MATHERIALS TO LABORATORY WORK № 1

Theme: Botanical microtehnics. Microscopic structure of vegetable cell. The main components of a plant cell. Types of plastids, plant cell wall structure and composition of cellular juice of plants

Light Microscopy

The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology. Yet, many students and teachers are unaware of the full range of features that are available in light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments are, unfortunately, unavailable to most academic programs. However, even the most inexpensive “student” microscopes can provide spectacular views of nature and can enable students to perform some reasonably sophisticated experiments.

A beginner tends to think that the challenge of viewing small objects lies in getting enough magnification. In fact, when it comes to looking at living things the biggest challenges are, in order,

• obtaining sufficient contrast
• finding the focal plane
• obtaining good resolution
• recognizing the subject when one sees it

The smallest objects that are considered to be living are the bacteria. The smallest bacteria can be observed and cell shape recognized at a mere 100x magnification. They are invisible in bright field microscopes, though. These pages will describe types of optics that are used to obtain contrast, suggestions for finding specimens and focusing on them, and advice on using measurement devices with a light microscope.

Types of light microscopes

The bright field microscope is best known to students and is most likely to be found in a classroom. Better equipped classrooms and labs may have dark field and/or phase contrast optics. Differential interference contrast, Nomarski, Hoffman modulation contrast and variations produce considerable depth of resolution and a three dimensional effect. Fluorescence and confocal microscopes are specialized instruments, used for research, clinical, and industrial applications.

Other than the compound microscope, a simpler instrument for low magnification use may also be found in the laboratory. The stereo microscope, or dissecting microscope usually has a binocular eyepiece tube, a long working distance, and a range of magnifications typically from 5x to 35 or 40x. Some instruments supply lenses for higher magnifications, but there is no improvement in resolution. Such “false magnification” is rarely worth the expense.

Bright Field Microscopy

With a conventional bright field microscope, light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. We see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light despite being colorless. A Paramecium should show up fairly well in a bright field microscope, although it will not be easy to see cilia or most organelles. Living bacteria won’t show up at all unless the viewer hits the focal plane by luck and distorts the image by using maximum contrast.

A good quality microscope has a built-in illuminator, adjustable condenser with aperture diaphragm (contrast) control, mechanical stage, and binocular eyepiece tube. The condenser is used to focus light on the specimen through an opening in the stage. After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. The magnification of the image is simply the objective lens magnification (usually stamped on the lens body) times the ocular magnification.

Students are usually aware of the use of the coarse and fine focus knobs, used to sharpen the image of the specimen. They are frequently unaware of adjustments to the condenser that can affect resolution and contrast. Some condensers are fixed in position, others are focusable, so that the quality of light can be adjusted. Usually the best position for a focusable condenser is as close to the stage as possible. The bright field condenser usually contains an aperture diaphragm, a device that controls the diameter of the light beam coming up through the condenser, so that when the diaphragm is stopped down (nearly closed) the light comes straight up through the center of the condenser lens and contrast is high. When the diaphragm is wide open the image is brighter and contrast is low.

A disadvantage of having to rely solely on an aperture diaphragm for contrast is that beyond an optimum point the more contrast you produce the more you distort the image. With a small, unstained, unpigmented specimen, you are usually past optimum contrast when you begin to see the image.

Using a bright field microscope

First, think about what you want to do with the microscope. What is the maximum magnification you will need? Are you looking at a stained specimen? How much contrast/resolution do you require? Next, start setting up for viewing.

Mount the specimen on the stage

The cover slip must be up if there is one. High magnification objective lenses can’t focus through a thick glass slide; they must be brought close to the specimen, which is why coverslips are so thin. The stage may be equipped with simple clips (less expensive microscopes), or with some type of slide holder. The slide may require manual positioning, or there may be a mechanical stage (preferred) that allows precise positioning without touching the slide.

Optimize the lighting

A light source should have a wide dynamic range, to provide high intensity illumination at high magnifications, and lower intensities so that the user can view comfortably at low magnifications. Better microscopes have a built-in illuminator, and the best microscopes have controls over light intensity and shape of the light beam. If your microscope requires an external light source, make sure that the light is aimed toward the middle of the condenser. Adjust illumination so that the field is bright without hurting the eyes.

Adjust the condenser

To adjust and align the microscope, start by reading the manual. If no manual is available, try using these guidelines. If the condenser is focusable, position it with the lens as close to the opening in the stage as you can get it. If the condenser has selectable options, set it to bright field. Start with the aperture diaphragm stopped down (high contrast). You should see the light that comes up through the specimen change brightness as you move the aperture diaphragm lever.

Think about what you are looking for

It is a lot harder to find something when you have no expectations as to its apprearance. How big is it? Will it be moving? Is it pigmented or stained, and if so what is its color? Where do you expect to find it on a slide? For example, students typically have a lot of trouble finding stained bacteria because with the unaided eye and at low magnifications the stuff looks like dirt. It helps to know that as smears dry down they usually leave rings so that the edge of a smear usually has the densest concentration of cells.

Focus, locate, and center the specimen

Start with the lowest magnification objective lens, to home in on the specimen and/or the part of the specimen you wish to examine. It is rather easy to find and focus on sections of tissues, especially if they are fixed and stained, as with most prepared slides. However it can be very difficult to locate living, minute specimens such as bacteria or unpigmented protists. A suspension of yeast cells makes a good practice specimen for finding difficult objects.

• Use dark field mode (if available) to find unstained specimens. If not, start with high contrast (aperture diaphragm closed down).
• Start with the specimen out of focus so that the stage and objective must be brought closer together. The first surface to come into focus as you bring stage and objective together is the top of the cover slip. With smears, a cover slip is frequently not used, so the first thing you see is the smear itself.
• If you are having trouble, focus on the edge of the cover slip or an air bubble, or something that you can readily recognize. The top edge of the cover slip comes into focus first, then the bottom, which should be in the same plane as your specimen.
• Once you have found the specimen, adjust contrast and intensity of illumination, and move the slide around until you have a good area for viewing.

Adjust eyepiece separation, focus

With a single ocular, there is nothing to do with the eyepiece except to keep it clean. With a binocular microscope (preferred) you need to adjust the eyepiece separation just like you do a pair of binoculars. Binocular vision is much more sensitive to light and detail than monocular vision, so if you have a binocular microscope, take advantage of it.

One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it. Since very few people have eyes that are perfectly matched, most of us need to focus one eyepiece to match the other image. Look with the appropriate eye into the fixed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope.

Select an objective lens for viewing

The lowest power lens is usually 3.5 or 4x, and is used primarily for initially finding specimens. We sometimes call it the scanning lens for that reason. The most frequently used objective lens is the 10x lens, which gives a final magnification of 100x with a 10x ocular lens. For very small protists and for details in prepared slides such as cell organelles or mitotic figures, you will need a higher magnification. Typical high magnification lenses are 40x and 97x or 100x. The latter two magnifications are used exclusively with oil in order to improve resolution.

Move up in magnification by steps. Each time you go to a higher power objective, re-focus and re-center the specimen. Higher magnification lenses must be physically closer to the specimen itself, which poses the risk of jamming the objective into the specimen. Be very cautious when focusing. By the way, good quality sets of lenses are parfocal, that is, when you switch magnifications the specimen remains in focus or close to focused.

Bigger is not always better. All specimens have three dimensions, and unless a specimen is extremely thin you will be unable to focus with a high magnification objective. The higher the magnification, the harder it is to “chase” a moving specimen.

When to use bright field microscopy

Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms. It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections. Here is a not-so-complete list of specimens that might be observed using bright-field microscopy, and appropriate magnifications (preferred final magnifications are emphasized).

• Prepared slides, stained – bacteria (1000x), thick tissue sections (100x, 400x), thin sections with condensed chromosomes or specially stained organelles (1000x), large protists or metazoans (100x).
• Smears, stained – blood (400x, 1000x), negative stained bacteria (400x, 1000x).
• Living preparations (wet mounts, unstained) – pond water (40x, 100x, 400x), living protists or metazoans (40x, 100x, 400x occasionally), algae and other microscopic plant material (40x, 100x, 400x). Smaller specimens will be difficult to observe without distortion, especially if they have no pigmentation.

