What is chlorophyll fluorescence?
The fate of absorbed light
Plants are green because they contain chlorophyll which captures photons and passes them to the photosynthetic reaction centres. Green plants contain two different reaction centres, photosystem I (PSI) and photosystem II (PSII).
Light absorbed by the light harvesting complexes associated with PSII has three possible fates:
1. it can be used to drive photochemistry providing the chemical energy (in the form of ATP and NADPH) for CO2 fixation in the Calvin cycle. PSII extracts electrons from water releasing oxygen in the process.
2. it can be dissipated by a variety of non-photochemical processes (principally as heat)
3. a small proportion is re-emitted as fluorescence. Because some energy is lost in this process the fluorescent light has a longer wavelength (it is redder). If we place appropriate filters in front of a camera we can screen out the illuminating (actinic) light and detect the chlorophyll fluorescence alone. /
These three processes compete with each other. If photochemistry and/or non-photochemical processes are very active then fluorescence will be low. Conversely if these processes are inactive or impaired then fluorescence will be high. Therefore we can use chlorophyll fluorescence as a probe of plant metabolism.
Chlorophyll fluorescence quenching
If a plant is placed in darkness for about an hour the photochemical and non-photochemical processes become inactive. If a light is then turned on chlorophyll fluorescence rises very quickly (within a few seconds) to a maximum value which we call Fm.
Over the next minute or so the photochemical and non-photochemical processes are activated and chlorophyll fluorescence falls quickly - this is called fluorescence quenching.
Eventually a steady state is reached (Fs) and the plant photosynthesises at a constant rate. This is known as photosynthetic induction.
The images opposite show the chlorophyll fluorescence emitted from a wheat leaf at different times following illumination.
Our imaging system uses a CCD camera attached to a microscope. This enables us to take images at very high resolution so that we can examine the responses of individual cells.
An image of chlorophyll fluorescence emitted from a wheat leaf taken at 10x magnification.
Click here for some movies of chlorophyll fluorescence quenching.
By varying the intensity of light falling on the plant from extremely dim (a moonlit night) to extremely bright (several times brighter than full summer sunshine) at different times during photosynthetic induction we can work out the relative proportions of light energy being used in photochemical and non-photochemical processes.
Each quantum of light absorbed by a chlorophyll molecule rises an electron from the ground state to an excited state. Upon de-excitation from a chlorophyll a molecule from excited state 1 to ground state, a small proportion (3-5% in vivo) of the excitation energy is dissipated as red fluorescence. The indicator function of chlorophyll fluorescence arises from the fact that fluorescence emission is complementary to alternative pathways of de-excitation which are primarily photochemistry and heat dissipation. Generally, fluorescence yield is highest when photochemistry and heat dissipation are lowest. Therefore, changes in the fluorescence yield reflect changes in photochemical efficiency and heat dissipation.
Light Absorption by Plant Pigments
We wanted to see if we could measure the absorption spectra of the pigments from various plants so here's what we did: We whipped up some dried leaves in a blender and used acetone to extract the chlorophyll. Then we put a sample in a spectrophotometer and measured how much light was absorbed by the chlorophyll at wavelengths from 400 to 720 nanometers. (Each wavelength of light is a different color.)
We used our TI graphing calculators to graph the data and here's what we got. The top line of this graph represents the absorption of the chlorophyll and it's acetone solvent. The bottom line represents the absorption of the acetone solvent alone. We learned how to subtract the data for the solvent from the data for the chlorophyll/solvent mixture to give us the graph below. (By the way these graphs were done with a TI-92 but the 82 and 83 worked just as well.)
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Here's a final graph for parsley. Find yourself a biology text somewhere and compare the textbook graph with ours. How did we do? (Pretty good we'd say!)
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After extracting pigments from several different types of leaves (green maple, maple leaves that turned yellow in fall, parsley, and red peppers) we connected the calculator to one of our classroom PCs and pulled all of the data into one graph.
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Now, to check your basic knowledge and your graph reading skills, try answering these questions...
