Study Questions for Test 3 of Plant Physiology 1) Respiration 1. Which two enzymes break down Sucrose? What are the products from each reaction? What is the difference in ATP requirement for glycolysis when these different enzymes are used? Process called glycolysis. Sucrose Synthase and Invertase are the enzymes that breaks down sucrose. Invertase hydrolyzes sucrose in the cell wall, vacuole or cytosol. Invertase forms fructose and glucose (hexoses) and later phosphorylated by hexokinase that uses ATP to form hexose phosphate. Sucrose synthase combines sucrose with UDP to form fructose and UDPglucose. Invertase uses energy as ATP to phosphorylate glucose and fructose into a hexose phosphate. 2. The action of hexokinase and phosphofructokinase produce which molecule? What happens to this molecule? Hexokinase uses ATP to make Fructose 6P and Glucose 6P from the invertase pathway. Phosphofructokinase converts Fructose 6P and Glucose 6P into Fructose 1,6bisphosphate. 3. How many ATP and NADH are produced in Glycolysis when Glyceraldehyde3P is oxidized to pyruvate? 2 ATP and 1 NADH is produced when a glyceraldehyde3phospate is oxidized to pyruvate. 4. What is the purpose of fermentation? What conditions would cause a plant to undergo fermentation? Fermentation: the metabolism of pyruvate in the absence of oxygen leading to the oxidation of the NADH generated in glycolysis to NAD+. Allows glycolytic ATP production to function in the absence of oxygen. 5. The Citric Acid Cycle (a.k.a. Kreb's Cycle; TCA cycle) fully oxidizes pyruvate to what? The Krebs cycle fully oxidizes 1 pyruvate into 3 CO2. It also produces 4 NADH, 1 FADH and 1 ATP. 6. Oxidative phosphorylation (mitochondrial electron transport) produces what energy storage product? What happens to create the proton gradient used? Where do the electrons come from? What is the final electron acceptor? What is the Alternative Oxidase and what does its use mean for overall ATP synthesis? Oxidative phosphorylation produces the energy storage molecule ATP from various reactions using NADH and FADH produced from the Krebs cycle. The proton gradient is produced by the electron transport chain which brings about the oxidation of NADH and FADH2. The electrons are held and transported by Ubiquinone. The final electron acceptor is Complex IV. Alternative Oxidase (AOD) does not pump protons, and are nonphosphorylating. The energy released from oxidizing NADH is released as heat. AOD oxidizes hydroubiquinol into ubiquinol and reduces oxygen. 7. What is the (oxidative) Pentose Phosphate Pathway? What energy storing compound is created and what is the starting molecule for the pathway? Which subcellular compartment(s) does it occur in? What are the uses of its products and intermediates in various cell types?The oxidative pentose phosphate pathway is another pathway for the oxidation of sugars in plant cells. It is a cytosolic and plastidic pathway that oxidizes glucose and produces NADPH and a number of sugar phosphates. RX [6 Glucose 6P+12 NADP+7 H2O5 Glucose 6P+6 CO2+Pi+12 NADPH+12 H. NADPH supply in this cytosol may therefore also contributes to cellular energy metabolism, electrons from NADPH may end up reducing O2 and generating ATP through a oxidative phosphorylation. NADPH supply in the plastids (root) and chloroplasts in the dark is a major supplier of NADPH. It is used in biosynthetic reactions such as lipid synthesis and nitrogen assimilation. The intermediates such as ribose5phosphate may be a precursor of ribose and deoxyribose synthesis of nucleic acids. Erythrose4phosphate combines with PEP and produces plant phenolic compounds including aromatic amino acids and the precursors of lignin, flavonoids and phytoalexins. 2) Nitrogen and Nutrient Assimilation 1. What (or who) are the two major producers of fixed N (i.e. NH4+ or NO3) in the world? Which forms of Nitrogen can plants use? Which form of fixed nitrogen is used by plants to build amino acids and other nitrogencontaining compounds? Two major producers of fixed nitrogen: Bacteria and Human Industry. The forms of Nitrogen plants can use are NH4+ (Ammonium) and/or NO3 (Nitrate) Plants assimilate nitrate into organic compounds, and ammonium into amino acids. 