leaves collected from
20 females and 15 males of the same age (20 weeks old). Because of the lack
of simultaneity in maturity and flowering, specific for this species, the plants were
in different developmental stages. The females were in the early fruit formation
phase and the male plants were in full bloom.
To obtain the crude extract for the determination of the enzymatic activities
and the amount of soluble protein, known quantities of well ground plant material
(fine powder) in 0.01 M sodium phosphate (pH 7) were homogenized. The homogenate
was maintained at 4oC for 4 hours, and then it was centrifuged at
22 0000 rpm for 10 minutes. The supernatant was used as extract.
Catalase activity was determined by the iodometric method (ARTENIE,
TÃNASE 1981). The principle of this method is based on potassium iodide oxidation
by undecomposed hydrogen peroxide, after an incubation interval with
catalase, followed by titration of delivered iodine with sodium thiosulfate, in
the presence of starch solution as an indicator. The mixture: 0.01 M phosphate
buffer pH 7, enzymatic extract and 3% hydrogen peroxide was incubated for
5 minutes. The reaction was blocked with 10% sulfuric acid. Then 10% potassium
iodide and 1% ammonium molybdate were added. Titration was made with 0.1 sodium
thiosulfate. The catalase activity was calculated knowing that one catalase
unit is equivalent to the amount of enzyme which decomposes 1 μmol (0.034 mg)
H2O2 during 1 minute. The results are expressed in mg H2O2/g fresh matter.
Peroxidase activity was quantified by the photometric method, based on
benzidine oxidation under the peroxidase activity, in the presence of H2O2/time
activities, as well as soluble proteins, leaves of female and male hemp plants were
used. These determinations were carried out individually, in leaves collected from
20 females and 15 males of the same age (20 weeks old). Because of the lack
of simultaneity in maturity and flowering, specific for this species, the plants were
in different developmental stages. The females were in the early fruit formation
phase and the male plants were in full bloom.
To obtain the crude extract for the determination of the enzymatic activities
and the amount of soluble protein, known quantities of well ground plant material
(fine powder) in 0.01 M sodium phosphate (pH 7) were homogenized. The homogenate
was maintained at 4oC for 4 hours, and then it was centrifuged at
22 0000 rpm for 10 minutes. The supernatant was used as extract.
Catalase activity was determined by the iodometric method (ARTENIE,
TÃNASE 1981). The principle of this method is based on potassium iodide oxidation
by undecomposed hydrogen peroxide, after an incubation interval with
catalase, followed by titration of delivered iodine with sodium thiosulfate, in
the presence of starch solution as an indicator. The mixture: 0.01 M phosphate
buffer pH 7, enzymatic extract and 3% hydrogen peroxide was incubated for
5 minutes. The reaction was blocked with 10% sulfuric acid. Then 10% potassium
iodide and 1% ammonium molybdate were added. Titration was made with 0.1 sodium
thiosulfate. The catalase activity was calculated knowing that one catalase
unit is equivalent to the amount of enzyme which decomposes 1 μmol (0.034 mg)
H2O2 during 1 minute. The results are expressed in mg H2O2/g fresh matter.
Peroxidase activity was quantified by the photometric method, based on
benzidine oxidation under the peroxidase activity, in the presence of H2O2/time
unit. The mixture, composed of 1% benzidine in glacial acetic acid, 3% hydrogen
peroxide, and the enzymatic extract, was incubated for 3 minutes. 30% NaOH was
used to stop the reaction. Absolute ethanol was added. The values of extinctions
were determined with a SPEKOL 20 spectrophotometer, at
peroxide, and the enzymatic extract, was incubated for 3 minutes. 30% NaOH was
used to stop the reaction. Absolute ethanol was added. The values of extinctions
were determined with a SPEKOL 20 spectrophotometer, at
= 470 nm. After
the estimation of the ratio: sample extinction/control extinction, the peroxidase
activity was expressed in mg H2O2 /g fresh matter.
