Cold treatment had significant effect on specific process of plant photosynthesis. significant degree of overlapping exists between metabolic alterations induced by, and/or in response to cold and heat short-term challenge. chloroplast and also plasma membrane/cytoskeleton were confirmed as early targets for temperature injury. Cold and heat influenced photosynthesis in opposition. major components of the early response to temperature stress are HSPs detoxification of ROS relied preferentially on the induction of GST under both cold or heat stress.

1. Impact Factor 3.35

2. Introduction
• Continuously challenged by unfavorable
conditions.
• Changes in gene expression &
metabolism.
• Low temperature.
Duration & Intensity
Gradual exposure to
low tem. For long
time: Adaptation
Short time exposure
to more severe cold
triggers a rapid
response
Partial
overlap

3. Basal thermotolerance Acquired thermotolerance
response to a sharp
temperature
increase (42–45 ⁰C) for a
short time (2–6 h ).
transcription factor
MBF1c and catalase
plants are first exposed to
moderately
high temperatures or to a
gradual increase, allowing
them to acclimate to
sHSPs or HSP70s, and APX
Partially shared
COLD vs HEAT

4. Materials and methods
22⁰C, 80% Humidity
( 16h light/ 8h dark )
Cold( 4⁰C)
6h
12h
Heat(42⁰C)
6h
12h
3 weeks old seedlings
Control were incubated at 21⁰C for 6 & 12 h

5. • Relative electrolyte leakage measurement.
– The leaves were cut into 1-cm segments and
washed three times with ultrapure water.
– The segments were placed in tubes containing 5
ml of ultrapure water and incubated at 28 C.
– Two hours later, the electrical conductivity of the
bathing solution (Lt) was measured.
– Then the tubes were incubated at 100 C for 20
min and subsequently at 28 C for 1 h, and the
electrical conductivity (L0) was measured again.
The relative electrolyte leakage was calculated by
the formula Lt/L0 ᵡ100%.

6. • Protein extraction
plant leaves were finely powdered in liquid N2
dried under vacuum.
One gram of dried leaves was suspended in 20 mL of ice cold extraction buffer (700
mM sucrose, 500 mM Tris-HCl, pH 7.5, 50 mM EDTA, 100 mM KCl, 2% w/v b-
mercaptoethanol, 1 mM
PMSF, 1% w/v PVP, 0.25% w/v CHAPS, 40 mM NaF, 1 mM okadaic acid).
After addition of an equal volume of phenol saturated-
500 mM Tris-HCl, pH 7.5, the mixture was stirred for 5 min in a
Waring blender and then centrifuged at 10 000 g for 10 min, at
4 ⁰C.
The upper phenol phase was removed and extracted once again with the extraction
buffer.
Proteins were precipitated from the phenol phase by addition of 5 vol of saturated
ammonium acetate in methanol, at 20 ⁰C, overnight.
Precipitated proteins were centrifuged at 10 000 g, for 30 min. Proteins were stored at
-80 ⁰C, until used.

7. • 2D electrophoresis and gel image acquisition
Protein pellets were dissolved in IEF buffer (9 M urea, 4% w/v
CHAPS, 0.5% v/v Triton X-100, 20 mM DTT, 1% w/v Bio-Rad
carrier ampholytes pH 4–7).
IPG strips (17 cm, pH 4–7, Bio-Rad ReadyStrip,
Bio-Rad) were rehydrated overnight with 300 mL of IEF buffer
containing 300 microgram of total proteins.
Proteins were focused using a Protean IEF Cell (Bio-Rad) at 12 ⁰C, by applying the following
voltages: 250 V (90 min), 500 V (90 min), 1000 V (180 min) and
8000 V for a total of 52 KVh.
After focusing, the proteins were reduced by incubating the IPG strips with 1% w/v DTT in 10 mL
of equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30%
w/v glycerol, 2% w/v SDS and a dash of bromophenol blue) for
15 min, and then alkylated with 2.5% w/v iodoacetamide in
10 mL of equilibration buffer, for 15 min.
Electrophoresis in the second dimension was carried out on 12% polyacrylamide gels
(180 240 1 mm) using a Protean apparatus (Bio-Rad), using
electrophoresis buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine and 0.1% w/v SDS), with 120 V
applied for 12 h.
2-DE gels were then
stained with colloidal Coomassie G250; resulting images were
acquired using a GS-800 imaging system (Bio-Rad).

8. • PD Quest (ver 7.4) 2-D analysis software (Bio-
Rad).
• Significant spots were identified by Student’s t
test.
• Excised and trypsin digested. Desalting before
MALDI-TOF PMF analysis. And/ or nanoLC-ESI-
LIT-MS/MS analysis
Results & discussion

9. • ATPase gamma chain ,isoforms of OEE protein 2-1 were up-regulated
• Calvin cycle enzymes, levels of RuBis carboxylase small chain 2B and of
specific isoforms of RuBis carboxylase large chain and RuBis
carboxylase/activase were significantly increased.
– Some other forms of these proteins were also constantly represented.
Cold treatment had significant effect on specific process of
plant photosynthesis
Effect of cold treatment

10. • HSP70s
• 20 kDa chaperonin, and
• GST F10
• abundance of chloroplastic TRX M4
• chloroplastic CYP20-3
• ANX D1

11. Effect of heat treatment
• Affected the abundance of leaf proteins involved
in photosynthetic electron transport chain &
carbon metabolism.Significant reduction
 OEE protein 2-1
 OEE protein 1-1
 subunit II-2 of the
photosystem I reaction center
 chloroplastic 29 kDa
 RuBis carboxylase small chain
2B, and specific isoforms of
RuBis carboxylase large
consistent increase of a specific
isoform of RuBis
carboxylase/oxygenase activase

12. • fructose-bisphosphate aldolase 2 ,cytosolic
triosephosphate
• isomerase ,ATPase gamma chain and
• soluble inorganic pyrophosphatase 1 (PPase1)
,whereas
an increase in the abundance of chloroplastic
glyceraldehyde 3-phosphate dehydrogenase
(GAPDH)

13. • HSP70s
• 20 kDa chaperonin, and
• GST F10
• abundance of chloroplastic TRX M4
• chloroplastic CYP20-3
• ANX D1

14. Conclusion
• significant degree of overlapping exists between
metabolic alterations induced by, and/or in response to
cold and heat short-term challenge.
• chloroplast and also plasma membrane/cytoskeleton
were confirmed as early targets for temperature injury.
• Cold and heat influenced photosynthesis in opposition.
• major components of the early response to temperature
stress are HSPs
• detoxification of ROS relied preferentially on the
induction of GST under both cold or heat stress.