2. What two enzymes are used to convert NO3 to NH4+? Is this a reduction or an oxidation reaction? What are the electron donors for each enzyme? In which cellular compartment/organelle does each reaction occur? Nitrate (NO3) is converted into nitrite (NO2) in the cytosol, a reduction reaction that involves the transfer of two electrons. The enzyme nitrate reductase catalyzes this reaction. The common form of nitrate reductase uses NADH as an electron donor while another form of the enzyme found in nongreen tissues can use either NADH or NADPH. [NO3+NAD(P)H+HNO2+NAD(P)+H2O] Nitrite (NO2) is highly reactive and a potentially toxic ion. Plant cells immediately transport the nitrite from the cytosol into chloroplasts in leaves and plastids in roots. In these organelles, the enzyme nitrite reductase reduces nitrite (NO2) into ammonium (NH4). Ferredoxin is used in this reaction, which transfers 6 electrons. [NO2+6 Fdred+8HNH4+6 Fdox+2H2O] 3. Are NO3 and NH4+ toxic to plants? Animals? If these are toxic, what is the reason? Ammonium if it accumulates to high levels in living tissue is toxic to most plants and animals because ammonium dissipates transmembrane proton ingredients that are required for Photosynthetic and respiratory electron transport. Plants assimilate ammonium near the site of absorption or generation and they rapidly store any excess in their vacuoles thus avoiding toxic effects on membranes and the cytosol. Plants can store high levels of nitrate and they can translocate it from tissue to tissue without deleterious effect. If livestock or humans consumes plant material that is high in nitrate, they may suffer methemoglobinemia, a disease in which the liver reduces nitrate to nitrite which combines with hemoglobin and renders hemoglobin unable to bind oxygen. Humans and other animals make also convert nitrates into nitrosamines which are potent carcinogens or into nitric oxide which is a potent signaling molecule involved in many physiological processes such as widening the blood vessels. 4. What molecules and enzymes are involved in the first two steps of ammonium assimilation? Where do the carbon skeletons come from to create amino acids? How many of the twenty amino acids used to build proteins can plants produce themselves? Glutamine synthetase combines ammonium with glutamate to form glutamine [Glutamate+NH4+ATPGlutamine+ADP+Pi] Elevated plastid levels of glutamine stimulate the activity of glutamate synthase. This enzyme transfers the amide group of glutamine to 2oxoglutarate, yielding two molecules of glutamate. Plants contain two types of glutamate synthase, one accepts electrons from NADH and the other accepts electrons from ferredoxin. [Glutamine+2oxoglutarate+NADHH+2glumate+NAD] [Glutamine+2oxoglutarate+Fedred2glutamate+Fdox] the NADH type of the enzyme is located in plastids of nonphotosynthetic tissues such as roots or the vascular bundles of developing leaves. The ferredoxin dependent type of glutamate synthase is found in chloroplasts and serves in respiratory nitrogen metabolism. The carbon skeleton backbone for amino acids comes from 3phosphoglycerate, phosphoenolpyruvate or pyruvate generated during glycolysis as well as 2oxoglutarate or oxaloacetate. plants synthesize all of the 20 amino acids that are common in proteins.5. What type of reaction would create Glutamate from Aspartate? Asparagine and Glutamine are similar molecules and are both highly abundant in plants? What purpose do they serve outside of being incorporated into proteins? Why would you expect more Glutamine in the light than in the dark? 6. What form of sulfur do plants take in? What is considered the final product of sulfur assimilation in plants? Most of the sulfur in higher plant cells derives from sulfate (SO42) which is transported via an H+ SO42 symporter from the soil solution. Sulfate in the soil comes from the weathering of parent rock material. The first steps in the synthesis of sulfurcontaining organic compounds involves the reduction of sulfate and the synthesis of the amino acid cysteine. 7. What is the problem that plants have with Fe3+? What are some ways that they modify their environment to gain access to iron? plants obtain iron from the soil where it is present primarily as ferric iron (Fe3+) in oxides such as Fe(OH)2+, Fe(OH)2 and Fe(OH)4 but at neutral pH, ferric iron is highly insoluble. To absorb sufficient amounts of iron from the soils solution, roots have developed several mechanisms that increase iron solubility and thus its availability. soil acidification which increases the solubility of ferric iron, followed by the reduction of ferric iron to the more soluble ferrous form (Fe2+) release of compounds that form stable, soluble complexes with iron. Such compounds are called iron chelators. 