Factors Affecting Rates of Respiration

Factors Affecting Rates of Respiration
Temperature- For every 10 degree C rise in temperature between 0-35 C the rate of respiration increases 2X – 4X. Storage temperature for harvested plant parts is often critical because these parts continue to respire after harvest ( a catabolic process) which causes a build up of heat, and the breakdown of the product.

Most plants grow better when night time temperatures are 5 degrees C lower than day time temperatures. This is because lower night time respiration reduces the use of carbohydrates and allows more carbohydrates to be stored or used for growth.

Oxygen concentration- Generally speaking, lower oxygen level results in the reduction of respiration. Controlled atmosphere (CA) storage in which oxygen is decreased is useful in storage of fruits and vegetables because of lower respiration rates.

Soil conditions- Compacted and/or wet soil conditions result in low oxygen in the root zone and reduced root respiration. Consequently, roots don’t function well in supplying mineral nutrients essential for the activity of respiratory enzymes which decreases overall respiration.

Light- Lower light intensities result in lower respiration rates. Lower photosynthesis rates in low light supply fewer carbohydrates essential for respiration. Plant growth- As a plant grows it depends on energy to be supplied by respiration. The more growth that is occurring, the higher the respiration rate must be.

Summary of Respiration
Aerobic Respiration Glycolysis Transition Rx. Kreb’s Cycle Electron Transport Chain Anaerobic Respiration Pyruvate  Lactic Acid Mixed Acids Alcohol + CO2 Recycle NADH 2 ATP / Glucose

Amino Acid Catabolism

Amino Acids Building blocks for polymers called proteins
Contain an amino group, –NH2, and a carboxylic acid, –COOH Can form zwitterions: have both positively charged and negatively charged groups on same molecule 20 required for humans

Peptide Bond Connect amino acids from carboxylic acid to amino group
Produce amide linkage: -CONH- Holds all proteins together Indicate proteins by 3-letter abbreviation

Sequence of Amino Acids
Amino acids need to be in correct order for protein to function correctly Similar to forming sentences out of words

Transaminase enzymes (aminotransferases)
Catalyze the reversible transfer of an amino group between two a-keto acids.

Example of a Transaminase reaction:
Aspartate donates its amino group, becoming the a-keto acid oxaloacetate. a-Ketoglutarate accepts the amino group, becoming the amino acid glutamate.

In another example, alanine becomes pyruvate as the amino group is transferred to a-ketoglutarate.

Essential amino acids must be consumed in the diet.
Mammalian cells lack enzymes to synthesize their carbon skeletons (a-keto acids). These include: Isoleucine, leucine, & valine Lysine Threonine Tryptophan Phenylalanine (Tyr can be made from Phe.) Methionine (Cys can be made from Met.) Histidine (Essential for infants.)

Amino Acid Metabolism Metabolism of the 20 common amino acids is considered from the origins and fates of their: (1) Nitrogen atoms (2) Carbon skeletons For mammals: Essential amino acids must be obtained from diet Nonessential amino acids - can be synthesized

The Nitrogen Cycle and Nitrogen Fixation
Nitrogen is needed for amino acids, nucleotides Atmospheric N2 is the ultimate source of biological nitrogen Nitrogen fixation: a few bacteria possess nitrogenase which can reduce N2 to ammonia Nitrogen is recycled in nature through the nitrogen cycle

Fig 17.1 The Nitrogen cycle

Nitrogenase An enzyme present in Rhizobium bacteria that live in root nodules of leguminous plants Some free-living soil and aquatic bacteria also possess nitrogenase Nitrogenase reaction: N2 + 8 H+ + 8 e ATP 2 NH3 + H ADP + 16 Pi

Assimilation of Ammonia
Ammonia generated from N2 is assimilated into low molecular weight metabolites such as glutamate or glutamine At pH 7 ammonium ion predominates (NH4+) At enzyme reactive centers unprotonated NH3 is the nucleophilic reactive species

A. Ammonia Is Incorporated into Glutamate
Reductive amination of a-ketoglutarate by glutamate dehydrogenase occurs in plants, animals and microorganisms

Glutamine Is a Nitrogen Carrier in Many Biosynthetic Reactions
A second important route in assimilation of ammonia is via glutamine synthetase

Glutamate synthase transfers a nitrogen to a-ketoglutarate
Prokaryotes & plants

Alternate amino acid production in prokaryotes
Especially used if [NH3] is low. Km of Gln synthetase lower than Km of Glu dehydrogenase.

The First Step in Amino Acid Degradation is the Removal of Nitrogen
Amino acids released from protein turnover can be resynthesized into proteins. Excess amino acids are degraded into specific compounds that can be used in other metabolic pathways. This process begins with the removal of the amino group, which can be converted to urea and excreted. The -ketoids that remain are metabolized so that their carbon skeletons can enter glycolysis, gluconeogenesis, or the TCA cycle.

