PLANT GENE & TRAIT

2014, Vol. 5, No.1, 1-10 http://pgt.sophiapublisher.com

Research Report                                                                                                          Open Access 
Leaf Photosynthesis and Antioxidant Defense in Male and Hermaphrodite Tree
of A Critically Endangered Legume, Gymnocladus assamicus Kanjilal ex P.C.
Kanjilal
Tulika Talukdar1 , Dibyendu Talukdar2
1. Department of Botany, Krishnagar Govt. College (University of Kalyani), Krishnanagar, Nadia, west Bengal, India
2. Plant Cell and Stress Biology Lab, Department of Botany, R.P.M. College (University of Calcutta), Uttarpara, Hooghly 712258, West Bengal, India
Correspondings author, dibyendutalukdar9@gmail.com; Authors
Plant Gene and Trait, 2014, Vol.5, No.1 doi: 10.5376/pgt.2014.05.0001
Copyright © 2014 Talukdar, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Photosynthetic pigment content, photosynthesis rate and antioxidant defense components were studied in leaves of male
and hermaphrodite trees of an endemic and critically endangered leguminous tree Gymnocladus assamicus Kanjilal ex P.C. Kanjilal
in Indian Himalayas to ascertain the differential physiological behavior between two different breeding types. Significant differences
were noticed between male and hermaphrodite trees during leaf flushing stage and between leaf flushing and seed setting stage in
hermaphrodite trees. During flushing period, pigment content and photosynthetic rates were significantly higher in hermaphrodite
tree compared with its male. Similarly, reduced ascorbate (AsA) and glutathione (GSH), and their redox states were substantially
higher in hermaphrodites than those in male tree undergoing flushing stage. By contrast, low levels of ascorbate peroxidase,
dehydroascorbate reductase and glutathione reductase jeopardized scavenging of H2O2 and regeneration of AsA and GSH in male
plant, consequently resulting in over-accumulation of H2O2 and high level of lipid peroxidation in leaves of male tress, marking the
onset of oxidative stress, despite the high level of superoxide dismutase, catalase and guaiacol-peroxidase in male. No such situation
was encountered in hermaphrodite trees during leaf flushing, although a significant decline of physiological fitness was observed
after its entry into seed setting stage. Concomitant with reduction in photosynthetic capacity, there was a marked decrease in
radical-scavenging capacity, leading to rise in H2O2 level in leaves. Accumulation of malondealdehyde suggested oxidative damage
in hermaphrodites, which may be instrumental for low photosynthetic capacity, high pollen sterility and reduction in number of
healthy reproductive population of G. assamicus.
Keywords Antioxidant defense; Breeding types; Gymnocladus assamicus; Hydrogen peroxide; Oxidative damage; Physiological
fitness

Background tribes, the population of G. assamicus is facing threats

Gymnocladus assamicus Kanjilal ex P.C. Kanjilal is a of extinction due to unstable germination rate, slow

member of the small archaic genus Gymnocladus
under the family Leguminosae or Fabaceae
(Caesalpinioideae), and is of great ecological and
economic importance (Ganeshaiah et al., 2005;
Choudhury et al., 2007; 2009). This medium-sized
(~17 m) deciduous tree is endemic to North-Eastern
part (formerly greater Assam state) of India
(Choudhury et al., 2007). The number and population
of this tree is alarmingly dwindling for which the
taxon has been declared as ‘critically endangered’ and
is now placed under the supervision of national
recovery programme in India (Ganeshaiah et al.,
2005). Besides its over- exploitation in diverse
medicinal and economic purposes by local ethnic
PLANT GENE AND TRAIT 
growth rate, absence of seed dispersing agents, and
heavy fungal damage to seeds (Choudhury et al.,
2007). Two breeding types have primarily been
identified; male tree and hermaphrodite with distinct
morphological variations (Lee et al., 1976; Choudhury
et al., 2007). Although some new population have
recently been discovered (Menon et al., 2010), limited
number of fruits in hermaphrodite tree and the low
natural regeneration capacity of G. assamicus are
adversely affecting in situ conservation strategy of the
plant (Singh et al., 2009).
Understanding of intrinsic physiological mechanism
of plant species may provide a mechanistic or
functional road map to draw effective conservation

Preferred citation for this article:

Talukdar, 2014, Leaf Photosynthesis and Antioxidant Defense in Male and Hermaphrodite Tree of A Critically Endangered Legume, Gymnocladus assamicus
Kanjilal ex P.C. Kanjilal, Plant Gene and Trait, Vol.5, No.1 1-10 (doi: 10.5376/pgt.2014.05.0001)
Received: 22 Jul., 2013 | Accepted: 02 Dec., 2013 | Published: 06 Dec.,2013
Leaf Photosynthesis and Antioxidant Defense in Male and Hermaphrodite Tree of A Critically Endangered Legume, 2
Gymnocladus assamicus Kanjilal ex P.C. Kanjilal

strategy (Dudley, 1996; Ackerly et al., 2000; Scarano, defense capability of endemic taxa (Bogdanović et al.,
2002). This approach is especially important for those 2005; Kukić et al., 2006), and no such reports are
taxa which are under the constant threat of extinction available on G. assamicus.