The specific activities for catalase and peroxidase were estimated by reporting
the quantity of substratum consumed by enzyme to the concentration of soluble
protein in 1 g of tissue. They were expressed in mg H2O2 /mg protein.
The obtaining of enzymatic extracts used in electrofocusing involved very fine
grinding of the plant material with a Potter homogenizer. The homogenate (1 : 3,
w/v, in 0.1 M Tris/HCl buffer pH 7.2) was centrifuged at 17 000 rpm, with a refrigerated
JANETZKI K-24 centrifuge. The supernatant was used to identify
the izoenzymatic patterns. To assess the isoenzymatic pattern for esterase
and peroxidase, we used electrofocusing on polyacrylamidic gel containing urea,
H2O, acrylamide, ampholine, and ammonium persulfate, according to the Wrigley
method (TÓTH 1992). Ampholine pH 3.5-10 (LKB) was used to establish the pH
gradient. The pH was controlled with a digital RADELKIS 20 pH meter. The sep-
the estimation of the ratio: sample extinction/control extinction, the peroxidase
activity was expressed in mg H2O2 /g fresh matter.
The specific activities for catalase and peroxidase were estimated by reporting
the quantity of substratum consumed by enzyme to the concentration of soluble
protein in 1 g of tissue. They were expressed in mg H2O2 /mg protein.
The obtaining of enzymatic extracts used in electrofocusing involved very fine
grinding of the plant material with a Potter homogenizer. The homogenate (1 : 3,
w/v, in 0.1 M Tris/HCl buffer pH 7.2) was centrifuged at 17 000 rpm, with a refrigerated
JANETZKI K-24 centrifuge. The supernatant was used to identify
the izoenzymatic patterns. To assess the isoenzymatic pattern for esterase
and peroxidase, we used electrofocusing on polyacrylamidic gel containing urea,
H2O, acrylamide, ampholine, and ammonium persulfate, according to the Wrigley
method (TÓTH 1992). Ampholine pH 3.5-10 (LKB) was used to establish the pH
gradient. The pH was controlled with a digital RADELKIS 20 pH meter. The sep-
Biochemical differences in
Cannabis sativa L. 453
aration was achieved in the glass test tubes 0.5 × 7.5 cm of the electrophoretic apparatus,
in a disk of SHANDON type. The peroxidase visualization was
performed with o-dianisidine (M
in a disk of SHANDON type. The peroxidase visualization was
performed with o-dianisidine (M
CDONALD, SMITH 1972), at pH 4.8, established
with 0.2 M sodium acetate buffer. The esterase visualization was conducted according
to SCANDALIOS (1969). In the solution for incubation (0.15Mphosphate
buffer, pH 7), 1% -naphthylacetate and Fast blue RR stain (2 mg/ml) were introduced.
with 0.2 M sodium acetate buffer. The esterase visualization was conducted according
to SCANDALIOS (1969). In the solution for incubation (0.15Mphosphate
buffer, pH 7), 1% -naphthylacetate and Fast blue RR stain (2 mg/ml) were introduced.
The isoenzymatic fractions separated by electrofocusing were drawn and represented
as zymograms. The stain intensity is indicated by hatching.
as zymograms. The stain intensity is indicated by hatching.
The phytochemical analyses were conducted on biological material dried at
40
40
oC, powdered and then subjected to extraction with different solvents. Determination
of polyholozidic content was realized in aqueous extract, the results being
expressed as 'absent', +, ++ or +++, depending on reaction intensity. Methanolic
extracts were processed to permit the quantification of flavones by colorimetric
method (on account of complexation with AlCl3) and of catechin-like
polyphenolic derivatives by the colorimetric method, in the presence
of 4-dimethyl-amino-antipyrine and ammonium persulfate, at pH = 8-9 (GRIGORESCU,
STÃNESCU 1982).
of polyholozidic content was realized in aqueous extract, the results being
expressed as 'absent', +, ++ or +++, depending on reaction intensity. Methanolic
extracts were processed to permit the quantification of flavones by colorimetric
method (on account of complexation with AlCl3) and of catechin-like
polyphenolic derivatives by the colorimetric method, in the presence
of 4-dimethyl-amino-antipyrine and ammonium persulfate, at pH = 8-9 (GRIGORESCU,
STÃNESCU 1982).
