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Summary of work done so far details
Post-Doctoral Research
Currently working as, a post-doc fellow in the Nam’s laboratory of complex biology, with
Professor Hong Gil Nam an eminent scientist, working in the area of plant senescence & aging
since long time. The lab research work has been published in “Science” and “Nature” thus
working in this lab is a great experience to think and to work in a new direction.
As a post-doctoral researcher I am currently engaged in a project to find out the role of a
plasma membrane LRR receptor like kinase (RPK1) in oxidative stress signaling during
senescence in Arabidopsis thaliana” using Molecular biology and Genetic Engineering
approaches. Our experimental results show delayed leaf senescence phenotype of the knock-out
mutants in comparison to WT indicating that RPK1 can function as a positive regulator of agedependent PCD. However, the data for ROS generation and accumulation suggests its role in
O2˙¯ generation during age-dependent PCD. In line, our further study show that RPK1 is
responsible for the generation of O2˙¯ at the plasma membrane, moreover, a critical observation
revealed formation of numerous vesicles like patches of O2˙¯ at the plasma membrane and also a
few in the cytoplasm in WT but not in rpk1 mutants, which seems to be a novel exciting cellular
mechanism mediated via RPK1. Hence, with the above interesting findings I am trying very
keenly to find out the downstream target molecules of RPK1 in ROS signaling pathway.
Along with this I am also working in another collaborative project on NAC transcription factor
regulatory network in Arabidopsis.
I hope these findings will soon be appear in a good impact reputed journals as the elucidation of
molecular regulatory mechanisms underlying plant senescence in the model plant Arabidopsis
thaliana will be of great significance.
Major research findings during Ph.D. and significance of the scientific contribution
A. First time demonstrated that plant cell wall bound Peroxidases have their role in cadmium
induced oxidative stress in Brassica juncea. Ionically Bound Cell Wall Peroxidases
(IBCWP) have their role in cadmium induced oxidative stress tolerance (Published in
Plant Cell Reports, 2008). After intensive investigation I found that IBCWP are age
specific and organ specific but not heavy metal specific.
B. Nitric oxide plays an important role in regulation of antioxidant system in Brassica
juncea during Cd induced oxidative stress. NO acts as an antioxidant and have its role in
cadmium tolerance in Brassica. As NO donor SNP decreased the level of antioxidants
induced in the presence of cadmium stress, however, in Brassica pretreated with NO
scavenger cPTIO the level of antioxidants again increased in comparison to SNP+Cd
treated plants demonstrating its role in metal tolerance (Revision under JEEB).
C. Brassica juncea has an enormous potential of stripping cadmium from heavy metal
contaminated areas. Studies were made to explore the antioxidant defense system of
Brassica juncea during Cd stress. By understanding the physiology and biochemistry of
Brassica at the level of antioxidant network and its antioxidant defense system under
heavy metal stress make Brassica sp. useful for phytoremediation processes in the areas
contaminated with heavy metals.
D. Brassica juncea can tolerate high Cd toxicity as it accumulates large amounts of Cd in its
harvestable parts but Cd causes oxidative stress in Brassica and reduces overall growth.
Studied the cadmium accumulation and toxicity in the Brassica juncea by determining
the intracellular metal accumulation (absorption) from hydroponic culture medium.
Measured reactive oxygen species level and oxidative stress markers along with
physiological growth parameters in Brassica juncea under Cd stress (Published in
Protoplasma).
Project Proposal:
Elucidation of Cadmium Induced Oxidative Stress Signaling Mechanism Mediated via Cell
Wall Bound Peroxidases in Arabidopsis thaliana
Origin of the proposal
During literature survey on cell wall proteins and cell wall enzymes mainly on cell wall bound
peroxidases (CWBPs), I found that for the very first time Bacon et al. (1997) reported that cell
wall peroxidase activity accounts for the observed reduction in leaf expansion rates during
drought stress. Since then, a very few studies has been conducted on the potential roles of CWPs
in other plant stress responses mainly in heavy metal stress responses. Bound to this curiosity, in
my doctoral work I investigated that ionically bound cell wall peroxidases induced during
cadmium (Cd2+) induced oxidative stress in Brassica juncea (Verma et al., 2008). However, at
that time due to lack of good research facilities we were unable to investigate its role at
molecular level to explain specific cell wall peroxidase functions in ROS signaling mechanism
induced via Cd. It has already been reported that plant metabolism must be highly regulated in
order to allow effective integration of a diverse spectrum of biosynthetic pathways that are
reductive in nature. This regulation does not completely avoid photodynamic or reductive
activation of molecular oxygen to produce reactive oxygen species (ROS) particularly
superoxide, H2O2 and singlet oxygen (Foyer and Noctor 2005). Plant cells produce ROS,
particularly superoxide and H2O2 as second messengers to mediate ROS signaling in many
processes associated with plant growth and development (Mittler et al., 2011). Cd triggers
common defense pathways in plant cells like other biotic or abiotic environmental stresses. A
joint initial event of these pathways is an accumulation of H2O2, which acts as a substrate in
cross-linking reactions catalyzed by cell wall peroxidases. Choi et al. (2007) reported that
extracellular peroxidases are involved in H2O2 generation during pathogen response; however,
the mechanism underlying is still unclear and to the best of my knowledge there are no reports
on ROS signaling pathway mediated via CWPs in Arabidopsis thaliana under Cd stress.