The Biosynthesis of Amino Acids
Amino acids are the building blocks of proteins and the nitrogen source of many other important molecules including nucleotides, neurotransmitters, and prosthetic groups such as porphyrins. Ammonia is the source of all nitrogen for all of the amino acids. The carbon backbones come from the glycolytic pathway, the pentose phosphate pathway, and/or the TCA cycle. Amino acid biosynthesis is feedback regulated to ensure that all amino acids are maintained in sufficient amounts for protein synthesis and other processes.

Summary of Protein and Amino Acid Degradation
Proteins are degraded to amino acids. Protein turnover is tightly regulated. The first step in amino acid degradation is the removal of nitrogen. Ammonium ion is converted into urea in most terrestrial vertebrates. Carbon atoms of degraded amino acids emerge as major metabolic intermediates. Inborn errors of metabolism can disrupt amino acid degradation.

Summary of Amino Acid Biosynthesis
Microorganisms use ATP and a powerful reductant to reduce atmospheric nitrogen to ammonia. Amino acids are made from intermediates of the TCA cycle and other major pathways. Amino acid metabolism is regulated by feedback inhibition. Amino acids are precursors of many molecules.

Overview of Nucleotide Biosynthesis
Nucleotides serve as active precursors of nucleic acids. ATP is the universal currency of energy. Nucleotide derivatives such as UDP-glucose participate in bioynthetic processes. Nucleotides are essential components of signal transduction pathways.

Two Classes of Pathways for the Synthesis of Nucleotides.
In the salvage pathway, a base is attached to a ribose, activated in the form of 5- phosphoribosyl-1-pyrophosphate (PRPP). In de novo synthesis, the base itself is synthesized from simpler starting materials, including amino acids. ATP hydrolysis is necessary for de novo synthesis.

Summary of Nucleotide Biosynthesis
In de novo synthesis, the pyrimidine ring is assembled from bicarbonate, aspartate, and glutamine. Purine bases can be synthesized de novo or recycled by salvage pathways. Deoxyribonucleotides are synthesized by the reduction of ribonucleotides. Key steps in nucleotide biosynthesis are feeback regulated. NAD+, FAD, and Coenzyme A are formed from ATP. Disruptions in nucleotide metabolism can cause pathological conditions.

Proteins

Structure of Proteins Four organizational levels
Primary structure: amino acid sequence Secondary structure: arrangement of chains around an axis Pleated sheet Alpha helix: right-handed helix

Pleated Sheets

Alpha Helix

Tertiary Structure Spatial relationships of amino acids relatively far apart in protein chain Globular proteins: compact spherical shape

Quaternary Structure Structure when two or more amino acid sequences are brought together Hemoglobin has four units arranged in a specific pattern

Intermolecular Forces in Proteins
Hydrogen bonding Ionic bonds Disulfide linkages Dispersion forces

Protein metabolism Transamination: use the essential AA to synthesize the others!

Protein metabolism Another route:
Intestinal bacteria -> ammonia (toxic) -> liver uses it to make amino acids

Protein metabolism Amino acids: C, H, O plus amine group with N

Protein metabolism Amino acids are broken down into:
a) ammonia -> urea b) pyruvate or molecules that are part of the krebs cycle -> respired for energy, or converted to fats or glucose

Proteins are degraded into amino acids.
Protein turnover is tightly regulated. First step in protein degradation is the removal of the nitrogen Ammonium ion is converted to urea in most mammals. Carbon atoms are converted to other major metabolic intermediates. Inborn errors in metabolism

Amino acids are also a source of nitrogen for other biomolecules.
Amino acids used for synthesizing proteins are obtained by degrading other proteins Proteins destined for degradation are labeled with ubiquitin. Polyubiquinated proteins are degraded by proteosomes. Amino acids are also a source of nitrogen for other biomolecules. - What is an example of another type of biomolecule that requires nitrogen?

Excess amino acids cannot be stored.
Surplus amino acids are used for fuel. Carbon skeleton is converted to Acetyl–CoA Acetoacetyl–CoA Pyruvate Citric acid cycle intermediate The amino group nitrogen is converted to urea and excreted. Glucose, fatty acids and ketone bodies can be formed from amino acids. - Unlike glucose and fatty acids, which can be stored.

1. Protein Degradation proteins are a vital source of amino acids.
Discarded cellular proteins are another source of amino acids. - Proteins a re hydrolyzed in the stomach and small intestines and the component amino acids absorbed into the bloodstream.

