ALGAL ORIGINS of LAND PLANTS Introduction The

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LAB 1: ALGAL ORIGINS OF LAND PLANTS Introduction The Class Charophyceae contains mostly freshwater green algae and is believed to be the line containing the ancestral stock from which land plants evolved. Multigene DNA phylogenetic analysis has shown that the Charales (Chara, Nitella) are the closest living relatives of green land plants (McCourt et al, 2004). Green algae classified in the Class Charophyceae have certain traits (features) also found in land plants. Such algae are believed to be in the evolutionary lineage from which land plants evolved. Land plants have uninucleate cells as their basic unit. The cells are arranged in tissue known as parenchyma; which is formed by cell division in three planes resulting in a solid mass of tissue. Typical land plants have functional differentiation with aboveground, green photosynthetic structures or shoots (differentiated into stems and leaves) and belowground anchoring structures (rhizoids or roots) for absorption of water and soil nutrients. We will look at examples of algae that are representative of the sister lineage to land plants. Morphological traits, combined with molecular phylogenetics, have helped to determine the algal lineage that is the closest relative of land plants, the Charophyceae. Shared features include a pattern of nuclear and cell division (mitosis and cytokinesis) similar to that of extant land plants and details of the ultrastructure of motile cells. In Charophyceae motile cells are asymmetrical with subapical flagella that extend at right angles from the cell; and nuclear and cell division combining an open spindle, a phragmoplast at nuclear separation and the cell plate type of cytokinesis (Fig. 1.1).

Figure 1.1 Mitosis and cytokinesis showing the open spindle, phragmoplast and cell plate that are shared by land plants and Charophyceae

Figure 1.2: Chara. A) “plant” showing nodes and internodes, B) node with whorl of branches, C) fertile branch, D) gametangia, oogonium above (female), antheridium below (male)

McCourt, R.M., Delwiche, C.F., and Karol, K.G. (2004). Charophyte algae and land plant origins. Trends in Ecology & Evolution 19, 661–666

Laboratory Exercise Goals: I. Become familiar with the use of the dissecting and compound microscopes. II. Observe representatives of the green algal lineage most closely related to land plants. III. Start spore cultures of the fern Ceratopteris richardii for Lab. 5. IV. Investigate key innovations of land plants.

I. INTRODUCTION TO MICROSCOPY: DISSECTING MICROSCOPE

1. Adjust seat height. Always begin a lab by adjusting seat height. Place the dissecting scope on the bench in front of you and adjust your seat height so you are looking comfortably into the eyepieces without stretching your neck up or bending over too much.

2. Fresh plant material may not be placed directly on the stage. If you are examining fresh plant material it will gum up the stage and will also tend to quickly dry up. Work on a damp piece of towel paper or kimwipe. Or, in some cases, you may wish to place small specimens in a Petri dish with a few drops of water.

3. Adjust the light. For thin specimens which light will pass through, you can use both transmitted light (from below) and reflected light (from above). For opaque objects, the light must be from above.

4. Adjusting the eyepieces. Start by roughly focusing and then make the following adjustments. This is an important step to reduce fatigue and eyestrain.

a. Interocular distance: The eyepieces can be moved closer together or farther apart to accommodate the distance between your eyes, which varies from person to person. You may also want to experiment with how close you want your eyes to the eyepieces. Sometimes it helps to move back a bit.

b. Focusing the 2 eyepieces separately: The vision in your two eyes is probably not identical and, in any case, whoever used the scope before you will have adjusted the eyepieces to suit themselves - and it won't suit you. Proceed as follows:

i. Look through the right eyepiece (close left eye or simply ignore what it sees or put your hand over it). Focus with the focusing knob.

ii. Now look through the left eyepiece, closing the right eye. Focus using the eyepiece itself (it should have a knurled ring which you can turn).

If the image is not sharp when you have finished the above, you may have to make some more minor adjustments or try cleaning the eyepieces and objective lens (see T.A. for how to proceed). Additional suggestions. When studying microscope slides, it is a good idea to examine them with the dissecting scope before using the compound scope. The reason for doing this, particularly with large specimens, is that you may see only a small part of the specimen with the compound scope. Looking at the entire thing first will help in getting oriented. Use transmitted light (from below) when looking at a slide on the dissecting scope.

COMPOUND MICROSCOPE

1. You will be using an Olympus microscope, so the basic set of instructions refers to the CH-2 Olympus compound scope.

2. Adjust seat height. Ideally, you will not be stretching your neck or bending your back excessively to look through the eyepieces.

