Alstonia scholaris R. Br. is an elegant evergreen tree of family Apocynaceae, generally bears leaf galls caused by
an insect Pauropsylla tuberculata Crawf. In the present investigation the electrophoretic protein analysis technique
was used, which revealed that some of the protein bands are varied and shown their presence and absence in gel
at different stages of gall formation. The amount of total protein increased during early development and young
stages of gall formation and falls down in older stages. It is due to a rapid enzymatic activity in gall tissue during early
and mature stages as a response to insect interaction. Pathogens inject some elicitors and lead to the synthesis of
different type of enzymes and some specific proteins at high amount in the plant. The insect switches the defense
mechanism of the plant which results increased amount of some specific proteins in gall visible as dark bands in the
gel. The degeneration of proteins during older stages shows the exit of insect and death of gall tissue.
Alstonia scholaris R. Br.; Leaf galls; SDS-PAGE; Pauropsylla
Alstonia scholaris is an elegant evergreen tree of family Apocynaceae,
commonly known as devil tree or saptparna grows to height of
30-40 m found in most of parts of India. The plant is used in Ayurvedic,
Unani and Sidhha/Tamil types of alternative medicinal systems .
Leaves and bark are rich in Echitamine, Echitamine chloride, Scholarine,
Scholaricine, monoterpenoid indole alkaloids, iridoids, coumarins,
flavonoids, simple phenolics, steroids, saponins and tannins were also
found in the plant . The plant is traditionally being used in fever ,
cancer, tumour, jaundice, hepatitis, malaria and skin diseases . One
of the important alkaloid present in the plant called alstonine  was
reported to have anticancerous property [6,7].
Leaves are slightly rounded, leathery, dark green, shortly stalked,
lanceolate or obovate, oblong upto 20 cms long and are in whorls of
5-7. Three types of galls have been reported, one each on leaf, fruit and
flower of A. scholaris . The leaf gall of A. scholaris induced by Pauropsylla
tuberculata crawf., which is an insect belongs to class Psyllidae of
order homoptera. Within the Psyllidae, there are nearly 350 widely distributed
gall inducing species occurring mainly on the leaves of dicotyledonous
plants . Among the insects various hemipteran are known
to be capable of inducing galls [10-13].
The development and structure of galls induced by homoptera are
generally correlated with the feeding habit of the insects and are predominantly
leaf galls. The insects are known to extract nutrients from
the phloem, xylem or non-conducting plant cell . Growth of gall
tissues are associated with the changes in the levels of their cellular
contents such as carbohydrates, proteins, nucleic acids, phenols, IAA
and enzymes . The insect activates a perturbation in growth mechanisms
and alters the differentiation processes in the host plant, modifying
the plant architecture to its advantage . The gall caused by the
insect occurs on both sides of the leaf blade of the plant , which are
covering growth pouch galls. They are semiglobose, conical on adaxial
surface of the leaf and trunculated conical on the abaxial side. The galls
are pale green in the young stages but become yellowish when mature.
The gall opens at abaxial side through an ostiole, which is very narrow
in immature galls but widens out as the development of the insect proceeds
and opens to get the mature insect free (Figure 1a and 1b).
Figure 1:(a) Healthy and (b) Gall bearing leaves of A. scholaris.
The sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) technique is a powerful tool for estimating the molecular
weights of proteins . A major advantage of electrophoresis over
morphological evaluation is the speed with which a large number of
test samples can be analyzed . It simultaneously exploits differences
in molecular size to resolve proteins differing by as little as 1% in their
electrophoretic mobility through the gel matrix . Usefulness of the
technique depends on the variations within and between test samples.
Electrophoretic banding pattern polypeptides can be an efficient approach
for assessment of different samples. The present study therefore,
explains the existing polymorphism of total proteins through SDSPAGE
to facilitate characterization of different gall stages of A. scholaris.
Materials and Methods
Periodic collection of the plant material is done for three months
after a period of every 15 days and leaf protein was extracted in an extraction
buffer (50 mM Tris HCl) followed by centrifugation at 10,000 rpm at 4°C for 15 min. Total protein content of the gall stages was estimated
in supernatant following Lowry's method  using BSA as
To study the SDS-PAGE protein profile, unidimentional SDS-PAGE
, (10% separating gel and 5% stacking gel) was carried out in a mini
vertical system. For this 100 μg of protein was loaded in each sample
well along with 10 μl sample buffer containing bromophenol blue as
tracking dye. A medium range marker was also incorporated into the
gel to determine the molecular weight of the bands.