Care of the microscope

• EVERYTHING on a good quality microscope is unbelievably expensive, so be careful.
• Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for example.
• Hold the plug (not the cable) when unplugging the illuminator.
• Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done.
• Always make sure the stage and lenses are clean before putting away the microscope.
• NEVER use a paper towel, a kimwipe, your shirt, or any material other than good quality lens tissue or a cotton swab (must be 100% natural cotton) to clean an optical surface. Be gentle! You may use an appropriate lens cleaner or distilled water to help remove dried material. Organic solvents may separate or damage the lens elements or coatings.
• Cover the instrument with a dust jacket wheot in use.
• Focus smoothly; don’t try to speed through the focusing process or force anything. For example if you encounter increased resistance when focusing then you’ve probably reached a limit and you are going in the wrong direction.

 

Anatomical structure of plant cells, tissues, and organs, and their morphology are very important for pharmacist’s practical work on identification of medicinal raw material.Due to investigation of microscopes we can master anatomical structure of plant cells, tissues, and organs. There are two types of microscopes: light and electron. Pharmacist usually use light microscopes to conduct analysis of medical herbal material.

 

Light microscopes are of two basic types: compound microscopes, which require material being examined to be sliced thiny enough for light to pass through, and dissecting microscopes, which permit the viewing of opaque objects.

The cell is the basic unit of life. Plant cells (unlike animal cells) are surrounded by a thick, rigid cell wall.

Several kinds of plastids are generally found in living cells, with chloroplasts of green organisms usually being the most conspicuous.

The following is a glossary of plant cell anatomy terms.

amyloplast – an organelle in some plant cells that stores starch. Amyloplasts are found in starchy plants like tubers and fruits.
ATP – ATP is short for adenosine triphosphate; it is a high-energy molecule used for energy storage by organisms. In plant cells, ATP is produced in the cristae of mitochondria and chloroplasts.

cell membrane – the thin layer of protein and fat that surrounds the cell, but is inside the cell wall. The cell membrane is semipermeable, allowing some substances to pass into the cell and blocking others.
cell wall – a thick, rigid membrane that surrounds a plant cell. This layer of cellulose fiber gives the cell most of its support and structure. The cell wall also bonds with other cell walls to form the structure of the plant.centrosome – (also called the “microtubule organizing center”) a small body located near the nucleus – it has a dense center and radiating tubules. The centrosomes is where microtubules are made. During cell division (mitosis), the centrosome divides and the two parts move to opposite sides of the dividing cell.
chlorophyll – chlorophyll is a molecule that can use light energy from sunlight to turn water and carbon dioxide gas into sugar and oxygen (this process is called photosynthesis). Chlorophyll is magnesium based and is usually green.
chloroplast – an elongated or disc-shaped organelle containing chlorophyll. Photosynthesis (in which energy from sunlight is converted into chemical energy – food) takes place in the chloroplasts.

christae – (singular crista) the multiply-folded inner membrane of a cell’s mitochondrion that are finger-like projections. The walls of the cristae are the site of the cell’s energy production (it is where ATP is generated).
cytoplasm – the jellylike material outside the cell nucleus in which the organelles are located.
Golgi body – (also called the golgi apparatus or golgi complex) a flattened, layered, sac-like organelle that looks like a stack of pancakes and is located near the nucleus. The golgi body packages proteins and carbohydrates into membrane-bound vesicles for “export” from the cell.

granum – (plural grana) A stack of thylakoid disks within the chloroplast is called a

granum.

mitochondrion – spherical to rod-shaped organelles with a double membrane. The inner membrane is infolded many times, forming a series of projections (called cristae). The mitochondrion converts the energy stored in glucose into ATP (adenosine triphosphate) for the cell.

nuclear membrane – the membrane that surrounds the nucleus.
nucleolus – an organelle within the nucleus – it is where ribosomal RNA is produced.
nucleus – spherical body containing many organelles, including the nucleolus. The nucleus controls many of the functions of the cell (by controlling protein synthesis) and contains DNA (in chromosomes). The nucleus is surrounded by the nuclear membrane

photosynthesis – a process in which plants convert sunlight, water, and carbon dioxide into food energy (sugars and starches), oxygen and water. Chlorophyll or closely-related pigments (substances that color the plant) are essential to the photosynthetic process.
ribosome – small organelles composed of RNA-rich cytoplasmic granules that are sites of protein synthesis.
rough endoplasmic reticulum – (rough ER) a vast system of interconnected, membranous, infolded and convoluted sacks that are located in the cell’s cytoplasm (the ER is continuous with the outer nuclear membrane). Rough ER is covered with ribosomes that give it a rough appearance. Rough ER transport materials through the cell and produces proteins in sacks called cisternae (which are sent to the Golgi body, or inserted into the cell membrane).
smooth endoplasmic reticulum – (smooth ER) a vast system of interconnected, membranous, infolded and convoluted tubes that are located in the cell’s cytoplasm (the ER is continuous with the outer nuclear membrane). The space within the ER is called the ER lumen. Smooth ER transport materials through the cell. It contains enzymes and produces and digests lipids (fats) and membrane proteins; smooth ER buds off from rough ER, moving the newly-made proteins and lipids to the Golgi

body and membranes

 

stroma – part of the chloroplasts in plant cells, located within the inner membrane of chloroplasts, between the grana.

thylakoid disk – thylakoid disks are disk-shaped membrane structures in chloroplasts that contain chlorophyll. Chloroplasts are made up of stacks of thylakoid disks; a stack of thylakoid disks is called a granum. Photosynthesis (the production of ATP molecules from sunlight) takes place on thylakoid disks.
vacuole – a large, membrane-bound space within a plant cell that is filled with fluid. Most plant cells have a single vacuole that takes up much of the cell. It helps maintain the shape of the cell.

Plant cell structure

Plant cells are quite different from the cells of the other eukaryotic kingdoms’ organisms. Their distinctive features include:

• A large central vacuole (enclosed by a membrane, the tonoplast), which maintains the cell’s turgor and controls movement of molecules between the cytosol and sap.
• A cell wall composed of cellulose and protein, and in many cases lignin, and deposited by the protoplast on the outside of the cell membrane. This contrasts with the cell walls of fungi, which are made of chitin, and prokaryotes, which are made of peptidoglycan.
• The plasmodesmata, linking pores in the cell wall that allow each plant cell to communicate with other adjacent cells. This is different from the network of hyphae used by fungi.
• Plastids, especially chloroplasts that contain chlorophyll, the pigment that gives plants their green color and allows them to perform photosynthesis.
• Plant groups without flagella (including conifers and flowering plants) also lack centrioles that are present in animal cells.

Cell membrane

Illustration of a Eukaryotic cell membrane

The cell membrane is a biological membrane that separates the interior of all cells from the outside environment.^[1]. The cell membrane is selectively-permeable to ions and organic molecules and controls the movement of substances in and out of cells.^[2]. It consists of the phospholipid bilayer with embedded proteins, which are involved in a variety of cellular processes such as cell adhesion, ion conductivity and cell signaling. The plasma membrane also serves as the attachment surface for the extracellular glycocalyx and cell wall and intracellular cytoskeleton.

The cell membrane surrounds the protoplasm of a cell and, in animal cells, physically separates the intracellular components from the extracellular environment. Fungi, bacteria and plants also have the cell wall which provides a mechanical support for the cell and precludes passage of the larger molecules. The cell membrane also plays a role in anchoring the cytoskeleton to provide shape to the cell, and in attaching to the extracellular matrix and other cells to help group cells together to form tissues. The barrier is differentially permeable and able to regulate what enters and exits the cell, thus facilitating the transport of materials needed for survival. The movement of substances across the membrane can be either passive, occurring without the input of cellular energy, or active, requiring the cell to expend energy in moving it. The membrane also maintains the cell potential.

According to the fluid mosaic model of S. J. Singer and Garth Nicolson 1972, the biological membranes can be considered as a two-dimensional liquid where all lipid and protein molecules diffuse more or less easily^[3]. This picture may be valid in the space scale of 10 nm. However, the plasma membranes contain different structures or domains that can be classified as (a) protein-protein complexes; (b) lipid rafts, and (c) pickets and fences formed by the actin-based cytoskeleton.

Lipid bilayer

Diagram of the arrangement of amphipathic lipid molecules to form a lipid bilayer. The yellow polar head groups separate the grey hydrophobic tails from the aqueous cytosolic and extracellular environments.

The cell membrane consists primarily of a thin layer of [amphipathic] phospholipids which spontaneously arrange so that the hydrophobic “tail” regions are shielded from the surrounding polar fluid, causing the more hydrophilic “head” regions to associate with the cytosolic and extracellular faces of the resulting bilayer. This forms a continuous, spherical lipid bilayer.

