Name of the Assignment:

Important Proteins Involved In Heat Stress Tolerance In Rice

The assignment is submitted as a partial fulfilment for the course of

GPB-502: Biotechnology and Molecular Genetics Practical

Submitted To Submitted By
Prof. Dr. Mohammad Anwar Jaber Bin Azim
Hossain Roll: 19GPBJJ25M

Department of Genetics and Plant Breeding

1 Introduction 1

2 Heat-Shock Proteins (HSPs) 1-4

3 Heat stress tolerance in rice 4-5

4 Conclusion 6

5 References 6
1. Introduction
Among the ever-changing components of the environment, the constantly rising ambient
temperature is considered one of the most detrimental stresses. The global air temperature is
predicted to rise by 0.2 °C per decade, which will lead to temperatures 1.8–4.0 °C higher than the
current level by 2100. Plants suffer the ups and downs of temperature of their environment, while
animals often regulate their temperature, either by movement or metabolism. Therefore, global
warming may affect plants more than animals and there are indications that plants experience
substantial damage from high temperature stress. Estimates range up to a 17% decrease in crop
yield for each degree Celsius increase in average growing season temperature. Heat stress due to
increased temperature is an agricultural problem in many areas in the world. Transitory or
constantly high temperatures cause an array of morpho-anatomical, physiological and biochemical
changes in plants, which affect plant growth and development and may lead to a drastic reduction
in economic yield. Because of the temperature sensitivity of the forces responsible for protein
folding, proteins are easily denatured by high temperature. Biological organisms have a suite of
proteins that are made in response to high temperature that appear to be designed to prevent or
reverse the effects of heat on protein denaturation. These are called heat shock proteins because
they are made in abundance when organisms are subjected to potentially damaging high
temperature. It is believed that these proteins help other proteins fold correctly or refold after
damage by heat stress.
2. Heat-Shock Proteins (HSPs): Master Players for Heat Stress Tolerance
In general, heat stress is responsible for the up-regulation of several heat inducible genes,
commonly referred as “heat shock genes” (HSGs) which encode HSPs and these active products
are very much necessary for plant’s survival under fatal HT. High temperature induced constitutive
expression of most of these proteins protect intracellular proteins from being denaturation and
preserve their stability and function through protein folding; thus it acts as chaperones. The HSPs
are extremely heterogeneous in nature and this dynamic protein family is expanding continuously
as per the recent researches are going on. The expression of HSPs is restricted to certain
developmental stages of plant like seed germination, embryogenesis, microsporogenesis and fruit
maturation. Heat shock protein (HSP) is an intracellular protein that increases in concentration
during metabolic stress, such as exposure to heat. HsPs affect protein assembly, folding, sorting,
and uptake into organelles. There are various kinds of HsPs, each performing different functions.
In 1962, Italian geneticist Ferruccio Ritossa reported that heat and the metabolic uncoupler 2,4-
dinitrophenol induced a characteristic pattern of puffing in the chromosomes of Drosophila. This
discovery eventually led to the identification of the heat-shock proteins (HSP) or stress proteins
whose expression these puffs represented. Increased synthesis of selected proteins in Drosophila
cells following stresses such as heat shock was first reported in 1974. HSPs play crucial roles in
folding/unfolding of proteins, assembly of multiprotein complexes, transport/sorting of proteins
into correct subcellular compartments, cell-cycle control and signaling, and protection of cells
against stress/apoptosis.

