Plant Physiology talk Five Basic Plant Biochemistry Carbohydrates

Plant Physiology talk Five Basic Plant Biochemistry

Carbohydrates • Of the macromolecules that we will cover in this class, those involving carbohydrates are the most abundant in nature. • Via photosynthesis, over 100 billion metric tons of CO 2 and H 2 O are converted into cellulose and other plant products. • The term carbohydrate is a generic one that refers primarily to carbon-containing compounds that contain hydroxyl, keto, or aldehydic functionalities. • Carbohydrates can range in sizes, from simple monosaccharides (sugars) to oligosaccharides, to polysaccharides.

Carbohydrates • Carbohydrates constitute more than 1/2 of organic molecules • Main role of carbos in nature · Storage of energy · Structural support · Lipid and protein modification: · membranes asymmetry, recognition by Ig. G/fertilization/virus recognition/cell communication Definition: Carbohydrates, Sugars and Saccharides- are all polyhydroxy · (at least 2 OH) Cn(H 20) n = hydrate of carbon • Notice that there are two distinct types of monosaccharides, ketoses and aldoses. • The number of carbons is important in general nomenclature (triose = 3, pentose = 5, hexose =6,

Basic facts Monosaccharides - Simple sugars • Single polyhydroxyl · Can’t be hydrolyzed to simpler form Trioses - Smallest monosaccharides have three carbon atoms Tetroses (4 C) Pentose (5 C) Hexoses (6 C) Heptoses (7 C) etc… Disaccharide - two sugars linked together. Can be the same molecule or two different sugars. Attached together via a glycosidic linkage Oligosaccharide - 2 to 6 monosaccharides Polysaccharides - straight or branched long chain monosaccharides. Bonded together by glycosidic linkages

The functional groups • Aldehyde: Consists of a carbon atom bonded to a hydrogen atom and doublebonded to an oxygen atom. – Polar. Oxygen, more electronegative than carbon, pulls the electrons in the carbon-oxygen bond towards itself, creating an electron deficiency at the carbon atom. • Ketone: Characterized by a carbonyl group (O=C) linked to two other carbon atoms or a chemical compound that contains a carbonyl group – A carbonyl carbon bonded to two carbon atoms distinguishes ketones from carboxylic acids, aldehydes, esters, amides, and other oxygen-containing compounds

Classification of monosaccharides • Monosaccharides are classified according to three different characteristics: – the placement of its carbonyl group, – the number of carbon atoms it contains – its chiral handedness. • If the carbonyl group is an aldehyde, the monosaccharide is an aldose • if the carbonyl group is a ketone, the monosaccharide is a ketose. • Monosaccharides with three carbon atoms are called trioses, those with four are called tetroses, five are called pentoses, six are hexoses, and so on. • These two systems of classification are often combined. – For example, glucose is an aldohexose (a six-carbon aldehyde)

carbonyl group • A functional group composed of a carbon atom double-bonded to an oxygen atom: C=O. • The term carbonyl can also refer to carbon monoxide as a ligand in an inorganic or organometallic complex.

Classification of monosaccharides • D-glucose • is an aldohexose with the formula (C·H 2 O)6. • The red atoms highlight the aldehyde group • the blue atoms highlight the asymmetric center furthest from the aldehyde; because this -OH is on the right of the Fischer projection, this is a D sugar.

Classification of monosaccharides • The and b anomers of glucose. • Note the position of the hydroxyl group (red or green) on the anomeric carbon relative to the CH 2 OH group bound to carbon 5: • Either on the opposite sides ( ) • Or the same side (b).

Important disaccharides • Sucrose • The osmotic effect of a substance is tied to the number of particles in solution, so a millilitre of sucrose solution with the same osmolarity as glucose will be have twice the number carbon atoms and therefore about twice the energy. – Thus, for the same osmolarity, twice the energy can be transported per ml. • As a non-reducing sugar, sucrose is less reactive and more likely to survive the journey in the phloem. • Invertase (sucrase) is the only enzyme that will touch it and this is unlikely to be present in the phloem sieve tubes.