(Werff and Consiglio, 2004) and show sexual
The present study investigated leaf pigment
dimorphism (Dawson and Geber, 1999; Caruso et al.,
composition, rate of photosynthesis, pollen sterility
2003). The endemic habitats of G. assamicus comprise
and foliar antioxidant defense activities of male and
fragile Himalayan ecology and climate change factors
hermaphrodite trees of G. assamicus to provide
(Pandit et al., 2007) for which plants have to adapt
firsthand information on physiological responses of
abiotic and biotic stresses to maintain a healthy
two different breeding types in their natural habitats.
reproductive population (Kearns et al., 1998; Attri and
The results revealed significant differences between
Nayyar, 2011). The low regeneration capacity and
the two types, and indicated significant alterations in
breeding failure of G. assamicus may be either due to
physiological processes of hermaphrodite trees during
genetic factors or intrinsic metabolic alterations or
its transition from leaf flushing/flowering stage to
both, as reported in other endemic taxa (Ackerly et al.,
seed setting stage. The result gives vital physiological
2000; Culley et al., 2005).
inputs in dimorphic sex, and discusses metabolic
Antioxidant defense compounds constitute an disturbances in andro-dioecious conditions of an
important part in adaptation of plant physiological endemic and endangered tree in Indian Himalayas.
processes to changing environment. While
1 Results
ascorbate and glutathione are low molecular weight
1.1 Photosynthetic pigment content and rate of

PLANT GENE AND TRAIT 
non-enzymatic compounds, the enzymatic defense is
photosynthesis
mainly composed of superoxide dismutase (SOD),
Chl a content was significantly higher in hermaphrodite
ascorbate peroxidase (APX), dehydroascorbate
trees compared with male type during leaf flushing
reductase (DHAR), glutathione reductase (GR) and
stage, while reverse situation was noticed for chl b
catalases (CAT) (Noctor and Foyer, 1998). The APX,
content at this stage (Table 1). In comparison to leaf
DHAR, and GR are predominant enzymes in
flushing stage, chl a content was decreased by about
ascorbate-glutathione cycle, in which APX scavenges
1.8-fold in hermaphrodite tree during seed setting
hydrogen peroxide (H2O2) generated by SOD using
stage. Marginal variation was noticed in chl b level.
reduced ascorbate (AsA) as its co-factor. The resulting
The chl a: chl b ratio in both male and hermaphrodite
dehydroascorbate (DHA) is recycled to AsA by
trees changed, accordingly (Table 1). Significant
DHAR, which in turn uses reduced glutathione (GSH)
change was also noticed for carotenoid content
as its electron donor and forms oxidized glutathione
between the two plant types and during transition
(GSSG). The enzymatic action of GR regenerated
from leaf flushing to seed setting stage of
GSH within AsA-GSH cycle. Outside this cycle, CAT
hermaphrodite tree. Compared to leaf flushing stage,
also decomposes H2O2 but without using any reducing
nearly two fold reductions in total carotenoid content
power (Noctor and Foyer, 1998). Both AsA and GSH
were observed in hermaphrodite trees during seed
interact with numerous cellular compounds, and their
setting stage, which was even below to that measured
redox states in favor of reducing environment are
in male tree at flushing (Table 1). The photosynthetic
crucial in modulating the reactive oxygen
rate was significantly higher in hermaphrodite tree in
species-antioxidant interactions as it ultimately
relation to male tree during flushing but decreased
determines plant growth and development (De Pinto
2-fold during seed setting stage (Table 1).
and De Gara, 2004). Significant roles of both AsA and
GSH in vital physiological processes of plants have 1.2 Pollen sterility
been elucidated (Noctor et al., 2011; Zechmann, 2011; Pollen sterility in hermaphrodite tree was significantly
Talukdar, 2012a; 2012b). Despite immense importance, lower than that in male tree at leaf flushing stage, but
not much work has been done regarding antioxidant increased markedly at seed setting stage (Table 1).