For the determination of soluble protein the Lowry method (L
OWRY et al.
1951) was used. The enzymatic extract was treated with Folin-Ciocâlteu solution.
The extinctions were registered with a SPEKOL 20 spectrophotometer, at
1951) was used. The enzymatic extract was treated with Folin-Ciocâlteu solution.
The extinctions were registered with a SPEKOL 20 spectrophotometer, at
= 500 nm. The amounts of soluble proteins are expressed in mg/g fresh matter.
These values are required to estimate the specific activities of the two
hydroperoxidases.
The statistical analysis of the obtained data was performed using the method
described by RAICU et al. (1973). The arithmetical mean (x), the standard deviation
(SD), the standard error of the mean (SE), the coefficient of variation (CV)
and the standard error of the mean, expressed in % (SE %), were calculated.
These values are required to estimate the specific activities of the two
hydroperoxidases.
The statistical analysis of the obtained data was performed using the method
described by RAICU et al. (1973). The arithmetical mean (x), the standard deviation
(SD), the standard error of the mean (SE), the coefficient of variation (CV)
and the standard error of the mean, expressed in % (SE %), were calculated.
Results and discussion
Quantitative analysis of catalase and peroxidase activities
As shown in Table 1, some differences are obvious between the two groups
of plants of different sex. First, the male individuals have a greater catalase activity
in relation to fresh biomass unit, as compared to females. The difference between
mean values for males and females was 2.27 mg H
of plants of different sex. First, the male individuals have a greater catalase activity
in relation to fresh biomass unit, as compared to females. The difference between
mean values for males and females was 2.27 mg H
2O2/g fresh matter.
The peroxidase activity registered superior values in female plants, but the difference
between mean values of female and male plants was smaller (0.9 mg H2O2/g
fresh matter). The specific activities of the two enzymes were also different. Thus,
the mean and the standard error of the mean of catalase were 4.88 ± 0.10 mg
H2O2/mg protein, for the group of female plants, and 6.02 ± 0.10 mgH2O2/mg pro-
The peroxidase activity registered superior values in female plants, but the difference
between mean values of female and male plants was smaller (0.9 mg H2O2/g
fresh matter). The specific activities of the two enzymes were also different. Thus,
the mean and the standard error of the mean of catalase were 4.88 ± 0.10 mg
H2O2/mg protein, for the group of female plants, and 6.02 ± 0.10 mgH2O2/mg pro-
454 E. Tru
et al.
Table 1.