Important disaccharides • Maltose • Malt sugar or corn sugar consists of two glucose molecules linked by an -1, 4 -glycosidic bond • It comes from partial hydrolysis of starch by the enzyme amylase, which is in saliva and also in grains (like barley) • Maltose is an important intermediate in the digestion of starch. Starch is used by plants as a way to store glucose. After cellulose, starch is the most abundant polysaccharide in plant cells.

Important plant saccharides • Raffinose is a trisaccharide composed of galactose, fructose, and glucose. • Raffinose can be hydrolyzed to Dgalactose and sucrose by the enzyme αgalactosidase ( -GAL), an enzyme not found in the human digestive tract. -GAL also hydrolyzes other -galactosides such asstachyose, verbascose, and galactinol, if present. The enzyme does not cleave βlinked galactose, as in lactose. • The raffinose family of oligosaccharides (RFOs) are alphagalactosyl derivatives of sucrose, and the most common are raffinose, stachyose, verbascose. RFOs are almost ubiquitous in the plant kingdom, being found in a large variety of seeds from many different families, and they rank second only to sucrose in abundance as soluble carbohydrates. •

Carbohydrates-make up 16 -25% of sap. • The major organic transport materials are sucrose, stachyose (sucrose-gal), raffinose (stachyosegal). • These are excellent choices for transport materials for two reasons: • (a) they are non-reducing sugars (the hydroxyl group on the anomeric carbon, the number one carbon, is tied up) which means that they are less reactive and more chemically stable. • (b) the linkage between sucrose and fructose is a "high-energy" linkage similar to that of ATP. Thus, sucrose is a good transport form that provides a high energy, yet stable packet of energy;

Important Polysaccharides: Starch - energy reservoir in plants - made of two polysaccharides Amylose -long unbranched glucose (1, 4) with open reducing end large tight helical forms. Test by iodination. .

Important Polysaccharides: Starch - energy reservoir in plants - made of two polysaccharides – Amylose -long unbranched glucose (1, 4) with open reducing end large tight helical forms. Test by iodination. – Amylopectin - polymer of (1, 4) and (1, 6) branches. Not helical.

Plant Starch (Amylose and Amylopectin) • Starch contains a mixture of amylose and amylopectin • Amylose is an unbranched polymer (forms -helix) of D-glucose molecules linked by 1, 4 -glycosidic bonds • Amylopectin is like amylose, but has extensive branching, with the branches using -1, 6 glycosidic bonds

Cellulose • Linear glucan chains of unbranched (1 -4)-b-linked -D-glucose in which every other glucose residue is rotated 180° with respect to its two neighbors and contrasts with other glucan polymers such as: • starch (1 -4 - -glucan) • callose (1 -3 -b-glucan).

Cellulose • This means that cellobiose, and not glucose, is the basic repeating unit of the cellulose molecule. Groups of 30 to 40 of these chains laterally hydrogen-bond to form crystalline or para-crystalline microfibrils.

Proteins Basic facts

Amino acids • -20 common amino acids there are others found naturally but much less frequently • Common structure for amino acid • COOH, -NH 2, H and R functional groups all attached to the alpha carbon

Proteins: Three-dimensional structure • Background on protein composition: • Two general classes of proteins · Fibrous - long rod-shaped, insoluble proteins. These proteins are strong (high tensile strength). · Globular - compact spherical shaped proteins usually water-soluble. Most hydrophobic amino acids found in the interior away from the water. Nearly all enzymes are globular… · Proteins can be simple - no added groups or modifications, just amino acids • Or proteins can be conjugated. Additional groups covalently bound to the amino acids. The naked protein is called the apoprotein and the added group is the prosthetic group. Together the protein and prosthetic group is called the holoprotein. Ex. chlorophyll

Four levels of protein structure • Primary structure: amino acid only. The actual amino acid sequence is specified by the DNA sequence. The primary structure is used to determine genetic relationships with other proteins - AKA homology. Amino acids that are not changed are considered invariant or conserved. Primary sequence is also used to determine important regions and functions of proteins domains.

