I sang of leaves, of leaves of gold, and
leaves of gold there grew;
Of wind I sang, a wind there came and in the branches blew.
Beyond the Sun, beyond the Moon, the foam was on the Sea
And by the Strand of Ilmarin there grew a golden Tree.
-the Lady Galadriel
-Lord of the Rings, J. R. R. Tolkien
In Lord of the Rings, Tolkien uses the imagery of colors to help paint the
picture he wants us to see. The good guys wear white, the bad guys wear black.
When he creates the enchanted forest, Lothlórien, he calls it the Golden
Wood, and makes the leaves gold. He stirs a fair amount of magic into the forest
as well, with Galadriel the wearer of one of the magic rings, but he plays on
our expectations as well. The name itself-The Golden Wood-is alluring on its
own. There are at least two reasons. First, there is the fascination humans
have with gold. Anything golden must be special. Second, the trees are the wrong
color. We are so accustomed to having plants be green that any one that differs
must be special.
So, what is it about green-ness that makes it so important
among plants? Why is it ubiquitous? Sure, there are a few plants with white
patterns amid the green, or with red or yellow superimposed on the green, but
the basic color is green. Why?
We can answer this question at two levels. One, at the "proximate" level, would address the physical/chemical mechanism that results in green-ness. The other, at the "ultimate" level, would address the evolutionary history that allowed this situation to develop. Let's think first about the ultimate level, and do so by imagining things that might have happened.
Once upon a time, long, long ago...
We are standing on the shore of a shallow sea in the Precambrian,
looking at the wild world around us. The earliest single-celled things have
long since disappeared, and have given rise to new, and fantastic, kinds of
cells. There are bacteria that can swim, and strange new cells that are huge
by contemporary standards, and that have a membrane around their DNA. Most amazingly,
some of the bacteria synthesize a light-absorbing pigment, from which they can
extract some of the sun's energy. Photosynthesis has begun.
But life is perilous. These huge, nucleated cells are predators,
and can surround and consume the smaller bacteria. Glup! A eukaryotic cells
swallows a bacterium. Glup! Another one. Glu...hmm. This one got stuck, and
didn't get digested. What's going to happen? Will it kill the cell that swallowed
it?
By some strange mechanism like this, one of the photosynthetic bacteria (cyanobacteria) became a denizen in the cytoplasm of a eukaryotic cell. The eukaryote divides with its extra baggage, and the prokaryotic symbionts divide to fill the cytoplasm with as many cells as they can, somehow reaching a stable truce. Is this novel cellular symbiosis going to be too much for either partner, and cause the strain to die out? Time will tell.
It proved, eventually, that the symbiosis was productive.
The symbiotic bacterium could harvest light energy, and use it for growth. It
also spilled some of the food it had produced using the light energy into the
surrounding cytoplasm, thereby helping its "host" cell grow, too.
This turned out to be so successful a partnership that the strain grew well,
indeed. In time, the prokaryotic symbionts lost a significant portion of their
DNA. Some of the prokaryotic genes were no longer needed; if they were lost
by accidental deletion during DNA replication, no harm was done. Some of the
genes were transferred-mechanism unknown-to the nucleus, where they reside to
this day. Not all of the genes were lost, however, so present-day chloroplasts
still retain some of their own genetic information, descended from the original
symbiotic bacteria.
If we imagine the history of the chloroplast, somewhat along
these lines, we make two rather striking, and conflicting observations. First,
the first ancestral photosynthetic eukaryotic cells diversified into the large
number of different plant and algal species that now exist. This implies that
a huge number of genetic changes have occurred. The second observation is that
all these species use the same green pigment to carry out photosynthesis-chlorophyll.
This implies that very few genetic changes have occurred. How can we reconcile
these different implications?
Remember how Natural Selection works. Populations of organisms
exhibit genetic variation. Some combinations of genetic traits are more successful
than others. Those individuals with more successful traits are more likely to
reproduce and leave more surviving offspring, while those with less successful
traits are more likely to leave fewer offspring. As the successful traits are
inherited, and the unsuccessful traits are lost, the overall character of the
population evolves.
In this context, let us think about the evolution of plants.
