Plant Physiology Midterm 3 PDF

MIDTERM 3 PLANT PHYSIOLOGY Lecture 19 Gibberellins (GAs) First discovered in Japan in 1920s and 1930s in association with ‘bakane’ – foolish seedling disease Abnormal elongation due to infection with fungus Gibberella fujikuroi Plants will fall over Isolated two compounds that...

MIDTERM 3 PLANT PHYSIOLOGY Lecture 19 Gibberellins (GAs) First discovered in Japan in 1920s and 1930s in association with ‘bakane’ – foolish seedling disease Abnormal elongation due to infection with fungus Gibberella fujikuroi Plants will fall over Isolated two compounds that will cause disease in absence of fungus World War II ensued Rediscovery In the 1950s two groups discovered the original work – one group in England, one in Illinois Purified the compound and named it Gibberellic Acid Large number of compounds (over 100), but only a few have biological activity GA1, GA3, GA4 and GA7 (numbers are in order of discovery) Synthesis of GA Three cellular compartments involved: chloroplast, ER and cytoplasm Geranylgeranyl phosphate is considered precursor Regulation is generally accomplished by converting an active form of GA to an inactive form Role of GA in plants and how we can use it to our advantage Regulation of height ○ Some dwarf plants are dwarfs because of interruption in GA biosynthetic pathway – treatment with GA will restore height (see B) ○ In rosette plants like lettuce (not a mutant), bolting precedes flowering. GA can induce bolting (see C) Growth in grapes – Thompson seedless GA treated grapes (GA3) have larger fruit and elongation of pedicels (fruit stalks) Not only is this more attractive to consumer – limits fungal growth in bunch Prevent russeting in apples Fruit “cosmetics” – left hand apple is treated with GA More cosmetics Red delicious apples are a ’type’ apple – people expect a certain size/shape Treatment with GA4 + GA7 accentuates the type More GA effects Influence floral initiation ○ Bolting ○ Substitute for inductive photoperiod (what is this?) Affect sex determination in some plants ○ Cucumber: Normally forms male flowers first on vine – then female ○ Treat with GA: Promote male flowers – use GA inhibitor to promote female flowers – important in commercial seed production Overcome dormancy – promote germination in seeds Enhance malting in barley Horticulturally anti-GAs (GA synthesis inhibitors) are important GA inhibitors keep plants compact Mechanism(s) of action Cell elongation – but not by acid growth – activates xyloglucan endotransglycosylase – XET ○ molecular rearrangements in cell wall leading to elongation GA can affect cell cycle progression – especially shorten G2 to M – but up to 30% decrease in total cycle time – more cells Mobilize endosperm reserves – classic experiment Classic Experiment – barley embryo and GA When barley seed imbibes water, endosperm liquifies and seed begins to germinate  Cut embryo portion (green) away from endosperm put both in separate containers of water – nothing happens Add GA to each – endosperm will begin to liquify Barley and GA – what happens in intact seed 1. Gibberellins are synthesized by the embryo and released into the starchy endosperm via the scutellum 2. Gibberellins diffuse to the aleurone layer 3. Aleurone layer cells are induced to synthesize and secrete α-amylase and other hydrolases into the endosperm 4. Starch and other macromolecules are broken down to small molecules 5. The endosperm solutes are absorbed by the scutellum and transported to the growing embryo Barley aleurone PSV=Protein storage vesicle GA receptor GA binds to receptor – leads to ubiquitin attachment and degradation of DELLA repressor by 26S proteasome {DELLA proteins are characterized by the presence of a DELLA motif (aspartate-glutamate-leucine-leucine-alanine or DELLA) in single letter aa code} Transcription factor activated and genes can be expressed Here’s how that plays out in barley aleurone system – LOL Lecture 20 Finishing GA and Intro to Cytokinins Question for the day ○ How do we currently describe the process of translocation in a plant? ○ What has caused so many of the problems in studying this process? Finishing ideas for GA, Assay for GA Bioassays are known – any speculation about how they might work? HPLC or GC-MS to detect specific GA and levels *Bioassay, hormone does elongation so for experiment I would get dwarf plants and then apply the GA hormone, the strongest concentration that the plant could withstand so we are able to measure growth and which could grow the fastest *We could also do this experiment with seed germination and how adding GA could seed germinating faster GA - Uses in agriculture/horticulture Synthetic forms of GA not available – must be produced by plant or microorganism Fairly expensive Used on high value crops (like grapes or apples) where input costs will be recovered GA synthesis inhibitors are inexpensive and widely used – control height in horticultural crops including bedding plants Prevent lodging in agricultural crops (grains). See photo at right. ○ Logging, not able to harvest, treat with GA inhibitor, makes it shorter for better harvesting. Cytokinins Reports of substances that would enhance cell division in early 1920s, but this class of hormones eluded investigators Search for a substance that would promote cell division in plant tissue culture  First - What about this technique (tissue culture)? Plant tissue culture Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used to produce clones of a plant in a method known as micropropagation. Used frequently in genetic engineering of plants. ○ Growth medium contains: ○ Plant nutrients ○ Sucrose ○ Agar ○ Plant hormones ○ Sometimes various selection factors (antibiotics) In examining tissue culture cells, early researchers found polyploid cells if only auxin were in the medium ○ Cells were polyploid, nucleus divided, multiple nuclei Add coconut water (sometimes called milk – not the stuff you cook with) and cells would divide Adenine like compound sought The search Tried various chemicals from various labs Autoclaved herring sperm DNA yielded a compound named kinetin ○ Kinetin, 1st cytokinin made, not found in plants Not found in plants but chemically similar compounds called cytokinins are found Zeatin best studied-> used as a model Zeatin/Zeatin riboside Ribose sugar makes the riboside Conjugate may be active or storage form Cytokinins do not share structural similarity Defined by what they do: Activities similar to zeatin including: ○ Promotion of cell division in tissue culture (with auxin) ○ Promote bud or root initiation in tissue culture (with auxin) ○ Delay leaf senescence ○ Promote cotyledon expansion Zeatin Dimethylallyl diphosphate Synthesized primarily in plastids Cytokinins enhance cell division and greening Auxin Auxin +Cytokinin, greening occurs Cytokinins in tissue culture Cytokinins and normal shoot apical meristem Normal apical meristem Wild Type: shoot apical meristem Cytokinin: Oxidase expression reduces the size of shoot meristems and number of dividing cells Cytokinin oxidase expression reduces size of shoot meristem and number of dividing cells Cytokinin oxidase breaks down cytokinins. Mutant overexpresses this enzyme ○ Levels need to be normal for plant to have normal apical meristems ○ Overexpression mutant ○ Express to much of something ○ Cytokinin oxidase ○ Breaks down cytokinin Recall the role in apical dominance Delay senescence Transgenic tobacco (on left) containing a cytokinin biosynthesis gene (ipt) fused to a senescence-induced promoter. The ipt gene is expressed in response to signals that induce senescence ○ Plant 1: plant expressing ipt gene remains green and photosynthetic (age determined senescence) ○ Plant 2: Age-matched control shows advanced senescence (promoter in senescence turns off) Exogenous cytokinins can change a source to a sink Mature source leaf-> Add cytokinin-> Radioactivity accumulate-> draws photosynthate toward it Mechanism(s) of action Regulates cell division, specifically acting on cell cycle Regulates many developmental genes Acts in concert with other hormones like auxin to promote effects ○ Greening of green part chlorophyll Receptor Model for the cytokinin signal transduction pathway – unlike the hormones studied so far, this is related to bacterial histidine kinase receptors. Transcription is turned on by Type-B ARR (Arabidopsis response regulator) ○ Cytokinin receptors are located in the endoplasmic reticulum Assay for cytokinin Bioassay? ○ Measure greening (speed) or measure cell division (harder to measure) Regularly assayed by RIA Agricultural/horticultural uses Many studies on delay of senescence, increased yield – conflicting or complex results – not widely used Lecture 21 Questions of the day You have found a plant that you suspect may be a cytokinin deficient mutant. What could you examine in this plant to determine if this was the case – be sure you justify your answer. ABA and Ethylene ABA – an “old friend” Abscisic Acid - ABA Studied earlier in semester with respect to stomatal closing Discovery in two different labs studying different aspects of plant physiology Despite its name, ABA does not usually promote abscission Does have a role in senescence and other processes Synthesis of ABA Synthesized in plastids Pathway includes other “old friends” as well as other important molecules Regulation by conjugation to a monosaccharide Sometimes stored in vacuole Roles of ABA Most important: Stomatal closure during water stress – in your notes Involved in seed dormancy – two kinds Coat-imposed ○ Physical prevention of water uptake – coat rigid Embryo-imposed ○ ABA high in embryo- GA low ○ Reverse this and seed will germinate Chemical prevention – ABA in coat Embryo-imposed ○ ABA high in embryo – GA low ○ Reverse this and seed will germinate Role in senescence – but not abscission Plant Physiol. 