Four levels of protein structure • Secondary structure: This level is only concerned with the local or close in structures on the protein - peptide backbone. The side chains are not considered here, even though they have an affect on the secondary structure. ·Two common secondary structures - alpha helix and beta pleated sheet ·Non- regular repeating structure is called a random coil. - no specific repeatable pattern

Four levels of protein structure Tertiary structure - the overall three-dimensional shape that a protein assumes. This includes all of the secondary structures and the side groups as well as any prosthetic groups. This level is also where one looks for native vs. denatured state. The hydrophobic effect, salt bridges And other molecular forces are responsible for maintaining the tertiary structure

Four levels of protein structure • Quaternary structure: The overall interactions of more than one peptide chain. Called subunits. Each of the sub units can be different or identical subunits, hetero or homo – x mers (ex. Heterodimer is a protein composed of two different subunits).

Lipids fats oils…. Greasy molecules, mmmmm donuts. Several levels of complexity: • Simple lipids - a lipid that cannot be broken down to smaller constituents by hydrolysis. – Fatty acids, waxes and cholesterol • Complex lipids - a lipid composed of different molecules held together mostly by ester linkages and susceptible to cleavage reactions. – acylglycerols - mono, di and triacyl glycerols ( fatty acids and glycerol) – phospholipids (also known as glycerophospholipids) - lipids which are made of fatty acids, glycerol, a phosphoryl group and an alcohol. Many also contain nitrogen – glycolipids (also known as glycosphingolipids): Lipids which have a spingosine and different backbone than the phospholipids

General Structure • glycerol (a type of alcohol with a hydroxyl group on each of its three carbons) • Three fatty acids joined by dehydration synthesis. • Since there are three fatty acids attached, these are known as triglycerides.

General Structure - The longer the fatty acids the higher the melting point. - Again the more hydrophobic interactions effects the more the energy it takes to break the order. Decreases in the packing efficiency decreases the mp - The van der Waals forces then come apart more easily at lower temperatures. - Animal alter the length and unsaturated level of the fatty acids in lipids (cholesterol too) to deal with the cold temps

Saturated or not – the power of H • The terms saturated, monounsaturated, and poly-unsaturated refer to the number of hydrogens attached to the hydrocarbon tails of the fatty acids as compared to the number of double bonds between carbon atoms in the tail. • Oils, mostly from plant sources, have some double bonds between some of the carbons in the hydrocarbon tail, causing bends or “kinks” in the shape of the molecules. • Because some of the carbons share double bonds, they’re not bonded to as many hydrogens as they could if they weren’t double bonded to each other.

Trans and Cis • In unsaturated fatty acids, there are two ways the pieces of the hydrocarbon tail can be arranged around a C=C double bond. • TRANS – The two pieces of the molecule are on opposite sides of the double bond, that is, one “up” and one “down” across from each other. • CIS – the two pieces of the carbon chain on either side of the double bond are either both “up” or both “down, ” such that both are on the same side of the molecule

Trans and Cis • Naturally-occurring unsaturated vegetable oils have almost all cis bonds – but using oil for frying causes some of the cis bonds to convert to trans bonds. • If oil is used only once like when you fry an egg, only a few of the bonds do this so it’s not too bad. • However, if oil is constantly reused, like in fast food French fry machines, more and more of the cis bonds are changed to trans until significant numbers of fatty acids with trans bonds build up. • The reason this is of concern is that fatty acids with trans bonds are carcinogenic!

Phospholipids • Made from glycerol, two fatty acids, and (in place of the third fatty acid) a phosphate group with some other molecule attached to its other end. • The hydrocarbon tails of the fatty acids are still hydrophobic, but the phosphate group end of the molecule is hydrophilic because of the oxygens with all of their pairs of unshared electrons. • This means that phospholipids are soluble in both water and oil. •

Plant Biotechnology, GMOs, and the Environment

Why GMOs? • “For centuries, humankind has made improvements to crop plants through selective breeding and hybridization — the controlled pollination of plants. • Plant biotechnology is an extension of this traditional plant breeding with one very important difference — – plant biotechnology allows for the transfer of a greater variety of genetic information in a more precise, controlled manner. ”

Indeed Figure why? 9. 1 • The Earth is currently experiencing the most population increase in Human history. • 2. 5 billion in 1955 to 6 billion in 1999 • At current rate, will double within 30 years! • Fastest growing nations have growth rates at or above 4% - this will double the countries population every 17 years

Indeed why? • Hunger, starvation, and malnutrition are endemic in many parts of the world today. • Rapid increases in the world population have intensified these problems! • ALL of the food we eat comes either directly or indirectly from plants. • Can’t just grow more plants, land for cultivation has geographic limits – Also, can destroy ecosystems!