Average values of the catalase and peroxidase activities and the amount of soluble proteins in male and female hemp plants
Plant sex n
Catalase Peroxidase Soluble proteins
mg H
Catalase Peroxidase Soluble proteins
mg H
2O2/g fresh matter mg H mg/g fresh matter 2O2/mg protein mg H2O2/g fresh matter mg H2O2/mg protein
x
± SE SD CV
%
SE
%
%
SE
%
x
± SE SD CV
%
SE
%
SE
%
x
± SE SD CV
%
SE
%
SE
%
x
± SE SD CV
%
SE
%
SE
%
x
± SE SD CV
%
SE
%
SE
%
Female 20
27.62
±
0.38
1.71 6.21 1.39
4.88
±
0.10
0.45 9.22 2.04
10.53
±
0.38
1.69 16.04 3.60
1.86
±
0.02
0.10 5.37 1.07
5.659
±
0.16
0.72 12.72 2.83
Male 15
29.89
±
0.61
2.39 7.99 2.04
6.02
±
0.10
0.39 6.47 1.66
9.63
±
0.40
1.55 16.09 4.15
1.94
±
0.05
0.21 10.82 2.57
4.961
±
0.07
0.30 6.04 1.41
27.62
±
0.38
1.71 6.21 1.39
4.88
±
0.10
0.45 9.22 2.04
10.53
±
0.38
1.69 16.04 3.60
1.86
±
0.02
0.10 5.37 1.07
5.659
±
0.16
0.72 12.72 2.83
Male 15
29.89
±
0.61
2.39 7.99 2.04
6.02
±
0.10
0.39 6.47 1.66
9.63
±
0.40
1.55 16.09 4.15
1.94
±
0.05
0.21 10.82 2.57
4.961
±
0.07
0.30 6.04 1.41
n = number of studied plants;
x = mean; SE = standard error of the mean; SD = standard deviation; CV = coefficient of variation; SE % = standard error of the mean, expressed in %
tein, for the male individuals. The specific peroxidase activities registered values
of 1.86 ± 0.02 mg H
of 1.86 ± 0.02 mg H
2O2/mg protein in pistillate plants and 1.94 ± 0.05 mg
H2O2/mg protein in staminate plants. For both catalase and peroxidase, the specific
activity was greater in males, but the increase is more important for catalase
(1.14 mg H2O2/mg protein), while for peroxidase the increase is only 0.08 mg
H2O2/mg protein.
The amount of soluble protein was greater in pistillate plants (5.659
± 0.16 mg/g fresh matter). In staminate plants, these values were 4.961
± 0.07 mg/g fresh matter, namely with 0.698 mg protein/g fresh matter smaller
than those noted for female sexual phenotypes (Table 1).
The standard deviation gives indications on the spread of the observations
around the mean. For the three analysed quantitative characters, the greatest concentration
of the observations around the mean was registered for proteins. In this
case, SD had the smallest values (0.30 for males, and 0.73 for females). The greatest
deviations were noted for catalase: SD = 2.39 in males, and SD = 1.71 in females.
The values of CV showed that the most variable character seems to be
the peroxidase activity (CV = 16.09% for males, and CV = 16.04% for females).
Besides that, the SE% had the highest values for this biochemical trait (4.15 for
males and 3.60 for female plants) (Table 1).
H2O2/mg protein in staminate plants. For both catalase and peroxidase, the specific
activity was greater in males, but the increase is more important for catalase
(1.14 mg H2O2/mg protein), while for peroxidase the increase is only 0.08 mg
H2O2/mg protein.
The amount of soluble protein was greater in pistillate plants (5.659
± 0.16 mg/g fresh matter). In staminate plants, these values were 4.961
± 0.07 mg/g fresh matter, namely with 0.698 mg protein/g fresh matter smaller
than those noted for female sexual phenotypes (Table 1).
The standard deviation gives indications on the spread of the observations
around the mean. For the three analysed quantitative characters, the greatest concentration
of the observations around the mean was registered for proteins. In this
case, SD had the smallest values (0.30 for males, and 0.73 for females). The greatest
deviations were noted for catalase: SD = 2.39 in males, and SD = 1.71 in females.
The values of CV showed that the most variable character seems to be
the peroxidase activity (CV = 16.09% for males, and CV = 16.04% for females).
Besides that, the SE% had the highest values for this biochemical trait (4.15 for
males and 3.60 for female plants) (Table 1).
