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Light-independent (dark) reactions occur in the stroma of the chloroplast (pH 8) Consumes ATP & NADPH from light reactions regenerates ADP, Pi and NADP+ fixing CO2 1) RuBP binds CO2 fixing CO2 1) RuBP binds CO2 2) rapidly splits into two 3-Phosphoglycerate • therefore called C3 photosynthesis Reversing glycolysis G3P has 2 possible fates 1) 1 in 6 becomes (CH2O)n 2) 5 in 6 regenerate RuBP Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell) or is converted to DHAP & exported to cytoplasm to make sucrose Pi/triosePO4 antiporter only trades DHAP for Pi Regenerating RuBP Basic problem: converting a 3C to a 5C compound must assemble intermediates that can be broken into 5 C sugars after adding 3C subunit Regulating the Calvin Cycle Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+] c) CO2 is an allosteric activator of rubisco that only binds at high pH and high [Mg2+] also: stomates open in the light Regulating the Calvin Cycle Several Calvin cycle enzymes (e.g.Fructose-1,6bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark in light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme How light reactions talk to the Calvin cycle SH SH light 2Fdox PSI + PSII 2e- 2Fdred reduced thioredoxin S-S oxidized thioredoxin S-S oxidized enzyme (inactive) SH SH reduced enzyme (active) PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate Releases CO2 without making ATP or NADH PHOTORESPIRATION Releases CO2 without making ATP or NADH Called photorespiration : undoes photosynthesis PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon Both rxns occur at same active site PHOTORESPIRATION C3 plants can lose 25%-50% of their fixed carbon phosphoglycolate is converted to glycolate : poison! Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine produce H2O2 Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 Why photorespiration loses CO2 Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 5) serine is returned to peroxisome Detoxifying Glycolate 1) glycolate is shuttled to peroxisomes 2) peroxisomes convert it to glycine 3) glycine is sent to mitochondria 4) mitochondria convert 2 glycine to 1 serine + 1 CO2 5) serine is returned to peroxisome 6) peroxisome converts it to glycerate & returns it to chloroplast Detoxifying Glycolate Why peroxisomes are next to cp and mito in C3 plants Mitochondrion Lipid metabolism Most are glycerolipids: fatty acids bonded to glycerol GLYCEROLIPIDS Triacylglycerols = FAs on all 3 C • store energy GLYCEROLIPIDS Bond FA to glycerol Diacylglycerols = FAs on 2 Cs, headgroup on C 3 GLYCEROLIPIDS Diacylglycerols = FAs on 2 Cs, headgroup on C 3 Form bilayers in water Lipid metabolism Unique aspects in plants Make fatty acids by same set of reactions, but in plastids with a prokaryotic fatty acid synthase 12 proteins, cf one multifunctional protein Lipid metabolism Make fatty acids in plastids with a prokaryotic FAS • 12 proteins, instead of one multifunctional protein • Assemble some lipids in CP, others in ER Lipid metabolism Make fatty acids in plastids with a prokaryotic FAS • 12 proteins, instead of one multifunctional protein • Assemble some lipids in CP, others in ER • Acetyl-CoA carboxylase is also prokaryotic = 4 subunits, except in grasses (profoxydim & other grass herbicides inhibit ACCase) Lipid metabolism • Acetyl-CoA carboxylase is also prokaryotic = 4 subunits, except in grasses (profoxydim & other grass herbicides inhibit ACCase) • Same biochem, but diff location and enzymes Lipid metabolism • Acetyl-CoA carboxylase is also prokaryotic = 4 subunits, except in grasses (profoxydim & other grass herbicides inhibit ACCase) • Same biochem, but diff location and enzymes • In light cp make lots of NADPH, and leaves are main sinks for FA Lipid metabolism • Acetyl-CoA carboxylase is also prokaryotic = 4 subunits, except in grasses (profoxydim & other herbicides inhibit ACCase) • Same biochem, but diff location and enzymes • In light cp make lots of NADPH, and