3 http://pgt.sophiapublisher.com

Table 1 Changes in leaf physiological and biochemical parameters of male and hermaphrodite trees of Gymnocladus assamicus plants
Parameters Male tree Hermaphrodite tree 
Leaf flushing stage Leaf flushing stage Seed setting stage
Chlorophyll a (mg/g fresh weight) 2.78±0.34b 4.56±0.34a 2.54±0.21b
Chlorophyll b (mg/g fresh weight) 1.93±0.20a 1.29±0.18a 1.31±0.21a
Chl a/chl b 1.43±0.45b 3.52±0.36a 1.94±0.25b
Carotenoids (mg/g fresh weight) 1.08±0.16b 1.92±0.23a 0.97±0.21b
Photosynthetic rate (μmol CO2 m–2 s-1) 17.41±0.39b 33.29±0.51a 16.65±0.42b
Pollen sterility (%) 51.33±1.65a 2.88±0.76c 43.77±1.37b
AsA (µmol/g dry weight) 32.65±5.87b 64.10±8.35a 23.08±5.75c
DHA (µmol/g dry weight) 19.54±4.48a 8.16±2.37b 25.17±3.54a
AsA redox (AsA/AsA+DHA) 0.62±0.65b 0.89±0.75a 0.47±0.84c
GSH (nmol/g dry weight) 40.37±2.5c 223.56±3.0a 63.12±1.9b
GSSG (nmol/g dry weight) 31.33±1.5b 27.04±1.2b 39.18±2.1a
GSH redox (GSH/GSH+GSSG) 0.56±0.04b 0.90±0.05a 0.61±0.04b
SOD [U/mg (protein)] 223.34±5.6a 97.8±3.6b 100.45±4.5b
APX (nmol ascorbate oxidized min−1 mg−1 protein) 98.18±6.1b 202.67±7.5a 104.1±2.6b
DHAR (nmol ascorbate formed min−1 mg−1 protein) 8.91±5.0b 33.89±7.1a 9.78±6.8b
GR (nmol NADPH oxidized min-1 mg-1 protein) 20.67±5.5b 39.11±3.8a 18.65±6.4b
CAT (nmol H2O2 degraded min-1 mg-1 protein) 90.65±5.4a 63.18±3.6b 52.56±4.2c
POX (μmol min−1 mg−1 protein) 198.87±8.0a 203.34±10.5a 50.63±4.0b
H2O2 (µmol g-1 dry weight) 21.31±2.8a 8.51±1.6b 17.20±0.9a

PLANT GENE AND TRAIT 
MDA (nmol MDA g-1 dry weight) 29.43±6.3a 8.93±2.8b 20.15±5.5a
EL (%) 16.67±6.6a 5.78±3.7b 19.23±5.2a

1.3 Foliar ascorbate content and the redox state seed setting stage (Table 1). AsA redox in male tree
Significant (P<0.05) differences were found between during flushing was measured intermediate between
leaves of male and hermaphrodite trees for total, these two values (Table 1).
reduced and redox state of ascorbate in G. assamicus
1.4 Foliar glutathione content and the redox state
tree (Table 1) during new leaf flushing/flowering and
Both total and reduced glutathione (GSH) content in
seed setting stage. Total ascorbate (AsA+DHA)
leaves of hermaphrodite tree was significantly higher
content was the highest in hermaphrodite tress during
than that in male tree during flushing stage (Table 1).
leaf flushing, followed by male tree. The reduced form
Marginal variation, however, was observed in the
of ascorbate (AsA) was measured about two-fold
oxidized glutathione or GSSG content between male
higher in hermaphrodite tree than that in male tree
and hermaphrodite types at this stage. During seed
during flushing stage. At this stage, the oxidized form
setting of hermaphrodite tree, foliar GSH level was
(dehydroascorbate or DHA) of ascorbate was
decreased by about 2.5-fold while GSSG content
significantly higher in male plant than hermaphrodite
increased compared to flushing period. The redox
plant (Table 1). During seed setting stage of
state of GSH, calculated as GSH/ (GSH+GSSG), was
hermaphrodite tree, foliar AsA content decreased by
significantly higher in hermaphrodite tree (0.90) than
about 3-fold while DHA content was increased
that in male tree (0.56) at flushing but the value
significantly in comparison to flushing stage (Table 1).
decreased (0.61) significantly in the hermaphrodite
Comparing leaf flushing stage, the total content was
tree during seed setting stage (Table 1).
considerably lower during seed setting. Redox state of
AsA, calculated as AsA/(AsA+DHA), was changed 1.5 Antioxidant enzyme activities
accordingly, hovering around 0.9 in hermaphrodite Significant differences between male and hermaphrodite
plants during flushing stage but reduced to 0.47 at trees were noticed for antioxidant enzyme activities
Leaf Photosynthesis and Antioxidant Defense in Male and Hermaphrodite Tree of A Critically Endangered Legume,
Gymnocladus assamicus Kanjilal ex P.C. Kanjilal
during leaf flushing stage (Table 1). Measurable SOD
and CAT activities in male tree were considerably
higher than that in hermaphrodite tree. By contrast,
activities of APX, DHAR and GR were significantly
lower in the former type compared with
hermaphrodite tree at this growth stage (Table 1).