In the early days of Life, before photosynthesis, most of the earth's oxygen
was tied up in water, CO2, and rocks. There was no atmospheric oxygen, and no
ozone. Ultraviolet light was intense, and could easily damage and mutate the
single cells that were there. Mutations must have occurred frequently, generating
the genetic diversity upon which Natural Selection could act. In terms of the
species diversity that we now see among plants and algae, we imagine a scenario
something like the following. In some population, appropriate mutations occurred
to allow single-celled photosynthetic eukaryotes to associate with one another
and cooperate. Cooperation was successful, and eventually gave rise to true
multicellular organisms. Similarly, mutations occurred that allowed some species
to withstand dessication, which eventually led to the ability of plants to invade
the land. One can easily imagine the rest of the scenario, with the virtual
explosion of different shapes and sizes of plants, as different genetic variations
were selected in different environments.
Through all of this, the appearance of the cyanobacteria, of green algae, of plants, and the diversification of plants, the mechanism of harvesting light energy remained the same. Apparently, the green pigment (chlorophyll) was good enough at this job that no significant changes were selected. Is this because the green pigment is the "best" or the only pigment that can absorb light and drive photosynthesis? No, it is not. There are photosynthetic bacteria that use red and purple pigments, and that photosynthesize just fine. Apparently, several different pigments work well to capture light energy, yet it is the green chlorophyll that is used in all plants, even though green cholorphyll is not necessarily the "best" photosynthetic pigment. What has constrained the course of evolution in this way?
First, there is the historical accident that the original
prokaryote that gave rise to the symbiotic eukaryote happened to be one with
a green pigment, rather than one of those with red or purple. Therefore, all
the descendents of this early strain of photosynthetic cells had only one pigment
to work with.
Second, this photosynthetic pigment could be altered only insofar as it was
possible to change its structure and still maintain its ability to function
in photosynthesis. This is an interesting thought: Natural Selection can't so
just anything. It can act only on the variation present within a population,
and this variation in turn is constrained by both the mechanisms of chemistry
and genetics.
Let us elaborate on this issue with an example. Suppose that
the green pigment works really well, but an orange pigment might work a whole
lot better. Can Natural Selection necessarily lead to the use of that orange
pigment instead? This depends on what has to be altered. If the chemistry of
the orange pigment is very similar to the chemistry of the green pigment, so
that only minor changes are required, then it may be possible through mutation
to alter the activity of a few of the enzymes involved in the synthesis of the
pigment so that the orange pigmant is now made rather than the green one. However,
if the chemistry is sufficiently different, then it may require many mutations
to change the pathway of chemical synthesis so that it creates the orange pigment
instead of the green one. Imagine that it takes four separate mutations to convert
the biosynthetic pathway making the green pigment to one making the orange.
It is inconceivable that all four will ever occur in a single step-the probability
is just too low. Instead, the four mutations are likely to occur independently.
But, if any one of them prevents photosynthesis from working, or even makes
photosynthesis work less well, then the mutant organism will be unfit, and will
be selected against before the next three mutations have a chance to occur.
So, there may be constraints that have maintained chlorophyll
as the photosynthetic pigment throughout plant evolution. Does this mean that
plants are stuck with only a fixed mechanism of photosynthesis, regardless of
the environment in which they find themselves, or are there other ways that
selection can operate, other kinds of adaptations that can arise, for plants
to maximize their ability to utilize light energy?
There are many such adaptations. Plants that are grown in dim light respond by significant morphological changes. For many plants, dim light causes them to extend, and grow long and spindly. They are in effect reaching up, trying to grow into a region where there is more light. For many plants, dim light causes the leaves to develop differently. Dim-light leaves are extra long, extra thin, and extra broad. This gives them the maximum surface area possible, so they can capture as much light per leaf as possible. Plants also bend and twist to face the direction from which the light shines on them most strongly. Again, this maximizes their ability to capture light. Plants are fascinating things, that display a huge variety of behavioral and developmental traits to maximize their fitness.
Now let's turn to the proximate issues: just how does chlorophyll
make plants green, and what good does it do for the plant? Let's design an experiment
or two to find out how this works. We'll ask several questions-
1. What pigments are in leaves and what is their relative
absorption of different colors (wavelengths) of light ?
2. Does chlorophyll really absorb light energy and use it
to drive a chemical reaction?
3. What wavelengths of light are most effective at driving
photosynthesis?
To answer these questions, we will need to do several different
kinds of experiments, but they will all work together to give us a good understanding
of what's going on inside leaves. The first question can be addressed fairly
easily. Just extract the pigments, separate them, and see what they are. The
second is much more complex.