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Figure 1: The role of heat shock proteins (Hsp) and small heat shock proteins (sHsps) in recognition and
refolding of unfolded and misfolded proteins. Unfolded or misfolded proteins are recognized by Hsps and
sHsps. Together with these unfolded or misfolded proteins, Hsps and sHsps form a complex. In addition,
Hsps recover unfolded or misfolded proteins back to their native form using ATP. If unfolded or misfolded
protein are not recognized by the Hsp/sHsps, these unfolded or misfolded proteins are capable of forming
aggregates
Heat shock proteins constitute a large family of proteins that are often classified based on their
molecular weight: hsp10, hsp40, hsp60, hsp70, hsp90, etc. HSPs have a dual function depending
on their intracellular or extracellular location. Heat shock proteins were discovered as proteins that
were not expressed or not very prominent in assays of plants grown in moderate temperature but
which became prominent when organisms were subjected to a heat shock. The following is a quick
tour of some of the important aspects of each class of heat shock proteins.
Table 1. An outline of basic function of major classes of heat shock proteins in plant system for
heat stress tolerance
Classes of HSP Intracellular localization Major Functions
HSP100 Cytosol, mitochondria, ATP-dependent dissociation and
Chloroplast degradation of aggregate protein
HSP90 Cytosol, mitochondria, Co-regulator of heat stress linked
Chloroplast, Endoplasmic signal transduction complexes and
reticulum manages protein folding. It requires
ATP for its function
HSP70, HSP40 Cytosol, mitochondria, Primary stabilization of newly formed
Chloroplast, Endoplasmic proteins, ATP-dependent binding and
reticulum release
HSP60, HSP10 Cytosol, mitochondria, ATP-dependent specialized folding
Chloroplast machinery
HSP20 or small Cytosol, mitochondria, Formation of high molecular weight
HSP (sHSP) Chloroplast, Endoplasmic oligomeric complexes, which serve as
reticulum, Membrane cellular matrix for stabilization of
unfolded proteins. HSP100, HSP70
and HSP40 are needed for its release

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Due to their thermotolerant nature, the expression of HSP can be induced by heat treatment in the
presence of conserved heat shock elements (HSEs) in the promoter region of HSGs, which
triggering transcription in response to heat. These cis-acting elements (HSE) consist of the
palindromic nucleotide sequence (5-AGAANNTTCT-3) that serve as recognizing as well as
binding site for heat shock transcription factors or simply heat shock factors (HSFs).
Thermotolerance against heat stress have been accomplished in plants transferred with heat shock
regulatory proteins. In most of the plant species, HSFs are constitutively expressed; in normal
conditions, these proteins (HSPs) are present as a monomer bound to one of the HSP70 in the
cytoplasm. Once the plant has sensed a heat stress (increase in temperature), HSP70 dissociates
from cytoplasmic monomeric HSFs and then it enters into the nucleus and form a trimer that can
bind with the HSEs. Heat shock factor binding recruits other transcriptional components, resulting
in gene expression within minutes in increased temperature. Since all HSGs contain HSE
conserved sequence, overexpression of HSF gene intern turned on almost all HSGs and
consequently provides protection against heat stress. Heat shock protein, singly or in form of
chaperone, has been implicated in plant cell protection mechanisms under heat stress; they are
responsible for protein synthesis, targeting, maturation and degradation, and function in protein
and membrane stabilization, and protein renaturation under heat damage condition. Protein
denaturation occurs under HT because decreased cellular volume increases the likelihood of
degradative molecular interactions. Heat shock proteins maintain and repair companion protein
structure, target incorrectly aggregated, and non-native proteins for degradation and removal from
cells. These proteins primarily function to control the proper folding and conformation of both
structural (i.e., cell membrane) and functional (i.e., enzyme) proteins, ensuring the correct function
of many cellular proteins under conditions of elevated temperature.