66:1164-68 ABA levels begin to rise while leaves are still green; steadily increase during senescence Mechanism of action for ABA Causes membrane depolarization (stomatal closure) Regulate gene expression at level of transcription Activation of enzymes (protein kinases) to phosphorylate proteins and activate them ABA receptor In the absence of ABA, the protein phosphatase PP2C keeps the protein kinase SnRK2 dephosphorylated and thereby inactivated When ABA is present, its receptor prevents dephosphorylation of SnSK2 or PP2C. Phosphorylated (active) SnRK2 phosphorylates downstream substrates, thereby invading ABA responses. Assay Bioassay? HPLC and RIA are standard means of assay Agricultural/horticultural uses Most applications involve prevention of drought stress What do you think this involves? Ethylene Effects of ethylene reported before people knew that ethylene was the “culprit” 1864 – reports that street lights (which used “illuminating gas”) would cause leaves to fall off street trees near the lights 1900-1910 – data from various agricultural offices recommended that certain fruits not be stored with bananas because the fruit would ripen too fast Old saying “one bad apple can spoil the whole bunch” How does all this relate? Ethylene 1901 – Neljubow clearly demonstrated that ethylene from illuminating gas could cause triple response in pea plants: ○ Inhibition of stem elongation ○ Increased stem thickening ○ Promotion of horizontal growth habit Exogenous effects Ethylene 1936 - Gane demonstrated that plants synthesize ethylene Resistance to calling a gas a plant hormone During 1930s the wide range of ethylene responses began to be elucidated Ethylene production in plants Produced in response to injury, infection, ripening Can regulate seed germination, cell differentiation, cell expansion Important in senescence and abscission Biotic and abiotic stress response Ethylene synthesis Ethylene biosynthesis and the Yang Cycle Knowledge of this pathway brought ways to control it Ethylene effects Promotes ripening in some fruit – more on this Promotes flowering in mango/pineapple Promotes abscission and senescence Responsible for leaf epinasty in waterlogging – more on this  May play a role in plant defense Ripening – will ethylene ripen all fruit? Ethylene will ripen climacteric fruit: like tomato, avocado, banana, peach, apple and mango Examples of non-climacteric fruit: citrus, grapes, watermelon, strawberries Important considerations Most fruit is shipped to distant markets Post harvest physiology How can the timing or release of ethylene be controlled? Control of ethylene in shipping fruit/flowers –post harvest physiology Raise concentration of CO2 and lower O2 – be careful here Lower temperature Ag+ will inhibit ethylene activity – so why is this not widely used? Range of chemicals will bind to ethylene receptor are used Treatment with Ag+ (STS) will delay senescence in flowers A great alternative – 1 Methylcyclopropene (1MCP) Brief description of 1-MCP activity Works by tightly binding to the ethylene receptor site in fruit tissues, thereby blocking the effects of ethylene. Once ethylene production is prevented, It no longer promotes ripening and senescence. Blocking of ethylene receptor by I-MCP gas causes fruits to be ripen and soften more slowly. Lecture 22 Ethylene – final ideas and introduction to the “new kids” Question of the morning We mentioned last time the saying “one bad apple can spoil the bunch” Using what we talked about on Monday, please explain this from a plant physiology perspective. Ethylene synthesis Ethylene biosynthesis and the Yang Cycle Knowledge of this pathway brought ways to control it Epinasty – often in response to waterlogged soil Note elongation on top of the petiole. Leaf has appearance of bending down Ethylene receptor - in ER ○ “Irrigation or drainage system” ○ Cellular respiration can no longer be performed, see it in the upper part of the plant Assay for ethylene Bioassay? ○ Ethylene causes avocado to ripen Unripe avocados and see faster high concentrations of of ethylene Leaf abscission take long for days GC (Gas Chromatography) -> easy way to measure gas Agriculture/horticulture Antagonizing ethylene – already mentioned several ways to do this Promoting ethylene – gas? Chemicals that can be applied to plants and then cells release ethylene – Florel ○ Synthetic product ○ Promotes mechanical harvest Mechanical harvest ○ Promote picking of the fruit Fruit thinning ○ Apples, fruit thinning ○ Source-> sink concept ○ All fruit allowed on tree One more example for ethylene application Example of use to thin or harvest apples (abscission) Cotton harvesting is example of use of ethylene to promote senescence prior to mechanical harvesting Chlorophyll will stain cotton and lower its value Treat with ethylene to promote senescence of plants Senescent plants are brown and easily harvested – dead plant material easily removed from bolls Brassinosteroids, jasmonic acid and salicylic acid (“the new kids on the block”) Brassinosteroids Steroid hormones are well known in animals – only recently described in plants Originally discovered in Brassica rapa pollen – a growth promoting property Called brassins in early literature 230 kg of pollen yielded 10 mg of brassins (551 lbs yielded 0.0003 oz) Activity similar to IAA so effects were difficult to discriminate Brassinosteroids or auxin responsible? IAA mutants were essential to this determination – what kind and why? ○ Auxin and brassinosteroids, have auxin mutant separates (exists or not), experimental results to brassinosteroids, this causes elongation, see mutant exposure ○ IAA mutant detected in IAA ○ Needed to use for testing growth ○ Plant cannot make IAA Experiment? Many brassinosteroids have been isolated, but two active forms are recognized: brassinolide and and its immediate precursor castasterone Brassinosteroids Synthesis mostly in pollen, immature seeds and fruit – but low levels in various plant organs Wide ranging effects including involvement in cell division, elongation, germination, senescence and stress responses  Note that all the brassinosteroid mutants in Arabidopsis are dwarfed ○ Classic plant material for genetics Uses in agriculture/horticulture Literature about various potential applications (seed germination, plant propagation, stress coping) – but use is scant ○ Little in regards for plants Actively marketed in Asia but not US Chemical synthesis difficult and costly Steroidal compounds are not easily absorbed by plants Assay GC-MS-> standard way for assay Jasmonic Acid and Salicylic Acid Part of plant defenses Jasmonic Acid (JA) Considered a lipidic plant hormone since it is synthesized from linolenic acid – via the octadecanoid pathway This pathway is a major signaling path in plant defense responses Involved in some plant carnivory responses ○ Get nitrogen by eating bugs ○ Able to capture insects JA has several roles, but defense in abiotic and biotic stress seems to be primary Parallels to oxylipins that are involved in mammalian inflammatory response ○ Mammalian defense system Synthesis First steps of synthesis are in chloroplast Peroxisome is where synthesis completed Plant insect herbivore interaction – JA has active role Many hormones can play a role in response to insect herbivory Ethylene is often detected When applied alone ethylene has little effect But with JA – response is enhanced ○ In response to herbivores Suggests synergy of several compounds important Jasmonic Acid role in defense against insects JA promotes biosynthesis of defense proteins/responses in many plants: ○ In legumes – a amylase inhibitors synthesized which block starch digestion in insects ○ In other plants - lectins which bind to carbohydrates in the epithelial cells of an insects’ digestive tracts and block nutrient absorption ○ Best studied system – proteinase inhibitors found in many plants including legumes and tomatoes ○ Bind to enzymes such as trypsin and chymotrypsin in insect digestive tract Signaling pathway Insect chews on leaf Wounded leaves synthesize prosystemin in phloem parenchyma – processed to systemin Binds to receptors in companion cells Activates a cascade that results in JA synthesis JA moves via STE to unaffected leaves Proteinase inhibitors synthesized Considered a systemic (whole plant) response ○ Insect chews on leaves, systemin, companion cells , JA, loaded to phloem, sink, plant movement, protease, advantage self unattracted for insect not to seed (JA in companion cells), Not attracted plants, growth, not dry out, limit photo Responses to herbivory involve JA JA can accumulate within minutes locally and remotely Plants lack a nervous system, but some evidence for electrical signals Wave of plasma membrane depolarization Experiments with leafworm feeding on Arabidopsis – electrical signals spread to unwounded leaves (9 cm/min) probably in vascular tissue JA mediated gene expression turned on ○ GLR = glutamate receptor like. This group of genes is being researched in conjunction with the electrical signaling path JA induces HIPV synthesis Insect herbivore induced plant volatiles (HIPV) HIPVs attract natural enemies of the attacking insect And they can signal to neighboring plants ○ Nematodes (feeding on plants), not all plants can do this ○ All have some sort of systemic defenses Insects come and attack Resistant plants naturally occurring Lecture 23 Two questions for you derived from grading tests What is acid growth? What is polar auxin transport and how does it relate to rooting? Acid Growth Cell walls extend faster at an acid pH Acid treatment will stimulate this as will auxin treatment Acidification of the wall is believed to occur through activation of H+-ATPase Salicylic Acid (SA) Originally discovered in extracts from willow (Salix) – active ingredient in aspirin Important role in plant stress response – especially with respect to phloem-feeding insects (aphids) and pathogens JA is more often seen in response to chewing insects that we looked at last time ○ Sucking insect-> aphids in the phloem Model for defense response to pathogens and salicylic acid involvement Several things going on here Signaling molecules: Ca+, NO (nitric oxide), ROS Various responses possible HR – hypersensitive response – cells around infection site “commit suicide” to prevent pathogen spread Systemic acquired resistance (SAR) – local pathogen attack enhancing resistance to further damage Salicylic acid needs to be methylated for response to occur – interfere with methylation – no SAR Much research in this area A note on HR and cell death in plant cells This type of cell death (cells die to accomplish a particular purpose) is called PCD (programmed cell death) in plants instead of apoptosis – animal cells Presence of cell wall and absence of phagocytes mean that