POX activity, however, was nearly similar in both
types. At seed setting stage of hermaphrodite tree,
activities of APX, DHAR, GR and POX decreased by
about 2-4-fold, while no noticeable change was
observed in SOD and CAT activity compared with
flushing stage (Table 1).
Values are means (±standard errors) of six replicates.
Values followed by the same superscript letters are not
significantly different (P<0.05) in Tukey’s multiple
range Test. AsA-reduced ascorbate, GSH-reduced
glutathione, DHA-dehydroascorbate, GSSG-glutathione
disulfide, SOD-superoxide dismutase, APX-ascorbate
peroxidase, DHAR-dehydroascorbare reductase,
GR-glutathione reductase, CAT-catalases, POX-guaiacol-
peroxidase, MDA-malondealdehyde, EL-electrolyte
leakage
1.6 H2O2 content, membrane lipid peroxidation
and electrolyte leakage
Foliar H2O2 content was significantly higher (2.5-fold)
in male tree compared with hermaphrodite tree during
flushing stage. The level, however, increased by about
2-fold in hermaphrodite tree during seed setting period.
Compared with male tree, foliar MDA content and
membrane leakage % was also considerably lower in
hermaphrodite plants under flushing stage but
increased significantly during seed setting period
(Table 1).
2 Discussion
2.1 Photosynthetic apparatus and antioxidant
defense activity in leaf flushing stage
Distinct differences were observed between male and
hermaphrodite plants for photosynthetic pigment
compositions and rate of photosynthesis during leaf
flushing stage of G. assamicus tree. Leaf flushing
stage usually occurs during onset of spring in both
types which also coincides with blooming period but
the male tree produced flower with smaller sizes than
that in hermaphrodite tree. High chl a:b ratio in
4
hermaphrodite tree during flushing period may be due
to its higher chl a content than chl b. This along with
high carotenoid levels suggested better pigment
composition in hermaphrodite trees, which may be
instrumental for enhancement of rate of
photosynthesis in bisexual type compared with male
plant. As photosynthesis is the cumulative action of
many vital physiological processes of a plant, the
higher rate of photosynthesis in hermaphrodite tree
suggested its physiological fitness superior to its male
partner during arrival of new leaf. The result is in
agreement with earlier reports on Lobelia siphilitica
and in the Hawaiian endemic genus Schiedea,
exhibiting higher photosynthesis and concomitantly
stronger physiological fitness of female than male and
indicating ecophysiological differences between the
sexes (Dawson and Geber, 1999; Caruso et al., 2003;
Schultz, 2003; Culley et al., 2005). Furthermore, it
must be noted that the male tree cannot produce fruit,
while formation of reproductive population is solely
dependent on hermaphrodite plant. Thus, partitioning
PLANT GENE AND TRAIT 
of photosynthate in hermaphrodite plant must attend
grain filling and pod development stage while the
reproductive process is usually terminated in
flowering stage in male tree. Although detail analysis
of photosynthetic product was not carried out, it seems
likely that the demand for photosynthate is much
higher in hermaphrodite tree than that in male plant.
The results indicated higher efficiency of the
hermaphrodite tree than its male partner in generation
of enough photosynthate for its efficient partitioning
to both vegetative and reproductive sinks, and
suggested modulation of source-sink relationship in
sex-specific physiology (Laporte and Delph, 1996).
The high photosynthetic rate strongly suggested
greater carbon uptake in hermaphrodites which can
also occur through the changes in biochemical pathways
(Geber and Dawson, 1997; Caruso et al., 2003).
Differential profiles of leaf photosynthesis between
male and hermaphrodite tree of G. assamicus was also
associated with significant variations in antioxidant
defense activity and generation of reactive oxygen
species (ROS) during flushing period. Although
ROS-mediated growth and development has been
reported in many plant species (De Pinto and De Gara,
5 http://pgt.sophiapublisher.com
2004), this is certainly not the situation in the present
case, particularly for male tree undergoing leaf
flushing. Higher redox state of AsA in hermaphrodite
tree could be attributed to enhanced AsA content and
concomitant low DHA content in leaves. Likewise,
elevated GSH content resulted in high GSH redox of
the plant. Completely reverse situation, however, was
encountered in male tree; low AsA and GSH might led
to reduced level of redox state in AsA and GSH,
respectively. The values of AsA redox around 0.6 and
that of GSH redox around 0.5 in male plants are far
lower than that reported as optimum in other plant
species, including crop and tree legumes (Talukdar,
2012a; 2012b; 2013c) and is quite surprising in a large
tree like Gymnocladus in its natural environment.