3. Starting light intensity. Most of the Olympus scopes have a light switch on the base of the scope in front. Turn the light on. On the right side of the base, you will find voltage control dial that sets the brightness. For starters set it about in the middle.

4. Substage condenser. The substage condenser focuses the light on your slide. There is a knob (condenser height adjustment knob) that drives the condenser up and down. Move it so that the condenser is all the way up, then back off slightly. The substage condenser should remain in this position.

5. Bright field illumination. On Olympus phase microscopes, the substage condenser has a plate that rotates displaying different numbers. The number (which shows in front) should always be in the zero position, which is the bright field position.

6. Using the revolving nosepiece rotate the 10X objective into place. Always start with the lowest magnification. Place the slide on the compound scope stage and roughly center the tissue section under the objective. With the lowest magnification only, you can start by moving the stage all the way up with the coarse focus knob. For a practice session, the T.A. will assign you to look at either a root tip (Allium) or a shoot tip (Salvia or Coleus).

7. Initial light adjustment. Look in the eyepieces. If the light is too bright, use the substage condenser iris diaphragm lever to adjust it.

Always make your initial light adjustment with the condenser diaphragm. Use the rheostat adjustment (dial at base) only if the condenser iris diaphragm does not suffice.

8. Find the tissue section and roughly focus on it. Use the coarse focus knob (the larger one) to slowly move the specimen into focus. If you don't see the tissue section, you will have to move the stage around as well to get the tissue section under the objective.

9. Adjusting the eyepieces. Once you have the light adjusted and have focused on the tissue, it is time to adjust interpupillary distance and individual eyepieces

a. Interpupillary distance: The eyepieces slide closer together and farther apart to accommodate the distance between your eyes. Make this adjustment while looking at the tissue section. Sometimes it helps to move your head back slightly so that your eyes are further away from the eyepieces. You will have to experiment to find the best position for you.

b. Focusing eyepieces: The vision in your two eyes is probably not identical and, in any case, whoever used the scope before you will have adjusted the eyepieces to suit themselves - and may not suit you. Proceed as follows:

i. Look through the right eyepiece (close left eye or simply ignore what it sees or put your hand over it). Focus with the focusing knob. 5

ii. Now look through the left eyepiece, closing the right eye. Focus using the eyepiece itself (using knurled ring which you can turn).

If the image is not sharp when you are done, you may have to make minor adjustments or perhaps try cleaning the eyepieces or objective lens.

These steps are very important for avoiding fatigue and eyestrain, so be sure to do them.

10. Now switch to the 43X objective and practice adjusting the light, something you will have to do each time you change objectives. In general, the light should not be too bright. Bright light produces light scatter and a fuzzy image. It would be better to have the light too dim than too bright.

Always start the light adjustment with the substage condenser iris diaphragm. Only adjust the rheostat current if the iris diaphragm does not allow enough latitude. Never lower the condenser itself to get more light. The best optics are obtained with the condenser nearly all the way up.

Lab policy on microscope slides. Most of the time you will be examining commercially prepared microscope slides. Sometimes we have only a few slides of each type for the entire class. It creates a problem when students collect more than one slide at a time and have them piled up waiting to look at. So take only one slide at time and return it as soon as you are done.

BIOL 441: MICROSCOPE PARTS

II. ALGAL ORIGIN OF LAND PLANTS You will examine the freshwater algae Chara and Nitella, which are evolutionarily closely related to land plants. While you won’t be able to observe their microscopic traits, they do have the unilateral type of motile cell organization and the land plant pattern of mitosis and cytokinesis. PHRAGMOPLAST. The phragmoplast is a complex assembly of microtubules, microfilaments, and elements of the endoplasmic reticulum that assemble in two opposing sets perpendicular to the plane of the future cell plate during anaphase and telophase. Examine the onion root tip slide showing the apical meristem: try to locate cells in telophase and look for the outline of the phragmoplast. This structure occurs in land plants and in some algae.

Microtubules assemble in the phragmoplast during late telophase; cell plate formation is marked by the presence of vesicles. (petiole of Echium judaeum).