processes are different Two distinct pathways of PCD in plants PCD in plants Vacuolar PCD - during normal development. Vacuole is storage site for proteases and other lytic enzymes – it swells and enzymes released – xylem development, leaf senescence ○ What we see in normal plant development ○ Lose all contents in cell Hypersensitive response PCD – defense mechanism against microbial attack – no vacuolar swelling or leakage – vacuole is small. Cell shows water loss and DNA degradation ○ Cellular debri left over ○ Cell dehydrates and DNA is degraded Loses water *Program cell, cell die for cell to be functionally unit of result, dead cell *Responds to invader cytoplasm collapses, results in cell death Selected whole plant developmental responses Xylem-> complete (dead) Phloem cells lack a nucleus, not death due to sieve tube elements (no nucleus, no DNA, no proteins) Senescence and Abscission Senescence Several factors affect the life of entire plant organs like leaves Genetics, nutritional status, water, other environmental cues ○ Temperature, day lengths Consider the process of leaf senescence developmentally and physiologically Three types of senescence Under normal growth, senescence is governed by developmental age of leaf Gradient of senescence on plant from oldest leaves at base to youngest leaves at tip (most important)  Sequential leaf senescence ○ Plant that keep their leaves most of the time Seasonal leaf senescence is also a normal process ○ Lose all leaves during the fall Seasonal leaf senescence – a timely topic In temperate climates leaves of deciduous trees (like aspen) senesce all at once in response to shorter days and cooler temperatures This is a coordinated process - discuss further with respect to abscission process ○ No leaves -> loss of leaves abscission Third type of senescence – stress-induced Not considered normal part of plant life Both sequential and seasonal senescence are variations of developmental senescence Under unfavorable conditions such as a stressful environment, leaf senescence can occur prematurely Stresses include: drought, nutrient deficiency, temperature extremes and pathogens Developmental leaf senescence Three phases: ○ Initiation: Transition from nitrogen sink to nitrogen source, Photosynthesis declines, Early signaling events ○ Degenerative phase: Dismantling of cellular constituents, degradation of macromolecules Phase we can see, yellowing, cellular constituents are being broken down ○ terminal phase: Loss of cellular integrity, Cell death, Leaf abscission Cells break down see cell death, chemicals that may be recycled Senescence as an active, programmed process Earliest cellular changes observed in chloroplast Before senescence and after about 50% loss of chlorophyll – gerontoplast Chlorophyll breakdown - mobilization ○ Swirling around ○ Losing ability to photosynthesize And in Arabidopsis leaves ○ Leaf numbers numbers indicate date after implanting Arabidopsis leaves Numbers on leaves indicate days after planting Design an experiment to test whether senescence in Arabidopsis can be delayed using ethylene inhibitors. What could you do and when? ○ Multiple parts Prevent ethylene detection (receptor), if we want day 30 then we treat the plant leaves with MCP in a bag, 27 or 24 day plants, stop/ slow synthesis (interfere with enzymes time w/ the receptor), cold temperature, slow metabolism, raise CO2 concentration, treatment, of AG+ (STS) (ethylene receptor vs. ethylene synthesis) delay, senescence in flowers MCP receptor, day 30 need to assume work day 20 or day 25 so we apply whatever (enzymes), treat plant to slow down Lecture 24 Question for today Choose two of the hormones we have discussed so far besides auxin. Describe one of the things that each of the hormones influences in plant growth – be as specific as possible. ○ Ethylene: influences ripening of fruits, one bad apple ○ GA: elongation, dwarfs (something wrong with GA) promote long elongation, add GA ○ JA: signaling path in plant defense responses, involved in some plant carnivory responses, defense in abiotic and biotic stress, response is enhanced Abscission Shedding of leaves, fruits, flowers and other plant parts Leaf abscission well understood – partly because of research funded during Vietnam War Abscission zone – located at base of petiole – often visible many weeks before separation takes place ○ Abscission layer: separation takes place ○ Protective layer: interior to abscission, leaf scar, scab bacteria, insects can’t get into layer Abscission – LM views Diagrams from text ○ Protective layer here in many plants Auxin and ethylene play roles -hormones play off each other Know that ethylene can promote abscission As leaves become “competent” to respond to this – recognize three stages: ○ Leaf maintenance – auxin reduces leaf sensitivity to ethylene High auxin, low sensitivity in leaf ○ Abscission induction – auxin goes down – ethylene sensitivity is increased, a reduction in the abscission zone, which triggers the abscission phase Reduction of auxin, ethylene rise Enzymes ○ Abscission phase – cellulases and pectinases break down walls and leaf separation results, synthesis of enzymes that hydrolyze the cell wall polysaccharides results in cell separation and leaf abscission Move, the leaf separates Other organs – flower petals, fruit etc. Less well studied Parallels with leaf abscission noted in the species examined Differential Growth and Plant Movement Many examples of differential growth Consider selected ones First ones will deal with plant movements  Nastic movements – external stimulus triggers, but direction of stimulus does not determine direction of response ○ Direction of stimulus does not determine the direction of response Tropisms – external stimulus triggers, but direction of stimulus does determine direction of response – positive or negative ○ Does determine the direction of response Nastic movements Epinasty – cells on top of petiole elongate more – waterlogging Nyctinasty – sleep movements ○ Sleep movement of a bean plant. The movements are caused by reversible changes in the turgor pressure of cells on opposing sides of the motor organs of the leaf. Continues even when leaves are “held down or up”. They return to “normal” position for that time of day. http://plantsinmotion.bio.indiana.edu/plantmotion/movements/leafmovements/clocks.html Sleep movements What do you think is involved in the water loss or gain? Circadian rhythms also involved Direction may be different in different plants – leaves may fold up ○ Extensors full of water during day ○ Flexors are full of water at night At night cells lose water Water potential involved, moving plant parts-> water moves from one region to other from height to day Thigmonasty – response to touch http://plantsinmotion.bio.indiana.edu/plantmotion/movements/nastic/nastic.html Thigmonasty Response to touch – Robert Hooke was one of the first to investigate this Mimosa – sensitive plant A mechanical or heat stimulus induces an electrical signal, similar to the electrical potentials in nerve cells, that can move from cell to cell at a high rate When the electrical potential reaches specialized "motor cells" in pulvini at the base of each leaflet, folding is caused by a rapid efflux of K+ followed by rapid water transport out of the motor cells Venus flytrap Another kind of plant studied by Darwin Carnivorous plants inhabit nitrogen poor environments Catching insects is a way to get nitrogen Closing is thought to involve an action potential and acid growth Followed by activity of digestive glands prior to reopening Reopening requires cell division and elongation on inside ○ Once plant closes trap ○ Insect triggers two hairs at the same time, action potential result of insect touch the hairs, cascades rapid closing, acid growth, influx of protons, cellulose microfibrils What is an action potential? An action potential is a rapid sequence of changes in the voltage across a membrane. ○ Animal example: measure spikes-> hook up electrodes, dissecting needle touch triggers hairs, reader sees spikes Cells potential travel Phloem, discussion Measure w/ instrument ○ Action potential measured Electrical signaling in Venus flytrap (Dionaea muscipula) – plant example Figure 4.39 Electrical signaling in Venus flytrap (Dionaea muscipula) (Part 1) ○ (A) Photograph of the snap-trap leaves with needlelike tines and touch-sensitive trigger hairs. ○ (B) Action potentials (APs) associated with Ca2+ influx and K+ efflux are triggered by movement of mechanosensitive channels in trigger hairs. ○ Mechanical stimulation of the trigger hairs twice by a prey raises Ca2+ levels to a threshold that results in flytrap closure. ○ Five triggering events are sufficient to induce jasmonate signaling that stimulates production of digestive enzymes and compounds that create the “green stomach” that digests the prey. Figure 4.39 Electrical signaling in Venus flytrap (Dionaea muscipula) (Part 2) ○ Action potentials (AP) associated with Ca2+ influx and K+ efflux are triggered by mechanosensitive channels in the trigger hairs. ○ Mechanical stimulation of the trigger hairs 2X by a prey raises calcium levels to a threshold that results in trap closure ○ Five trigger events are sufficient to induce JA signaling that stimulates production of digestive enzymes and compounds that create the “stomach” that digests the prey Thigmomorphogenesis Not a nastic movement – response to environmental stimulus – mechanical perturbation (touch) Generally, cells are shorter with increased collenchyma ○ Plants have defenses-> shorter cells, are shorter, cells are different collenchyma-> increase collenchyma Tropisms – direction matters Three areas to consider as we look at tropisms ○ Perception ○ Transduction ○ Response Sometimes these are difficult to delineate clearly Phototropism Looked at this before as we introduced plant hormones – Darwin experiments What is the perception in this case? ○ Perceives light This is a light mediated process – so how do you begin your investigation of it? Light mediated process action and absorption spectrum Need action spectrum to see wavelengths that are most active ○ How is the plant going to perceive-> phototropins perceive light change to conformation Need absorption spectra to try to identify pigment involved ○ How pigment is facilitated For many