The Green Revolution

Diversity of Photosynthetic Organisms

Introduction
Take a deep breath. The oxygen that you breathe to power your aerobic metabolism
was stripped from a water molecule by a photosynthetic organism. We learned in BIOL
1500 that photosynthesis can be broadly summarized with the equation:

6CO2 + 6H2O ------> C6H12O6 + 6O2

We also learned that photosynthesis begins when oxygen is stripped from a water
molecule, a process that requires energy- solar energy. The oxygen gets released as a waste
product- the remaining hydrogens and associated electrons are used to generate the
NADPH and ATP that ultimately power the assembly of carbohydrates during the Calvin
cycle. The cellular machinery associated with photosynthesis is complex and remains the
object of intense study- we still have a lot to learn about the process that made our
existence possible.

Photosynthesis is the basis for almost every ecosystem on Earth. It allows energy
from the sun to be captured stored chemically as biomass, which is then consumed,
transferred throughout food webs, decomposed, and recycled. As a heterotroph, you can
trace the energy that built your body and powers your metabolism to the organisms you
eat, who in turn built their bodies using energy from other organisms, and so on… until you
get to the origin of all that energy: sunlight, captured by the photosynthetic organisms that
are the foundation of most food webs. In the end, we are all powered by solar energy.

Photosynthesis has also transformed the Earth. Geological evidence suggests that
the atmosphere of early Earth contained almost no oxygen. Early photosynthetic organisms
diversified and became so abundant that oxygen began to accumulate in the atmosphere,
which likely led to the mass extinction of obligate anaerobic organisms worldwide. This
Great Oxygenation Event (GOE) also triggered a period of prolonged cooling, nicknamed
Snowball Earth, which lasted more than 300 million years. From our perspective, the GOE
had two major consequences. First, it resulted in the marginalization of the anaerobic
organisms that had previously dominated Earth, confining them to isolated anoxic habitats.
Second, it created an evolutionary advantage for organisms that could not only tolerate
oxygen, but use it metabolically to their great advantage: the stage was set for the adaptive
radiation of aerobic organisms, including early eukaryotes.

BIOL 1510 | Lab 03: The Green Revolution –Lab Manual 1

 The ability to use solar energy to split water molecules evolved once, in the
ancestors of modern-day cyanobacteria, at least 2.5 billion years ago (and possibly longer).
All other photosynthetic organisms- from algae to Euglena to plants- acquired
photosynthesis by forming a symbiotic association with cyanobacteria, or with organisms
that had previously formed such an association. The chloroplasts of green algae, red algae,
and plants are derived from formerly free living cyanobacteria. The plastids of brown algae
and several other protists (including Euglena) are derived from formerly free-living
photosynthetic algae.

In this laboratory, we survey some of the major photosynthetic taxa, with a special
emphasis on the land plants. We also look closely at some of the major adaptations that
have marked their evolution.

Learning outcomes for this laboratory exercise

1. List and describe the key photosynthetic taxa.

2. Identify the major features of these taxa.
3. List the adaptations that have contributed to the success of the angiosperms
(flowering plants).

Possible Pre-lab readings

Bateman, Richard M., et al. "Early evolution of land plants: phylogeny, physiology, and
ecology of the primary terrestrial radiation." Annual Review of Ecology and Systematics
(1998): 263-292.

Materials needed
Compound and dissecting microscopes Mnium sp. (moss) spore capsule cross
Glass slides and cover slips section
Light source (use detached lights from Pine pollen
dissecting scopes)
Food coloring (or methylene blue) Dried or preserved specimens
Scalpel Dried moss sporophyte
Beakers Seeds (various)
Tweezers Dry Fruits (various)

Prepared slides: Live organisms:

Nostoc sp. (use the Carolina slides w/dark Cultured Volvox sp., Chara sp.
blue writing b/c those have heterocysts) Celery (fresh)
Merismopdeia Leaves from Parker gardens
Monocot and dicot stems in cross section Live Mnium sp. (moss)
Lilac and / or corn leaf in cross section Fresh flowers
Mnium sp. (moss) antheridium Fleshy fruits (various)
Mnium sp. (moss) archegonium

BIOL 1510 | The Green Revolution –Lab Manual 2

Lab Methods

Exercise 1. The first photosynthetic organisms: cyanobacteria

Cyanobacteria photosynthesize like plants- water is the electron donor, their primary
pigment is chlorophyll a, they have two photosystems (II and I), and so on. One difference
is that cyanobacteria use accessory pigments such as blue phycobilin and red
phycoerythrins that make them appear reddish, green, or blue green (hence their former
name, bluegreen algae). Cyanobacteria can be found worldwide in marine, freshwater, and
terrestrial habitats. The various species can occur as single cells, or in multicellular lines
(filaments) or clumps (colonies).