Thus, it would be of great interest to hypothesize that how these CWPs, alone or in combination
with other interaction partners that are responsible for ROS generation and stress tolerance,
mediate this oxidative stress signaling pathway in Arabidopsis thaliana, a model plant for
research under different intensities of Cd. The study would be scientifically significant in relation
to detailed understanding of CWPs role in ROS signaling, plant heavy metal tolerance and
phytoremediation aspects.
Objectives of the study
1. Collection and selection of CWP genes and ordering knock out lines from Arabidopsis data
base (TAIR).
2. To study phenotype, physiological growth, ROS generation and signaling in WT and cwp
mutant lines in control and Cd treated plants
3. To study the CWP localization, gene and protein expression and enzyme activity under Cd
induced oxidative stress in Arabidopsis thaliana and its relationship with antioxidant
regulatory network
4. To study CWP role in Cd induced ROS signaling via its over-expression
Brief outline of methodology to be adopted
For the fulfillment of above objectives, following methodology will be adopted:
For the fulfillment of the first objective the data on CWPs will be collected regarding previous
studies and reports and selection of CWP genes will be done keeping in mind its functional role
in ROS generation. After selection of CWP genes of our interest knock out lines will be ordered
from TAIR/INRA for the further study.
For second objective to select working concentrations of Cd non-lethal, sub-lethal and lethal
concentrations of Cd for Arabidopsis thaliana will be screened via analyzing growth parameters.
Phenotype (morphological changes in plants) and physiological growth parameters (fresh weight,
root length, cell death, Ion leakage, chlorophyll content and Photosynthetic efficiency) will be
studies in WT and cwp mutant lines in control and Cd treated plants. To investigate CWP role in
ROS signaling ROS generation (O2˙¯ and H2O2 level) will be determined biochemically as well
as localization studies using NBT and DAB staining for O2˙¯ and H2O2 respectively. Oxidative
stress caused via Cd will be studied via lipid peroxidation measurement in WT and cwp
knockouts.
For third objective, to check CWP gene expression total RNA will be isolated from different
parts of control and Cd treated plants. cDNA will be synthesized. CWP gene specific primers
will be designed and ordered. Expression analysis will be done via qRT-PCR. For protein
expression and CWP activity analysis SDS-PAGE and native-PAGE will be done. To study
CWP localization, CWP gene cloning will be done in a suitable gateway vector (e.g. pCRCCDF)
and finally in destination vector (e.g. gCsVMV-eGFP-N-999) and transient expression of this
CWP in vivo will be visualized under fluorescence microscope. To study antioxidant regulatory
network relationship with CWP, non-enzymatic (GSH, NPT, proline) and enzymatic antioxidants
(SOD, CAT, APX) will be studied in the same above lines.
For the fulfillment of the fourth objective, to confirm CWP role in Cd induced ROS signaling,
CWP over-expressing lines will be generated. Agrobacterium mediated transformation technique
will be used for the generation of transgenic lines over-expressing specific CWP. Confirmation
of the above study will be done in these lines by examining the effect of exogenously applied
inhibitors of CWP on the O2˙¯, H2O2 generation, lipid peroxidation and CWP activities
Significance of the proposal in the context of current status in the field
The contamination of water, soil, and sediments with toxic metals has been and will continue to
be a major environmental problem that needs to be dealt with. In more recent years, the studies
with metals have concentrated on the antioxidant stress response characterization in a wide range
of plant species. The effects of many different metals have been published, but all have
consistently addressed very similar problems and investigated basic parameters. These kinds of
studies are important as they allow the verification of the sensitivities of different plant species to
distinct metals, eventually indicating specific biomarkers to stressful situations. However, metalinduced stress studies need new approaches that are likely to increase our understanding as how
these elements affect plant metabolism, signaling pathways and to identify the modifications that
are needed to improve plant adaptation and tolerance.