The commentary on the obtained results must start from the fact that
peroxidase activity is related to the developmental processes. Thus, in
organogenesis, the role of peroxidase is frequently explained by the double function
of this enzyme, involved both in oxidizing of some substrata and in auxine catabolism
(L
peroxidase activity is related to the developmental processes. Thus, in
organogenesis, the role of peroxidase is frequently explained by the double function
of this enzyme, involved both in oxidizing of some substrata and in auxine catabolism
(L
EGRAND, BOUAZZA 1991).The latter function enables the modulation
of morphogenesis by peroxidase, as a result of intervention on endogenous hormonal
balance. In hemp, as well as in other monoecious or dioecious plants,
the gibberellins, auxins, ethylene and cytokinins have an important contribution to
sex expression (MOHAN RAM, SETT 1982a, b, DURAND, DURAND 1984,
CHAÏLAKHYAN 1985). These hormones generally intervene in the derepression of
reglator genes, which enable the synthesis of specific proteins that control flower
organogenesis. Because of its intervention in the regulation of IAA
(indole-3-acetic acid) level, peroxidase has an indirect role in the sex-determining
mechanism in hemp, more exactly in stamenogenesis and carpellogenesis. Hemp
is one of the species in which a high level of IAA induces the female sex
phenotypisation. The idea of a strong peroxidase activity associated with an increased
auxine catabolism is generally accepted. Concerning catalase, the specific
reaction catalysed by this enzyme is the direct degradation of the toxic H2O2
of morphogenesis by peroxidase, as a result of intervention on endogenous hormonal
balance. In hemp, as well as in other monoecious or dioecious plants,
the gibberellins, auxins, ethylene and cytokinins have an important contribution to
sex expression (MOHAN RAM, SETT 1982a, b, DURAND, DURAND 1984,
CHAÏLAKHYAN 1985). These hormones generally intervene in the derepression of
reglator genes, which enable the synthesis of specific proteins that control flower
organogenesis. Because of its intervention in the regulation of IAA
(indole-3-acetic acid) level, peroxidase has an indirect role in the sex-determining
mechanism in hemp, more exactly in stamenogenesis and carpellogenesis. Hemp
is one of the species in which a high level of IAA induces the female sex
phenotypisation. The idea of a strong peroxidase activity associated with an increased
auxine catabolism is generally accepted. Concerning catalase, the specific
reaction catalysed by this enzyme is the direct degradation of the toxic H2O2
(the final product of biological oxidization), with release of water and oxygen, that
is taken over to oxygenate the tissues. It seems, however, that the peroxidative activity
of catalases (the reason for which the two enzymes are known as
“hydroperoxidases”) prevails in tissue. In the case of a smallerH
is taken over to oxygenate the tissues. It seems, however, that the peroxidative activity
of catalases (the reason for which the two enzymes are known as
“hydroperoxidases”) prevails in tissue. In the case of a smallerH
2O2 concentration
and of greater quantities of other substrata, catalase can use a hydrogen donor
other than H2O2.
and of greater quantities of other substrata, catalase can use a hydrogen donor
other than H2O2.
456 E. Tru
et al.
The isoesterase and isoperoxidase patterns
The isoenyzmatic patterns were done for single individuals. The differences between
individuals of the same sex were not significant. Therefore, the schematic
zymograms of izoesterases and isoperoxidases, for one male and one female, are
compared in Figure 1. The esterases are hydrolases that catalyse the hydrolytic
splitting of molecules of substrata at the level of esteric bonds, with formation of
one alcohol molecule and one acid molecule. For both esterase (A) and peroxidase
(B), the isoenzymatic spectrum is richer for staminate plants. Thus, in female
plants, eight multiple isoesterase forms appear, six of them being placed in the domain
of pH = 5.5-6.0 and the other two in the interval of pH = 6.5-7.0. The most
active isoesterase band is placed in the weak acid domain, having pI (isoelectric
point) at pH = 6.0. The eleven isoesterase forms of staminate plants are situated in
the interval of pH = 4.5-7.2, the most active esterase forms having pI situated in
the range of pH = 4.5-5.5, namely more acid that in the case of female plants.