What kind of experimental approach might work for the second
question? Well, we need a way to measure a chemical reaction that depends on
light. To do this, perhaps we can isolate some chloroplasts, which are the organelles
that actually carry out the reaction, and then incubate them in an appropriate
mix of chemicals to "visualize" the light-dependent reaction. The
Lab Manual describes in some detail how to do this; we'll leave the details
there for now, and just work with the concept. Suppose you do this experiment,
and, indeed, observe a light-dependent reaction. Does this answer the question?
No. What it tells us is that isolated chloroplasts can carry out a light-dependent
reaction. This is only half of what we want to know. We also need to know whether
chlorophyll itself absorbs light energy. To address this question, we need to
isolate chlorophyll away from the rest of the chloroplast, and assay its ability
to absorb light energy.
Unless you have done sonmething like it before, it is not
obvious how to assay the absorption of light energy by chlorophyll. Let's think
about what chlorophyll does, and see if we can figure it out. First, the chlorophyll
molecule absorbs a photon of light. This increases the energy state of the chlorophyll
molecule, putting it into what is called an "excited state." From
the excited state, chlorophyll can either transfer the energy to the photosynthetic
system (i.e., start a chemical reaction), or it can release the energy again
and do nothing. In the chloroplast, the photosynthetic system is present and
properly arranged; the energy drives the chemical reaction. With isolated chlorophyll,
there is nothing to accept the energy, so it must be released.
When molecules that have been excited by absorbing light
release the energy, they do so by a reversal of light absorption: they emit
light, or "fluoresce." Isolated chlorophyll, exposed to light, fluoresces.
Just shine a light on it and look-it glows red. This shows that it has absorbed
the light, because it can re-emit it. Are whole chloroplasts fluorescent? No.
The chlorophyll absorbs the light, but instead of re-emitting the energy as
fluorescence, it passes the energy on to the other components in the chloroplast.
Here, then, in these two kinds of experimental results, we have the data to answer the second of our questions. Think this through, and be sure the reasoning is sound.
The third question requires only a simple variation on the
technique mentioned above to measure the light-dependent reaction: use light
of different wavelengths. It should be pretty easy. If you are going to do it,
though, you need to remember that a critical requirement of a good experiment
is that you are able to isolate the effect of a single factor (e.g., the wavelength
of light). To illustrate this, consider two students who set out to test whether
blue light or red light is more effective in driving photosynthesis. One of
the students exposes the chloroplasts that she extracted from a spinach leaf
to red light, while the second student exposes the chloroplasts that he extracted
from a spinach leaf to blue light. When the two students compare results they
find that under red light, the reaction proceeded more rapidly. After thinking
about their results, however, the two students realize that the student who
tested the effectiveness of red light used a more concentrated suspension of
chloroplasts. In addition, the student using blue light had let his solution
warm up to room temperature during the isolation process, so that the enzymes
in his solution may have been partially inactivated and, therefore, may have
had lower activity. To further complicate interpretation of the results, they
realize that the red light was brighter than the blue light!
If you think about this example for a few minutes, it should be clear that the results obtained by the two students give absolutely no indication of whether blue or red light is more effective in driving photosynthesis. The more rapid rate of reaction in the sample exposed to red light could have been due to any one of the following factors: a) the difference in the wavelength or color of the two lights, b) the difference in the brightness of the two lights, c) the difference in the concentrations of the two suspensions of chloroplasts, or d) the difference in the activity of the two chloroplast suspensions . In other words, there are at least four different hypotheses that are consistant with the results. So, for example, the hypothesis that brighter light increases the rate of photosynthesis can explain the results just as well as the hypothesis that red light is more effective than blue light in driving photosynthesis. How would you design an experiment so that any difference in the rate of photosynthesis in the two solutions could be attributed only to the difference in the wavelength of light, rather than to some other factor? Consider what variables will need to be held constant, or " controlled," during the experiment.
We have still not learned why plants are green. We've talked about what chlorophyll does, and we've talked about the evolutionary history of green-ness, but there is one thing left unsaid. If a plant is green, it must have green pigments. If the pigments are green, is it because they absorb green light? Or, is it because they absorb every color except green light? Before your discussion session, design an experiment to find out.
This Discussion Topic, like the others this semester, is
designed to relate to this week's laboratory experiment in a very specific way.
Here, the connections are of two kinds: direct experimental logic (eg. the two
students and their chloroplast suspensions), and extrapolation to the evolutionary
context in which these experiments fit. Other discussions may have more of "the
big picture" and less direct experimental logic, or may present a different
example of the same principles upon which the experiments are based. If it is
not entirely clear to you what the relationship is to the lab, ask your AI or
the professor.