Biotechnology

What Is Biotechnology? Using scientific methods with organisms to produce new products or new forms of organisms Any technique that uses living organisms or substances from those organisms to make or modify a product, to improve plants or animals, or to develop microorganisms for specific uses

What Is Biotechnology? GMO- genetically modified organisms.
GEO- genetically enhanced organisms. With both, the natural genetic material of the organism has been altered. Roots in bread making, wine brewing, cheese and yogurt fermentation, and classical plant and animal breeding

What Is Biotechnology? Manipulation of genes is called genetic engineering or recombinant DNA technology Genetic engineering involves taking one or more genes from a location in one organism and either Transferring them to another organism Putting them back into the original organism in different combinations

What is the career outlook in biotechnology?
Biotech in 1998 1,300 companies in the US 2/3 have less than 135 employees 140,000 jobs Jobs will continue to increase exponentially Jobs are available to high school graduates through PhD’s

What Subjects Are Involved With Biotechnology?
Multidisciplinary- involving a number of disciplines that are coordinated for a desired outcome Science Life sciences Physical sciences Social sciences

What Subjects Are Involved With Biotechnology?
Mathematics Applied sciences Computer applications Engineering Agriculture

What Are the Stages of Biotechnology Development
Ancient biotechnology- early history as related to food and shelter; Includes domestication Classical biotechnology- built on ancient biotechnology; Fermentation promoted food production, and medicine Modern biotechnology- manipulates genetic information in organism; Genetic engineering

What Are the Areas of Biotechnology?
Organismic biotechnology- uses intact organisms; Does not alter genetic material Molecular biotechnology- alters genetic makeup to achieve specific goals Transgenic organism- an organism with artificially altered genetic material

What Are the Benefits of Biotechnology?
Medicine Human Veterinary Biopharming Environment Agriculture Food products Industry and manufacturing

What Is Molecular Biology?
Molecular biology- study of molecules in cells Metabolism- processes by which organisms use nutrients Anabolism- building tissues from smaller materials Catabolism- breaking down materials into smaller components

What Is a Cell? Cell- a discrete unit of life
Unicellular organism- organism of one cell Multicellular organism- organism of many cells Prokaryote- cells that lack specific nucleus Eukaryote- cells with well-defined nucleus

What Is a Cell? Cells are building blocks:
Tissue- collection of cells with specific functions Organs- collections of tissues with specific functions Organ systems- collections of organs with specific functions

What Are the Structures in Molecular Genetics?
Molecular genetics- study of genes and how they are expressed Chromosome- part of cell nucleus that contains heredity information and promotes protein synthesis Gene- basic unit of heredity on a chromosome DNA- molecule in a chromosome that codes genetic information

Deoxyribonucleic Acid (DNA)

What Is Ribonucleic Acid (RNA)?
Transcription- process of RNA production by DNA DNA-thread-like molecule which decodes DNA information

What Is Ribonucleic Acid (RNA)?
Kinds of RNA: mRNA- RNA molecules that carry information that specifies amino acid sequence of a protein molecule during translation rRNA- RNA molecules that form the ribosomal subunits; Mediate the translation of mRNA into proteins tRNA- molecules that decode sequence information in and mRNA snRNA- very short RNA that interconnects with to promote formation of mRNA

What Are Genetic Engineering Organisms?
Genetic engineering- artificially changing the genetic information in the cells of organisms Transgenic- an organism that has been genetically modified GMO- a genetically modified organism GEO- a genetically enhanced organism

How Can Genetically Engineered Plants Be Used?
Agriculture Horticulture Forestry Environment Food Quality

How Do We Create Transgenic Organisms?
Donor cell- cell that provides DNA Recipient cell- cell that receives DNA Protocol- procedure for a scientific process Three methods used in gene transfer Agrobacterium gene transfer- plasmid Ballistic gene transfer- gene gun Direct gene transfer- enzymes

How Does Agrobacterium Gene Transfer Work?
Extract DNA from donor Cut DNA into fragments Sort DNA fragments Recombine DNA fragments Transfer plasmids with bonded DNA Grow transformed (recipient) cells

What Are Methods of Classical Biotechnology?
Plant breeding- improvement of plants by breeding selected individuals to achieve desired goals Cultivar- a cultivated crop variety

What Are Methods of Classical Biotechnology?
Plant breeding methods; Line breeding- breeding successive generations of plants among themselves Crossbreeding- breeding plants of different varieties or species Hybridization- breeding individuals from two distinctly different varieties Selection

Why Are Plants Genetically Engineered?
Resist pests Resist herbicides Improved product quality Pharmaceuticals Industrial products

These definitions imply biotechnology
is needed because: Nature has a rich source of variation Here we see bean has many seedcoat colors and patterns in nature As we are all aware, most species have an abundance of variation. The photograph of bean seeds is a great illustration of the variation in nature. You can notice not only many different colors, but also many different patterns. A large array of interacting genes are responsible for this variation. But man can always dream of a new use for an organism. These dreams often involve asking the species to do something it does not now normally do. Biotechnology involves added new traits to a species. But we know nature does not have all of the traits we need

What controls this natural variation?
Allelic differences at genes control a specific trait Definitions are needed for this statement: Gene - a piece of DNA that controls the expression of a trait Allele - the alternate forms of a gene Before we can understand who man goes about using biotechnology approaches to modify a species, we must understand basic genetic principles and terminology. All traits are controlled by genes. A gene can have different forms. These forms are called alleles. It is important that you become fluent with these terms and the differences they imply. It is simple as remember that genes have alleles. Or, alleles are alternate forms of a gene.

