The ups and downs of auxin A presentation on the action of auxin prepared by GTAC staff

Hormones signal cells to divide, elongate and differentiate to form tissues in plants Meristem = zone of actively dividing cells In dicots, the meristem is found at root and shoot tips Different in monocots (grasses). Where is the meristem? Does grass continue to grow even after mowing? Do you think the zone of division is in the tip or lower in grasses? Just use this slide to discuss different things that plant cells can do in response to hormones. It’s also good for students to think that this happens in zones in plant tissues as per the diagram. Dicotyledonous plant

This discovery forms the basis of plant tissue culture Plant tumours Henri-Louis Duhamel (1700–1782) French agriculturalist and tree expert Hmmm….. The wound has filled with cells forming a tumour A little history here. This handsome chap was the first to discover the essence of tissue culture through viewing cells clumping in response to wounding plants. This discovery forms the basis of plant tissue culture

Establishing a tissue culture Using sterile technique a piece Of plant is cut = explant This mass of dividing cells forms a callus. Cells in the callus are totipotent – they can differentiate into any sort of plant tissue depending upon the signal we give them. Small wound area. How can we increase the wound area? This slide is to help students understand what plant tissue culture is. It’s good to discuss the need to use sterile technique. We cut a piece of plant that becomes known as explant tissue. This is placed onto growth media to stimulate cells to grow and divide from wounded areas. These cells are totipotent (similar to stem cells in us) and can differentiate into different tissues given different hormones and hormone concentrations in the growth media. Make a longitudinal cut MS medium – contains all of the salts and organic molecules that plants need to grow

Effect of hormones on plant cell differentiation What will I be? Auxin (IAA) Cytokinin (kinetin) Totipotent cell Set up what the experiment is about. Adding different amounts of auxin and then adding in some cytokinin. They will need to look at concentrations and determine effects on the plant callus. The results are shown on the following slide. Callus Observe the tissue cultured cells on the following slide to determine the effects auxin can induce in cells

How auxin affects plant cells Complete Q. 1 - 3 This is what students need to analyse. You may need to also give them a printed pic of this. Tube 1 is the control – no hormone added. This is used to compare hormone treatments to. Tube 2 = some callus expansion but you will notice cell differentiation has taken place with root tissue forming as a result. Tube 3 = Cell differentiation into shoots Tube 4 = no differentiation but we see massive calllus expansion (due to cell division and cell elongation) I’m also providing the photos on the bionet as they may be clearer for students to observe differences. 1. No Hormones added 2. Low auxin No Cytokinin 3. Low auxin High cytokinin 4. High Auxin Low cytokinin

Plants responding to gravity Gravitropism Plants responding to gravity Watch the you tube clip (it should already be downloaded on the computer for you) While watching the video Discuss the direction of gravity on the plant and that the shoot is bending in the opposite direction to gravity = negative gravitropism, while the root is bending in the same direction as gravity = positive gravitropism. Answer Q 3. View and complete Q 4

How shoots respond to gravity Statoliths are heavy organelles that sediment at the base of cells in response to gravity. This is how a plant knows which way is down. Statoliths are heavy organelles in plant cells that sediment at the base of cells in response to gravity. They let the plant sense which way is up and which is down. If the pot falls over the plant shoot will respond to the gravity signal. You can see the statoliths move to the base of the cell.

Auxin enters and leaves cells through channels Auxin influx channel Auxin efflux channel Auxin can only enter and leave plant cells through transporters. In this case point out that there are influx transporters to let auxin in AND Efflux transporters to let auxin out of the cell. This is useful for plants accumulating auxin in specific tissues as they can move their transporters to specific parts of the membrane in response to signals.

A pathway for auxin accumulation in shoot tissue 1. Statoliths fall to bottom of cell 2. The enzyme, Phospholipase, is activated to snip off some lipid heads 3. Lipid head second messengers stimulate auxin efflux channels to move to base of cell This slide provides information for completing the stimulus response pathway for auxin accumulation in plant shoot tissue. Stimulus is gravity Receptors (receiving the signal) are statoliths The statoliths falling to the base of the cell activate Phopholipase enzymes (trigger them to change shape). These phopholipase enzymes then cut of the heads of some lipids in the cell membrane (off with their heads). These lipid heads act as secondary messengers. The secondary messengers travel through the cell and stimulate efflux transporters to relocate to the base of the cell. The efflux transporters are effectors as they cause auxin to accumulate directionally in shoot tissue as auxin leaves cells in one direction. This auxin accumulation on the lower side of shoot tissue is the Response Enzyme

Auxin leaves cells through efflux transporters and accumulates at base of shoot tissues This shows many cells in the plant shoot tissue that have relocated the efflux transporters to the base of the cell in response to gravity. You can see the auxin is now moving directionally through the shoot tissue so it will accumulate in the lower parts of the tissue. Complete Q. 5