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Figure 3. Schematic diagram showing the molecular regulatory mechanism of heat shock proteins based
on a hypothetical cellular model. Upon heat stress perceived by the plant cell, (a) monomeric heat shock
factors (HSFs) are entering into the nucleus; (b) from the cytoplasm. In the nucleus, HSF monomers are
form active trimer; (c) that will bind; (d) to the specific genomic region (promoter or heat shock element,
HSE) of the respective heat shock gene (HSG). Molecular dissection of the HSF binding region of HSE
showing that it is consists of one DNA binding domain and two domains for trimerization of HSFs.
Successful transcription (e) translation and post-translational modification; (f) lead to produce functional
HSP to protect the plant cell and responsible for heat stress tolerance.
In essence, expression of stress proteins is an important adaptation toward heat-stress tolerance by
plants. Of these, expression of low and high molecular weight HSPs, widely reported in a number
of plant species, is the most important one. These proteins show organelle- and tissue-specific
expression with deduced function like chaperones, folding and unfolding of cellular proteins, and
protection of functional sites from the adverse effects of high temperature.Amongother stress
proteins, expression of ubiquitin, Pir proteins, LEA and dehydrins has also been established under
heat stress. A main function of these proteins appears to be protection of cellular and sub-cellular
structures against oxidative damage and dehydrative forces.
3. Heat stress tolerance in rice
Rice (Oryza sativa L.) is grown on around one 10th of the arable land (Wang and Peng 2017), and
sustains the lives of three billion people. Major share of the rice production and consumption is
centered in Asia (growing on ~90% acreage). Rice is also a source of protein (~14%) and fat (2%).
Rice is an attractive model plant species due to its small genome size (~440 Mb), suitability for
doing efficient transformation and genetic analysis, availability of genome sequence of both indica
and japonica subspecies and close genetic relationship with other cereals.
Heat stress (excessive heat) can cause irreversible damage by retarding the plant growth, metabolic
activities, and pollen fertility and seed setting, thus reducing the rice production. In another report,
it has been shown that excessive heat accelerates reduction in rate of photosynthesis, leaf area,
reduces shoot and grain mass as well as seed weight, and water-use efficiency. High temperature
may impede the vegetative as well as reproductive stages (from emergence till maturity). However,
booting and flowering are the most critical stages, which may lead to complete sterility in rice.
Oryza meridionalis Ng. a wild relative of the cultivated O. sativa, maintains high photosynthetic
rate, elongated leaf and maintains levels of protective proteins Hsp70, Hsp90, and Cpn60, all these
attributes make it heat tolerant.
Like many other crop species, response to heat stress in rice is very complicated. For instance, it
involved the up and down regulation of various proteins including proteins involved in protection,
biosynthesis and degradation of proteins, carbohydrate and energy metabolism and redox
homeostasis. A HSP acts as molecular chaperons, which repair stress-damaged proteins, as well
as protects cells from damage by stresses. Heat tolerant rice over expressing the sHSP17.7 was
developed through deploying transgenic approaches. Over expression of mitochondrial HSP70
(mthsp70) protects rice protoplasts from heat induced programmed cell death and ROS,

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consequently it sustains the mitochondrial membrane potential and retards the synthesis of ROS.
Importance of HSPs is increasing as their expression occur in nature, fluctuating number of genes
are present in all species, and their expression and tolerance to stress are positively correlated.
For mitigating the damaging effects of ROS, plants synthesize antioxidants. These antioxidants
are superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT),
dehydroascorbate reductase (DHAR), glutathione reductase (GR), monodehydroascorbate
reductase (MDHAR), glutathione-S-transferase (GST) glutathione peroxidase (GPX), and guaicol
peroxidase (GOPX). The role of antioxidants in conferring tolerance to heat was compared in O.
sativa, and it was proved that the role of antioxidant defense system is evident in imparting
tolerance to temperature stress. APX, GR, GST, and GPX enzymes protect seedling from the
oxidative stress induced after exposing them to high temperature. Generally, the elevation in
temperature leads to enhanced production of antioxidative enzymes upto a certain temperature
limit, and beyond this the production is reduced. The SOD converts the superoxide (produced on
membrane surface) into H2O2 followed by its detoxification by APX. Thus, these enzymatic
antioxidants can be used as biochemical markers for heat tolerance in rice.
In total, 48 heat responsive proteins identified in rice, which were cataloged in classes, related to
energy and metabolism, regulatory actions and heat shock proteins. Four proteins corresponding
to antioxidants can play role in heat tolerance. In addition, the heat shock inhibits the protein
synthesis, induces thermo-tolerance, increases the expression of heat shock proteins, and often
results in apoptotic cell death. Heat shock also initiates the process of phosphorylation of proteins
- involved in signaling the heat shock.
Table 2. Genes related to heat tolerance in rice
Gene Protein Function
hsp101 Heat shock protein Plants expressing this protein exhibited heat tolerance
ZFP Protein Heat tolerance at seedling stage
OsWRKY11 WRKY11 Responsible for heat tolerance
OsGSK1 Glycogen synthase Knockout mutant of OsGSK1 gene showed tolerance to heat
kinase3 stress.
OsHsfA2e Heat stress Over expression of OsHsfA2e improved tolerance to heat.
transcription
factor
(HSF)
mtHsp70 HSP70 Overexpression of mthsp70 suppresses the programmed
cell death and ROS
sHSP17.7 HSP17.7 Transgenic rice expressing sHSP17.7 is thermotolerant
FAD7 Dienoic fatty acids Transgenic rice with FAD7 gene has shown heat tolerance
by increasing the growth rate and chlorophyll content
SBPase SBPase Over-expression of SBPase gene confers heat tolerance by
CO2 assimilation and enhancing photosynthesis