years a flavonoid pigment was suspected since these compounds have absorption spectra that are close matches Phototropins are the pigments Use of mutants that were defective in phototropism led to identification of phototropins 1 and 2 (PHOT1, PHOT2) ○ Found mutants defective to phototopism Identify PHOT 1, PHOT 2 ○ Action-> pigments attracted to ○ Single receptor during two different things Phototropins Phototropins are also involved in other processes mediated by blue light including chloroplast movement and leaf movements Actin filaments are involved in pushing the chloroplasts around! ○ Pushed around actin filaments Phototropism (perception of things, detect the light) Need action spectrum to see wavelengths that are most active Need absorption spectra to try to identify pigment involved For many years a flavonoid pigment was suspected since these compounds have absorption spectra that are close matches Now know that phototropins are the pigments responsible ○ Equal and unequal distribution ○ Not chloroplast, Auxin movement Curvature from away from light Auxin moving lighted side to shaded side PIN proteins move auxin from unequal to equal Place w/ more auxin and more curving, that’s the response Lecture 25 Tropisms continued Phototropism Need action spectrum to see wavelengths that are most active Need absorption spectra to try to identify pigment involved For many years a flavonoid pigment was suspected since these compounds have absorption spectra that are close matches Now know that phototropins are the pigments responsible PHOT1 response 1. Light with blue wavelengths strikes plant cell membrane with phototropin 1 (PHOT1).  2. Blue light is absorbed by PHOT1, causing a change in conformation. 3. This conformational change results in auto-phosphorylation, triggering a signal transduction. Blue light receptor: Embedded in cell membrane When blue light detected, changes conformation, signal transduction → differential elongation Transduction What happens next? Precise sites of auxin production and lateral transport have been difficult to define PIN 1 and PIN 3 are thought to be involved in auxin redistribution 1. In the dark, auxin primarily moves from the shoot to the root through the vascular tissues in the petioles and hypocotyl, and through the epidermis. 2. After exposure to unidirectional blue light, auxin movement briefly stops at the cotyledonary node and the seedling stops growing vertically. 3. Auxin is redistributed to the shaded side and polar transport resumes. 4. The cells on the shaded side of the hypocotyl elongate, resulzing in differential growth, and the seedling bends toward the light source. Response: Differential growth Total growth is about the same But shaded side grows more as lighted side grows less ○ Phototropism- transduction, response Gravitropism Plants Response to gravity Roots are positively gravitropic Shoots are negatively gravitropic How to explain this – the same mechanism? Or different mechanisms? Most research on roots - begin there Perception – root cap Root cap is required-> cap is required w/out not able to grow properly ○ (A) Vertically oriented control root with cap ○ (A) Removal of the cap from the vertical root slightly stimulates elongation growth. ○ (A) Removal of half of the cap causes a vertical root to bend toward the side with the remaining half-cap. ○ Vertically oriented control root with cap ○ (B) Removal of the cap from the vertical root slightly stimulates elongation growth. ○ (B) Removal of half of the cap causes a vertical root to bend toward the side with the remaining half-cap. Perception Occurs in root cap If you remove root cap, roots will grow in random directions until cap regenerates Central cells – columella – primary area for sensing Cells are often called statocytes – amyloplasts in these cells are called statoliths  Sense gravity So what is happening? Gravistimulate a root – turn it sideways Follow what changes in the plant High auxin is red-orange in illustration Auxin is moved to lower portion of the root And the root begins to grow down (see lower panel at left) How is this possible? Be able to explain this… ○ Root: auxin causes elongation but high auxin causes plant to go downward ○ Redistribution of auxin PIN proteins 3 & 7 Auxin increases at bottom of root Differential tissue sensitivity Yes, we have high auxin but then we also get roots are sensitive to auxin get growth inhibition Gravitropism - roots (not light mediated) Transduction – IAA moves to “bottom” – growth inhibited here Response – differential growth – top can elongate more So how about shoots? Not as dramatic as in the root, but starch sheath is visible outside vasculature - amyloplasts are found here ○ Vascular bundle In mutants without amyloplasts – no response to gravity What happens? ○ Disadvantageous position, plant advantageous auxin accumulate bottom IAA transported via PIN proteins to “bottom”, Shoot grows upward via differential growth Be sure you know why this is Stem: tissue to sensitivity is different Root: auxin causes elongation but high auxin causes plant to go downward Only two tropisms to talk about Photomorphogenesis Change in plant form with respect to light Three major categories of receptors known: ○ Blue light ○ Protochlorophyllide a ○ Phytochrome Increasing pigment, seed germination, flowering, Focus on phytochrome ○ Seed germination ○ flowering Early experiments in two different systems – different systems – but idea of measuring involved in both Seed germination  1907 Kinzel examined seeds from 1964 species of plants – showed that 674 required light to germinate Most of these were small, undomesticated ○ No selection for agriculture Why a light requirement? ○ Required for plant growth even though the plant is small Evolutionary adaptation Seeds – light requirement for germination 1950s Hendricks and Borthwick looked at germination in lettuce seed Found light stimulated germination Action spectrum? Red light was specifically involved Far-red light exposure immediately following red inhibited germination The second line of experimentation – flowering Many plants flower when the reach a certain age, size and nutritional status – no further signal required – day neutral ○ Flowering at any time Others require an additional signal – photoperiod Short day plants (SDP) flower in late summer or early fall (soybean, chrysanthemum) ○ Days get shorter (soybean,chrysanthemum) Long day plants (LDP) flower in spring or early summer (lettuce, pea, barley) ○ Some plant need photoperiod (lettuce, pea, barley) What is the plant measuring? Original hypothesis – age/accumulated photosynthate ○ Was triggered- assumed Experiment: Soybeans Planting date Flowers Days May 2 Sept 4 125 June 2 Sept 4 94 June 30 Sept 15 77 Hypothesis not supported ○ No evidence that time led to flowering Experiments with Xanthium (SDP) 8 h light/16 h dark – flowers 4 h light/15 min dark/4 h light/16 h dark – flowers 8 h light/8 h dark/15 min light/8 h dark – no flowers What does the plant appear to be measuring? ○ Uninterrupted dark And an interesting parallel to seed experiments What wavelength of light is most effective in the night break? Red Follow it by far-red and what happens? What (if anything) does this have to do with seed germination? We are looking at a light mediated response – what should we measure?  What if anything does this have to do with seed germination, we are looking at a light mediated response- what should we measure? ○ phytochrome - seed germination , first they though flowering was similar- not true for flowering, circadian rhythm (when does the plant perceive light) ○ Circadian rhythm (plant defenses), applied to phytochrome and flowering Putting ideas together – action spectrum Bold idea – looking for a pigment 1 pigment that could exist in 2 forms 1959 Phytochrome was purified Exists in 2 forms ○ Synthesized as Pr – bluish in color – absorption max at 668 nm ○ Red light exposure will convert to Pfr – olive green in color – absorption max at 730 nm - has morphogenetic effect – can revert to Pr with 730 light ○ Pr ⇆Pfr (Photoreversibility) ○ Photoreversibility: goes back to reversibility form Structure This is the physiologically active form in most seed germination Phytochromobilin Phytochromobilin is synthesized in the chloroplast Apoprotein is added Exposure to red light – Pfr formed and most of it moves into nucleus ○ Pfr moving, interesting in the nucleus Photomorphogenesis, need to go to transcription and translation ○ Can be used for other things ○ Go through red, infrared experiments 1. The PoB chromo-phore attaches to the GAF domain at a conserved cysteine residue to produce the holoprotein, 2. Upon activation by red light, the D ring of PộB rotates, causing a conformational change in the holoprotein and exposing the nuclear localization sequence (NLS) within the PRD domain of phyB. The protein FHY provides the NLS for phyA. 3. Most of the phytochrome pool moves into the nucleus where it regulates gene expression. 