1. Which of the colors absorbed by green chlorophyll-bearing leaves is least visible to the human eye?
2. What is the approximate wavelength of this hard-to-see color?
3. What percentage of the violet/blue light is absorbed?
4. How much violet/blue light is being reflected?
5. What percentage of light energy is absorbed in the orange/red spectrum peak?
6. Why, would you say, are there no peaks in the range between about 500 and 600nm for all but the red peppers?
7. Which range of wavelengths/colors is reflected by the leaf? Does this explain the way leaves look?
The first website cited describes an interesting idea that plants are green
because purple Halobacterium evolved before chlorophyll-containing organisms.
The photosynthetic Halobacterium absorbed green light so other organisms
possibly evolved chlorophyll to absorb nongreen wavelengths and fill an
ecological niche. The first two websites both argue that a black chlorophyll
might absorb too much radiation and either overheat the plant or harm the plant
by absorbing destructive UV and x-rays.
It is important to remember that leaves often absorb more than half the green
wavelengths and use them in photosynthesis. It is a widespread misconception
that leaves reflect all green light. That misconception is based on looking at
a chlorophyll absorption spectrum (see second website cited), which is obtained
by extracting chlorophyll into an organic solvent, such as acetone, and
measuring its absorption in a spectrophotometer at wavelengths between 400 and
700 nanometers. The chlorophyll absorption spectrum does show that chlorophyll
in a test tube absorbs only about 2 to 3% of the green light. However, that is
very artificial because a leaf is highly structured. Salisbury and Ross (1985)
note that in the intact leaf, a green photon may not initially be absorbed by a
particular chlorophyll molecule but it is reflected and then gets another
chance to be absorbed, and perhaps another, and another, etc. within the
complex leaf structure that does not exist in a test tube. Thus, each green
photon has many opportunities to be absorbed in the leaf so the total
absorption of green light by chlorophyll is much higher in the leaf than in a
test tube of extracted chlorophyll. Accessory pigments, such as carotenoids,
also absorb green light (see second website cited) and funnel the energy to
chlorophyll.
Instead of a chlorophyll absorption spectrum, people should be looking at a
photosynthesis action spectrum (see third and fourth websites cited) which
shows the amount of photosynthesis at each wavelength. Because plants do absorb
substantial green and yellow wavelengths and use them in photosynthesis, plants
are more efficient than they seem by assuming they only use the red and blue
wavelengths that chlorophyll absorbs in a chlorophyll absorption spectrum.
Photosynthesis is the most widely taught plant biology concept but it is often
taught with numerous misconceptions.
In this endothermic transformation, the energy of the light absorbed by chlorophyll is converted into chemical energy stored in carbohydrates (sugars and starches). This chemical energy drives the biochemical reactions that cause plants to grow, flower, and produce seed.
Chlorophyll is not a very stable compound; bright sunlight causes it to decompose. To maintain the amount of chlorophyll in their leaves, plants continuously synthesize it. The synthesis of chlorophyll in plants requires sunlight and warm temperatures. Therefore, during summer chlorophyll is continuously broken down and regenerated in the leaves of trees.
Paper birch
Another pigment found in the leaves of many plants is carotene. Carotene absorbs blue-green and blue light. The light reflected from carotene appears yellow.
When carotene and chlorophyll occur in the same leaf, together they remove red, blue-green, and blue light from sunlight that falls on the leaf. The light reflected by the leaf appears green. Carotene functions as an accessory absorber. The energy of the light absorbed by carotene is transferred to chlorophyll, which uses the energy in photosynthesis. Carotene is a much more stable compound than chlorophyll. Carotene persists in leaves even when chlorophyll has disappeared. When chlorophyll disappears from a leaf, the remaining carotene causes the leaf to appear yellow.
Low temperatures destroy chlorophyll, and if they stay above freezing, promote the formation of anthocyanins. Bright sunshine also destroys chlorophyll and enhances anthocyanin production. Dry weather, by increasing sugar concentration in sap, also increases the amount of anthocyanin. So the brightest autumn colors are produced when dry, sunny days are followed by cool, dry nights.
When separation is completed, identify the pigment bands by their colors and relative positions on the chromatogram. The major pigments appear in 5 bands: in order, from the origin to the solvent front, they are chl b (olive-green), chl a (blue-green), violaxanthin (yellow), lutein (yellow), and b-carotene (yellow-orange). Click here to view images of the major photosynthetic pigments found in spinach.
