mRNA Analysis On Seed Development of MR7-3 Rice (Oryza Sativa)

College of Arts and Sciences
NOTRE DAME OF MARBEL UNIVERSITY

Alunan Avenue, City of Koronadal, 9506
South Cotabato, Philippines
mRNA Analysis on Seed Development of

MR7-3 Rice (Oryza sativa)
A paper submitted to:
Mr. Joni Rey Campilan

Natural Sciences and Mathematics Department
Notre Dame of Marbel University
In partial fulfillment of the requirements in
BIO FC 108
(Cell and Molecular Biology)
By:
Bernabe, Nico
Divinagracia, Arven Dale
Estember, Daryl Roie
Narvas, Michelle
September 2021
Introduction
Rice (Oryza sativa L.), as a major cereal crop consumed globally, holds great significance
in the food security of at least half of the total global population. As the population grows
exponentially, the improvement of rice grain quality has become a pioneering consideration in
resolving the increasing demand for food production and the decreasing number of arable lands
(Marathi B., Guleria S., Mohapatra T., et al., 2015). Rice grain quality refers to the physical
appearance and biochemical properties of the rice grain which includes but is not limited to grain
shape, grain appearance, milling efficiency and nutritional value. Rice chalkiness, a parameter
related to grain appearance, is an undesirable trait that posits a major problem to many rice-
producing countries in the world as it contains lower density starch granules. To lessen this
occurrence, studies have suggested the investigation of the role of genes in encoding transcription
factors during rice seed development and starch biosynthesis to understand how it alters the
appearance of the endosperm which affects the characteristics of starch (Wang et al., 2021).
Seed is the main storage organ in plants for storing nutrients such as starch, proteins, and
lipids. It is of great value for understanding the mechanism and regulatory networks during seed
development. Seed development especially in higher plants is a complex process (Juan Huang et.
al., 2017). Rice in particular incorporates the development of the endosperm and the embryo and
occurs in a sequential and systematic manner. The sequential process of rice seed development
can be summarized into five stages: post-fertilization to middle globular embryo in stage one;
embryo patterning and endosperm cellularization in stage two; embryo morphogenesis,
endoreduplication initiation, and milky endosperm formation in stage three; milky endosperm
maturation phase in stage four; and dormancy and desiccation in stage five (Mahto & Mathew,
2017). These changes in the development of rice are imparted through the operation of genes and
regulatory networks when exposed to internal and external stimuli. Understanding how these
networks administer the process is the key to understanding grain yield and grain quality (Agarwal
P., 2017).
Seed development also act as a safeguard in helping plants to retain certain genes to fight
against environmental factors as well for growth and development (Mahto, 2017). With use of
recent technological advances such as high-throughput mRNA profiling studies, significant genes
and pathways responsible for facilitating in various steps of rice seed development has been
identified. It has provided useful information with regards to the molecular changes that happen
in the different tissues associated with rice seed development (Agarwal P., 2017). The
transcriptional changes brought about by differentially expressed genes during rice seed
development which includes levels, patterns and molecular functions, has attributed to the changes
in endosperm appearance which eventually altered the characteristics of starch. Since fine
structures and physicochemical properties of starch serve as major determinants of rice grain
quality, it is important to determine the classifications of the expressed genes and evaluate its
possible correlation to starch biosynthesis and rice seed development (Mahto et. al., 2017).
The study “mRNA analysis on Seed Development of MR7-3 Rice” hopes to determine and
analyze what genes are present in rice during seed development in different ontological categories
such as molecular function, biological process, cellular component, protein class and pathway and
to determine the functions of these genes present and as well as to identify what genes are
prominently express and determining how it affects the quality of the grain. The researchers were
given a list of DEGs (differentially expressed genes) ranging from 5DAP, 10DAP, 15DAP and
20DAP. This data is to be fed to the PANTHER Classification System in order to determine what
genes are express.
Statement of the Problem

The research paper aimed to study the seed development of MR7-3 rice (Oryza sativa).
Particularly, the research sought to:
1. Determine the functional classification of 5DAP, 10DAP, 15DAP, and 20DAP MR7-3
DEGs.
2. Compare the functional classification of MR7-3 DEGs from 5DAP, 10DAP, 15DAP, up to
20DAP in terms of:
a) Molecular function
b) Biological process
c) Cellular Component
d) Protein Class
e) Pathway
Materials and Methods