• Examine prepared slides of Nostoc and Merismopedia. Draw them in the appropriate
box in your worksheet. What color are they? Are they unicellular, colonial, or
filamentous? Some filamentous cyanobacteria have specialized, enlarged
nonphotosynthetic cells called heterocysts that contain the enzyme nitrogenase,
allowing them to fix atmospheric nitrogen (N2) into a more usable form (NH4+). Do
either Nostoc or Merismopedia have heterocysts? If so, clearly identify them on your
drawings.

Photosynthetic protists
The Euglena you examined in the Microbial World lab was an example of a unicellular
photosynthetic protist. Other taxa of global significance include the dinoflagellates,
notorious for their occasional population spikes called "red tides", and the diatoms, that
live in intricate glassy shells made of silica (glass). These two taxa are ecologically
important, forming the basis of many food webs, particularly in marine environments. In
this exercise, however, we will focus on the colonial and multicellular photosynthetic
protists.

Volvox is a freshwater genus of green algae that forms spherical colonies of thousands of
cells. They are part of an Order, the Volvocales, whose members show differing degrees of
the cell specialization and division of labor that characterizes truly multicellular organisms.
They form colonies of four to many thousands of cells, and the larger forms exhibit some
division of labor and coordination. Flagella beat synchronously, colonies have a clear
"front" and "back", and only a subset of the cells are capable of reproduction. Daughter
colonies can be produced asexually or sexually and are protected within the larger mother
colony, giving these organisms the appearance of "spheres within spheres".

• Prepare and examine a wet mount from a living culture of Volvox. Observe the mode
of locomotion, and look for developing daughter colonies. To observe coordinated

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 movement, turn off the light source for your microscope, and place a strong light
source to one side of the colonies. Observe and describe their swimming
behavior.

Chara sp. are also freshwater green algae, but unlike Volvox they are truly multicellular. Of
the photosynthetic protists, they are the most closely related to the land plants (they are
considered representative of the plants' green algae ancestors). The body form is
superficially similar to that of early land plants, and they have many features in common
(e.g., identical chloroplasts, similar cell walls). However, Chara stems consist of giant
multinucleated cells, and the leaves are actually clusters of small "branchlets" appearing at
distinct nodes along the main axis of the body. Root-like rhizoids anchor Chara to the
substrate. The thallus often displays specialized structures located at the nodes that produce
male or female gametes: the male structures (antheridia) are round and produce flagellated
sperm, whereas the female structures (oogonia) are oval and produce eggs.

• Examine the Chara under a dissecting microscope. Draw the specimen, indicating any
structural details outlined above. Can you see antheridia or oogonia?

The major significance of Chara is that it possesses many ancestral features of the earliest
land plants, and is therefore considered a model organism for what the earliest land plants
may have looked like. Indeed, some of the oldest plant lineages have members that still
superficially resemble Chara, most notably the horsetails (Equisetum) in the prehistoric
gardens outside Parker.

Land plants
The earliest photosynthetic organisms to move onto land benefited from direct access to
sunlight, elevated CO2 levels, and shelter from aquatic herbivores. However, they did face
two main challenges: they had less access to the water needed for the first critical steps of
photosynthesis, and the lack of buoyancy meant that the earliest plants had little to no
structural support. The earliest plants, like their aquatic ancestors, reproduced using motile,
flagellated sperm that could only move through wet habitats. Initially, these limitations
constrained the first plants to remain small and live in damp environments. Modern land
plants have largely overcome these challenges, and the evolutionary history of the land
plants is marked by several key adaptations that deal with them. In this section we will
review these adaptations by examining prepared slides and live material.

1. Cell walls made of cellulose to help buffer cells against deformation as water
availability (and osmotic pressure) changes over time.
2. Vascular tissue: the evolution of vascular tissue allowed plants to transport water
throughout their bodies more effectively, allowing them to grow taller.

BIOL 1510 | The Green Revolution –Lab Manual 4

 • With a scalpel, cut a cross-section of the celery stick a few millimeters from the
bottom, avoiding the region which has become deeply stained with the dye. Try to
make your slice as thin as possible. Examine the cut surface of the stem with a
dissecting microscope. If your slice is thin enough, examine it at low magnification
under a compound scope.
• Draw your celery cross section, noting how individual cells are isolated from each
other by rigid cell walls. Also note how the vascular tissue is arranged in celery.
Vascular tissue includes the xylem, which transports water (and, in this case, dye!)
from the roots to the leaves, and phloem, which transports sugars throughout the
plant.
• Obtain prepared slides of monocot and dicot stems. Monocot and dicot plants differ
in the arrangement of their vascular system. Based on your observations, is celery a
monocot or a dicot? Briefly explain your answer in the space provided.