4. A small pool of phytochrome remains in the cytosol where it mediates rapid responses. A note about “escape” A response may be photoreversible if far red light is given soon enough But this is not the same amount of time for every response Few minutes to hours depending on pathway involved This has been referred to as the escape time ○ Photoreversibility will be escaped, run away photomorphogenetically Several different responses – many ways to categorize Rapid biochemical responses – organelle movement Slower morphological responses including movements and growth Also classify by the amount of light required: ○ VLFR – very low fluence rate response  ○ LFR – low fluence rate response ○ HIR – high irradiance response ○ Fluence – number of photons absorbed per unit surface area ○ Irradiance – Amount of energy that falls on a sensor of known area per unit time – Watts per meter2 VLFRs 0.0001 µmol/m2 – seconds of starlight or 1/10 the light of firefly flash Seed germination in some plants Interestingly many of these seeds are either those that have been buried for some time and/or agriculturally important LFRs Lots of seed germination Regulation of leaf movements – leaf unrolling Enhanced chlorophyll accumulation Classic responses ○ Most seed germination falls into this HIR Prolonged or continuous exposure to strong light Response is proportional to light until saturation is reached ○ Have little light-> more response ○ Saturation no response-> no matter how much nothing extra can be produced A little bit about genetics In Arabidopsis there are 5 genes that encode phytochromes designated PHYA-PHYE PhyA is involved in VLFRs and HIRs ○ VLFRs and HIR-> flowering most important It also is important in photoperiodic control of flowering – more on that later PhyB plays a role in LFRs – seed germination ○ We know a lot about, mainly on seed germination PhyC, D, and E are less well understood Phytochrome’s other “discovery area” - flowering What wavelength of light is most effective in the night break? Red Follow it by far-red and what happens? So what is going on? Not as simple as seed germination ○ Light break experiments Lecture 26 Phytochrome and Flowering Questions for today If you apply a protein synthesis inhibitor to a leaf you can delay senescence and abscission for a few days. Explain why this can happen. You have a plant root that is slow to respond to gravistimulation. Describe two things that could be going on in this plant. Bold idea – a pigment with two forms 1 pigment that could exist in 2 forms 1959 Phytochrome was purified Exists in 2 forms ○ Synthesized as Pr – bluish in color – absorption max at 668 nm ○ Red light exposure will convert to Pfr – olive green in color – absorption max at 730 nm - has morphogenetic effect – can revert to Pr with 730 light A note about photoreversibility A response may be photoreversible if far red light is given soon enough But this is not the same amount of time for every response Few minutes to hours depending on pathway involved This has been referred to as the escape time Saw this before and related it to seed germination – but… What wavelength of light is most effective in the night break? Red Follow it by far-red and what happens? So what is going on? Not as simple as seed germination Most research on SDP During the night, Pfr slowly reverts to Pr If you expose plant to red light – undo this reversion Length of unbroken dark period is important Also important is circadian clock ○ Also plays a role ○ Unbroken period of dark is important Another variation on night break in SDP – coincidence model Flashes of red light to plant as soon as it gets to the dark period, able to suppress flower in sensitive time period ○ Circadian clock part (flash light during light period) Coincidence model and beyond The point: Plants have a different physiology depending on light and dark periods As a result, the ability of light to either inhibit or promote flowering depends on the phase (in circadian clock) in which the light is given Flowering in both SDP and LDP is induced when the light exposure is coincident with the appropriate phase of the rhythm Putting the story together: Rice and Arabidopsis as models Plant needs to be coincident Putting this all together – revising the text order – start with SDP (bottom right) Start with D in the illustration (Bottom right) ○ Rice is a SDP. ○ Under long days (short nights) Hd1 mRNA during the light phase (sensed by phyA) leads to synthesis of Hd1 protein. ○ (transcription and translation) ○ This represses Hd3a mRNA synthesis – no flowering. ○ Hd = heading date Now C (Top Right) ○ With short days, Hd1 mRNA peak does not coincide with daylight, Hd1 is not made. ○ Repression of Hd3a mRNA lifted. Hd3a protein made. Flowering is initiated at apex LPDS Now A (Top Left) ○ In Arabidopsis (LDP) under short days – little overlap between CO mRNA and daylight. Not enough CO protein to promote flowering. Now B (Bottom Left) ○ In long days, peak of CO mRNA overlaps with daylight (phyA senses) and CO protein accumulates. CO activates FT mRNA which moves to apex to trigger flowering. ○ FT and Hd3a are homologous as are CO and Hd1 ○ CO = constans; FT = flowering locus T Summary Hd3a and FT both promote flowering Hd1 and CO are considered homologous, but Hd1 acts as inhibitor while CO is a promoter Revisit seeds – phytochrome is sensing light, but response much simpler Found light stimulated germination Red light was specifically involved Accumulation of Pfr will promote germination Pr inhibits it ○ Red light to turn Pr->PFr Back to flowering Day length is sensed in leaves Multiple events in flowering in Arabidopsis initiated as signal travels to apex Bottom panel shows expression of FT-GFP (green fluorescent protein) fusion in companion cells in leaf vein ○ Hormone: Giberellum Treat plants, goes straight to apex, by passing normal growing to promote flowering GAS Can not make in lab, expensive Photoperiod, shade cloths to trick plants no use of hormones 1. FT mRNA is expressed in companion cells of the leaf vein in response to multiple signals, including day length, light quality, and temperature. 2. FTIP1 mediates the transport of FT through a continuous ER network between the companion cells and the sieve tube elements. 3. FT moves in the phloem from leaves to the apical meristem. 4. FT is unloaded from the phloem in the meristem and interacts with FD. 5. The FT-FD complex activates SOCI in the inflorescence meristem and AP1 in the floral meristem, which triggers LFY gene expression. 6. LFY and AP1 trigger expression of the floral homeotic genes. The autonomous and vernalization pathways negatively regulate FLC, which acts as a negative regulator of SOCI in the meristem and as a negative regulator of FT in the leaves Flowering stimulus is transmissible by grafting -taking one plant part, and sticking it onto another plant SDP exposed to short days and grafted to non-induced plant will induce flowering An induced SDP can promote flowering in an LDP Girdling the petiole will inhibit transmission (why?) Induced leaf ○ Graft it to uninduced plant and induce flowering ○ phloem-> apex-> promote flowering Short day plant can induce a long day plant ○ Inhibit the transition-> cut off phloem no longer able to move Lecture 27 Finishing flowering But first - questions Your roommate sees a leaf on one of the houseplants that is beginning to turn yellow. They want to cut it off. What do you tell them and why?  The graph to the right was explained in the last class. What specifically did this experiment reveal about flowering and photoperiod? And more grafting Amplifies experiment ○ Stimulus is persistent and could move through the phloem Indirect induction can be demonstrated in serial grafting experiments in Xanthium Grafting of induced leaf to uninduced shoot causes flowering in multiple grafts in Perilla Florigen Long history of the use of this word to describe a putative flowering hormone that was translocated in phloem Now know that florigen is not a hormone per se, but a flowering stimulus that travels in the phloem ○ Stimulus Grafting studies indicate transmissibility of stimulus even between different species ○ Can be transmitted Plants talking the same language Apex transitions from production of leaves to floral parts – modified leaves Longitudinal section through developing flower Cross section of developing flower showing floral whorls Schematic diagram of developmental fields Flowering locus T (FT) is florigen FT is a small globular protein related to Hd3a in rice Will cause flowering in day neutral plant when gene for FT is introduced ○ Not only in rice ○ Day neutral plant with respond Activates “floral identity genes” in apex that set in motion the transition of the apex from vegetative to floral ○ Transition of apical meristem in shoot from Vegetative (A) to flowering (E) But what about other hormones? Application of GA can cause bolting in rosette plants – like Arabidopsis – this will lead to flowering Bypasses the internal signals Ethylene is also capable of promoting flowering in members of the pineapple family – genetics not well understood ○ SOC1 = (Suppression of Overexpression of CO1): A multifunctional protein which regulates not only flowering time but also floral patterning and floral meristem determination ○ Homeotic genes: regulate development of anatomical structures Flowering structures Abiotic and biotic interactions Potentials for Stress and Stress Relief -stress physiology (how they cope) Abiotic stress -water stress in plants (not all plants respond the same)  ○ Atmospheric Demands: Humidity, Air Temperature, Radiation, Wind, Leaf Temperature, Stomata Opening Plant Regulation Soil Supply ○ Soil moisture, Soil temperature, Soil Depth, Soil Texture Stress Any environmental condition that prevents a plant from achieving its genetic potential Primary abiotic parameters that have been examined are water, light, temperature and toxins (salinity and heavy metals) Soil nutrients and air quality have also been researched Plant genome controls the trade-offs Vegetative program may terminate early under stress so that reproduction may occur ○ See frequently, may flower before its suppose to Fewer and smaller seeds may result because the plant is likely to be smaller with fewer leaves The balance of these trade-offs will vary with the type of plant Responses – in general Acclimation (hardening) – response of the plant improves with repeated exposure to an environmental stress. Non-permanent change in physiology or morphology of individual ○ Improve with stress of the plant Adaptation – genetic changes in a plant over several generations by selective environmental pressure – stress resistance is inherited ○ Natural selection will occur ○ More permanent Need to also distinguish between avoidance (reduce the impact of the stress) and tolerance (endure the stress) ○ Have different way of avoiding stress and tolerating stress Reactive oxygen species (ROS) -talked about how they can be toxic if they accumulate Mentioned previously with respect to pathogens Toxic intermediates may be produced in times of stress Act as signals to induce acclimation mechanisms Also interact with and oxidize cellular constituents including proteins and nucleic acids Water stress You already know a lot about the importance of water and how water moves in a plant Three big categories classify plants with respect to water: ○ Hydrophytes – water is always available ○ Mesophytes – water availability varies (most aware of) ○ Xerophytes – water is scarce (live where water is not available) Hydrophytes Floating plants Large air spaces (aerenchyma) Stomates on leaves usually