The arrangement of hydrophilic heads and hydrophobic tails of the lipid bilayer prevent polar solutes (e.g. amino acids, nucleic acids, carbohydrates, proteins, and ions) from diffusing across the membrane, but generally allows for the passive diffusion of hydrophobic molecules. This affords the cell the ability to control the movement of these substances via transmembrane protein complexes such as pores and gates.

Flippases and Scramblases concentrate phosphatidyl serine, which carries a negative charge, on the inner membrane. Along with NANA, this creates an extra barrier to charged moieties moving through the membrane.

Membranes serve diverse functions in eukaryotic and prokaryotic cells. One important role is to regulate the movement of materials into and out of cells. The phospholipid bilayer structure (fluid mosaic model) with specific membrane proteins accounts for the selective permeability of the membrane and passive and active transport mechanisms. In addition, membranes in prokaryotes and in the mitochondria and chloroplasts of eukaryotes facilitate the synthesis of ATP through chemiosmosis.

Membrane polarity

Alpha intercalated cell

The apical membrane of a polarized cell is the surface of the plasma membrane that faces the lumen. This is particularly evident in epithelial and endothelial cells, but also describes other polarized cells, such as neurons.

The basolateral membrane of a polarized cell is the surface of the plasma membrane that forms its basal and lateral surfaces. It faces towards the interstitium, and away from the lumen.

“Basolateral membrane” is a compound phrase referring to the terms basal (base) membrane and lateral (side) membrane, which, especially in epithelial cells, are identical in composition and activity. Proteins (such as ion channels and pumps) are free to move from the basal to the lateral surface of the cell or vice versa in accordance with the fluid mosaic model.

Tight junctions that join epithelial cells near their apical surface prevent the migration of proteins from the basolateral membrane to the apical membrane. The basal and lateral surfaces thus remain roughly equivalent to one another, yet distinct from the apical surface.

Integral membrane proteins

The cell membrane contains many integral membrane proteins, which pepper the entire surface. These structures, which can be visualized by electron microscopy or fluorescence microscopy, can be found on the inside of the membrane, the outside, or membrane spanning. These may include integrins, cadherins, desmosomes, clathrin-coated pits, caveolaes, and different structures involved in cell adhesion.

Membrane skeleton

The cytoskeleton is found underlying the cell membrane in the cytoplasm and provides a scaffolding for membrane proteins to anchor to, as well as forming organelles that extend from the cell. Indeed, cytoskeletal elements interact extensively and intimately with the cell membrane.^[4] Anchoring proteins restricts them to a particular cell surface — for example, the apical surface of epithelial cells that line the vertebrate gut — and limits how far they may diffuse within the bilayer. The cytoskeleton is able to form appendage-like organelles, such as cilia, which are microtubule-based extensions covered by the cell membrane, and filopodia, which are actin-based extensions. These extensions are ensheathed in membrane and project from the surface of the cell in order to sense the external environment and/or make contact with the substrate or other cells. The apical surfaces of epithelial cells are dense with actin-based finger-like projections known as microvilli, which increase cell surface area and thereby increase the absorption rate of nutrients. Localized decoupling of the cytoskeleton and cell membrane results in formation of a bleb.

Carbohydrates

Plasma membranes also contain carbohydrates, predominantly glycoproteins, but with some glycolipids (cerebrosides and gangliosides). For the most part, no glycosylation occurs on membranes within the cell; rather generally glycosylation occurs on the extracellular surface of the plasma membrane.

The glycocalyx is an important feature in all cells, especially epithelia with microvilli. Recent data suggest the glycocalyx participates in cell adhesion, lymphocyte homing, and many others.

The penultimate sugar is galactose and the terminal sugar is sialic acid, as the sugar backbone is modified in the golgi apparatus. Sialic acid carries a negative charge, providing an external barrier to charged particles.

Proteins

Type	Description	Examples
Integral proteins or transmembrane proteins	Span the membrane and have a hydrophilic cytosolic domain, which interacts with internal molecules, a hydrophobic membrane-spanning domain that anchors it within the cell membrane, and a hydrophilic extracellular domain that interacts with external molecules. The hydrophobic domain consists of one, multiple, or a combination of α-helices and β sheet protein motifs.	Ion channels, proton pumps, G protein-coupled receptor
Lipid anchored proteins	Covalently-bound to single or multiple lipid molecules; hydrophobically insert into the cell membrane and anchor the protein. The protein itself is not in contact with the membrane.	G proteins
Peripheral proteins	Attached to integral membrane proteins, or associated with peripheral regions of the lipid bilayer. These proteins tend to have only temporary interactions with biological membranes, and, once reacted the molecule, dissociates to carry on its work in the cytoplasm.	Some enzymes, some hormones

The cell membrane plays host to a large amount of protein that is responsible for its various activities. The amount of protein differs between species and according to function, however the typical amount in a cell membrane is 50%.^[6] These proteins are undoubtedly important to a cell: Approximately a third of the genes in yeast code specifically for them, and this number is even higher in multicellular organisms.^[5]

The cell membrane, being exposed to the outside environment, is an important site of cell-cell communication. As such, a large variety of protein receptors and identification proteins, such as antigens, are present on the surface of the membrane. Functions of membrane proteins can also include cell-cell contact, surface recognition, cytoskeleton contact, signaling, enzymatic activity, or transporting substances across the membrane.

Most membrane proteins must be inserted in some way into the membrane. For this to occur, an N-terminus “signal sequence” of amino acids directs proteins to the endoplasmic reticulum, which inserts the proteins into a lipid bilayer. Once inserted, the proteins are then transported to their final destination in vesicles, where the vesicle fuses with the target membrane.

Plant Cell Structure

Plants are unique among the eukaryotes, organisms whose cells have membrane-enclosed nuclei and organelles, because they can manufacture their own food. Chlorophyll, which gives plants their green color, enables them to use sunlight to convert water and carbon dioxide into sugars and carbohydrates, chemicals the cell uses for fuel.

 

Like the fungi, another kingdom of eukaryotes, plant cells have retained the protective cell wall structure of their prokaryotic ancestors. The basic plant cell shares a similar construction motif with the typical eukaryote cell, but does not have centrioles, lysosomes, intermediate filaments, cilia, or flagella, as does the animal cell. Plant cells do, however, have a number of other specialized structures, including a rigid cell wall, central vacuole, plasmodesmata, and chloroplasts. Although plants (and their typical cells) are non-motile, some species produce gametes that do exhibit flagella and are, therefore, able to move about.

Plants can be broadly categorized into two basic types: vascular and nonvascular. Vascular plants are considered to be more advanced thaonvascular plants because they have evolved specialized tissues, namely xylem, which is involved in structural support and water conduction, and phloem, which functions in food conduction. Consequently, they also possess roots, stems, and leaves, representing a higher form of organization that is characteristically absent in plants lacking vascular tissues. The nonvascular plants, members of the division Bryophyta, are usually no more than an inch or two in height because they do not have adequate support, which is provided by vascular tissues to other plants, to grow bigger. They also are more dependent on the environment that surrounds them to maintain appropriate amounts of moisture and, therefore, tend to inhabit damp, shady areas.

It is estimated that there are at least 260,000 species of plants in the world today. They range in size and complexity from small, nonvascular mosses to giant sequoia trees, the largest living organisms, growing as tall as 330 feet (100 meters). Only a tiny percentage of those species are directly used by people for food, shelter, fiber, and medicine. Nonetheless, plants are the basis for the Earth’s ecosystem and food web, and without them complex animal life forms (such as humans) could never have evolved. Indeed, all living organisms are dependent either directly or indirectly on the energy produced by photosynthesis, and the byproduct of this process, oxygen, is essential to animals. Plants also reduce the amount of carbon dioxide present in the atmosphere, hinder soil erosion, and influence water levels and quality.

Plants exhibit life cycles that involve alternating generations of diploid forms, which contain paired chromosome sets in their cell nuclei, and haploid forms, which only possess a single set. Generally these two forms of a plant are very dissimilar in appearance. In higher plants, the diploid generation, the members of which are known as sporophytes due to their ability to produce spores, is usually dominant and more recognizable than the haploid gametophyte generation. In Bryophytes, however, the gametophyte form is dominant and physiologically necessary to the sporophyte form.