• Increasing crop yields Figure 11. 13 To feed the increasing population we have to increase crop yields. • Fertilizers - are compounds to promote growth; usually applied either via the soil, for uptake by plant roots, or by uptake through leaves. Can be organic or inorganic • Have caused many problems!! • Algal blooms pollute lakes near areas of agriculture

Increasing crop yields Figure 11. 13 • Algal blooms - a relatively rapid increase in the population of (usually) phytoplankton algae in an aquatic system. • Causes the death of fish and disruption to the whole ecosystem of the lake. • International regulations has led to a reduction in the occurrences of these blooms.

Chemical pest control Figure 11. 17 • Each year, 30% of crops are lost to insects and other crop pests. • The insects leave larva, which damage the plants further. • Fungi damage or kill a further 25% of crop plants each year. • Any substance that kills organisms that we consider undesirable are known as a pesticide. • An ideal pesticide would: – – Kill only the target species Have no effect on the non-target species Avoid the development of resistance Breakdown to harmless compounds after a short time

Chemical pest control Figure 11. 17 • DDT was first developed in the 1930 s • Very expensive, toxic to both harmful and beneficial species alike. • Over 400 insect species are now DDT resistant. • As with fertilizers, there are run-off problems. • Affects the food pyramid. – Persist in the environment

• Chemical pest control Figure 11. 18 DDT persists in the food chain. • It concentrates in fish and fisheating birds. • Interfere with calcium metabolism, causing a thinning in the eggs laid by the birds – break before incubation is finished – decrease in population. • Although DDT is now banned, it is still used in some parts of the world.

Plant Biotechnology • The use of living cells to make products such as pharmaceuticals, foods, and beverages • The use of organisms such as bacteria to protect the environment • The use of DNA science for the production of products, diagnostics, and research

Genetically modified crops • All plant characteristics, such as size, texture, and sweetness, are determined on the genetic level. • • • Also: The hardiness of crop plants. Their drought resistance. Rate of growth under different soil conditions. Dependence on fertilizers. Resistance to various pests and diseases. • Used to do this by selective breeding

Why would we want to modify an organism? • Better crop yield, especially under harsh conditions. • Herbicide or disease resistance • Nutrition or pharmaceuticals, vaccine delivery • “In 2010, approximately 89% of soy and 69% of corn grown in the U. S. were grown from Roundup Ready® seed. ” http: //www. oercommons. org/courses/detecting-genetically-modified-food-by-pcr/

Roundup Ready Gene • The glyphosate resistance gene protects food plants against the broad-spectrum herbicide Glyphosate - N-(phosphonomethyl) glycine [Roundup®], which efficiently kills invasive weeds in the field. • The major advantages of the "Roundup Ready®” system include better weed control, reduction of crop injury, higher yield, and lower environmental impact than traditional weed control systems. • Notably, fields treated with Roundup® require less tilling; this preserves soil fertility by lessening soil run-off and oxidation. ”

Glyphosate - N-(phosphonomethyl) glycine • An aminophosphonic analogue of the natural amino acid glycine. • It is absorbed through foliage and translocated to actively growing points. (Meristems!!!) • Mode of action is to inhibit an enzyme involved in the synthesis of the aromatic amino acids: • tyrosine, • tryptophan • phenylalanine Glyphosate Glycine

Glyphosate - N-(phosphonomethyl) glycine • It does this by inhibiting the enzyme 5 -enolpyruvylshikimate 3 -phosphate synthase (EPSPS), which catalyzes the reaction of shikimate-3 -phosphate (S 3 P) and phosphoenol pyruvate to form 5 enolpyruvyl-shikimate-3 -phosphate (ESP). • ESP subsequently dephosphorylated to chorismate, an essential precursor in plants for these aromatic amino acids. Glyphosate Glycine

Roundup Ready Gene • Glyphosate functions by occupying the binding site of the phosphoenol pyruvate, mimicking an intermediate state of the enzyme substrates complex. • The "Roundup Ready®” system introduces a stable gene alteration which prevents Glyphosate binding and allowing the formation of the essential aromatic amino acids