Present study indicated formation of sterile pollen
grains in sizable frequency by male tree. GSH plays
prominent role in prevention of pollen sterility,
abnormal germination and development of
gametophytes (Zechmann, 2011). Therefore, its
depleting level in the male plants may adversely affect
male reproductive potential in G. assamicus tree. The
lowering of redox state of AsA and GSH strongly
suggested that the balance between ROS production
and its scavenging is perturbed in male tree and tilted
in favor of oxidative cellular environment. Rising
oxidative imbalance has cascading effect on key
physiological processes of plants (Mittler, 2002;
Talukdar, 2011b), and leaf photosynthesis being one of
them is severely disturbed due to loss of pigment
apparatus, degradation of chlorophyll and other
reasons attributable to oxidative stress (Li et al., 2006;
Talukdar, 2013a; 2013b). Quite understandably, this
was not experienced by the present hermaphrodite tree
during leaf flushing.
A dissection through total measurable activities of six
prominent antioxidant enzymes primarily revealed
significantly different capability of antioxidant
defense between male and hermaphrodite tree during
leaf flushing stage. High SOD activity in male tree
indicated generation of superoxide radicals and its
dismutation to H2O2, another ROS. It is noteworthy
that the scavenging of excess H2O2 is usually
performed by enhanced activities of APX, CAT and
POX (Noctor et al., 2011). The H2O2 is a diffusible
ROS, and its dual roles as an inducer of stress and a
signaling molecule to stress have been revealed
(Kreslavski et al., 2012; Talukdar, 2013a). However, a
balance between its formation and scavenging is
necessary to prevent its toxicity and to function
properly in ROS-mediated plant growth and
development (Mittler, 2002; Kreslavski et al., 2012).
In the present study, extensive increase in SOD
activity was accompanied with steep decline in APX,
DHAR, and GR activities along with high level of
H2O2 in male plants. Obviously, low APX activity
could not contain high level of H2O2, and capability of
CAT and POX was not enough to cope with it.
Decreased activity of APX might be either due to low
availability of reduced ascorbate, its co-factor, or
inhibition of its isoform/s by excess H2O2 or both
(Hiner et al., 2000; Talukdar, 2012b). Furthermore,
due to low activity of GR and DHAR, regeneration of
glutathione and ascorbate to their reduced forms might
be severely hampered. The failure of CAT and POX to
contain high H2O2 level during low APX activity
PLANT GENE AND TRAIT 
indicated absences of compensatory H2O2-scavenging
machinery in the male plants. By contrast, low level of
CAT activity was possibly compensated by high
expression of APX during scavenging of H2O2 in
hermaphrodite plant. This along with enhanced
DHAR and GR level ensured more efficient
scavenging of H2O2 in hermaphrodite tree compared
with male tree. Existence of peroxidase/ catalase
compensatory mechanism to maintain ROS in a level
favorable to plant growth has been revealed in plants,
including endemic taxa experiencing stresses (De
Pinto and De Gara, 2004; Bogdanović et al., 2005;
Talukdar 2012a; 2013a).
Higher capability of H2O2-scavenging by
hermaphrodite plants compared with male tree was
also evidenced by comparatively lower accumulation
of MDA, a cytotoxic aldehyde of lipid peroxidation
product, in leaves of hermaphrodite plants. In many
instances including leguminous plants, rising H2O2
content often leads to enhanced rate of membrane
lipid peroxidation (Sandalio et al., 2001; Talukdar,
2011a, 2013b). Severe damage of membrane integrity
by lipid peroxidation in male plants was suggested by
nearly 2-fold higher electrolyte leakage in its leaves
Leaf Photosynthesis and Antioxidant Defense in Male and Hermaphrodite Tree of A Critically Endangered Legume,
Gymnocladus assamicus Kanjilal ex P.C. Kanjilal
than that measured in hermaphrodite tree. High lipid
peroxidation has the potential to damage
photosynthetic pigment apparatus (Li et al., 2006).
Significant increase in MDA content and loss in
photosynthetic apparatus have been recognized as the
marks of oxidative stress (Li et al., 2006; Talukdar,
2011b; 2012a; 2013a) and may be one of the prime
reasons for low rate of photosynthesis in male plants.
2.2 Photosynthesis and antioxidant defense in seed
setting stage of hermaphrodite tree
Staminate tree was not capable to produce fruits.