Animals are required to consume protein in order to obtaiitrogen, but plants are able to utilize inorganic forms of the element and, therefore, do not need an outside source of protein. Plants do, however, usually require significant amounts of water, which is needed for the photosynthetic process, to maintain cell structure and facilitate growth, and as a means of bringing nutrients to plant cells. The amount of nutrients needed by plant species varies significantly, but nine elements are generally considered to be necessary in relatively large amounts. Termed macroelements, these nutrients include calcium, carbon, hydrogen, magnesium, nitrogen, oxygen, phosphorus, potassium, and sulfur. Seven microelements, which are required by plants in smaller quantities, have also been identified: boron, chlorine, copper, iron, manganese, molybdenum, and zinc.

Thought to have evolved from the green algae, plants have been around since the early Paleozoic era, more than 500 million years ago. The earliest fossil evidence of land plants dates to the Ordovician Period (505 to 438 million years ago). By the Carboniferous Period, about 355 million years ago, most of the Earth was covered by forests of primitive vascular plants, such as lycopods (scale trees) and gymnosperms (pine trees, ginkgos). Angiosperms, the flowering plants, didn’t develop until the end of the Cretaceous Period, about 65 million years ago—just as the dinosaurs became extinct.

Protoplasm, Cytoplasm and Cytosol: The Cell’s Content

The “living” content of a cell, the protoplasm , is surrounded by a membrane called plasmamembrane or plasmalemma. The protoplasm is usually next to the cell wall, so that the plasmalemma can hardly be seen. To display it, the cells are transferred into a high salt or sugar solution. As a result the protoplasm shrinks and detaches itself from the wall. The process is reversible and is called plasmolysis. This behavior is due to the properties of the membrane and the plasma. It is reviewed in more detail elsewhere. A substance that causes plasmolysis is called plasmolyticum and – depending on its chemical composition (potassium ions or calcium ions, for example) – the protoplasm takes on different shapes. The plasmolyticum has accordingly an influence on the properties of the membrane. The properties of the plasmamembrane differ from that of the tonoplast. The tonoplast is the membrane that surrounds the vacuole. The difference is especially striking if cells with a colored vacuole content are used. Often the vacuole is criss-crossed by numerous plasma cords. The plasma cannot therefore not simply be viewed as a solution that is influenced by the rules of hydrodynamics alone. Rather, it contains viscous, structure-determining components, whose chemical, physicochemical and structural properties have only been recognized recently and in fragments.

Cytoplasm and Caryoplasm

The nucleus is a rather conspicuous part of nearly every living plant cell. Its structure separated from the rest of the cell by the nuclear envelope, a membrane system that consists of two discrete membranes as can be seen on electromicroscopic images. The nuclear content is called the caryoplasm while that of the rest of the cell is called cytoplasm. But these terms are only valid at certain stages of a cell’s life cycle. In the course of cell-division and mitosis, the nuclear envelope disintegrates and the nucleus is replaced by the chromosomes. It makes consequently no sense to speak of caryo- and cytoplasm during these stages.

The nucleus of plant cells is usually of a round or elliptic appearance, sometimes it is also shaped like a spindle. One nucleus per cell is the rule, but cells with two or more nuclei are no rare exception. The cells of certain algae of the genus Chladophora have many nuclei, they are polyenergid. The nucleoli that can often be perceived after staining are substructures of the nucleus. They, too, disintegrate during cell division and mitosis and do not reshape before a new nucleus has been formed.

Plastids

Plastids are organelles that occur only in plants. Their most prominent members are the chloroplasts. Others plastids are the colored chromoplasts and the colorless leucoplasts as well as their proplastids. Proplastids are vestigial bodies that are generated during germ cell development due to degeneration of plastids, for example. They may differentiate into complete plastids during the development of the plant embryo. Their ripening into chloroplasts occurs usually only after light exposure.

Chloroplasts contain the green plant color chlorophyll. They are the places where photosynthesis takes place. Chloroplasts enable the plants to convert solar energy into chemical energy. Because of this process, plants are called primary producers. The existence of consumers, like most animals, depends on them. Chloroplasts occur in most cell types, but only in organs above ground. They can be especially well observed in tissues consisting of a single layer as in the leaflike structures of some mosses (Funaria hygrometrica or Mnium hornum) or in the water plant Vallisneria). Here they are rather large and of a lens-shaped appearance. During daytime, when the light is diffuse, they occur mainly at the upper and lower surface of the chloroplast. They appear to be round under top view. If exposed to strong light, they gather in parallel to the lateral sides of the cell which gives them an elleptic appearance upon top view.

Chloroplasts are the site of starch production and -storage. Starch can easily be detected with the aid of potassium iodide (LUGOL’s reagent). The starch-iodine complex is deeply blue-violet.

 

Starch production during photosynthesis can be made visible by placing a mask at a leaf that covers it partially while leaving some places exposed to sunlight. After one day of exposure, the leaf is first bleached to get rid of other pigments and afterwards treated with potassium iodide. An image is gained that is the exact replication of the mask and at the same time represents starch synthesis in the leaf. This experiment has first been done by J. v SACHS, probably the most outstanding plant physiologist of the 20th century. He thought that starch was the primary product of photosynthesis. This assumption proved wrong. It is well-known today that the first products of photosynthesis are monomeric sugars (glucose and others) and that only part of them is used for starch production.

The structures of the chloroplasts of higher cells resemble largely that of mosses. Their average diameter is 4 – 8 mm, an average cells contains 10 through 50. Their chlorophyll is unevenly distributed. At high resolutions chlorophyll-rich and chlorophyll-poor areas can be distinguished. This is due to the inner structure of the chloroplast: it is organized into grana (chlorophyll-rich) and stroma (chlorophyll-poor). Upon stimulation with short-waved light (blue or violet) the chlorophyll emits an intensive red autofluorescence that looks especially impressive in a fluorescence microscope. The differences between grana and stroma become very obvious. The uniformity of the chloroplasts of all higher plants points out that the optimal form has been found rather early in evolution and has not been changed since. This is different with algae. The chloroplasts of green algae (Chlorophyceens) are very varied in shape. Many species have just one chloroplast that covers nearly the whole space of the cell’s interior. It is screw-like in Spirogyra-species, star-shaped in Zygnema and Zygnemopsis and netlike in Oedogonium.

The disc-shaped chloroplast of Mougeotia can be viewed either from above or in profile depending on the amount of light used. Its rotation is a well-analyzed example of an induced chloroplast movement. The chloroplasts of many species of algae contain often well-visible pyrenoids, structures, that produce and structure starch.

Chromoplasts are red, orange or yellow plastids. The color is usually the result of yellow xantophyll and yellow to red carotinoids. Both compounds do also exist in chloroplasts, but are concealed by chlorophyll. Chlorophyll is broken down much faster than carotinoids as can be observed in the colored leaves in autumn. Fluid transitions between chromo- and chloroplasts exist, just as between chromo- and leucoplasts. Typical chromoplasts cause the orange color of the carrot, the red color of the ripe pimento and tomato as well as the color of numerous flowers. Carotinoids are not very water-soluble and do therefore often crystallize within the chromoplasts. Their crystals can be disc-shaped, needle-like, jagged or sickle-like.

In many cases, flower and leaf colors are caused by the colored content of the vacuole. The color of the vacuole and that of the plastids may lead to a mixed color. The leaves of the copper beech, where the vacuole’s content is red and that of the chloroplasts is green are a typical example. The plastids of the red and brown algae are traditionally counted among the chromoplasts although they contain chlorophyll.

The green colour is concealed by the red phycoerythrin (Rhodophyceae) or the brown fucoxanthin (Phaeophyta).

Leucoplasts are common, colorless plastids. They develop from proplastids, but form no homogeneous group of their own. A part of them can differentiate into chloroplasts or chromoplasts at light exposure, while this is not due for others. The guard cells, for example, contain leucoplasts, that are permanently exposed to light without developing into chloroplasts. Leucoplasts do also occur within colorless leaves (variegated leaves) or plant parts. There exists a number of examples which show that they developed from chloroplasts that lost their ability to produce chlorophyll. There are even species, like Neottia, an orchid that cannot produce chlorophyll at all and are thus dependent on a parasitic or saprophytic lifestyle (saprophy is the feeding from dead organic material).

A second class of leucoplasts occurs within the non-green tissues of otherwise green plants. It is especially common within roots. Though these leucoplasts are capable to become green, they do usually not since they are not exposed to light. The leucoplasts of the calyptra (a calyptra is any hood or cap of cells protecting a plant part) contain starch and are therefore counted among the amyloplasts (starch-containing leucoplasts). They have, as is explained later, the function of statolithes, that have an important part in the perception of gravity (geotropism).

