The study revealed that the proteins seem similar in structure but functionally they are much more diverse. This analysis can help to identify the molecular basis of phenotypic differences and select gene expression targets for in-depth study. The regulation of gene expression in plants, as in other higher eukaryotes, is a subject of daunting complexity.

1. RICE STRESS RELATED GENE EXPRESSION
ANALYSIS
Dissertation submitted to the Department of Biotechnology
In the fulfilment of the requirement for the Degree of
Masters of Science (M. Sc.)In Biotechnology 2018-2020
By
Ron Hazarika
Enrollment number: A91700218003
Under the Guidance of: Dr. Chittabrata Mal
Assistant Professor
Amity Institute of Biotechnology
Amity University Kolkata

2. 2
DECLARATION
This is to declare that the project report entitled “Rice stress related gene expression
analysis” for the total fulfilment of M.Sc. Biotechnology 4th
semester, 2020 is a bonafide
record of work which is carried out by Ron Hazarika, student of M.Sc. Biotechnology (4th
semester) bearing Enrollment no.- A91700218003 under my guidance and supervision. The
results that have been presented here have not been published elsewhere by him.
Date-
Dr. Chittabrata Mal
Assistant Professor
Amity Institute of Biotechnology
Amity University Kolkata

3. 3
FACULTY GUIDE APPROVAL

4. 4
ACKNOWLEDGEMENT
It is my privilege to express my deep sense of gratitude to my guide, Dr.Chittabrata
Mal, Faculty of Amity Institute of Biotechnology, Amity University Kolkata for his valuable
guidance & support at each and every stage of my project work.
I express my sincere thanks to Dr.Swatilekha Ghosh, for her efficient supervision,
help and support during the course.
Last but not the least; I would like to offer special thanks to my friends for immense
help and support throughout the work.
Ron Hazarika

5. 5
CONTENTS
1. Introduction
1.1 Plant stress and its cause.
1.2 Plants taking in consideration.
1.3 Abiotic stress.
1.4 Biotic Stress.
2. Materials and methods.
3.Results and discussion.
4.Conclusion.
5.References.

6. 6
ABSTRACT
Gene expression analysis involves the determination of the pattern of genes
expressed at the level of genetic transcription, under specific circumstances or in a specific
cell. The measurement of gene expression is a critical tool employed across drug discovery,
life science research and the optimization of bio production. Various structural properties,
functional importance, phylogeny and expression pattern of all Stress Proteins were
determined using various bioinformatics tools. Most of the proteins are stable in the
cellular environment with a prominent expression in the extracellular region and plasma
membrane. Structurally, these proteinsare similar but functionally they are diverse with
novel enzymatic activities of oxalate decarboxylase, lyase, peroxidase, and oxidoreductase.

7. 7
1. INTRODUCTION
In recent years, the world has experienced significant challenges from Mother
Nature. Tragic wildfires, severe droughts, heavy rains, massive flooding, hurricanes, and
more have wreaked havoc throughout the states. These environmental threats have ruined
crops, harmed livestock, destroyed vegetations and even normal ecosystem, but especially
those in key agricultural regions.Lately, these natural disasters have taken the news stage
due to their intensity and frequency and represent the impacts of a changing climate.
Farmers around the world often bear the brunt of these disasters and feel the impact of
climate change especially close to home and in their business bank accounts.External
environmental impacts like those plaguing news headlines as of late pose significant risks to
plant and crop health and often stress plants beyond their tolerance limits and can lead to
diminished marketable yields. Natural disasters are an obvious cause of plant stress, even
to the naked eye. But, did you ever realize that plant stress comes in many other forms,
some even invisible to the naked eye.
1.1 Plant Stress and its causes
Plant stress is a state where a plant is growing in non-ideal growth conditions and has
increased demands put on it. Plant stress refers to any unfavourable condition or substance
that affects a plant’s metabolism, reproduction, root development, or growth. Plant stress
can come in different forms and durations. Some plant stressors are naturally occurring,
like drought or wind, while others may be the result of human activity, like over irrigation or
root disturbance.
Plant stress is caused by a variety of factors, some of which are obvious (like natural
disasters), while others occur on a micro scale in the soil. Recent natural disasters represent
one type of plant stress factors, called abiotic factors, which usually occur above ground. A
second type of plant stress factors are called biotic factors, which mostly occur
underground, and can cause plant stress through pathogens and pests.