Leaves
The earliest plants looked like mosses, whose “leaves” are more technically called
microphylls. Modern plants, including the ferns and the seed plants, have larger leaves
(megaphylls). In both cases, leaves evolved as flattened stems. Leaves are specialized
photosynthetic organs with clear functional specialization, so that the various tasks of
photosynthesis are carried out in different parts of the leaf.
• Examine a prepared slide of a cross section of a lilac or corn leaf. The epidermis is the
protective outer layer of cells; it is covered by a waxy cuticle that blocks water loss via
evaporation. The epidermis on the underside of the leaf has pores called stoma
surrounded by two guard cells. The stomates allow CO2 and O2 into / out of the inner
plant tissues. Beneath the epidermis on the upper side of the leaf, the palisades layer
consists of tightly packed cylindrical cells full of chloroplasts: this is where most
photosynthesis takes place. Beneath this is the spongy layer, loosely packed with cells.
Vascular tissue circulates throughout the leaf, usually in distinct bundles.
• Based on your observations and the information given above, label the leaf cross-section
diagram in your worksheet.

Alteration of generations
In animals, development typically starts with a fertilized zygote, which grows by mitosis into
a large diploid organism; upon maturation, a subset of cells undergo meiosis, producing
haploid sperm and eggs. In animals, the haploid cells (i.e. the gametes) have a very short life
span relative to that of the diploid organism that produced them. Plants, however, exhibit
alternation of generations, alternating between a diploid sporophyte and an haploid
gametophyte. Figure 2 depicts a typical plant life cycle (the numbers correspond to those in
the image): a diploid multicellular sporophyte (1) produces haploid spores (5) in structures

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called sporangia (3) by meiosis (4);
these spores develop into a multicellular
gametophyte (7), which produces
gametes (9) by mitosis (8). These
gametes unite during fertilization (10)
to form a zygote (11). As with animals,
plants increase their body size via
mitosis (12, 2, 6). The sporophyte was a
major evolutionary innovation because
it drastically enhanced the dispersal of
spores in terrestrial environments. One Figure 1. Diagram of plant life cycle. Note that the life cycle
alternates between haploid (n) gametophyte and a diploid (2n)
of the most important general patterns sporophyte stages.
in plant evolution is an increasing
dominance of the sporophyte generation, and a reduction of the gametophyte generation; in
some plants the gametophyte is microscopic, reduced to a small cluster of cells.

Parental care: we don’t tend to think of plants as providing parental care, but they do! In
fact, all of the most familiar plants are part of Subkingdom Embryophyta (within Kingdom
Plantae) that, as the name suggests, produce multicellular embryos that are dependent on
maternal tissues of the plant for support during the early stages of their development. If that
sounds familiar, it should: mammals like us do this too.

To explore the alternation of generations and parental care in plants, we will study the stellar
calcareous moss Mnium. Mosses such as Mnium are part of an ancient group of plants called
the Bryophyta. Mnium has many ancestral plant features: it has no vascular tissues, no seeds,
flowers, or fruit; the male gametophyte produces motile sperm that require damp habitats,
and the sporophyte develops from an embryo that remains wholly dependent on the female
gametophyte.

• Obtain a small sample of live Mnium and observe it under a dissecting microscope.
Note the tiny microphylls (leaflike structures). The green leafy parts of the plant are
the male and female gametophytes. They are both haploid. The male gametophyte has
an antheridium (pl.: antheridia) that produces sperm via mitosis, while the female
gametophyte has an archegonium (pl.: archegonia) that produces eggs and nourishes
the zygote after fertilization, supporting it as it develops into an embryo and
ultimately into the diploid sporophyte, which looks like a curved twig with a capsule
at its end. This is the sporangium (pl.: sporangia) that will produce haploid spores via
meiosis. Draw your live Mnium in the space provided on your worksheet, and identify
gametophytes, sporophytes (if present), and any other features you can identify.

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 • Note: we frequently have dried sporophytes on display. Observe and draw these if
your live Mnium don't have any.
• Obtain prepared slides of Mnium antheridia and archegonia. Note the presence of
sperm or egg. Note that some of them are surrounded by microphylls (as in Figure 3,
below) while others are dissected out. Also examine prepared slides of cross-sections
through the sporophyte's capsule, and note the dense layer of spores. Roughly sketch
these three structures in your worksheets and annotate.

Figure 2. Cross sections through Mnium gametophyte and sporophyte structures.

• Based on the information above, complete and annotate the blank Mnium life cycle
diagram in the worksheet.