The gels were run at a constant voltage of 100 V for 3 hrs followed
by staining in coomassie brilliant blue  over night. Relative mobility
(Rm) of the protein bands was determined and Zymograms were
constructed. The gel was photographed and stored in 3% acetic acid.
Results and Discussions
The total protein content in the crude extract among the studied
healthy and gall stages ranged from 1.8 mg/gm (6th stage) to 3.1 mg/gm
(3rd stage) fresh weight of healthy and gall tissue of leaf (Table 1).
Table 1:Total Protein content in leaf tissue of different leaf gall stages of A. scholaris.
SDS protein profile
SDS denatured protein gels could resolve a total of 29 bands in
6 samples of different stages of leaf gall with a sample of healthy leaf
which were grouped as 5 distinct SDS protein bands. These SDS protein
bands belong to different molecular weight ranging from 44 kDa to 97
kDa. The relative mobility of the bands varied from .23 to .53 in studied
stages. Low, medium and high mobility bands were observed in all the
cases. One polypeptide band exhibiting relative mobility of .53 representing
MW 44 kDa was present in all the stages. Healthy, Ist, 3rd and 4th
stages exhibited maximum number of bands i.e. all the five bands were
visible, followed by 2nd, 5th and 6th stages which showed 4, 4 and 1 band
respectively. A low molecular weight polypeptide band of medium to
light intensity with Rm 0.53 (MW 44 kDa) was unique to all gall stages
and healthy one. 3rd stage can be differentiated by all other stages due to
presence of a strong dark protein band of approximately 97 kDa. 2nd,
5th and 6th stage could be distinguished from healthy, 1st, 3rd and 4th stage
by the absence of a protein band of 97 kDa. Variability of protein bands
was well expressed in the middle of the gel. The bands in the lower side
of the gel were mostly common to all stages. Similarly the protein subunit
of 97 kDa was not resolved in 2nd, 5th and 6th stage, which helped in
differentiating these stages from the rest. The presence versus absence
type of polymorphism of SDS protein and these varied intensities was
revealed through this study (Figure 2).
Figure 2:Analysis of total protein content on SDS-PAGE (10 %) showing the
variable protein levels at different stages of leaf gall formation in A. scholaris (* Showing differences in protein levels at early and older stages).
Electrophoresis of proteins has been successfully used for the characterization
of different taxonomic, evolutionary and genetic relationship
In the present study, the electrophoratic banding profile of total
soluble proteins of 6 stages of leaf gall of A. scholaris exhibited presence
versus absence type of polymorphism, reflecting thereby, and differential
synthesis of proteins in the gall at different stages  (Table 2). The
present investigation on SDS denatured proteins showed differences in
number of bands, band width and intensity among different stages of
leaf galls of A. scholaris. In initial stages of gall the proteins are showing
the same banding pattern in the zymogram but in mature stages some
extra dark bands are visible, in older stages the number of bands becomes
reduced and in last stage only one band is visible  (Figure 3).
Table 2:SDS-PAGE Protein Profile of Different s of Leaf Gall of A. scholaris
Figure 3:Zymogram of SDS-PAGE protein profile of different stages of leaf
gall of A. scholaris.
The present findings confirm the presence of polypeptide bands of
heterogenous molecular weight and varying intensity in A. scholaris at
different leaf gall stages while undergoing the biotic stress. The unburst galled tissue showed almost two fold increases in the protein content.
The protein content showed an initial increase and registered the highest
level during the young galled stage of their development and declined
thereafter in the mature burst galled tissue where in the insect
had already exited out from the chamber . Synthesis of diverse plant
proteins are believed to be important in defense mechanism .
It can be concluded from the above study that due to interaction
between insect and plant tissue certain physiological and biochemical
changes occur which lead to hypertrophy and hyperplasia and gall formation
takes place. Generation of number of cells requires high amount
of protein so the young and mature gall tissue shows high difference in
protein concentration as compared to the normal leaf tissue.
This study confirms that when plant is attacked by the pathogens,
they inject some elicitors and lead to the synthesis of different type of
enzymes and some specific proteins at high amount which is a response
of plant against the biotic stress to overcome with it . Insects trigger
the defense mechanism of the plant which results the gall formation due
to initiation of some biochemical reactions and physiological activities.
Authors are grateful to Ms. Vipula Sharma for her support in compilation of
results and writing the manuscript.
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