However, marked deterioration of pigment
composition, rate of photosynthesis and antioxidant
defense capabilities were observed during the
transition of hermaphrodite plants to seed setting stage.
Significantly low level of chl a led to lowering of chl a:
b ratio which was accompanied by reduced
carotenoids and lower magnitude of photosynthesis
rate at this stage. This may have adverse effect on
investments of photosynthates in grain filling and pod
development, and may be one of the prime reasons for
the low number of mature pods and dormant
under-filled seed in hermaphrodite tree, as reported
earlier (Choudhury et al., 2007). Similar situation was
observed in the dioecious species, Aciphylla
glaucescens (Hogan et al., 1998) and Silene latifolia
(Laporte and Delph, 1996),
Significant reduction of AsA, GSH and their redox
states during seed setting stage of hermaphrodite
plants may lead to serious alterations in free radical
scavenging capacity of the plant. Although SOD and
CAT activity was comparable to flushing period,
substantial reduction in APX, DHAR and GR levels
during seed setting indicated crippling of AsA-GSH
cycle enzymes, badly hampering H2O2-scavenging
and regeneration of AsA and GSH in hermaphrodite
plants. The importance of DHAR and GR in
maintaining redox state of AsA and GSH during the
onset of reproductive stages has been revealed in
AsA-deficient legume crop, Lathyrus sativus L
(Talukdar, 2012a), and in the mutants showing
alterations in GSH pool (Talukdar, 2012b). Pertinent
to these reports is the possible involvement of GSH in
development and germination of pollen grains in
hermaphrodite plants during reproductive phase.
6
Substantial increase in frequency of defective and
sterile pollens at late flowering and early pod bearing
stages of hermaphrodite tree and high sterile pollen in
its male partner may be related to low redox pool of
GSH, presumably hampering effective pollination and
post-pollination development in andro-dioecious
population, and consequently, resulting in low number
of pod formation in hermaphrodite trees. Although
induction of both DHAR and GR activity was
reported under depleting pool of AsA and GSH
(Talukdar, 2012a), no such situation was encountered
in hermaphrodite tree. This indicated constitutive
lower expressions of AsA-GSH cycle enzymes in
hermaphrodites undergoing sink (seeds) maturation.
Outside this cycle, the H2O2 reportedly has the capacity
to induce activity of POX in many plants (De Pinto and
De Gara, 2004) but it also seems unlikely in the present
case. The failure of antioxidant defense enzymes to
up-regulate their activities presumably led to abnormal
accumulation of H2O2 during seed setting period in
comparison to flushing stage. The rise of MDA level
PLANT GENE AND TRAIT 
and high level of electrolyte leakage suggested
ROS-induced oxidative damage in the hermaphrodites
during vital physiological stage of life cycle.
G. assamicus shows sexual dimorphism, leading to
sex-specific differences in flower size, and other
morphological traits (Choudhury et al., 2007).
Present results strongly indicate differences in
physiological traits between two breeding types.
Relatively few studies have examined sexual
dimorphism in physiological traits (Dawson and
Geber, 1999). The outcome of the present result has
immense significance as females are absent with
hermaphrodites in Gymnocladus population, and thus
selection may not favor greater male function in
hermaphrodites (Charlesworth, 1989; Caruso et al.,
2003; Culley et al., 2005). The changes in
physiological traits are inevitable events with the
establishment of separate sexes (Ackerly et al., 2000;
Culley et al., 2005) and are often correlated with
adaptations of endemic plants (Petit et al., 2001). As
successful mating is necessary for maintenance of
andro-dioecy (Pannell, 2002), it seems likely that the
weak physiological fitness manifested by present male
sex is one of the bottlenecks to produce a healthy
7 http://pgt.sophiapublisher.com
reproductive population of G. assamicus in
andro-dioecious condition. Obviously, the remarkable
capability shown by hermaphrodite tree at flushing
state must be sustained to achieve a high population
evolutionary potential, considered essential in
conservation biology of an endemic taxon (Petit et
al., 2001).
In conclusion, the present results primarily indicated
that antioxidant defense capability in male tree of G.
assamicus is substantially low, adversely affecting its
ROS-scavenging capacity and ultimately the prospects
of photosynthetic capability. The hermaphrodite tree,
by contrast, exhibited increased activities of ROS-
scavenging machinery and high photosynthetic rate,
which are generally considered as adaptive traits in
plants, bringing about a tight regulation in H2O2
content and oxidative damage of membrane at leaf
flushing stage. The failure to keep this regulation up
during seed setting stage may impede the plant to
maintain a healthy reproductive population. To what
extent these physiological factors are affecting the
prospects of natural regeneration capacity of this
critically endangered endemic legume needs further
study. The present result indicated need of more
holistic approach for designing of successful
conservation strategy of this endangered tree.