leaves are main sinks for FA • But, each cell makes its own FA, so NADPH in other cells comes from Pentose-Pi shunt Lipid metabolism Source of acetyl-CoA is controversial • Most comes from plastid PDH Lipid metabolism Source of acetyl-CoA is controversial • Most comes from plastid PDH • Some comes from cytoplasmic acetate; activated in cp • Also used to make sterols, some amino acids, many others Lipid metabolism Source of acetyl-CoA is controversial • Most comes from plastid PDH • Some comes from cytoplasmic acetate; activated in cp • Also used to make sterols, some amino acids, many others • Why ACCase is “committed step” Lipid metabolism Assemble some lipids in CP, others in ER • “16:3 plants” assemble lipids in cp using FA-ACP = prokaryotic pathway (“primitive”) Lipid metabolism “16:3 plants” assemble lipids in cp using FA-ACP = prokaryotic pathway (“primitive”) “18:3 plants” export FA, assemble lipids in ER using FA-CoA = eukaryotic pathway (“advanced”) Lipid metabolism “16:3 plants” assemble lipids in cp using FA-ACP = prokaryotic pathway (“primitive”) “18:3 plants” export FA, assemble lipids in ER using FA-CoA = eukaryotic pathway (“advanced”) Substrates for most desaturases are lipids, not FA! Lipid metabolism Unique aspects in plants Chloroplasts have lots of galactolipids: sugar linked directly to diacylglycerol Lipid metabolism Unique aspects in plants Chloroplasts have lots of galactolipids: sugar linked directly to diacylglycerol: saves PO4 Lipid metabolism Unique aspects in plants Chloroplasts have lots of galactolipids: sugar linked directly to diacylglycerol : saves PO4 A) MGDG (Monogalactosyl diacylglycerol) 50% cp -> most abundant lipid on earth! Lipid metabolism Unique aspects in plants Chloroplasts have lots of galactolipids: sugar linked directly to diacylglycerol : saves PO4 A) MGDG (Monogalactosyl diacylglycerol) 50% cp B) DGDG (Digalactosyl diacylglycerol) 28% cp Lipid metabolism Chloroplasts have lots of galactolipids: sugar linked directly to diacylglycerol : saves PO4 A) MGDG (Monogalactosyl diacylglycerol) 50% cp B) DGDG (Digalactosyl diacylglycerol) 28% cp C) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cp Lipid metabolism Chloroplasts have lots of galactolipids: sugar linked directly to diacylglycerol : saves PO4 A) MGDG (Monogalactosyl diacylglycerol) 50% cp B) DGDG (Digalactosyl diacylglycerol) 28% cp C) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cp • Very unsaturated! Lipid metabolism Chloroplasts have lots of galactolipids: sugar linked directly to diacylglycerol : saves PO4 A) MGDG (Monogalactosyl diacylglycerol) 50% cp B) DGDG (Digalactosyl diacylglycerol) 28% cp C) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cp • Very unsaturated! • Makes membranes very fluid Lipid metabolism Chloroplasts have lots of galactolipids: sugar linked directly to diacylglycerol : saves PO4 A) MGDG (Monogalactosyl diacylglycerol) 50% cp B) DGDG (Digalactosyl diacylglycerol) 28% cp C) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cp • Very unsaturated! • Makes membranes very fluid • Source of 3 FA Lipid metabolism Unique aspects in plants 1) Make fatty acids in plastids 2) large amounts of galactolipids 3) Oleosomes: oil-storing organelles with only outer leaflet Lipid metabolism Oleosomes: oil-storing organelles with only outer leaflet • Put oils between the leaflets as they are made Lipid metabolism Oleosomes: oil-storing organelles with only outer leaflet • Put oils between the leaflets as they are made • Add oleosin proteins to outside: curve the membrane Lipid metabolism Oleosomes: oil-storing organelles with only outer leaflet • Put oils between the leaflets as they are made • Add oleosin proteins to outside: curve the membrane • Oils often have unusual fatty acids Lipid metabolism Use fats to store carbon as well as energy!!?? • Recover C via glyoxylate cycle & gluconeogenesis Lipid metabolism Biological roles • Plasma membrane lipids help survive freezing Lipid metabolism Biological roles • Plasma membrane