What is the difference between genes and alleles for Mendel’s Traits?
Mendel’s Genes Plant height Seed shape Smooth Wrinkled Allele This slide is intended to help you understand the difference between genes and alleles. Genes control specific traits. Here are two traits of pea that Gregor Mendel, the father of genetics, studied. Plant height is a trait. That trait is controlled by the plant height gene. The plant can be either tall or short. Different alleles of the plant height gene determine if the plant will be tall or short. If you remember from your genetics class, the allele for tall plant height is dominant to the short allele. This means that heterozygous individuals carrying both the tall and short allele will appear to be tall. Using the genetic terminology, this also means that the short allele is recessive to the tall allele. Similarly, seed shape is controlled by a specific gene. The alternate shapes, smooth or wrinkled, are controlled by different allelic forms of the seed shape gene. Similarly, smooth is dominant to the recessive wrinkled phenotype. Tall Short Allele

Central Dogma of Molecular Genetics
(The guiding principle that controls trait expression) Now that we understand the relationships between alleles at a gene, it is time to place this understanding in a large context. You should become very fluent with this concept: the Central Dogma of Molecular Genetics. This concept is a unifying principle that describes the manner in which the sequence information in the gene is eventually expressed as a trait (or phenotype). DNA is the biochemical molecule of all genes. DNA contains the genetic code that will eventually be converted into the protein that will control the phenotype expression. But DNA is not directly used for phenotypic expression. Instead, the information in the DNA molecule is used to create an intermediary molecule (RNA). The process to produce RNA is called transcription. RNA is an active molecule in another process called translation that is used to create the protein. The protein itself can have many different functions. It could be an enzyme in a metabolic pathway. Alternatively, it could act as a regulator transcription. Finally, it could serve as a structural component of the cell. Whatever it role is, it will control the final phenotype or the outward appearance of plant. Plant height Seed shape

In General, Plant Biotechnology Techniques
Fall Into Two Classes Identify a gene from another species which controls a trait of interest Or modify an existing gene (create a new allele) Gene Manipulation Introduces that gene into an organism Technique called transformation Forms transgenic organisms Gene Introduction Remember our definition of biotechnology??? Here is the detailed one again: The application of the technology to modify the biological function of an organism by adding genes from another organism. As the definition implies, we first need to isolate a gene that will be added to our organism of interest. For example, we may wish to add a gene from a bacteria into a plant species. Another approach would be to isolate a gene from our species of interest, modify that gene to change its function, and then reinsert the modified form back into the species. This is the first major biotechnology step, and it is called gene manipulation. Once we have isolated or modified the gene of interest, the next step is gene introduction. This step involves the addition of the gene to our species of interest. In all cases, this introduction must be accompanied by stable integration of that gene into the genome of the target species. Once the gene is stably integrated, it is passed along to all subsequent generation in the same manner as all other genes in the genome. The technique used to integrate the gene into the species is called transformation, and the modified organism is called a transgenic organism.

Gene Manipulation Starts
At the DNA Level The nucleus We will discuss gene manipulation and gene introduction separately. The Central Dogma of Molecular Genetics was introduced several slides back. As a remember, it states that the information stored in DNA is transcribed into RNA, the RNA molecule is used in a process called translation to produce translation to produce a protein, and the protein is involved in some process that actually produces the final phenotype of the organism. The dogma implies we need to manipulate the DNA of gene that encodes for the protein if we are going to develop a transgenic organism. The DNA itself is a double-helix molecule that is stored inside the nucleus of the cell. Every cell inside the organism has exactly the same DNA molecule. contains DNA Source: Access Excellence

DNA Is Packaged Double-stranded DNA is condensed into Chromosomes
We normally think of the DNA in the form of a chromosome. Chromosomes are the condensed form of DNA. The simplest form of DNA is the double-stranded molecule. These two strands are complementary to each other. This complementarity is based on the fact that if one strand has an adenine at one nucleotide residue, the complementary strand has a thymine residue at the same location. And if one strand has a guanine at a specific residue, the complemenntary strand has a cytosine residue. This is an important concept because it is the basis of an important screening process called hybridization. The double-strand molecule then undergoes a series of condensation steps to produce the chromosome. Each of this different steps are illustrated here in this slide. It is important to remember that throughout the life-cycle of the cell, DNA is in an uncondensed form. The chromosome only appears during the process of cell division. Chromosomes Source: Access Excellence

PCR Animation Denaturation: DNA melts Annealing: Primers bind
This is an animation of one step in the PCR process. Take a few minutes and let the animation run through a number of times. It will recycle on its own. This step will show the denaturation (converting the DNA from single- to double-stranded state). The second step is annealing (the binding of the primer to the single-stranded DNA). The final step is extension (the duplication of a strand from the end of the primer). Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated

