This presentation based on heat shock proteins.... Which is more important in regulation of different stages of life cycle of organisms. This presentation based on my project work. Isolation of heat shock Proteins from xerophyte....

1. DEPARTMENT OF BOTANY RTMNU
NAGPUR UNIVERSITY
SEMINAR BASED ON
ISOLATION OF HEAT SHOCK PROTEIN FROM
XEROPHYTES
( Carthamus tinctorius) (SAFFLOWER)
SUBMITTED TO
Dr. Madhuri Thakre

2. About myself
 NAME:- SUHEL ANEESH ANSARI
 CLASS:-MSC IVth SEMESTER
 SEMINAR BASED ON:- ISOLATION OF HEAT
SHOCK PROTEIN FROM XEROPHYTE
( Carthamus tinctorius) (SAFFLOWER)

3. Why I am Choose this
Subject for my Project
?????

4. “ Mujee Hmesha Se Interest thaa….
Ye Samajne kaa kii….
“ How this Universe or Cosmos are
so Stable???”

5. Content
 Review of Literature
 About Discovery of Heat Shock Protein
 Introduction about Heat shock protein
 Heat shock protein Classification
 Role of Hsps
 Abstract
 Results and Discussion
 Concluding remarks
 About my project of interest
 References

6. Review of Literature

7. Review of Literature

8. Review of Literature

9. About Discovery of Heat shock protein
 This story begins in the early 1960s at the genetics Institute in
Pavia when Adriano Buzzati Traverso organized a course on
the biological action of radiation with the aim of introducing
10 young people to modern genetics.
 During the second year of the course organized by Buzzati,
Ritossa decided to work on Drosophila.
 Ritossa choose Drosophila because she felt that this organism
was some how between bacteria and man.
 Ritossa established set up a method for autoradiography with
tritium and started what type of nucleic acid was synthesized
in puffs of the salivary glands of Drosophila.
In 1960, there was still some confusion on this matter.
 Ritossa cannot remember whether was John Pulizer OR Inge
Or Clara Ghini OR Giordano who shifted the temperature of
incubator.
 One day Ritossa noticed a different puffing's.

10. About Discovery of Heat shock protein
 Ritossa calculated the right conditions for the shifts and
observed new RNA synthesis.
 Ritossa was impressed by the rapidity with which new RNA
was synthesized just 2-3 minutes.
 Ritossa remembered the Szent Gyorgyi book and wondered
whether heat might destroy the ice structure of water around
the proteins of the electrons transport chain, which cause
uncoupling of oxidative phosphorylation

11. Introduction About Heat shock Protein
 Basics Stresses such as drought, salinity, tempreture and
chemical pollutants are simultaneously acting on the plants
causing cell injury and producing secondary stress such as
osmotic and oxidative ones.
 Plants could not change their sites to avoid such stresses, but
have different ways and morphologically adaptation to
tolerate these stress.
 Other ways of defense at the molecular level are very
important for survival and growth of plants. Plants show a
series of molecular response to these stress.
 Heat stress as well as other stresses can trigger some
mechanism of defense such as the obvious gene expression
that was not expressed under normal condition.

12. Introduction About Heat shock Protein
 In fact, this response to stresses on the molecular level found in
all living things, especially the sudden changes in genotypes
expression resulting in an increase in the synthesis of protein
group.
 These groups are called heat shock protein OR stress
protein.
 Almost all kind of stresses induce gene expression and
synthesis of heat shock protein in cells that are subjected to
stress.
 In Arabidopsis and some other plant species low temperature,
osmotic, salinity, oxidation, desiccation, high intensity
irradiation, wounding, and heavy metals stresses were found to
induce the synthesis of Hsps.

13. Introduction About Heat shock Protein
 Heat stress- High temperature affected the metabolism and
structure of plants, especially cell membrane and many basics
physiological process such as photosynthesis, respiration, and
water relation.
 On the molecular level, this effect of heat stress reflects the
temp. dependence of Michaelis-Menton constant (Km) of
every enzyme participating in the process.
 Plants must cope with heat stress for survival, so they
developed different mechanism including the maintenance of
cell membrane stability capturing the reactive oxygen
species(ROS), synthesis of antioxidant, accumulation of
some kinases that respond to stress, Ca-dependent kinase
protein, and enhancing the transcription and signal transfer of
chaperon

14. Heat shock protein classification
 Many types Hsps have been identified in almost all organism.
 All Hsps are characterized by the presence of a carboxylic
terminal called Heat shock domain.
 Heat shock protein having molecular weight ranging from 10 to
200 KD are characterized as a chaperon where they participate
in the induction of signal during heat stress.
 Heat shock protein of archea have been classified on the basics
of their appropriate molecular weight into:
1) Hsp100
2) Hsp90
3) Hsp70
4) Hsp60
5) sHsps
 Gupta et.al(2010) Put Hsps into families according to their
molecular weight, amino acid sequence homology, and
function.

17. Role of Hsps
 The role of Hsps is the folding of other proteins is important.
1. Induce (Refold) denature protein.
2. Participate in the finalization of the de novo synthesized
protein.
3. Reduce the protein aggregation.
 Simply, the Hsps are known for their roles in the maturation of
protein complexes and the degradation of damaged or
misfolded peptides, and for regulating the activity of many
signal transduction proteins.