8. 8
Abiotic stresses originate from the surrounding environment of the plant. One of the
most important abiotic factors affecting plants is water stress. A plant requires a certain
amount of water for optimal growth, too much water can cause plant cells to swell and
burst, whereas too little water can lead to desiccation. Temperature stresses can also
negatively impact a plants growth and livelihood. Cold weather may affect the amount and
rate of uptake of water and nutrients, and hot weather can affect the permeability of plant
membranes. Abiotic factors come in other forms as well such as wind, toxins, and light.
Biotic stresses can cause damage to plants through living organisms that may cause
disease. In agriculture, biotic stresses are most often responsible for pre or post-harvest
losses. Soil is filled with fungi and bacteria – 1 teaspoon of soil can hold billions of
microorganisms. Just like microorganisms found in humans, some can be beneficial, and
others can be detrimental. Healthy soil biological systems showcase an appropriate,
harmonious balance between beneficial microorganisms that protect against biotic stresses
and detrimental microorganisms – that if not held in check can result in biotic plant
stresses. Examples of common biotic plant stress factors include pathogens, insects, and
weeds but the exact types of factors depend on the environment and differ from region to
region.
1.2 About the plants
Rice is the seed of the grass species Oryza glaberrima (African rice) or Oryza sativa (Asian
rice). As a cereal grain, it is the most widely consumed staple food for a large part of the
world's human population, especially in Asia. It is the agricultural commodity with the third-
highest worldwide production (rice, 741.5 million tones in 2014), after sugarcane (1.9 billion
tones) and maize (1.0 billion tones).
Since sizable portions of sugarcane and maize crops are used for purposes other than
human consumption, rice is the most important grain with regard to human nutrition and

9. 9
caloric intake, providing more than one-fifth of the calories consumed worldwide by
humans. There are many varieties of rice and culinary preferences tend to vary regionally.
Rice, a monocot, is normally grown as an annual plant, although in tropical areas it can
survive as a perennial and can produce a ration crop for up to 30 years. Rice cultivation is
well-suited to countries and regions with low labor costs and high rainfall, as it is labor-
intensive to cultivate and requires ample water. However, rice can be grown practically
anywhere, even on a steep hill or mountain area with the use of water-controlling terrace
systems. Although its parent species are native to Asia and certain parts of Africa, centuries
of trade and exportation have made it commonplace in many cultures worldwide.
Figure 1. Rice Plant (Oryza sativa )
The traditional method for cultivating rice is flooding the fields while, or after, setting the
young seedlings. This simple method requires sound planning and servicing of the water
damming and channeling, but reduces the growth of less robust weed and pest plants that
have no submerged growth state, and deters vermin. While flooding is not mandatory for
the cultivation of rice, all other methods of irrigation require higher effort in weed and pest
control during growth periods and a different approach for fertilizing the soil.

10. 10
The name wild rice is usually used for species of the genera Zizania and Porteresia, both
wild and domesticated, although the term may also be used for primitive or uncultivated
varieties of Oryza.
The rice plant can grow to 1–1.8 m (3.3–5.9 ft) tall, occasionally more depending on the
variety and soil fertility. It has long, slender leaves 50–100 cm (20–39 in) long and 2–2.5 cm
(0.79–0.98 in) broad. The small wind-pollinated flowers are produced in a branched arching
to pendulous inflorescence 30–50 cm (12–20 in) long. The edible seed is a grain (caryopsis)
5–12 mm (0.20–0.47 in) long and 2–3 mm (0.079–0.118 in) thick.
High-yielding varieties
The high-yielding varieties are a group of crops created intentionally during the Green
Revolution to increase global food production. This project enabled labor markets in Asia to
shift away from agriculture, and into industrial sectors. The first "Rice Car", IR8 was
produced in 1966 at the International Rice Research Institute which is based in the
Philippines at the University of the Philippines' Los Baños site. IR8 was created through a
cross between an Indonesian variety named "Peta" and a Chinese variety named "Dee Geo
Woo Gen."
Scientists have identified and cloned many genes involved in the gibberellin signaling
pathway, including GAI1 (Gibberellin Insensitive) and SLR1 (Slender Rice). Disruption
of gibberellin signaling can lead to significantly reduced stem growth leading to a dwarf
phenotype. Photosynthetic investment in the stem is reduced dramatically as the shorter
plants are inherently more stable mechanically. Assimilates become redirected to grain
production, amplifying in particular the effect of chemical fertilizers on commercial yield. In
the presence of nitrogen fertilizers, and intensive crop management, these varieties
increase their yield two to three times.
Future potential
As the UN Millennium Development project seeks to spread global economic development
to Africa, the "Green Revolution" is cited as the model for economic development. With the