Complementary Genetics
(cont.) 4. Gene fragment used to screen library Clones transferred to filter Human clone library PCR fragment probe added to filter The final step in using complementary genetics for cloning involves screening a library. The steps are exactly the same as we described for homology cloning. The only difference is that we now use the PCR synthesized DNA as our probe. The final result will be the isolation of a DNA clone from the library. That clone will contain DNA sequences that will encode the gene for the protein in which we are interested. Hot-spots are human gene of interest

Map-based Cloning 1. Use genetic techniques to find marker near gene
2. Find cosegregating marker Gene/Marker 3. Discover overlapping clones (or contig) that contains the marker Gene/Marker The final technique of method of obtaining genes is called map-based cloning. This procedure combines genetic information that locates a gene to a small region of a chromosome. The position of the gene is located to that region by the use of a DNA marker that resides very close to the gene. The first two steps illustrate this point. Typically a marker is discovered at a short distance away from the gene of interest. That marker is then used to discover another marker that is very close that cosegregates with the gene of interest. That cosegregating marker is very close to the gene (closer than the first marker) and is used to isolate a series of overlapping clones or contig. A series of steps is then used to identify ORFs or open reading frames. An ORF is a DNA sequence that has all the characteristics of a gene. Since the DNA marker (and by association the gene of interest) resides on the contig, one of the ORFs will be the gene. We won’t go into the details, but the ORF that is a gene is eventually identified by one of two procedures. Transformation is one approach. As defined earlier, transformation involves the addition of DNA to an organism and changing that organism’s phenotype. For map-based cloning, the ORF is added to a mutant organism, and if the resulting transgenic plant expresses the wild type phenotype then the ORF is the gene you are trying to clone. For example, if you had and ORF that encoded the gene responsible for plant height in pea, that gene could be added to a short pea plant. If the addition of the ORF is the gene for plant height, that transgenic plant would be tall. An alternative approach is to sequence many mutant phenotypes of a specific ORF. That analysis may provide useful information that would allow you to determine a particular ORF is the gene of interest. This is the approach used in human genetics. 4. Find ORFs on contig Gene/Marker 5. Prove one ORF is the gene by transformation or mutant analysis Mutant + ORF = Wild type? Yes? ORF = Gene

Gene Manipulation It is now routine to isolate genes
But the target gene must be carefully chosen Target gene is chosen based on desired phenotype Function: Glyphosate (RoundUp) resistance EPSP synthase enzyme The development of the transgenic organism uses some gene isolated by the procedures that were just outlined. But it is important that the appropriate gene is used to obtain the specific phenotype you wish to develop. We are going to spend a bit of time concentrating on two important phenotypes: glyphosate (RoundUp) resistance and increased vitamin A content. Each of the phenotypes can be achieved by adding one or several genes to a plant. Increased Vitamin A content Vitamin A biosynthetic pathway enzymes

The RoundUp Ready Story
Glyphosate is a broad-spectrum herbicide Active ingredient in RoundUp herbicide Kills all plants it come in contact with Inhibits a key enzyme (EPSP synthase) in an amino acid pathway Plants die because they lack the key amino acids The RoundUp Ready technology is the most visible plant biotechnology product on the market. To better understand plant biotechnology in general, it is important to understand the development of these transgenic organisms. RoundUp is a brand name herbicide manufactured by Monsanto Corp. The active ingredient in this herbicide is glyphosate. The chemical binds to the active site of the EPSP synthase enzyme. This enzyme is a key to the development of a group of amino acids called the aromatic amino acids. When this enzyme is bound by glyphosate, it can not synthesize those amino acids, and the plants die because protein synthesis is severely disrupted. Glyphosate will not bind the to a particular genetically-engineered version of EPSP synthase. Therefore RoundUp Ready crops with this altered enzyme will survive when sprayed with the herbicide. A resistant EPSP synthase gene allows crops to survive spraying

3-Enolpyruvyl shikimic acid-5-phosphate
RoundUp Sensitive Plants Shikimic acid + Phosphoenol pyruvate 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Plant EPSP synthase Aromatic amino acids + Glyphosate X X This slide shows the actual biochemical pathway that we discussed in the previous slide. EPSP synthase synthesizes 3-enolpyruvly shikimic acid-5-phosphate. This is the essential precursor to aromatic amino acids. When plants are sprayed with a glyphosate-containing herbicide, such as RoundUp, this important precursor is not synthesized, and consequently the plant is starved of aromatic amino acids. The result is plant death. Without amino acids, plant dies X X

RoundUp Resistant Plants
Shikimic acid + Phosphoenol pyruvate + Glyphosate RoundUp has no effect; enzyme is resistant to herbicide Bacterial EPSP synthase 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) RoundUp Resistant plants have a very simple solution. An engineered version of EPSP synthase, one that was discovered in a bacteria, is introduced into the plant. This enzyme can not be bound by glphosate. Therefore, if a field is sprayed with the herbicide, the introduced version of the gene produces a functional enzyme. The 3-enolpyruvl shikimic acid-5-phosphate precursor is synthesized normally, and the plant produces enough aromatic amino acids to survive. With amino acids, plant lives Aromatic amino acids