3 Materials and methods
3.1 Collection of leaf samples
The G. assamicus tree undergoes leaf flushing stage
during early March to May, flowering during late
March to mid May and attains seed setting stage
during May-June of every year. Fresh leaf samples
were collected from six small trees of approximately
10-year-old grown in Khasi hill region of Meghalaya
state (91° 21˝ E/25°07˝ N) of North-East India during
several study trips on 2010-2012. Fresh leaf samples
were collected during leaf flushing and seed setting
stage of four individual hermaphrodite trees and only
during leaf flushing stage of male tree, washed with
distilled water, dried and stored in -80 °C until
further use.
3.2 Estimation of chlorophyll, carotenoids, leaf
photosynthetic rate and pollen sterility
Leaf chlorophyll (Chl) and carotenoid contents were
determined by the method of Lichtenthaler (1987).
Leaf tissue (50 mg) was homogenized in 10 ml chilled
acetone (80%). The homogenate was centrifuged at
4,000 g for 12 min. Absorbance of the supernatant was
recorded at 663 nm, 647 nm and 470 nm for
chlorophyll a, chlorophyll b and carotenoids, respe-
ctively. The contents were expressed as mg chl or
carotenoids g−1 fresh weight. Leaf photosynthetic rate
was assayed following the methods of Coombs et al.,
(1985) using a portable photosynthesis system
(LI-6400XT, LI- COR, Inc. USA). Pollen sterility was
studied from randomly selected fresh anthers during
anthesis following staining with 1% acetocarmine
solution, and was expressed in percentage (Talukdar,
2010; 2012c).
3.3 Estimation of ascorbate and glutathione content
Reduced and oxidized form of ascorbate and
glutathione were measured following the methods of
Law et al (1983) and Griffith (1985), respectively.
3.4 Antioxidant enzyme assays
Leaves stored at -80°C (0.3~0.5 g fresh weight) were
PLANT GENE AND TRAIT 
ground to a fine powder in liquid nitrogen with a
chilled mortar and pestle under ice-cold conditions.
They were subsequently homogenized in 1 ml of 50
mM K-phosphate buffer (pH 7.8) containing 1 mM
EDTA, 1 mM dithiotreitol and 2 % (w/ v) polyvinyl
pyrrolidone (PVP). The homogenate was centrifuged
at 15 000 g for 30 min at 4°C. Clear supernatant was
used for enzyme assays. For measuring APX
(ascorbate peroxidase) activity, the tissue was
separately ground in homogenizing medium
containing 2.0 mM AsA in addition to the other
ingredients. All assays were done at 25°C. Soluble
protein content was determined according to Bradford
(1976) using BSA as a standard
SOD (EC 1.15.1.1) activity was determined by nitro
blue tetrazolium (NBT) photochemical assay
following Beyer and Fridovich (1987). In this method
1 ml of solution containing 50 mM K- phosphate
buffer (pH 7.8), 9.9 mM L-methionine, 57 μM NBT,
0.025% triton-X-100 was added into small glass tubes,
and was followed by 20 μl of enzyme extract.
Reaction was started by adding 10 μl of riboflavin
solution (0.044 mg ml−1) and placing the tubes in an
Leaf Photosynthesis and Antioxidant Defense in Male and Hermaphrodite Tree of A Critically Endangered Legume,
Gymnocladus assamicus Kanjilal ex P.C. Kanjilal
aluminium foil-lined box having two 20-W
fluorescent lamps for 7 min. A parallel control was run
where buffer was used instead of sample. After
illumination, the absorbance of solution was measured
at 560 nm. A non-irradiated complete reaction mixture
was served as a blank. SOD activity was expressed as
U (unit) mg−1 protein. One unit of SOD was equal to
that amount which causes a 50% decrease of
SOD-inhibited NBT reduction.
APX (EC 1.11.1.11) activity was assayed following
methods of Nakano and Asada (1981). Three
milliliters of the reaction mixture contained 50 mM
K-phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM
AsA, 0.1 mM H2O2 and 0.1 ml enzyme extract. The
H2O2-dependent oxidation of AsA was followed by a
decrease in the absorbance at 290 nm (ε=2.8 mM−1
cm−1). APX activity was expressed as nmol AsA
oxidized min−1 mg−1 protein.
DHAR (EC 1.8.5.1) activity was measured following
the protocol of Nakano and Asada (1981). The
complete reaction mixture contained 50 mM
K-phosphate buffer (pH 7.0), 2.5 mM GSH, 0.2 mM
DHA and 0.1 mM EDTA in a final volume of 1 ml.