3) Light Signaling Phytochrome 1. What is photoreversibility and how does phytochrome achieve this? What colors of light are important for phytochrome activity? Are Very Low Fluence Responses or Low Fluence Responses said to be photoreversible? What is the difference between Fluence and Fluence Rate (irradiation)?photoreversibility: the interconversion of the Pr (phytochrome red light absorbing form) and Pfr (phytochrome farred light absorbing form) forms of phytochrome. Phytochrome is present in a red light absorbing from in darkgrown seedlings (etiolated). The cyan blue colored inactive form is converted by far red light absorbing form which is pale cyan green in color and is considered to be the active form of phytochrome. Pfr can revert back to being active Pr in darkness but this is a relatively slow process. However, Pfr can be rapidly converted to PR by irradiation with far red light. Fluence: a measurement of the number of photons, irrespective of time Fluence rate: a measure of the number of photons, per time period (thought as brightness 2. What is the molecular nature of phytochrome? Is it a monomer or dimer? What is the basic structure of the chromophore of phytochrome? How does the chromophore’s shape change after absorption of light? What does this do to the phytochrome protein to allow for a different function? What does the nuclear localization signal do? It is a dimer; interaction of the phytochrome protein with other cellular components ultimately brings about changes in the growth, development, or position of an organ. Responses fall into two general categories. Ion fluxes, which cause relatively rapid turgor responses and altered gene expression, which typically results in a slower, longer term response. A nuclear localization signal is needed, which is a specific amino acid sequence required for a protein to gain entrance into the nucleus. 3. What are PIFs? How do PIFs and Phytochromes act to control the expression of light responsive genes? PIF: phytochrome interacting factors; families of phytochrome interacting proteins that may activate and repress gene transcription, Some are targets for phytochrome mediated degradation. They are negative regulators of photomorphogenic responses. PIF regulates various aspects of of phytochrome mediated photomorphogenesis, including see germination, chlorophyll biosynthesis, shade avoidance, and hypocotl elongation, while promoting etiolated development in the dark, by serving as transcriptional activators of dark induced genes. 4. What are the morphological differences between Photomorphogenesis and Skotomorphogenesis? What does “etiolated” mean? What are etiolated seedlings trying to do? What does a red light flash do to the etiolated seedling? photomorphogenesis: the influence and specific roles of light on plants development. In the seedling, light induced changes in gene expression to support aboveground growth in the light rather than belowground growth in the dark. skotomorphognesis: is the developmental program plants follow when seeds are germinated and grown in the dark. etiolation: effects of seedling growth in the dark, in which the hypocotyl and stems are more elongated, cotyledons and leaves do not expand and chloroplasts do not mature. 5. What do the Red HIR and FarRed HIR responses do to an etiolated seedling in terms of hypocotyl growth? Loss of phyA destroys which HIR response? Loss of phyB? RHIR makes the hypocotyl grow normally, and is mediated by phythochrome A, allowing for normal growth. Loss of phytochrome A makes the hypocotyl grow longer in far red light and unable to open the cotyledons. In far red light, seedlings grow small in response to the low fluence rate of light, while seedlings exposed to red light grow longer than those exposed to far red light, and the absence of phytochrome A in red light grown seedlings have a longer hair root, while the absence of phytochrome B makes the hypocotl grow longer. Phytochrome A seems to be correlated with leave unrolling, and cotyledon opening, while phytochrome b plants lacks pigment and makes the hypocotl grow longer. Phytochrome A mediates responses from far red light, while phytochrome b mediates responses from red and white light. 