11. 11
intent of replicating the successful Asian boom in agronomic productivity, groups like
the Earth Institute are doing research on African agricultural systems, hoping to increase
productivity. An important way this can happen is the production of "New Rices for Africa"
(NERICA). These rices, selected to tolerate the low input and harsh growing conditions of
African agriculture, are produced by the African Rice Center, and billed as technology "from
Africa, for Africa". The NERICA have appeared in The New York Times (October 10, 2007)
and International Herald Tribune (October 9, 2007), trumpeted as miracle crops that will
dramatically increase rice yield in Africa and enable an economic resurgence. Ongoing
research in China to develop perennial rice could result in enhanced sustainability and food
security.
Golden rice
Rice kernels do not contain vitamin A, so people who obtain most of their calories from rice
are at risk of vitamin A deficiency. German and Swiss researchers have genetically
engineered rice to produce beta-carotene, the precursor to vitamin A, in the rice kernel.
The beta-carotene turns the processed (white) rice a "gold" color, hence the name "golden
rice." The beta-carotene is converted to vitamin A in humans who consume the
rice. Although some rice strains produce beta-carotene in the hull, no non-genetically
engineered strains have been found that produce beta-carotene in the kernel, despite the
testing of thousands of strains. Additional efforts are being made to improve the quantity
and quality of other nutrients in golden rice.
The International Rice Research Institute is currently further developing and evaluating
Golden Rice as a potential new way to help address vitamin A deficiency.
Expression of human proteins
Ventria Bioscience has genetically modified rice to express lactoferrin, lysozyme which
are proteins usually found in breast milk, and human serum albumin, These proteins
have antiviral, antibacterial, and antifungal effects.

12. 12
Rice containing these added proteins can be used as a component in oral rehydration
solutions which are used to treat diarrheal diseases, thereby shortening their duration and
reducing recurrence. Such supplements may also help reverse anemia.
Flood-tolerant rice
Due to the varying levels that water can reach in regions of cultivation, flood tolerant
varieties have long been developed and used. Flooding is an issue that many rice growers
face, especially in South and South East Asia where flooding annually affects 20 million
hectares. Standard rice varieties cannot withstand stagnant flooding of more than about a
week, mainly as it disallows the plant access to necessary requirements such as sunlight
and essential gas exchanges, inevitably leading to plants being unable to recover. In the
past, this has led to massive losses in yields, such as in the Philippines, where in 2006; rice
crops worth $65 million were lost to flooding. Recently developed cultivars seek to improve
flood tolerance.
Drought-tolerant rice
Drought represents a significant environmental stress for rice production, with 19–
23 million hectares of rained rice production in South and South East Asia often at
risk. Under drought conditions, without sufficient water to afford them the ability to obtain
the required levels of nutrients from the soil, conventional commercial rice varieties can be
severely affected—for example, yield losses as high as 40% have affected some parts of
India, with resulting losses of around US$800 million annually.
The International Rice Research Institute conducts research into developing drought-
tolerant rice varieties, including the varieties 5411 and Sookha dhan, currently being
employed by farmers in the Philippines and Nepal respectively. In addition, in 2013 the
Japanese National Institute for Agrobiological Sciences led a team which successfully
inserted the DEEPER ROOTING 1 (DRO1) gene, from the Philippine upland rice variety
Kinandang Patong, into the popular commercial rice variety IR64, giving rise to a far deeper
root system in the resulting plants. This facilitates an improved ability for the rice plant to

13. 13
derive its required nutrients in times of drought via accessing deeper layers of soil, a
feature demonstrated by trials which saw the IR64 + DRO1 rice yields drop by 10% under
moderate drought conditions, compared to 60% for the unmodified IR64 variety.
Salt-tolerant rice
Soil salinity poses a major threat to rice crop productivity, particularly along low-lying
coastal areas during the dry season. For example, roughly 1 million hectares of the coastal
areas of Bangladesh are affected by saline soils. These high concentrations of salt can
severely affect rice plants' normal physiology, especially during early stages of growth, and
as such farmers are often forced to abandon these otherwise potentially usable areas.
Progress has been made, however, in developing rice varieties capable of tolerating such
conditions; the hybrid created from the cross between the commercial rice variety IR56 and
the wild rice species Oryza coarctata is one example. O. coarctata is capable of successful
growth in soils with double the limit of salinity of normal varieties, but lacks the ability to
produce edible rice. Developed by the International Rice Research Institute,
the hybrid variety can utilise specialised leaf glands that allow for the removal of salt into
the atmosphere. It was initially produced from one successful embryo out of 34,000 crosses
between the two species; this was then backcrossed to IR56 with the aim of preserving the
genes responsible for salt tolerances that were inherited from O. coarctata.]
Extensive trials
are planned prior to the new variety being available to farmers by approximately 2017–18.