on top  Can’t grow if water is not available Plants that live near water that may change in level can have different leaf morphologies depending on whether the apical meristem was under water or above water when the leaf was initiated Again, if water dries up – plant will not survive ○ Air and water leaves ○ 2 different leaf formats: heterophylly ○ Determined by: where the vegetative meristem is at the shoot When a leaf is initiated Leaf underwater, water leaf, responds to when leaf is initiated Xerophytes – the other extreme (from hydrophytes) Many ways to classify these – one strategy: ○ Drought escape ○ Desiccation postponement ○ Desiccation tolerance Drought escape True avoiders Spring flowers in the desert Complete their life cycle when water is available – before drought becomes an issue again Spring ephemerals ○ Flower bloom ○ Drought escapers ○ Life cycles is complete before it gets hot ○ Avoid the drought Dependent on water to complete short life cycle Desiccation postponement Ability to maintain tissue hydration – several strategies ○ Store water – cacti and succulents ○ Reduce water loss – CAM, sunken stomates, thick cuticle ○ Deep tap root ○ Sometimes called “water spenders” Strategies to avoid dedication (cati) Stores water Reduces water loss (CAM) and sunken stomates Large cuticle Very deep, tap root Euxerophytes – desiccation tolerance About 300 species that have been reported Selaginella lepidophylla – a well-known example Desert fern in spike moss family Curls completely with desiccation – can withstand desiccation of nearly 95% ○ Most cells 25%-90% ○ Can take down to 5% and still exist “Semi-botanical” history and fame The ability of this plant to survive extreme desiccation was noted by Spanish missionaries when they reached the New World The missionaries used the plant to demonstrate to potential native American converts the concept of being reborn An infusion (tea) can be made by steeping a tablespoon of dried material in hot water and the tea is used as an antimicrobial in cases of colds and sore throat ○ Botanical medicinal properties Plant strategies for surviving desiccation Increase in sugars including sucrose and trehalose Increase in sugar alcohols and phenolics Rapid reproduction when water becomes available Heteroblasty of seeds Increase in secondary metabolites – these are being studied actively for anti-cancer, antifungal and antibacterial use – we will discuss in upcoming lecture what they can do in plants ○ Secondary metabolism Plants secondary metabolise for a function Energize What about mesophytes and water stress? Environmental factor ○ Water deficit Primary effects ○ Water potential reduction ○ Cell dehydration ○ Hydraulic resistance Secondary effects ○ Reduced cell leaf expansion, Reduced cellular and metabolic activities ○ Stomatal closure, Photosynthetic inhibition ○ Leaf abscission, Altered carbon partitioning, Cytorrhysis ○ Cavitation, Membrane and protein destabilization ○ ROS production, lon cytotoxicity, Cell death Important ideas to remember Cell wall growth appears to be most sensitive – mild stress will slow growth – so when do plants do most growing? ○ Design an experiment Cell wall growth Hypothesis: plant will grow in high water availability day in life of plant, most growth happens at night, plants are less stressed at night Prolonged water stress will result in less leaf area, less branching – remember cell expansion is driven by water movement Possible generation of ethylene Root expansion is usually enhanced ○ Prioritize roots as sink when stressed  Stomates close – ABA levels are high ○ Stomatal regulation ○ Cell expansion: is affected by water movement Root growth ○ Root to shoot ratio Length or biomass ○ More root mass than shoot mass when less water Benefit of fewer shoots Less leaves, less transpiration ○ Stressed, stuck, so have to have plant growth strategy or a coping strategy Lecture 28 Abiotic stress - continued What did this tell you about water stress in sunflower? Phloem transport Photosynthesis Leaf expansion is super sensitive to water stress ○ Going down when water stress ○ Plateau Another sunflower experiment Two groups of plants: One never exposed to stress (red), one grown under continuous stress (olive green) Water both sets of plants Then begin water stress What does this experiment show? ○ showed-> plant growth on both sets of plants, showed stressed, remembered, they allowed to be stressed, had experienced more likely to handle ○ Do the tests respond the same? No the lines would be closer together ○ Plant with stress, how do they cope with stress Use water ○ Who stops/ slows growth sooner The one experiences water stressed ○ Plant grown under continuous water stress Shut things down sooner Might survive better than the one never experienced stress ○ Turgor (MPa), Ψp High water potential-> increase Turgor-> happier cell ○ Plants never exposed to water stress Allows to be able not to grow more Less recovery GR=m(Ψp-Y) Remember: too much water is also a stress for some plants ○ Stunted growth-> root phenomenon Salt stress – Na+ -sodium-> worst defender-> stays in soil Sources of salt in soils – accumulation from irrigation water, seepage Classify plants as: ○ Glycophytes (sweet plants) – what we have looked at so far – plants not resistant to salt ○ Halophytes – plants variously adapted to salty areas Salt marshes High salt in water similar to water stress Problems with high salt – similar to water stress – see table 15.1 in current edition of text Figure 15.2 Impact of abiotic stress on plant growth and reproduction (Part 2) (A) Whereas moderate water-deficit stress does not have a significant effect on the growth of rice plants, severe water-deficit stress reduces growth. (B) Effect of salt stress on maize ear size. (C) Effect of salt stress on maize kernel size. Consider: Osmotic stress – water potential gradient between root and soil hard to maintain since soil water potential is very negative - plant needs to get water into the root Trying to establish and maintain a low solute potential within plant usually compromises growth Ion effects – sodium accumulation can inactivate some enzymes and inhibit protein synthesis ○ Salty soil: difficult for plant to be maintained soil-> root plant cells have to be more negative, plant cells more negative, more negative due to sodium potential ○ Sorghum plants treated with various levels of NaCl 50mM: presence in height and leaf 100mM: leaf color change 200mM: see ion effects, inactivates enzymes Sodium-> proteins deprotonates proteins by salts + What does Na do to proteins? More considerations Sodium can displace calcium from membranes Photosynthesis inhibited ○ Light harvesting inhibited Cell division and expansion can be compromised ○ compromised-> lower Classification and coping III-> very salt sensitive II-> more resistant to salt IA-> considered true halophytes, salt concentrations enhance growth at high concentrations-> due to genetics, selective trait  Group IA (halophytes) includes sea blite (Suaeda maritima) and salt bush (Atriplex nummularia). These species show growth stimulation with Cl- levels below 400 mM. Group IB (halophytes) includes Townsend's cordgrass (Spartina x townsendiï ) and sugar beet (Beta vulgaris). These plants tolerate salt, but their growth is retarded. Group II (halophytes and nonhalophytes) includes salt-tolerant halophytic grasses that lack salt glands, such as red fescue (Festuca rubra subsp. littoralis) and Puccinellia peisonis, and nonhalophytes, such as cotton (Gaoypium spp.) and barley (Hordeum vulgare). All are inhibited by high salt concentrations. Within this group, tomato (Lycopersicon esculentum) is intermediate, and common bean (Phaseolus vulgaris) and soybean (Glycine max) are sensitive. The species in Group III (very salt-sensitive nonhalophytes) are severely inhibited or killed by even low salt concentrations. Included are many fruit trees, such as citrus, avocado, and stone fruits. Coping: Salt glands to excrete salt White spots, salt glands, secrete salt out of plants ○ Very thick cuticle to prevent salt from seeping in Type 1: Bladder Type 2: Multicellular gland More strategies Exclude salt at root – requires energy Decrease solute potential in cells ○ Accumulation of sucrose, proline, sorbitol in cells to lower solute potential ○ Solute potentials may be as low as -17 MPa in some leaves (-1 to -3 MPa in glycophytes) 5 full increase in solute potential (coping with salt) ○ This will generally reduce plant growth and yield – energy allocation to coping rather than growth Stress, limit stress and ability to perform How to ameliorate salt stress Treat soil to minimize salt – add calcium or acid to leach sodium Breed plants for salt tolerance ○ Why would acid leach sodium Add bumps on ions ○ Damper things Weak acid-> then put back what we want Temperature stress – low temperatures Put leaf in freezer – then thaw How will it look and why? Water expands when frozen ○ Let all water and end up with a dead leaf when thawed Freezing Primary Effects ○ Water potential reduction, Cell dehydration, Symplasmic ice crystal formation Secondary Effects  ○ Same as for water deficient, physical destruction How can a plant cope with low temperatures? Some plants can’t live in areas where the temperatures get outside a certain range Plants are classified by hardiness and “hardiness zone maps” are published and easily available for professionals and other growers What factors contribute to being cold hardy – or cold tolerant In general plants respond to shortening days and gradually cooling temperatures that tend to precede very cold or freezing weather ○ Shortening days Sense the day, use circadian, light sensitivity pigment To receive phytochrome Several changes can potentially occur Loss of leaves Membrane changes similar to those that occur in animals ○ Increase in unsaturated fatty acid composition in membranes – what does this do? Unsaturated fatty acids Increase in sugars – primarily sucrose in cytoplasm may act as cryoprotectant – prevent freezing in cells ○ Slow freezing in the cell Several gene products may contribute to process Antifreeze proteins – inhibit ice formation which can damage membranes LEA proteins (late embryogenesis abundant) – first identified in seeds associate with seeds drying as they mature – protect membranes Side