The Golden Rice Story Vitamin A deficiency is a major health problem
Causes blindness Influences severity of diarrhea, measles >100 million children suffer from the problem For many countries, the infrastructure doesn’t exist to deliver vitamin pills The second major plant biotechnology product is more recent and was developed to address the vitamin A deficiency problems prevalent throughout the world. This vitamin deficiency is very critical because it can cause blindness and affects the severity of many diseases including diarrhea and measles. This is a severe problem that affects more than 100 million children worldwide. A simple solution would be to distribute vitamins to the affected children. Unfortunately, many countries where the deficiency is chronic do not have the necessary infrastructure to deliver the vitamin tablets to the most needed. The solution that is currently being promoted is to improve the vitamin content in widely-consumed, and readily available to the consumer. Transgenic rice plants were developed that contain elevated levels of the precursor to vitamin A. This GMO is called “Golden Rice” because of its color: it is yellow rather than white. It is yellow because β-carotene, a yellow precursor to vitamin A is abundant in the seed. Improved vitamin A content in widely consumed crops an attractive alternative

Lycopene-beta-cyclase
-Carotene Pathway in Plants IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Problem: Rice lacks these enzymes Unlike the single-step RoundUp Ready pathway, the β–carotene synthesis pathway involves multiple enzymes. This important vitamin A precursor cannot be synthsized in rice because it lacks four of the key enzymes. Therefore, the precursor is not made, and the plant contains white kernels. Normal Vitamin A “Deficient” Rice

The Golden Rice Solution
-Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Vitamin A Pathway is complete and functional Daffodil gene Single bacterial gene; performs both functions In a major feat of genetic engineering, scientists inserted a complete functioning -carotene biosynthetic pathway into the rice plant. They did this by inserting genes from daffodil the produce functioniong versions of the first and last enzymes of the pathway. In addition, a single bacterial gene that provides the same function as the second and third enzymes of the pathway, was also introduced. With a functioning pathway, the transgenic rice is able to produce the vitamin A precursor β-carotene. It is this product that gives "Golden Rice" its characteristic yellow color. Daffodil gene Golden Rice

Metabolic Pathways are Complex and Interrelated
Understanding pathways is critical to developing new products The “Golden Rice” story illustrates a key point: it is very important to industry metabolic pathways. These pathways are very important for our understanding of specific products are produced in the organism. Only by understanding this pathways will we be able to create novel new products.

Modifying Pathway Components Can Produce New Products
Turn On Vitamin Genes = Relieve Deficiency Modified Lipids = New Industrial Oils This diagram shows in general the interrelationship between the many different pathways. A key point to understand is that the different sub-pathways interconnect. Therefore modify one component of the pathway may affect the production of a product in a separate sub-pathway. Keeping this in mind, we can now envision how to engineer plants so they produce novel products. We have already seen how modifying a vitamin biosynthetic pathway can positively affect vitamin production. We could also improve nutrition in other ways. For example, if we were to focus our attention on the amino acid pathways we could, for example, increase the lysine content in typically lysine-poor grains. Conversely, someday we might be able to improve legumes by introducing the correct genes necessary to enrich the metionine content. We could also envision new products if we modify other pathways. Oils are a key component to both the food and manufacturing industries. A better understanding of the genes in the complex lipid pathway may allow us to produce better industrial oils. Increase amino acids = Improved Nutrition

Trait/Gene Examples Gene Trait RoundUp Ready Bacterial EPSP
Golden Rice Complete Pathway Plant Virus Resistance Viral Coat Protein Male Sterility Barnase This slide illustrates the variety of different traits that have been modified in plants. It also shows the particular gene that was introduced into the plant to obtain the specific trait. As you can see, scientists have successfully introduced many different genes and produced many different results. For example, it was discovered that expressing the in the plant a particular protein of a virus, the coat protein, the plant would then become resistant to that virus. This technique has been widely credited with saving the papaya industry in Hawaii, where the papaya ringspot virus nearly eliminated the papaya growing industry. This is a success story that is often overlooked, probably because the problem was to a crop of limited production value. Plant Bacterial Resistance p35 Salt tolerance AtNHX1

Introducing the Gene or Developing Transgenics
Steps 1. Create transformation cassette 2. Introduce and select for transformants It is now time to cover the development of transgenic crops in greater depth. The two major steps are creating a transformation cassette that contains the gene of interest, and then successfully introducing the cassette into the plant.