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 Sp17 (rice HSFA4d (heat Sp17 mutant showed spotted leaf phenotype under high
spotted stress transcription temperature
leaf gene) factor protein)

4. Conclusion
Increasing temperature would adversely threaten the global production of different crop species
including rice. Plants cope such stresses by adapting various mechanisms in cells by altering gene
expression as well as altering morphology and physiology of various parts. There is a need to
explore these genes, which are involved in conferring heat tolerance. Also, the genes from well-
studied plant species can be transferred to rice for improving its tolerance to heat. Individual
members of each class of Hsps/chaperones have particular functions, but the co-operation between
different Hsp/chaperone networks appears to be a central principle of the integrated Hsp/chaperone
machinery. Under normal growth conditions or during and after stress, the fate of a particular
denatured or non-native protein is determined by the entire Hsp/chaperone system. Many questions
about this issue have still to be answered. For example, how does a specific chaperone recognize
its particular substrate (a nascent peptide or a malfunction protein) in a crowded cellular
environment? How and when is the decision on the fate of each denatured/non-native protein
determined? What is the fate of a denatured/non-native protein: is it stabilized or protected from
aggregation, refolded, or eventually aggregated and targeted for degradation? We are still far from
understanding how Hsps/chaperones, as regulatory molecules, participate in stress sensing, signal
transduction and transcription activation of stress genes. An integrated approach using multiple
techniques for improving complex traits like heat tolerance would be a more suitable route towards
a swift success.
5. References
Zafar, S. A., Hameed, A., Nawaz, M. A., Wei, M. A., Noor, M. A., & Hussain, M. (2018).
Mechanisms and molecular approaches for heat tolerance in rice (Oryza sativa L.) under
climate change scenario. Journal of Integrative Agriculture, 17(4), 726-738.
Hasanuzzaman, M., Nahar, K., Alam, M., Roychowdhury, R., & Fujita, M. (2013). Physiological,
biochemical, and molecular mechanisms of heat stress tolerance in plants. International
journal of molecular sciences, 14(5), 9643-9684.
Maestri, E., Klueva, N., Perrotta, C., Gulli, M., Nguyen, H. T., & Marmiroli, N. (2002). Molecular
genetics of heat tolerance and heat shock proteins in cereals. Plant molecular biology, 48(5-
6), 667-681.
Wahid, A., Gelani, S., Ashraf, M., & Foolad, M. R. (2007). Heat tolerance in plants: an overview.
Environmental and experimental botany, 61(3), 199-223.
Wang, W., Vinocur, B., Shoseyov, O., & Altman, A. (2004). Role of plant heat-shock proteins and
molecular chaperones in the abiotic stress response. Trends in plant science, 9(5), 244-252.