temperature stress responsive proteins of Arabidopsis.
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temperature stress responsive proteins of Arabidopsis.

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Cold treatment had significant effect on specific process of plant photosynthesis. ...

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.

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  • Plants being sessile they are… con challenged by unf conditions.. In order to cope with these plants bring modifications. Now if we talk about the temp stress pplants response to change in tem. Depends upon
  • Impo key factor.. Impo. Fctors ARE the proteins from chaperone family. In the present study researchers have tried to figure out whether there is significant overlap b/t the plants response to heat or cold
  • Incubated in the dark. Leaves were samples.
  • 6 biological replicates for each samples were used.
  • Phenol extraction method. Three biological replicates.
  • triplicate
  • In gel reduced and S-alkylated.
  • Cold treatment had significant effect on specific process of plant photosynthesis. Stabilizes Mn cluster of Ph II where H2o is split to give electrns.
  • Can be catogorised in major category of pro. That protects from aboitic stress. glucose-6-phosphate dehydrogenase pentose phosphate pathway. NADPH form. May be due to cells neccessitytomaintain reducing environment to fight oxidative stress. Cyclophillins primary role to assist protein folding. Unction in membrane association.
  • Unlikely to cold stress a general protein down representation is seen. the same trend was also observed for.. It has been suggested that a RuBis carboxylase/oxygenase activase over-representation may be part of the adaptative response, in order to maintain CO2 fixation under stress conditions
  • heat stress negatively affects the glycolytic pathway and reduces energy production in rice, bringing about a marked decrease in the levels of different glycolytic enzymes or proteins involved in energy-generating reactions.23 In agreement with this scenario… cytosolic form invoved in glysolysis while two chloroplastic form are involved in photosyntheis.
  • Clearly matched if we talk about the proteins involved in stress response. Can be catogorised in major category of pro. That protects from aboitic stress. glucose-6-phosphate dehydrogenase pentose phosphate pathway. NADPH form. May be due to cells neccessitytomaintain reducing environment to fight oxidative stress. Cyclophillins primary role to assist protein folding. Unction in membrane association.
  • Over-representation of enzymes involved in electron transport and Calvin cycle seems to be distinctive of the cold response, aimed at protection of plant from photoinhibition. This response is apparently hampered by heat stress, which probably affects thylakoidal structures more severely; on the other hand, the shared proteomic signatures we observed may be part of a common cellular strategy to sustain carbon fixation under general stress conditions.

temperature stress responsive proteins of Arabidopsis. temperature stress responsive proteins of Arabidopsis. Presentation Transcript

  • 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 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 View slide
  • 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 View slide
  • • 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 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.
  • • 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).
  • • 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) ,whereas an 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.