Impact Factor 3.35

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

Basal thermotolerance Acquired thermotolerance

response to a sharp temperatureincrease (42–45 ⁰C) for a short time (2–6 h ).

transcription factor MBF1c and catalase

plants are first exposed to moderatelyhigh temperatures or to a gradual increase, allowing them to acclimate to

sHSPs or HSP70s, and APX

Partially shared

COLD vs HEAT

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

• 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%.

• Protein extractionplant 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 aWaring 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.

• 2D electrophoresis and gel image acquisitionProtein 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-Radcarrier 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 followingvoltages: 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 mLof 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 in10 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 thenstained with colloidal Coomassie G250; resulting images were

acquired using a GS-800 imaging system (Bio-Rad).

• 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

• 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

• HSP70s• 20 kDa chaperonin, and• GST F10

• abundance of chloroplastic TRX M4• chloroplastic CYP20-3 • ANX D1

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

• fructose-bisphosphate aldolase 2 ,cytosolic triosephosphate

• isomerase ,ATPase gamma chain and• soluble inorganic pyrophosphatase 1

(PPase1) ,whereasan increase in the abundance of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

• HSP70s• 20 kDa chaperonin, and• GST F10

• abundance of chloroplastic TRX M4• chloroplastic CYP20-3 • ANX D1

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.