6. Why does the Red:Farred ratio change in the shade? Do shade plants or sun plants have a response to changes in this R:FR ratio?The relative amount of red and farred light will be different when shaded, because the plants doing the shading will absorb much of the red light for photosynthesis. Thus, there will be a higher proportion of far red light when shaded. BLUE LIGHT RECEPTORS 7. What are the two types of blue light receptors in plants? What do the blue light receptors do, in terms of their physiological responses to light signals? Cryptochromes: like phytochromes, play a major regulatory role in plant morphogenesis Phototropins: are involved in directing organ, chloroplast, and nuclear movement, solar tracking, and stomatal opening. These are light dependent processes that optimizes the photosynthetic efficiency of plants. Zeitlupe proteins: shown to participate in the control of circadian clocks and flowering. COP PROTEINS 1. What is the effect of a loss of COP1 activity through a mutation? What does a cop1 mutant look like if it is grown in the dark? What does COP1 normally do to prevent photomorphogenesis in the dark? In darkness, COP1 works with SPA1 and other factors work to degrade transcription factors such as HY5 which also induces the expression of genes required for photomorphogenesis. A COP1 mutant grown in the dark will induce transcription photomorphogenesis, and look like a regular plant grown in the light. 1. In the dark, COP1/SPA1 acts to degrade transcription factors such as HY5 which is required for photomorphogenesis. 2. In the light, cry1is activated directly by blue light and indirectly by blue lightinduced phosphorylation. Activated cry1 forms a complex with COP1 and SPA1, via the Cterminal domain preventing them from degrading protein targets such as HY5. 3. In the absence of the photosensory N terminus as in the truncation mutant, the CCT region can adopt an active conformation that sequesters COP1/SPA1 in the absence of light, thereby promoting an increase in HY5 protein levels and transcription of key morphogenic genes. UVB RECEPTOR 1. How does the UVR8 protein function in UVB light signaling? What is the role of COP1 in this pathway?UVR8 is responsible for several UVB induced phosphotomorphogenic responses. UVR8 lacks a chromophore (pigment molecule) and its two identical subunits are linked in the dimer by a network of salt bridges formed between tryptophan residues, which serve as the primary UVB sensors and nearby arginine residues. Upon absorbing UVB photons, the tryptophans undergo structural changes that break the salt bridges leading to the disassociation of the two functionally active monomers. The monomers then interact with COP1/SPA complexes to activate gene expression. During phytochrome and crytochrome responses, COP1/SPA complexes acts as a negative regulator that targets transcription factors for degradation but COP1/SPA as a positive regulator during UVB signaling by interacting with the cterminal region of the UVR8 complex in the nucleus. The UVR8 complex then activates the transcription of the major transcription factor HY5, which controls the expression of many of the genes induced by UVB. 4) Auxin and Hormones A. Auxin 1. What did Darwin do to discover that there was some message sent from the coleoptile tips to the growth region of the coleoptiles? What tropism was involved in bending of the coleoptile? If you cut and displaced a tip to one half of the coleoptile, what would happen in terms of bending? “a matter which transmits its effects from one part of the plant to another” Darwin cut the tip off of coleoptile seedlings and predicted that a growth stimulus is produced in the coleoptile tip and is transmitted to the growth zone. Phototropic bending occurs towards directional light. 2. Auxin is predominantly made in what two types of tissues? Auxin synthesis is primarily found in shoot apical meristems and young leaves. As well as young fruit. 3. What is the generalized direction of auxin transport in a plant? How does the plant transport auxin in one direction? Which type of cells is involved in the polar transport of auxin in the vasculature? What happens to auxin at the root tip to promote growth of the root? generally, auxin moves from the peak of auxin production, away from peak down. This type of transport becomes canalized in a way that promotes vascular development. vascular parenchyma cells are involved in the polar transport of auxin, resulting in a positive feedback loop. These cells precedes the production of vasculature (xylem and phloem). auxin at the root tip moves up the sides of root, causing cells to elongate and grow 4. How do the channels of auxin flow that develop during leaf development relate to the vasculature pattern in the mature leaf? Does this make sense with where the auxin transporting cells are located in a mature tissue? 