note here – seeds are usually really resistant to cold – why? ○ All stuff is inside protective shell, less resistant HSP – heat shock proteins – first identified in Drosophila during studies of heat stress, but protect membranes of plants, animals and microbes in both cold and heat THPs – thermal hysteresis proteins – lower the temperature at which fluid in the cell goes from liquid to solid ○ Least frequently seen Seeds are more resistant to low temps-> very cold like formation of cells, cells die to expansions of water ○ Leaves and roots-> slow or prevent water in cells to not freeze High temperatures – the other extreme Hard to separate from drought effects in many cases Most plants are unable to survive extended exposure to temperatures above 45oC – CAM plants may be able to survive in some cases Several issues for the plant: ○ Transpiration can’t sufficiently cool leaf ○ Photosynthesis is inhibited before respiration ○ At high temperatures photosynthesis can’t replace carbon used by respiration so carbohydrates are lower in plant – fruit and vegetable crops less sweet  ○ Membrane may be too fluid – hydrogen bonding and electrostatic attractions affected to transport can also be affected Lecture 29 More defenses Question of the day Temperature stress is a factor that frequently limits where plants can grow successfully. Given a plant gradually subjected to cold, explain the changes you might expect to see in the following as the plant adapts: a) cell membranes: cell membranes more saturated, see change in animals b) types of proteins present in the cells: denatured ○ Heat shock proteins, anti freezing proteins ○ Protein confor or get an advantage as its getting cold Wrap up abiotic stress biotic interactions and various defenses High temperatures Hard to separate from drought effects in many cases Most plants are unable to survive extended exposure to temperatures above 45oC – CAM plants may be able to survive in some cases Several issues for the plant: ○ Transpiration can’t sufficiently cool leaf ○ Photosynthesis is inhibited before respiration ○ At high temperatures photosynthesis can’t replace carbon used by respiration (fast enough) so carbohydrates are lower in plant – fruit and vegetable crops less sweet (less desirable, less market value) ○ Membrane may be too fluid – hydrogen bonding and electrostatic attractions affected to transport can also be affected (can be disrupted) Makes sugar for various plant needs, cellular respiration-> uses sugar to get energy Cellular respiration->uses sugar to get energy ○ In stress-this can overwhelm photosynthesis BIG DANGER, ice forming in cells, poke cells in cell membrane water freezes Photosynthesis Makes sugar for various plant needs Cellular Respiration Uses sugar to get energy In stress- this can overwhelm photosynthesis How to cope with high temperatures - if you’re a plant Plants adapted to cool temperatures may not be able to cope HSP – some are constitutive others are synthesized in response to stress – mRNA synthesis for these can be measured 3-5 minutes after stress begins ○ Constitutive Always made, housekeeping enzymes other will be induced ABA and salicylic acid increase – appears to be independent of HSPs ○ ABA: increase, close stomates, limits transportation in high temp  ○ Salicylic acid: increase, good stress regulator Plant interactions with environment - biotic We have already mentioned some plant interactions with organisms – both helpful and harmful Recall mycorrhizae and discussions about herbivore defenses Other interactions as well Focus on two additional biotic interactions – not always stressful ○ Sucking and chewing insects ○ Getting nutrients, insects, interactions with pathogens, how a plant defend about those Revisiting nitrogen fixation and plants Nitrogen is 80% of our atmosphere ○ Nitrogen fixation-> use nitrogen from air Limits plant growth Yet nitrogen is the most limiting nutrient for plant growth What if plants could use nitrogen in the air to get “fertilized”? Nitrogen fixation Capturing nitrogen from the air to use on Earth Can do this industrially – expend oil energy to fuel the process Can do it biologically via a catalyst – and cellular energy Enzyme is called nitrogenase ○ Only synthesized by prokaryotes Synthesized only by prokaryotes Symbiosis for nitrogen fixation Some plants are capable of forming a symbiosis with bacteria that can fix atmospheric nitrogen Bacteria enter roots – infection thread - and form root nodules Often seen in legumes (pea, bean, alfalfa) Bacteria of the genus Rhizobium ○ Other genes as well ○ Big bumps nodules (forming a nodule-> bacteria enters root) Within nodule bacteria synthesize nitrogenase enzyme Atmospheric nitrogen to ammonium (symbiosis) Plants provide carbohydrates from photosynthesis – bacteria use these for nutrition – plant makes amino acids ○ Proteins etc. Nitrogen fixation Fixed nitrogen “fertilizes” the plant Extra nitrogen fixed goes into the soil enriching it As we mentioned earlier, this is the basis of “crop rotation” – alternating a legume and non-legume crop to reduce fertilizer costs and improve soil Amazing coevolution (symbiosis) Nitrogenase enzyme is broken down by high levels of oxygen  Plants need oxygen to live – so how does symbiosis work? Leghemoglobin synthesized to protect nitrogenase ○ Binds oxygen Part of molecule is made by plant (apoprotein), part by bacteria (heme moiety) ○ Self assemble, function is similar, coevolution as a biotic interaction ○ Plant cells need cellular respiration Another biotic interaction Not so beneficial to the plant, but useful to us Infection by Agrobacterium tumefaciens Causes “crown gall” -> caused by agar bacteria Progression of crown gall and use in molecular biology 1. The tumor is initiated when bacteria enter a lesion and attach themselves to cells. 2. A virulent bacterium carries a Ti plasmid in addition to its own chromosomal DNA. The plasmid’s T-DNA enters a cell and integrates into the cell’s chromosomal DNA 3. Transformed cells proliferate to form a crown gall tumor. 4. Tumor tissue can be “cured” of bacteria by incubation at 42°C. The bacteria-free tumor can be clustered indefinitely in the absence of hormones Agrobacterium mediated transformation –in the lab -can get the plant to do what we want due to gene change in bacteria Agrobacterium can be used to transform plant cells Hormone and opine genes removed – gene of interest and selectable reporter inserted Plasmid will integrate into plant chromosome Tissue culture can be used to regenerate whole plants from transformed cells ○ Add a protein resistance gene, plate on antibiotic, gene of interest with antibiotic, gives us what we want This is called stable transformation as opposed to the transient transformation the 472L students examined Some successes in genetically engineering plants Gene manipulation offers some advantages, characteristics (useful to some in agriculture; interest for people in medicine) ○ Insulin 2006 insulin available for pig or cow pancreas, animal produced products, insulin produced by bacteria and plant cells, beef and pork insulin still used in other countries ○ Vaccine production Places where refrigeration are not available, most countries don’t have, some plant being used as genes of interest, grow up plant, freeze and dried an produces an edible vaccine And applications for animals (humans) Insulin production Vaccines Vaccines in edible form are also possible – consider why this is important 1. A gene encoding a β-cell autoantigen is placed in a plasmid and introduced into agrobacterium  2. Agrobacterium transfer the gene into the plant cells 3. Plant cells are regenerated to plants 4. Selection of the plants with the highest levels of antigen 5. Elite transgenic plants are transferred to greenhouse 6. Fruits or leaves are harvested (depending on the host plant) and the ones with higher antigen expression are chosen 7. Plant material is freeze-dried 8. Dried plant material containing the antigen are pulverized and made into capsules 9. Oral plant-made vaccine candidates are tested in animal and clinical traits Vaccines in edible form are also possible-consider why this is important \ ○ Problem: get a product to last in stomach due to acid, would need the edible vaccine to get past stomach digestive properties Secondary metabolites – previously unrecognized helpers Secondary metabolites are located at the interface between primary metabolism and the interaction of organisms with their environment ○ Interactions w/ external environment (biotic stress, abiotic stress responses, beneficial interactions) ○ Secondary metabolites (phenolics, terpenoids, alkaloids) ○ Primary metabolism First, a little more on plants and defense systems -hormones, jasmonic acid, salicylic acid When we look at all the ways that plants have to defend themselves, not all are mediated by hormones (jasmonic and salicylic acid) Herbivory defenses also include antixenosis (from the Greek for ‘guest’) factors which discourage feeding: ○ Movement (as we saw in mimosa)-> not other plants ○ Thick cuticle ○ Hairs – stinging hairs (discourage feeding) Antibiosis factors are chemicals those that actually can harm an herbivore Secondary metabolites Once considered plant “waste products” or byproducts Increasingly recognized as having an important role in metabolism and defense Three categories: ○ Terpenes ○ Phenolics ○ Nitrogen containing compounds (alkaloids) Terpenes Pyrethroids from leaves and flowers of chrysanthemum (Pyrethrum) – insecticidal properties Natural and synthetic forms available Not toxic to animals ○ Very easy to harvest Can make synthetic form, safe way to eliminate insects Essential oils Another example of terpenes We tend to think of essential oils as beneficial in cooking or perfumes Many oils have more wide-ranging applications both for people and for plants Known and used for insect repellent properties Oils are frequently found in hairs that protrude from leaf surfaces, but also in fruits and leaf tissues (sources of essential oils) Can be extracted by steam distillation Also have well known antimicrobial and antifungal properties Insecticidal properties of some essential oils Citrus sinensis is orange Citrus aurantium is bitter orange Triterpenes Azadirachtin (from neem tree) affects more than 200 species of insects and is