Reaction was started by addition of suitable aliquots
of enzyme extract and the increase in absorbance was
recorded at 30 s intervals for 3 min at 265 nm.
Enzyme activity was expressed as nmol AsA formed
min−1 mg−1 protein.
GR (EC 1.6.4.2) activity was determined by
monitoring the glutathione dependant oxidation of
NADPH, as described earlier (Carlberg and
Mannervik, 1985). In a cuvette, 0.75 ml 0.2 M K-
phosphate buffer (pH 7.0) containing 2 mM EDTA,
75 μL NADPH (2 mM), and 75 μL oxidized
glutathione (20 mM) were mixed. Reaction was
initiated by adding 0.1 mL enzyme extract to the
cuvette and the decrease in absorbance at 340 nm was
monitored for 2 min. GR specific activity was
expressed as nmol NADPH oxidized min-1 mg-1
protein.
CAT (EC 1.11.1.6) activity was measured according to
Chance and Maehly (1955) with slight modifications.
Enzymatic activity was initiated by adding 50 μL of
8
enzyme extract into the reaction mixture containing
500 μL of K-phosphate buffer (0.1 M, pH 6.5), 250 μL
of distilled water, and 200 μL of 75 mM H2O2. CAT
activity was monitored at 240 nm for 2 min at 25 °C
after initiation of the reaction, and was measured
against a blank reaction mixture containing no enzyme
extract. CAT specific activity (nmol H2O2 degraded
min-1 mg-1 protein) was calculated using the molar
absorptivity of 43.6 M-1 cm-1for H2O2 at 240 nm.
POX (EC 1.11.1.7) was measured by monitoring
oxidation of 4 mM guaiacol at 470 nm (ε=22.6
mM −1 cm−1 ) in 50 mM K-phosphate buffer (pH 6.5),
following addition of 1 mM H2 O2 , and expressed
as μmol min −1 mg −1 protein (Veljovic-Jovanovic et
al., 2001).
3.5 H2O2 content and lipid peroxidation assay
H2O2 content was estimated following the methods of
Wang et al (2007). Leaf tissue of 0.1 g was powdered
and blended with 3 ml acetone for 30 min at 4°C.
Then the sample was filtered through eight layers of
gauze cloth. After addition of 0.15 g active carbon, the
sample was centrifuged twice at 3,000 g for 20 min at
PLANT GENE AND TRAIT 
4°C, then 0.2 ml 20% TiCl4 in HCl, and 0.2 ml
ammonia was added to 1 ml of the supernatant. After
reaction, the compound was centrifuged at 3, 000 g for
10 min, the supernatant was discarded, and the pellet
was dissolved in 3 ml of 1 M H2SO4 and spectrum
measurement was taken at 410 nm. H2O2 content was
measured from the absorbance at 410 nm using a
standard curve. Lipid peroxidation rates were
determined by measuring the malondialdehyde (MDA)
equivalents following the earlier method (Hodges et
al., 1999) with necessary modifications (Talukdar,
2011a, 2012c). About 0.5 g of tissue was homogenized
in a mortar with 80% ethanol. The homogenate was
centrifuged at 3,000 g for 12 min at 4 °C. The pellet
was extracted twice with the same solvent. The
supernatants were pooled and 1 ml of this sample was
added to a test tube with an equal volume of either the
solution comprised of 20% TCA and 0.01% butylated
hydroxy toluene (BHT) or a solution of 20% TCA,
0.01% BHT, and 0.65% TBA. Samples were heated at
95°C for 25 min and cooled to room temperature.
Absorbance was measured at 450 nm, 532 nm, and
600 nm. Level of lipid peroxides was calculated
9 http://pgt.sophiapublisher.com
following Hodges et al (1999) and expressed as nmol
MDA g-1 dry weight.
3.6 Determination of electrolyte leakage
Electrolyte leakage (EL) was assayed by measuring
the ions leaching from the tissue into deionised water
(Dionisio-Sese and Tobita, 1998). The EL was
expressed as a percentage by the formula, EL%=(EC1)
/(EC2)×100, where EC1 denotes the initial and EC2
indicates the final electrical conductivity.
3.7 Statistical analyses
The results presented are the mean values±standard
errors of at least six replicates (n=10). Multiple
comparisons of means among plant types were
performed by ANOVA using SPSS v. 10 (SPS Inc.,
USA) and separated by Tukey’s Multiple Range Test.
A probability of P<0.05 was considered significant.
Authors' contributions
Both authors contributed equally in the manuscript. TT designed the
experiment, and measured leaf photosynthesis and other non-enzymatic
biochemical parameters, while DT assayed antioxidant enzyme activities.
Both authors jointly collected samples, conducted statistical analysis, read
and approved the final manuscript.
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