A specific aspect is the presence of three well outlined isoesterase bands at pH
= 6.1-6.5 in male plants – bands that have no correspondence in the isoesterase
pattern of pistillate plants.
individuals of the same sex were not significant. Therefore, the schematic
zymograms of izoesterases and isoperoxidases, for one male and one female, are
compared in Figure 1. The esterases are hydrolases that catalyse the hydrolytic
splitting of molecules of substrata at the level of esteric bonds, with formation of
one alcohol molecule and one acid molecule. For both esterase (A) and peroxidase
(B), the isoenzymatic spectrum is richer for staminate plants. Thus, in female
plants, eight multiple isoesterase forms appear, six of them being placed in the domain
of pH = 5.5-6.0 and the other two in the interval of pH = 6.5-7.0. The most
active isoesterase band is placed in the weak acid domain, having pI (isoelectric
point) at pH = 6.0. The eleven isoesterase forms of staminate plants are situated in
the interval of pH = 4.5-7.2, the most active esterase forms having pI situated in
the range of pH = 4.5-5.5, namely more acid that in the case of female plants.
A specific aspect is the presence of three well outlined isoesterase bands at pH
= 6.1-6.5 in male plants – bands that have no correspondence in the isoesterase
pattern of pistillate plants.
Differences also exist for the isoperoxidase pattern. Thus, the female plant has,
as in the case of esterase, fewer bands, two isoforms being in the range of
pH = 4.5-5.5, one at pH = 7.4 and one at pH = 8.9 (extremely basic). The ten fractions
of male genotypes were distributed in the following manner: seven at
pH = 4.7-6.1 (acid), one isoform at pH = 7.4-7.6 (weakly basic) and two bands at
the extremely basic value (pH = 8.9). The presence of four isoperoxidase fractions
at pH = 5.8-6.1 confers a strong physiological advantage of male genotypes over
the female genotypes. The isoesterase and isoperoxidase patterns reveal some differences
at the metabolic level, related to a specific multigenic determinism.
Peroxidase is distributed both in the cytosol and cell wall, as different genetic
isoenzymatic forms (O
as in the case of esterase, fewer bands, two isoforms being in the range of
pH = 4.5-5.5, one at pH = 7.4 and one at pH = 8.9 (extremely basic). The ten fractions
of male genotypes were distributed in the following manner: seven at
pH = 4.7-6.1 (acid), one isoform at pH = 7.4-7.6 (weakly basic) and two bands at
the extremely basic value (pH = 8.9). The presence of four isoperoxidase fractions
at pH = 5.8-6.1 confers a strong physiological advantage of male genotypes over
the female genotypes. The isoesterase and isoperoxidase patterns reveal some differences
at the metabolic level, related to a specific multigenic determinism.
Peroxidase is distributed both in the cytosol and cell wall, as different genetic
isoenzymatic forms (O
KEY et al. 1997). Cell wall is regarded as the site of primary
plant peroxidase activity (FRY 1986). Generally it is agreed that acid peroxidases
(especially those found in the cell wall) intervene in lignin biosynthesis, while
the basic ones (with cytoplasmic and vacuolar distribution) are involved in IAA
catabolism, through a decarboxylation step (LIMAM et al. 1998). It is obvious
(Figure 1) that, although there are fewer isoenzymatic bands in the female genotype,
the bands associated with cell walls (active in the acid range) prevail in both
analysed genotypes – a fact in accordance with data suggesting that cell walls
are a principal site of plant peroxidases. Concerning acid peroxidases it is not clear
if they are the products of different genes or if they are modified post-translational
products of a small number of genes (ROS BARCELO et al. 1987). In hemp, like in
other plant species, peroxidase has several isoforms, each with a well-defined
role. The isoperoxidase pattern is complex, just this complexity being the element
amplifying the difficulty to decipher all specific functions of this enzyme
(CLEMENTE 1998, YUN et al. 1998). The genetic determinism of these isoforms is
multigenic. If there are still unexplained details for hemp, for Brassica napus
plant peroxidase activity (FRY 1986). Generally it is agreed that acid peroxidases
(especially those found in the cell wall) intervene in lignin biosynthesis, while
the basic ones (with cytoplasmic and vacuolar distribution) are involved in IAA
catabolism, through a decarboxylation step (LIMAM et al. 1998). It is obvious
(Figure 1) that, although there are fewer isoenzymatic bands in the female genotype,
the bands associated with cell walls (active in the acid range) prevail in both
analysed genotypes – a fact in accordance with data suggesting that cell walls
are a principal site of plant peroxidases. Concerning acid peroxidases it is not clear
if they are the products of different genes or if they are modified post-translational
products of a small number of genes (ROS BARCELO et al. 1987). In hemp, like in
other plant species, peroxidase has several isoforms, each with a well-defined
role. The isoperoxidase pattern is complex, just this complexity being the element
amplifying the difficulty to decipher all specific functions of this enzyme
(CLEMENTE 1998, YUN et al. 1998). The genetic determinism of these isoforms is
multigenic. If there are still unexplained details for hemp, for Brassica napus
Biochemical differences in
Cannabis sativa L. 457
the existence of at least four distinct genes has been established (H
AMED et al.