Roundup Ready Gene • The shikimate pathway is not present in animals, which instead obtain aromatic amino acids from their diet. • Glyphosate has also been shown to inhibit other plant enzymes • Also has been found to affect animal enzymes. • The United States Environmental Protection Agencyconsiders glyphosate to be relatively low in toxicity, and without carcinogenic or teratogenic effects • However, some farm workers have reported chemical burns and contact skin burns

Environmental degradation • When glyphosate comes into contact with the soil, it can be rapidly bound to soil particles and be inactivated. • Unbound glyphosate can be degraded by bacteria. – However, glyphosate has been shown to increase the infection rate of wheat by fusarium head blight in fields that have been treated with glyphosate. • In soils, half-lives vary from as little as 3 days at a site in Texas to 141 days at a site in Iowa. • In addition, the glyphosate metabolite amino methyl phosphonic acid has been shown to persist up to 2 years in Swedish forest soils. • Glyphosate absorption varies depending on the kind of soil.

Insect Resistance • B. thuringiensis (commonly known as 'Bt') is an insecticidal bacterium, marketed worldwide for control of many important plant pests - mainly caterpillars of the Lepidoptera (butterflies and moths) but also mosquito larvae, and simuliid blackflies that vector river blindness in Africa. • Bt products represent about 1% of the total ‘agrochemical’ market (fungicides, herbicides and insecticides)

Genetically modified crops • 1992 - The first commercially grown genetically modified food crop was a tomato was made more resistant to rotting, by adding an anti-sense gene which interfered with the production of the enzyme polygalacturonase. – The enzyme polygalacturonase breaks down part of the plant cell wall, which is what happens when fruit begins to rot.

Genetically modified crops • Need to build in a: • Promoter • Stop signal ON/OFF Switch Makes Protein PROMOTER INTRON CODING SEQUENCE stop sign poly A signal

Genetically modified crops • So to modify a plant: • Need to know the DNA sequence of the gene of interest • Need to put an easily identifiable maker gene near or next to the gene of interest • Have to insert both of these into the plant nuclear genome • Good screen process to find successful insertion

Building the Transgenes ON/OFF Switch Makes Protein PROMOTER INTRON CODING SEQUENCE Plant Transgene Plant Selectable Marker Gene bacterial genes • antibiotic marker • replication origin Plasmid DNA Construct stop sign poly A signal

Cloning into a Plasmid • The plasmid carrying genes for antibiotic resistance, and a DNA strand, which contains the gene of interest, are both cut with the same restriction endonuclease. • The plasmid is opened up and the gene is freed from its parent DNA strand. They have complementary "sticky ends. " The opened plasmid and the freed gene are mixed with DNA ligase, which reforms the two pieces as recombinant DNA.

Cloning into a Plasmid • Plasmids + copies of the DNA fragment produce quantities of recombinant DNA. • This recombinant DNA stew is allowed to transform a bacterial culture, which is then exposed to antibiotics. • All the cells except those which have been encoded by the plasmid DNA recombinant are killed, leaving a cell culture containing the desired recombinant DNA.

So, how do you get the DNA into the Plant?

Meristems Injections • The tissue in most plants consisting of undifferentiated cells (meristematic cells), found in zones of the plant where growth can take place. • Meristematic cells are analogous in function to stem cells in animals, are incompletely or not differentiated, and are capable of continued cellular division. • First method of DNA transfer to a plant. • Inject DNA into the tip containing the most undifferentiated cells – more chance of DNA being incorporated in plant Genome • Worked about 1 in 10, 000 times! Tunica-Corpus model of the apical meristem (growing tip). The epidermal(L 1) and subepidermal (L 2) layers form the outer layers called the tunica. The inner L 3 layer is called the corpus. Cells in the L 1 and L 2 layers divide in a sideways fashion which keeps these layers distinct, while the L 3 layer divides in a more random fashion.

Particle Bombardment

Particle Bombardment Particle-Gun Bombardment • DNA- or RNA-coated gold/tungsten particles are loaded into the gun and you pull the trigger. • Selected DNA sticks to surface of metal pellets in a salt solution (Ca. Cl 2).