Chara (Figure 1.2) and Nitella (Stoneworts): Observe cultured material to see the general organization of these algae. Note the long internodal cells with whorled branches at the nodes. These large cells are multinucleate. These two algae are anchored to the substrate by branching rhizoids, which may give rise to additional “plants”. Both algae live in ponds and lakes where they form extensive underwater meadows. Clear bodies of hard water are common habitats for them and many species of stoneworts deposit bands of calcium carbonate on the walls of their long cells. As a result of this calcium deposition, they are more likely to fossilize and are therefore known from the fossil record. a. Observe and draw a Chara plant. Label nodes, internodes and rhizoids.

b. Look for the male and female gametangia; these gametangia produce sperm and eggs. Prepared slides of Chara gametangia, cross sections of oogonium and antheridium, will be on demonstration. Note the layer of cells surrounding the gametes.

Draw Chara gametangia based on your observations.

c. Diagram the life cycle for Chara. Indicate meiosis, fertilization, gametes, spores and the ploidy of every stage.

How much of the Chara/Nitella life cycle is haploid vs. diploid?

What is the meaning and consequence of zygotic meiosis?

III. SOWING FERN SPORES: Follow instructions in C-fern manual pE3-E5 (Day 0) to start spores of Ceratopteris richardii, you will follow your culture in subsequent labs.

IV-LAND PLANT KEY INNOVATIONS (major synapomorphies)

Introduction Alternation of Generations Land plants are characterized by a life cycle in which there are two kinds of mature generations. Thus, to be strictly accurate, if we wished to show what a particular species of fern looked like, we would have to produce two pictures. One picture would show the spore-producing phase of the life cycle, the sporophyte (the one you are more likely familiar with). It is usually diploid and eventually develops organs known as sporangia (singular, sporangium). Certain cells in the sporangia divide by meiosis to produce spores. Spores grow into the second generation of the life cycle, the gametophyte, which is usually haploid (one set of chromosomes). A picture of the gametophyte reveals that it does not resemble the sporophyte. As its name implies, the gametophyte eventually develops organs (gametangia) that produce gametes (sperm and eggs) by mitosis. Land plant gametes fuse to form a zygote, which divides by mitosis to produce the diploid sporophyte generation.

(sporophyte may or may not remain attached to gametophyte, see fig. 2.2)

MEIOSIS

MITOSIS DIPLOID SPOROPHYTE

MULTICELLULAR EMBRYO (develops while still attached SPORES to gametophyte) (haploid)

MITOSIS ZYGOTE (diploid)

MITOSIS

EGG

SPERM

HAPLOID GAMETOPHYTE EGG AND SPERM FORM BY MITOSIS Figure 2.1: Diagrammatic representation of alternation of generations in Land Plants.

Note that in the fern the sporophyte will become a free-living plant with roots and shoots, while the moss sporophyte (right) remains permanently attached to the gametophyte (Fig. 2.2).

Sporophyte

Gametophyte

FERN MOSS Figure 2.2: Comparison of gametophyte and sporophyte generations in a fern and a moss.

It is important to note that there are also many algal species that have a life cycle involving alternation of generations. Most algae, however, produce gametes that are shed into the water, where fertilization occurs. In some algal species, the egg is fertilized while it is still attached to the parental gametophyte, but even in those cases the fertilized egg is always released into the water before further embryonic development occurs. In all land plants, the egg is retained by the parental gametophyte, is fertilized in situ and develops into a multicellular embryonic sporophyte while still attached to and nutritionally dependent upon the parental gametophyte. Thus, land plants are often referred to as embryophytes to distinguish them from algae in which a zygote is released from the parental plant before developing into a multicellular embryo.

After the initial stages of development of a sporophyte or gametophyte, the land plant body always becomes parenchymatous (Fig. 2.3). The term as used here denotes tissues that have three-dimensional thickness as a consequence of mitoses in at least three planes. The parenchymatous body of land plants contrasts strongly with algae. The vast majority of algal species are unicellular, filamentous, or colonial. Each generation in land plants begins as a single cell (spore or zygote) and may go through distinct filamentous and two-dimensional stages, but continued growth soon produces a parenchymatous body. Exceptions occur only in the seed plants where gametophytes have been reduced to unique structures that consist of only a small number of cells.

Figure 2.3: Types of growth. A. Unicells (daughter cells separate after mitosis). B. Filaments (mitosis occurs in one plane). C. 2-D Sheets (mitosis in 2 planes). D. Parenchyma (mitosis in 3 planes). E. Pseudoparenchyma A B C D E (massed filaments).

Distinguishing Land Plants from Algae.