Starch

We got to know starch in the section above as a content of chloroplasts and leucoplasts (amyloplasts). It is produced by the polymerization of glucose residues, which again are products of photosynthesis. Since the plant is able to transport sugars from leaf to root or from leaf to seed and fruit, starch production can also take place in these organs. Different species produce starch grains of different shape. Since the shape of starch grains informs about their origin, they are helpful in the identification of seeds and other starch-containing plant parts. The following numbers show the variations in their diameters. Starch grains from potato tubers have a diameter of 70 – 100 µm, that of the endosperm of wheat 30 – 45 µm and that of corn endosperm 12 – 18 µm. Their shape reflects their development. The starch molecules are long-stretched and only sparsely branched. They are deposited within the plastids and their development begins at a so-called formation center from where it proceeds radially. Layer follows layer and the thickness of one layer is dependent on the average molecular length. A starch grain is therefore organized like a crystal (semi-crystalline). This can be shown very impressively with a polarization microscope. Within the moistened specimen can a layering be viewed that is dependent on the water content of the single molecular parts.

A. Model of a starch grain structure. The single lines symbolize starch molecules. They are arranged in a radial pattern. B. Layering of the starch grains. a. Formation center and layer borders, b. Diagram of the refraction conditions. The ordinate shows the refraction index. (according to A. FREY-WYSSLING, 1938)

The denser the molecular packaging, the less water is deposited. Layers with less water content refract light stronger than those with much water. After drying of the preparatioo layering can be perceived any more. Depending the central or peripheral placing of the formation center, starch grains with either concentric or eccentric layering develop. The starch grains of graminaceous plants (wheat, corn, etc.) are usually concentric, while those of the potato are always eccentric.

Sometimes plastids with two to three formation centres occur in graminaceous plants. This leads to the development of several starch grains. During growth common outer layers may be formed.

Compound starch grains are typical for oat, they are built from a large number of smaller grains.

Starch grains in bean seeds (Phaseolus vulgaris) are very big, their shape is round or oval, the spacing of the layers is very regular. Their centers can easily be hollowed out by addition of water displaying radial ruptures in microscopic images. In the sap of Euphorbia splendens dumb-bell shaped starch grains can be found.

Thus

                                         Plant Cell Structure

Plants are unique among the eukaryotes, organisms whose cells have membrane-enclosed nuclei and organelles, because they can manufacture their own food. Chlorophyll, which gives plants their green color, enables them to use sunlight to convert water and carbon dioxide into sugars and carbohydrates, chemicals the cell uses for fuel.

Like the fungi, another kingdom of eukaryotes, plant cells have retained the protective cell wall structure of their prokaryotic ancestors. The basic plant cell shares a similar construction motif with the typical eukaryote cell, but does not have centrioles, lysosomes, intermediate filaments, cilia, or flagella, as does the animal cell. Plant cells do, however, have a number of other specialized structures, including a rigid cell wall, central vacuole, plasmodesmata, and chloroplasts. Although plants (and their typical cells) are non-motile, some species produce gametes that do exhibit flagella and are, therefore, able to move about.

Plants can be broadly categorized into two basic types: vascular and nonvascular. Vascular plants are considered to be more advanced thaonvascular plants because they have evolved specialized tissues, namely xylem, which is involved in structural support and water conduction, and phloem, which functions in food conduction. Consequently, they also possess roots, stems, and leaves, representing a higher form of organization that is characteristically absent in plants lacking vascular tissues. The nonvascular plants, members of the division Bryophyta, are usually no more than an inch or two in height because they do not have adequate support, which is provided by vascular tissues to other plants, to grow bigger. They also are more dependent on the environment that surrounds them to maintain appropriate amounts of moisture and, therefore, tend to inhabit damp, shady areas.

It is estimated that there are at least 260,000 species of plants in the world today. They range in size and complexity from small, nonvascular mosses to giant sequoia trees, the largest living organisms, growing as tall as 330 feet (100 meters). Only a tiny percentage of those species are directly used by people for food, shelter, fiber, and medicine. Nonetheless, plants are the basis for the Earth’s ecosystem and food web, and without them complex animal life forms (such as humans) could never have evolved. Indeed, all living organisms are dependent either directly or indirectly on the energy produced by photosynthesis, and the byproduct of this process, oxygen, is essential to animals. Plants also reduce the amount of carbon dioxide present in the atmosphere, hinder soil erosion, and influence water levels and quality.

Plants exhibit life cycles that involve alternating generations of diploid forms, which contain paired chromosome sets in their cell nuclei, and haploid forms, which only possess a single set. Generally these two forms of a plant are very dissimilar in appearance. In higher plants, the diploid generation, the members of which are known as sporophytes due to their ability to produce spores, is usually dominant and more recognizable than the haploidgametophyte generation. In Bryophytes, however, the gametophyte form is dominant and physiologically necessary to the sporophyte form.

Animals are required to consume protein in order to obtaiitrogen, but plants are able to utilize inorganic forms of the element and, therefore, do not need an outside source of protein. Plants do, however, usually require significant amounts of water, which is needed for the photosynthetic process, to maintain cell structure and facilitate growth, and as a means of bringing nutrients to plant cells. The amount of nutrients needed by plant species varies significantly, but nine elements are generally considered to be necessary in relatively large amounts. Termed macroelements, these nutrients include calcium, carbon, hydrogen, magnesium, nitrogen, oxygen, phosphorus, potassium, and sulfur. Sevenmicroelements, which are required by plants in smaller quantities, have also been identified: boron, chlorine, copper, iron, manganese, molybdenum, and zinc.

Thought to have evolved from the green algae, plants have been around since the earlyPaleozoic era, more than 500 million years ago. The earliest fossil evidence of land plants dates to the Ordovician Period (505 to 438 million years ago). By the Carboniferous Period, about 355 million years ago, most of the Earth was covered by forests of primitive vascular plants, such as lycopods (scale trees) and gymnosperms (pine trees, ginkgos).Angiosperms, the flowering plants, didn’t develop until the end of the Cretaceous Period, about 65 million years ago—just as the dinosaurs became extinct.

·           Cell Wall – Like their prokaryotic ancestors, plant cells have a rigid wall surrounding the plasma membrane. It is a far more complex structure, however, and serves a variety of functions, from protecting the cell to regulating the life cycle of the plant organism.

·           Chloroplasts – The most important characteristic of plants is their ability to photosynthesize, in effect, to make their own food by converting light energy into chemical energy. This process is carried out in specialized organelles called chloroplasts.

·           Endoplasmic Reticulum – The endoplasmic reticulum is a network of sacs that manufactures, processes, and transports chemical compounds for use inside and outside of the cell. It is connected to the double-layered nuclear envelope, providing a pipeline between the nucleus and the cytoplasm. In plants, the endoplasmic reticulum also connects between cells via the plasmodesmata.

·           Golgi Apparatus – The Golgi apparatus is the distribution and shipping department for the cell’s chemical products. It modifies proteins and fats built in the endoplasmic reticulum and prepares them for export as outside of the cell.

·           Microfilaments – Microfilaments are solid rods made of globular proteins called actin. These filaments are primarily structural in function and are an important component of the cytoskeleton.

·           Microtubules – These straight, hollow cylinders are found throughout the cytoplasm of all eukaryotic cells (prokaryotes don’t have them) and carry out a variety of functions, ranging from transport to structural support.

·           Mitochondria – Mitochondria are oblong shaped organelles found in the cytoplasm of all eukaryotic cells. In plant cells, they break down carbohydrate and sugar molecules to provide energy, particularly when light isn’t available for the chloroplasts to produce energy.

·           Nucleus – The nucleus is a highly specialized organelle that serves as the information processing and administrative center of the cell. This organelle has two major functions: it stores the cell’s hereditary material, or DNA, and it coordinates the cell’s activities, which include growth, intermediary metabolism, protein synthesis, and reproduction (cell division).

·           Peroxisomes – Microbodies are a diverse group of organelles that are found in the cytoplasm, roughly spherical and bound by a single membrane. There are several types of microbodies but peroxisomes are the most common.

·           Plasmodesmata – Plasmodesmata are small tubes that connect plant cells to each other, providing living bridges between cells.

·           Plasma Membrane – All living cells have a plasma membrane that encloses their contents. In prokaryotes and plants, the membrane is the inner layer of protection surrounded by a rigid cell wall. These membranes also regulate the passage of molecules in and out of the cells.