19. Isolation and characterization of the main
small heat shock proteins induced in tomato
pericarp by thermal treatment.
-Gustavo A. Polenta
-Juan J. Calvete
-Claudia B. Gonzalez

20. Abstract
 In recent years, heat treatment has been used to prevent the
development of chilling injury in fruits and vegetables. The
acquired tolerance to chilling seen in treated fruit is related to
the accumulation of heat shock proteins (HSPs).
 The positive effect of heat treatment has generally been
verified for only a narrow range of treatment intensities and
more reliable methods of determining optimal conditions are
therefore needed.
 In this regard, quantitation of HSPs would seem to be an
interesting tool for monitoring purposes.
 As a step toward the development of analytical methodology,
the objective of this study was the isolation a characterization
of relevant HSPs from plant tissues.
 Tomato fruits were exposed to a temperature of 380C for
0, 3, 20 and 27 h, and protein extracts from pericarp were
analysed using SDS⁄PAGE.

21.  Analysis revealed the appearance of an intense 21 kDa protein
band in treated samples.
 IEF of this band showed the presence of four major proteins
(HSPC1, HSPC2, HSPC3 and HSPC4) with similar pI values.
 A monospecific polyclonal antiserum was raised in rabbits
against purified HSPC1 protein, which cross-reacted with other
small heat shock proteins.
 The antiserum obtained proved suitable for detecting increased
amounts of small heat shock proteins in tomatoes and
grapefruits subjected to heat treatment for 24 and 48 h; these
treatments were successful in preventing the development of
chilling injury symptoms during cold storage.
 Regarding the biochemical basis of heat protection, it has been
reported that exposure of most live tissues to a transient
temperature increment of 5–10 0C induces the synthesis of a
specific group of proteins referred to as heat shock proteins
(HSPs).

22. Results and Discussion
Biochemical characterization of sHSP

23. Immunological characterization of sHSP
 The main overexpressed sHSP (HSPC1) was used as an
immunogen to produce polyclonal antibodies for further
characterization of the heat response.
 The antiserum obtained is a valuable tool for identifying heat
shock-induced proteins and ⁄ or developing reliable quantitative
methods.
 Interestingly, western blot analysis revealed that the obtained
antibodies cross-reacted with the four main proteins detected
by IEF in heat-treated tomatoes (Fig. 1B). This is probably due
to the presence of a primary domain (known as an a-crystallyn
domain) that is shared by different sHSPs.
 Even more, antibodies against a synthetic sHSP-oligopeptide
antigen were also able to recognize sHSP from tomato leaf.
 This study shows that the augmented synthesis of proteins
reactive to anti-HSPC1 serum reflects the increased intensity of
the treatments applied (Fig. 2B).

24. • Increased accumulation of HSPs in treated fruits has been linked
to the acquisition of a tolerance to low temperatures

25. Concluding remarks
 Heat treatments applied to tomatoes induced the synthesis of
two major (HSPC1 and HSPC2) and two minor (HSPC3
and HSPC4) class I sHSPs.
 The antiserum obtained against HSPC1 cross-reacted with
other members of this family, and the increased amounts of
sHSP detected in heat-treated tomatoes and grapefruits showed
a positive correlation with the tolerance to the development of
chilling injury after cold storage.
 Thus, we proposed the quantification of sHSPs as a tool to
predict treatment performance.
 In this regard, the described electrophoretic method may be a
helpful tool to purify individual sHSPs and obtain specific
antiserum in laboratory animals considering that, to the best of
our knowledge, there is no available commercial antibodies
against fruits sHSPs.

26. About my project of interest

27.  Same as previous one…….
 But lack of technical instrument. I used classical method to
isolate Hsps. i.e SDS PAGE Electrophoresis (1D).
 And Carry Out Enzyme essay.
 But Material is Different. In this review experimental material
tomato and grapefruit.
 But I use Carthamus tinctorius (Safflower) Which is xerophytic
plant.

28. References
 Ferruccio Ritossa, Discovery of the Heat shock protein,
Reflection, 1996.
 Polenta G.A, J.J Calvete, C.B Gonzalez, Isolation and
characterization of small heat shock protiens induced in
tomato pericarp by thermal treatment, The FEBS Journal
(2007).
 Mohammed H. Al Whaibi, Plant heat shock protein, Journal of
king Saud University Science.
 Wang W, B. Vinocur, O Shoseyov, A. Altman, Role of Plant
Heat-Shock protein and molecular chaperons in the abiotic
stress response, Elsevier (2004).
 Mayer M.P, B. Bukau, Hsp70 Chaperones: Cellular function
and molecular mechanism, Springer (2005).
 Mishra D, S. Shekhar, D. Singh, S. Chakraborty, N.
Chakraborty, Heat shock protein and abiotic stress tolerance in
plants, Springer (2010).

29. “Good thought of Life”
Don’tcompareyourselftoanyoneelse,
Becausenobodyisperfect.Andforthe
successitisnotnecessarytobeaperfect
person……
-MissB.R
Bisen

30. Lot of Thanks
1. Miss B.R Bisen
2. Punit Patle
3. Umesh Chandewar
4. Aadesh Walde
5. And All My Sisters

31. “Dedicatedto my Best Teachers of entire Life”

32. If any question??????