1998). The isoenzymes of Petunia are under the control of three genes, while in
wheat isoperoxidases are controlled by different genomes and the environmental
conditions do not modify the isoenzymatic pattern (HAMED et al. 1998).
1998). The isoenzymes of Petunia are under the control of three genes, while in
wheat isoperoxidases are controlled by different genomes and the environmental
conditions do not modify the isoenzymatic pattern (HAMED et al. 1998).
Quantitative analysis of polyphenols, flavones and crude polyholozides
The data regarding the amount of some secondary metabolites, depending on sex
and organ of the plant, were obtained from the individuals whose zymograms are
presented in Figure 1. For every quantitative essay, two replications were made.
Table 2 presents the average values of these phytochemical determinations, depending
on sexual phenotype and on analysed organ, in dry and fresh matter.
The comparative analysis of these results exhibits important differences. Thus,
leaves from female plants have an increased level of polyphenols (1.560 mg%, expressed
in catechin) and flavones (1.084 mg%, expressed in rutosid), while in
male leaves polyphenols are not present, and the quantity of flavones represents
2/3 of the value for female leaves (0.680 mg%). Regarding the stem, no
polyphenols were identified in the terminal (top) part of male plants. In female
plants, however, their level was high (0.940 mg%, expressed in catechin). For flavones,
the situation is inversed: in pistillate plants these compounds are not found,
and organ of the plant, were obtained from the individuals whose zymograms are
presented in Figure 1. For every quantitative essay, two replications were made.
Table 2 presents the average values of these phytochemical determinations, depending
on sexual phenotype and on analysed organ, in dry and fresh matter.
The comparative analysis of these results exhibits important differences. Thus,
leaves from female plants have an increased level of polyphenols (1.560 mg%, expressed
in catechin) and flavones (1.084 mg%, expressed in rutosid), while in
male leaves polyphenols are not present, and the quantity of flavones represents
2/3 of the value for female leaves (0.680 mg%). Regarding the stem, no
polyphenols were identified in the terminal (top) part of male plants. In female
plants, however, their level was high (0.940 mg%, expressed in catechin). For flavones,
the situation is inversed: in pistillate plants these compounds are not found,
458 E. Tru
et al.
Figure 1. Zymograms of isoesterases (A) and isoperoxidases (B), in female (1) and male (2)
of individuals Cannabis sativa L.
Figure 1. Zymograms of isoesterases (A) and isoperoxidases (B), in female (1) and male (2)
of individuals Cannabis sativa L.
and in staminate plants their level was 0.460 mg%. In middle parts of stems,
the results were negative for all three categories of tested compounds in female
plants, whereas in male plants, polyphenols are lacking. The flavones of
the rutosid type have a value of 0.525 mg%, and the crude polyholozides have
a good representation (+++). Generally, higher levels of polyholozides were present
in male plants (for example, ++ for terminal part of stem, +++ for leaves, +++
for middle part of stem, and for female plants, respectively: +, ++, absent).
