Presentation on theme: "Light Reactions of Photosynthesis"— Presentation transcript:
1 Light Reactions of Photosynthesis Lab 7: The Light Reactions of PhotosynthesisRationale:Today you will investigate some of the fundamental principles underlying photosynthesis, one of the most important physical/chemical processes responsible for life on earth.Biology 171L, Fall 2000
2 Purpose of the lab exercises: Study the Hill Reaction and the effects of DCMU on electron transportDetermine absorption spectrum of chlorophyllObserve fluorescence in chlorophyll
3 Properties of Light Source of energy wave and particle (photons) Wavelength of light: Peak to PeakDifferent wavelengths have different characteristics and energies (wavelength)
4 The Electromagnetic Spectrum Visible portion between 380 and 750 nm Different wavelengths = different colors.Short wavelengths have high energiesLong wavelengths have lower energies
5 Photosynthesis Today, you will examine the Hill Reaction The chemical equation for photosynthesis is:6CO2 + 6 H2O + ENERGY C6H12O6 + 6O2
6 Photosynthesis Two sets of reactions: (1) The light reactions Light energy trapped by chlorophyll(NADPH) and (ATP) are formed in thylakoid membranes2)The Dark ReactionsCalvin Cycle Combines H2O and CO2 to produce sugars in stroma
7 Light Reactions of Photosynthesis Complexes embedded in thylakoid membrane Organized cluster of chlorophyll and proteinsHarvest light energy, resonance transferReaction centers = chlorophyll a + primary electron acceptorTwo Photosystems: PSII and PSIContain chlorophyll a in reaction centerPSII chlorophyll a is P680 (Absorbs 680)PSI chlorophyll a is P700 (Absorbs 700)
8 Light Reactions of Photosynthesis Primary electron acceptorsassociated w/ chlorophyll a of reaction centertraps high-energy electrons (excited)prevent return to ground state.
9 Light Reactions of Photosynthesis Photosystem II (P680) Electrons lost to primary electron acceptorHow are they replaced?Splitting of waterEach water molecule: provides 2 electronsAn atom of oxygenTwo atoms of oxygen form O2What happens to electrons at the primary electron acceptor?
10 Light Reactions of Photosynthesis Electrons move from PS II to PS IElectron Transport ChainPlastoquinone (Pq) Complex of two cytochromes Plastocyanin (Pc)As electrons move through electron transport chainLose energy Lower energy level Produce ATP
11 Light Reactions of Photosynthesis Hill ReactionNamed after Robin HillChloroplast preparations can split water
12 Study of Hill Reaction: DCPIP Colorimetric indicator (DCPIP)Intercepts electrons in electron transport chainBetween Pq and cytochrome complexReduced (gains electrons)
13 Study of Hill Reaction: DCPIP As DCPIP becomes reduced, gradually turns from blue to colorlessOver the 30s intervals, drop in absorbance and readings in spectrophotometer
14 Light Reactions of Photosynthesis Study of Hill Reaction: DCMUDCMUDCPIPDCMU inhibitor of electron transportBlocks passage of electrons from primary acceptor of PS II - plastoquinonePrevents DCPIP from being reducedDegree of inhibition depends on concentrationBiology 171L, Fall 2000
15 Study of Hill Reaction: DCMU High concentrations of DCMU, electrons are almost completely blocked from passing to Pq very little reduction of DCPIP, little change in spec readingsLower concentrations of DCMU, electrons are only moderately inhibited from passing to Pq,DCPIP continues to be reduced
16 Interaction of Light with Matter Light can bereflected transmitted absorbedColor of objects due to reflected or transmitted light Chlorophylls absorb red and blue light Reflects and transmits green light.
17 Pigments: Chlorophyll Pigments Absorb visible lightChlorophyll a and b:Two primary pigments in photosynthesisDiffer slightly in chemical structureChlorophyll molecule
18 Absorption Spectrum Graph of light absorbence vs. wavelength Today you will create your own absorbance spectrum using isolated chlorophyll.
19 Fluorescence in Isolated Chlorophyll But what happens if chlorophyll is isolated from the intact structure of chloroplast, and then illuminated with light?Fluorescence!Isolated chlorophyll molecules
20 Fluorescence in Isolated Chlorophyll Electrons still boosted to higher energy levelsNo electron acceptorThey quickly drop back down to ground stateEnergy released as light and heat.Why does it fluoresce red?
21 Fluorescence in Isolated Chlorophyll Looking at the spectrum, red is associated with lower energyIn returning back to ground state, some energy is lost as heatEnergy of fluorescing light is less than that which illuminated itLonger wavelengths have lower energy
22 Fluorescence in Isolated Chlorophyll Today you will illuminate isolated chlorophyll with different wavelengths (colors) from the visible portions of the spectrumObserve the INTENSITY and red fluorescence.Must use absorption spectrum.Would you expect the intensity of fluorescence to be high, moderate, or low if chlorophyll was exposed to blue light?
23 Fluorescence in Isolated Chlorophyll Chlorophyll absorbs most of the blue light Electrons boosted to higher orbitals fall back to ground state: “ High Fluorescence”What about green light nm) on isolated chlorophyll, the intensity of fluorescence will be low, moderate, or high?Little energy from green light is absorbedMost is reflected or transmittedFew electrons boostedLow!
24 Experiment 1: The Hill Reaction DCPIP (e- acceptor; blue to non-blue)DCMU (e- inhibitor)Experiment 2: Determining the Absorption Spectrum of ChlorophyllSpinach leaf pigment extractExperiment 3: Fluorescence of Chlorophyll Extract in Acetone