Now days, scientists trying to explore novel molecules in oxidative stress induced ROS
signaling. Recent studies in the area of extracellular peroxidases reveals that it can induce H2O2
production and can perform a significant function in responses to various environmental stresses
via the regulation of ROS in plants. In tobacco plants over-expression of sweet potato swpa4
peroxidase results in increased H2O2 production and enhances stress tolerance in tobacco plants
functioning as a positive defense signal in the H2O2-regulated stress response signaling pathway
(Kim et al. 2008) A capsicum extracellular peroxidase (CaPO2) is involved in ROS generation,
both locally and systemically, to activate cell death and PR gene induction during the defense
response to pathogen invasion (Choi et al. 2007). Pshenichnov et al. (2011) reported that specific
chitin-binding isozymes of peroxidase play an important role in pathogenesis of plant diseases
caused by fungi and suggested potential utilization of chitin binding peroxidases as a
biochemical tool to guide breeding programs to increase resistance to V. dahliae. However there
are only a few reports explaining the role of these extracellular peroxidases in plants under heavy
metal stress. Since Cd toxicity limits the crop productivity in economically important crops by
inducing oxidative stress leading to cell death. In order to find out the solution of this problem,
mechanism of Cd toxicity and its detoxification mechanisms are needed to be elucidated. In line,
it becomes very important to explore the role of these CWPs in plants under metal stress
conditions. Present study would increase our knowledge to understand heavy metal stress
induced signaling pathway and metabolic adaptation mechanisms in plants with novel insight.
The outcome of the present study may find its application in manipulation of plants to develop
better heavy metal-tolerant high biomass yielding crops for use in phytoremediation of metal
contaminated soils and will come up with new suggestions and a detail insight of molecular,
physiological and biochemical role of cell wall peroxidases in Cd induced oxidative stress and its
detoxification mechanisms at cellular level. Moreover, taking Arabidopsis thaliana as a model
plant to investigate CWPs role in ROS signaling induced via Cd is of significance because it is a
powerful model system for plant cell wall Research (Liepman et al. 2010) This kind of study
need the availability of the genome sequence and the tools for functional analysis that are
available in Arabidopsis. Furthermore, knowledge gained from these kind of studies will have
important practical applications, including enabling the engineering of plant tailored for various
uses under stressful environments.
Prospects of this work for extending it as a long-term project
As many more discoveries are needed to increase our understanding of cell wall mediated
processes in heavy metal induced oxidative stress signaling and the functional roles of various
cell-wall components, this study will be continued further depending on the above research
outcomes. In line of the above study it would be of great significance to answer the further
questions like
What may be the direct or indirect targets of the CWPs in ROS signaling pathway?
How CWPs mediate ROS signaling via integrating with other signaling mechanisms/pathways?
As there are 73 genes of class III peroxidases in Arabidopsis and among these around 6 genes are
specific for cell wall. Thus, there is a high possibility of peroxidase functional redundancy in the
cell. To overcome this problem this study will further need to generate CWP double or triple
mutants which is a time taking process and need more than 3 years for the complete study.
Details of Professional Training and Research Experience
1. Professional Training Experience
Name of Supervisor
Period
and Address of the
Training details
From
To
Institution
Dr. S.K. Raj (scientist
F), Molecular Plant
Virology Lab, National
Botanical Research
Institute (NBRI),
Summer Training Project entitled:
“Molecular detection of potyvirus,
01.06.2005
30.06.2005
01.01.2006
30.04.2006
infecting cardamom plant”
Lucknow, India
Dr. Nilima Kumari
(Associate Professor),
Banasthali University,
Banasthali, Rajasthan,
India
Dissertation project entitled:
“Photosynthetic performance and
certain biochemical changes in
Brassica juncea (L.) cv Varuna
under abiotic stress”
2. Research Experience
Post
Institution
Period
From
Pohang University of
Post-Doctoral
Science & Technology
Fellow
(POSTECH), Pohang,
July 1, 2011
Fellow of CSIR
India
June 30,
2012
Banasthali University,
Banasthali, Rajasthan,
To
Cont….till
South Korea
Senior Research
Total
April 1, 2009
March 31,
2011
experience
Will be of 1
year
2 years
3. Teaching Experience
Name and address
of the employer
Banasthali
University,
Banasthali, Rajasthan,
India
Period
Level/Students
Post graduate
and Under
graduate
From
Total
To
Course Taught
experience
Biostatistics subject
July 3,
Dec. 31,
classes and
2008
2008
Biotechnology practical
classes
½ Year
Teaching Plan for Pre-doctoral, Postgraduate and Undergraduate programs
To maintain standards for teaching and research, I would like to involve in courses related to my
area of specialization i.e. plant sciences (plant physiology, plant biochemistry and plant
molecular biology). Teaching plan in pre-doctoral will include lessons creating awareness for
novel and applied research, journal clubs, students research progress presentations and
organization of seminars. However, for undergraduate and postgraduate teaching I would like to
clear basic subject fundamentals and improve students overall personality. Including objective, I
will consider best author subject texts, materials required including scientific figures & models
explaining the related subject, session for question-answers or discussions, class tests and
assignments and some space for independent practice.