The study analyzes 4 datasets of mRNA on the seed development of MR7-3 rice. The
datasets include 5DAP, 10DAP, 15DAP, and 20DAP. To determine the functional classification
of the identified MR7-3 DEGs (Differentially Expressed Gene), we have categorized each set of
data by their gene ontology using the Protein ANalysis THrough Evolutionary Relationships
(PANTHER v.13.0) Classification System. The gene ontology runs and visualizes us the basic
terms subdivided into 5 important categories, namely, molecular function, biological process,
cellular component, protein class, and pathway among these groups. Each ontology describes a
particular aspect of a gene or gene product functionality, as well as the relations between the terms.
As the PANTHER Classification System displays the gene list analysis, a bar chart is being shown
wherein the bar chart category will drill down to its subcategories. Each gene is associated with
the most specific set of terms that describe its functionality. By definition, if a gene is associated
with a term, it is also associated with all the parents of that term. The annotation process is
presented in a more detailed mannered.
Results and Discussion

Analysis on Gene Ontology of DEGs (Differentially Expressed Genes) at Different DAP (Days
After Pollination)
Among the 79 categories, a total of 43, 245, 338, and 661 DEGs were annotated using the
PANTHER Classification System in order to analyze and identify their possible involvement
towards molecular function, biological process, cellular component, protein class, and pathways
(Figure 1). There are 43 DEGs in 5DAP, 245 DEGs in 10DAP, 338 DEGs in 15DAP, and 661
DEGs in 20DAP.
Molecular Function
Transporter activity
Structural molecule activity

Categories
Molecular transducer activity
Molecular function regulator
Catalytic activity 7 38 59 121
Binding
0 50 100 150 200 250

Total # of Genes
5DAP 10DAP 15DAP 20DAP
Figure 1. Categorical classification on molecular function of DEGs from 5DAP, 10DAP,

15DAP, up to 20DAP.
• In molecular function, most Differentially Expressed Genes (DEGs) are involved in
catalytic activity with 225 total number of genes. Catalytic activity is the catalysis of a
biochemical reaction at physiological temperatures wherein it aids in increasing the
chemical reaction rates without altering the chemical equilibrium between reactants and
products, and without permanently altering or consuming themselves by the reaction.
• Catalytic activity is a category of molecular function that has a following subcategories
namely; catalytic activity acting on RNA, catalytic activity acting on protein, hydrolase
activity, isomerase activity, ligase activity, lyase activity, oxidoreductase activity, and
transferase activity.
Biological Process
Signaling
Response to stimulus
Reproductive process
Reproduction
Multicellular organismal process
Categories
Metabolic process
Localization
Interspecies interaction between organisms
Developmental process
Cellular process 2 46 71 137
Biological Regulation
Biological phase
Biological regulation
0 50 100 150 200 250 300

Total # of Genes
Figure 2. Categorical classification on biological process of DEGs from 5DAP, 10DAP, 15DAP,
up to 20DAP.
• In biological process, most Differentially Expressed Genes (DEGs) are involved in cellular
process with 256 total number of genes. Cellular process is any process which are not
necessarily restricted to a single cell, and is carried out at the cellular level. Cellular
processes that occur in plants is the utilization of sunlight, carbon dioxide, and water
wherein the energy is being produced by the form of ATP.
• Cellular process is a category of biological process that has a following subcategories
namely; cell communication, cell cycle checkpoint, cell cycle process, cell cycle, cell wall
organization or biogenesis, cellular component organization or biogenesis, cellular
homeostasis, cellular localization, cellular metabolic process, cellular response to stimulus,
exocytic process, export from cell, gene silencing, maintenance of location in cell,
microtubule-based process, signal transduction, transmembrane transport, vacuolar
transport, and vesicle-mediated transport.
Cellular Component
Cellular anatomical quantity

Categories
Protein-containing complex
Intracellular 3 46 63 130
Cellular anatomical entity
0 50 100 150 200 250 300

Total # of Genes
Figure 3. Categorical classification on cellular component of DEGs from 5DAP, 10DAP,