Particle Bombardment • A low pressure helium pulse delivers the coated gold/tungsten particles into virtually any target cell or tissue. • The particles carry the DNA cells do not have to be removed from tissue in order to transform the cells • As the cells repair their injuries, they integrate their DNA into their genome, thus allowing for the host cell to transcribe and translate the transgene.

Particle Bombardment The DNA sometimes was incorporated into the nuclear genome of the plant Gene has to be incorporated into cell’s DNA where it will be transcribed Also inserted gene must not break up some other necessary gene sequence

Overview of the Infection Process

Agrobacterium chromosomal DNA psc. A chv. B T-DNA-inserts into plant genome for transfer to the plant vir genes p. Ti tra bacterial conjugation opine catabolism ori. V

Agrobacterium tumefaciens • Agrobacterium tumefaciens chromosomal genes: chv. A, chv. B, psc. A required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface. • Virulence region (vir) carried on p. Ti, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer.

Agrobacterium tumefaciens • T-DNA encodes genes for opine synthesis and for tumor production. • occ (opine catabolism) genes carried on the p. Ti and allows the bacterium to utilize opines as nutrient

Agrobacterium can be used to transfer DNA into plants

Overall process – Uses the natural infection mechanism of a plant pathogen – Agrobacterium tumefaciens naturally infects the wound sites in dicotyledonous plant causing the formation of the crown gall tumors. – Capable to transfer a particular DNA segment (T-DNA) of the tumor-inducing (Ti) plasmid into the nucleus of infected cells where it is integrated fully into the host genome and transcribed, causing the crown gall disease. • So the pathogen inserts the new DNA with great success!!!

Agrobacterium tumafaciens senses Acetosyringone via a 3 -component-like system 3 components: Chv. E, Vir. A, Vir. G

Genetically modified crops • The vir region on the plasmid inserts DNA between the T-region into plant nuclear genome • Insert gene of interest and marker in the T-region by restriction enzymes – the pathogen will then “infect” the plant material • Works fantastically well with all dicot plant species – tomatoes, potatoes, cucumbers, etc – Does not work as well with monocot plant species - corn • As Agrobacterium tumefaciens do not naturally infect monocots

Mini. Ti T-DNA based vector for plants a binary vector system kanr polylinker LB 11 RB ori T-DNA deleted bom modified Ti plasmid vir mini. Ti bom = basis of mobilization ori. V

Mini. Ti T-DNA based vector for plants Disarmed vectors: do not produce tumors; can be used to regenerate normal plants containing the foreign gene. • Binary vector: the vir genes required for mobilization and transfer to the plant reside on a modified p. Ti. • consists of the right and left border sequences, a selectable marker (kanomycin resistance) and a polylinker for insertion of a foreign gene. mini. Ti

1. Chv. E periplasmic protein binds to sugars, arabinose, glucose binds to Vir. A periplasmic domain amplifies the signal Vir. A Periplasmic domain acetosyringone Transmitter Inhibitory domain sugars Chv. E receiver Vir. G DNAbinding

2. Vir. A : Receptor kinase 1. Membrane protein five functional domains: a) Periplasmic binds Chv. E-sugar complex does NOT bind acetosyringone b) Transmembrane domain c) Linker region BINDS acetosyringone NOTE this is on the cytoplasmic side! Vir. A Periplasmic domain acetosyringone Transmitter Inhibitory domain sugars Chv. E receiver Vir. G DNAbinding

2. Vir. A : Receptor kinase d) Transmitter domain (His) auto- phosphorylates and then transfers to the response regulator protein Vir. G e) Inhibitory domain will bleed off the phosphate from the His in the transmitter domain (to an Asp) Vir. A Periplasmic domain acetosyringone Transmitter Inhibitory domain sugars Chv. E receiver Vir. G DNAbinding

3. Vir. G : Response Regulator • Receiver domain that is phosphorylated on an Asp residue by the His on the transmitter domain of Vir. A • Activates the DNA binding domain to promote transcription from Vir-box containing promoter sequences (on the Ti plasmid) Vir. A Periplasmic domain acetosyringone Transmitter Inhibitory domain sugars Chv. E receiver Vir. G DNAbinding

sugars Vir. A Periplasmic domain Chv. E receiver acetosyringone Transmitter Inhibitory domain Vir. G DNAbinding

Summary Agrobacteria are biological vectors for introduction of genes into plants. • Agrobacteria attach to plant cell surfaces at wound sites. • The plant releases wound signal compounds, such as acetosyringone. • The signal binds to vir. A on the Agrobacterium membrane. • Vir. A with signal bound activates vir. G.

