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A Mutant of Arabidopsis Lacking a Chloroplast – Specific Lipid By Browse J McCourt P Somerville CR What are lipids ? (Bile Salt) Lipid classification Fatty acids eg. Palmitic acid Trans C16:1 Storage lipids Triacylglycerols Waxes Glycerol phospholipids lipids Membrane lipids eg. Phosphatidyl glycerol Sphingolipids Cholesterol Other functions Nutrition: Vitamins Signaling: Steroid hormones Eicosanoids Emulsification: Bile salt Lipid classification Fatty acids eg. Palmitic acid Trans C16:1 Storage lipids Triacylglycerols Waxes Hydrophobic Fatty acyl chain lipids Palmitic acid ( C16:0) Hydrophilic Carboxylate 3 (Trans- C16:1 Δ3 t) Lipid classification Glycerol phospholipids eg. Phosphatidyl glycerol lipids Membrane lipids Sphingolipids Cholesterol Head group Phosphatidyl glycerol (PG) R1 = saturated FA R2 = saturated/unsaturated FA Phosphatidyl glycerol (PG) CH2 OH CHOH CH2 Lipid classification Fatty acids eg. Palmitic acid Trans C16:1 Storage lipids Triacylglycerols Waxes Glycerol phospholipids lipids Membrane lipids eg. Phosphatidyl glycerol Sphingolipids Cholesterol Other functions Nutrition: Vitamins Signaling: Steroid hormones Eicosanoids Emulsification: Bile salt Fatty acids Chain Length Fatty acids Degree of unsaturation Short chain =< 6C Medium chain 8 – 12C Long chain 14 - 18C Very long chain >= 20C Saturated - Straight chain Unsaturated – double bond (s) ie. Cis vs. Trans H H H H Fatty acids Packed acyl chains, Semicrystalline -High energy required to separate -Less fluidity Less packed, Liquid crystalline -Less energy required to separate -More fluidity Fluid Mosaic Model of Plasma Membrane Unsaturation on plasma membrane Packed acyl chains, Semicrystalline -High energy required to separate -Less fluidity / trans -Less packed, Liquid crystalline -Less energy required to separate -More fluidity Cis Major hypotheses about compositions of membrane lipids before this paper: 1. Lipid unsaturation increases chilling resistance 2. Lipid unsaturation differences in thermal tolerance between species 3. Trans unsaturation photosynthetic functions Chilling (~ 0-12 C) causes: -Reduced growth and development -Phase transition from the liquid crystalline state to the semicrystalline So, elevated level of cisunsaturation to maintain membrane fluidity in some species, such as Arabidopsis. Chromatography background Thin-layer chromatography (TLC) Gas-liquid chromatography (GC) TLC Mobile phase: solvent Stationary phase: a sheet of glass coated with silica gel Silica gel Less polar More polar Thin-layer chromatography Rf value Silica gel Thin-layer chromatography Recycle the component we want degreaser Silica gel Gas-liquid chromatography Mobile phase: gas Stationary phase: a high boiling point liquid absorbed onto a solid Gas-liquid chromatography retention time TB Solubility TLC GC Advantages: More efficient (time and money) Recycle the sample Advantages: Easy to use get the amount of the sample Disadvantage Detector is limited Disadvantage Destroy the sample Mutant What type of mutant? Alterations in leaf membrane fatty acyl composition. Material Arabidopsis thaliana (L.) Heynh 0.3% EMS (Ethyl methanesulfonate) 16h X 200 μ E/m2/sec 23℃ 70%humidity GC Gas-liquid chromatography Column: 2m 170℃ 10% diethylene glycol succinate Detector: flame ionization detector recessive? heritable? 0.3% EMS 16h X X X genetic complementation X 200 μ E/m2/sec 23℃ 70%humidity One gene? X GC Result No difference of Morphology No difference of growth rate Fatty acid composition changes JB60: no trans-C16:1 A chloroplast-specific mutant Biochemical Phenotypes: - Lacks trans-C16:1 - Elevated level of C16:0 A single nuclear mutation ? A chloroplast-specific mutant P: F1: JB60 No trans-C16:1 X Wild type Trans-C16:1 ½ amount of trans-C16:1 -codominant -suggesting single nuclear gene A chloroplast-specific mutant P: F1: F2: JB60 No trans-C16:1 X Wild type Trans-C16:1 ½ amount of trans-C16:1 trans-C16:1 3 : So, Single nuclear mutant -Mendelian ratio for one gene no trans-C16:1 1 A chloroplast-specific mutant What is unusual about trans-C16:1? 1. Trans-configuration 2. Delta-3 double bond 3. It only attaches to position two of PG !! => compare WT-PG vs. Mut-PG Fig. 1 FA composition of PG Wild type Mutant C16:0 < 34% C16:0 34% Trans -C16:1 20% TransC16:1 0% http://www.wfu.edu/chem/courses/organic/GC/index.html Table 1. FA composition of leaves A chloroplast-specific mutant Proposal: a desaturase specifically converts C16:0 at position two of PG to trans-C16:1 is involved However…no gene was cloned by that time… Name the locus: fadA (=FAD4) Thylakoid membrane Stroma S PS II Appressed membrane of grana LHC II trimer Is fadA a chloroplast-encoded gene ? Exp 1. mutant in chloroplast ribosome No trans-C16:1 Exp 2. add chloroplast protein synthesis inhibitor No trans-C16:1 Exp 3. Mendelian segregation => Not supportive evidence Other evidence Trans-C16:1 Location Etiolated tissue Light-induced chloroplast development LHCP Why are we focusing on the association of LHCP (Light-harvesting chlorophyll a/b protein complex) with trans-C16:1 ? light-induced chloroplast development The structure (trimer): each subunit associates with trans-C16:1-PG The main function of LHCP Contribute to formation of the grana Enhance the capture of light energy the thylakoid ultrastructure Wild type mutant The main function of LHCP Contribute to formation of the grana Enhance the capture of light energy The effects of light intensity PS ΙΙ PS Ι Sensitive fluorescence techniques Aim: Extensive analysis of energy transfer from LHCP to photosystem ΙΙ Result: NO functional difference Since 1985… Specials about trans-C16:1 of PG -only found in thylakoid membranes -always esterified specifically to the sn-2 position of the glycerol backbone of PG -found exclusively in eukaryotic, Chl a/b-containing photoautotrophs Functions of trans-C16:1 of PG -phosphatidylglycerol (PG) and its fatty acid composition play a crucial role in stabilizing the oligomeric state of LHCII (1983). -not involved in energy transfer (1985) -the apparent stability of oligomeric LHCII is dependent not on PG content, but rather on the molecular species composition of PG such that oligomeric LHCII was stabilized when thylakoids exhibit high levels of PG 16:0/trans-Δ3-hexadecenoic acid (trans-16:1) relative to PG 16:0/16:0 (1998) Formation of trans-C16:1 of PG -by a trans-desaturase located within the inner recesses of the thylakoid compartment. (1991) Gene cloned -FAD4 (2009)