considered a natural insecticide Phytoecdysones-> insect molting process Similar in structure to insect molting hormones Disrupts molting and other processes ○ Frequently lethal Latex (milky secretion) from oleander, mulberry and other plants is made of terpenes. Latex is stored in cells call laticifers. Discourages herbivores – can be poisonous Lecture 30 More Defense Strategies Questions of the day Outline a general method that could be used to introduce a gene of interest into a plant. We discussed a variety of terpenes in the last lecture. Other than all being of a similar chemical class, what is another property that all of them have in common? Phenolics Huge variety – more than 8000 structures known Include primary as well as secondary metabolites Recent interest in human health since plant polyphenols have antioxidant properties ○ Also reports of anticancer properties (diverse properties (huge group)) Phenolics and plants Various phenolics are responsible for a phenomenon called allelopathy ○ Chemical inhibition of neighboring plants due to release into environment acting as germination Allelopathy is chemical inhibition of one plant (or other organism) by another, due to the release into the environment of substances acting as germination or growth inhibitors. Secondary metabolites like phenolics can be released into soil or air and can deter the growth of nearby plants ○ Allelopathy-> plant release phenolics-> prevents plants around Allelopathy Spotted knapweed – an invasive species produces catechin which displaces native species Other compounds include cinnamic acid and ferulic acid Interesting research on possible allelopathy to weedy species ○ Conservation of native species Flavenoids -mentioned before (purple cabbage, color) Largest group of phenolics Include anthocyanins – red, pink and purple color – not harmful Isoflavenoids – rotenone from legumes is active as insecticide and pesticide (rat poison) – affects mitochondrial ETS – you saw an example of this type of activity in Biol 240 ○ Cyanide: mitochondria-> anything inhibits, binds to 4 major complexes Prevents oxygen from binding ○ Not harmful isoflavenoids-> harmful, rat poison Best known flavenoids (from a plant point of view) - phytoalexins Used to be thought of as plant equivalent of antibodies – but aren’t specific to infecting organism Chemically diverse group of secondary metabolites with strong antimicrobial activity Not detectable prior to infection (see no phytoalexins) Vary by plant family (no consistency) Defensive significance not completely understood ○ They are actively defensively but do not target things like we expect Tannins -very widespread in plants Another phenolic Widespread in plants Water soluble Feeding repellant to many insects and some animals because of astringency Pleasing to many humans: tea and red wine Nitrogen containing compounds – alkaloids Alkaloids include: ○ Nicotine ○ Atropine ○ Cocaine (naturally occurring plant products) ○ Codeine ○ Morphine Many good drug applications as well as the possibly harmful or illegal ones ○ Drug products-> some have insecticidal properties Alkaloids These were once thought to be nitrogenous waste – exact role in plants unclear, but current research supports defense against herbivores – especially insects and mammals  Large numbers of livestock deaths each year are due to grazing on alkaloid containing plants – lupine, larkspur and groundsel Toxic in high concentrations Various effects on animal physiology at the cellular level – particularly the nervous system Current research: potential anti-cancer agents – vinblastine/vincristine ○ Plant deviates/ used as less toxic version of chemotherapies Alkaloids and cancer Vinca: A periwinkle Leaves contain alkaloids, vincristine and vinblastine, that are useful in treating various forms of leukemia (blood related cancer) and lymphoma (cancer of the lymphatic system) Also being tested on other forms of cancer ○ With both blood related Want to know if works in other parts of the body Plant defense systems Hormonal regulation Secondary metabolites Another player – circadian rhythms ○ How they contribute to flowering and Jasmonic Acid (JA) damage during the day, salicylic acid pathogen entry, cabbage cirvadian fluctuation Bring all together ○ flowering-> phytochrome day length circadian rhythms-> plant defenses-> defense, herbivore pathogen-> JA phenolics-> salicylic acid resistance for pathogens Circadian rhythms Actually nearly 1/3 of all plant genes exhibit some circadian aspect to their expression In addition to those involved in photosynthesis, flowering and metabolism – genes involved in plant defense Circadian influence Arabidopsis and cabbage looper – a generalist lepidopteran herbivore Research by Goodspeed et al. 2012 Observation: cabbage looper herbivory and jasmonate defense in Arabidopsis appear to be under circadian influence Hypothesis? ○ When do you think most herbivores feed? In the day JA increases Reverse circadian rhythm JA increase at night, kills the plant Hypothesis and experiment Timing of the jasmonate-mediated defense response may be an adaptation that maximizes protection against herbivory ○ Adaptation for preventing quivery Compare herbivory in Arabidopsis plants that are either in phase or out of phase with respect to circadian rhythm of cabbage looper  Important to remember that both organisms have circadian rhythms Circadian rhythms in and out of phase Caterpillars feed day ○ JA ↑- day Experiment and Results Loopers fed for 72 hours Out of phase plants clearly suffered more feeding damage than in phase (normal) plants And cabbage loopers reaped the benefits in terms of weight gain Jasmonates and salicylates cycle, but may be complementary – not synergistic Jasmonates peak during the day – herbivore protection Salicylates (gray line) peak in evening – may protect more against pathogenic bacteria which tend to infect in the early morning ○ May help target against pathogens ○ Pathogens Sensitive to moisture levels (stomates closed at night) ○ Is plant more stressed in Day or Night Day: temperature effects accumulate ○ Peak at night Reverse the problems Lecture 31 More defense strategies Questions for today We will mention the hypersensitive response in lecture today. What is this and how does it help a plant? Continuing to recall – what is systemin and what does it do for a plant? What hormone should be part of your answer here? Focus so far primarily on herbivores – now look at microbes Microbes are also a source of stress for plants Examine plant pathogens and look at strategies for entering plant We have talked about some of these previously ○ Fungi penetrating cells directly ○ Bacteria penetrating the plant through a wounding site ○ Fungi penetrating the plant through stomata ○ Bacteria penetrating the plant through stomata Plant defense cascade – you have seen this previously with respect to hypersensitive response Pathogen strategy Remember that pathogens are trying to feed on the plant – as are herbivores – just different mechanisms Interestingly, successful pathogenic attack is less prevalent than herbivory because plants have evolved some very effective defenses to pathogens  ○ As new way, select for pathogens that are more resistant, plant pathology, what has increased in bacteria for the pathogen Microbe Attack Strategies Secrete cell wall degrading enzymes or toxins – necrotrophic pathogens Feed on host with minimal damage to plant tissue – biotrophic pathogens (coexist) Initially keep host cells alive – then damage them – hemibiotrophic pathogens Attack mode Plant pathogens produce effectors that change the plant’s structure or physiology to the advantage of the pathogen These effectors can be ○ Enzymes – cellulase, pectinase, cutinase are examples ○ Toxins – target specific proteins ○ Growth regulators – you already know an example of this from the fungus Gibberella Growth rate inhibitors can mess up physiology Plant responses Plants have pattern recognition receptors (PRRs) that detect microbes through “microbe associated molecular patterns” MAMPs (microbe presence) MAMPs are conserved among microbes, but absent in plant hosts - distinguish self from non-self ○ Conserve against microbes, cannot be something secreted Molecular “alarm bells” – damage associated molecular patterns – DAMPs are set off in the plant (actual damage has occurred) Systemin (response to herbivores) is now classified as a DAMP Note: DAMPs is also used in animal immunology for molecules that activate the immune system ○ DAMPs in animal vs plants are similar due to something being damaged Just to remind you Defenses in response to “danger signals” MAMPS – microbe associated molecular patterns (produced by microbes) DAMPS – danger associated molecular patterns (released from plant in response to degradation of cutin and cell wall by microbe attack) Bind to pattern recognition receptors (PRR) PRR triggered immunity (PTI) – left side of illustration – initiated Calcium signalling and transcription Inhibits growth of nonadapted pathogens RLCK = receptor-like cytoplasmic kinase ○ These danger signals include microbe-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs), and effectors. Extracellular MAMPs produced by microbes, and DAMPs released by microbial enzymes, bind to pattern recognition receptors (PRRs) on the cell surface. As plants coevolved with pathogens, the pathogens acquired effectors as virulence factors that mainly function to suppress PRR signaling. When MAMPs, DAMPs, and effectors bind to their PRRs and resistance (R) proteins, two types of defense responses are induced: PRR-triggered immunity and effector-triggered immunity. Receptor-like cytoplasmic kinases (RLCKs) are phosphorylated by receptor-like kinases, which are part of the PRR complex. NLR, nucleotide-binding site–leucine rich repeat protein; RBOH, respiratory burst oxidase homolog What about the right side of diagram? The evolution of R (resistance) proteins in pathogens has put selection pressure on plants to devise “back-up” pathways Effector-triggered immunity The presence of effectors blocks some of the PTI steps But an independent calcium channel is potentially responsible for HR (mechanisms here are not understood yet) NLR = nucleotide-binding site-leucine rich repeat protein Coevolution an important idea to keep in mind