14. 14
1.3 ABIOTIC STRESS
Abiotic stress is the negative impact of non-living factors on the living organisms in a
specific environment. The non-living variable must influence the environment beyond its
normal range of variation to adversely affect the population performance or individual
physiology of the organism in a significant way.
Whereas a biotic stress would include living disturbances such as fungi or harmful insects,
abiotic stress factors, or stressors, are naturally occurring, often intangible and inanimate
factors such as intense sunlight, temperature or wind that may cause harm to the plants
and animals in the area affected. Abiotic stress is essentially unavoidable. Abiotic stress
affects animals, but plants are especially dependent, if not solely dependent, on
environmental factors, so it is particularly constraining. Abiotic stress is the most harmful
factor concerning the growth and productivity of crops worldwide. Research has also shown
that abiotic stressors are at their most harmful when they occur together, in combinations
of abiotic stress factors.
Stress Consequences Plant Responses
Heat stress
High temperature
leads to high
evaporation and water
deficit. The
consequent increased
turnover of enzymes
leads to plant death.
Efficient protein repair
systems and general
protein stability support
survival, temperature
can lead to acclimation.

15. 15
Chilling and cold stress
Biochemical reactions
proceed at slower rate,
photosynthesis
proceeds, carbon
dioxide fixation lags,
leading to oxygen
radical damage.
Indeed, freezing lead
to ice crystal formation
that can distrupt cells
membranes.
Cessation of growth in
adaptable species may
be overcome by changes
in metabolism. Ice
crystal formation can be
prevented by osmolyte
accumulation and
synthesis of hydrophilic
proteins.
Drought
Inability to water
transport to leaves
leads to
photosynthesis
declines.
Leaf rolling and other
morphological
adaptations. Stoma
closure reduces
evaporative
transpiration induced by
ABA. Accumulation of
metabolities,
consequently lower
internal water potential
and water attracting

16. 16
Flooding and
submergence
Generates anoxic or
micro aerobic
conditions
Interfering with
mitochondrial
respiration.
Development of cavities
mostly in the roots that
facilitate the exchange
of Oxygen and ethylene
between shoot and root
(aerenchyma).
Heavy metal
accumulation and
metal stress
In excess,
detoxification
reactions may be
insufficient or storage
capacity may exceed.
Excess of metal ions may
be countered by export
or vacuolar deposition
but metal ions may also
generate oxygen
radicals.
High light stress
Excess light can lead to
increased production
of highly reactive
intermediates and by-
products that can
potentially cause
photo-oxidative
damageand inhibit
photosynthesis
Exposure of a plant to
light exceeding what is
utilized in
photochemistry leads to
inactivation of
photosynthetic functions
and the production of
reactive oxygen species
(ROS). The effects of
these ROS can be the
oxidation of lipids,
proteins, and enzymes
necessary for the proper
functioning of the
chloroplast and the cell
as a whole.

17. 17
1.31 Abiotic stress-inducible genes
The complex plant response to abiotic stress involves many genes and biochemical
molecular mechanisms. The analyze of the functions of stress-inducible genes is an
important tool not only to understand the molecular mechanisms of stress tolerance and
the responses of higher plants, but also to improve the stress tolerance of crops by gene
manipulation. Hundreds of genes are thought to be involved in abiotic stress responses.
Many drought-inducible genes are also induced by salt stress and cold, which suggests the
existence of similar mechanisms of stress responses.
These genes are classified into three major groups:
1.Those that encode products that directly protect plant cells against stresses such as heat
stress proteins (HSPs) or chaperones, LEA proteins, osmo protectants, antifreeze proteins,
detoxification enzymes and free-radical scavengers.
2.Those that are involved in signalling cascades and in transcriptional control, such as
Mitogen-activated protein kinase (MAPK),Calcium-dependent protein kinase (CDPK) and
SOS kinase, phospholipases and transcriptional factors.
3. Those that are involved in water and ion uptake and transport such as aquaporins and
ion transporters.
1.4 BIOTIC STRESS
Biotic stress is stress that occurs as a result of damage done to an organism by other
living organisms, such as bacteria, viruses, fungi, parasites, beneficial and harmful
insects, weeds, and cultivated or native plants. It is different from abiotic stress, which is
the negative impact of non-living factors on the organisms such as temperature, sunlight,
wind, salinity, flooding and drought. The types of biotic stresses imposed on an organism
depend the climate where it lives as well as the species' ability to resist particular stresses.
Biotic stress remains a broadly defined term and those who study it face many challenges,