15DAP, up to 20DAP.
• In cellular component, most Differentially Expressed Genes (DEGs) are involved in
intracellular with 242 total number of genes. Intracellular component is a protein adjacent
network to the postsynaptic membrane. Proteins that functionally and spatially organize
neurotransmitter receptors in the adjacent membrane, such as scaffolding and anchoring
molecules, signaling enzymes and cytoskeletal components are major components of
intracellular.
• Intracellular category belongs to cellular component classification which has a following
subcategories namely; CCR-NOT complex, RNA polymerase complex, cyclin-dependent
protein kinase holoenzyme complex, cytoplasm, intracellular organelle, proteasome
complex, proton-transporting ATP synthase complex, thylakoid, and ubiquitin ligase
complex.
Protein Class
Viral or transposable element protein
Transporter
Transmembrane signal receptor
Translational protein
Scaffold/ adaptor protein
Transfer carrier protein
Protein- binding activity modulator
Protein modifying enzyme
Categories
Nucleic acid metabolism protein

Metabolite interconversion enzyme 12 40 57 105
Membrane traffic protein
Gene- specific transcriptional regulator
Extracellular matrix protein
Defense immunity protein
Cytoskeletal protein
Chromatin/chromatin binding, or -regulatory protein
Chaperone
Cell adhesion molecule
Calcium- binding protein
0 50 100 150 200 250

Total # of Genes
Figure 4. Categorical classification on protein class of DEGs from 5DAP, 10DAP, 15DAP, up to
20DAP.
• In protein class, most Differentially Expressed Genes (DEGs) are involved in metabolite
interconversion enzyme with a total of 214 genes. Metabolite interconversion enzyme
represent a series of enzymatic reactions wherein it synthesizes activated monosaccharides
for the integration into cell wall that regulates complex metabolic pathways. In addition, it
is a category belonging to protein class which has a following subcategories namely;
hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase.
Pathway
Wnt signaling pathway
Ubiquitin proteasome pathway 1 3 1
Valine biosynthesis
Tyrosine biosynthesis
Tryptophan biosynthesis
Transcription regulation by bZIP transcription factor
Ras pathway
Phenylalanine biosynthesis
P13 kinase pathway
PDGF signaling pathway
Notch signaling pathway
Parkinson disease
Nicotinic acetylcholine receptor signaling pathway
Interleukin signaling pathway
Insulin/IGF pathway- mitogen activated protein kinase…
Leucine biosynthesis
Isoleucine biosynthesis
Categories
Insulin/IGF pathway- protein kinase B signaling cascade

Inflammation mediated by chemokine and cytokine signaling…
Mannose metabolism
Huntington disease 1 1 2
Histidine biosynthesis
Glycolysis
Glutamine glutamate conversion
General transcription by RNA polymerase 1
Fructose galactose metabolism
FGF signaling pathway
EGF receptor signaling pathway
De novo purine biosynthesis 1 1 2
Cytoskeletal regulation by Rho GTP ase
Cholesterol biosynthesis
Cadherin signaling pathway
Cell cycle
Asparagine and aspartate biosynthesis
Angiogenesis
lanine biosynthesis
Alzheimer disease- presenilin pathway
0 1 2 3 4 5 6
Total # of Genes
Figure 5. Pathway categorical classification of DEGs from 5DAP, 10DAP, 15DAP, up to

20DAP.
• In pathways classification, most Differentially Expressed Genes (DEGs) are involved in