Transformation Cassettes
Contains 1. Gene of interest The coding region and its controlling elements 2. Selectable marker Distinguishes transformed/untransformed plants All transformation cassettes contain three regions. The “gene of interest” region contains the actual gene that is being introduced into the plant. This is the gene that provides the new function to the plant. In this diagram, the region is shown in red. Many plant tissues are treated with the transformation cassette during the transformation step. Not all of these tissues actually receive the cassette. To distinguish those that contain the gene from those that don’t, it is necessary to use a selection process. The selectable marker is a gene that provides the ability to distinguish transformed from non-transformed plants. This is shown by green. The most common method to introduce the transformation cassette is by using the plant pathogen Agrobacterium. For this system to work it is necessary that the cassette contain insertion sequences that are used by the bacteria. These are shown by the gray. 3. Insertion sequences Aids Agrobacterium insertion

Gene of Interest Promoter Region
Coding Region TP Promoter Region Controls when, where and how much the gene is expressed ex.: CaMV35S (constitutive; on always) Glutelin 1 (only in rice endosperm during seed development) Transit Peptide Targets protein to correct organelle ex.: RbCS (RUBISCO small subunit; choloroplast target All of these components of the transformation cassette contain multiple components. In addition to the coding region that encodes the protein product, the gene of interest region also contains two important controlling regions. The promoter region resides just before the coding region and determines when, where, and to what degree the gene of interest will be expressed. In general, two types of promoter regions are used. A constitutive promoter turns the gene on in all tissues at all times. In general, this leads to a relatively high level of gene expression. The most often used constitutive promoter controls the expression of the 35S RNA of the cauliflower mosaic virus. It is abbreviated as CaMV35S promoter. Other promoters direct a very specific expression pattern. For example, the glutelin 1 promoter directs that the expression of the glutelin storage protein at a specific time of seed development. It also ensures the protein is only expressed in the rice endosperm. If the gene of interest is preceded by the CaMV35S promoter, it will be expressed in all tissues at all times. Conversely, the expression of the target gene could be limited to the endosperm if it is controlled by the glutelin 1 promoter. Some, but not all genes, encode protein that function in the plant organelles. These organelles are the chloroplast and the mitochondria. For example, photosynthesis, and part of the carbon and lipid metabolism pathways are carried out in the organelles. To ensure these protein are delivered to the appropriate organelle, a transit peptide is required. This is a short amino acid sequence that is found directly before the coding region. This sequence is recognized by proteins in the outer membranes of the appropriate organelle. This recognition process leads to the import of the protein into the organelle. Therefore, if you are gene of interest functions in the organelle, an appropriate transit peptide must be included in the transformation cassette Coding Region Encodes protein product ex.: EPSP -carotene genes

Selectable Marker Promoter Region Normally constitutive Coding Region
ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter Coding Region Gene that breaks down a toxic compound; non-transgenic plants die ex.: nptII [kanamycin (bacterial antibiotic) resistance] aphIV [hygromycin (bacterial antibiotic) resistance] Bar [glufosinate (herbicide) resistance] As stated above, the selectable marker is a gene that encodes a protein product. For it to be expressed, it also needs a promoter region. It is typical to use the constitutive CaMV35S RNA promoter. The gene it controls encodes a protein that enables a transformed plant to survive in the presence of normally toxic compound. The most often used selective agents are kanamycin and hygromyin, two bacterial antibiotics, and the herbicide glufosinate. The protein encoded by the selectable marker genes generally renders these selective agents harmless to the transgenic plant.

X Effect of Selectable Marker Non-transgenic = Lacks Kan or Bar Gene
Plant dies in presence of selective compound X Transgenic = Has Kan or Bar Gene Plant grows in presence of selective compound This slide shows the effect of the selectable marker.

Insertion Sequences Required for proper gene insertions
TL TR Used for Agrobacterium-transformation ex.: Right and Left borders of T-DNA Required for proper gene insertions The insertion sequences straddle the gene-of-interest coding region and the selectable marker. These are use by Agrobacteria to create a DNA molecule that is sent out of the bacteria into the plant where it is eventually inserted into the nucleus of a cell in the recipient plant tissue. If the cell follows the proper developmental pathway that leads to a new plant, every cell in that plant will contain the sequences in between the insertion sequences.

Let’s Build A Complex Cassette
pB19hpc (Golden Rice Cassette) aphIV 35S Gt1 psy 35S rbcS crtl TL TR T-DNA Border Hygromycin Resistance Phytoene Synthase Phytoene Desaturase Insertion Sequence Selectable Marker Gene of Interest Gene of Interest This slide demonstrates one of the transformation cassettes used to develop “Golden Rice” was developed. Slowly click through this slide, and you will see each of the components of the cassette.