Plant shoot bends upwards in response This is recapping what we know so far. The auxin accumulates at the base of shoot tissues. In response the shoot bends upwards. In response to gravity, auxin accumulates in cells on lower side of shoot Complete Q. 6

Formulate a hypothesis For a shoot to bend upwards it needs to grow longer on the lower side. What can happen to make one side grow longer? Is Auxin giving these cells a message to divide? OR B. Is Auxin giving these cells a message to elongate? A Why does the shoot bend upwards? Get them to decide from the two options in this powerpoint. We know the lower side must get longer than the upper side to make the shoot turn upwards. How can it get longer = growth. The question is…..is auxin stimulating the lower part of the shoot to grow through cell elongation OR through cell division? B Complete Q. 7

Design an experiment to explore action of auxin on oat shoots Cut 10 mm section here Complete Q. 8 Design your experiment Now try to get them to design and experiment using 10mm long pieces of oat shoot. They are provided with 10 in their materials (this experiment has been done for them as it has a long incubation time but they should think about the design before viewing the results) Why use 5 coleoptiles in each treatment? If we’re testing the effect of auxin on growth of the shoot tissue, what will the two treatment be? Control = Water Test = AUXIN solution When they have done their design, give them the results and they can get the average length of shoots in water compared to shoots in auxin. Put this in their results table and answer Q9. Treatment 1 Treatment 2 Incubate for 18 hours then find out average length of shoots in each treatment Complete Q. 9 - 11

Preparing a thin longitudinal section of coleoptile cells Use a scalpel to slice thin length-wise sections of a coleoptile that was immersed in water and one that was immersed in auxin. Add 2 drops of methylene blue stain onto a slide. Place a coleoptile section from each treatment on each drop, and label treatments W and A. Lower a coverslip onto the slide and view under the microscope at 100X. Now students use microscopy to test their hypothesis. Get them to try to see if cells in auxin are elongated in comparison to water. It’s tough. Spend 10 mins max on this microscopy task and then hand them the stickers of cells from each treatment to do their calculations. They get the average length of 3 cells from each test. It is obvious from this that auxin is stimulating cell elongation in these shoots. W A Cover slip Slide Coleoptile section Methylene blue Complete Q. 12 - 15

What’s happening in the roots? Where is auxin accumulating? Complete Q. 14 This will really challenge them. Show the animation and ask where auxin is accumulating in roots. Which way do they expect the root to bend based on: The video earlier What they just learned about auxin in shoot cells. Yes – it just doesn't figure. Show the animation for what the root does do. Ask if the effect of auxin on root cells is the same as the effect on shoot cells? I hope they say NO. Is the effect of auxin on root cells the same as the effect on shoot cells? Complete Q. 16 & 17

These proteins promote cell elongation Complete Q. 18 & 19 Auxin These proteins promote cell elongation These proteins Inhibit cell elongation Auxin transporter Root-specific proteins Degradation complex Shoot-specific proteins Gene regulator + inhibitor Auxin receptor This slide explains how it is possible for auxin to have so many different effects on cells…..signal transduction at the cellular level Auxin is involved in gene regulation. In order to get specific proteins produced, the genes for these proteins must be switched on. Genes are regulated in a number of ways. One way to stop a gene from being expressed is to block the action of a gene regulator (or block an “on switch”). Auxin is involved in breaking down the molecules that block these gene regulators (remove the molecule blocking gene “on switches”). Auxin removes inhibitory molecules from specific gene regulators. As a result the gene regulator can then go and bind to the DNA of the gene that it regulates so that gene expression is switched on and proteins are made. FOR EXAMPLE In the shoot cell: Gene Regulator + inhibitor cannot activate any genes. Auxin enters the cell via a transporter. (NB, this is active transport of ionised auxin. Unionised IAA can passively enter the cell by diffusion but, once inside, it immediately ionises and is trapped there.) The auxin binds its receptor causing the receptor to change shape. The auxin-receptor complex is now capable of binding a protein degradation complex. The supra-complex targets the inhibitor bound to the gene regulator for destruction. (NB, this simplifies a lot of steps involving a ubiquitin-based degradation pathway.) The gene regulator is now free to bind DNA at precise sites to switch on shoot-specific genes. Shoot-specific proteins are produced leading to a suite of cellular responses that cause the shoot cell to elongate. In the root cell: Analogous processes occur but act instead on a root-specific gene regulator leading to production of root-specific proteins. In this case, the response is the opposite leading to inhibition of cell elongation. Shoot-specific genes Root-specific genes Shoot-specific genes Root-specific genes Nucleus Nucleus Cytoplasm Cytoplasm Shoot cell Root cell