ubiquitin proteasome pathway with a total of 5 DEGs, followed by Huntington disease and
De novo purine biosynthesis which are both containing 4 DEGs.
• The category of ubiquitin proteasome pathway belongs to pathway classification which has
the following subcategories namely; 26S proteasome ubiquitin-conjugating enzyme, and
TATA binding protein, wherein these genes contribute significantly to the developmental
processes including embryogenesis, hormone signaling, and senescence among plants.
• Pathway classification in Huntington disease is the neurodegenerative disease that has a
material basis in autosomal dominant inheritance and characterized by the unwanted
choreatic movements, behavioral and psychiatric disturbances which results in
degeneration of neuron affecting cognitive abilities and muscle coordination. It has a
following subcategories namely; microtubule, beta-tubulin, histidinol phosphate
aminotransferase, phosphoribosyl AMP cyclohydrolase, phosphoribosyl ATP
pyrophosphatase, and TATA binding protein.
• De novo purine biosynthesis is the chemical reactions and pathways leading to the
development of inosine monophosphate, by the gradual assembly of a purine ring on ribose
5-phosphate, wherein the purine nucleotides are essential components for sustaining plant
growth and development. This category has only one subcategory which are identified as
adenylate kinase.
Conclusion:
• mRNA Analysis on seed development discusses the Differentially Expressed Genes
(DEGs) occurring in MR7-3 Rice (Oryza sativa). Functional classification of MR7-3 Rice
(Oryza sativa) showed that most of the genes involved is metabolite interconversion
enzyme in 5DAP, cellular anatomical entity in 10DAP, cellular process in 15DAP, and
cellular anatomical quantity in 20DAP.
• Functional classification of DEGs based on its gene ontology presented that most DEGs
involved was catalytic activity in terms of molecular function, cellular process for
biological process, intracellular in terms of cellular component, metabolite interconversion
enzyme for protein class, and ubiquitin proteasome in terms of pathway. Most of these
DEGs suggests the variations in emergence of organic substances towards seed
development.
• Analysis on pathway classification under of starch and sucrose affects the chalkiness on
grain quality of rice which suggests that the unusual degradation of starch is involved in
the occurrence of chalky grains during grain filling period of rice, and this could be
correlated in the biosynthesis of amino acid. The biosynthesis of amino acids shows that is
it is controlled by various genes, wherein histidine biosynthesis can be correlated for the
cause of Huntington’s disease which are found in 20DAP of MR7-3 rice. Thus, affecting
the grain quality and chalkiness of MR7-3 rice and plays a crucial role in the growth and
development of plants.
Contribution of Authors:
Authors Contribution
Bernabe, Nico Introduction
Divinagracia, Arven Dale Summary of DEGs Table, Introduction, Conclusion
Estember, Daryl Roie Results
Narvas, Michelle Summary of DEGs Table, SOP, Materials and Methods,
Discussion, Conclusion
References:
Bao, J. (2015) Genes and QTLs for Rice Grain Quality Improvement.
http://dx.doi.org/10.5772/56621
Dhurai, S., Reddy, D. M., & Ravi, S. (2016). Correlation and Path Analysis for Yield and Quality
Characters in Rice (Oryza sativa L.). Rice Genomics and Genetics. Published.
https://doi.org/10.5376/rgg.2016.07.0004
Mahto A., Mathew I.E., & Agarwal P. (2017). Decoding the Transcriptome of Rice Seed During
Development, Advances in Seed Biology, Jose C. Jimenez-Lopez, IntechOpen,
DOI:10.5772/intechopen.70574
Marathi B., Guleria S., Mohapatra T. et al. (2015). “QTL analysis of novel genomic regions
associated with yield and yield related traits in new plant type based recombinant inbred
lines of rice (Oryza sativa L.),” BMC Plant Biology, vol. 12, article no. 137, 2015
Wang, C., Zhu, C., Lu Y., Yang, Y.; Li, Q., Zhang, C. Grain Quality and Starch Physicochemical
Properties of Chalky Rice Mutant. Agronomy 2021, 11, 1575.
https://doi.org/10.3390/agronomy11081575

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College of Arts and Sciences

NOTRE DAME OF MARBEL UNIVERSITY

mRNA Analysis on Seed Development of

A paper submitted to:

Mr. Joni Rey Campilan

In partial fulfillment of the requirements in

Statement of the Problem

Materials and Methods

Results and Discussion

Structural molecule activity

Molecular transducer activity

Molecular function regulator

Catalytic activity 7 38 59 121

0 50 100 150 200 250

5DAP 10DAP 15DAP 20DAP

Figure 1. Categorical classification on molecular function of DEGs from 5DAP, 10DAP,

0 50 100 150 200 250 300

5DAP 10DAP 15DAP 20DAP

Cellular anatomical quantity

Cellular anatomical entity

0 50 100 150 200 250 300

5DAP 10DAP 15DAP 20DAP

Figure 3. Categorical classification on cellular component of DEGs from 5DAP, 10DAP,

Nucleic acid metabolism protein

0 50 100 150 200 250

5DAP 10DAP 15DAP 20DAP

Insulin/IGF pathway- protein kinase B signaling cascade

5DAP 10DAP 15DAP 20DAP

Figure 5. Pathway categorical classification of DEGs from 5DAP, 10DAP, 15DAP, up to

• In pathways classification, most Differentially Expressed Genes (DEGs) are involved in

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