Sporopollenin, a compound that (like lignin) resists chemical or enzymatic breakdown is found in the cell walls of spores and pollen grains. Sporopollenin also has been found in the zygote walls of Charophycean algae, the Green algal group that is the indisputable ancestor of land plants. However, the presence of sporopollenin in meiotic products is restricted to land plants. The evolution of dessication-resistant spores must have been an important first step in the transformation of fresh water algae into partially terrestrial organisms with wind-dispersed spores.

In nearly all extant land plants, the meiotically-produced tetrads break up in the sporangia before dehiscence - so that spores are dispersed as single cells. But a few land plant species do disperse their spores as intact tetrads. No algal species are known to do this. Thus the presence of sporopollenin-containing tetrads in Mid-Ordovician rocks constitutes convincing evidence that land plants were present at that time. In land plants, the epidermal cells secrete cutin - a fatty acid that combines with waxes and impedes water loss from the surface of the plant, forming the cuticle (Fig. 2.4b). Cutin is another land plant chemical compound that is resistant to biodegradation. Because cuticle inhibits the gas exchange necessary to carry out respiration and photosynthesis, the epidermis needs openings. The earliest fossil cuticles lack pores, presumably because the small size of the plants allowed a sufficiently rapid diffusion of gases through the cuticularized plant surface to meet the needs of internal tissues. The earliest fossil cuticles that exhibit pores show up in the early Devonian and have pores of the stomatal type. Stomata (or stomates, Fig. 2.4a) are pores in which the size of the opening can be altered by shape changes in a pair of surrounding guard cells. Guard cells (Fig. 2.4a) change shape in response to environmental conditions and to the water status of the plant.

A B

Figure 2.4: A. Stomata, guard cells and epidermis cells of lower leaf surface. B. Cuticle on epidermis (surface cell layer) of stem section.

When stomata are present, they occur only in the sporophyte generation. Virtually all vascular plants have stomata (a few taxa lack them, undoubtedly due to loss rather than to retention of an ancestral condition). Some bryophytes (non-vascular plants) have stomata, some have pores in which the aperture is not controllable (i.e. guard cells are lacking) and many are entirely lacking pores of any kind. When bryophytes possess non-stomatal pores, they are found in the gametophyte generation only. A cuticle is unknown in algae, although a layer of material in certain Charophytes is structurally similar to early developmental stages of vascular plant cuticles. Whether or not this material is biochemically similar to the land plant cuticle is not clear yet. Although the presence of a cuticle and stomata are often cited as characteristics of land plants it is important to keep in mind that this generalization is not true for many bryophytes. In summary, if we focus on the vegetative body of land plants, it tends to be distinguished from algae on the basis of a cuticularized epidermis with pores and a vascular system for moving water out of the soil and food downward from the top of the plant to subterranean organs. This is certainly true of the dominant stage (the sporophyte) in the life cycle of all tracheophytes, but not of the gametophyte stage. However, it is only partially true of bryophytes. In both the sporophyte and gametophyte of some bryophyte taxa, the cuticle is rudimentary or absent and there are no epidermal pores. In other bryophyte taxa, the sporophyte is well cuticularized and stomata are present. Likewise, some bryophytes have vascular tissue and others lack it entirely.

Reproductive characteristics of land plants. 1. Land plants produce aerial spores that are wind-dispersed. Although spore dispersal in seed plants has been extensively modified, it is clear that this is a derived trait. 2. Spores of land plants have a wall that contains sporopollenin, a complex polymer that is resistant to degradation. Although sporopollenin has been found in some Charophycean algae, it does not occur in meiotic products there. 3. Land plants are embryophytes. This term indicates that the egg and zygote are retained by the gametophyte and a multicellular sporophytic embryo develops while still attached to the parental gametophyte. The developing embryo depends on the parental gametophyte for its nutrition. In the Charophycean algae the egg and zygote are retained, but the zygote is released before a multicellular embryo develops. 4. In land plants, reproductive organs (gametangia and sporangia) are covered by a sterile jacket, a layer of cells that has protective or dispersal functions.

IV-LAND PLANT KEY INNOVATIONS-Laboratory exercise

A. LAND PLANTS ARE EMBRYOPHYTES

All land plants are embryophytes. To demonstrate this you will observe bean seeds that have been soaked and are getting ready to germinate. Inside each seed is a small plant embryo, the young sporophyte. Select a seed, remove the seed coat and carefully separate the cotyledons from one another, the embryonic sporophyte should remain attached to one of the cotyledons. View this under the dissecting scope and identify the embryonic leaves in the embryonic shoot (hypocotyl) and the embryonic root (radicle). Find the structures shown below.