·           Ribosomes – All living cells contain ribosomes, tiny organelles composed of approximately 60 percent RNA and 40 percent protein. In eukaryotes, ribosomes are made of four strands of RNA. In prokaryotes, they consist of three strands of RNA.

·           Vacuole – Each plant cell has a large, single vacuole that stores compounds, helps in plant growth, and plays an important structural role for the plant.

Leaf Tissue Organization – The plant body is divided into several organs: roots, stems, and leaves. The leaves are the primary photosynthetic organs of plants, serving as key sites where energy from light is converted into chemical energy. Similar to the other organs of a plant, a leaf is comprised of three basic tissue systems, including the dermal,vascular, and ground tissue systems. These three motifs are continuous throughout an entire plant, but their properties vary significantly based upon the organ type in which they are located. All three tissue systems are discussed in this section.

In reviewing plant physiology, plant cells are grouped into tissues based on similar characteristics, then into five distinct structures (organs).

Cells – individual building blocks for life processes and growth.  Common cells contain genetic matter (deoxyribonuecleic acid or DNA) and metabolic organelles but they are mostly water.  In green plants, they are the site of sugar production (photosynthesis).

lant Metabolism

Introduction

Plants are responsible for incredible feats of molecular transformation. The processes are always being studied, but there are a few basic things that are well understood at this point in history. We will be looking at photosynthesis and respiration in some detail, as always, if you have additional questions please post them on the forum.

Photosynthesis

Photosynthesis is the process by which light energy is captured, converted and stored in simple sugar molecule. This process occurs in chloroplasts and other parts of green organisms.  It is a backbone process, in the sense that all life on earth depends on it’s functioning. The following equation sums up the process:

6CO₂ + 12 H₂O + light energy -> C₆H₁₂O₆ + 6O₂ +6H₂O

carbon        water                              glucose     oxygen   water

As you see from the equation, this process is vital to us as humans, because it transforms carbon dioxide into oxygen—which we enjoy with every breath!

Carbon Dioxide (CO₂)

The earth’s atmosphere contains approximately 79% nitrogen, 20% oxygen and the remaining 1% is a mixture of less common gases—including 0.039% carbon dioxide. Carbon dioxide in the atmosphere reaches plant mesophyll via the stomata. The carbon dioxide dissolves on the thin film of water that covers the outside of cells. The carbon dioxide then diffuses through the cell wall into the cytoplasm in order to reach the chloroplasts. The oceans hold a large reservoir of carbon dioxide, which keeps the atmospheric levels essentially constant. Although there are some indicators that the atmospheric levels of CO₂ are rising and adding to the global warming issue. That is a whole other topic though.

Water

Water is plentiful on earth, however, it may or may not be plentiful at the location of each individual plant. Therefore, plants will close their stomata, if need be, which reduces the CO₂ supply to the mesophyll. Not even 1% of the water that is absorbed by plants is used in photosynthesis, the remainder is either transpired or incorporated into protoplasm, vacuoles or other cell materials. The water utilized in photosynthesis is the source of oxygen released as a photosynthetic byproduct.

Light

Light has a dual nature, in that it exhibits properties of both waves and particles. The energy from the sun comes to earth in various wavelengths, the longest being radio waves and the shortest are gamma rays.  Approximately 40% of the radiant energy the earth receives from the sun is visible light. Visible light ranges from red, 780 nanometers to violet, 390 nanometers. The violet to blue and red to orange ranges are the most often used in photosynthesis. Most light in the green range is reflected. Of the visible light that reaches a leaf, approximately 80% is absorbed. Light intensity varies widely. Time of day, temperature, season of year, altitude, latitude and other atmospheric conditions all play roles in the intensity of the radiant energy that will reach the earth and it’s organisms. High intensity light isn’t necessarily a beneficial thing for plants. In high intensity light, photorespiration may occur, which is a type of respiration that uses oxygen and releases carbon dioxide but differs from standard aerobic respiration in the pathways that it utilizes.

Chlorophyll

A few things to know about chlorophyll before we get into the nitty gritty of photosynthesis and respiration. There are more than one type of chlorophyll, however, they all have one atom of magnesium in the center. In some ways the chlorophyll is quite analogous to the heme structure in hemoglobin (the iron containing pigment that carries oxygen in blood). Chlorophyll has a long lipid tail that anchors the molecule in the lipid layers of the thylakoid membranes—recall that thylakoids are coin-like discs in stacks within the stroma of the chloroplasts.  The chloroplasts of most plants contain two types of chlorophyll imbedded in the thylakoid membranes. The formula for bluish-green chlorophyll a is C₅₅H₇₂MgN₄O₅ and the formula for yellow-green chlorophyll b is C₅₅H₇₀MgN₄O₆. In general, most a chloroplast has about three times as much chlorophyll a than b. The main role of chlorophyll b is to broaden the spectrum of light available for photosynthesis: chlorophyll b absorbs light energy and transfers the energy to a chlorophyll a molecule. Other pigments are contained in chlorophyll c, d, and e and take the place for chlorophyll b in some cases. Note that all the chlorophyll molecules are related to each other and differ only slightly in molecular structure.  Light-harvesting complexes contain 250 to 400 pigment molecules and are referred to as a photosynthetic unit. There are countless numbers of these units spread throughout the grana of a chloroplast. In the chloroplasts of green plants, two types of these harvesting units operate together in order to bring about the first phase of photosynthesis.

The photosynthetic process occurs in two successive processes: the light reactions and the carbon-fixing reactions.

1.     The light reactions

The light reactions involve light striking the chlorophyll molecules embedded in the thylakoids of chloroplasts. The subsequent reaction results in the conversion of some light energy to chemical energy. In the light reactions, water molecules are split apart into hydrogen ions and electrons and oxygen gas is released. In addition, ATP (adenosine triphosphate) molecules are created and the hydrogen ions derived from the water molecules are involved in “loading” NADP which carries the hydrogen as NADPH. NADPH is integral in providing the hydrogen ions used in the second series of major photosynthetic reactions: the carbon-fixing reactions.

1.     The carbon-fixing reactions

The carbon-fixing reactions used to be called the dark reactions because light does not play a direct role in their functioning. The reactions take place in series outside of the grana in the stroma of the chloroplast. These reactions only occur if the end products of the light reactions are available for use. Depending on the plant involved, the carbon-fixing reactions may develop or progress in different ways. The most common type of carbon-fixing reactions in plants is the process called the Calvin cycle. In the Calvin cycle, carbon dioxide from the atmosphere is combined with a 5-carbon sugar—RuBP, or ribulose bisphosphate. The combined molecules are converted via several steps into a 6-carbon sugar, such as glucose. The ATP and NADPH molecules from the light reactions provide the energy and resources for the reactions. Some of the sugars produced are further combined into polysaccharides (strings of simple sugars) or are stored as starch within the plant. There are other variations, including the 4-carbon pathway which is usually found in desert plants (C₄ plants).

Before getting into respiration let’s take a closer look at what happens in both the light reactions and the carbon-fixing reactions.

Nitty-gritty of Light Reactions

Einstein called the discrete particles of light photons.  Particles (photons) and waves are both currently accepted aspects of light. The quantum (energy) of photons is different depending on what kind of light they are in. Longer wavelength light has lower photon energies, while light with shorter wavelengths have higher photon energies. As mentioned earlier, every pigment color has a different distinctive pattern of light absorption—called the pigment’s absorption spectrum. The energy levels of some of the pigment’s electrons are raised when the pigment absorbs light. If energy is emitted immediately upon absorption, the effect is called fluorescence. The red part of light does this characteristically, as demonstrated when chlorophyll is placed in light it will appear red. If the absorbed energy is emitted as light after a delay, then the effect is called phosphorescence. The energy may be converted to heat or stored, as in photosynthesis within chemical bonds.

Oxidation-reduction reactions

OIL RIG, a cute little mnemonic device to remember that oxidation is loss and reduction is gain. Perhaps better put, oxidation results in the net loss of an electron or electrons, while reduction results in a net gain of an electron or electrons. The electrons come from  compounds within the process or donated in from previous processes. These types of chemical reactions are found scattered throughout the processes within photosynthesis and respiration.

Photosystems

The two types of photosynthetic units in most chloroplasts are what constitute photosystem I and photosystem II.