18. 18
such as the greater difficulty in controlling biotic stresses in an experimental context
compared to abiotic stress.
The damage caused by these various living and nonliving agents can appear very
similar. Even with close observation, accurate diagnosis can be difficult. For
example, browning of leaves on an oak tree caused by drought stress may appear similar to
leaf browning caused by oak wilt, a serious vascular disease caused by a fungus, or the
browning caused by anthracnose, a fairly minor leaf disease.
Biotic stress in plants is caused by living organisms, specifically viruses, bacteria,
fungi, nematodes, insects, arachnids, and weeds. In contrast to abiotic stress caused by
environmental factors such as drought and heat, biotic stress agents directly deprive their
host of its nutrients leading to reduced plant vigor and, in extreme cases, death of the host
plant. In agriculture, biotic stress is a major cause of pre- and postharvest losses.
In contrast to vertebrates, plants lack an adaptive immune system, or the ability to adapt to
new diseases and memorize past infections. Though lacking an adaptive immune system,
plants have evolved a plethora of sophisticated strategies to counteract biotic stresses. The
genetic basis of these defense mechanisms is stored in the plant's genetic code. Plant
genomes encode hundreds of biotic stress resistance genes. With the completion of several
plant genome sequences during the past decade – among them are important agricultural
crops such as maize, sorghum, and rice – we obtained a first glimpse into the wealth of
biotic stress resistance genes encoded within plant genomes. However, we have just
started to uncover the molecular mechanisms and networks controlling biotic stress
resistance in cereals.
Biotic stress which is often called decay is caused by infectious diseases that develop in
harvested fruit and is usually caused by bacteria, fungi, or yeasts. Plants respond to biotic
stress through a defense system. The defense mechanism is classified as an innate and
systemic response. After infection, reactive oxygen species (ROS) are generated and
oxidative bursts limit pathogen spread (Atkinson and Urwin, 2012). Also, in response to

19. 19
pathogen attack, plants increase cell lignification. This mechanism blocks invasion of
parasites and reduces host susceptibility.
The defenses to biotic stress include morphological and structural barriers, chemical
compounds, and proteins and enzymes. These confer tolerance or resistance to biotic
stresses by protecting products and by giving them strength and rigidity. The resistance to
biotic stress can be induced through specific chemical compounds such as β-aminobutyric
acid (BABA) or benzothiadiazole (BTH).
Plant hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene play central roles in
biotic stress signaling. Several transcription factors (TFs) are mediators in multiple hormone
signaling. Plant defenses against biotic stresses involve numerous signal
transduction pathways. Abscisic acid (ABA) is reflected as the main hormone involved in the
perception of many abiotic stresses (Cramer et al., 2011). However, ABA has a positive
effect on biotic stress resistance (Rejeb et al., 2014). Under abiotic and biotic stress, ABA
acts antagonistically with ethylene, which induces liability of the plant against disease
attack. However, under abiotic stress ABA increases and induces stomatal closure. As a
result, the entry of biotic attackers through stomata is prevented. Therefore, under such
situations, the plant is protected from abiotic and biotic stress (Rejeb et al., 2014). Kinase
protein signals also interact with ROS and ABA leads to plant defense enhancement (Rejeb
et al., 2014).
Pathogenesis-related (PR) proteins are critical for plant resistance against pathogens and
when plants are attacked; their expression is strongly upregulated. It is suggested that with
an increase in ABA expression of specific TFs like C-repeat binding factors (CBFs), and cup-
shaped cotyledon mediated by ABA could be enhanced, which induces upregulation of PR
genes (Rejeb et al., 2014).

20. 20
2. MATERIALS AND METHODS
2.1. Selection of stress response proteins
All the proteins responsible for regulation of heat, drought and salinity stress of
Oryza sativa were collected from PlantPReS (Mousavi et al., 2016). PlantPReS can be used
to explore a large collection of plant stress proteins, identified by experiments that can be
easily accessed along with the protein accession number, protein name, stress types,
tissues and respective organelles in which the proteins resides. Furthermore, their
regulation in different tissues and different stresses can be selected and analyzed (Mousavi
et al., 2016). A list of non-redundant S.R.Ps was retrieved by mapping the PlantPReS ID to
Uniprot ID using Uniprot Id converter.
2.2. Search for the proteins in stress response of drought, heat, and salinity
Common sets of stress response proteins responsible for heat, drought and salinity,
obtained from PlantPReS database. An interactive tool ‘VENNY’ was utilized to compare
different proteins and generate a Venn diagram (Oliveros, 2007). The common proteins
were found in different stresses by identifying the proteins present in different overlapping
zones of the Venn diagram. The protein that showed a response to three major abiotic
stresses - draught, heat’ and ‘salinity was found out and its name was obtained from
UniProt.
2.3 Choosing of the highest interaction of both UP and DOWN protein
From the VENNY tool, the common proteins were indentified respectively for both
UP REGULATORY and DOWN REGULATROY. Now these proteins were individually run on
STRING DATABASE (https://string-db.org/). The protein with highest interactions from both
sides was chosen.