Summary • Activated vir. G turns on other vir genes, including vir D and E. • vir D cuts at a specific site in the Ti plasmid (tumor-inducing), the left border. The left border and a similar sequence, the right border, delineate the T-DNA, the DNA that will be transferred from the bacterium to the plant cell • Single stranded T-DNA is bound by vir E product as the DNA unwinds from the vir D cut site. Binding and unwinding stop at the right border.

Summary • The T-DNA is transferred to the plant cell, where it integrates in nuclear DNA. • T-DNA codes for proteins that produce hormones and opines. Hormones encourage growth of the transformed plant tissue. Opines feed bacteria a carbon and nitrogen source.

And then? . . . . • What is the last step? . . . Tissue culture The basics!

What is Plant Tissue Culture? Of all the terms which have been applied to the process, "micropropagation" is the term which best conveys the message of the tissue culture technique most widely in use today. The prefix "micro" generally refers to the small size of the tissue taken for propagation, but could equally refer to the size of the plants which are produced as a result. Relies on two plant hormones Auxin Cytokinin

Biosynthesis of cellulose • synthesized at the plasma membrane by rosette terminal complexes (RTCs). • RTC - hexameric protein structures, approximately 25 nm in diameter • Contain the Cellulose synthase enzymes that synthesise the individual cellulose chains

Biosynthesis of cellulose • Each RTC floats in the cell's plasma membrane and "spins" a microfibril into the cell wall. • RTCs contain at least three different cellulose synthases, encoded by Ces. A genes

Biosynthesis of cellulose • Requires chain initiation and elongation, and the two processes are separate. • Ces. A glucosyl transferase initiates cellulose polymerization using a steroid primer, sitosterol-betaglucoside, and UDP-glucose. • Cellulose synthase utilizes UDP-Dglucose precursors to elongate the growing cellulose chain. • A cellulase functions to cleave the primer from the mature chain.

Protoplast to Plant • Callus: Induced by • 2, 4 dichlorphenoxyacetic acid (2, 4 D) • Unorganized, growing mass of cells • Dedifferentiation of explant – Loosely arranged thinned walled, outgrowths – No predictable site of organization or differentiation

Protoplast to Plant • 2, 4 dichlorphenoxyacetic acid (2, 4 D) • Stops synthesis of cellulose • Knocks out every other rosette • Makes b 1, 3 linked glucose – Callose • Temporarily alters the cell wall

Auxin (indoleacetic acid) Produced in apical and root meristems, young leaves, seeds in developing fruits • cell elongation and expansion • suppression of lateral bud growth • initiation of adventitious roots • stimulation of abscission (young fruits) or delay of abscission • hormone implicated in tropisms (photo-, gravi-, thigmo-)

Cytokinin (zeatin, ZR, IPA) Produced in root meristems, young leaves, fruits, seeds • cell division factor • stimulates adventitious bud formation • delays senescence • promotes some stages of root development

Organogenesis The formation of organs from a callus • Rule of thumb: Auxin/cytokinin 10: 1100: 1 induces roots. • 1: 10 -1: 100 induces shoots • Intermediate ratios around 1: 1 favor callus growth.

Edible Vaccines Transgenic Plants Serving Human Health Needs • Works like any vaccine • A transgenic plant with a pathogen protein gene is developed • Potato, banana, and tomato are targets • Humans eat the plant • The body produces antibodies against pathogen protein • Humans are “immunized” against the pathogen • Examples: üDiarrhea üHepatitis B üMeasles

Genetically modified crops • Issues: • Destroying ecosystems – tomatoes are now growing in the artic tundra with fish antifreeze in them! • Destroying ecosystems – will the toxin now being produced by pest-resistance stains kill “friendly” insects such as butterflies. • Altering nature – should we be swapping genes between species?

Genetically modified crops • Issues: • Vegetarians – what about those tomatoes? • Religious dietary laws – anything from a pig? • Cross-pollination – producing a superweed

The End! Any Questions?