1.     Photosystem I contains photosynthetic units with 200 or more molecules of chlorophyll a, small amounts of chlorophyll b, protein saddled carotenoid pigment, and a pair of specialized reaction-center molecules of chlorophyll called P₇₀₀. All pigments in a photosystem are capable of absorbing photons, however, only the reaction-center molecules can really utilize the light energy.  The other pigments aren’t worthless in the system, as they act sort of like an antenna in gathering and passing light energy along to the reaction-center. Iron-sulphur complexed proteins initially receive electrons from P₇₀₀ and serve as primary electron acceptors for the unit.

2.     Photosystem II contains chlorophyll a, protein saddled beta-carotene, a small amount of chlorophyll b and special pair of reaction-center molecules of chlorophyll a otherwise called, P₆₈₀. The photosystem has a primary electron acceptor called pheophytin or Pheo.

For the record, the 680 and 700 in the names of the reaction-center molecules stands for the peaks in the absorption spectra of light waves of 680 nm and 700 nm.

Photolysis

A photon of light strikes the photosystem II reaction-center, the P₆₈₀ molecule to be exact near the inner surface of a thylakoid membrane. The received light energy excites an electron (boosts it to a higher energy level) which is an unstable reaction and thus most of the energy is lost to heat. Up to four photons at a time can strike the P680 molecule, however, it can only accept one electron at a time.  The molecule of pheophytin picks up the excited electron, which then crosses the thylakoid membrane and is passed along to another acceptor called plastoquinone or Pq near the outside surface of the thylakoid membrane. Protein Z extracts electrons from water and replaces the ones lost by the P680 molecule. Protein Z contains manganese which is required in order to split water molecules. Simultaneously, as two water molecules are split and molecule of oxygen and four protons are produced. This enzyme-mediated water splitting process is called photolysis.

Photophosphorylation

Pq, the acceptor molecule, releases the excited electron into the care of an electron transport system that is sort of like a downhill bucket brigade. The transport system moves electrons extracted from water temporarily to a high-energy storage molecule called nicotinamide adenine dinucleotide phosphate (NADP⁺). NADP⁺ is an electron acceptor for photosystem. The transport chain is essentially iron-containing pigments, cytochromes, a copper containing protein called plastocyanin and other electron transferring molecules. As electrons are passed through the chain and protons are being shuffled through a coupling factor, ATP molecules are assembled from ADP and phosphate in a process called photophosphorylation.

A similar series of events occurs in photosystem I. After a photon of light strikes a P₇₀₀ molecule, the resulting excited electron is passed along to an iron-sulphur molecule Fe-S which in turn passes it to another acceptor molecule ferrodoxin, (Fd). The ferrodoxin molecule releases the electron to a carrier molecule called flavin adenine dinucleotide (FAD) and then eventually on to NADP⁺. A reduction occurs and NADP+ becomes NADPH.  Electrons from photosystem II and the activities of the electron transport system replace any electrons removed from the P₇₀₀ molecule. Because the electrons move in one direction, the movement of electrons from water to photosystem II to photosystem I to NADP⁺ are said to be part of noncyclic electron flow. Any ATP that is produced are designated noncyclic phosphorylation.

It should be noted that photosystem I can operate independently of photosystem II. When this occurs, the electrons boosted from P₇₀₀ reaction-center molecules (photosystem I) are passed through an intermediary acceptor molecule called P₄₃₀ and then on to the electron transport chain. This is rather then to the ferrodoxin and NADP⁺. After being passed through the electron transport chain, the electron is dumped back into the reaction-center of photosystem I. This process demonstrates cyclic electron flow and any ATP generated by cyclic electron flow is termed cyclic phosphorylation. Note, that no water molecules are split and no NADPH or oxygen is produced.

Chemiosmosis

Earlier we mentioned in passing a coupling factor. The enzyme necessary for mediation of the splitting of water molecules is on the inside of the thylakoid membrane. As a result of this, a proton gradient forms across the membrane and the movement of these protons is thought to be a source of energy for generating ATP. The motion is thought to be similar to molecular movement during osmosis and has hence been termed chemiosmosis. As the protons move across the membrane, they are assisted in crossing by protein channels called ATPase or coupling factor.  Because of the proton movement, ADP and phosphate combine which produces ATP.

Nitty-gritty of Carbon-Fixing Reactions

Both ATP and NADPH are important products of the light reactions and both of them play roles in the synthesis of carbohydrates from atmospheric carbon dioxide. Although the carbon-fixing reactions do not require daylight, they generally are conducted during daylight hours as there is some indication that some of the enzymes required for the processes in carbon-fixing may require some level of light.  These reactions take place in the stroma of the chloroplast.

Three known mechanisms of converting carbon dioxide to sugar.

1.     The Calvin Cycle or the 3-carbon pathway—The Calvin cycle is the most common of the three mechanisms and has four main results:

1.     With the assistance of the enzyme rubisco (RuBP carboxylase), six molecules of atmospheric carbon dioxide combine with six molecules of ribulose 1, 5-bisphosphate (RuBP)

2.     The result of the first step is six unstable 6-carbon complexes, which immediately split into two 3-carbon molecules of 3-phosphoglyceric acid or 3PGA. This is the first stable compound in photosynthesis.

3.     NADPH and ATP from the light-reactions, supply the energy required to convert the 3PGA to 12 molecules of glyceraldehydes 3-phosphate (GA3P), which is a 3-carbon sugar phosphate complex.

4.     Finally, of the 12 molecules formed; 10 are restructured into six 5-carbon molecules of RuBP—the sugar that the process started with.

 The sugars produced can either add to an increase in the sugar content (carbohydrate content) of the plant or they can be used in pathways that lead to the production of lipids and amino acids.

1.     4-Carbon Pathway—C₄ plants: These plants use a 4-carbon molecule called oxaloacetic acid in place of the 3-carbon 3-phosphoglyceric acid used in step two of the Calvin cycle. Oxaloacetic acid is produced from a 3-carbon compound PEP—phosphoenolpyruvate and carbon dioxide. This process is enzyme mediated and occurs in the mesophyll cells of the leaf. Some species will convert the resulting oxaloacetic acid to aspartic, malic or other acids.

Note that the acids do not substitute for 3PGA. The 4-carbon acids migrate to the bundle sheaths surrounding the vascular bundles, where they are further converted to pyruvic acid and carbon dioxide. In returning to the mesophyll cells and interacting with ATP molecules, the pyruvic acids molecules are able to produce additional PEP. In the bundle sheath cells, the carbon dioxide formed converts into 3PGA and other molecules, by combining with RuBP. The other molecules formed are similar to the other ones formed in the Calvin cycle. The C₄ cycle furnishes carbon dioxide to the Calvin cycle in a more roundabout way than the C₃ pathway, but there is an advantage to this extra pathway. The extra pathway greatly reduces photorespiration in C₄ plants, and this is a good thing because photorespiration is in direct competition with the Calvin cycle and even takes place in the light while the Calvin cycle is functioning. During photorespiration, RuBP reacts with oxygen to create carbon dioxide; in contrast, during photosynthesis RuBP and carbon dioxide are used to form carbohydrates. C₄ plants are able to pick up carbon dioxide in very low concentrations via the mesophyll cells. The Calvin cycle occurs in the bundle sheath where carbon dioxide is readily available. Because of the location, the enzyme rubisco will be in a prime spot to catalyze the reaction between RuBP and carbon dioxide, rather than oxygen. As a result C₄ plants have photosynthetic rates that are two to three times higher than C₃ plants. There are a few other characteristic features of C₄ plants worth noting:

1.     C₄ plants have two types of chloroplasts and an alternate pathway for using carbon dioxide. C₃ plants only have one type of chloroplast and one pathway. Chloroplasts with starch grains and are large with very little grana, and sometimes none, in the bundle sheath cells. In the mesophyll, the small, but numerous chloroplasts have no starch grains and contain highly developed grana.

2.     PEP carboxylase is found in high concentration in the mesophyll cells which permits ready conversion of carbon dioxide to carbohydrate at lower concentrations than does rubisco (in bundle sheath cells) of the Calvin cycle.

3.     The temperature ranges for C₄ plants are much higher than C₃ plants which enables C₄ plants to live well in conditions that would likely kill a C₃ plant.