21. 21
2.4 finding of similar proteins of the same family
From the Interpro Database, Similar 10 proteins of the same family were identified
and their sequences were saved.
2.5 Sequences and Database Search
Full-length coding sequences of the 10 genes were retrieved. Their peptide
sequences were predicted using the online server of “Emboss Trasseq” which was accessed
through the European Molecular Biology Laboratory (EMBL-EBI) server
(http://www.ebi.ac.uk/Tools/st/emboss_ transeq/)
2.6 Conserved Motives and Domain Analysis
The cupin-domain of these genes was confirmed via “NCBI CD-search” (conserved
domain-search) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Multiple
sequence alignment (Clustal-W) of the peptide sequences was performed using BioEdit
(Ver. 7.1.9). Furthermore, to get more insight into the multiple conserved motives of these
genes, the peptide sequences were analyzed via “Multiple Em for Motif Elicitation” (MEME)
software (http://meme.nbcr.net/meme/cgi-bin/meme.cgi) for the possible occurrence of 3
motives.
2.7 Protein Sequence Analysis
Various physicochemical properties such as molecular weight (M.wt), atomic mass,
total number of positive (+R) and negative (-R) residues, extinction coefficient (EC),
instability index (II), aliphatic index (AI), and grand average of hydropathicity (GRAVY) were
predicted with “ExPASY-ProtParam” (http://web.expasy.org/protparam/)

22. 22
2.8 The 3D structural models
The 3D structural models of all 10 proteins were obtained using Swiss modelling server
(http://swissmodel.expasy.org/interactive) and their quality and authenticity were
confirmed via “Ramachandran plot analysis (RPA)” using Rampage server
(http://mordred.bioc.cam.ac.uk/~rapper/rampage.php). Every analysis was performed
twice for authentication.

23. 23
3. RESULTS AND DISCUSSION
3.1 Identification of common stress proteins
Common proteins were indentified separately for UP-regulation and DOWN- regulation
from the stress taken in consideration.
Common protein among biotic stress was also checked out, but there was no common
protein there. So the stress protein analysis in terms of biotic stress was compiled and
completed. These data are for abiotic stresses (draught, salinity and heat).
17 proteins were common in UP-regulation: - figure 3.1
1. Q6Z7L1 number of edges 54
2. Q65XH8 number of edges 55
3. Q65XA0 number of edges 29
4. Q0D840 number of edges 36
5. Q7XPY2 number of edges 14
6. Q9MB31 number of edges 29
7. Q0DG48 number of edges 45
8. Q5QMK7 number of edges 33
9. Q9FE01 number of edges 39
10.Q7FAH2 number of edges 54
11.P48494 number of edges 53
12.Q42971 number of edges 53
13.P12085 number of edges 45
14.Q7XDC8 number of edges 48
15.Q0J8G4 number of edges 28
16.P93431 number of edges 39
17.Q10N21 number of edges 39

24. 24
Figure 3.1 common protein of UP-REGULATION
3 proteins were common in DOWN-regulation: - figure 3.2
1. Q650W6 number of edges 50
2. Q93X08 number of edges 29
3. P12085 number of edges 45

25. 25
Figure 3.2 common protein of DOWN-REGULATION
3.2 Protein with highest interaction and their similar proteins
For UP-regulation- protein with highest interaction - Q65XH8; this protein belongs to the
actin family.
10 Similar proteins of the same family (actin)-
1) A0A010NJQ4
2) A0A010QE55
3) A0A010QP73
4) A0A010QW85
5) A0A010R4Z7
6) A0A010R6I6
7) A0A010R6Z6
8) A0A010RFL5
9) A0A010RHW1
10) A0A010RZ88

26. 26
For DOWN-regulation- protein with highest interaction - Q650W6; this protein belongs to
the actin family.
6 Similar proteins of the same family (actin)-
1. A0A010QIG0
2. A0A010RAG2
3. A0A010RE11
4. A0A010RQD0
5. A0A010RVI2
6. A0A010RY01
3.3 Multiple Sequence Alignment
Proteins sequences of the proteins were aligned and searched for conserved motives to
uncover their common features. Possible occurrences of 3 motives were determined.
Results of CLUSTAL-W analysis- 10 UP- regulated proteins (figure 3.31 and 3.32)
Figure 3.31 Phylogram (midpoint rooted tree) with branch length

27. 27
Figure 3.32 Figure 3.31 Phylogram (midpoint rooted tree) without branch length
Results of CLUSTAL-W analysis- 6 DOWN- regulated proteins (figure 3.33 and 3.34)
Figure 3.33 Phylogram (midpoint rooted tree) with branch length