The quantitative analyses conducted in fresh matter were negative for
polyphenols for all tested organs, both for male and female plants, but the flavones
and crude polyholozides were present, the latter in high levels (+++) in all samples.
Thus, between sexual phenotypes as well as between the organs of hemp
plants, visible differences exist. It is also important that in the fresh and dry matter
of the male plants, polyphenols were absent – an aspect possibly related to the fact
that in hemp species, in which a high IAA level favours the phenotypisation of female
sex, the capacity to degrade IAA is counterbalanced by auxine protectors
(phenols).
the results were negative for all three categories of tested compounds in female
plants, whereas in male plants, polyphenols are lacking. The flavones of
the rutosid type have a value of 0.525 mg%, and the crude polyholozides have
a good representation (+++). Generally, higher levels of polyholozides were present
in male plants (for example, ++ for terminal part of stem, +++ for leaves, +++
for middle part of stem, and for female plants, respectively: +, ++, absent).
The quantitative analyses conducted in fresh matter were negative for
polyphenols for all tested organs, both for male and female plants, but the flavones
and crude polyholozides were present, the latter in high levels (+++) in all samples.
Thus, between sexual phenotypes as well as between the organs of hemp
plants, visible differences exist. It is also important that in the fresh and dry matter
of the male plants, polyphenols were absent – an aspect possibly related to the fact
that in hemp species, in which a high IAA level favours the phenotypisation of female
sex, the capacity to degrade IAA is counterbalanced by auxine protectors
(phenols).
As in the case of other secondary metabolites, the hemp callus was unable to
synthetize polyphenols and flavones (T
synthetize polyphenols and flavones (T
RUÞÃ, unpublished), which is in accordance
with results of other studies (BRAEMER et al. 1985, BRAUT-BOUCHER et al.
1981, GILLE 1996).
with results of other studies (BRAEMER et al. 1985, BRAUT-BOUCHER et al.
1981, GILLE 1996).
Biochemical differences in
Cannabis sativa L. 459
Table 2
. Average values registered for polyphenols, flavones and crude polyholozides,
depending on sexual phenotype and on analysed organ in hemp
depending on sexual phenotype and on analysed organ in hemp
Plant sex Organ
Polyphenols
mg % catechin
Flavones
mg % rutosid
Polyholozides
Polyphenols
mg % catechin
Flavones
mg % rutosid
Polyholozides
Dry matter
Male terminal part of stem 0
Male terminal part of stem 0
0.460 ++
Female terminal part of stem 0.940
0 +
Male leaves 0
0.680 +++
Female leaves 1.560
1.084 ++
Male middle part of stem 0
0.525 +++
Female middle part of stem 0
0 absent
Male inflorescence 0
1.006 absent
Fresh matter
Male leaves 0
Male leaves 0
0.340 +++
Female leaves 0
0.525 +++
Male inflorescence 0
0.444 +++
Conclusions
In this study, the isoenzymatic pattern of esterase and peroxidase is richer in hemp
male plants, as compared to female plants. For both analysed sexes,
the isoperoxidase bands localized in acid domain are prevalent. The relative
and specific activity of catalase have more reduced values in female plants.
The specific peroxidasic activity is greater in male plants. The average level
of soluble protein was higher in female plants. Significant differences are registered,
depending not only on sex, but also on tested organ in the same plant, in respect
of level of polyphenols, flavones and polyholozides. In male plant,
polyphenols were absent.
male plants, as compared to female plants. For both analysed sexes,
the isoperoxidase bands localized in acid domain are prevalent. The relative
and specific activity of catalase have more reduced values in female plants.
The specific peroxidasic activity is greater in male plants. The average level
of soluble protein was higher in female plants. Significant differences are registered,
depending not only on sex, but also on tested organ in the same plant, in respect
of level of polyphenols, flavones and polyholozides. In male plant,
polyphenols were absent.
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