Delivering the Gene to the Plant
Transformation cassettes are developed in the lab They are then introduced into a plant Two major delivery methods Agrobacterium Two techniques are used to deliver DNA found in the transformation cassette into plant tissues during the plant transformation process. One is a biological system based on the plant pathogen Agrobacterium tumefaciens. The second is a mechanical method where the DNA is “shot” into plant cells using a gene gun. Regardless of the delivery method, the delivery system must use a plant tissue source that can be manipulated to produce new plants. Tissue culture required to generate transgenic plants Gene Gun

A Requirement for Transgenic Development plant grows to maturity
Plant Tissue Culture A Requirement for Transgenic Development Callus grows This slide shows the basic steps of plant tissue culture. Some plant part is placed is on a defined culture media. That media induces the the tissue to develop callus. Callus is an undifferentiated mass of cells. These cells then grow into plant shoots, which are later rooted. The small seedling will then grown into a mature, seed-producing plant. When developing transgenic plants, the transformation cassette is introduced into that plant part that can be induced to grow new plants. A plant part Is cultured Shoots develop Shoots are rooted; plant grows to maturity

But Nature’s Agrobacterium
Has Problems Infected tissues cannot be regenerated (via tissue culture) into new plants Why? Phytohormone balance incorrect regeneration Solution? Transferred DNA (T-DNA) modified by Removing phytohormone genes Early on it was known that tissues infected with Agrobacterium could not be coaxed to regenerate new plants. Soon it was realized that the plant hormone balance was not correct. To over come this effect, the genes encoding the phytohormones were removed. Once removed, plant tissues infected with the modified Agrobacterium could produce the regenerated plant. With this realization, it was a simple step to envision how to deliver genes of interest into a plant: include these genes in the cassette. Retaining essential transfer sequences Adding cloning site for gene of interest

The Gene Gun DNA vector is coated onto gold or tungsten particles
Particles are accelerated at high speeds by the gun Particles enter plant tissue DNA enters the nucleus and incorporates into chromosome The second method currently in use is the gene gun. The principle is very simple. The transformation cassette DNA is coated onto a particle. That particle is then accelerated (using ballistics or an air stream). The particle then enters the plant cell. At that point, the transformation cassette DNA is eluted off the particle, and by a process that is not known, the DNA becomes integrated into the nucleus of the cell. The same basic principles guiding plant transformation with Agrobacterium is used with the gene gun. A tissue source that is capable of being manipulated to produce new plants is treated; in this case it is shot with particles containing the transformation cassette. The tissue is then placed under selection, and those shoots that develop contain the gene of interest. Integration process unknown

Transformation Steps Prepare tissue for transformation Introduce DNA
Leaf, germinating seed, immature embryos Tissue must be capable of developing into normal plants Introduce DNA Agrobacterium or gene gun Culture plant tissue Develop shoots Root the shoots This slide summarizes the steps necessary for plant transformation. Field test the plants Multiple sites, multiple years

The Lab Steps And this slide illustrates those steps.

CBF transcription factors
Lab Testing The Transgenics Insect Resistance Cold Tolerance This and the next slide illustrate the types of traits that can be obtain using genetic engineering of plants. Notice the particular types of genes that were used to obtain these traits. Some genes encode a protein that directly provides the trait. This is illustrated by the gene that encodes the Bt-toxin protein that is harmful to plant insect pests. Other genes encode protein that regulate the expression of a trait. Cold tolerance can be added to a crop by introducing gene that encode a transcription factor. These factors interact with other genes to turn on their expression. Transgene= Bt-toxin protein Transgene= CBF transcription factors

What is plant biotechnology?
Desired gene Traditional plant breeding DNA is a strand of genes, much like a strand of pearls. Traditional plant breeding combines many genes at once. Traditional donor Commercial variety New variety Desired Gene X = (crosses) (many genes are transferred) Plant biotechnology Using plant biotechnology, a single gene may be added to the strand. Desired gene Commercial variety New variety (transfers) = (only desired gene is transferred) Plant biotechnology is just a much more precise tool than selective breeding or crossbreeding. It’s like using a scalpel instead of a bush knife to perform surgery. As you can see from this diagram, traditional plant breeding transfers many genes to create a new plant variety — some of them desired, some not. (Click) Plant biotechnology allows scientists to select just the gene they want that has the desired traits. What is plant biotechnology?

Benefits of biotechnology
More food Better food It allows farmers to grow more food, better food, in ways that are better for the environment. More and more studies are quantifying the benefits of biotechnology. Better for the environment

What Is Cloning? Clone- new organism that has been produced asexually from a single parent Genotype is identical to parent Cells or tissues are cultured

What Is Bioremediation?
Bioremediation- using biological processes to solve environmental problems Biodegradation- natural processes of microbes in breaking down hydrocarbon materials Biodegradable- capable of being decomposed by microbes

How Can Bioremediation Be Used?
Oil spills Wastewater treatment Heavy metal removal Chemical degradation

What Is Phytoremediation?
Phytoremediation- process of plants being used to solve pollution problems Plants absorb and break down pollutants Used with heavy metals, pesticides, explosives, and leachate

References

Referance

references www.stcsc.edu/anatomy/210/Chapter%202%20part%202.ppt
METABOLISM ww.ims.uni-stuttgart.de/lehre/teaching/2005-SS/BioNLP/CoreferenceAndClassification.ppt

References in Plants

References

Factors Affecting Rates of Respiration

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