5. What does it mean that auxin responses are concentration dependent? What concentrations promote root growth or shoot growth? How are root and shoot bending similar and different with respect to auxin localization? low concentrations of auxin promotes the growth of intact roots, whereas higher concentrations inhibits root growth, but encourages stem and coleoptile elongation.Auxin (IAA) is dependent on the pH of the plant. A lower pH promotes growth. Most auxin influences H+ATPases which promotes acidification of the apoplast, important for cell wall growth. 6. How does auxin rapidly promote cell elongation/ growth? using H+ gradients. Hydrogen ions are act as an intermediate between auxin and cell wall loosening. The source of the hydrogen ions is the plasma membrane H+ATP synthase whose activity is thought to increase in response to auxin concentration. 7. What types of cells are involved in gravitropism in the shoot and the root? What is special about the amyloplasts of these cells? statocytes have larger amyoplasts and are capable of sensing gravity. The amyoplasts are of sufficient density relative to the cytosol that they readily sediment to the bottom of the cell. Amyoplats that function as gravity sensors are statoliths. central cells (columella) also have large dense amyoplasts that sediment thorugh the cytosol in response to gravity. 8. What is apical dominance and how does auxin play a role in species that have it? How does auxin influence fruit size and shape? Which part of a fruit is producing the auxin? in most higher plants, the growing apical bud’s inhibition of the growth of lateral buds (auxillary buds). Auxin regulates auxillary bud growth. Plants with strong apical dominance (no auxillary bud growth) means that auxin is regulating growth. seeds in fruit produces auxin, and when they’re removed, they stop growing. 9. Does auxin regulate gene expression? How does the TIR1 protein (the auxin receptor) influence gene expression in plants? What are the roles of AUXIN RESPONSE FACTORS (ARFs) and the Aux/IAA proteins in this signaling? Auxin affects gene expression, in the absence of the auxin hormone, the AUX/IAA proteins prevent the ARF proteins from promoting the expression of auxinregulated genes. Auxin makes the TIR1 protein bind very tightly to the AUX/IAA proteins. Auxin acts like a glue to hold the AUX/IAA protein to a TIR1 protein. 10. What are PIN proteins and what are the AUX1 proteins used for in Auxin transport? How are these proteins localized with the cells to promote the polar transport of Auxin? How does Auxin enter and exit a cell? B. Ethylene1. What is the chemical structure of this hormone? H2C=CH2 2. Why did coal lights in the late 1800s cause plants living near them to defoliate? Coal lights produced ethylene, which is used for regulation of leaf abcision. 3. What is the triple response of etiolated seedlings exposed to ethylene? Not natural, and happens when plants are exposed high levels of ethylene. 1. shorter hypocotyl 2. horizontal growth, and curved apical hook 3. increased lateral growth (wider stems) 4. What are the roles of Auxin and Ethylene in leaf abscission? Higher concentrations of ethylene messes with auxin 5. Why would a banana ripen faster in a closed container that contained an apple than in the same container without an apple? Why wouldn’t an orange ripen faster? Apples produce ethylene, and ethylene ripens fruit faster and oranges and other citruses don’t ripen with more ethylene. C. Cytokinins 1. Why would the thermal breakdown of DNA create a bioactive, although unnatural, cytokinin hormone? Transzeatin (cytokinin) is structurally similar to adenine (DNA sugar). Heat can break apart hydrogen bonds. 2. What is the bellcurve like celldivision response to cytokinin in roots and shoots? 3. What would a cytokinin insensitive plant look like? A cytokinin insensitive plant would be dwarfed compared to a normal version. 4. In cultured callus tissue, how can you induce the formation of roots or shoots? Yes, increasing only auxin concentration will make new roots, while increasing only cytokinin concentration will give new shoots. 5. Why do many pathogens make cytokinins? What happens to plants that have been infected with Agrobacteria? What neat thing does Agrobacteria do to the genome of the plant cell that it infects? What types of genes does it add?To feed and grow and reproduce, it makes a special food for the bacterium (sugar with nitrogen). Plants that have been infected develops a crown gall tumor. Agrobateria cuts a small specific set of DNA from its plastid, and injects its cut piece into the plant cell, where the plant will add it to its own genome.