Pollen
As stated above, the earliest plants had flagellated sperm
that could only swim in aquatic environments, so that
fertilization could only take place in damp habitats, with
adjacent plants. Pollen grains consist of the
microgametophyte- the male gametophyte of pollen-
producing plants- surrounding a generative cell that will
eventually become sperm. The major evolutionary
advantages of pollen are that it frees plants from damp
Figure 3. Structure of pine pollen.
habitats and allows them to pollinate plants that are very far
away, as the pollen is carried by air currents or by pollinating
animals (e.g. bees, butterflies).
• Obtain prepared slides of pine pollen and observe under high magnification. Pine trees
use pollen designed to be carried on air currents. Note the pronounced "wings".

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• If any of the cut flowers in the lab have anthers bearing pollen, shake some onto a slide
and observe / draw them in your worksheet. In your worksheet, hypothesize why pine
and flower pollen might have drastically different shapes.

Seeds

The earliest plants dispersed using spores that, while resistant to environmental changes,
provided little support to the developing plant. Seeds consist of three major parts: a plant
embryo, a starchy food supply for that embryo, and a protective coat to protect it until
environmental conditions are suitable for germination.

• Examine the seeds on display. Some of the seeds are split open; observe them under a
dissecting scope. In addition to the three major parts listed above, you may also observe
distinct root, stem, shoot, and one or two cotyledons (leaflets) depending on the type of
plant. Based on your observations, annotate the basic seed diagram in the worksheet,
modifying the drawing if necessary.

Angiosperms: Those with Flowers and Fruits

Flowers are highly modified leaves that function as sexual organs in flowering plants. Their
purpose is to improve the effectiveness of fertilization, whether by wind or via pollinating
animals depending on the plant. In most flowering plants, fruits are the fertilized ovaries of
the plant- the seeds of the plant are held within this chamber (hence the scientific name of
this group, the angiosperms, which translates to container seed). The purpose of the fruit
depends on the dispersal strategy used by the plant: some are designed to be carried on the
wind, others are intended to be eaten and subsequently deposited in feces, and others still
are designed to nourish the seeds within. The evolutionary significance of flowers and fruits
cannot be overstated: the great majority of plant species produce them, including almost all
of our key food and medicinal plants. Flowering plants are the most diverse photosynthetic
organisms on Earth.

• Examine the flowers on display,

particularly those that have been
partially dissected to reveal key
anatomical details. Identify the major
features using the diagram in Figure 5,
and complete the diagram in the
worksheet. Note the ovary at the base of
Figure 4. Anatomy of a flower.
the flower: this is the structure that will
mature into a fruit after pollination.

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 A fruit is the matured ovary of a flower. It consists of the pericarp (the wall of the overy),
wrapped around one or more seeds. As with pollen, note the difference in design. Fruits can
promote the reproductive success of the plant in different ways: by enhancing dispersal by
wind, water, by clinging to animals, or via the feces of animals that ingest the fruit. They can
discourage herbivory, or provide additional nutrition to the seeds. Examine the fruits on
display. Fruits fall into two broad categories: dry fruit (nuts, grains, peas, beans) and fleshy
fruit. Fleshy fruit include berries (grape, tomato), pomes (apple, pear), drupes (cherry,
peach), and hesperidia (oranges, limes). Simple fruits (peas, peach) develop from flowers
with a single pistil; aggregate fruits (raspberry) develop from individual flowers with
multiple carpels; multiple fruits (pineapple) derive from clusters of flowers; accessory fruits
(strawberry, apple, cucumber, melon, pumpkin, blueberry). For the fruits on display
determine if each fruit is dry or fleshy and determine the function of each fruit.

Types of fruit
Aggregate fruit– develops from the merging of several ovaries.

Achene– simple dry fruit contain a single seed that nearly fills the pericarp, but does not
adhere to it.

Berry– fleshy fruit, without a stone; that develops form a single ovary. Berries are extremely
diverse and may be the most common type of edible fruit.

Capsule– a dry fruit that splits open to release its many seeds.

Drup– fleshy fruit usually developing from one carpel; stony endocarp (pit), fleshy mesocarp,
thin skinned.

Multiple fruit– develops from an inflorescence (cluster of flowers). Each flower produces a
fruit, that merges into a single mass.

Nut– dry fruit with a hard ovary wall, and the seed remains unattached or free within the
ovary wall.

Pome– fleshy edible part is ripened tissue surrounding ovary, which matures into "core";
contains multiple seeds.

Pulse (Legume)– a simple dry fruit that develops from a single carpel. Usually opens along a
"seam".

Samara– a winged achene

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Additional exercise: your professor may opt to guide you on a tour of the plants on display
around the Parker Building. Such tours are subject to limitations due to weather and
darkness, so the decision on whether they happen is left to your instructor. Of course
students are encouraged to visit the various gardens around Parker any time outside of class
hours!

Gastropodcast Museums and the Mafia: The Secret History of Citrus

BIOL 1510 | The Green Revolution –Lab Manual 10