lipids help survive freezing • Unacclimated cells vesiculate as they lose water & pop when it returns Lipid metabolism Biological roles • Plasma membrane lipids help survive freezing • Unacclimated cells vesiculate as they lose water & pop when it returns • Acclimated cells shrivel & reswell Lipid metabolism Biological roles • Plasma membrane lipids help survive freezing • Mito lipid composition may also influence chilling sensitivity • CS plants (eg bananas) are damaged at 10˚ C Lipid metabolism log respiration (nmol O2/min/mg protein) Biological roles • Plasma membrane lipids help survive freezing • Mito lipid composition may also influence chilling sensitivity • CS plants (eg bananas) are damaged at 10˚ C • Mito show defects at <10˚ C not seen in other plants 3.0 B) SB-H mitochondria A) fr2 x fr1 mitochondria 34° 2.8 2.6 18° 2.4 35° 25° 24° 2.2 2.0 16° Control 17:0 18:1 Control 17:0 18:1 1.8 3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.15 3.60 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60 1/T (°K x 1000) 1/T (°K x 1000) Lipid metabolism CS plants (eg bananas) are damaged at 10˚ C • Mito show defects at <10˚ C not seen in other plants • Membrane lipids show phase changes at these T Lipid metabolism CS plants (eg bananas) are damaged at 10˚ C • Mito show defects at <10˚ C not seen in other plants • Membrane lipids show phase changes at these T • Blamed on saturated PG log respiration (nmol O2/min/mg protein) Lipid metabolism Biological (& commercial) roles • Plasma membrane lipids help survive freezing • Mito lipid composition influences chilling sensitivity • Mito show defects at <10˚ C not seen in other plants • unsaturated FA did not fix CS, but saturated FA made it worse: reason for GM desaturases 3.0 B) SB-H mitochondria A) fr2 x fr1 mitochondria 34° 2.8 2.6 18° 2.4 35° 25° 24° 2.2 2.0 16° Control 17:0 18:1 Control 17:0 18:1 1.8 3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.15 3.60 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60 1/T (°K x 1000) 1/T (°K x 1000) Lipid metabolism Other commercial aspects • Yield and quality of seed oil is very important:12 million tons/year Lipid metabolism Other commercial aspects • Yield and quality (especially unsaturation) of seed oil is very important:12 million tons/year • Want more double bonds, especially -3, for health • Want less double bonds for shelf life and taste • Each double bond increases p(oxidation) 40x Lipid metabolism Other commercial aspects • Yield and quality (especially unsaturation) of seed oil is very important:12 million tons/year • Want more double bonds, especially -3, for health • Want less double bonds for shelf life and taste • Each double bond increases p(oxidation) 40x • Have GM oils with more & less double bonds Lipid metabolism Other commercial aspects • Yield and quality of seed oil is very important:12 million tons/year • Also have markets for many specialized oils Lipid metabolism Other commercial aspects • Yield and quality of seed oil is very important • Also have markets for many specialized oils • Have genetically-engineered many crops to alter seed oils or produce specific fats Lipid metabolism Biofuels are now very fashionable • Biodiesel = fatty acid methyl esters • Trans-esterify oils to make them volatile Lipid metabolism Biofuels are now very fashionable • Biodiesel = fatty acid methyl esters • Trans-esterify oils to make them volatile • Projected to be 10% of european diesel in 2015 Lipid metabolism Biofuels are now very fashionable • Biodiesel = fatty acid methyl esters • Trans-esterify oils, used cooking oil, etc • Projected to be 10% of european diesel in 2010 • Also use coconut & other oils directly in diesel engines Lipid metabolism Also use coconut & other oils directly in diesel engines • Just need to be sufficiently fluid to reach cylinder Lipid metabolism Also use coconut & other oils directly in diesel engines • Just need to be sufficiently fluid to reach cylinder • Add double bonds to fatty acids or make them shorter