Also remember: because the discussion topics are written to prepare you for the laboratory experiment that follows, it may be quite helpful if you read the Lab Manual before you come to the discussion section. Even if you don't read it before discussion, you absolutely must read the Lab Manual before you come to the lab. There just won't be time to do the experiments if you are reading the Lab Manual for the first time.
Answer the following questions before coming to discussion.
This is imporant: for some of the discussions, you will be asked to write out
an answer to one of the questions as a "5-minute essay." If you have
written out answers before you come to the discussion section, you will be able
to do this easily. We also encourage you to get together with your friends to
talk about these questions. They can help you, and you can help them. For additional
advice, feel free to talk to your AI or to the professor; we are here to help.
Questions aimed at understanding the reading:
Note: these questions are here to help you see what we think are the "important
points" of the material you have just read. You should be able to answer
these without having to look up any additional information. However, if you
feel it would be helpful to check your L111/L112 textbook, check the syllabus
for the page numbers that are relevant.
1. What kinds of constraints can influence the direction of evolution? (This is sophisticated phrasing. Try breaking it down into these smaller questions: (a) how can chemistry enable some kinds of evolutionary changes but not others, and (b) is it always easy to evolve from a "good" trait to a "better" trait when multiple mutations are required?)
2. What evidence would be needed to determine whether the chlorophyll in chloroplasts absorbs light, and transfers the light energy to other chloroplast components to drive a chemical reaction?
3. For the experiments you will do in the lab that is coming up, identify as many variables as you can that need to be controlled carefully. Indicate how each one might change the results to make them hard to interpret.
More interesting questions:
Note: these questions are here to help you relate the reading to the laboratory
experiment itself, or to "the big picture." You will find that these
"extrapolation" questions may not have a single right answer; rather,
you can answer the questions in several different ways. You will do best with
these questions if you present your ideas and support them with explanations
and logic. Your assignment here is to think about the science, and make connections
among things you have learned, not just list facts you may have memorized.
4. Why is it necessary to convert light energy to chemical energy in order for energy to be useful to the living cell?
5. If chlorophyll, with its specific chemical properties, allows only certain kinds of chemical adaptations, then what other adaptations might plants use to maximize their light uptake? Below is a list of several plant traits that may, or may not, be familiar to you. Think as carefully as you can about each of them, and write out a discussion of one of them, answering the question, "If this trait is an adaptation for maximizing light uptake, how would it help increase the fitness of a plant in the wild?" (Feel free to write discussions of all them!)
a) Plants bend toward, and grow toward, light. (Phototropism)
b) Germinating seeds elongate dramatically in the dark. (Etiolation)
c) Plants grown in shade elongate their stems between leaf nodes-particularly true of vines.
d) Some plants secrete toxic compounds that kill other plants nearby, or inhibit germination of other plants' seeds. (Allelopathy)
e) "Weedy" plants (e.g., milkweed, dandelion) have seeds that disperse long distances, and grow and produce seeds very rapidly, usually within one growing season.
f) "Ephemeral" species, like toothwort and rattlesnake lilies can be found in the woods of Southern Indiana every spring. They grow leaves very early in the spring, well before the trees do, then flower and die back in only a few weeks. Then they go dormant.
You might notice that, for some of these traits, there could be additional selective pressures besides maximal utilization of sunlight that might have influenced their evolution. Consider these in your discussion.
6. Plants are green. What colors of light do you expect them to be able to absorb? What colors do you expect them to reflect? Why? On the basis of this information, what hypothesis can you propose about how the color of light should affect photosynthesis?
Note: You may think that this last question is nutty, as
it asks you to figure out the result of the experiment before you have done
the experiment. Actually, this is not nutty, as is explained in Section A of
your Lab Manual under the subheading "Designing an Experiment." To
design an experiment, it is necessary to have a good idea of the hypothesis
you intend to test. You need to predict the result of the experiment in order
to assess whether the way you intend to do it would, or would not, work well.
This also helps you determine which variables it is essential to control to
avoid an ambiguous answer. It is very easy to design an experiment that gives
an ambiguous answer; designing one that is clear-cut is more difficult. Questions
like these that ask you to think about the experiment before you do it are aimed
at helping you learn how to design experiments that are clear-cut, rather than
ambiguous. Remember, later in the semester you will have the opportunity to
do an experiment of your own design (your independent project). It would be
disappointing if your experiment turned out to be ambiguous.