28. 28
Figure 3.34 Phylogram (midpoint rooted tree) without branch length
3.4 Physicochemical Properties
Various physicochemical properties of the protein exhibited significant variations
(Table 1 and table 2).
For UP-regulatory proteins— Table 1
Their sizes and molecular weights varied considerably resulting in variations in other
properties as well. Molecular weight ranged from 37705.87(A0A010NJQ4) Dalton to
113894.05(A0A010QW85) Dalton suggesting variations in their structure and physico-
chemical properties. The isoelectric point (pI) ranged from 4.93 to 9.34 which are
considered very important for the estimation of solubility, electrophoresis and
electrophoresis separation of the protein. The instability index (II) ranged from 29.18 to
69.51 for A0A010NJQ4 and A0A010R6Z6 respectively. Stability of the protein is crucial for
its proper functioning in the cellular environment. 40% (4 in number) of the protein are
stable in the cellular environment as their II value is less than 40, while the rest are

29. 29
unstable. The aliphatic index (AI) ranged from 75.14 to 108.93 as shown by A0A010RFL5
and A0A010QE55 respectively. High AI value is considered as a positive indicator of the
protein thermal stability. Proteins with the highest AI values include A0A010NJQ4,
A0A010QE55, A0A010QP73, A0A010QW85, A0A010RHW1 and A0A010RZ88 indicating that
they might be stable at a wide range of temperature. But proteins with lower AI values
indicate their structural flexibility at various temperatures which are directly associated
with the presence of aliphatic AAs (Ala, Val, Ile, and Leu) with aliphatic side chains. Values
for GRAVY ranged from -0.752 to -0.009 for A0A0A10R616 and A0A10NJQ4, respectively. All
proteins (100%) were hydrophilic in nature as their GRAVY values were negative (below
zero).
For DOWN-regulatory proteins— Table 2
Their sizes and molecular weights varied considerably resulting in variations in other
properties as well. Molecular weight ranged from 36420 (A0A010QIG0) Dalton to 87525.22
(A0A010RQD0) Dalton suggesting variations in their structure and physicochemical
properties. The isoelectric point (pI) ranged from 4.99 to 9.47 which are considered very
important for the estimation of solubility, electrophoresis and electrophoresis separation of
the protein. The instability index (II) ranged from 34.64 to 58.62 for A0A010RE11 and
A0A010RQD0 respectively. Stability of the protein is crucial for its proper functioning in the
cellular environment. 20% (2 in number) of the protein are stable in the cellular
environment as their II value is less than 40, while the rest are unstable. The aliphatic index
(AI) ranged from 29.58 to 81.40 as shown by A0A010RY01 and A0A010RE11 respectively.
High AI value is considered as a positive indicator of the protein thermal stability. Proteins
with lower AI values indicate their structural flexibility at various temperatures which are
directly associated with the presence of aliphatic AAs (Ala, Val, Ile, and Leu) with aliphatic
side chains. Values for GRAVY ranged from 1.316 to -0.466 for A0A010QIG0 and
A0A010RE11, respectively. Most of the proteins (90%) were hydrophilic in nature as their
GRAVY values were negative (below zero). Interestingly, only one protein had GRAVY of
1.316 which is hydrophobic in nature as their GRAVY values were positive (above zero).

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Table 1. Computational analyses based various structural and functional properties
of the Oryza sativa (var. Japonica) stress Protein family, M. wt: Molecular weight, pI:
Isoelectric point, +R: positive charged residues, -R: Negative Charged residues, EC:
Extinction Coefficient, II: Instability Index, Al: Aliphatic Index values, GRAVY: Grand
Average of Hydropathicity.

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Table 2. Computational analyses based various structural and functional properties
of the Oryza sativa (var. Japonica) stress Protein family, M. wt: Molecular weight, pI:
Isoelectric point, +R: positive charged residues, -R: Negative Charged residues, EC:
Extinction Coefficient, II: Instability Index, Al: Aliphatic Index values, GRAVY: Grand
Average of Hydropathicity.
3.4 Functional analysis
A detailed picture of the proteins enzymatic activities and their corresponding
roles in various plant processes were taken. Functional analysis with STRING
predicted that all proteins play important role in plant defence by offering a broad-
spectrum disease resistance. At molecular level, 98% of the proteins require a
metal/man- ganese ion for proper functioning and exhibit a nutrient reservoir-
specific (endosperm) expression which showed their crucial role in germination and
early plant processes. At the cellular level, they function by interacting with various
other proteins in apoplast by assisting in the transport of various materials through
the plant body. However, no function was predicted for 2 proteins. The analysis
predicted novel enzymatic activities for these proteins which include
methyltransferases, peroxidases, laccases and proteases representing their diverse