B. LIFE CYCLE HAS (a special type of) ALTERNATION OF GENERATIONS

Find an angiosperm with flowers from the ones provided in lab and locate sporophyte and gametophyte. Compare the size, temporal predominance and photosynthetic ability of the sporophyte and gametophyte of the available moss and the flowering plant. Write your conclusions below. Add diagrams of moss and angiosperm gametophytes and sporophytes to help you remember your observations: ______

C. Plants are protected by a waxy CUTICLE with PORES or STOMATA for gas exchange Make a thin cross sections of a leaf or petiole of plants available in lab. The section will be surrounded by an epidermis, a surface layer of cells that is covered by a waxy cuticle. Use the space below to illustrate your cross section.

Observe and illustrate the pores on the surface of a liverwort gametophyte. Note the diamond- shaped pattern, each "diamond" indicating the presence of an air chamber below. Notice the pore at the center of each chamber. These pores are used for gas exchange. At the base of the air chambers are photosynthetic filaments that can be seen in a thin cross-section. Liverworts are the only lineage of plants that has pores rather than stomata. As you examine the epidermis of Angiosperms, contrast the stomata to the pore shown below. In what ways are these pores different from stomata?

Section through a pore into the thallus (lower large cells) of the local liverwort Conocephalum. Note chamber with photosynthetic filaments in the bottom.

Make a thin hand-section through a pore, observe tiers of cells. Illustrate below

Epidermal replica: Obtain a leaf from Tradescantia zebrina, a Monocot, and paint the underside of the leaf with clear fingernail polish. Allow the polish to dry (5 min). Now peel a small strip of the dried fingernail polish from the leaf. You may have to break the leaf to free the polish. Apply the sliver of finger nail polish to a clean glass slide. Flatten the polish strip out on the slide, add a drop of water, and then lower a cover slip over the preparation. Sketch a few guard and epidermal cells in the space below.

D. THE THREE-DIMENSONAL PARENCHYMATOUS STRUCTURE OF PLANT TISSUE

To help illustrate the three-dimensional parenchymatous structure of plant tissue, you will prepare a thin section through the gametophyte of a thalloid liverwort (an early-diverging land plant with little tissue differentiation) using a razor blade. As you make your observations, think about how cell division might develop a three-dimensional structure (Fig 2.3). View the section beginning at the lowest power and then increase the magnification as needed. Illustrate your section in below.

E. APICAL MERISTEMS –Observe examples of the apical meristems of a shoot and a root of a vascular plant. These are areas of active cell division, where the plant achieves primary growth.

*Plant meristems have evolved from a single apical cell (bryophytes), to a few cells (lycopods), to a mantle-core (Gymnosperms), to a tunica-corpus organization (angiosperms, as seen below). More on this later in the course!*

(1) Shoot apical meristem (SAM). Obtain one Brussels sprout, a very large axillary bud. Cut lengthwise directly through the point where it was attached to the stem. Label the SAM, leaf primordia, and the axillary buds associated with the leaf primordia.

(2) Long section of angiosperm (Coleus/Salvia) shoot tip. Locate the apical dome (SAM), adjacent leaf primordia (developing leaves), the axillary buds associated with the older leaves below the shoot apex, protoderm, ground meristem and procambium. Examine the SAM in more detail to identify the tunica (2 layers of cells dividing only anticlinally) and corpus (dividing both anti and periclinally, looking more disorganized)

(3) Root apical meristem (RAM). Long section onion (Allium) root tip. Find cells at different stages of mitosis. The RAM produces cells in two directions, toward the inside to become part of the root proper, and toward the outside to form the root cap. Label root cap, RAM, protoderm, ground meristem and procambium (provascular tissue).

F. GAMETANGIA AND SPORANGIA HAVE A STERILE JACKET OF CELLS

Gametangia and sporangia of land plants have an outer covering of sterile cells that protect the developing gametes and spores, respectively. Observe the demonstration slides of moss antheridium, the male gametangium that produces sperm, an archegonium, the female gametangium that produces an egg, and a sporangium that produces spores. Look for the jacket of sterile cells forming the outer covering of these structures. Look also for the thick and tough coat of sporopollenin surrounding spores. Use the space below to record your observations and illustrations.

Compare these multicellular gametangia to those of the Charophycean algae Chara or Nitella. Discuss the hypothesis of homology between these structures.