Calories in Spinach:
4 oz/100g = 25 calories
Chlorophyll as a Photoreceptor
Chlorophyll is the molecule that traps this 'most elusive of all powers' - and is called a photoreceptor. It is found in the chloroplasts of green plants, and is what makes green plants, green. The basic structure of a chlorophyll molecule is a porphyrin ring, co-ordinated to a central atom. This is very similar in structure to the heme group found in hemoglobin, except that in heme the central atom is iron, whereas in chlorophyll it is magnesium.Click for 3D structure file
There are actually 2 types of chlorophyll, named a and b. They differ only slightly, in the composition of a sidechain (in a it is -CH3, in b it is CHO). Both of these two chlorophylls are very effective photoreceptors because they contain a network of alternating single and double bonds, and the orbitals can delocalise stabilising the structure. Such delocalised polyenes have very strong absorption bands in the visible regions of the spectrum, allowing the plant to absorb the energy from sunlight.
The different sidegroups in the 2 chlorophylls 'tune' the absorption spectrum to slightly different wavelengths, so that light that is not significantly absorbed by chlorophyll a, at, say, 460nm, will instead be captured by chlorophyll b, which absorbs strongly at that wavelength. Thus these two kinds of chlorophyll complement each other in absorbing sunlight. Plants can obtain all their energy requirements from the blue and red parts of the spectrum, however, there is still a large spectral region, between 500-600nm, where very little light is absorbed. This light is in the green region of the spectrum, and since it is reflected, this is the reason plants appear green. Chlorophyll absorbs so strongly that it can mask other less intense colours. Some of these more delicate colours (from molecules such as carotene and quercetin) are revealed when the chlorophyll molecule decays in the Autumn, and the woodlands turn red, orange, and golden brown. Chlorophyll can also be damaged when vegetation is cooked, since the central Mg atom is replaced by hydrogen ions. This affects the energy levels within the molecule, causing its absorbance spectrum to alter. Thus cooked leaves change colour - often becoming a paler, insipid yellowy green.
The electromagnetic spectrum. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
The action spectrum of photosynthesis is the relative effectiveness of different wavelengths of light at generating electrons. If a pigment absorbs light energy, one of three things will occur. Energy is dissipated as heat. The energy may be emitted immediately as a longer wavelength, a phenomenon known as fluorescence. Energy may trigger a chemical reaction, as in photosynthesis. Chlorophyll only triggers a chemical reaction when it is associated with proteins embedded in a membrane (as in a chloroplast) or the membrane infoldings found in photosynthetic prokaryotes such as cyanobacteria and prochlorobacteria.
Absorption spectrum of several plant pigments (left) and action spectrum of elodea (right), a common aquarium plant used in lab experiments about photosynthesis. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Figure 2. Absorption spectrum of isolated chlorophyll and carotenoid species. The color associated with the various wavelengths is indicated above the graph.
Other photosynthetic organisms, such as cyanobacteria (formerly known as blue-green algae) and red algae, have additional pigments called phycobilins that are red or blue and that absorb the colors of visible light that are not effectively absorbed by chlorophyll and carotenoids. Yet other organisms, such as the purple and green bacteria (which, by the way, look fairly brown under many growth conditions), contain bacteriochlorophyll that absorbs in the infrared, in addition to in the blue part of the spectrum. These bacteria do not evolve oxygen, but perform photosynthesis under anaerobic (oxygen-less) conditions. These bacteria efficiently use infrared light for photosynthesis. Infrared is light with wavelengths above 700 nm that cannot be seen by the human eye; some bacterial species can use infrared light with wavelengths of up to 1000 nm. However, most pigments are not very effective in absorbing ultraviolet light (<400 nm), which also cannot be seen by the human eye. Light with wavelengths below 330 nm becomes increasingly damaging to cells, but virtually all light at these short wavelengths is filtered out by the atmosphere (most prominently the ozone layer) before reaching the earth. Even though most plants are capable of producing compounds that absorb ultraviolet light, an increased exposure to light around 300 nm has detrimental effects on plant productivity.