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role. Closely occurring genes showed similar functional properties due to
duplication. These genes not only interact with each other but may co-express with
other disease resistance genes. Such genes include proton-dependent oligopeptide
transporter (POT) (also known as the peptide transport [PTR] family), peroxidases,
laccases, methyl- transferases and SHR5-receptor-like kinases which have
considerable importance. The expression of protein at reproductive stages probably
indicates their involvement in reproductive processes, including maturation and
growth of gametes. However, further study will be required to shed more light on
proteins function during reproductive stages.
3.5 3D Structural Analysis
Previously, crystallography and non-magnetic resonance (NMR) data were
considered essential for the determination of the 3D structure; but now, it can be
predicted for an unknown protein by aligning and blasting with the known protein
structure. Three models were obtained for each protein. The best model was selected
based on its Global model quality estimation (GMQE) and Q mean (Z-score) estimation
scores in which high value indicates the higher reliability of the results. (Figure 3.51-
3.510)
Figure 3.51 protein name – A0A010NJQ4

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Figure 3.52 Protein name – A0A010QE55
Figure 3.53 Protein name – A0A010QP73

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Figure 3.54 Protein name – A0A010QW85
Figure 3.55 Protein name – A0A010R427

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Figure 3.56 Protein name – A0A010QP73
Figure 3.57 Protein name – A0A010RHW1

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Figure 3.58 Protein name – A0A010R6I6
Figure 3.59 Protein name – A0A010R6Z6

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Figure 3.510 Protein name – A0A010RFL5

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4. CONCLUSION
The study revealed that the proteins seem similar in structure but functionally
they are much more diverse. Genes located on the same CHR possess similar
physiochemical properties, subcellular localization, functional properties, expression
pattern and close phylogenetic relationship confirming their origin through duplication.
Through the course of evolution, the proteins have gone through considerable changes
in their domain architecture which resulted in the arousal of various novel enzymatic
activities. Functionally, the proteins are interlinked with each other or with the genes
of other families to cope with various stresses. This analysis can help to identify the
molecular basis of phenotypic differences and select gene expression targets for in-depth
study. Plant gene expression, in response to stress cues, is tightly controlled by
transcriptional regulators. Posttranslational modifications are a key mechanism to control
the activities of transcription factors (TFs). The regulation of gene expression in plants, as in
other higher eukaryotes, is a subject of daunting complexity. Nevertheless, even a partial
understanding of how plant genes work, in conjunction with the methods of molecular
biology and plant tissue cultures, opens the door to a dazzling array of techniques for
manipulating various aspects of the phenotypes of plants.

39. 39
5. References
1. Gene expression in plant https://www.sciencedirect.com/topics/biochemistry-
genetics-and-molecular-biology/gene-expression-in-plant
2. Plant genes for abiotic stress https://www.intechopen.com/books/abiotic-stress-in-
plants-mechanisms-and-adaptations/plant-genes-for-abiotic-stress
3. Plant stress. What causes it –how to reduce it
https://www.coolplanet.com/blog/plant-stress-what-causes-plant-stress-and-how-
to-reduce-it/
4. Introduction to plant stress https://link.springer.com/chapter/10.1007/978-3-319-
59379-1_1
5. Water Stress in Plants: Causes, Effects and Responses
https://www.researchgate.net/publication/221921924_Water_Stress_in_Plants_Cau
ses_Effects_and_Responses
6. Response of plants to water stress
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3952189/
7. Lin C, Chen W, Qiu C, Wu Y, Krishnan S, Zou Q. LibD3C: Ensemble classifiers
with a clustering and dynamic selection strategy. Neurocomputing 2014;
123:424-435. https://doi.org/10.1016/j.neucom.2013.08.004
8. Nagy T, Yosa Reyes J, Meuwly M. Multisurface Adiabatic Reactive Molecular
Dynamics. J. Chem. Theory Comput. 2014; 10: 1366-1375.
https://doi.org/10.1021/ct400953f
9. Tong X, Nagy T, Reyes JY, Germann M, Meuwly M, Willitsch S. State-selected
ion -molecule reactions with Coulomb-crystallized molecular ions in traps.
Chem. Phys. Lett. 2012; 547: 1-8. https://doi.org/10.1016/j.cplett.2012.06.042
10.Hollebeek T, Ho TS, Rabitz H. Constructing multidimensional molecular
potential energy surfaces from ab initio data. Annu. Rev. Phys. Chem. 1999; 50:
537-570. https://doi.org/10.1146/annurev.physchem.50.1.537

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