1.     CAM Photosynthesis—Crassulacean acid metabolism is a modified photosynthetic system that is somewhat similar to C₄ photosynthesis in that 4-carbon compounds are produced during the carbon-fixing reactions. CAM plants accumulate malic acid in their chlorenchyma tissues at night, which is converted back to carbon dioxide during the day. In the daytime, malic acid diffuses out of the vacuoles and is converted to carbon dioxide for use in the Calvin cycle. PEP carboxylase is responsible for converting the carbon dioxide plus PEP to malic acid at night. This modification allows for a greater amount of carbon dioxide to be converted to carbohydrate during the day than would be otherwise converted given the conditions CAM plants generally grow in. CAM plants generally close their stomata during the day in order to reduce water loss. There are more than 20 families that contain CAM plants, including cacti, stonecrops, orchids, bromeliads and many succulents growing in regions of high light intensity. There are some succulents that do not have CAM photosynthetic capabilities, as well as non-succulents that do have the ability. 

There are great resources available that go into even greater detail on these reactions. If you are interested in these titles, please don’t be afraid to ask on the forum for direction.

Respiration

Respiration is the group of processes that utilizes the energy that is stored through the photosynthetic processes. The steps in respiration are small enzyme-mediated steps tha release tiny amounts of immediately available energy, the energy released is usually stored in ATP molecules which allow for even more efficient use of an organism’s energy. Respiration occurs in the mitochondria and cytoplasm of cells.

There are several forms of respiration: aerobic—which requires oxygen, anaerobic—which occurs in the absence of oxygen, and fermentation—which also occurs in the absence of oxygen.

Aerobic respiration is the most common form of respiration and cannot be completed without oxygen gas. The controlled release of energy is the main event in aerobic respiration.

Certain types of bacteria and other organisms without oxygen gas carry on anaerobic respiration and fermentation. Compared to aerobic respiration the amount of energy released is quite small. The main difference between aerobic respiration and fermentation is in the way hydrogen is released and combined with other substances. Two very common forms of fermentation are summed up by the following equations:

C₆H₁₂O₆ -> (with enzymes)-> 2C₂H₅OH + 2CO₂ + energy (ATP)

glucose                                       ethyl alcohol   carbon dioxide

C₆H₁₂O₆ -> (with enzymes) -> 2C₃H₆O₃ + energy (ATP)

glucose                                         lactic acid

Note the first equation is particularly valuable to the brewing industry.

Major Steps in Respiration:

Glycolysis—the first step does not require oxygen gas (O₂) and takes place in the cytoplasm. The glycolytic phase is subdivided into three main steps and several smaller ones. Each step is mediated by an enzyme. A small amount of energy is released and hydrogen atoms are removed from compounds derived from glucose. The main gist of the steps are:

A.     the glucose molecules goes through several steps and becomes a double phosphorylated fructose molecule.

B.     The 6-carbon fructose molecule is split into two 3-carbon fragments, each with a phosphate, GA3P

C.     Hydorgen, energy and water are removed from the GA3P molecules leaving pyruvic acid.

Glycolysis requires two molecules of ATP to get the process started. In the processes, four ATP molecules are created, with a net gain of 2 ATP molecules at the end of glycolysis. The hydrogen ions and electrons that are released are held by an acceptor molecule called NAD—nicotinamide adenine dinucleotide.  The overall end products of gylcolysis is: 2-ATP molecules, 2-NADH molecules, and pyruvic acid.

The next step depends on the kind of respiration involved—aerobic, true anaerobic or fermentation.

Aerobic Respiration (with oxygen present)

1.     The Krebs Cycle (or citric acid cycle)—The Krebs cycle takes place in the fluid matrix of the cristae compartments of the mitochondria. It is called the citric acid cycle because of all the intermediate acids in the cycle. The pyruvic acid product of glycolysis is restructured, some of the CO₂ is lost and becomes acetyl CoA which then dumps into the Krebs cycle. During the restructuring of pyruvic acid, a molecule of NADH is produced.  The Krebs cycle removes energy, CO₂ and hydrogen from acetyl CoA via enzyme mediated reactions of organic acids.

The hydrogen removed during the Krebs cycle is picked up by FAD and NAD acceptor molecules. The end result of the metabolizing of two acetyl CoA molecules in the Krebs cycle is: 2-ATP molecule, oxaloacetic acid (to further drive the cycle), 6-NADH₂ molecules, 2-FADH₂ molecules and 2CO₂ molecules.

The NAD and FAD molecules and the hydrogens that they carry will be dumped into the next step in respiration in order to extract the energy from the molecules.

1.     The Electron Transport Chain—The electron transport chain is a bit like a bucket brigade in that the chain passes the molecules along until the job is done. Energy is released as the hydrogen and electrons from the NAD⁺ and FAD⁺ carrier molecules is dumped into the system. When the electrons reach the end of the chain they pick up an oxygen and water is released. ATP is produced by oxidative phosphorylation during the action of the electron transport chain. This occurs essentially like chemiosmosis.

As a whole, from glycolysis to finish aerobic respiration yields the following:

Glycolysis:
  4-molecules of ATP
 +2-molecules of NADH—which yields 4-ATP in the ETS
 ————
  8-molecules of ATP net
  -2 ATP molecules to start the glycolysis process
 ————-
  6- ATP molecules
 Conversion of pyruvic acid to acetyl CoA:
  2-molecules of NADH—yields 6 ATP in the ETS

Krebs Cycle:
  2-molecules of ATP
 2-molecules of FADH₂—which yields 4-ATP in the ETS
 6-molecules of NADH₂—which yields 18-ATP in the ETS
 ————
Total ATP yield: 36

The 36 resulting ATP molecules represent approximately 39% of the energy in a molecule of glucose. Compared to each other, aerobic respiration is about six times as efficient as anaerobic respiration.

Anaerobic respiration and fermentation result in a net gain of 2-ATP molecules from glycolysis. It should be noted, that the by-products of these processes, lactic acid and alcohol, will eventually kill the organism if the products are not digested.

Factors regulating rate of respiration

Temperature—To a point, the higher the temperature the faster respiration occurs. At some temperature, enzymes will become inactivated, although there are thermophilic (heat-loving) organisms that do quite well in high-temperature environments. Energy from sugar is released faster as the rate of respiration increases which results in a net weight loss. Plants offset the weight loss by increasing photosynthetic production of sugar. Note that during respiration, some of the energy is lost as heat, which results in an overall increase in organism temperature—not necessarily detectible by human hands. 

Water—Enzymes generally operate in the presence of water, and reduced water in a plant will reduce the rate of respiration. Seeds usually have a water content of less than 10%, while mature living cells usually are in excess of 90% water. Seeds keep better if they are kept dry as the respiration rate remains quite low. However, if a seed comes into contact with water and via imbibition swells, the respiration rate will skyrocket. The temperature could increase to the point of killing the seeds. Spontaneous combustion can occur from the respiration generated heat when a fungi or bacterium is permitted to grow on wet seeds. Kind of a neat little trivia fact to tuck away.

Oxygen—Oxygen is an important regulator of respiration. If oxygen is drastically reduced, respiration may drop off to the point of retarding growth or death.  Low oxygen concentrations can lead to the onset of fermentation processes.

Assimilation and Digestion

Assimilation is the conversion of the sugar produced by photosynthesis to fats, proteins, complex carbohydrates and other substances. While digestion is the breakdown of large insoluble molecules by hydrolysis to smaller soluble forms that can be transported to various parts of the plant.

Summary of key differences between photosynthesis and respiration:

Photosynthesis

Energy stored in sugar molecules

Carbon dioxide and water used

Increases weight

Requires light

Occurs in chlorophyll

In green organisms, produces oxygen

With light energy, produces ATP

Respiration

Energy released from sugar molecules

Carbon dioxide and water released

Decreases weight

Can occur in light or darkness

Occurs in all living cells

Uses oxygen (aerobic respiration)

With energy released from sugar, produces ATP.

                                  A) Basic:

1. Botany / Randy Moore, W.Denis Clark, Kingsley R.Stern, Darrell Vodopich. – Dubuque, IA, Bogota, Boston, Buenos Aires, Caracas, Chicago, Guilford, CT, London, Madrid, Mexico City, Sydney, Toronto: Wm.C.Brown Publishers.- 1994. -P.44 -95.

2. Gulko R.M. Explanatory Dictionary of Medicinal Botany. – Lviv: LSMU, 2003.-200 p.

                                  b) Additional:

1) Kindsley R. Stern. Introductory plant biology- Dubuque, Ajowa,
Melburne and Australia, Oxford, England: Wm.C.Brown Publishers-
1994.-P.23-38.

2) Ткаченко H.M., Cep6iн  A..Г. Ботаніка.- Xapків: Ocновa, 1997. – C.9-39.          

Prepared by ass.-prof. Shanayda M.I.